Difference between revisions of "Journal of Morphology 26 (1915)"

From Embryology
m (The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day)
m
 
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J. S. KINGSLEY
 
J. S. KINGSLEY
  
University of Illinois Urbana, 111.
+
University of Illinois Urbana, Ill.
  
 
with the collaboration of Gary N. Calkins Edwin G. Conklin C. E. McClung
 
with the collaboration of Gary N. Calkins Edwin G. Conklin C. E. McClung
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PHILADELPHIA
 
PHILADELPHIA
  
COMPOSED AND PRINTED AT THE WAVKRLY PRESS
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COMPOSED AND PRINTED AT THE WAVERLY PRESS
  
 
By the Williams & Wilkins Company
 
By the Williams & Wilkins Company
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E. A. Baumgartner. The development of the hypophysis in SqualusAcanthias. Forty-three figures ,391
 
E. A. Baumgartner. The development of the hypophysis in SqualusAcanthias. Forty-three figures ,391
  
J. Frank Daniel. The anatomy of Heterodontus francisci. II. The en doskeleton. Thirtj^-one figures (eight plates) 447
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J. Frank Daniel. The anatomy of Heterodontus francisci. II. The endoskeleton. Thirty-one figures (eight plates) 447
  
 
Robert W. Hegner. Studies on germ cells. IV. Protoplasmic differentiation in the oocytes of certain Hymenoptera. Ninety-seven figures (thirteen plates) 495
 
Robert W. Hegner. Studies on germ cells. IV. Protoplasmic differentiation in the oocytes of certain Hymenoptera. Ninety-seven figures (thirteen plates) 495
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James L. Kellogg. Ciliary mechanisms of lamellibranchs with descriptions of anatomy. Seventy-two figures 625
 
James L. Kellogg. Ciliary mechanisms of lamellibranchs with descriptions of anatomy. Seventy-two figures 625
  
A. T. Evans. The morphology of the frontal appendage of the male in the Phyllopod crustacean Thamnocephalus platyurus Packard. Nine figures. . 703
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A. T. Evans. The morphology of the frontal appendage of the male in the Phyllopod crustacean Thamnocephalus platyurus Packard. Nine figures. 703
  
 
==Oogenesis In Philosamia Cynthia==
 
==Oogenesis In Philosamia Cynthia==
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3 . Literature on the early development of eggs and nurse cells 22
 
3 . Literature on the early development of eggs and nurse cells 22
  
Summary • • 25
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Summary 25
  
 
Literature cited 27
 
Literature cited 27
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INTRODUCTION
 
INTRODUCTION
  
From the standpoint of sex production the Lepidoptera are of especial interest as compared with other insects, because the experimental evidence of Doncaster ('06, '08) and Raynor ('06), Punnett and Bateson ('08) seems to demand the assumption that there are two kinds of eggs in the moth. The absence of visible dimorphism in the spermatozoa of the Lepidoptera also lends probability to this hypothesis. In other groups of insects studied, the spermatozoa are often dimorphic. The eggs have been assumed to be all ahke, and this condition has
+
From the standpoint of sex production the Lepidoptera are of especial interest as compared with other insects, because the experimental evidence of Doncaster ('06, '08) and Raynor ('06), Punnett and Bateson ('08) seems to demand the assumption that there are two kinds of eggs in the moth. The absence of visible dimorphism in the spermatozoa of the Lepidoptera also lends probability to this hypothesis. In other groups of insects studied, the spermatozoa are often dimorphic. The eggs have been assumed to be all ahke, and this condition has been demonstrated by Morrill ('09) for certain coreid Hemiptera, by Morgan ('09) for phylloxerans, by Stevens ('06 a, '09) and von Baehr ('08, '09) for aphids.
 
 
1
 
 
 
JOnRNAI^ OF MOUPHOLOar, VOL. 2(5, NO. 1 MARCH, 1915
 
 
 
 
 
 
 
2 PAULINE H, DEDERER
 
 
 
been demonstrated by Morrill ('09) for certain coreid Hemiptera, by Morgan ('09) for phylloxerans, by Stevens ('06 a, '09) and von Baehr ('08, '09) for aphids.
 
  
 
In studying the history of the male germ cells in the moth Philosamia cynthia (Dederer '07), the spermatocytes were found to contain exactly similar groups of chromosomes. The same facts had been determined by Stevens ('06 b) and Cook ('10) for various other Saturniidae. Doncaster ('12) believes that in Pieris brassicae there is no dimorphism, either in the male or female germ cells. Recently, however. Seller ('13) has decribed two kinds of eggs in a lepidopteran. These two papers will be discussed later. The present work was undertaken with special reference to the question of dimorphism in the eggs. I wish to express my indebtedness to Prof. E. B. Wilson for valuable advice and criticism during the course of the investigation.
 
In studying the history of the male germ cells in the moth Philosamia cynthia (Dederer '07), the spermatocytes were found to contain exactly similar groups of chromosomes. The same facts had been determined by Stevens ('06 b) and Cook ('10) for various other Saturniidae. Doncaster ('12) believes that in Pieris brassicae there is no dimorphism, either in the male or female germ cells. Recently, however. Seller ('13) has decribed two kinds of eggs in a lepidopteran. These two papers will be discussed later. The present work was undertaken with special reference to the question of dimorphism in the eggs. I wish to express my indebtedness to Prof. E. B. Wilson for valuable advice and criticism during the course of the investigation.
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Eggs laid 2 to 2j hours fusion of pronuclei
 
Eggs laid 2 to 2j hours fusion of pronuclei
  
 
 
. OOGENESIS IN PHILOSAMIA 6
 
  
 
In the study of the early stages of oogenesis, both caterpillars and pupae were used. The ovaries in the early pupal stage lie enveloped in the fat bodies just beneath the dorsal wall of the abdomen in the fifth segment. Upon removing the wall, the ovaries were located and transferred immediately to the fixing fluid. Flemming's, Bouin's and Carnoy's fluids were used, the latter being the only one good for all stages. The stains employed were safranin and iron hematoxylin.
 
In the study of the early stages of oogenesis, both caterpillars and pupae were used. The ovaries in the early pupal stage lie enveloped in the fat bodies just beneath the dorsal wall of the abdomen in the fifth segment. Upon removing the wall, the ovaries were located and transferred immediately to the fixing fluid. Flemming's, Bouin's and Carnoy's fluids were used, the latter being the only one good for all stages. The stains employed were safranin and iron hematoxylin.
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Figure 32 is from a longitudinal section through the animal pole of an egg, showing a spindle with chromosomes in anaphase, within a dense granular mass, whose long protoplasmic processes reach out into the more reticular portion of the cytoplasm. This latter region is free from yolk, and is cone-shaped in form, the apex pointing inward, and extending as a sort of narrow vacuolated core, into the center of the egg. The remainder of the egg is filled with large yolk spheres. At the periphery of the egg appears a thin layer of dark granular protoplasm.
 
Figure 32 is from a longitudinal section through the animal pole of an egg, showing a spindle with chromosomes in anaphase, within a dense granular mass, whose long protoplasmic processes reach out into the more reticular portion of the cytoplasm. This latter region is free from yolk, and is cone-shaped in form, the apex pointing inward, and extending as a sort of narrow vacuolated core, into the center of the egg. The remainder of the egg is filled with large yolk spheres. At the periphery of the egg appears a thin layer of dark granular protoplasm.
 
 
 
4 PAULINE H. DEDERER
 
  
 
In earlier eggs, before or at the time of laying, a clear pale vitelline membrane may be seen beyond this. The polar bodies are formed in the dense granular layer, very near the middle of the anterior end, just within the cone-shaped area.
 
In earlier eggs, before or at the time of laying, a clear pale vitelline membrane may be seen beyond this. The polar bodies are formed in the dense granular layer, very near the middle of the anterior end, just within the cone-shaped area.
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The earliest nuclear stage obtained after the growth of the egg, is the late prophase, found in eggs which had not been laid (fig. 3). The chromosomes, 13 in number, lie enclosed within the nuclear membrane, near the surface of the egg, in the same position as the first maturation spindle. The chromosomes are smooth, elliptical or dumb-bell shaped bodies, almost equal in size. Later the nuclear wall breaks down, the spindle fibers appear and the chromosomes become arranged upon them preparatory to division (figs. 4-5). When first placed upon the spindle, the chromosomes do not all show a dyad form, but later a median constriction appears in each one. The spindle lies obliquely to the surface of the egg. The spindle fibers can rarely be traced to a point of convergence, and no centrosomes nor asters appear. Various cytoplasmic bodies lie near or attached to the spindle (fig. 4), but they are not constant in size or number, and often cannot be detected. They are present only during the metaphase.
 
The earliest nuclear stage obtained after the growth of the egg, is the late prophase, found in eggs which had not been laid (fig. 3). The chromosomes, 13 in number, lie enclosed within the nuclear membrane, near the surface of the egg, in the same position as the first maturation spindle. The chromosomes are smooth, elliptical or dumb-bell shaped bodies, almost equal in size. Later the nuclear wall breaks down, the spindle fibers appear and the chromosomes become arranged upon them preparatory to division (figs. 4-5). When first placed upon the spindle, the chromosomes do not all show a dyad form, but later a median constriction appears in each one. The spindle lies obliquely to the surface of the egg. The spindle fibers can rarely be traced to a point of convergence, and no centrosomes nor asters appear. Various cytoplasmic bodies lie near or attached to the spindle (fig. 4), but they are not constant in size or number, and often cannot be detected. They are present only during the metaphase.
  
In figures 6 and 7 are shown two first division groups. There is but slight difference in the size of the chromosomes, and each one appears to be separated into two equal parts. On account of their small size they were at first interpreted as chroraosomes of the second division, but a further study showed that this was not the case. There was no trace of a first polar body, nor of sperm within the egg. Moreover, the eggs had not been laid, but were taken from a moth which had just begun laying. Figures 4 and 5 and numerous other undoubted first metaphase stages, with larger chromosomes, were obtained from the same lot of eggs. Restaining and extraction had practically no effect in altering the size difference which remains unexplained. In twelve cases I found chromosome groups similar in size to those shown in figures 6 and 7. These figures seem to indicate that the chromosomes divide equally in the first division but a
+
In figures 6 and 7 are shown two first division groups. There is but slight difference in the size of the chromosomes, and each one appears to be separated into two equal parts. On account of their small size they were at first interpreted as chroraosomes of the second division, but a further study showed that this was not the case. There was no trace of a first polar body, nor of sperm within the egg. Moreover, the eggs had not been laid, but were taken from a moth which had just begun laying. Figures 4 and 5 and numerous other undoubted first metaphase stages, with larger chromosomes, were obtained from the same lot of eggs. Restaining and extraction had practically no effect in altering the size difference which remains unexplained. In twelve cases I found chromosome groups similar in size to those shown in figures 6 and 7. These figures seem to indicate that the chromosomes divide equally in the first division but a definite statement is unwarranted, for the chromosomes are so small that a shght size difference might easily escape detection; moreover the variability in size in different groups, as seen in figures 5 and 6 would render any deductions in this respect extremely hazardous.
 
 
 
 
 
 
OOGENESIS IN PHILOSAMIA 5
 
 
 
definite statement is unwarranted, for the chromosomes are so small that a shght size difference might easily escape detection; moreover the variability in size in different groups, as seen in figures 5 and 6 would render any deductions in this respect extremely hazardous.
 
  
 
As the chromosomes approach the ends of the spindle, the fibers thicken enormously in the middle, forming a deeply staining cell plate, which in side view, gives the appearance of a band encircling the spindle (fig. 8). Henking ('92) described similar bodies in Pieris, which he considered as waste achromatic substance. With long extraction the cell plate appears very faint, while the chromosomes remain dark. Figure 9 shows another anaphase, in which 13 chromosomes may be counted at each pole. No lagging chromosomes were observed. In a late anaphase (fig. 10) another size peculiarity is observed, each chromosome being after division approximately as large as those of the metaphase stage. The irregularity in the form of the chromosomes in late anaphase was characteristic of this stage, and was equally apparent with either dark or light staining, although in the latter case the chromosomes appeared slightly smaller. Figure 11 is an oblique polar view of a similar stage; on account of the plane of the section the spindle cannot be seen. In figures 12 to 14 are shown polar views of spindles, the groups lettered a in each case being those entering the first polar body. Four chromosomes are in the center of each group, surrounded by a ring of nine. The polar body groups sometimes appear slightly smoother in outline than the egg groups (fig. 12), smaller, and bipartite in preparation for a second division. In attempting to compare chromosomes in the polar body with those in a similar position in the egg group, it is impossible to obtain any evidence either for or against an equal division of chromosomes. The variability is extreme, within both egg and polar groups, and in many cases it is very difficult to be sure of the actual chromosome outlines.
 
As the chromosomes approach the ends of the spindle, the fibers thicken enormously in the middle, forming a deeply staining cell plate, which in side view, gives the appearance of a band encircling the spindle (fig. 8). Henking ('92) described similar bodies in Pieris, which he considered as waste achromatic substance. With long extraction the cell plate appears very faint, while the chromosomes remain dark. Figure 9 shows another anaphase, in which 13 chromosomes may be counted at each pole. No lagging chromosomes were observed. In a late anaphase (fig. 10) another size peculiarity is observed, each chromosome being after division approximately as large as those of the metaphase stage. The irregularity in the form of the chromosomes in late anaphase was characteristic of this stage, and was equally apparent with either dark or light staining, although in the latter case the chromosomes appeared slightly smaller. Figure 11 is an oblique polar view of a similar stage; on account of the plane of the section the spindle cannot be seen. In figures 12 to 14 are shown polar views of spindles, the groups lettered a in each case being those entering the first polar body. Four chromosomes are in the center of each group, surrounded by a ring of nine. The polar body groups sometimes appear slightly smoother in outline than the egg groups (fig. 12), smaller, and bipartite in preparation for a second division. In attempting to compare chromosomes in the polar body with those in a similar position in the egg group, it is impossible to obtain any evidence either for or against an equal division of chromosomes. The variability is extreme, within both egg and polar groups, and in many cases it is very difficult to be sure of the actual chromosome outlines.
  
 
During the formation of the first polar body the spindle fibers elongate considerably, and the granular cytoplasm forms a
 
During the formation of the first polar body the spindle fibers elongate considerably, and the granular cytoplasm forms a
 
 
 
6 PAULINE H. DEDERER
 
 
 
conspicuous projection on the surface of the egg (fig. 33). There is apparently no first telophase, for no loss of contour or massing of the chromosomes was observed between the late anaphase and the second metaphase.
 
conspicuous projection on the surface of the egg (fig. 33). There is apparently no first telophase, for no loss of contour or massing of the chromosomes was observed between the late anaphase and the second metaphase.
  
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In figure 15 A, B, drawn from adjacent sections, are shown 13 approximately equal chromosomes, arranged upon the two spindles, preparatory to a second division. It will be observed that the groups in the egg B and the first polar body A are at this time similar in the form and size of the chromosomes. The remnants of the cell plate are composed of deep-staining bodies, so large and definite as to give the appearance of chromosomes, but they are very irregular in form, and vary in size from large masses to very small granules. Figure 16 is another view of a similar stage. In figure 17, a polar view from three succeeding sections, the cell plate is composed of 15 large bodies, and numerous granules. One chromosome is missing from the egg group, B,
 
In figure 15 A, B, drawn from adjacent sections, are shown 13 approximately equal chromosomes, arranged upon the two spindles, preparatory to a second division. It will be observed that the groups in the egg B and the first polar body A are at this time similar in the form and size of the chromosomes. The remnants of the cell plate are composed of deep-staining bodies, so large and definite as to give the appearance of chromosomes, but they are very irregular in form, and vary in size from large masses to very small granules. Figure 16 is another view of a similar stage. In figure 17, a polar view from three succeeding sections, the cell plate is composed of 15 large bodies, and numerous granules. One chromosome is missing from the egg group, B,
  
For various reasons second anaphase stages were extremely diflficult to find. A few cases, however, were obtained, which seem fairly clear. In figure 19 is shown a spindle in which 13 chromosomes are seen at one pole, 11 at the other. Thi^ latter group, which is incomplete, enters the second polar body. The 13 chromosomes of the egg nucleus are very small rounded bodies nearly equal in size. There is nothing to indicate a pe
+
For various reasons second anaphase stages were extremely diflficult to find. A few cases, however, were obtained, which seem fairly clear. In figure 19 is shown a spindle in which 13 chromosomes are seen at one pole, 11 at the other. Thi^ latter group, which is incomplete, enters the second polar body. The 13 chromosomes of the egg nucleus are very small rounded bodies nearly equal in size. There is nothing to indicate a peculiarity in behavior of any of the chromosomes. In figure 20 polar views of two groups in anaphase are shown; here 13 approximately similar chromosomes appear in each. In figure 21, a polar view of an egg group, 13 chromosomes may be counted. Figures 23 and 24 are two oblique sections through spindles in anaphase. The groups in each case have been slightly displaced. There are 13 chromosomes in each group. These examples, while not numerous, are sufficient to show that the second polar body receives a group of chromosomes similar in number to those remaining in the egg. The small size of the chromosomes, and the lack of early anaphase stages, make it impossible, as in the case of the first division, to draw any conclusions as to the equal or unequal division of the chromosomes.
 
 
 
 
OOGENESIS IN PHILOSAMIA 7
 
 
 
culiarity in behavior of any of the chromosomes. In figure 20 polar views of two groups in anaphase are shown; here 13 approximately similar chromosomes appear in each. In figure 21, a polar view of an egg group, 13 chromosomes may be counted. Figures 23 and 24 are two oblique sections through spindles in anaphase. The groups in each case have been slightly displaced. There are 13 chromosomes in each group. These examples, while not numerous, are sufficient to show that the second polar body receives a group of chromosomes similar in number to those remaining in the egg. The small size of the chromosomes, and the lack of early anaphase stages, make it impossible, as in the case of the first division, to draw any conclusions as to the equal or unequal division of the chromosomes.
 
  
 
The first polar body was frequently observed in anaphase, during the stage figured above. After the second anaphase the egg chromosomes show a tendency to fusion (fig. 25) and it is impossible to distinguish separate chromosomes at either pole.
 
The first polar body was frequently observed in anaphase, during the stage figured above. After the second anaphase the egg chromosomes show a tendency to fusion (fig. 25) and it is impossible to distinguish separate chromosomes at either pole.
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In sections through eggs in the first metaphase stage, several spermatozoa may be seen within the vitelline membrane, but only occasionally within the egg. In late anaphase, sections show that the spermatozoon has penetrated into the egg and is enveloped in a dark granular island of cytoplasm. Numerous eggs were found containing two or three spermatozoa. Thus in P. cynthia, as in many other insects, polyspermy appears to be normal. Shortly after entering the egg, the sperm appears as a long tapering rod. Later, it has the form of an oval, deepstaining vesicle surrounded by a clear area, and in contact with the female pronucleus, which lies nearer the surface of the egg. Subsequently the male pronucleus becomes spherical, the clear area disappearing. The chromatin in both nuclei is in the form of irregular flocculent masses, at first darker in the male pronucleus. Later it has the same staining capacity in both, so that it is impossible to distinguish male and female except by position.
 
In sections through eggs in the first metaphase stage, several spermatozoa may be seen within the vitelline membrane, but only occasionally within the egg. In late anaphase, sections show that the spermatozoon has penetrated into the egg and is enveloped in a dark granular island of cytoplasm. Numerous eggs were found containing two or three spermatozoa. Thus in P. cynthia, as in many other insects, polyspermy appears to be normal. Shortly after entering the egg, the sperm appears as a long tapering rod. Later, it has the form of an oval, deepstaining vesicle surrounded by a clear area, and in contact with the female pronucleus, which lies nearer the surface of the egg. Subsequently the male pronucleus becomes spherical, the clear area disappearing. The chromatin in both nuclei is in the form of irregular flocculent masses, at first darker in the male pronucleus. Later it has the same staining capacity in both, so that it is impossible to distinguish male and female except by position.
 
 
 
8 PAULINE H. DEDERER
 
  
 
At a later period, it is possible to count the chromosomes in each nucleus. In figure 35 the pronuclei lie near the surface of the egg where the second polar body appears. The surface cytoplasm merges with the remnants of the vitelline membrane, in which the polar body lies. Figure 27 is the same section enlarged. The pronuclear walls appear broken at the region of contact, or are so thin as to be invisible. Nine chromosomes are seen in the first section of the outer nucleus, 11 in the first section of the inner; they differ s ightly in size, and some are noticeably dyad in form. To the right of these are drawn portions of the nuclei from a succeeding section, showing 4 more chromosomes in the outer nucleus, 2 more n the inner, making 13 in each. No nucleoli are present. Within the polar body figured here remains of spindle fibers and a nuclear membrane are seen. Here too, 13 chromosomes appear. This is probably the second polar body, for the first becomes very vague after the second anaphase.
 
At a later period, it is possible to count the chromosomes in each nucleus. In figure 35 the pronuclei lie near the surface of the egg where the second polar body appears. The surface cytoplasm merges with the remnants of the vitelline membrane, in which the polar body lies. Figure 27 is the same section enlarged. The pronuclear walls appear broken at the region of contact, or are so thin as to be invisible. Nine chromosomes are seen in the first section of the outer nucleus, 11 in the first section of the inner; they differ s ightly in size, and some are noticeably dyad in form. To the right of these are drawn portions of the nuclei from a succeeding section, showing 4 more chromosomes in the outer nucleus, 2 more n the inner, making 13 in each. No nucleoli are present. Within the polar body figured here remains of spindle fibers and a nuclear membrane are seen. Here too, 13 chromosomes appear. This is probably the second polar body, for the first becomes very vague after the second anaphase.
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2. CONCLUSIONS AND COMPARISONS
 
2. CONCLUSIONS AND COMPARISONS
  
The evidence obtained from the foregoing study indicates that in Philosamia cynthia the 13 chromosomes seen in the late prophase of the egg all divide in both maturation divisions. The male and female pronuclei at the time of their union each contain 13 chromosomes, giving the somatic number 26, which is found in the nuclei of the blastoderm. It appears to be certain that all of the eggs contain the same number of chromosomes, but the evidence for either the presence or absence of an XY-pair is not conclusive, on account of the variability in the size of the chromosomes in the metaphase and anaphase plates. In the early oogenesis, to be described later, there is no indication of
+
The evidence obtained from the foregoing study indicates that in Philosamia cynthia the 13 chromosomes seen in the late prophase of the egg all divide in both maturation divisions. The male and female pronuclei at the time of their union each contain 13 chromosomes, giving the somatic number 26, which is found in the nuclei of the blastoderm. It appears to be certain that all of the eggs contain the same number of chromosomes, but the evidence for either the presence or absence of an XY-pair is not conclusive, on account of the variability in the size of the chromosomes in the metaphase and anaphase plates. In the early oogenesis, to be described later, there is no indication of the presence of a heterochromosome, either of equal or unequal parts, and from this we might suspect its absence in later stages. Although from the totality of the evidence, it appears probable that there is no difference in the chromosome groups, the matter will have to be left an open question.
  
 +
Doncaster ('12) found that in Pieris brassicae both the male and female germ cells contain an equally paired heterochromosome which constitutes a chromatin nucleus during the growth period. He believed that in Abraxas a similar condition probably prevailed, and concluded that the chromosomes here do not provide any visible basis for the sex-limited transmission of characters." More recently, however ('13) he has found some females of Abraxas with 56 chromosomes, some w'th 55, and he beheves that there s a possibility of two kinds of eggs in this form.
  
 +
Until the past year, the only recorded case of nuclear dimorphism in eggs (exclusive of parthenogenetic and sexual eggs) was that of the sea-urchin described by Baltzer ('09), in which the female appeared to be the heterogametic sex. Tennant, ('12) however, discovered that in other forms the male is heterogametic. Baltzer has recently ('13) announced that the results described in his former paper are erroneous, and he is convinced that the male is the heterogametic sex. This solves the apparent contradiction within the echinoderm group, the females being homozygous for sex in all cases described.
  
OOGENESIS IN PHILOSAMIA 9
+
The latest case of heterogamy in the female is that recently described by Seller ('13) for the lepidopteran Phragmatobia fuliginosa. In the spermatocyte divisions, 27 small chromosomes are present, and a large one, which, though lagging somewhat, divides equally in both maturation divisions. In the first metaphase plate of the egg, 27 small chromosomes and a large chromosome, slightly segmented or lobed appear. After the first division, at one pole of the spindle are seen 27 small chromosomes and a large one; at the other pole, 28 small chromosomes and a large one. It is a matter of chance whether the polar body or the egg nucleus receives the extra chromosome. Seller interprets the extra small chromosome as a lobe of the large autosome which has separated from it during division, since in anaphase a small chromatin mass lies near one end of the large chromosome as if detached from it. Second divisions were not observed but Seller believes they are probably equational. He suggests the tentative interpretation that the extra small chromosome is the X chromosome. Unfortunately, only polar views of the first division are given, and these only of late anaphase, so it is impossible to determine how the extra chromosome arises. It is possible that the separation of this chromosome (described by Seller) inay be merely a temporary condition, followed by a union with the large one at the second metaphase, thus giving similar groups of chromosomes in all the oocytes. In view of the fact that in echinoderm eggs an apparently clear case of dimorphism has been found to be incorrect, it seems particularly necessary "to scrutinize carefully any evidence along this line.
  
the presence of a heterochromosome, either of equal or unequal parts, and from this we might suspect its absence in later stages. Although from the totality of the evidence, it appears probable that there is no difference in the chromosome groups, the matter will have to be left an open question.
+
EARLY OOGENESIS
 
 
Doncaster ('12) found that in Pieris brassicae both the male and female germ cells contain an equally paired heterochromosome which constitutes a chromatin nucleus during the growth period. He believed that in Abraxas a similar condition probably prevailed, and concluded that the chromosomes here do not provide any visible basis for the sex-limited transmission of characters." More recently, however ('13) he has found some females of Abraxas with 56 chromosomes, some w'th 55, and he beheves that there s a possibility of two kinds of eggs in this form.
 
 
 
Until the past year, the only recorded case of nuclear dimorphism in eggs (exclusive of parthenogenetic and sexual eggs) was that of the sea-urchin described by Baltzer ('09), in which the female appeared to be the heterogametic sex. Tennant, ('12) however, discovered that in other forms the male is heterogametic. Baltzer has recently ('13) announced that the results described in his former paper are erroneous, and he is convinced that the male is the heterogametic sex. This solves the apparent contradiction within the echinoderm group, the females being homozygous for sex in all cases described.
 
 
 
The latest case of heterogamy in the female is that recently described by Seller ('13) for the lepidopteran Phragmatobia fuliginosa. In the spermatocyte divisions, 27 small chromosomes are present, and a large one, which, though lagging somewhat, divides equally in both maturation divisions. In the first metaphase plate of the egg, 27 small chromosomes and a large chromosome, slightly segmented or lobed appear. After the first division, at one pole of the spindle are seen 27 small chromosomes and a large one; at the other pole, 28 small chromosomes and a large one. It is a matter of chance whether the polar body or the egg nucleus receives the extra chromosome. Seller interprets the extra small chromosome as a lobe of the large
 
 
 
 
 
 
 
10 PAULINE H. DEDERER
 
 
 
autosome which has separated from it during division, since in anaphase a small chromatin mass lies near one end of the large chromosome as if detached from it. Second divisions were not observed but Seller believes they are probably equational. He suggests the tentative interpretation that the extra small chromosome is the X chromosome. Unfortunately, only polar views of the first division are given, and these only of late anaphase, so it is impossible to determine how the extra chromosome arises. It is possible that the separation of this chromosome (described by Seller) inay be merely a temporary condition, followed by a union with the large one at the second metaphase, thus giving similar groups of chromosomes in all the oocytes. In view of the fact that in echinoderm eggs an apparently clear case of dimorphism has been found to be incorrect, it seems particularly necessary "to scrutinize carefully any evidence along this line.
 
 
 
EARLY OOGENESIS
 
  
 
A study of the early oogenesis of P. cynthia was undertaken in order to determine the origin of the haploid groups of chromosomes which enter the first polar metaphase. By analogy with spermatogenesis, pairing of the chromosomes in the egg should occur before the growth period. Although the material is unfavorable for the study of oogenesis as a whole, a seriation of stages was obtained, and several points of interest were observed in regard to the tlifferentiation of primitive ovarian -cells into eggs and nurse cells, and their later relation to each other.
 
A study of the early oogenesis of P. cynthia was undertaken in order to determine the origin of the haploid groups of chromosomes which enter the first polar metaphase. By analogy with spermatogenesis, pairing of the chromosomes in the egg should occur before the growth period. Although the material is unfavorable for the study of oogenesis as a whole, a seriation of stages was obtained, and several points of interest were observed in regard to the tlifferentiation of primitive ovarian -cells into eggs and nurse cells, and their later relation to each other.
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The earliest ovaries obtained were from larvae fixed the latter part of August, a few days before the spinning of the cocoon. They are pear-shaped bodies, about 1 mm. in length, slightly smaller than a mature egg. Figure 29 is a lengthwise section through a larval ovary. The oval mass of connective tissue surrounds four egg strings which take a complicated course
 
The earliest ovaries obtained were from larvae fixed the latter part of August, a few days before the spinning of the cocoon. They are pear-shaped bodies, about 1 mm. in length, slightly smaller than a mature egg. Figure 29 is a lengthwise section through a larval ovary. The oval mass of connective tissue surrounds four egg strings which take a complicated course
 
 
 
OOGENESIS IN PHILOSAMIA 11
 
 
 
within the capsule. The strings open into a single slightlyexpanded chamber at the surface of the ovary, from which the oviduct arises. The earliest eggs are found near the opposite end of the ovary. Oogonial stages to very early eggs were found in this and similar ovaries. Figure 30 is of a January ovary, showing an increase in size and the growth of the egg strings. Two strings are broken away from the oviduct, but their points of attachment may be seen. The stages in this ovary ranged from a few spiremes to well-developed eggs, each with its five nurse cells contained in a separate chamber in the string. The ovaries of early June were practically identical in size with those of January. AH the cells by this time have differentiated into eggs and nurse cells. In early July, the ovaries are markedly different. Figure 31 shows portions of three egg strings from a pupa about three days before the time of emergence. The ovary now consists entirely of egg strings with a decidedly beaded appearance due to the growth of the eggs.
 
within the capsule. The strings open into a single slightlyexpanded chamber at the surface of the ovary, from which the oviduct arises. The earliest eggs are found near the opposite end of the ovary. Oogonial stages to very early eggs were found in this and similar ovaries. Figure 30 is of a January ovary, showing an increase in size and the growth of the egg strings. Two strings are broken away from the oviduct, but their points of attachment may be seen. The stages in this ovary ranged from a few spiremes to well-developed eggs, each with its five nurse cells contained in a separate chamber in the string. The ovaries of early June were practically identical in size with those of January. AH the cells by this time have differentiated into eggs and nurse cells. In early July, the ovaries are markedly different. Figure 31 shows portions of three egg strings from a pupa about three days before the time of emergence. The ovary now consists entirely of egg strings with a decidedly beaded appearance due to the growth of the eggs.
  
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, Stage a {fig. 37 a): The oogonial region containing cells in various stages of final oogonial divisions and in rest before these divisions. A polar view (fig. 2) of a metaphase plate shows 26 chromosomes. In anaphase stages no lagging chromosomes were observed, nor differential divisions, such as have been described in several insects (Buchner '09, Gtinthert '10).
 
, Stage a {fig. 37 a): The oogonial region containing cells in various stages of final oogonial divisions and in rest before these divisions. A polar view (fig. 2) of a metaphase plate shows 26 chromosomes. In anaphase stages no lagging chromosomes were observed, nor differential divisions, such as have been described in several insects (Buchner '09, Gtinthert '10).
  
Stage b {fig. 36): Post-oogonial nuclei. Here the chromatin assumes the form of deep-staining bodies with ragged and irregular outhnes. No constant number can be counted. The cells are connected by dense protoplasmic strands or tubes, which appear to originate from the spindle remains of the oogonial divisions. Gtinthert ('10) figured similar connections between eggs and nurse cells in the oogonia of Dytiscus. In figures 41 and 42 are shown two tubes with their branches appearing to terminate in rounded knobs, which are merely the upturned.
+
Stage b {fig. 36): Post-oogonial nuclei. Here the chromatin assumes the form of deep-staining bodies with ragged and irregular outhnes. No constant number can be counted. The cells are connected by dense protoplasmic strands or tubes, which appear to originate from the spindle remains of the oogonial divisions. Gtinthert ('10) figured similar connections between eggs and nurse cells in the oogonia of Dytiscus. In figures 41 and 42 are shown two tubes with their branches appearing to terminate in rounded knobs, which are merely the upturned ends of the branches. The largest number of branches observed was 6 and this is probably the correct number as sections of later ovaries show that each egg cell is connected with five nurse cells.
 
 
 
 
 
 
12 PAULINE H. DEDERER
 
 
 
ends of the branches. The largest number of branches observed was 6 and this is probably the correct number as sections of later ovaries show that each egg cell is connected with five nurse cells.
 
  
 
Stage c (figs. 38 and 39). The chromosomes are transformed into smaller irregular fragments which later assume the form of pale delicate threads. There is no trace of the uncoiling, of convoluted threads from the chromatin masses to form the leptotene stage, as described by Davis ('08) and Wilson ('12) in the spermatogenesis of insects.
 
Stage c (figs. 38 and 39). The chromosomes are transformed into smaller irregular fragments which later assume the form of pale delicate threads. There is no trace of the uncoiling, of convoluted threads from the chromatin masses to form the leptotene stage, as described by Davis ('08) and Wilson ('12) in the spermatogenesis of insects.
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From this point on*, a gradual differentiation occurs between eggs and nurse cells, so that it is convenient to treat the two separately. The further nuclear changes in the nurse cells will be considered first.
 
From this point on*, a gradual differentiation occurs between eggs and nurse cells, so that it is convenient to treat the two separately. The further nuclear changes in the nurse cells will be considered first.
  
 
 
OOGENESIS IN PHILOSAMIA 13
 
  
 
c. Development of nurse cells
 
c. Development of nurse cells
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Stage g (figure 48): The diplotene stage. A longitudinal split now appears for the first time in all the chroniosomes, and shows very clearly in cells which lie directly in contact with those of the preceding period which show no split. They are differentiated from them also in length of the chromosomes, for the split threads are considerably shorter. Doncaster ('12) describes in Abraxas the double thi'ead as arising probably by a bending over of the chromosome, with a separation later at the bend, but this is certainly not the case in P. cynthia. There is no clue whatever to the relation of these double threads to
 
Stage g (figure 48): The diplotene stage. A longitudinal split now appears for the first time in all the chroniosomes, and shows very clearly in cells which lie directly in contact with those of the preceding period which show no split. They are differentiated from them also in length of the chromosomes, for the split threads are considerably shorter. Doncaster ('12) describes in Abraxas the double thi'ead as arising probably by a bending over of the chromosome, with a separation later at the bend, but this is certainly not the case in P. cynthia. There is no clue whatever to the relation of these double threads to
 
 
 
14 PAULINE H. DEDERER
 
 
 
the chromosomes of the oogonia, since the spht appears de novo, and also since there is no direct evidence that the chromatin threads have conjugated in synapsis. Unfortunately, therefore, P. cynthia cannot be added to the list of forms in which either parasynapsis or telosynapsis has been observed. The impression gained from a study of the material is that reduction is accomplished by a simple segmentation of a continuous spireme into the haploid number of threads.
 
the chromosomes of the oogonia, since the spht appears de novo, and also since there is no direct evidence that the chromatin threads have conjugated in synapsis. Unfortunately, therefore, P. cynthia cannot be added to the list of forms in which either parasynapsis or telosynapsis has been observed. The impression gained from a study of the material is that reduction is accomplished by a simple segmentation of a continuous spireme into the haploid number of threads.
  
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With further growth of the cell, the nucleus increases in size, and the chromatin fragments multiply considerably. Giardina ('01), Debaisieux ('09) and Gunthert ('10) have figured in the
 
With further growth of the cell, the nucleus increases in size, and the chromatin fragments multiply considerably. Giardina ('01), Debaisieux ('09) and Gunthert ('10) have figured in the
 +
Dytiscidae a markedly regular division of tetrads, each part giving rise to a whole tetrad, this process being repeated several times. In P. cynthia there is no evidence of any order in the fragmentation, for there is the greatest irregularity in the size and shape of the pieces.
  
 +
The period of fragmentation marks the first broad phase in the history of the nurse cells. It is interesting to note that the cells have passed through a cycle of changes as if for maturation divisions, since they show the reduced number of chromosomes. These are destined, however, only for disintegration.
  
 
+
Stages j to I. At the beginning of this phase, the eggs and nurse cells are practically similar in size (fig. 37 e). The egg cell increases steadily in size during the growth period, the nurse cells, although increasing for a time, do not keep pace with the growth of the egg, and become relatively smaller as development proceeds.
OOGENESIS IN PHILOSAMIA 15
 
 
 
Dytiscidae a markedly regular division of tetrads, each part giving rise to a whole tetrad, this process being repeated several times. In P. cynthia there is no evidence of any order in the fragmentation, for there is the greatest irregularity in the size and shape of the pieces.
 
 
 
The period of fragmentation marks the first broad phase in the history of the nurse cells. It is interesting to note that the cells have passed through a cycle of changes as if for maturation divisions, since they show the reduced number of chromosomes. These are destined, however, only for disintegration.
 
 
 
Stages j to I. At the beginning of this phase, the eggs and nurse cells are practically similar in size (fig. 37 e). The egg cell increases steadily in size during the growth period, the nurse cells, although increasing for a time, do not keep pace with the growth of the egg, and become relatively smaller as development proceeds.
 
  
 
Stage j (figs. 37 e and 53): At the close of fragmentation, numerous small dark granules lie within the nucleus near the periphery, together with a variable number of larger round bodies, which, although stained very deeply in some sections, are very pale in others, and appear to be of the nature of plasmosomes. The cell contents appear shghtly granular, or reticular, with a very darkly granular, flask-shaped area extending from the nuclei toward one end of the cell, appearing to perforate the cell wall in the form of a curved tube which enters the egg cell. Marshall ('07), Giinthert ('10) and others describe similar areas, but the tubular portion is not apparent. A later stage is shown in figure 54. The nuclear wall is less easily seen on the side nearest the tubes, for the granules are thickest at this point, and lie close to the dark granular cytoplasm. In addition to this mass of granules, the nucleus contains much smaller masses scattered near the periphery, and several small plasmosomes. A thin dark band of cytoplasm often encircles the nucleus, merging with the flask-shaped portion. Beyond this the cytoplasm appears reticular. Figure 55 A shows another cell in which the nuclear cavity is indented in two regions, giving a somewhat dumb-bell shape. During these changes in the shape of the
 
Stage j (figs. 37 e and 53): At the close of fragmentation, numerous small dark granules lie within the nucleus near the periphery, together with a variable number of larger round bodies, which, although stained very deeply in some sections, are very pale in others, and appear to be of the nature of plasmosomes. The cell contents appear shghtly granular, or reticular, with a very darkly granular, flask-shaped area extending from the nuclei toward one end of the cell, appearing to perforate the cell wall in the form of a curved tube which enters the egg cell. Marshall ('07), Giinthert ('10) and others describe similar areas, but the tubular portion is not apparent. A later stage is shown in figure 54. The nuclear wall is less easily seen on the side nearest the tubes, for the granules are thickest at this point, and lie close to the dark granular cytoplasm. In addition to this mass of granules, the nucleus contains much smaller masses scattered near the periphery, and several small plasmosomes. A thin dark band of cytoplasm often encircles the nucleus, merging with the flask-shaped portion. Beyond this the cytoplasm appears reticular. Figure 55 A shows another cell in which the nuclear cavity is indented in two regions, giving a somewhat dumb-bell shape. During these changes in the shape of the
 
 
 
16 PAULINE H. DEDERER
 
 
 
nucleus, the plasmosomes are extremely variable. Figure 55 B is a plasmosome from a similar nucleus, very irregular in form and encrusted with chromatin granules. In some cases the granules adhere in such numbers as practically to obscure the plasmosome.
 
nucleus, the plasmosomes are extremely variable. Figure 55 B is a plasmosome from a similar nucleus, very irregular in form and encrusted with chromatin granules. In some cases the granules adhere in such numbers as practically to obscure the plasmosome.
  
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In figure 57 a later stage of the nurse cells is shown, drawn to the same scale as figure 56. Here the nurse cells are considerably larger than before, yet smaller than the egg. The flaskshaped region, circular granular region and nuclear cavity appear as previously indicated. The plasmosomes are covered with granules, and single strands of more prominent granules partly line and extend, down into the circular region. Only two nurse cells are figured here. The total number for each egg is five, which can be readily determined by following through a series of transverse sections. Gross ('03) also found five in other Lepidoptera. In figure 57 and other similar sections, the follicle cells are arranged in a layer around the groups of eggs and
 
In figure 57 a later stage of the nurse cells is shown, drawn to the same scale as figure 56. Here the nurse cells are considerably larger than before, yet smaller than the egg. The flaskshaped region, circular granular region and nuclear cavity appear as previously indicated. The plasmosomes are covered with granules, and single strands of more prominent granules partly line and extend, down into the circular region. Only two nurse cells are figured here. The total number for each egg is five, which can be readily determined by following through a series of transverse sections. Gross ('03) also found five in other Lepidoptera. In figure 57 and other similar sections, the follicle cells are arranged in a layer around the groups of eggs and
 
 
 
OOGENESIS IN FHILOSAMIA 17
 
 
 
nurse cells, first definitely formed at the periphery of the egg string, then growing in at the base of the egg cell. Later they grow in between the egg and its nurse cells, separating them except in the region of the tubes.
 
nurse cells, first definitely formed at the periphery of the egg string, then growing in at the base of the egg cell. Later they grow in between the egg and its nurse cells, separating them except in the region of the tubes.
  
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After the nurse cells are well differentiated, the spireme of the egg cell appears much less convoluted (fig. 59 A), spreading out through the nuclear cavity, which has increased considerably in size. In figure 59 B is shown a portion of the spireme which was not included in the first section. It is not possible to determine accurately if the spireme is continuous, but it is my belief that this is the case. During this period, one or two large plasmosomes may appear, and frequently two smaller bodies, probably of the same nature. The entire nucleus has the yellow tinge noted in the earliest stage of its differentiation.
 
After the nurse cells are well differentiated, the spireme of the egg cell appears much less convoluted (fig. 59 A), spreading out through the nuclear cavity, which has increased considerably in size. In figure 59 B is shown a portion of the spireme which was not included in the first section. It is not possible to determine accurately if the spireme is continuous, but it is my belief that this is the case. During this period, one or two large plasmosomes may appear, and frequently two smaller bodies, probably of the same nature. The entire nucleus has the yellow tinge noted in the earliest stage of its differentiation.
 
JODRNAI, of .\rOUPHOLOGY, VOL. 26, NO. 1
 
 
  
 
18 PAULINE H. DEDERER
 
  
 
The cells next to be described are taken from sections of ovaries fixed in January, later than the preceding sections. Figure 57, already referred to, shows a portion of an egg string just beyond its point of emergence from the ovary proper. Figure 60 is a nucleus from a similar egg. The spireme is typical for the eggs at this period; it is still convoluted as before, with no trace of a longitudinal split. The plasmosome varies considerably in form, consisting usually of a dark spherical portion, and a light portion, sometimes lobed and vacuolated. In this figure the plasmosome gives the appearance of breaking through the nuclear membrane, and in another egg near by a similar body was observed lying in the reticular cytoplasm at a little distance from the nucleus. A few other cases were observed on the same slide. The material appeared to be unusually well fixed, but as other ovaries failed to show a similar condition, this is probably not a normal occurrence.
 
The cells next to be described are taken from sections of ovaries fixed in January, later than the preceding sections. Figure 57, already referred to, shows a portion of an egg string just beyond its point of emergence from the ovary proper. Figure 60 is a nucleus from a similar egg. The spireme is typical for the eggs at this period; it is still convoluted as before, with no trace of a longitudinal split. The plasmosome varies considerably in form, consisting usually of a dark spherical portion, and a light portion, sometimes lobed and vacuolated. In this figure the plasmosome gives the appearance of breaking through the nuclear membrane, and in another egg near by a similar body was observed lying in the reticular cytoplasm at a little distance from the nucleus. A few other cases were observed on the same slide. The material appeared to be unusually well fixed, but as other ovaries failed to show a similar condition, this is probably not a normal occurrence.
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Stage g: Disappearance of the spireme in the later growth period. The next stages figured are sections from the ovary shown in figure 31, from a moth fixed a few days before the time
 
Stage g: Disappearance of the spireme in the later growth period. The next stages figured are sections from the ovary shown in figure 31, from a moth fixed a few days before the time
 
 
 
OOGENESIS IN PHILOSAMIA 19
 
 
 
of emergence. In a few of the youngest eggs in this material, the spireme is still vaguely discernible (fig. 61) in the form of a pale network of irregular threads joined together, not the coiled spireme of earlier eggs. Several dark bodies of irregular size and shape are characteristic of this period. In figure 62 — a slightly older nucleus in the same string — all traces of the spireme have disappeared. The nuclear cavity contains a pale body with a large vacuole, and numerous smaller rounded masses, which frequently stain veiy deeply. These are probably all plasmosomes. There is extreme variability in respect to their number, size and appearance, some being apparently homogeneous, others filled with vacuoles. Figure 63 is a nucleus of about the same age as figure 62.
 
of emergence. In a few of the youngest eggs in this material, the spireme is still vaguely discernible (fig. 61) in the form of a pale network of irregular threads joined together, not the coiled spireme of earlier eggs. Several dark bodies of irregular size and shape are characteristic of this period. In figure 62 — a slightly older nucleus in the same string — all traces of the spireme have disappeared. The nuclear cavity contains a pale body with a large vacuole, and numerous smaller rounded masses, which frequently stain veiy deeply. These are probably all plasmosomes. There is extreme variability in respect to their number, size and appearance, some being apparently homogeneous, others filled with vacuoles. Figure 63 is a nucleus of about the same age as figure 62.
  
 
As the eggs increase in size, the nuclei appear to have at one side a darker region, frequently crescentic (fig. 64), which seems to be connected with a dark granular protoplasmic strand running down into the cytoplasmic region of the egg, now coneshaped, as in the mature egg. The nucleus is party surrounded by yolk spheres, lying in faintly granular cytoplasm. The crescentic region merges gradually into the lighter granular portion of the nucleus, and suggests merely a condensation of the nucleoplasm here. Over twenty-five nuclei of this stage were examined, after varying degrees of extraction. In many cases the contents of the darker region were visible, and all showed the same condition of darker granules merging into lighter ones. No plasmosomes were to be seen, nor any trace of chromatin. The nuclei lie near the periphery of the eggs in the cytoplasmic region near the nurse cells, which at this time are reduced to shrunken remnants. Later the nuclear wall seems to fade out at the side nearest the periphery, and several bipartite rod-like chromosomes may be seen within the nucleus. At a slightly later period, the chromosomes, now shorter and more dumb-bell-shaped, appear to lie in a rounded area in which no distinct nuclear boundary is discernible. In the latest prophase (fig. 3) the nuclear boundary reappears, very faint, and very much smaller than the former nuclear area.
 
As the eggs increase in size, the nuclei appear to have at one side a darker region, frequently crescentic (fig. 64), which seems to be connected with a dark granular protoplasmic strand running down into the cytoplasmic region of the egg, now coneshaped, as in the mature egg. The nucleus is party surrounded by yolk spheres, lying in faintly granular cytoplasm. The crescentic region merges gradually into the lighter granular portion of the nucleus, and suggests merely a condensation of the nucleoplasm here. Over twenty-five nuclei of this stage were examined, after varying degrees of extraction. In many cases the contents of the darker region were visible, and all showed the same condition of darker granules merging into lighter ones. No plasmosomes were to be seen, nor any trace of chromatin. The nuclei lie near the periphery of the eggs in the cytoplasmic region near the nurse cells, which at this time are reduced to shrunken remnants. Later the nuclear wall seems to fade out at the side nearest the periphery, and several bipartite rod-like chromosomes may be seen within the nucleus. At a slightly later period, the chromosomes, now shorter and more dumb-bell-shaped, appear to lie in a rounded area in which no distinct nuclear boundary is discernible. In the latest prophase (fig. 3) the nuclear boundary reappears, very faint, and very much smaller than the former nuclear area.
 
 
 
20 PAULINE H. DEDERER
 
  
 
e. Degenerating cells in the ovary
 
e. Degenerating cells in the ovary
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Differential divisions in the oogonia, which have been described for the Dytiscidae, are not found in P. c^ynthia. The germ cells all appear similar in size until the post-synaptic spireme stage, agreeing in this respect with Pieris and Abraxas (Griinberg '03, Doncaster '12), the bee (Paulcke '00), and the di^.gon-fly (McGill '06, Marshall '07). Doncaster finds a differentiation appearing a little later than in P. cynthia, when the chromatin threads shorten to form bipartite chromosomes in the nurse
 
Differential divisions in the oogonia, which have been described for the Dytiscidae, are not found in P. c^ynthia. The germ cells all appear similar in size until the post-synaptic spireme stage, agreeing in this respect with Pieris and Abraxas (Griinberg '03, Doncaster '12), the bee (Paulcke '00), and the di^.gon-fly (McGill '06, Marshall '07). Doncaster finds a differentiation appearing a little later than in P. cynthia, when the chromatin threads shorten to form bipartite chromosomes in the nurse
 +
cells. The egg spireme is not continuous, but is composed of the haploid number of interlaced threads, which have not yet contracted. Marshall ('07) found a still later dilTerentiation in Platyphylax, a neuropteran, in which the tetrad stage is common to both kinds of cells, but the egg cell is larger, and the tetrads persist longer before disintegration.
  
 +
The haploid number of chromatin segments is present in the nurse cells of P. cynthia, as in Pieris (Doncaster '12), indicating a preparation for division in these cells whose function is only nutritive. A number of writers, including Griinberg ('03), Gross ('03), Marshall ('07), and Woltereck ('98), figure tetrads in the nurse cells of various animals, but do not state whether the haploid number is present. They agree, however, that differentiation of eggs and nurse cells occurs after synapsis, which would imply that nurse cells as well as eggs must have undergone pseudo-reduction.
  
 
+
A transfer of material takes place from the nurse cells and the egg through the connecting tubes which in P. cynthia have very prominent walls. A markedly similar condition was observed by Giinthert ('10) in Dytiscus, where converging bundles of fibrils appear, beset with chromidia or chromatin granules which enter the egg. In this case there is no definite wall to the tubes, Griinberg ('03j states that in Pieris the egg sends a large blunt process up between the nearest nurse cells. Evidently there is considerable variation in the relation of eggs and nurse cells within the Lepidoptera, for in P. cynthia it is the nurse cells which send processes into the egg.
OOGENESIS IN PHILOSAMIA 21
 
 
 
cells. The egg spireme is not continuous, but is composed of the haploid number of interlaced threads, which have not yet contracted. Marshall ('07) found a still later dilTerentiation in Platyphylax, a neuropteran, in which the tetrad stage is common to both kinds of cells, but the egg cell is larger, and the tetrads persist longer before disintegration.
 
 
 
The haploid number of chromatin segments is present in the nurse cells of P. cynthia, as in Pieris (Doncaster '12), indicating a preparation for division in these cells whose function is only nutritive. A number of writers, including Griinberg ('03), Gross ('03), Marshall ('07), and Woltereck ('98), figure tetrads in the nurse cells of various animals, but do not state whether the haploid number is present. They agree, however, that differentiation of eggs and nurse cells occurs after synapsis, which would imply that nurse cells as well as eggs must have undergone pseudo-reduction.
 
 
 
A transfer of material takes place from the nurse cells and the egg through the connecting tubes which in P. cynthia have very prominent walls. A markedly similar condition was observed by Giinthert ('10) in Dytiscus, where converging bundles of fibrils appear, beset with chromidia or chromatin granules which enter the egg. In this case there is no definite wall to the tubes, Griinberg ('03j states that in Pieris the egg sends a large blunt process up between the nearest nurse cells. Evidently there is considerable variation in the relation of eggs and nurse cells within the Lepidoptera, for in P. cynthia it is the nurse cells which send processes into the egg.
 
  
 
The history of the egg nucleus seems to show that the chromosomes lose their visible identity during the growth period. I am convinced of the accuracy of the results in this particular, on account of the very careful study given to this stage. More than half the nuclei from one individual were examined, and only in the very earliest eggs were traces of spiremes to be found. I examined also egg strings of other moths. In Clisiocampa the spireme persists relatively longer, being found in large eggs. As in P. cynthia, it becomes gradually fainter and more broken the older the eggs become, and finally disappears altogether.
 
The history of the egg nucleus seems to show that the chromosomes lose their visible identity during the growth period. I am convinced of the accuracy of the results in this particular, on account of the very careful study given to this stage. More than half the nuclei from one individual were examined, and only in the very earliest eggs were traces of spiremes to be found. I examined also egg strings of other moths. In Clisiocampa the spireme persists relatively longer, being found in large eggs. As in P. cynthia, it becomes gradually fainter and more broken the older the eggs become, and finally disappears altogether.
  
 
 
22 PAULINE H. DEDERER
 
  
 
Throughout the growth period the nucleus contains many small non-chromatic bodies, in a pale flocculent nucleoplasm, but no trace of a spireme. Sections of Rothschildia jorulla eggs and Actias luna showed an essentially similar condition.
 
Throughout the growth period the nucleus contains many small non-chromatic bodies, in a pale flocculent nucleoplasm, but no trace of a spireme. Sections of Rothschildia jorulla eggs and Actias luna showed an essentially similar condition.
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In this list are included only a few of the papers dealing with the various early stages in the growth of the eggs and nurse cells in insects.
 
In this list are included only a few of the papers dealing with the various early stages in the growth of the eggs and nurse cells in insects.
  
Lepidoptera. Doncaster ('12) describes the early oogenesis in Pieris brassicae and Abraxas grossulariata. In Pieris, after the oogonial divisions, when 30 chromosomes are seen in the equatorial plate, the nucleus enlarges and forms a reticulum, followed suddenly by the synizesis stage, in which a chromatin nucleolus appears. In the ensuing stage, a broken spireme of 14 separate threads is seen, the fifteenth element being repre
+
Lepidoptera. Doncaster ('12) describes the early oogenesis in Pieris brassicae and Abraxas grossulariata. In Pieris, after the oogonial divisions, when 30 chromosomes are seen in the equatorial plate, the nucleus enlarges and forms a reticulum, followed suddenly by the synizesis stage, in which a chromatin nucleolus appears. In the ensuing stage, a broken spireme of 14 separate threads is seen, the fifteenth element being represented by the double chromatin nucleolus, which is interpreted as an equally paired heterochromosome. When the threads shorten to chromosomes, this is indistinguishable from the others. In Abraxas these stages are similar, A distinction is noted here between eggs and nurse cells. In the former "the bivalent threads persist to the latest stage observed — possibly till the prophase of the polar divisions;" in the latter, the bivalent threads shorten into loops to form chromosomes. In Bombyx and Pieris, as described by Griinberg ('03), the germ cells are at first all alike, with nucleolus and granules in the nucleus. The next zone in the ovary shows spiremes, in which stage synapsis occurs. Cell boundaries are not figured here. This is followed by a differentiation zone, in which the egg nucleus is distinguished by a nucleolus and threads, the nurse cells by tetrads. Details of their origin are not given. Fragmentation of the tetrads is described, and the arrangement of nurse cells near the egg, followed by a transfer of granular material to the egg cell.
 
 
 
 
OOGENESIS IN PHILOSAMIA 23
 
 
 
sented by the double chromatin nucleolus, which is interpreted as an equally paired heterochromosome. When the threads shorten to chromosomes, this is indistinguishable from the others. In Abraxas these stages are similar, A distinction is noted here between eggs and nurse cells. In the former "the bivalent threads persist to the latest stage observed — possibly till the prophase of the polar divisions;" in the latter, the bivalent threads shorten into loops to form chromosomes. In Bombyx and Pieris, as described by Griinberg ('03), the germ cells are at first all alike, with nucleolus and granules in the nucleus. The next zone in the ovary shows spiremes, in which stage synapsis occurs. Cell boundaries are not figured here. This is followed by a differentiation zone, in which the egg nucleus is distinguished by a nucleolus and threads, the nurse cells by tetrads. Details of their origin are not given. Fragmentation of the tetrads is described, and the arrangement of nurse cells near the egg, followed by a transfer of granular material to the egg cell.
 
  
 
Neuroptera. The observations of Marshall ('07) on the ovary of Platyphylax are meagre as regards chromatin changes. From synapsis, beaded threads appear, showing a lengthwise split. These threads give rise to tetrads, which fragment. In cells destined to form eggs, the tetrads disappear later and the nuclei are slightly larger. The further history of the eggs and nurse cells is not given.
 
Neuroptera. The observations of Marshall ('07) on the ovary of Platyphylax are meagre as regards chromatin changes. From synapsis, beaded threads appear, showing a lengthwise split. These threads give rise to tetrads, which fragment. In cells destined to form eggs, the tetrads disappear later and the nuclei are slightly larger. The further history of the eggs and nurse cells is not given.
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Hemiptera. In a study of the early ovary of Protenor, Foot and Strobell ('11) distinguish three zones in the ovary: Zone A consists of nuclei with numerous granules; zone B of larger nuclei with granules and a nucleolus, arising by growth from A;
 
Hemiptera. In a study of the early ovary of Protenor, Foot and Strobell ('11) distinguish three zones in the ovary: Zone A consists of nuclei with numerous granules; zone B of larger nuclei with granules and a nucleolus, arising by growth from A;
 
 
 
24 PAULINE H. DEDERER
 
  
 
zone C contains very small nuclei similar to A, arising chiefly from the cells of zone B by amitosis, and giving rise to the ova. They believe amitosis plays an important role. No cell bomidaries appear in these zones. In young ova, leptotene threads -are seen, followed by a stage of broken spireme threads. These gradually disappear, and reappear later to form chromosomes. The figures given are chiefly photographs and do not adequately illustrate the points mentioned in their paper. The importance of amitosis has been questioned b}^ Gross ('01), who concluded from his studies on Hemiptera that nuclei which divide amitotically never divide again by mitosis.
 
zone C contains very small nuclei similar to A, arising chiefly from the cells of zone B by amitosis, and giving rise to the ova. They believe amitosis plays an important role. No cell bomidaries appear in these zones. In young ova, leptotene threads -are seen, followed by a stage of broken spireme threads. These gradually disappear, and reappear later to form chromosomes. The figures given are chiefly photographs and do not adequately illustrate the points mentioned in their paper. The importance of amitosis has been questioned b}^ Gross ('01), who concluded from his studies on Hemiptera that nuclei which divide amitotically never divide again by mitosis.
Line 446: Line 332:
  
 
Coleoptera. Debaisieux ('09) has described the early oogenesis in Dytiscus marginalis, amplifying Giardina's work on the same form. Debaisieux discovered a synaptic and a diplotene stage between the zone of differentiation of eggs and nurse cells and the growth zone. In the latter, the chromatin of the nurse cells gives rise to tetrads which fragment, the chromatin of the egg cell remaining in the diplotene stage as before. His main conclusions are, (1) that the 'chromatic mass', which Giardina believed to be derived from certain chromosomes is not true chromatin, but a condensation of the reticulum left in the nucleus after the chromosomes of the last oogonial division are formed; (2) that the chromosomes persist autonomously up to the maturation divisions. In Giinthert's paper ('10) the chief point of interest is the description of difi'erential mitosis in the oogonia. When a cell divides, a 'chromatic mass' and the spherical remains of the spindle pass into one cell undivided. This becomes the egg cell, which again divides differentially
 
Coleoptera. Debaisieux ('09) has described the early oogenesis in Dytiscus marginalis, amplifying Giardina's work on the same form. Debaisieux discovered a synaptic and a diplotene stage between the zone of differentiation of eggs and nurse cells and the growth zone. In the latter, the chromatin of the nurse cells gives rise to tetrads which fragment, the chromatin of the egg cell remaining in the diplotene stage as before. His main conclusions are, (1) that the 'chromatic mass', which Giardina believed to be derived from certain chromosomes is not true chromatin, but a condensation of the reticulum left in the nucleus after the chromosomes of the last oogonial division are formed; (2) that the chromosomes persist autonomously up to the maturation divisions. In Giinthert's paper ('10) the chief point of interest is the description of difi'erential mitosis in the oogonia. When a cell divides, a 'chromatic mass' and the spherical remains of the spindle pass into one cell undivided. This becomes the egg cell, which again divides differentially
 
 
 
OOGENESIS IN PHILOSAMIA 25
 
 
 
as before. At the end of the fourth differential mitosis, there are 15 nurse cells and one egg cell, which enters the resting stage. The spindle remains of the nurse cells join that of the egg, forming a protoplasmic bridge between them. Gtinthert believes that differential divisions occur in many animals; and that the 'accessory body' described by Buchner ('09) in Gryllus is merely a 'chromatic mass' indicating a differential mitosis. The origin of nurse cells from the egg by a process of budding, as described by Will, is probably to be interpreted in the same way. In the later nurse cells of Dytiscus Gtinthert finds tetrads which subdivide regularly several times, freeing thousands of granules in the nucleus. When the nuclear wall breaks down, they migrate into the cytoplasm, where they increase by division, eventualh^ entering the egg.
 
as before. At the end of the fourth differential mitosis, there are 15 nurse cells and one egg cell, which enters the resting stage. The spindle remains of the nurse cells join that of the egg, forming a protoplasmic bridge between them. Gtinthert believes that differential divisions occur in many animals; and that the 'accessory body' described by Buchner ('09) in Gryllus is merely a 'chromatic mass' indicating a differential mitosis. The origin of nurse cells from the egg by a process of budding, as described by Will, is probably to be interpreted in the same way. In the later nurse cells of Dytiscus Gtinthert finds tetrads which subdivide regularly several times, freeing thousands of granules in the nucleus. When the nuclear wall breaks down, they migrate into the cytoplasm, where they increase by division, eventualh^ entering the egg.
  
Line 464: Line 345:
  
 
4. In the oogonia no differential divisions occur. The germ cells all appear similar through the presynaptic and synizesis stages.
 
4. In the oogonia no differential divisions occur. The germ cells all appear similar through the presynaptic and synizesis stages.
 
 
 
26 PAULINE H. DEDERER
 
  
 
5. In the post-synaptic spireme stage, the nuclei of the future nurse cells show the haploid number of threads, indicating a preparation for division, although the chromosomes are destined only for disintegration. In the egg cell the spireme is probably continuous. A plasmosome is present in both cases.
 
5. In the post-synaptic spireme stage, the nuclei of the future nurse cells show the haploid number of threads, indicating a preparation for division, although the chromosomes are destined only for disintegration. In the egg cell the spireme is probably continuous. A plasmosome is present in both cases.
Line 484: Line 361:
  
 
March, 1914.
 
March, 1914.
 
 
 
OOGENESIS IN PHILOSAMIA 27
 
  
 
LITERATURE CITED
 
LITERATURE CITED
Line 526: Line 399:
  
 
Foot, K., and Strobell, E. C. 1911 Amitosis in the ovary of Protenor belfragei and a study of the chromatin nucleolus. Arch. f. Zellf., Bd. 7, no. 2.
 
Foot, K., and Strobell, E. C. 1911 Amitosis in the ovary of Protenor belfragei and a study of the chromatin nucleolus. Arch. f. Zellf., Bd. 7, no. 2.
 
 
 
28 PAULINE H. DEDEKER
 
  
 
GiARDiNA, A. 1901 Origine dell' oocite e delle cellule nutrici nel Dytiscus. Internat. Monatschr. f. Anat. u. Phys., Bd. 18.
 
GiARDiNA, A. 1901 Origine dell' oocite e delle cellule nutrici nel Dytiscus. Internat. Monatschr. f. Anat. u. Phys., Bd. 18.
Line 573: Line 442:
 
Punnett, R. C, and Bateson, W. 1908 The heredity of sex. Science, N. S., vol. 27, no. 698.
 
Punnett, R. C, and Bateson, W. 1908 The heredity of sex. Science, N. S., vol. 27, no. 698.
  
 
 
OOGENESIS IN PHILOSAMIA 29
 
  
 
RucKERT, J. 1892 Zur Entwickelungsgeschichte des Ovarialeies bei Selachiorn. Anat. Anz., Bd. 7.
 
RucKERT, J. 1892 Zur Entwickelungsgeschichte des Ovarialeies bei Selachiorn. Anat. Anz., Bd. 7.
Line 628: Line 494:
  
  
 +
PLATE 2
  
30
+
EXPLANATION OF FIGT'RES .
  
 +
15 Metaphase of second oocyte division, side view, from serial sections, showing cell plate between .4, 13 chromosomes of first polar boch', and B, 13 chromosomes in the egg.
  
 +
16 Same, without cell plate.
  
OOGENESIS IN PHILOSAMTA
+
17 Same, with cell plate; polar view.
  
PAULINE H. DEDERER
+
18, 19, 22, 26 Anaphases of second oocA^te division, all incomplete except lower group in figure 19.
  
 +
20 Sister anaphase groups of second division, showing a, 13 chromosomes of second polar body; h, 13 chromosomes in the egg.
  
 +
21 Second anaphase group of 13 chromosomes in the egg.
  
PLATE 1
+
23 and 24 Sister anaphase groups of second division; oblique polar view, the groups slightly displaced.
  
 +
25 Telophase of second division; incomplete.
  
  
1\^^
+
PLATE 3
  
 +
EXPLANATION OF FIGURES
  
 +
27 Copulation of the pronuclei, from serial sections, showing 13 chromosomes in each pronucleus B, C, and 13 in second polar body, A.
  
 +
28 Polar bodies from egg of similar stage; the first one has divided, and shows shadowy chromosome outlines.
  
 +
29 Ovary from a larva, longitudinal section. X 16.
  
 +
30 Ovary from a pupa, fixed in January; total, X 16.
  
^^»»
+
31 Same, fixed in July; total, X 16.
  
  
  
•* %'
+
PLATE 4
  
 +
EXPLANATION OF FIGURES
  
 +
32 and 33 Anaphases of first oocyte division. X 700.
  
 +
34 Metaphase of second oocyte division. X 700.
  
 +
35 Copulation of pronuclei; same egg as figure 27. X 700.
  
 +
36 Post-oogonial nuclei with chromatin masses. The cells are connected by protoplasmic strands or tubes.
  
 +
37 Longitudinal section through portion of an egg string of a larval ovary. X 400. a. Stage a, oogonial region; b, Stage e, synizesis; c, Stage f, post-synaptic spireme; d, Stage h, dyad or tetrad chromosomes in nurse cells; e, eggs and nurse cells well differentiated.
  
 +
38 and 39 Stage c; the chromatin masses are transformed into small irregular fragments which later assume a thread-like form.
  
 +
40 Stage d; presynaptic leptotene.
  
 +
41 and 42 Two groups of protoplasmic tubes with branches.
  
  
 +
PLATE 5
  
i^l^t
+
EXPLANATION OF FIGURES
  
 +
43 Stage e; synizesis.
  
 +
44 to 47 Stage f ; post-sjoiaptic spireme of nurse cells. 48 and 49 Stage g; diplotene stage; 13 split rods.
  
If
+
50 to 52 Stage h; chromosomes begin to fragment.
  
^ % • 4
+
53 and 54 Stage j ; young nurse cells, showing tubes entering egg cell. X 700.
  
 +
55 A, nucleus of nurse cell with plasmosomes and chromatin granules; X 700. B, plasmosome enlarged.
  
 +
56 Older nurse cells, with tubes. X 400.
  
il^^
 
  
 +
PLATE 6
  
 +
EXPLANATION OF FIGUKES
  
•;
+
57 Portion of an egg string, showing an egg and two nurse cells. X 400.
  
 +
58 and 59 Stage f; early and later post-synaptic stages of egg cell; spireme probably continuous.
  
 +
60 Nucleus from an egg cell similar to the one shown in figure 57. X 700.
  
 +
61 Stage g; spireme disappearing in the later-growth period; plasmosomes of varying size and form.
  
• t
+
62 Slightly older nucleus; all traces of the spireme have disappeared.
  
 +
63 Same. X 850.
  
 +
64 Nucleus from a nearly mature egg, showing dark crescentic region. The nuclear cavity is filled with granules. X 400.
  
  
  
  
^
 
  
 +
==The Structure And Growth Of The Incisor Teeth Of The Albino Rat==
  
 +
William H. F. Addison And J. L. Appleton, Jr.
  
11
+
From the Anatomical Laboratory of the University of Pennsylvania and The Wistar Institute of Anatomy, Philadelphia
  
 +
TWENTY-NINE FIGURES
  
 +
CONTENTS
  
 +
Introduction 43
  
 +
Historical survey 44
  
 +
Material and methods 46
  
b 12
+
Dentition of adult animal 46
  
 +
Minute description of the incisors 52
  
 +
Microscopic structure of enamel and dentine 55
  
.•/.•
+
Development of the incisors 59
  
•::•
+
Detailed description of development up to time of eruption 61
  
 +
Eruption of the teeth 78
  
 +
Changes in apex of tooth by use 81
  
 +
Description of mature tooth and tooth-forming organs, in 5-month animal. . 83
  
 +
Rate of growth of incisor teeth 88
  
 +
Overgrowth of incisor teeth 89
  
13
+
Summary 91
  
 +
Literature cited 95
  
 +
INTRODUCTION
  
9t
+
The incisor teeth of the Rodentia have long been regarded by the zoologist as having a high value for the understanding of many of the characteristics of this order. For instance, in 1888, Cope wrote "nearly all the peculiarities of the rodent dental system and manner of mastication are the mechanical consequences of an increase in length of the incisor teeth." Tullberg ('98-'99) gives the taxonomic position of the genus Mus, proceeding from the more general to the more specific grouping: Rodentia, Simplicidentati, Sciurognathi, Myomorphi, Myoidei, Muriformes, Myodontes, Muridae, Murini, Mus. A consideration of these terms merely from an etymological view suggests the importance of the teeth and jaws in the classification of the gnawing animals. The observations here recorded are based on the study of the celhilar processes involved in the formation, eruption and growth of the incisor teeth in a single rodent form — Mus norvegicus albinus. An additional interest was lent to the work by the fact of the increasing use of this animal for laboratory purposes, which makes it desirable to learn the time-relations of its life-processes, as a basis of comparison in various forms of experimental studies. Although the rodent incisors have been the object of much study, few observers have carried out their observations through the complete life-history, including developmental stages and adult structure, in one form of animal and this it has been our aim to do.
  
 +
HISTORICAL SURVEY
  
 +
Oudet ('23) proved the phenomenon of permanent growth in the incisor teeth of rodents by cutting off the teeth at the gingival margin and observing that they were regenerated. Retzius ('37) and others noted the overgrowth of these teeth in cases of malocclusion. MacGillavry ('76) observed the rate of growth of the incisors of a rabbit by making marks on the teeth and noting the gradual advance and disappearance of these marks, as the teeth grew out and were worn away.
  
4 .%*
+
Questions which have called forth much study and controversy are (1) does the rodent incisor belong to the milk or to the permanent dentition; and (2) which of the three incisors of the typical mammalian dental formula does it represent. Without exception, all who have studied the first question agree that the large rodent incisor belongs to the second or permanent dentition. These same studies show that abortive milk incisors occur in a varying degree in the several families of the Rodentia; and that they are slightly, if at all, represented in the Muridae. As to the second question. Cope on palaeontological evidence decided that the large rodent incisor was h. Adloff ('98) on embryological evidence confirmed this view. Freund ('92), Woodward ('94) and Stach ('10) beheved it to be I,. Weber ('04) has given a resume and extended bibliography of this work, up to the date of his writing.
  
  
 +
The histology of the incisor was briefly described by Owen ('40-'45) and more completely studied by J. Tomes ('50). The latter found a considerable diversity of arrangement of the enamel prisms in the different families of the order, so that in many cases he was able correctly to refer a tooth to a particular family by a simple inspection of thin sections of its enamel. Von Brunn ('87) showed that at eruption the tip of the incisor of the albino rat is free from enamel, and Sachse ('94) confirmed this on Mus musculus. J. L. Williams ('96), in a comparative study of the formation of enamel, gives a number of good illustrations of the structure of the enamel and enamel-organ of the rat, prepared from microphotographs.
  
+
Ryder ('78) and Cope ('88), in harmony with their views on the "Origin of the Fittest," described the form and position of the rodent incisor as manifestations of a most efficient mechanical system; and studied the various effects on skull topography, necessitated by adaptation to this system.
  
 +
The enamel organ of the albino rat was studied by von Brunn ('87) who described in some detail the differences in structure between its functional labial portion and its non-functional lingual side. He also described the early continuity of the lingual side of the enamel-organ and its later penetration by the surrounding connective tissue. Roetter ('89), studying Mus musculus, denied von Brunn's position in regard to the invasion of the lingual side of the enamel-organ by connective tissue, and Sachse ('94), also using Mus musculus, agreed with Roetter and described the continuity of the lingual portion as persisting through life.
  
14
+
The development of the rodent incisor has been studied especially by Roetter ('89), Sachse ('96) and Meyerheim ('98). Burckhardt ('06), in his description of the development of the persistently growing rodent incisor in O. Hertwig's Handbuch der Entwickelungslehre has followed chiefly Sachse's work upon Mus musculus. In both Weber ('04) and Hertwig ('06) are extensive bibliographies and in these may be found all references not fully given in our appended list of literature cited.
  
 +
MATERIAL AND METHODS
  
 +
The albino rat is a variety of Mus norvegicus, the common gray rat (Donaldson '12). This has been shown by similarity of skull measurements (Hatai '07) and of hemoglobin crystals (Reichert and Brown '10) and also by the fact that the two interbreed freely.
  
31
+
The material used was obtained from the rat colony of The Wistar Institute. Serial sections in paraffin or in paraffincelloidin were made of decalcified heads of fetuses taken at daily intervals from the 16th day onwards until birth, and of jaws of animals newly-born and at short intervals until one month, and of several older stages. Serial sections of fetuses younger than 16 days were examined in the collection of The Wistar Institute. Ground sections were made of the isolated teeth, and the petrifaction method of imbedding in Canada balsam was used to prepare the teeth and adjacent soft parts in situ. Also a series of prepared crania, some entire and some disarticulated, was made at selected ages, varying from birth to old age. The 'gold dust' method of Davison, as tested out for different ages at The Wistar Institute was used for the preparation of the former, and maceration in tap water for the latter. Schultze's clearing method was found useful in studying the early periods of calcification.
  
 +
DENTITION OF ADULT ANIMAL
  
 +
The dental formula of the albino rat is I ., C ^, P „, M ' .
  
PLATE 2
+
There is only one set of teeth, and hence the dentition is monophyodont. The time of eruption of the various teeth extends over a period of 3^ weeks. The incisors are the first to appear, viz., at 8 to 10 days after bhth. The first and second molars erupt at about the 19th and 21st days respectively, and it is after this period that the young animals may be weaned and are able to maintain an independent existence, as far as food is concerned. The third molars are delayed until 2 weeks later and do not appear until about the 35th day.
  
EXPLANATION OF FIGT'RES .
 
  
15 Metaphase of second oocyte division, side view, from serial sections, showing cell plate between .4, 13 chromosomes of first polar boch', and B, 13 chromosomes in the egg.
 
  
16 Same, without cell plate.
+
Fig. 1 Cranium of a 5-month albino rat. X 2.
  
17 Same, with cell plate; polar view.
+
Fig. 2 Cranium of a 5-month albino rat, with the bony alveoli dissected away to show the entire length of the incisor teeth. X 2.
  
18, 19, 22, 26 Anaphases of second oocA^te division, all incomplete except lower group in figure 19.
+
The incisors are permanently-growing (or rootless) teeth, while the molars have a definite limited period of development and acquire roots. A wide diastema separates the incisors from the molars as may be seen by reference to figm-e 1. The incisors are strongly curved and Owen ('40-'45) has described the lower incisor as being the smaller segment of a larger circle, and the upper incisor as the larger segment of a smaller circle. In the lower incisor of the albino rat this statement needs a slight modification. For while the curvature of the upper incisor is in one plane only, the lower incisor is a portion of a flattened spiral, possessing a curve in three planes. The upper incisor is a segment of a true circle (at 5 months about 210°) and in cases of overgrowth it has often been known to complete the cu'cle. In the case of the lower incisor, however, when we project it on the sagittal, frontal or coronal planes, it gives in each case a curve. It was the very evident curved projection seen on the sagittal plane to which Owen referred. Considering only this view, the lower incisor of a 5-month animal forms a segment of about four-fifths of a semicircle (140-145°).
  
20 Sister anaphase groups of second division, showing a, 13 chromosomes of second polar body; h, 13 chromosomes in the egg.
+
TABLE 1 23 1 41 ! 10 15 , 5 ; 8 10
  
21 Second anaphase group of 13 chromosomes in the egg.
+
DAYS j DAYS I WEEKS WEEKS MONTHS MONTHS MONTHS
  
23 and 24 Sister anaphase groups of second division; oblique polar view, the groups slightly displaced.
+
imii. ! m7n. : mm. \ mm. mm. mm. mm.
  
25 Telophase of second division; incomplete.
+
Xaso-occipital length 29 .7 32 .5 39 40 43 44 46 .5
  
 +
Interzvgomatic 13.7 14 14.5 14.6 15. -> 15.1 15.5
  
 +
Uppe/diastema 7.4 9.5 10 11.4 12 .3 12 .5 13
  
32
+
Upper incisor— total length 12.8 15 18.3 20.3 23.3 23.7 26.2
  
 +
Upper incisor — extra-alveolar
  
 +
length ! 5.1 i 5.5 j 7 8.4 8.7 9 9 3
  
OOGENESIS IN PHILOSAMIA
+
Lower diastema ' 4.6 I 5 I 5.6 6 6.7 7 6.8
  
PAULINE H. DEDERER
+
Lower incisor— totallength 18.1 21.7 25.5 26.4 29.4 29.9 31.3
  
 +
Lower incisor — extra-alveolar
  
 +
length 6.5: 7 I 10.5 11.4 11.6 12 12.4
  
  
111
 
  
 +
Measurements of the incisors and skulls of animals of different ages, were made as shown in table 1.
  
 +
The teeth were measm-ed along their convex surfaces by means of silk thread wet with water, and applied to the object to be measured. The thread was then cut with scissors at the end of the object, straightened on paper and measured to tenths of millimeters.
  
t ; V » \ t *
+
A consideration of table 1, shows in a definite way the peculiarities characteristic of the dentition, not only of the rat but of rodents in general. As is well known, these are the great development of the incisors, the wide diastema, and the consequent posterior position of the molar teeth as related to the rest of the skull. Cope ('88) wrote that he considered "the increase in the length of these teeth has been due to their continued use, as beUeved by Ryder." The effects of this increased elongation upon surrounding parts he described under several different headings, but reference will be made here only to one, viz., upon the shape of the glenoid cavity. "A peculiarity of the masticating apparatus is the lack of a postglenoid process, and the consequent freedom of the lower jaw to slide backward and forward in mastication. Appropriately to this motion, the condyle of the mandible is extended antero-posteriorly and the glenoid ca^dty is a longitudinal instead of a transverse groove."
  
  
  
'V
+
Fig. 3 Thimble-shaped portion of the maxiUa bone, in which the basal end of the upper incisor is located. X 2.
  
 +
The lower incisors are longer and more slender than the upper and extend far back in the mandible, beneath the lower molars, to near the sigmoid notch. The upper incisors are contained within the premaxilla and maxilla, the basal end occupying a thin-walled, thimble-shaped recess of bone (fig. 3) to be seen best in the disarticulated skull, and which is attached at only one limited region to the rest of the maxilla. In both upper and lower teeth, the intra-alveolar portions are longer than the extra-alveolar. When one compares the extra-alveolar lengths of the upper and lower teeth of the mature animal, the latter are always greater, and, as may be seen by reference to table 1, the difference in lengths becomes greater with increased age and size.
  
 +
In both upper and lower incisors the bone is so contoured around their imbedded portions that their course may be easilj^ recognized. The basal end or foraminal apex of the lower incisor forms on the outer aspect of the mandible a marked rounded projection, directed upwards and backwards beneath the coronoid process, and sometimes extending sUghtly posteriorly beneath the sigmoid notch. Almost directly opposite this projection on the mesial aspect of the mandible is the inferior dental foramen. This projection marks the position of the growing end of the formative organs of the incisor in the adult. In the new-born anunal it is not present, nor at the end of the first month. By the age of 2| months it may be recognized, and thereafter it increases in prominence and constitutes a very evident feature of the bone. This region of the growing end of the tooth is protected b}'^ the zygomatic arch, and also by the overlying muscles.
  
 +
The course of the upper incisor may also be readily followed in the prepared skull. Laterally it is covered with a thin rounded layer of bone. Mesially it forms an elevated, distinct ridge projecting markedly into the nasal fossa. In the adult the position of its basal or growing end is not so prominent as that of the lower incisor. As these incisor teeth are an indispensable part of the rodents' existence their importance demands protection from traumatism which might injure their growing pulp. Here in the upper incisors, this protection is afforded by a flange of the maxilla running parallel to the lateral wall of the cranium, as shown in figure 1 , as well as being encased in a separate thimble-shaped recess of bone (fig. 3), beneath, and separated by a narrow interval from, the outer layer of the maxilla. These details are in harmony with Cope's idea ('88) of the influence of the incisors in moulding the general topography of the rodent skull.
  
16
+
The diastema in the upper jaw is always longer than in the lower (fig. 1). By reference to table 1 it may be seen that in the mature animal the upper is nearly twice as long as the lower, but that in the younger stages the difference is not so great. The upper hair-covered lips are infolded into the diastema, dividing the oral cavity into an anterior and posterior compartment. This arrangement probably prevents the debris and splinters of gnawing from entering the main oral cavity.
  
  
 +
The mandibular symphysis is formed of fibrous tissue and allows independent rotation of either ramus with its contained tooth. This lateral movement of the lower incisors appears to be under the control of the will of the animal. According to the observations of Jolyet and Chaker (75) this mobility has a definite purpose in mastication. They observed a rapid alter
  
PLATE 2
 
  
  
  
/
+
Fig. 4 Cross-sections of the (a) upper and (b) lower incisor teeth of a 5-month albino rat, taken near the alveolar margins. These show the arrangement of the enamel and the dentine, and the difference in contour of the enamel in the upper and lower teeth. The mesial surface cf each tooth is towards the right side. X 15.
  
 +
nate separation and approximation of the tips of the lower incisors in the act of attempting to bite into a match or other slender object offered to the animal. At the same time the upper incisors were held stationary.
  
 +
Mention may be made here of a point of variation among the Rodentia in the relation of the angle of the lower jaw to the sheath of bone around the lower incisor. In the Myomorphi and Sciuromorphi the angle arises from the lower surface of the incisive sheath, while in Hystrix the angle arises entirely on the outer side.
  
17
+
Ryder (77) suggested a classification of rodents based on the shape of their incisors as seen in cross-section. In some genera the diameter of the teeth is less from side to side, than in the antero-posterior direction, while in others the reverse condition is found. The present form belongs to the former group, as is shown in figure 4. From the consideration of many rodents, Ryder deduced the general principle, that where the incisors are thicker in the antero-posterior direction, the gnawing habit is greatly developed.
  
 +
MINUTE DESCRIPTION OF THE INCISORS
  
 +
Enamel and dentine make up the hard tooth substance, enclosing the pulp. Owen, in his "Odontography" ('40-'45, p. 399) said that there existed a general investment of cementum over the whole tooth structure. J. Tomes ('50, p. 533) was not able to agree entirely but said that in most, if not in all, incisors of rodents cementum could be seen investing the posterior surface. In the rat, it is not apparent that there is any cementum at all. The enamel is usually colored with a pigment which is yellowish in the young but becomes orange-colored with age, and is usually more pronounced in the upper than in the lower incisors. At 13 days, there is as yet no color, but at 21 days a slight tinge of yellow is perceptible in the uppers, but none in the lowers. At 25 days the uppers are distinctly yellow, and the lowers have now acquired a slight color. At 38 days, these colors have intensified, the uppers having more pigment than the lowers; and in the mature animal the same relation continues, the uppers being orange-colored and the lowers yellow. The enamel is found principally on the labial side, and this accounts for the shape of the occlusal surface. For, the enamel being harder than the dentine, the latter is more easily worn away by the action of the opposing tooth, and the more resistant enamel remains as the cutting edge or point. The shape of the incisal end of the upper and lower teeth is different, being chisellike (scalpriform) in the upper, and more rounded and narrower in the lower. The incisal line is also usually different in the upper and lower teeth. In the former, it is often sHghtly concave from side to side, while in the latter it is convex (fig. 5). As is shown in figures 1 and 5 the occlusal surface is an elongated concave area on the lingual aspect of the teeth, and in the living animal extends practically to the gingival margin. Due to the difference in the curve of the upper and lower teeth, the occlusal surface of the lower teeth is always longer than that of the upper, and in the mature animal it is usually found to be nearly twice as long.
  
B
 
  
  
  
15
 
  
 +
Fig. 5 Labial and lingual aspects of the extra-alveolar portions of the (a) upper and (b) lower incisors of a 5-month albino rat, showing the occlusal surfaces and incisal edges of the teeth, and the outline of the bony alveolar margins. X 2.
  
  
•••:.
 
  
.•V.
+
It follows that because these teeth are constantly growing, the occlusal surfaces are constantly being worn away. As we shall see, when discussing the growth of the teeth, the elongated temporo-mandibular articulation is important, in allowing the teeth to have either the position pictured in figure 1 or to have the opposite relation, with the lower teeth outside of the upper. Thus the very important factor in the animal's economy — the proper regulation of the length of the opposing incisors — is controlled by their own inter-action.
  
  
  
 +
The pulp-chamber has the characteristic shape found in all permanently growing teeth, as is well seen, for instance, in the elephant's incisor. Its cross-area is greatest at the basal end of the tooth, and gradually diminishes anteriorly. The pulpchamber is found to extend in the tooth beyond the line of the gingivus, and very nearly to the occlusal surface. The shape
  
  
 +
Fig. 6 Upper incisor of a o-month albino rat (X o) and cross-sections of it at different points (X 8), to show the relative cross-area of the dentine and of the pulp chamber at these regions. The dotted line indicates the position of the margin of the alveolus.
  
20
 
  
  
 +
in cross-section of the pulp-chamber at different levels may be seen by reference to figure 6. The position of the filled-in pulpchamber is usually well marked on the occlusal surfaces as a line (fig. 5). In weathered specimens of rats' teeth from recent geological formations this last-formed part which fills in the pulpchamber at the end of the tooth, is usually found to be lacking, and is evidently not of the same hardness as the surrounding parts of the tooth.
  
••^%
 
  
 +
MICROSCOPIC STRUCTURE OF ENAMEL AND DENTINE
  
 +
Sections of enamel show two layers; an outer thin and an inner thicker layer, as noted by Owen ('40-'45, p. 399). The enamel rods run in different directions in the two layers as fully described by J. Tomes in 1850. In the inner layer the enamel rods appear to run in two sets, obliquely to one another, while in the outer layer the rods are all parallel. The outer layer has also been called the fibrous layer, and in its superficial part is situated the yellow or orange pigment which gives the color to the enamel.
  
gGl^i
+
Figures 7 and 8 show the arrangement of the enamel rods in the two la3^ers. In the inner or plexiform layer, when examined in cross section, the alternating series of enamel rods decussate, forming an angle varying between 70 and 90°. In longitudinal sections (fig. 26) these rods are slightly S-shaped, running outwards from the enamel-dentine surface at an angle of 50 to 54°, and inclining towards the anterior end of the tooth. Figure 8 is from a ground-section in which the enamel was broken during the process of preparation, and the broken edge shows distinctly the two sets of rods running at nearly right angles to each other. Under high magnification the rods are slightly notched.
  
 +
In cross-sections of the outer fibrous layer, the rods are parallel and form in the mid-line of the tooth an angle of 90° with the outer surface. As one proceeds away from the mid-line of the tooth, whether mesially or laterally, the general tendency of the long axis of the rods as they pass from the dentine junction to the periphery, is to incline in the direction away from the mid-line of the tooth. The ameloblasts usually form an obtuse angle with the rods of the outer layer and seldom coincide in direction with them (fig. 7). In longitudinal sections the rods of the outer layer are not usually so distinctly seen as in cross-sections. In favorable longitudinal sections, however, they are seen to run quite obliquely, inclining towards the apex of the tooth, and forming an angle of 20 to 25° with the plane of the enamel-dentine junction. The pigment, as will be seen
  
  
18
 
  
  
 +
Fig. 7 Portion of cross-section of lower incisor with enamel-organ, prepared by the petrifaction method, showing the decussation of the enamel-rods in the inner or plexiform layer and their jiarallel arrangement in the outer or fibrous layer. X 350.
  
• • •
+
Fig. 8 Small piece of enamel, showing the rods of the inner or plexiform layer running in two directions nearly at right angles to one another. X 350.
  
  
 +
below, is confined to the outermost part of the fibrous layer. There appears to be no Nasmyth's membrane over the enamel, which means that there has been a complete transformation of the enamel matrix into enamel rods. The pigment extends about two-thirds of the total length of the upper tooth, and about one-half of the total length of the lower tooth, and hence it follows that the deposition of enamel is completed within the basal third of the upper and the basal half of the lower tooth. By examining cross-sections of the tooth at different regions (fig. 6) it would seem that the full thickness of the enamel is attained within even a smaller area at the basal end of the tooth.
  
\\\
+
The arrangement of the enamel over the labial aspect of the upper and lower teeth is shown in figure 4, drawn from crosssections of the teeth of a 5-month animal. In both teeth the sections were made just posterior to the alveolar border. In both upper and lower teeth the enamel is thickest over the labial aspect, and is continued over the adjacent mesial and lateral surfaces. In both, the enamel is continued farther on the lateral than on the mesial surfaces, and relatively farther on the lateral surface in the lower than in the upper tooth. In the upper tooth the enamel has a flattened external surface labially, while in the lower it has a rounded contour. In the upper there is a distinct labio-mesial and a labio-lateral angle, the enamel being somewhat thicker at the former. In the lower there is a labio-mesial angle, though less prominent than in the upper, and the labio-lateral angle is practically absent.
  
 +
In a 5-month animal the thickness of the enamel and its constituent layers was measured in the mid-line of the teeth, as follows:
  
 +
Upper Lower
  
 +
Total thickness 100-110 lJO-150
  
 +
Outer fibrous layer 30-40 20-30
  
 +
Pigmented portion of outer fibrous layer 8-10-12 6-8
  
19
+
Inner plexiforni layer 70 120-125
  
 +
It will be observed, however, in figure 4 that the enamel is not thickest in the mid-line of the upper tooth, but at the lateral and mesial angles. While the enamel of the upper tooth measures only 100 to llO^t in the mid-line, it measures 160 to 180/x at the region of these angles, and is, therefore, thicker here than the enamel of the lower tooth. The increased thickness at the angles is principally in the inner plexiform layer, the other layer being increased only slightly or not at all. The outer fibrous layer is distinctly thicker in the uppers and has a slightly wider band of pigment in it superficially. This, no doubt, is the basis of the more deeply pigmented appearance of the labial surface of the upper as compared with the lower teeth.
  
 +
The dentine, unlike the enamel, grows continually thicker as one passes towards the outer end of the tooth. At the basal, growing, end it begins as an extremely thin layer. The thickness at different points is seen in figure 6. As the dentine increases in thickness, the pulp-chamber is in consequence proportionately reduced. At the distal end there is no longer any pulp-chamber and the site of its previous position has been filled in by the formation of a kind of secondary dentine. C. Tomes ('14) notes that "in some rodents the final closure of the axial tract takes place almost by a continuance of the formation of normal fine-tubed dentine, with very little secondary dentine of different structure, while in others there is a large area of dentine with vascular tracts in it." In the rat there is relatively little of this secondary dentine. It is laid down in irregular trabeculae, with the pulp tissue, including bloodvessels, at first within it. At the exposed surface, however, it forms a continuous granular mass with apparently no soft tissues in it (fig. 27). The ordinary dentine of the tooth is quite typical in structure, with numerous parallel dentinal tubules, each having many fine lateral branches. The tubules are slightly sinuous, and the lateral branches anastomose with those of neighboring tubules. Sometimes a tubule sends off at an acute angle a branch nearly equal in diameter to the continuation of the main tubule. This is usually in the dentine not covered by enamel. Where these large branches come off the diameter of the tubule is greater than elsewhere, measuring nearly 2^. Elsewhere the diameter varies from 1 to l.T/z. Slight differences may be seen between the tubules (a) in the dentine covered by enamel, and (b) in the dentine free from enamel. The tubules of the anterior region (a) of the dentine, covered by enamel, are more regularly parallel and have finer lateral branches than elsewhere. They also seem to taper slightly as one follows them towards the enamel. In the dentine not covered by enamel (b) the tubules are more sinuous and irregular, the irregularities marking the position of origin of the larger lateral branches. In all parts at the periphery of the dentine the tubules end in a great number of very fine anastomosing arching branches. As a consequence of the smaller diameter of the little tubules here, a narrow zone at the periphery of the dentine has usually a more homogeneous appearance than has the remainder. Towards the anterior end of the tooth, in the vicinity of the pulp-chamber, are vascular channels in the form of loops within the dentine. The tubules must necessarily take a curved course around these vascular channels, and thus the position of the vessels is more easily seen.
  
••V
+
In the dentinal tubules Mummery ('12), Fritsch ('14) and others have demonstrated not only the processes of the odontoblasts, but also fine non-medullated nerve fibers. As to why the exposed dentine on the lingual aspect of the teeth is insensitive, there are no definite observations to decide. A contributing factor may be the compression which the pulp tissues undergo at the anterior end of the pulp-chamber, leading to the physiological cutting off of the nerve supply to the dentinal tubules.
  
 +
DEVELOPMENT OF THE INCISORS
  
 +
The times of the early stages of development of the incisors were seen as follows:
  
21
+
14-day fetus — slight thickening of oral epithelium
  
 +
15-day fetus — distinct thickening and growth inwards of oral epithelium
  
 +
16-day fetus — dental ledge and beginning of flask-shaped enamel organ
  
•»'
+
17-day fetus — dental papilla with crescentic enamel organ capping it
  
 +
19-day fetus — both ameloblasts and odontoblasts differentiated
  
 +
new-born animal — enamel and dentine formation begun
  
22
+
8 to 10 days — eruption of the tooth
  
 +
Throughout life growth continues, and in the adult animal is on the average 2.2 mm. per week in the upper and 2.8 mm. per week in the lower incisor.
  
  
• • •% • • .
+
The structures to be described here, as in the development of the crowns of all teeth, are the enamel-organ with the ameloblasts, and the dental papilla (which becomes the pulp-substance) with the odontoblasts. There are two factors, however, which alter the usual history of the development of these structures, and especially of the enamel-organ. First, in permanently growing teeth of which these are examples, all these structures continue functional throughout life, so that the enamelorgan is also a persistent structure. The other factor and one correlated to some extent with the first, is that the enamel is formed on one side of the tooth only, and here only does the enamel-organ develop to its most highly differentiated functional condition.
  
 +
The history of the development and growth of the tooth may be conveniently considered in two stages (1) pre-eruptive, and (2) post-eruptive. The pre-eruptive stage extends from the 14th or 15th day of fetal life until eruption of the tooth takes place between the 8th and 10th post-natal days. Until near the time of birth there is no formation of enamel and dentine, but from birth onwards these substances are laid down rapidly, so that at eruption, the teeth have their characteristic elongated narrow form. This pre-eruptive stage is characterized by the rapid elongation of the tooth-forming organs, and by the teeth attaining very similar relations to the other structures of the jaw which the imbedded portions of the erupted teeth possess. Thus, the anlage of the lower incisor appears under the oral epithelium in the anterior region of the mandible, and grows continually backwards, until its growing end reaches the region beneath the developing molars. At this time the growing end presumably reaches a region which, by reason of its increasing calcification, offers resistance to further progress. The result of the ever-continuing mitotic division and cell growth at the basal end, is the pushing of the whole tooth and its formative organs, in the opposite direction, and the consequent eruption of the tooth. During the latter half of this pre-eruptive stage, the anterior tip of the developing tooth structure is immediately beneath the oral epithelium, and remains at a fixed point, while the posterior end is continually growing backwards and changing its relations. At eruption this condition changes, and the posterior extremity becomes practically a fixed point from which the whole tooth moves forward. That there is, however, a gradual change in the position of the posterior end of the tooth may be seen in figure 9. As the jaw grows, the entire tooth not only grows to keep the same general relative position, to surrounding structures, but it may be seen that the growing end progresses gradually posteriorly. In the full-grown animal this end occupies a distinct outpushing of the bone (fig. 1).
  
 +
During the post-eruptive period, which continues throughout life, this outward growth is continued at a regular rate, and at the same rate the outer end has to be worn away. This wearing-away process would soon result in the pulp becoming exposed were not the occlusal end of the pulp-chamber also being continually filled in. As may be seen from figure 6 the dentine continues to increase in thickness until near the end of the tooth. This means that the odontoblasts continue their regular functional activity until near the end of the tooth. However, the final fiUing-in of the pulp-chamber to form a continuous hard occlusal surface is accomplished by the deposition of a hard matrix between the pulp elements and by the probable calcification of the latter. The result is, that as the tooth is worn away, the soft pulp never becomes exposed. Although the pulp reaches very near to the end of the tooth, a hard substance always fills in the end of the pulp-chamber, and so protects the pulp beneath.
  
23
+
DETAILED DESCRIPTION OF DEVELOPMENT UP TO THE TIME OF ERUPTION
  
 +
The anlage of the enamel-organ of each incisor arises as an epithelial ingrowth, distinct and separate from that for the molars.
  
 +
In frontal sections of the 14-day fetus, there are slight diffuse thickenings of the oral epithelium in the four positions, which represent the sites of the future tooth-formations.
  
  
  
 +
Fig. 9 Series of mandibles of the albino rat at ages \arying from birth to ten months, viewed from the lateral aspect. These show the changing relation of the basal end of the incisor to the rest of the mandible during this period.
  
  
  
 +
At 15 days these thickenings have become more definite, and in the lower jaws especially have begun to push into the underlying mesenchyme, and may be described as the dental ledges or dental laminae.
  
24
+
At 16 days the ingrowths have continued to increase as broad masses of cells, pushing deeper into the underlying mesenchyme, and in the lower jaws the enamel organs may be distinguished as expanded structures, each connected by a slightly narrower mass of cells with the oral epithelium. In the upper jaws the differentiation of the enamel-organs from the remainder of the epithelial ingrowth is not so marked.
  
 +
At 17 daj^s (fig. 10) the dental papillae are beginning, and the enamel-organs in both upper and lower jaws have a crescentic outline. In the enamel-organs there is already an indication of the differentiation into three layers. As seen in sagittal sections, the papillae develop on the posterior side of the enamelorgans, thus foreshadowing the axis of growth of the toothforming organs in the antero-posterior direction.
  
 +
Eighteen-day fetus
  
 +
Series of frontal sections of 18-day lower jaws, show that the enamel-organs are growing over the dental papillae more rapidly on the labial and lingual surfaces than elsewhere, and extend more posteriorly on these surfaces. There are thus two projections of the posterior margin of each enamel-organ as already noted by Meyerheim ('98). The labial process is broad and thin and extends more posteriorly than the lingual process, which is somewhat narrower and thicker. One may her-e remark, therefore, an early difference between the labial and lingual part of the enamel-organ. Other differences which will soon appear have not yet developed. Thus, the inner layer of the enamel-organ is made up of columnar elements which are still similar in all parts, both labially and lingually. In the dental papilla no columnar odontoblasts are yet seen.
  
33
+
The enamel-organ remains connected with the surface epithelium by a broad band of epithelial cells. In the lower jaw,
 +
immediately laterad to the line of j miction of this stalk of the enamel-organ to the surface epitheliimi, the lip furrow is a depression, the plane of which is continued into the underlying mesenchyme by an ingrowth of surface epithelium several layers of cells in thickness. It is by the subsequent splitting of this epithelial layer into two, that the separation of the lip will be effected.
  
 +
Nineteen-day fetus
  
 +
At 19 days, the enamel-organ in the upper jaw (fig. 11) is crescentic in outline in sagittal section, and in the lower jaw (fig. 12) is more elongated and conical in shape. At this age odontoblasts are first seen as columnar cells on the labial aspect of the mesodermal papilla. Three layers are recognizable in the enamel-organ, but the middle layer (enamel pulp), as has been also described by Sachse ('94) for the mouse, is extremely thin, and therefore is not present in the great quantity tj^pically seen in the development of rooted teeth. It appears as a more lightly stained zone between the inner and outer layers, and is thickest at the basal end of the enamel-organ as shown in figures 11 and 12. It averages about 20^i in thickness and is made up of stellate cells loosely arranged. Already there is an indication of a compact arrangement of the two or three rows of cells next the inner layer of the enamel-organ, which will result in the so-called stratum intermedium seen at later ages. This middle layer is also slightly more abundant at the anterior end in the region where the enamel-organ is continuous with the stalk which joins it to the oral epithelium.
  
1^
+
At this age the enamel-organs in the lower jaws have a greater total length than those in the upper, and especially in the lower
  
 +
Fig. 10 Longitudinal section of upper jaw of 17-day fetus, showing tooth anlage of incisor, with the enamel-organ longer labially than lingually when measured from the point of junction of the stalk of the enamel-organ. The dental papilla is on the posterior aspect of the enamel-organ. X 70.
  
 +
Fig. 11 Longitudinal section of upper incisor anlage of 19-day fetus of albino rat, showing the crescentic outline of the enamel-organ, its greater length labially than lingually, and its thickened basal margin. X 70.
  
/
+
Fig. 12 Longitudinal section of lower incisor anlage of 19-day fetus of albino rat, showing the conical outline of the enamel-organ and its greater length than in the upper jaw^ at the same age. X 70.
  
  
?:*:
 
  
 +
jaws distinct differences may be made out between the oral and labial sides of the tooth-forming organs. These differences are:
  
 +
(1) The enamel-organ is longer labially than on the oral side.
  
^•'••*
+
(2) The staining of the inner layer of the enamel-organ on the labial side is more intense, and here the cells are slightly longer than in other parts of the enamel-organ, measuring 24/x in length and assuming the typical appearance of ameloblasts. Measurements show the similar cells on the lingual side to be about 20m in length. It is also to be noted at this age that the site of the most advanced cells which are differentiating to become ameloblasts is not at the apex of the enamel-organ, as is the case in the development of rooted teeth. For as one follows the cells of the labial side of the enamel-organ from the apex towards the base, while at the apex the cells are columnar they become longer as one goes posteriorly, and then towards the base of the enamel-organ diminish again. So that the site of most advanced differentiation here is a short distance posterior to the apex on the labial side. This is true also of the developing odontoblasts which are longest opposite the tallest ameloblasts.
  
»
+
(3) The outer layer of the enamel-organ on the labial side is becoming slightly wavy in outline, and this denotes the beginning of the papillae, which form such a characteristic part of the mature functional enamel-organ (fig. 26).
  
 +
(4) The odontoblasts are seen only on the labial side of the dental papilla.
  
26
+
Mitoses are abundant in all parts of the developing tissues.
  
 +
Twenty-one-day fetus
  
 +
Thus the anlage of the rodent incisor begins in the usual way, and for a short time continues along the typical mammalian course. From 19 days onward, however, the differences which have already begun, become more distinct and definite. At 21 days (end of gestation) the enamel-organ has become more definitely differentiated into a labial and a lingual region. Of the three constituent layers, the inner especially is strikingly different in these two parts. On the labial side at the anterior end, the organ has advanced to the condition where functional activity is beginning, while the oral side has remained stationary, or has actually retrogressed. Thus in the innermost layer on the labial side of the lower incisor, where the ameloblasts have begun to form enamel, these cells measure 30 to 34ju in length, while the non-functional cells on the oral side of the innermost layer are low columnar or cubical in shape and measure only 12 /j. in length (fig. 13). Comparison of these measurements with those at 19 days shows that the cells of the inner layer of the labial side of the enamel-organ have advanced in length from 24/i to 30 : or 34m, while the cells on the lingual side have decreased from 20 to 12ju. There is, therefore, a primary tendency for the cells of the inner layer to develop equally in all parts, but very soon the nonenamel-forming cells of the lingual side begin to retrogress, while the functional cells of the labial side continue to grow. This constitutes another point of contrast with the development of the crowns of rooted teeth. For here in the 21 -day fetus, when the enamel and dentine formation has just begun, these substances are thickest, not over the apex of the tooth-forming organs, as in the usual method, but at a short distance posterior to this point, on the labial surface. Thus, not only are the odontoblasts and the ameloblasts first differentiated on the labial side, posterior to the apex, but at this region enamel and dentine formation is also evidently first begun.
  
JOURNAL OF MORPBOLOQY, VOL. 26, NO. I
+
Over the apex of the dental papilla there is apparently a very thin outline of dentine deposited, but within this, in the tissues of the apex of the dental papilla, there is also beginning an irregular formation of a hard matrix. Between the cells of the pulp, trabeculae of a bone-like material are appearing. As development proceeds this substance increases until the final result is, as seen in figure 20, that the primary apex of the tooth has a bone-like structure, consisting of cells imbedded in lacunae within a dense matrix. This has been called by Tomes ('04) 'osteo-dentine.'
  
 +
A similar difference between the labial and oral sides is noted in the cells on the margin of the dental papilla, which are becoming odontoblasts. In the basal half of the papilla (fig. 14), odontoblasts occur only on the labial side opposite the tall ameloblasts, the peripheral cells of the other sides being still irregular or cuboidal in shape. Farther forwards the odontoblasts are found also on the lateral and mesial surfaces of the dental papilla, but not on the lingual. In the apical one-fourth of the dental papilla odontoblasts occur all round, measuring 20 to 24/x in length, and are engaged in the formation of dentine (fig. 13). The dentine is thickest on the labial side.
  
 +
In the region where enamel and dentine formation has begun no mitoses were seen in the formative ameloblastic and odontoblastic cells, but posteriorly, where the deposition of enamel and dentine has not yet commenced, many mitoses occur in the layers of developing ameloblasts and odontoblasts, as well as elsewhere. The nearer one approaches the basal margin of the enamel-organ the more numerous are the mitoses and it is apparent that it is principally in this region that growth by addition of new cells is taking place.
  
PLATE 3
+
One day old
  
EXPLANATION OF FIGURES
+
At the end of the first day of post-natal life, there has been great progress in the enamel and dentine formation, and the narrow, pointed outline of the tooth has been already laid down. In the upper jaw the teeth measure about 2.3 mm. in length and in the lower jaw about 3 mm. Definite changes in its relation to the oral epithelium have occurred also at the anterior end of the tooth. The original epithelial stalk connecting the enamelorgan with the oral epithelium has increased in size and the end
  
27 Copulation of the pronuclei, from serial sections, showing 13 chromosomes in each pronucleus B, C, and 13 in second polar body, A.
+
Fig. 13 Cross-section of developing lower incisor of 21-day fetus of albino rat, nearer the anterior extremity of the tooth than figure 14. Shows the greater thickness of the labial side of the enamel organ, as compared with that of the other sides, and shows odontoblasts around the entire periphery of the pulp. Enamel and dentine formation has begun. X 110.
  
28 Polar bodies from egg of similar stage; the first one has divided, and shows shadowy chromosome outlines.
+
Fig. 14 Cross-section of developing lower incisor of 21-day fetus of albino rat, posterior to the region shown in figure 13. No enamel or dentine yet formed at this point. Odontoblasts highest on the labial aspect, decreasing in height laterally but not yet differentiated as columnar elements on the lingual side. Enamel-organ thickest on the labial side. X 110.
  
29 Ovary from a larva, longitudinal section. X 16.
 
  
30 Ovary from a pupa, fixed in January; total, X 16.
+
of the tooth has apparently advanced somewhat into it. So this thick stratified layer of epithelium forms a close-fitting investment about the tooth apex, and is continuous posteriorly with the remainder of the enamel-organ. But in this epithelial cap there are no ameloblasts and consequently there can be no enamel over the osteodentine which forms the tip of the primitive tooth. This substance forming the tip of the unerupted tooth is a form of secondary dentine with its cells located in the lacunae of the matrix. Passing backwards, one comes to the ordinary dentine containing the vascular pulp with odontoblasts situated at the periphery of the pulp-chamber in a regular manner.
  
31 Same, fixed in July; total, X 16.
+
As the odontoblasts were first differentiated labially, and dentine formation began there before on the other side, the dentine of the labial side is thicker than on the lingual side. Thus at a point about the middle of the entire tooth structure, the dentine measured 54/^ labially and 20/x orally (fig. 15). Between the odontoblasts are numerous fine capillary loops. At this region may also be seen the characteristic structure of the enamel-organ (fig. 15). This extends all around the tooth, but is much thicker on the labial side than elsewhere. This difference in thickness is seen in all the constituent layers. In the inner layer, the tall ameloblasts of the labial surface measure iOfj., while the similarly situated cells on the other surfaces are cubical and measure only 10^. Comparing these with the previous stage described, it is seen that the cells on the labial surface have increased and those on the other surfaces have decreased. Of the middle layer on the labial side, the stratum intermedium is a distinct line of cuboidal cells, one to two rows in thickness, lying behind the ameloblasts. The other constituent — the original enamel pulp — is small in amount and is principally within the elevations of the outer layer, which form the beginning of the epithelial papillae. The cells of the outer layer, somewhat irregular in shape with round nuclei, are in a single row. Between the developing papillae (called by Sachse StLitzpapillen) are numerous capillary blood-vessels. On the other surfaces, practically nothing remains of the middle layer,
 +
although the outer layer still persists as a layer of flattened cells. Thus lingually the enamel-organ is represented by only two rows of cells — one representing the inner, the other the outer layer of the enamel-organ.
  
  
  
34
 
  
 +
Fig. 15 Cross-section of developing lower incisor of 1-day albino rat, showing the great development of the ameloblasts on the labial side, and the thinness of the enamel-organ elsewhere. The space between the ameloblasts and the dentine is an artefect, and was formerly partly filled by the enamel, which has disappeared in the pi'ocess of decalcification. In the layer of odontoblasts are seen the nuclei of the endothelial cells of the walls of capillaries. X 110.
  
  
OOGENESIS IN PHILOSAMIA
 
  
PAULINE H. DEDERER
+
Two days old
  
 +
Figure 16 shows a longitudinal section of a 2-day upper incisor. The epithelial enamel-organ is continuous over the whole tooth, but only shows its specialized functioning structure on the labial side. On the lingual side it is still intact and consists only of two rows of cuboidal or flattened epithelial cells. On the labial side, along the region where enamel has been formed (fig. 17) the
  
  
PLATE 3
 
  
 +
Fig. 16 Longitudinal section of upper incisor of 2-day albino rat showing the enamel-organ continuous over the labial surface and terminating posteriorly in the thickened margin. X 18.
  
 +
Fig. 17 Small portion of preceding figure more highly magnified, to show the structure of the enamel-organ and the odontoblasts, a, outermost layer of enamel-organ and epithelial papillae; b, enamel pulp; c, stratum intermedium; d, layer of ameloblasts; e, layer of dentine; f, layer of odontoblasts. X 175.
  
  
,".
+
ameloblasts measure about 40^. These are backed by two rows of darkly staining flattened cells composing the stratum intermedium. Next to these is the looser arrangement of stellate cells, comparable to the enamel pulp of ordinary tooth development, but with much smaller spaces between the cells. This tissue is covered by the layer of cells constituting the outer layer of the enamel-organ, and the two together constitute the epithelial papillae. At the summit of each of these papillae the cells of the outer layer are grouped in a more compact manner. With higher magnification processes can be seen running from the ameloblasts into the developing enamel — the so-called enamel processes of Tomes.
  
 +
At the basal formative part of the enamel-organ the three original layers show distinctly. At the thickened basal margin of the enamel-organ, around its entire circumference, is a mass of rapidly dividing cells. As seen in figure 16 this thickened margin is more noticeable on the labial side. Its peripheral zone as seen in longitudinal sections is deeply staining and its cells, more or less columnar in shape, are compacted together. The interior, of more lightly stained appearance, is composed of oval or elongated cells, irregularly parallel, but more loosely arranged than the cells of the periphery. This region constitutes the site of origin of the cells of the ever-forming enamelorgan. From this pass forward the outer and inner layers, and between them, in larger quantity than is found more anteriorly, the tissue of the middle layer. This for a short distance is all enamel pulp and shows no differentiated layer of stratum intermedium.
  
 +
In this formative region on the labial side, the inner layer consists of columnar cells, the future ameloblasts, in which many mitoses are seen. While the outer layer consists of cells which are columnar near the margin, a short distance anterior to this (150/x) they change shape, first to cubical, then to flattened cuboidal. Between the two layers are cells representing the enamel pulp. At this region there are no papillae, although numerous blood-vessels are seen alongside the outer layer of the enamel-organ. About 0.5 nmi. from the basal end this outer layer of the enamel-organ becomes sinuous, and low papillae are being formed.
  
 +
On the lingual side, the structure of the basal end of the enamelorgan is similar, but somewhat simpler. Thus there are three layers at and near the basal margin, but soon, proceeding anteriorly, these become reduced to two by the disappearance of the middle layer. The lingual side then consists of two rows of cuboidal or flattened cells, one constituting the outer and the other the inner layer of the enamel-organ in this situation.
  
 +
The dental papilla is made up of closely packed small stellate cells, with rounded nuclei. The mesenchymal cells which lie against the basal margin of the enamel-organ are rounded or irregular in shape, but within a short distance (0.5 mm.) anterior to this margin, the peripheral cells become first cubical and then columnar in shape. Where they are beginning to form dentine they measure 30m in length. From the odontoblasts processes enter the dentinal tubules of the dentine. The outer surfaces of the odontoblasts from which these processes arise show a distinct cuticular margin. Between the odontoblasts at short intervals capillaries form loops around the cells. These are evidently for the purpose of insuring an ample blood supply to these functionally active cells.
  
 +
Four days old
  
27
+
By 4 days of age there has been continued growth, and deposition of enamel and dentine. The upper incisor measures 3.6 mm. in length and the lower 5 mm. The position of the apex of the tooth is in close relation to the oral epithelium (fig. 18). A thickened mass of epithelium, partly a derivative of the original stalk of the enamel-organ, and partly an ingrowth from the
  
 +
Fig. 18 Longitudinal section of upper incisor of 4-day albino rat, showing the increased curvature of the outline of the tooth and the relation of the apex of the tooth to the ingrowth of the oral epithelium. X 16.
  
 +
Fig. 19 Longitudinal section through basal end of labial side of enamelorgan of 4-day albino rat showing the region of the thickened margin, a, margin composed of mass of proliferating cells; b, region where three layers are seen; c, region where stratum intermedium becomes differentiated from rest of middle layer. Anterior to the region of this figure the epithelial papillae appear and the ameloblasts begin to form enamel. X 80.
  
30
 
  
  
 +
surface epithelium surrounding the tip of the tooth, is a preparation for the eruption of the tooth, and will serve as a resistant ling of tissue through which the tooth will be pushed at eruption. It may be looked upon as a protective device, to prevent adjacent tissues from being carried out by the erupting tooth.
  
 +
The typical enamel-organ seen on the labial side does not cover the apex, for the tall columnar cells give place here, first to cubical and then to flat squamous epithelial cells, which form but a part of the thick mass of stratified epithelium, constituting the epithelial sheath over the end of the dentine. The other layers of the functioning enamel-organ also lose their identity at the region where the ameloblasts cease to have their characteristic elongated form. As maintained by von Brunn ('87) and Sachse ('94), there is no enamel apparent over the dentine at the apex of the tooth.
  
35
+
The cells representing the enamel-organ on the lingual side can be traced forward for a short distance as a two-layered stratum. These cells are flattened, with oval nuclei. Beyond this point only a single regular row of cells is apparent, and about half way along the length of the tooth-structure, even this ceases to be definite, and apparently here the mesenchymal cells of the peridental tissues have grown between and scattered these cells. As a result of this activity of the mesenchymal cells in this region, the enamel-organ now ceases to exist as a complete conical investment of the tooth. Approaching the apex of the tooth on the lingual side, one finds the prolongation of the epithelial sheath as a thin layer of flattened cells which thickens as it passes forwards into the epithelial sheath.
  
 +
The basal formative end of the enamel-organ consists of a thickened band of tissue, as shown in figure 18, and under higher magnification in figure 19. This end is thicker on the labial side than elsewhere and it curves inwards, as seen in longitudinal sections, thus considerably diminishing the diameter of the entrance to the pulp-chamber. The extremity of this mass of tissue (fig. 19, a), constitutes a common origin for the several layers of the enamel-organ and contains many dividing cells. A short distance (0.1 to 0.2 mm.) from the extremity (fig. 19, b)
  
  
PLATE 4
+
the cells form three layers, inner, middle and outer. The inner and outer layers, made up of columnar elements, stain more darkly than the middle layer, and the inner is thicker than the outer. The middle layer consists of elongated cells with oval nuclei, arranged for the most part with theii* long axes parallel to the surface of the enamel-organ. Frequent mitoses are also to be seen here, especially in the inner layer.
  
EXPLANATION OF FIGURES
+
In the region about 0.6 mm. anterior to this (fig. 19, c), where enamel formation has not yet begun, the innermost layer shows a single row of distinct tall columnar cells, the ameloblasts. The middle layer now shows two subdivisions (a) two or three layers of compacted flattened cells lying against the ameloblasts, and composing the stratum intermedium, and (b) a somewhat thicker stratum, lightly staining, of more loosely arranged cells, constituting the enamel pulp. The outermost layer is a single row of cubical cells, which form a straight continuous surface for the enamel-organ. Beyond this layer and in contact with it are numerous small blood-vessels. Passing still farther forwards, the outermost layer becomes more sinuous in outline, and blood-vessels occupy the depressions between the elevations. This arrangement shows the beginning formation of the typical epithelial papillae.
  
32 and 33 Anaphases of first oocyte division. X 700.
+
Seven days old
  
34 Metaphase of second oocyte division. X 700.
+
At 7 days the tip of the tooth is in the oral epithelium (fig. 20), and ready for eruption, being separated from the outside by only a thin layer of superficial cornified epithelium. The epithelial tissues immediately about the apex of the tooth show the appearance of pressure atrophy. The cell boundaries are more indistinct than elsewhere, the tissue takes the acid stain deeply, and there is increased granularity — evidently degenerative effects due to the pressure of the advancing tooth.
  
35 Copulation of pronuclei; same egg as figure 27. X 700.
+
In the upper jaw, the basal end of the tooth in its backward growth has reached the region of the maxilla, into which it continues to grow, pushing before it a little pocket of thin bone. The average length of the upper teeth at this age is 5 mm., and of the lower teeth, 7 to 8 mm. Their pointed apices, and their comparatively slight curvature are shown in figure 24.
  
36 Post-oogonial nuclei with chromatin masses. The cells are connected by protoplasmic strands or tubes.
 
  
37 Longitudinal section through portion of an egg string of a larval ovary. X 400. a. Stage a, oogonial region; b, Stage e, synizesis; c, Stage f, post-synaptic spireme; d, Stage h, dyad or tetrad chromosomes in nurse cells; e, eggs and nurse cells well differentiated.
 
  
38 and 39 Stage c; the chromatin masses are transformed into small irregular fragments which later assume a thread-like form.
 
  
40 Stage d; presynaptic leptotene.
+
Fig. 20 Osteodentine of apex of tooth of 7-day albino rat imbedded in the surface epithelium, showing cells in the lacunae in the matrix. X 175.
  
41 and 42 Two groups of protoplasmic tubes with branches.
+
ERUPTION OF TEETH
  
 +
Eight to ten days
  
 +
During the process of eruption (fig. 21), the tooth and its formative organs gradually move forward as a whole, and the apex of the dentine forming the anterior end of the tooth pierces the surface epithelium. This procedure is accompanied by new changes in the tooth-forming organs. For while the same process of cell-division continues at the basal end of the dental papilla and enamel-organ, these structures are subjected to
  
36
+
Fig. 21 Longitudinal section of the upper tooth of an 8-day albino rat, showing the apex of the tooth piercing the surface epithelium. X 10.
  
 +
Fig. 22 Longitudinal section of the upper tooth of a 12-day albino rat, showing the increased size and curvature of the tooth, the basal end directed more towards the palatal surface and the progression of the apex of the tooth through the epithelium. X 10.
  
 +
Fig. 23 Longitudinal section of the upper incisor of a 26-day albino rat, showing the well-established occlusal surface, the approximation of the basal end towards the palatal surface, abundant blood-vessels in the pulp, and the position of the granular osteodentine filling in apex of the pulp chamber. X 10.
  
OOGENESIS IN PHILOSAMIA
+
new conditions at the erupting end of the tooth. Before detailing these changes, it may be advisable to state, in a general way, the changing circumstances attendant upon eruption. Up to this time the anterior end of the tooth has been nearly stationary, but there has been continued growth backward of the posterior extremity. At this time the rate of progression foi-ward is greatly increased, and the rate of progression backward much reduced. As suggested before, the process of eruption may depend largely upon the fact of increasing calcification in the bones, rendering them more resistant to the backward growth of the developing tooth. Whatever may be the causal factors, from now on the tooth continues to grow out at a regular rate, through the development of new cells at the basal end of the formative organs, these cells in turn giving rise to the hard parts of the tooth. Within a few days after eruption, the use of the tooth involves the process of attrition by which, in spite of the regular rate of growth, the exposed length is kept nearly constant for any age.
  
PAULINE H. DEDERER
+
It is generally agreed that, by reason of the protoplasmic processes which extend into enamel and dentine from ameloblasts and odontoblasts respectively, these cells must be carried along with the tooth as it moves. Thus, as there is constantly a regeneration of these cells at the basal end of the tooth, there must be an opposite process of some nature by which these cells are eventually lost at the apical end, when carried thither by the outward progress of the tooth. First we may follow the history of the ameloblasts in this locality. Before eruption, the enamel-organ is continuous with the stratified epithelium forming the sheath around the gingival margin, and this relation continues at and after eruption. As the tooth moves forward during eruption the ameloblasts must move along with it and, when those at the anterior end approach the gingival margin, they must either be held there, or be carried out on the enamel until detached. On examining longitudinal sections at 12 days (fig. 22) it is seen that the ameloblasts, as they approach the gingival margin, become shorter and shorter, until, beneath the thickened sheath of epithelium forming the gingival margin they acquire a flattened form. As a continuation of these flattened cells next the tooth is seen, extending out into the space between the erupted tip of the tooth and the epithelial gingival margin, a thin layer of tissue, which must be looked upon as the portion of the enamelorgan which has been carried out during eruption. At later stages this same appearance occurs — a thin layer of flattened cells continuous with the enamel-organ lying in the space between the tooth and the epithelium of the gingival border. It may be that some of the cells are added to the epithelium of the gingival margin, but the majority appear to be continually carried out, and eventually detached.
  
 +
The mesenchymal tissues of the pulp at the anterior end are little afl'ected by the mere act of eruption and not until some days later when attrition begins, do we see definite changes. At eruption the anterior conical extremity of the tooth is formed of osteodentine, containing within its matrix the remains of scattered cells and blood-vessels. Immediately posterior to this begins the true fine-tubed dentine with a central pulp-chamber. The cells at the anterior end of the pulp-chamber are irregularly arranged, but following backwards one soon sees the odontoblasts in parallel arrangement at the periphery of the chamber. At 10 days, when the apex of the tooth has pierced the epithelium and is easily seen from the outside, the measurements of the upper and lower teeth are 7 and 11 mm, respectively. At 12 days, they have increased to 7.5 and 11.8 mm.
  
 +
CHANGES IN APEX OF TOOTH BY USE
  
PLATE 4
+
Already at 12 days, when one examines the exposed ends of the teeth, they show little pits, which have been caused by the pressure of the opposing teeth. At 14 days, the ends are flattened, and at 16 days, because of the increased obliquity of this flattened surface due to the wearing away of the lingual side of the dentine, they are acquiring a cutting edge. The length of these occlusal surfaces continues to increase so that by 19 or 21 days (fig. 24), they have nearly the appearance typically seen in the fully developed teeth. The osteodentine of the tip of the tooth is softer than is true dentine, for when the young tooth is dried this end shrivels and darkens in color. This cap of osteodentine on the end of the tooth may be useful, as suggested by Sachse ('94), because of its softness, in allowing the early formation of the functional occlusal surface. When this soft substance begins to wear away the tissues of the pulp would soon become exposed were there not a provision for the filling in of the apex of the pulp-chamber. This is effected by the formation of an irregular hard matrix, which may also be called osteodentine, within the extremity of the pulp-chamber. As
  
  
  
frt>?.rV>.T,v:V.
 
  
 +
Fig. 24 Isolated upper and lower incisors of several ages of young albino rats. The pointed shape just before eruption is seen at 7 days. At 12 days, there is yet very slight change in the apices. At 21 days the occlusal surfaces are concave, and at 26 days they have nearly the typical mature appearance. X 2.
  
 +
the outer surface of the tooth wears away, this formation is constantly taking place a short distance from the occlusal -surface.
  
^^^
+
Thus in examining a longitudinal section of the tooth at an age when the process of attrition has begun, and the typical occlusal surface has been formed (e.g., 26 days, fig. 23), we find this form of secondary dentine or osteodentine filling in the distal extremity of the pulp-chamber. As one approaches the anterior end of the pulp-chamber, the pulp becomes more and more restricted and the blood-vessels appear congested. Proceeding distally, the irregular matrix formation is seen between the cells and blood-vessels and finally near the occlusal worn surface is a granular mass of osteodentine with no circulating blood in it, but spaces are still seen containing the remains of the pulp elements. Here the living elements have disappeared, but by staining (e.g., with acid fuchsin), the remains of these may be made out in less calcified spots in the matrix. Evidently the odontoblasts and other tissues of the pulp which move with the dentine, become more and more compressed at the narrowing apex of the pulp-chamber, and finally there is this irregular deposit of secondary dentine between them, which serves to obliterate the pulp-chamber. As the tooth moves out, this process is constantly going on, just in advance of the occlusal surface, and keeps pace with the process of attrition.
  
 +
It is interesting to note the rate at which the teeth are increased in length during their formative period and prior to attrition.
  
 +
Up per Lower mm. mm.
  
?&^->-^
+
1 day old 2.3 3
  
 +
4 days old 3.6 5
  
 +
7 days old 5 7-8
  
32
+
10 days old 7 11
  
 +
Average growth 0.52 nun. and 0.88 mm per day
  
 +
As will be seen later, this exceeds the rate at which the mature tooth continues to grow out.
  
 +
DESCRIPTION OF MATURE TOOTH AND TOOTH-FORMING ORGANS, IN FIVE-MONTH ANIMAL
  
33
+
In the mature tooth, the general relations are shown in figure 25, made from a photograph of a decalcified section of the upper tooth of a 5-month animal. The regular curved outline is seen, with the greater proportion of the length imbedded within the jaw, and only a small part projecting. The formative end lies witliin an investment of bone belonging to the maxilla. At this end the dentine is very thin and the pulp greatest in amount. As one goes forward, the dentine increases regularly in thickness while the pulp-chamber becomes smaller and smaller. The vacuolated appearance at the anterior end of the chamber is due to shrinkage of the pulp tissue during fixation. The enamel has been lost in the process of decalcification except over the basal third. Numerous blood-vessels are seen within the pulp.
  
  
  
 +
Fig. 25 Longitudinal section of upper incisor of a 5-month albino rat. The letter a shows where the next illustration (fig. 26) is taken. X 6.
  
 +
Fig. 26 Small portion of the preceding, more highly magnified, to show the enamel-organ and the enamel, and the odontoblasts and dentine. X 135.
  
 +
The enamel-organ is continuous over the convex labial surface of the imbedded portion of the tooth but is restricted to the most posterior region of the other surfaces, extending only 1 mm. forward from the basal margin. The enamel-organ differs in its structure in three regions of the labial side, and may be described separately in these three parts: (1) at the basal formative end, (2) near the gingival margin and (3) in the long intervening region. In (1) the enamel-organ is being constantly regenerated by the addition and growth of new cells. In (2) the enamel-organ is undergoing a retrograde process, while (3) represents the region where the enamel-organ is at its highest functional development, although its activity in increasing the thickness of the enamel is restricted, as noted before, to the basal third or less in the upper and to the basal half in the lower tooth.
  
 +
Considering first the region (3), as shown in figure 26, the enamel-organ is conspicuous by reason of its tall ameloblasts and the high, narrow papillae. The enamel-organ is described in three layers — inner, middle and outer. The inner layer consists of the ameloblasts, which measure about 40 /x in height, with nuclei situated towards the outer end of the cells. The middle is composed of two strata (a) stratum intermedium, and (b) enamel pulp. The stratum intermedium is formed of 1 or 2 rows of fairly regular cuboida cells resting upon the outer ends of the ameloblasts, but the enamel pulp is not now recognizable as a distinct layer and exists principally within the papillae. The outer layer of the enamel-organ consisted originally of a single layer of cells, but these are no longer regular in form or arrangement. Together with the remains of the enamel pulp, the outer layer forms the papillary elevations, 60 to 70 M in height. These papillae are surrounded by an abundant capillary blood supply for the nourishment of the cells engaged in the formation of the enamel, and the purpose of the elevations is apparently to increase the surface area through which absorption may take place from the blood-stream.
  
 +
The enamel is in two layers (fig. 26), the rods while traversing the inner layer being very distinct, and inclining towards the apex of the tooth at an angle of from 50 to 54° with the dentine surface. The continuations of these rods in the outer layer are not so distinctly seen, but the inclination, as made out in thin sections, is still greater towards the apex, forming an angle of from 20 to 25° with the plane of the surface of the dentine. As noted before in the study of enamel, the rods in the inner layer, when observed in cross-sections of the tooth, decussate at an angle of from 70 to 90°, but when they reach the outer layer all run parallel. The fact that the rods run in these various directions seems incontrovertible, but in the light of our present knowledge of enamel formation it is difficult to understand how this condition is arrived at. If each ameloblast is responsible for an enamel-rod, then it follows that because the alternate layers of rods are oblique to one another, the ameloblasts responsible for these series of rods must have changed their relative positions during the process of formation of these rods. No such phenomenon has been observed, or even suggested. The other possibility is that the matrix of the rods is formed in a regular manner, but that afterwards, before calcification is complete, the rods become re-arranged owing to pressure strains.
  
 +
The plane of cUrection of the rods is suggestive of the importance of the enamel-organ in the persistent growth. For always the general plane of the rods, as they leave the enameldentine junction, is towards the outer end of the tooth, as if the ameloblasts, while engaged in enamel-formation were always held back by the enamel, in which their processes were imbedded.
  
 +
The basal formative end of the enamel-organ (region 1) in the adult animal corresponds very closely in structure with what has already been described for earlier ages, e.g., 4 days. This is the region where the enamel-organ is constantly being renewed, and it retains the same embryonal character at all stages of development.
  
34
+
At the anterior end where the enamel-organ is continuous with the surface epithelium (region 2), a gradual transition occurs between the typical enamel-organ and the stratified squamous epithelium (fig, 27). As one follows the innermost layer of the enamel-organ forward, the cells become shorter, until they are cubical and finally flattened in shape. Here the other layers also lose their regular arrangement, and form, with the preceding, a thin layer of stratified cells. This layer can be followed directly into contact with the epithelium of the gingivus.
  
  
  
  
 +
Fig. 27 Longitudinal section of the apex of the tooth of a 5-month albino rat, showing at a the position of the outward prolongation of the remains of the enamel-organ, and at h the more granular osteodentine filling in the apex of the pulp chamber. X 10.
  
  
  
 +
The cells, however, do not lose their identity in the surface epithelium but remain separate as a thin layer lying against the enamel (fig. 27, a). This thin layer of epithelium, therefore, represents the ultimate fate of the enamel-organ after it has completed its functional activity. It is being continually pushed out and its most anterior part must be continually being lost.
  
  
  
 +
RATE OF GROWTH OF THE INCISOR TEETH
  
jP?
+
Two methods were used for determining the rate of growth of the incisor teeth (a) cutting off one or more teeth at the gingival margin and (b) making marks upon the enamel. The results here given are based on the latter method, as giving more nearlj^ the normal rate of growth. By means of a dental engine, the animal always having been anesthetized, a fine transverse notch was made on the enamel of the incisors a short distance from the gingival margin. The interval between this mark and the tip of the tooth was then measured. At the end of about a week the distance between these two limits was again taken, and the difference between the two measurements showed the amount of wearing away. Two series of experiments were made by this method upon adult animals. In the first series six animals were used and in the second four animals, and measurements were made for several consecutive weeks. The longest period that one individual was studied was six weeks.
  
 +
The two series gave very similar results. The lower tooth was always found to grow more rapidly than the upper. The upper tooth averaged 0.31 to 0.32 mm. per day, or 2.2 mm. per week, while the lower tooth averaged 0.4 mm. per day, or 2.8 mm. per week. No doubt there are many variations of the rate of growth under different circumstances, so that these figures must be taken as representing the average rate under one particular set of conditions. The food of these animals was the mixed diet now in use in the rat colony of The Wistar Institute. A short series of trials was made with animals kept in a large glass jar and given only soft food. In these animals the rate of wearing away corresponded very closely to that seen in animals which have also hard food and have the opportunity of gnawing. In these, therefore, the interaction of the opposing teeth must have caused the attrition.
  
 +
For assistance with these experiments we wish to thank Dr. Stotsenburg, who greatly aided us in carrying out our observations.
  
kf^N
 
  
 +
It is interesting to compare these results with those obtained on the rabbit — the only other rodent which has been carefully studied in respect to the growth of its incisors. MacGillavry ('75), using a young adult rabbit, made marks upon its lower incisors 2.5 mm. and 3 mm. from the tip. After five to seven days the marks had disappeared. Evidently the rate of growth was about 2.5 to 3 mm. per week. Noe ('02) used a rabbit which happened to possess overgrown teeth. The animal accidentally broke off the lower incisors in the bars of its cage, and Noe made observations upon the rate of their growing out. This he found to be .615 mm. per day, or 4.3 mm. per week. This is larger than MacGillavry's results and may have been due to the unopposed growth and to the other abnormal conditions which may have been present in the formative organs.
  
 +
Using MacGillavry's figures for comparison, it would seem that the lower teeth of the albino rat and of the rabbit grow out at about the same rate.
  
.;^
+
OVERGROWTH OF INCISORS
  
 +
Examples of overgrowth of the incisors of rodents, especially in rabbits and hares, which were hunted as food, must have been observed from early times. In the older literature, they are referred to principally as curiosities, which have excited the interest of whoever has found them. later the causes of the malformations were also considered. Thus Jenyns ('29), to cite only one observer, found several examples in wild rabbits, and has given a good illustration of the curved aspect of the teeth. He also clearly states the several causes which, in his opinion, may give rise to the condition. In addition to the one usually accepted at his time — accidental breaking off of one tooth — he considered also as causes (a) too soft food, (b) morbid or too rapid secretion of the osseous matter of the teeth, and (c) dislocation of one of the condyles.
  
 +
Wiedersheim ('02-'03) has reported a case occurring in a rat, where he found an associated assymmetry of the cranium. He is in doubt as to which was cause and which was effect — the overgrowth of the teeth or the assymmetry of the cranium.
  
>^.
 
  
 +
Fig. 28 Cranium of albino rat, showing the overgrown upper incisors recurving to the left side. The left incisor passes to the outer side of the skull, while the apex of the right incisor has penetrated the bone of the maxilla in the region of the basal end of the left incisor. X 1.
  
 +
Fig. 29 Cranium of the same albino rat shown in the preceding figure, viewed from the right side. It shows the overgrown lower incisors recurving to the right side, and the cavity which the right incisor has worn in the palate bone.
  
^s#
+
Figures 28 and 29 show a skull obtained some years ago from the rat colony of The Wistar Institute by Dr. Stotsenburg, and prepared in the Histological Laboratory by Miss E. F. Brooks. The upper teeth curved to the left side of the head and the lower to the right side. As seen in figure 29, the right lower has penetrated through the bone of the palate into the nasal chamber, while the right upper (fig. 28) has recurved and grown into the maxilla.
  
 +
In The Wistar Institute rat colony, at the time when the animals were fed on bread and milk, frequent examples of this and similar conditions were found, but now under a more varied mixed diet they practically never occur.
  
 +
Beretta ('13) has recently made an analysis of these abnormalities and has classified them in three groups.
  
 +
(1) Overgrowth of the upper and lower incisors through lack of an opposing tooth.
  
 +
(2) Overgrowth of the incisors of the upper and lower jaws through deviation of the jaws.
  
38
+
(3) Prognathism of the lower jaw, and as a result, overgrowth of the incisor of the lower jaw.
  
 +
In the present instance, diet seemed to be the controlling factor, probably by reason of its influence on the hardness of the bone of the alveoli from which the teeth grew out.
  
 +
SUMMARY
  
39
+
The rate of growth of the upper and lower incisor teeth of Mus norvegicus albinus, in the mature animal, averages 2.2 and 2.8 mm. per week, or 12.5 cm. and 14.5 cm. per year, respectively.
  
 +
Growth is due primarily to the proliferation and growth of cells at the basal end of the enamel-organ, where new enamelforming cells arise, and at the basal end of the dental papilla where new dentine-forming cells develop.
  
 +
The enamel-organ of the adult forms a narrow circular band around the basal end of the tooth, and extends forward from this on the labial side only. It coincides in its lateral boundaries with the enamel, and extends along the entire imbedded portion of the tooth. Anteriorly, it comes in contact with the epithelium of the gingival margin, and is carried out continually as a narrow band of cells lying on the enamel, between the latter and the gingival epithelial tissue.
  
40
+
The first indication of the anlage of the incisors appears in 14-day-old fetuses. In fetuses, 21 daj^s of age (just before birth), enamel and dentine formation is beginning. In animals 1 day old the upper and lower teeth measure 2.3 and 3 nrni. At 8 to 10 days the teeth erupt, and at 10 days measure 7 and 11 mm. respectively. This period is therefore characterized by the rapid elongation of the teeth.
  
 +
The process of attrition begins within a few days after eruption, so that by 19 or 21 days of age, the typical occlusal surface is formed. Up to the time of eruption the anterior end or apex of the tooth is immediately under the oral epithelium, while the basal or growing end is continually progressing posteriorly. After eruption, the basal end becomes nearly stationary in position, while the whole tooth structure is continually moving forward. The extra-gingival length of the tooth is kept constant, however, by the attrition of the occlusal surface, either through use in gnawing or by the action of the opposing teeth.
  
 +
The histogenesis of the enamel-organ is practically completed by the 4th day after birth, although it does not attain its final relations to the tooth as a whole, until after eruption. In the 18-day fetus the enamel-organ is similar in all parts, and the cells of the inner layer measure the same, both lingually and labially. From this period forwards, however, the labial portion continues to progress towards its fully differentiated functional structure, while the lingual portion retrogresses, until at 4 days after birth the latter is disrupted, by the ingrowth of the surrounding connective tissue. Contrasting the cells of the inner layer — the potential ameloblasts — on the labial and lingual sides, thej^ are practically the same in the 18-day fetus, but at 19 days they are found to measure 24 and 20 m respectively. In the 21-day fetus, they measure 30 to 34 and 12 fx, and 1 day after birth the true ameloblasts on the labial side have increased to 40 n, while the non-functional cells of the lingual side are only 10 fx in height. At 4 days, the latter cease to form a continuous layer, by reason of the dispersion of the cells by the surrounding connective tissue, except at the basal formative region.
  
41
+
Characteristic of the permanently-growing enamel-organ are the epithelial papillae, formed by the elevations of the outer layer of the enamel-organ, and the cells of the enamel pulp. Between these elevations are numerous capillaries which insure a rich blood supply to the enamel-forming cells.
  
 +
There are three layers in the functional enamel-organ — inner, middle and outer. The inner is constituted of the tall ameloblasts, and the middle is made up of two divisions, (a) stratum intermedium and (b) enamel pulp. The latter unites with the single layer of cuboidal cells which compose the outer layer, to form the epithelial papillae (fig. 26).
  
 +
The apex of the primitive tooth is formed of a variety of secondary dentine — 'osteo-dentine' of Tomes — which is softer than true dentine, and differs in its structural arrangement (fig. 20). After eruption, this terminal portion of osteodentine is soon worn away by attrition, and the typical occlusal surface is developed, as seen at 19 or 21 days. At 21 and 23 days the first two molars erupt in both upper and lower jaws, and from now on the animal is able to secure food for itself, and if necessary can maintain an independent existence.
  
42
+
As the tooth continues to be worn away there is a provision for the continual filling in of the apex of the pulp-chamber by the formation of what may also be called osteodentine. This is a form of secondary dentine, containing, when first formed, cells and blood-vessels. This is always at a little distance, about 1 mm., from the occlusal surface, but as any part of the tooth, in its outward progression, approaches the occlusal surface, the soft elements disappear within the osteodentine, and the latter forms a hard continuous surface with the adjoining true dentine. The position of this osteodentine is marked as a line on the occlusal surface of the teeth (fig. 5).
  
 +
Prior to eruption there develops around the apex of the tooth, as it lies in contact with the surface epithelium, a thickened ring of stratified epithelium. This ring of tissue is pierced by the apex of the tooth at eruption, and would seem to have the function of serving as a resistant margin for the soft tissues, and of preventing other tissues being carried along with the erupting tooth.
  
  
37
+
The length of the teeth varies with the size of the cranium (table 1) so that the persistent growth is not only sufficient to offset the continual attrition, but also serves to keep the length of the teeth in a definite relation to the length of the skull, as the latter increases in size.
  
 +
The lower tooth is always longer than the upper, and this difference manifests itself even in the anlagen of these structures in the 19-day fetus (figs. 11 and 12).
  
 +
The contour of the enamel, as seen in cross-sections, is characteristically different in the upper and lower teeth (fig. 4).
  
PLATE 5
+
The enamel of the tooth is composed of two layers which are different in appearance. The enamel rods run in two sets which decussate with each other in the inner or plexiform layer, but they change their direction as they continue into the outer layer, so that in it they are all parallel. In longitudinal sections, the general direction of the rods (fig. 26), is to incline towards the apex of the tooth, as they run from the enamel-dentine boundary to the outer surface of the enamel.
  
EXPLANATION OF FIGURES
+
In conclusion, we wish to thank Professor Piersol for generous assistance in many ways, and Professor Donaldson for his constant interest in the study. We also wish to acknowledge the kind assistance of Mr. E. F. Faber in the preparation of the drawings.
  
43 Stage e; synizesis.
 
  
44 to 47 Stage f ; post-sjoiaptic spireme of nurse cells. 48 and 49 Stage g; diplotene stage; 13 split rods.
+
LITERATURE CITED
  
50 to 52 Stage h; chromosomes begin to fragment.
+
Adloff, p. 1898 Zur Entwicklungsgeschichte des Nagetiergebisses. Jena. Zeitschr. fiir Naturwissenschaft, Bd. 32, ss. 347-410.
  
53 and 54 Stage j ; young nurse cells, showing tubes entering egg cell. X 700.
+
Beretta, a. 1913 La normala dentatura dei roditori in rapporto alle anomalie dentali in questi osservate. La Stomatologia, t. 10. Abstract in Deutsche Monatsschrift fiir Zahnheilkunde, April, s. 287.
  
55 A, nucleus of nurse cell with plasmosomes and chromatin granules; X 700. B, plasmosome enlarged.
+
VON Brunn, a. 1887 Ueber die Ausdehnung des Schmelzorganes und seine Bedeutung fiir die Zahnbildung. Arch. f. mikr. Anat.. Bd. 29, ss. 367-383.
  
56 Older nurse cells, with tubes. X 400.
+
BuRCKHARDT, R. 1906 In Hertwig's Handbuch der Entwickelungslehre der Wirbeltiere, Bd. 2, Teil 1, Kapitel 4, ss. 349-456. "Die Entwickelungsgeschichte der Verknockerungen des Integuments und der Mundhohle der Wirbeltiere."
  
 +
Cope, E. D. 1888 The mechanical causes of the origin of the dentition of the Rodentia. Amer. Nat., vol. 22, pp. 3 11.
  
 +
Donaldson, H. H. 1912 The history and zoological position of the albino rat. Proceed. Acad. Nat. Sci., Philadelphia.
  
38
+
Freund, p. 1892 Beitriige zur Entwicklungsgeschichte der Zahnanlagen bei Nagethieren. Arch. f. mikr. Anat., Bd. 39, ss. 525-5c6.
  
 +
Fritsch, C. 1914 Untersuchungen iiber den Bau und die Innervierung des Dentins. Arch, fur mikr. Anat., Bd. 84, ss. 307-20.
  
 +
Hatai, S. 1907 On the zoological position of the albino rat. Biol. Bull., vol. 12, pp. 266-273.
  
OOGENESIS IN PHILOSAMIA
+
Jenyns, L. 1829 Observations on a preternatural growth of the incisor teeth occasionally observed in certain of the Mammalia Rodentia. Loudon's Magazine Nat. Hist., London, vol. 2, pp. 134-137.
  
PAULINE H. DEDEREB
+
Jolyet et Chaker 1875 De I'acte de ronger, etudie chez les rats. Comptes Rendus et Memoires de la Soc. de Biol., pp. 73-74.
  
 +
Lowe, L. 1881 Beitrage zur Kenntniss des Zahnes und seiner Befestigungsweise im Kiefer. Arch. f. mikr. Anat., Bd. 19, ss. 703-719.
  
 +
MacGillavry, T. H. 1875 Les dents incisives du Mus decumanus. Arch. Neerl. Sc. exact, et nat., Haarlem.
  
PLATE 5
+
Meyerheim, M. 1898 Beitrage zur Kenntnis der Entwicklung der Schneideziihne bei Mus decumanus. Dissertation. Leipzig.
  
 +
Mummery, J. H. 1912 On the distribution of the nerves of the dental pulp. Philos. Trans. Roy. Soc, London, vol. 202, B., pp. 337-349.
  
 +
NoE, J. 1902 Vitesse de croissance des incisives chez les Leporides. Comptes Rendus, hebd. des Seances et Memoires de la Soc. de Biol., pp. 531-532.
  
 +
OuDET, J. E. 1823 Experiences sur I'accroissement continue et la reproduction des dents chez les lapins. Jour, de Physiol. Exper. et Patholog., Tomes 3 et 4.
  
43
+
Owen, R. 1840-45 Odontography. London.
  
 +
Reichert, E. T., and Brown, A. P. 1910 The crystallography of hemoglobin. Pub. Carnegie Inst, of Washington.
  
 +
Retzius, a. 1838 Bemerkungen iiber den inneren Bau der Zahne mit besonderer Rlicksicht auf den in Zahn vorkommenden Rohrenbau. Mliller's Archiv.
  
 +
Roetter, F. 1889 TJeber Entwicklung und Wachstum der Schneidezahne bei jNIus musculus. Morphol. Jahrb., Bd. 15, ss. 457-477.
  
 +
Ryder, J. A. 1877 The significance of the diameters of the incisors in Rodentia. Proc. Acad. Nat. Sci., Philadelphia, vol. 29, pp. 314-318.
  
 +
1878 On the mechanical genesis of tooth-forms. Proc. Acad. Nat. Sci., Philadelphia, vol. 30, pp. 45-80.
  
 +
Sachse, B. 1894 Entwicklung der Schneidezahne bei Mus musculus. Dissertation. Leipzig.
  
. I. «K^
+
Stach, J. 1910 Die Ontogenie der Schneidezahne bei Lepus cuniculus. Extrait. Bui. d'Acad. Sc, Cracovie.
  
 +
Tomes, C. S. 1914 A manual of dental anatomy; human and comparative. 7th Ed. Edited by H. W. Marett-Tims and A. Hopewell-Smith.
  
 +
Tomes, J. 1850 Structure of the dental tissues of the order Rodentia. Phil. Trans. Royal Society of London, pp. 529-567.
  
 +
Tullberg, T. 1898-99 Ueber das System der Nagethiere. Nova Acta Reg. Soc. Sc. Upsaliensis, Series 3, ss. 1-514.
  
\*..^£j'.
+
Weber, M. 1904 Die Saugetiere.
  
 +
Wiedersheim, R. 1902-03 Ein abnormes Rattengebiss. Anat. Anz., Bd. 22, ss. 569-573.
  
 +
Williams, J. L. 1896 The formation and structure of dental enamel. Dental Cosmos, vol. 38.
  
48
+
Woodward, M. J. 1894 On the milk dentition of the Rodentia with a description of a vestigial milk incisor in the mouse (Alus musculus). Anat. Anz., Bd. 9, ss. 619-631.
  
  
  
^
+
==A Peculiar Structure In The Electroplax Of The Stargazer, Astroscopus Guttatus==
  
 +
James G. Hughes, Jr.
  
 +
From the Histological Laboratory of Princeton University, U. S. A.
  
49
+
THREE FIGURES
  
 +
The purpose of this paper is to determine the function and composition of the pecuhar pointed fibers and long pointed rods lying in the electric layer of the electroplaxes of the stargazer, Astroscopus guttatus.
  
 +
Before proceeding with a discussion of these rods, a brief description of the electric organ of this fish (according to Dahlgren)^ will be given.
  
 +
The electric apparatus is composed of two organs, which form two vertical columns roughly oval in horizontal section, and placed behind and somewhat under each eye. Each organ extends from the peculiar bare spot on the top of the head down to the tissues which form the roof of the oval cavity; and is composed of about 200 thin layers of electric tissue, which extend horizontally all the way across the organ. These layers of tissue are flat, and alwaj^s at the same distance from one another. Each layer contains about 20 electroplaxes, the outlines of which present a very irregular or scalloped appearance. The electric tissue in which the electroplaxes are imbedded is in appearance a jelly-like or mucous-like tissue, usually known as electric connective tissue, and which I have shown in the course of my work to be of the same composition as white fibrous connective tissue. The nerve and blood supply runs in the above tissue. The general form of a vertical section of an electroplax is shown by figure 1, which is a drawing of part of a section of a single electroplax.
  
/
 
  
 +
1 Anat. Anz., Bd. 29, S. 387, 1906.
  
  
^
+
Each electroplax is composed of three principal layers, a nervous or electric layer which forms the upper surface, a middle layer, and a lower or nutritive layer which along with the middle layer is evaginated into a large number of long papillae. All three layers are deeply marked with a dense series of fine striations, which are peculiar to the electroplaxes of several other fishes. The upper or electric surface is flat and smooth and receives the nerve endings. The current of electricity runs downward through the organ which produces it, and thus the nerve endings in accordance with Pacini's law are found on the negative pole of the electroplax.
  
 +
Proceeding directly to the subject of this paper, we may say that one of the most interesting of the points noted in the electroplaxes, when properly fixed and stained with iron hematoxylin, is a series of rod-like or thread-like objects running horizontally in the electric layer, among, above and below the nuclei and without any apparent connection with them (figs. 1 and 2).^ These rods are of various sizes and shapes, and in form are said to resemble the classic thunderbolts seen in the hand of representations of Jove. They usually taper slowly and branch extensively at one or both ends. Some of these branches sometimes seem to be mere lines, while others are wide and heavily pointed; at their other ends the rods are usually rounded; this latter appearance may be due, however, to the cut ends of the rods, for as noted above they sometimes branch at both ends. Some are short and heavy in appearance while others are long and thread-like. Peculiar looping, twisting, or knot-like bends are sometimes found at points on the longer rods. The outlines and contour of these rods are always smooth. Their size may vary from thick or thin rods of over 300 /x in length down to small ones that do not exceed 1 m- In those electroplaxes where the rods are few they sometimes lie parallel and point in a definite parallel direction, while in others where the rods are very numerous they do not seem to have any definite arrangement. In this latter condition the rods present a very wavy appearance. Their form may be seen in figures 1 and 2, which are drawings of the electric layer of an electroplax when
  
 +
^ All the figures are drawings of sections of electroplaxes of Astroscopus guttatus.
  
A
 
  
  
 +
Fig. 2 Horizontal section through an electroplax. Only the electric layer is seen; a, an extensively branching rod; h, fine branches of the above rod; c, a large characteristic loop in a rod; d, a cut end of a rod; e, nuclei of the electric layer. X 1200.
  
r
 
  
  
 +
stained with iron hematoxylin. These drawings show the electric layer in which the rods are found in horizontal section (fig. 2) and in vertical section (fig. 1).
  
If *ZM
+
The purpose, function, and chemical composition of these rods have been previously unknown to histologists. In order to determine anything in respect to their function or purpose, a knowledge as to the class of organic substance to which they belong, whether muscle, connective tissue, nervous, or chitinous,
 
 
 
 
 
 
 
 
 
 
 
 
 
 
53
 
 
 
 
 
 
 
^w.O?
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
-f;-'^
 
 
 
 
 
 
 
■*■
 
 
 
 
 
55
 
 
 
 
 
 
 
B
 
 
 
 
 
 
 
 
 
 
 
 
 
54
 
 
 
 
 
 
 
^^««Bi^-?>
 
 
 
 
 
 
 
56
 
 
 
 
 
 
 
39
 
 
 
 
 
 
 
PLATE 6
 
 
 
EXPLANATION OF FIGUKES
 
 
 
57 Portion of an egg string, showing an egg and two nurse cells. X 400.
 
 
 
58 and 59 Stage f; early and later post-synaptic stages of egg cell; spireme probably continuous.
 
 
 
60 Nucleus from an egg cell similar to the one shown in figure 57. X 700.
 
 
 
61 Stage g; spireme disappearing in the later-growth period; plasmosomes of varying size and form.
 
 
 
62 Slightly older nucleus; all traces of the spireme have disappeared.
 
 
 
63 Same. X 850.
 
 
 
64 Nucleus from a nearly mature egg, showing dark crescentic region. The nuclear cavity is filled with granules. X 400.
 
 
 
 
 
 
 
40
 
 
 
 
 
 
 
OOGENESIS IN PHILOSAMIA
 
 
 
PAULINE H. DEDETIER
 
 
 
 
 
 
 
PLATE
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ri
 
 
 
 
 
 
 
\0i
 
 
 
 
 
 
 
^«9 /.--'*
 
 
 
 
 
 
 
■^ -®J
 
 
 
 
 
 
 
 
 
 
 
 
 
57
 
 
 
 
 
 
 
 
 
 
 
V/^
 
 
 
 
 
 
 
60
 
 
 
 
 
 
 
 
 
59
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
• •
 
 
 
 
 
 
 
o^.
 
 
 
 
 
 
 
61
 
 
 
 
 
 
 
62
 
 
 
 
 
 
 
o
 
 
 
 
 
 
 
 
 
63
 
 
 
 
 
 
 
 
 
64
 
 
 
 
 
 
 
41
 
 
 
 
 
 
 
THE STRUCTURE AND GROWTH OF THE INCISOR TEETH OF THE ALBINO RAT
 
 
 
WILLIAM H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
From the Anatomical Laboratory of the University of Pennsylvania and The Wistar Institute of Anatomy, Philadelphia
 
 
 
TWENTY-NINE FIGURES
 
 
 
CONTENTS
 
 
 
Introduction 43
 
 
 
Historical survey 44
 
 
 
Material and methods 46
 
 
 
Dentition of adult animal 46
 
 
 
Minute description of the incisors 52
 
 
 
Microscopic structure of enamel and dentine 55
 
 
 
Development of the incisors 59
 
 
 
Detailed description of development up to time of eruption 61
 
 
 
Eruption of the teeth 78
 
 
 
Changes in apex of tooth by use 81
 
 
 
Description of mature tooth and tooth-forming organs, in 5-month animal. . 83
 
 
 
Rate of growth of incisor teeth 88
 
 
 
Overgrowth of incisor teeth 89
 
 
 
Summary 91
 
 
 
Literature cited 95
 
 
 
INTRODUCTION
 
 
 
The incisor teeth of the Rodentia have long been regarded by the zoologist as having a high value for the understanding of many of the characteristics of this order. For instance, in 1888, Cope wrote "nearly all the peculiarities of the rodent dental system and manner of mastication are the mechanical consequences of an increase in length of the incisor teeth." Tullberg ('98-'99) gives the taxonomic position of the genus Mus, proceeding from the more general to the more specific grouping: Rodentia, Simplicidentati, Sciurognathi, Myomorphi, Myoidei, Muriformes, Myodontes, Muridae, Murini, Mus. A consideration of these terms merely from an etymological view suggests the importance of the teeth and jaws in the classifi. 43
 
 
 
JOURNAL OF MORPHOLOGY, VOL. 26. NO 1
 
 
 
 
 
 
 
44 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
cation of the gnawing animals. The observations here recorded are based on the study of the celhilar processes involved in the formation, eruption and growth of the incisor teeth in a single rodent form — Mus norvegicus albinus. An additional interest was lent to the work by the fact of the increasing use of this animal for laboratory purposes, which makes it desirable to learn the time-relations of its life-processes, as a basis of comparison in various forms of experimental studies. Although the rodent incisors have been the object of much study, few observers have carried out their observations through the complete life-history, including developmental stages and adult structure, in one form of animal and this it has been our aim to do.
 
 
 
HISTORICAL SURVEY
 
 
 
Oudet ('23) proved the phenomenon of permanent growth in the incisor teeth of rodents by cutting off the teeth at the gingival margin and observing that they were regenerated. Retzius ('37) and others noted the overgrowth of these teeth in cases of malocclusion. MacGillavry ('76) observed the rate of growth of the incisors of a rabbit by making marks on the teeth and noting the gradual advance and disappearance of these marks, as the teeth grew out and were worn away.
 
 
 
Questions which have called forth much study and controversy are (1) does the rodent incisor belong to the milk or to the permanent dentition; and (2) which of the three incisors of the typical mammalian dental formula does it represent. Without exception, all who have studied the first question agree that the large rodent incisor belongs to the second or permanent dentition. These same studies show that abortive milk incisors occur in a varying degree in the several families of the Rodentia; and that they are slightly, if at all, represented in the Muridae. As to the second question. Cope on palaeontological evidence decided that the large rodent incisor was h. Adloff ('98) on embryological evidence confirmed this view. Freund ('92), Woodward ('94) and Stach ('10) beheved it to be I,. Weber ('04) has given a resume and extended bibliography of this work, up to the date of his writing.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 45
 
 
 
The histology of the incisor was briefly described by Owen ('40-'45) and more completely studied by J. Tomes ('50). The latter found a considerable diversity of arrangement of the enamel prisms in the different families of the order, so that in many cases he was able correctly to refer a tooth to a particular family by a simple inspection of thin sections of its enamel. Von Brunn ('87) showed that at eruption the tip of the incisor of the albino rat is free from enamel, and Sachse ('94) confirmed this on Mus musculus. J. L. Williams ('96), in a comparative study of the formation of enamel, gives a number of good illustrations of the structure of the enamel and enamel-organ of the rat, prepared from microphotographs.
 
 
 
Ryder ('78) and Cope ('88), in harmony with their views on the "Origin of the Fittest," described the form and position of the rodent incisor as manifestations of a most efficient mechanical system; and studied the various effects on skull topography, necessitated by adaptation to this system.
 
 
 
The enamel organ of the albino rat was studied by von Brunn ('87) who described in some detail the differences in structure between its functional labial portion and its non-functional lingual side. He also described the early continuity of the lingual side of the enamel-organ and its later penetration by the surrounding connective tissue. Roetter ('89), studying Mus musculus, denied von Brunn's position in regard to the invasion of the lingual side of the enamel-organ by connective tissue, and Sachse ('94), also using Mus musculus, agreed with Roetter and described the continuity of the lingual portion as persisting through life.
 
 
 
The development of the rodent incisor has been studied especially by Roetter ('89), Sachse ('96) and Meyerheim ('98). Burckhardt ('06), in his description of the development of the persistently growing rodent incisor in O. Hertwig's Handbuch der Entwickelungslehre has followed chiefly Sachse's work upon Mus musculus. In both Weber ('04) and Hertwig ('06) are extensive bibliographies and in these may be found all references not fully given in our appended list of literature cited.
 
 
 
 
 
 
 
46 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
MATERIAL AND METHODS
 
 
 
The albino rat is a variety of Mus norvegicus, the common gray rat (Donaldson '12). This has been shown by similarity of skull measurements (Hatai '07) and of hemoglobin crystals (Reichert and Brown '10) and also by the fact that the two interbreed freely.
 
 
 
The material used was obtained from the rat colony of The Wistar Institute. Serial sections in paraffin or in paraffincelloidin were made of decalcified heads of fetuses taken at daily intervals from the 16th day onwards until birth, and of jaws of animals newly-born and at short intervals until one month, and of several older stages. Serial sections of fetuses younger than 16 days were examined in the collection of The Wistar Institute. Ground sections were made of the isolated teeth, and the petrifaction method of imbedding in Canada balsam was used to prepare the teeth and adjacent soft parts in situ. Also a series of prepared crania, some entire and some disarticulated, was made at selected ages, varying from birth to old age. The 'gold dust' method of Davison, as tested out for different ages at The Wistar Institute was used for the preparation of the former, and maceration in tap water for the latter. Schultze's clearing method was found useful in studying the early periods of calcification.
 
 
 
DENTITION OF ADULT ANIMAL
 
 
 
The dental formula of the albino rat is I ., C ^, P „, M ' .
 
 
 
There is only one set of teeth, and hence the dentition is monophyodont. The time of eruption of the various teeth extends over a period of 3^ weeks. The incisors are the first to appear, viz., at 8 to 10 days after bhth. The first and second molars erupt at about the 19th and 21st days respectively, and it is after this period that the young animals may be weaned and are able to maintain an independent existence, as far as food is concerned. The third molars are delayed until 2 weeks later and do not appear until about the 35th day.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
47
 
 
 
 
 
 
 
 
 
,Xt®^",
 
 
 
 
 
 
 
 
 
 
 
Fig. 1 Cranium of a 5-month albino rat. X 2.
 
 
 
Fig. 2 Cranium of a 5-month albino rat, with the bony alveoli dissected away to show the entire length of the incisor teeth. X 2.
 
 
 
The incisors are permanently-growing (or rootless) teeth, while the molars have a definite limited period of development and acquire roots. A wide diastema separates the incisors from the molars as may be seen by reference to figm-e 1. The incisors are strongly curved and Owen ('40-'45) has described the lower incisor as being the smaller segment of a larger circle, and the upper incisor as the larger segment of a smaller circle. In the lower incisor of the albino rat this statement needs a slight modification. For while the curvature of the upper in
 
 
 
 
 
48 W. H.'F. ADDISON AND J. L. APPLETON, JR.
 
 
 
cisor is in one plane only, the lower incisor is a portion of a flattened spiral, possessing a curve in three planes. The upper incisor is a segment of a true circle (at 5 months about 210°) and in cases of overgrowth it has often been known to complete the cu'cle. In the case of the lower incisor, however, when we project it on the sagittal, frontal or coronal planes, it gives in each case a curve. It was the very evident curved projection seen on the sagittal plane to which Owen referred. Considering only this view, the lower incisor of a 5-month animal forms a segment of about four-fifths of a semicircle (140-145°).
 
 
 
TABLE 1 23 1 41 ! 10 15 , 5 ; 8 10
 
 
 
DAYS j DAYS I WEEKS WEEKS MONTHS MONTHS MONTHS
 
 
 
imii. ! m7n. : mm. \ mm. mm. mm. mm.
 
 
 
Xaso-occipital length 29 .7 32 .5 39 40 43 44 46 .5
 
 
 
Interzvgomatic 13.7 14 14.5 14.6 15. -> 15.1 15.5
 
 
 
Uppe/diastema 7.4 9.5 10 11.4 12 .3 12 .5 13
 
 
 
Upper incisor— total length 12.8 15 18.3 20.3 23.3 23.7 26.2
 
 
 
Upper incisor — extra-alveolar
 
 
 
length ! 5.1 i 5.5 j 7 8.4 8.7 9 9 3
 
 
 
Lower diastema ' 4.6 I 5 I 5.6 6 6.7 7 6.8
 
 
 
Lower incisor— totallength 18.1 21.7 25.5 26.4 29.4 29.9 31.3
 
 
 
Lower incisor — extra-alveolar
 
 
 
length 6.5: 7 I 10.5 11.4 11.6 12 12.4
 
 
 
 
 
 
 
Measurements of the incisors and skulls of animals of different ages, were made as shown in table 1.
 
 
 
The teeth were measm-ed along their convex surfaces by means of silk thread wet with water, and applied to the object to be measured. The thread was then cut with scissors at the end of the object, straightened on paper and measured to tenths of millimeters.
 
 
 
A consideration of table 1, shows in a definite way the peculiarities characteristic of the dentition, not only of the rat but of rodents in general. As is well known, these are the great development of the incisors, the wide diastema, and the consequent posterior position of the molar teeth as related to the rest of the skull. Cope ('88) wrote that he considered "the
 
 
 
 
 
 
 
STRUCTUEE AND GROWTH OF INCISOR TEETH 49
 
 
 
increase in the length of these teeth has been due to their continued use, as beUeved by Ryder." The effects of this increased elongation upon surrounding parts he described under several different headings, but reference will be made here only to one, viz., upon the shape of the glenoid cavity. "A peculiarity of the masticating apparatus is the lack of a postglenoid process, and the consequent freedom of the lower jaw to slide backward and forward in mastication. Appropriately to this motion, the condyle of the mandible is extended antero-posteriorly and the glenoid ca^dty is a longitudinal instead of a transverse groove."
 
 
 
 
 
 
 
Fig. 3 Thimble-shaped portion of the maxiUa bone, in which the basal end of the upper incisor is located. X 2.
 
 
 
The lower incisors are longer and more slender than the upper and extend far back in the mandible, beneath the lower molars, to near the sigmoid notch. The upper incisors are contained within the premaxilla and maxilla, the basal end occupying a thin-walled, thimble-shaped recess of bone (fig. 3) to be seen best in the disarticulated skull, and which is attached at only one limited region to the rest of the maxilla. In both upper and lower teeth, the intra-alveolar portions are longer than the extra-alveolar. When one compares the extra-alveolar lengths of the upper and lower teeth of the mature animal, the latter are always greater, and, as may be seen by reference to table 1, the difference in lengths becomes greater with increased age and size.
 
 
 
In both upper and lower incisors the bone is so contoured around their imbedded portions that their course may be easilj^ recognized. The basal end or foraminal apex of the lower
 
 
 
 
 
 
 
50 W. H. F. ADDISON AND J. L, APPLETON, JR.
 
 
 
incisor forms on the outer aspect of the mandible a marked rounded projection, directed upwards and backwards beneath the coronoid process, and sometimes extending sUghtly posteriorly beneath the sigmoid notch. Almost directly opposite this projection on the mesial aspect of the mandible is the inferior dental foramen. This projection marks the position of the growing end of the formative organs of the incisor in the adult. In the new-born anunal it is not present, nor at the end of the first month. By the age of 2| months it may be recognized, and thereafter it increases in prominence and constitutes a very evident feature of the bone. This region of the growing end of the tooth is protected b}'^ the zygomatic arch, and also by the overlying muscles.
 
 
 
The course of the upper incisor may also be readily followed in the prepared skull. Laterally it is covered with a thin rounded layer of bone. Mesially it forms an elevated, distinct ridge projecting markedly into the nasal fossa. In the adult the position of its basal or growing end is not so prominent as that of the lower incisor. As these incisor teeth are an indispensable part of the rodents' existence their importance demands protection from traumatism which might injure their growing pulp. Here in the upper incisors, this protection is afforded by a flange of the maxilla running parallel to the lateral wall of the cranium, as shown in figure 1 , as well as being encased in a separate thimble-shaped recess of bone (fig. 3), beneath, and separated by a narrow interval from, the outer layer of the maxilla. These details are in harmony with Cope's idea ('88) of the influence of the incisors in moulding the general topography of the rodent skull.
 
 
 
The diastema in the upper jaw is always longer than in the lower (fig. 1). By reference to table 1 it may be seen that in the mature animal the upper is nearly twice as long as the lower, but that in the younger stages the difference is not so great. The upper hair-covered lips are infolded into the diastema, dividing the oral cavity into an anterior and posterior compartment. This arrangement probably prevents the debris and splinters of gnawing from entering the main oral cavity.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
51
 
 
 
 
 
 
 
The mandibular symphysis is formed of fibrous tissue and allows independent rotation of either ramus with its contained tooth. This lateral movement of the lower incisors appears to be under the control of the will of the animal. According to the observations of Jolyet and Chaker (75) this mobility has a definite purpose in mastication. They observed a rapid alter
 
 
 
 
 
 
 
 
 
Fig. 4 Cross-sections of the (a) upper and (b) lower incisor teeth of a 5-month albino rat, taken near the alveolar margins. These show the arrangement of the enamel and the dentine, and the difference in contour of the enamel in the upper and lower teeth. The mesial surface cf each tooth is towards the right side. X 15.
 
 
 
nate separation and approximation of the tips of the lower incisors in the act of attempting to bite into a match or other slender object offered to the animal. At the same time the upper incisors were held stationary.
 
 
 
Mention may be made here of a point of variation among the Rodentia in the relation of the angle of the lower jaw to the sheath of bone around the lower incisor. In the Myomorphi and Sciuromorphi the angle arises from the lower surface of the incisive sheath, while in Hystrix the angle arises entirely on the outer side.
 
 
 
 
 
 
 
52 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
Ryder (77) suggested a classification of rodents based on the shape of their incisors as seen in cross-section. In some genera the diameter of the teeth is less from side to side, than in the antero-posterior direction, while in others the reverse condition is found. The present form belongs to the former group, as is shown in figure 4. From the consideration of many rodents, Ryder deduced the general principle, that where the incisors are thicker in the antero-posterior direction, the gnawing habit is greatly developed.
 
 
 
MINUTE DESCRIPTION OF THE INCISORS
 
 
 
Enamel and dentine make up the hard tooth substance, enclosing the pulp. Owen, in his "Odontography" ('40-'45, p. 399) said that there existed a general investment of cementum over the whole tooth structure. J. Tomes ('50, p. 533) was not able to agree entirely but said that in most, if not in all, incisors of rodents cementum could be seen investing the posterior surface. In the rat, it is not apparent that there is any cementum at all. The enamel is usually colored with a pigment which is yellowish in the young but becomes orange-colored with age, and is usually more pronounced in the upper than in the lower incisors. At 13 days, there is as yet no color, but at 21 days a slight tinge of yellow is perceptible in the uppers, but none in the lowers. At 25 days the uppers are distinctly yellow, and the lowers have now acquired a slight color. At 38 days, these colors have intensified, the uppers having more pigment than the lowers; and in the mature animal the same relation continues, the uppers being orange-colored and the lowers yellow. The enamel is found principally on the labial side, and this accounts for the shape of the occlusal surface. For, the enamel being harder than the dentine, the latter is more easily worn away by the action of the opposing tooth, and the more resistant enamel remains as the cutting edge or point. The shape of the incisal end of the upper and lower teeth is different, being chisellike (scalpriform) in the upper, and more rounded and narrower in the lower. The incisal line is also usuallv different in the
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
53
 
 
 
 
 
 
 
upper and lower teeth. In the former, it is often sHghtly concave from side to side, while in the latter it is convex (fig. 5). As is shown in figures 1 and 5 the occlusal surface is an elongated concave area on the lingual aspect of the teeth, and in the living animal extends practically to the gingival margin. Due to the difference in the curve of the upper and lower teeth, the occlusal surface of the lower teeth is always longer than that of the upper, and in the mature animal it is usually found to be nearly twice as long.
 
 
 
 
 
 
 
 
 
 
 
Fig. 5 Labial and lingual aspects of the extra-alveolar portions of the (a) upper and (b) lower incisors of a 5-month albino rat, showing the occlusal surfaces and incisal edges of the teeth, and the outline of the bony alveolar margins. X 2.
 
 
 
 
 
 
 
It follows that because these teeth are constantly growing, the occlusal surfaces are constantly being worn away. As we shall see, when discussing the growth of the teeth, the elongated temporo-mandibular articulation is important, in allowing the teeth to have either the position pictured in figure 1 or to have the opposite relation, with the lower teeth outside of the upper. Thus the very important factor in the animal's economy — the proper regulation of the length of the opposing incisors — is controlled by their own inter-action.
 
 
 
 
 
 
 
54
 
 
 
 
 
 
 
W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
 
 
 
 
The pulp-chamber has the characteristic shape found in all permanently growing teeth, as is well seen, for instance, in the elephant's incisor. Its cross-area is greatest at the basal end of the tooth, and gradually diminishes anteriorly. The pulpchamber is found to extend in the tooth beyond the line of the gingivus, and very nearly to the occlusal surface. The shape
 
 
 
 
 
 
 
 
 
anterior end
 
 
 
 
 
 
 
•■ E D C B A
 
 
 
Fig. 6 Upper incisor of a o-month albino rat (X o) and cross-sections of it at different points (X 8), to show the relative cross-area of the dentine and of the pulp chamber at these regions. The dotted line indicates the position of the margin of the alveolus.
 
 
 
 
 
 
 
in cross-section of the pulp-chamber at different levels may be seen by reference to figure 6. The position of the filled-in pulpchamber is usually well marked on the occlusal surfaces as a line (fig. 5). In weathered specimens of rats' teeth from recent geological formations this last-formed part which fills in the pulpchamber at the end of the tooth, is usually found to be lacking, and is evidently not of the same hardness as the surrounding parts of the tooth.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 55
 
 
 
MICROSCOPIC STRUCTURE OF ENAMEL AND DENTINE
 
 
 
Sections of enamel show two layers; an outer thin and an inner thicker layer, as noted by Owen ('40-'45, p. 399). The enamel rods run in different directions in the two layers as fully described by J. Tomes in 1850. In the inner layer the enamel rods appear to run in two sets, obliquely to one another, while in the outer layer the rods are all parallel. The outer layer has also been called the fibrous layer, and in its superficial part is situated the yellow or orange pigment which gives the color to the enamel.
 
 
 
Figures 7 and 8 show the arrangement of the enamel rods in the two la3^ers. In the inner or plexiform layer, when examined in cross section, the alternating series of enamel rods decussate, forming an angle varying between 70 and 90°. In longitudinal sections (fig. 26) these rods are slightly S-shaped, running outwards from the enamel-dentine surface at an angle of 50 to 54°, and inclining towards the anterior end of the tooth. Figure 8 is from a ground-section in which the enamel was broken during the process of preparation, and the broken edge shows distinctly the two sets of rods running at nearly right angles to each other. Under high magnification the rods are slightly notched.
 
 
 
In cross-sections of the outer fibrous layer, the rods are parallel and form in the mid-line of the tooth an angle of 90° with the outer surface. As one proceeds away from the mid-line of the tooth, whether mesially or laterally, the general tendency of the long axis of the rods as they pass from the dentine junction to the periphery, is to incline in the direction away from the mid-line of the tooth. The ameloblasts usually form an obtuse angle with the rods of the outer layer and seldom coincide in direction with them (fig. 7). In longitudinal sections the rods of the outer layer are not usually so distinctly seen as in cross-sections. In favorable longitudinal sections, however, they are seen to run quite obliquely, inclining towards the apex of the tooth, and forming an angle of 20 to 25° with the plane of the enamel-dentine junction. The pigment, as will be seen
 
 
 
 
 
 
 
 
 
ayer of tTieloblasts
 
 
 
 
 
 
 
uter layer f enamel
 
 
 
 
 
 
 
nner layer enamel
 
 
 
 
 
 
 
 
 
Fig. 7 Portion of cross-section of lower incisor with enamel-organ, prepared by the petrifaction method, showing the decussation of the enamel-rods in the inner or plexiform layer and their jiarallel arrangement in the outer or fibrous layer. X 350.
 
 
 
Fig. 8 Small piece of enamel, showing the rods of the inner or plexiform layer running in two directions nearly at right angles to one another. X 350.
 
 
 
56
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 57
 
 
 
below, is confined to the outermost part of the fibrous layer. There appears to be no Nasmyth's membrane over the enamel, which means that there has been a complete transformation of the enamel matrix into enamel rods. The pigment extends about two-thirds of the total length of the upper tooth, and about one-half of the total length of the lower tooth, and hence it follows that the deposition of enamel is completed within the basal third of the upper and the basal half of the lower tooth. By examining cross-sections of the tooth at different regions (fig. 6) it would seem that the full thickness of the enamel is attained within even a smaller area at the basal end of the tooth.
 
 
 
The arrangement of the enamel over the labial aspect of the upper and lower teeth is shown in figure 4, drawn from crosssections of the teeth of a 5-month animal. In both teeth the sections were made just posterior to the alveolar border. In both upper and lower teeth the enamel is thickest over the labial aspect, and is continued over the adjacent mesial and lateral surfaces. In both, the enamel is continued farther on the lateral than on the mesial surfaces, and relatively farther on the lateral surface in the lower than in the upper tooth. In the upper tooth the enamel has a flattened external surface labially, while in the lower it has a rounded contour. In the upper there is a distinct labio-mesial and a labio-lateral angle, the enamel being somewhat thicker at the former. In the lower there is a labio-mesial angle, though less prominent than in the upper, and the labio-lateral angle is practically absent.
 
 
 
In a 5-month animal the thickness of the enamel and its constituent layers was measured in the mid-line of the teeth, as follows:
 
 
 
Upper Lower
 
 
 
Total thickness 100-110 lJO-150
 
 
 
Outer fibrous layer 30-40 20-30
 
 
 
Pigmented portion of outer fibrous layer 8-10-12 6-8
 
 
 
Inner plexiforni layer 70 120-125
 
 
 
It will be observed, however, in figure 4 that the enamel is not thickest in the mid-line of the upper tooth, but at the lateral and mesial angles. While the enamel of the upper tooth meas
 
 
 
 
 
58 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
ures only 100 to llO^t in the mid-line, it measures 160 to 180/x at the region of these angles, and is, therefore, thicker here than the enamel of the lower tooth. The increased thickness at the angles is principally in the inner plexiform layer, the other layer being increased only slightly or not at all. The outer fibrous layer is distinctly thicker in the uppers and has a slightly wider band of pigment in it superficially. This, no doubt, is the basis of the more deeply pigmented appearance of the labial surface of the upper as compared with the lower teeth.
 
 
 
The dentine, unlike the enamel, grows continually thicker as one passes towards the outer end of the tooth. At the basal, growing, end it begins as an extremely thin layer. The thickness at different points is seen in figure 6. As the dentine increases in thickness, the pulp-chamber is in consequence proportionately reduced. At the distal end there is no longer any pulp-chamber and the site of its previous position has been filled in by the formation of a kind of secondary dentine. C. Tomes ('14) notes that "in some rodents the final closure of the axial tract takes place almost by a continuance of the formation of normal fine-tubed dentine, with very little secondary dentine of different structure, while in others there is a large area of dentine with vascular tracts in it." In the rat there is relatively little of this secondary dentine. It is laid down in irregular trabeculae, with the pulp tissue, including bloodvessels, at first within it. At the exposed surface, however, it forms a continuous granular mass with apparently no soft tissues in it (fig. 27). The ordinary dentine of the tooth is quite typical in structure, with numerous parallel dentinal tubules, each having many fine lateral branches. The tubules are slightly sinuous, and the lateral branches anastomose with those of neighboring tubules. Sometimes a tubule sends off at an acute angle a branch nearly equal in diameter to the continuation of the main tubule. This is usually in the dentine not covered by enamel. Where these large branches come off the diameter of the tubule is greater than elsewhere, measuring nearly 2^. Elsewhere the diameter varies from 1 to l.T/z. Slight differences may be seen between the tubules (a) in the dentine covered by enamel, and (b) in the dentine free from
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 59
 
 
 
enamel. The tubules of the anterior region (a) of the dentine, covered by enamel, are more regularly parallel and have finer lateral branches than elsewhere. They also seem to taper slightly as one follows them towards the enamel. In the dentine not covered by enamel (b) the tubules are more sinuous and irregular, the irregularities marking the position of origin of the larger lateral branches. In all parts at the periphery of the dentine the tubules end in a great number of very fine anastomosing arching branches. As a consequence of the smaller diameter of the little tubules here, a narrow zone at the periphery of the dentine has usually a more homogeneous appearance than has the remainder. Towards the anterior end of the tooth, in the vicinity of the pulp-chamber, are vascular channels in the form of loops within the dentine. The tubules must necessarily take a curved course around these vascular channels, and thus the position of the vessels is more easily seen.
 
 
 
In the dentinal tubules Mummery ('12), Fritsch ('14) and others have demonstrated not only the processes of the odontoblasts, but also fine non-medullated nerve fibers. As to why the exposed dentine on the lingual aspect of the teeth is insensitive, there are no definite observations to decide. A contributing factor may be the compression which the pulp tissues undergo at the anterior end of the pulp-chamber, leading to the physiological cutting off of the nerve supply to the dentinal tubules.
 
 
 
DEVELOPMENT OF THE INCISORS
 
 
 
The times of the early stages of development of the incisors were seen as follows:
 
 
 
14-day fetus — slight thickening of oral epithelium
 
 
 
15-day fetus — distinct thickening and growth inwards of oral epithelium
 
 
 
16-day fetus — dental ledge and beginning of flask-shaped enamel organ
 
 
 
17-day fetus — dental papilla with crescentic enamel organ capping it
 
 
 
19-day fetus — both ameloblasts and odontoblasts differentiated
 
 
 
new-born animal — enamel and dentine formation begun
 
 
 
8 to 10 days — eruption of the tooth
 
 
 
Throughout life growth continues, and in the adult animal is on the average 2.2 mm. per week in the upper and 2.8 mm. per week in the lower incisor.
 
 
 
JOURNAL OF MORPHOLOGY, VOL. 26, NO. 1
 
 
 
 
 
 
 
60 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
The structures to be described here, as in the development of the crowns of all teeth, are the enamel-organ with the ameloblasts, and the dental papilla (which becomes the pulp-substance) with the odontoblasts. There are two factors, however, which alter the usual history of the development of these structures, and especially of the enamel-organ. First, in permanently growing teeth of which these are examples, all these structures continue functional throughout life, so that the enamelorgan is also a persistent structure. The other factor and one correlated to some extent with the first, is that the enamel is formed on one side of the tooth only, and here only does the enamel-organ develop to its most highly differentiated functional condition.
 
 
 
The history of the development and growth of the tooth may be conveniently considered in two stages (1) pre-eruptive, and (2) post-eruptive. The pre-eruptive stage extends from the 14th or 15th day of fetal life until eruption of the tooth takes place between the 8th and 10th post-natal days. Until near the time of birth there is no formation of enamel and dentine, but from birth onwards these substances are laid down rapidly, so that at eruption, the teeth have their characteristic elongated narrow form. This pre-eruptive stage is characterized by the rapid elongation of the tooth-forming organs, and by the teeth attaining very similar relations to the other structures of the jaw which the imbedded portions of the erupted teeth possess. Thus, the anlage of the lower incisor appears under the oral epithelium in the anterior region of the mandible, and grows continually backwards, until its growing end reaches the region beneath the developing molars. At this time the growing end presumably reaches a region which, by reason of its increasing calcification, offers resistance to further progress. The result of the ever-continuing mitotic division and cell growth at the basal end, is the pushing of the whole tooth and its formative organs, in the opposite direction, and the consequent eruption of the tooth. During the latter half of this pre-eruptive stage, the anterior tip of the developing tooth structure is immediately beneath the oral epithelium, and remains at a fixed
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 61
 
 
 
point, while the posterior end is continually growing backwards and changing its relations. At eruption this condition changes, and the posterior extremity becomes practically a fixed point from which the whole tooth moves forward. That there is, however, a gradual change in the position of the posterior end of the tooth may be seen in figure 9. As the jaw grows, the entire tooth not only grows to keep the same general relative position, to surrounding structures, but it may be seen that the growing end progresses gradually posteriorly. In the full-grown animal this end occupies a distinct outpushing of the bone (fig. 1).
 
 
 
During the post-eruptive period, which continues throughout life, this outward growth is continued at a regular rate, and at the same rate the outer end has to be worn away. This wearing-away process would soon result in the pulp becoming exposed were not the occlusal end of the pulp-chamber also being continually filled in. As may be seen from figure 6 the dentine continues to increase in thickness until near the end of the tooth. This means that the odontoblasts continue their regular functional activity until near the end of the tooth. However, the final fiUing-in of the pulp-chamber to form a continuous hard occlusal surface is accomplished by the deposition of a hard matrix between the pulp elements and by the probable calcification of the latter. The result is, that as the tooth is worn away, the soft pulp never becomes exposed. Although the pulp reaches very near to the end of the tooth, a hard substance always fills in the end of the pulp-chamber, and so protects the pulp beneath.
 
 
 
DETAILED DESCRIPTION OF DEVELOPMENT UP TO THE TIME
 
 
 
OF ERUPTION
 
 
 
The anlage of the enamel-organ of each incisor arises as an epithelial ingrowth, distinct and separate from that for the molars.
 
 
 
In frontal sections of the 14-day fetus, there are slight diffuse thickenings of the oral epithelium in the four positions, which represent the sites of the future tooth-formations.
 
 
 
 
 
 
 
62
 
 
 
 
 
 
 
W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
 
 
 
 
6 day
 
 
 
 
 
 
 
 
 
23 day
 
 
 
 
 
 
 
 
 
10 months
 
 
 
 
 
 
 
Fig. 9 Series of mandibles of the albino rat at ages \arying from birth to ten months, viewed from the lateral aspect. These show the changing relation of the basal end of the incisor to the rest of the mandible during this period.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 63
 
 
 
At 15 days these thickenings have become more definite, and in the lower jaws especially have begun to push into the underlying mesenchyme, and may be described as the dental ledges or dental laminae.
 
 
 
At 16 days the ingrowths have continued to increase as broad masses of cells, pushing deeper into the underlying mesenchyme, and in the lower jaws the enamel organs may be distinguished as expanded structures, each connected by a slightly narrower mass of cells with the oral epithelium. In the upper jaws the differentiation of the enamel-organs from the remainder of the epithelial ingrowth is not so marked.
 
 
 
At 17 daj^s (fig. 10) the dental papillae are beginning, and the enamel-organs in both upper and lower jaws have a crescentic outline. In the enamel-organs there is already an indication of the differentiation into three layers. As seen in sagittal sections, the papillae develop on the posterior side of the enamelorgans, thus foreshadowing the axis of growth of the toothforming organs in the antero-posterior direction.
 
 
 
Eighteen-day fetus
 
 
 
Series of frontal sections of 18-day lower jaws, show that the enamel-organs are growing over the dental papillae more rapidly on the labial and lingual surfaces than elsewhere, and extend more posteriorly on these surfaces. There are thus two projections of the posterior margin of each enamel-organ as already noted by Meyerheim ('98). The labial process is broad and thin and extends more posteriorly than the lingual process, which is somewhat narrower and thicker. One may her-e remark, therefore, an early difference between the labial and lingual part of the enamel-organ. Other differences which will soon appear have not yet developed. Thus, the inner layer of the enamel-organ is made up of columnar elements which are still similar in all parts, both labially and lingually. In the dental papilla no columnar odontoblasts are yet seen.
 
 
 
The enamel-organ remains connected with the surface epithelium by a broad band of epithelial cells. In the lower jaw,
 
 
 
 
 
 
 
64 W. H. F. ADDISON AND J. L. APPLETON, JE.
 
 
 
immediately laterad to the line of j miction of this stalk of the enamel-organ to the surface epitheliimi, the lip furrow is a depression, the plane of which is continued into the underlying mesenchyme by an ingrowth of surface epithelium several layers of cells in thickness. It is by the subsequent splitting of this epithelial layer into two, that the separation of the lip will be effected.
 
 
 
Nineteen-day fetus
 
 
 
At 19 days, the enamel-organ in the upper jaw (fig. 11) is crescentic in outline in sagittal section, and in the lower jaw (fig. 12) is more elongated and conical in shape. At this age odontoblasts are first seen as columnar cells on the labial aspect of the mesodermal papilla. Three layers are recognizable in the enamel-organ, but the middle layer (enamel pulp), as has been also described by Sachse ('94) for the mouse, is extremely thin, and therefore is not present in the great quantity tj^pically seen in the development of rooted teeth. It appears as a more lightly stained zone between the inner and outer layers, and is thickest at the basal end of the enamel-organ as shown in figures 11 and 12. It averages about 20^i in thickness and is made up of stellate cells loosely arranged. Already there is an indication of a compact arrangement of the two or three rows of cells next the inner layer of the enamel-organ, which will result in the so-called stratum intermedium seen at later ages. This middle layer is also slightly more abundant at the anterior end in the region where the enamel-organ is continuous with the stalk which joins it to the oral epithelium.
 
 
 
At this age the enamel-organs in the lower jaws have a greater total length than those in the upper, and especially in the lower
 
 
 
Fig. 10 Longitudinal section of upper jaw of 17-day fetus, showing tooth anlage of incisor, with the enamel-organ longer labially than lingually when measured from the point of junction of the stalk of the enamel-organ. The dental papilla is on the posterior aspect of the enamel-organ. X 70.
 
 
 
Fig. 11 Longitudinal section of upper incisor anlage of 19-day fetus of albino rat, showing the crescentic outline of the enamel-organ, its greater length labially than lingually, and its thickened basal margin. X 70.
 
 
 
Fig. 12 Longitudinal section of lower incisor anlage of 19-day fetus of albino rat, showing the conical outline of the enamel-organ and its greater length than in the upper jaw^ at the same age. X 70.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
65
 
 
 
 
 
 
 
 
 
 
 
10
 
 
 
 
 
 
 
11
 
 
 
 
 
 
 
 
 
66 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
jaws distinct differences may be made out between the oral and labial sides of the tooth-forming organs. These differences are:
 
 
 
(1) The enamel-organ is longer labially than on the oral side.
 
 
 
(2) The staining of the inner layer of the enamel-organ on the labial side is more intense, and here the cells are slightly longer than in other parts of the enamel-organ, measuring 24/x in length and assuming the typical appearance of ameloblasts. Measurements show the similar cells on the lingual side to be about 20m in length. It is also to be noted at this age that the site of the most advanced cells which are differentiating to become ameloblasts is not at the apex of the enamel-organ, as is the case in the development of rooted teeth. For as one follows the cells of the labial side of the enamel-organ from the apex towards the base, while at the apex the cells are columnar they become longer as one goes posteriorly, and then towards the base of the enamel-organ diminish again. So that the site of most advanced differentiation here is a short distance posterior to the apex on the labial side. This is true also of the developing odontoblasts which are longest opposite the tallest ameloblasts.
 
 
 
(3) The outer layer of the enamel-organ on the labial side is becoming slightly wavy in outline, and this denotes the beginning of the papillae, which form such a characteristic part of the mature functional enamel-organ (fig. 26).
 
 
 
(4) The odontoblasts are seen only on the labial side of the dental papilla.
 
 
 
Mitoses are abundant in all parts of the developing tissues.
 
 
 
Twenty-one-daij fetus
 
 
 
Thus the anlage of the rodent incisor begins in the usual way, and for a short time continues along the typical mammalian course. From 19 days onward, however, the differences which have already begun, become more distinct and definite. At 21 days (end of gestation) the enamel-organ has become more definitely differentiated into a labial and a lingual region. Of the three constituent layers, the inner especially is strikingly different
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 67
 
 
 
in these two parts. On the labial side at the anterior end, the organ has advanced to the condition where functional activity is beginning, while the oral side has remained stationary, or has actually retrogressed. Thus in the innermost layer on the labial side of the lower incisor, where the ameloblasts have begun to form enamel, these cells measure 30 to 34ju in length, while the non-functional cells on the oral side of the innermost layer are low columnar or cubical in shape and measure only 12 /j. in length (fig. 13). Comparison of these measurements with those at 19 days shows that the cells of the inner layer of the labial side of the enamel-organ have advanced in length from 24/i to 30 : or 34m, while the cells on the lingual side have decreased from 20 to 12ju. There is, therefore, a primary tendency for the cells of the inner layer to develop equally in all parts, but very soon the nonenamel-forming cells of the lingual side begin to retrogress, while the functional cells of the labial side continue to grow. This constitutes another point of contrast with the development of the crowns of rooted teeth. For here in the 21 -day fetus, when the enamel and dentine formation has just begun, these substances are thickest, not over the apex of the tooth-forming organs, as in the usual method, but at a short distance posterior to this point, on the labial surface. Thus, not only are the odontoblasts and the ameloblasts first differentiated on the labial side, posterior to the apex, but at this region enamel and dentine formation is also evidently first begun.
 
 
 
Over the apex of the dental papilla there is apparently a very thin outline of dentine deposited, but within this, in the tissues of the apex of the dental papilla, there is also beginning an irregular formation of a hard matrix. Between the cells of the pulp, trabeculae of a bone-like material are appearing. As development proceeds this substance increases until the final result is, as seen in figure 20, that the primary apex of the tooth has a bone-like structure, consisting of cells imbedded in lacunae within a dense matrix. This has been called by Tomes ('04) 'osteo-dentine.'
 
 
 
A similar difference between the labial and oral sides is noted in the cells on the margin of the dental papilla, which are be
 
 
 
 
 
68 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
coming odontoblasts. In the basal half of the papilla (fig. 14), odontoblasts occur only on the labial side opposite the tall ameloblasts, the peripheral cells of the other sides being still irregular or cuboidal in shape. Farther forwards the odontoblasts are found also on the lateral and mesial surfaces of the dental papilla, but not on the lingual. In the apical one-fourth of the dental papilla odontoblasts occur all round, measuring 20 to 24/x in length, and are engaged in the formation of dentine (fig. 13). The dentine is thickest on the labial side.
 
 
 
In the region where enamel and dentine formation has begun no mitoses were seen in the formative ameloblastic and odontoblastic cells, but posteriorly, where the deposition of enamel and dentine has not yet commenced, many mitoses occur in the layers of developing ameloblasts and odontoblasts, as well as elsewhere. The nearer one approaches the basal margin of the enamel-organ the more numerous are the mitoses and it is apparent that it is principally in this region that growth by addition of new cells is taking place.
 
 
 
One day old
 
 
 
At the end of the first day of post-natal life, there has been great progress in the enamel and dentine formation, and the narrow, pointed outline of the tooth has been already laid down. In the upper jaw the teeth measure about 2.3 mm. in length and in the lower jaw about 3 mm. Definite changes in its relation to the oral epithelium have occurred also at the anterior end of the tooth. The original epithelial stalk connecting the enamelorgan with the oral epithelium has increased in size and the end
 
 
 
Fig. 13 Cross-section of developing lower incisor of 21-day fetus of albino rat, nearer the anterior extremity of the tooth than figure 14. Shows the greater thickness of the labial side of the enamel organ, as compared with that of the other sides, and shows odontoblasts around the entire periphery of the pulp. Enamel and dentine formation has begun. X 110.
 
 
 
Fig. 14 Cross-section of developing lower incisor of 21-day fetus of albino rat, posterior to the region shown in figure 13. No enamel or dentine yet formed at this point. Odontoblasts highest on the labial aspect, decreasing in height laterally but not yet differentiated as columnar elements on the lingual side. Enamel-organ thickest on the labial side. X 110.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
69
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
u
 
 
 
 
 
 
 
70 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
of the tooth has apparently advanced somewhat into it. So this thick stratified layer of epithelium forms a close-fitting investment about the tooth apex, and is continuous posteriorly with the remainder of the enamel-organ. But in this epithelial cap there are no ameloblasts and consequently there can be no enamel over the osteodentine which forms the tip of the primitive tooth. This substance forming the tip of the unerupted tooth is a form of secondary dentine with its cells located in the lacunae of the matrix. Passing backwards, one comes to the ordinary dentine containing the vascular pulp with odontoblasts situated at the periphery of the pulp-chamber in a regular manner.
 
 
 
As the odontoblasts were first differentiated labially, and dentine formation began there before on the other side, the dentine of the labial side is thicker than on the lingual side. Thus at a point about the middle of the entire tooth structure, the dentine measured 54/^ labially and 20/x orally (fig. 15). Between the odontoblasts are numerous fine capillary loops. At this region may also be seen the characteristic structure of the enamel-organ (fig. 15). This extends all around the tooth, but is much thicker on the labial side than elsewhere. This difference in thickness is seen in all the constituent layers. In the inner layer, the tall ameloblasts of the labial surface measure iOfj., while the similarly situated cells on the other surfaces are cubical and measure only 10^. Comparing these with the previous stage described, it is seen that the cells on the labial surface have increased and those on the other surfaces have decreased. Of the middle layer on the labial side, the stratum intermedium is a distinct line of cuboidal cells, one to two rows in thickness, lying behind the ameloblasts. The other constituent — the original enamel pulp — is small in amount and is principally within the elevations of the outer layer, which form the beginning of the epithelial papillae. The cells of the outer layer, somewhat irregular in shape with round nuclei, are in a single row. Between the developing papillae (called by Sachse StLitzpapillen) are numerous capillary blood-vessels. On the other surfaces, practically nothing remains of the middle layer,
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
71
 
 
 
 
 
 
 
although the outer layer still persists as a layer of flattened cells. Thus lingually the enamel-organ is represented by only two rows of cells — one representing the inner, the other the outer layer of the enamel-organ.
 
 
 
 
 
 
 
M
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 15 Cross-section of developing lower incisor of 1-day albino rat, showing the great development of the ameloblasts on the labial side, and the thinness of the enamel-organ elsewhere. The space between the ameloblasts and the dentine is an artefect, and was formerly partly filled by the enamel, which has disappeared in the pi'ocess of decalcification. In the layer of odontoblasts are seen the nuclei of the endothelial cells of the walls of capillaries. X 110.
 
 
 
 
 
 
 
Tivo days old
 
 
 
Figure 16 shows a longitudinal section of a 2-day upper incisor. The epithelial enamel-organ is continuous over the whole tooth, but only shows its specialized functioning structure on the labial side. On the lingual side it is still intact and consists only of two rows of cuboidal or flattened epithelial cells. On the labial side, along the region where enamel has been formed (fig. 17) the
 
 
 
 
 
 
 
 
 
16
 
 
 
 
 
 
 
 
 
Fig. 16 Longitudinal section of upper incisor of 2-day albino rat showing the enamel-organ continuous over the labial surface and terminating posteriorly in the thickened margin. X 18.
 
 
 
Fig. 17 Small portion of preceding figure more highly magnified, to show the structure of the enamel-organ and the odontoblasts, a, outermost layer of enamel-organ and epithelial papillae; b, enamel pulp; c, stratum intermedium; d, layer of ameloblasts; e, layer of dentine; f, layer of odontoblasts. X 175.
 
 
 
72
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 73
 
 
 
ameloblasts measure about 40^. These are backed by two rows of darkly staining flattened cells composing the stratum intermedium. Next to these is the looser arrangement of stellate cells, comparable to the enamel pulp of ordinary tooth development, but with much smaller spaces between the cells. This tissue is covered by the layer of cells constituting the outer layer of the enamel-organ, and the two together constitute the epithelial papillae. At the summit of each of these papillae the cells of the outer layer are grouped in a more compact manner. With higher magnification processes can be seen running from the ameloblasts into the developing enamel — the so-called enamel processes of Tomes.
 
 
 
At the basal formative part of the enamel-organ the three original layers show distinctly. At the thickened basal margin of the enamel-organ, around its entire circumference, is a mass of rapidly dividing cells. As seen in figure 16 this thickened margin is more noticeable on the labial side. Its peripheral zone as seen in longitudinal sections is deeply staining and its cells, more or less columnar in shape, are compacted together. The interior, of more lightly stained appearance, is composed of oval or elongated cells, irregularly parallel, but more loosely arranged than the cells of the periphery. This region constitutes the site of origin of the cells of the ever-forming enamelorgan. From this pass forward the outer and inner layers, and between them, in larger quantity than is found more anteriorly, the tissue of the middle layer. This for a short distance is all enamel pulp and shows no differentiated layer of stratum intermedium.
 
 
 
In this formative region on the labial side, the inner layer consists of columnar cells, the future ameloblasts, in which many mitoses are seen. While the outer layer consists of cells which are columnar near the margin, a short distance anterior to this (150/x) they change shape, first to cubical, then to flattened cuboidal. Between the two layers are cells representing the enamel pulp. At this region there are no papillae, although numerous blood-vessels are seen alongside the outer layer of the enamel-organ. About 0.5 nmi. from the basal end this outer
 
 
 
 
 
 
 
74 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
layer of the enamel-organ becomes sinuous, and low papillae are being formed.
 
 
 
On the lingual side, the structure of the basal end of the enamelorgan is similar, but somewhat simpler. Thus there are three layers at and near the basal margin, but soon, proceeding anteriorly, these become reduced to two by the disappearance of the middle layer. The lingual side then consists of two rows of cuboidal or flattened cells, one constituting the outer and the other the inner layer of the enamel-organ in this situation.
 
 
 
The dental papilla is made up of closely packed small stellate cells, with rounded nuclei. The mesenchymal cells which lie against the basal margin of the enamel-organ are rounded or irregular in shape, but within a short distance (0.5 mm.) anterior to this margin, the peripheral cells become first cubical and then columnar in shape. Where they are beginning to form dentine they measure 30m in length. From the odontoblasts processes enter the dentinal tubules of the dentine. The outer surfaces of the odontoblasts from which these processes arise show a distinct cuticular margin. Between the odontoblasts at short intervals capillaries form loops around the cells. These are evidently for the purpose of insuring an ample blood supply to these functionally active cells.
 
 
 
Four days old
 
 
 
By 4 days of age there has been continued growth, and deposition of enamel and dentine. The upper incisor measures 3.6 mm. in length and the lower 5 mm. The position of the apex of the tooth is in close relation to the oral epithelium (fig. 18). A thickened mass of epithelium, partly a derivative of the original stalk of the enamel-organ, and partly an ingrowth from the
 
 
 
Fig. 18 Longitudinal section of upper incisor of 4-day albino rat, showing the increased curvature of the outline of the tooth and the relation of the apex of the tooth to the ingrowth of the oral epithelium. X 16.
 
 
 
Fig. 19 Longitudinal section through basal end of labial side of enamelorgan of 4-day albino rat showing the region of the thickened margin, a, margin composed of mass of proliferating cells; b, region where three layers are seen; c, region where stratum intermedium becomes differentiated from rest of middle layer. Anterior to the region of this figure the epithelial papillae appear and the ameloblasts begin to form enamel. X 80.
 
 
 
 
 
 
 
STEUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
75
 
 
 
 
 
 
 
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tfl»*«vvf ^ ,
 
 
 
 
 
 
 
m
 
 
 
 
 
 
 
18
 
 
 
 
 
 
 
 
 
76 W. H. F. ADDISON AND J. F. APPLETON, JR.
 
 
 
surface epithelium surrounding the tip of the tooth, is a preparation for the eruption of the tooth, and will serve as a resistant ling of tissue through which the tooth will be pushed at eruption. It may be looked upon as a protective device, to prevent adjacent tissues from being carried out by the erupting tooth.
 
 
 
The typical enamel-organ seen on the labial side does not cover the apex, for the tall columnar cells give place here, first to cubical and then to flat squamous epithelial cells, which form but a part of the thick mass of stratified epithelium, constituting the epithelial sheath over the end of the dentine. The other layers of the functioning enamel-organ also lose their identity at the region where the ameloblasts cease to have their characteristic elongated form. As maintained by von Brunn ('87) and Sachse ('94), there is no enamel apparent over the dentine at the apex of the tooth.
 
 
 
The cells representing the enamel-organ on the lingual side can be traced forward for a short distance as a two-layered stratum. These cells are flattened, with oval nuclei. Beyond this point only a single regular row of cells is apparent, and about half way along the length of the tooth-structure, even this ceases to be definite, and apparently here the mesenchymal cells of the peridental tissues have grown between and scattered these cells. As a result of this activity of the mesenchymal cells in this region, the enamel-organ now ceases to exist as a complete conical investment of the tooth. Approaching the apex of the tooth on the lingual side, one finds the prolongation of the epithelial sheath as a thin layer of flattened cells which thickens as it passes forwards into the epithelial sheath.
 
 
 
The basal formative end of the enamel-organ consists of a thickened band of tissue, as shown in figure 18, and under higher magnification in figure 19. This end is thicker on the labial side than elsewhere and it curves inwards, as seen in longitudinal sections, thus considerably diminishing the diameter of the entrance to the pulp-chamber. The extremity of this mass of tissue (fig. 19, a), constitutes a common origin for the several layers of the enamel-organ and contains many dividing cells. A short distance (0.1 to 0.2 mm.) from the extremity (fig. 19, b)
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 77
 
 
 
the cells form three layers, inner, middle and outer. The inner and outer layers, made up of columnar elements, stain more darkly than the middle layer, and the inner is thicker than the outer. The middle layer consists of elongated cells with oval nuclei, arranged for the most part with theii* long axes parallel to the surface of the enamel-organ. Frequent mitoses are also to be seen here, especially in the inner layer.
 
 
 
In the region about 0.6 mm. anterior to this (fig. 19, c), where enamel formation has not yet begun, the innermost layer shows a single row of distinct tall columnar cells, the ameloblasts. The middle layer now shows two subdivisions (a) two or three layers of compacted flattened cells lying against the ameloblasts, and composing the stratum intermedium, and (b) a somewhat thicker stratum, lightly staining, of more loosely arranged cells, constituting the enamel pulp. The outermost layer is a single row of cubical cells, which form a straight continuous surface for the enamel-organ. Beyond this layer and in contact with it are numerous small blood-vessels. Passing still farther forwards, the outermost layer becomes more sinuous in outline, and blood-vessels occupy the depressions between the elevations. This arrangement shows the beginning formation of the typical epithelial papillae.
 
 
 
Seven days old
 
 
 
At 7 days the tip of the tooth is in the oral epithelium (fig. 20), and ready for eruption, being separated from the outside by only a thin layer of superficial cornified epithelium. The epithelial tissues immediately about the apex of the tooth show the appearance of pressure atrophy. The cell boundaries are more indistinct than elsewhere, the tissue takes the acid stain deeply, and there is increased granularity — evidently degenerative effects due to the pressure of the advancing tooth.
 
 
 
In the upper jaw, the basal end of the tooth in its backward growth has reached the region of the maxilla, into which it continues to grow, pushing before it a little pocket of thin bone. The average length of the upper teeth at this age is 5 mm., and of the lower teeth, 7 to 8 mm. Their pointed apices, and their comparatively slight curvature are shown in figure 24.
 
 
 
 
 
 
 
78 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
 
 
 
 
 
 
Fig. 20 Osteodentine of apex of tooth of 7-day albino rat imbedded in the surface epithelium, showing cells in the lacunae in the matrix. X 175.
 
 
 
ERUPTION OF TEETH
 
 
 
Eight to ten days
 
 
 
During the process of eruption (fig. 21), the tooth and its formative organs gradually move forward as a whole, and the apex of the dentine forming the anterior end of the tooth pierces the surface epithelium. This procedure is accompanied by new changes in the tooth-forming organs. For while the same process of cell-division continues at the basal end of the dental papilla and enamel-organ, these structures are subjected to
 
 
 
Fig. 21 Longitudinal section of the upper tooth of an 8-day albino rat, showing the apex of the tooth piercing the surface epithelium. X 10.
 
 
 
Fig. 22 Longitudinal section of the upper tooth of a 12-day albino rat, showing the increased size and curvature of the tooth, the basal end directed more towards the palatal surface and the progression of the apex of the tooth through the epithelium. X 10.
 
 
 
Fig. 23 Longitudinal section of the upper incisor of a 26-day albino rat, showing the well-established occlusal surface, the approximation of the basal end towards the palatal surface, abundant blood-vessels in the pulp, and the position of the granular osteodentine filling in apex of the pulp chamber. X 10.
 
 
 
 
 
 
 
IW^'"^;
 
 
 
 
 
 
 
 
 
23
 
 
 
79
 
 
 
 
 
 
 
80 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
new conditions at the erupting end of the tooth. Before detailing these changes, it may be advisable to state, in a general way, the changing circumstances attendant upon eruption. Up to this time the anterior end of the tooth has been nearly stationary, but there has been continued growth backward of the posterior extremity. At this time the rate of progression foi-ward is greatly increased, and the rate of progression backward much reduced. As suggested before, the process of eruption may depend largely upon the fact of increasing calcification in the bones, rendering them more resistant to the backward growth of the developing tooth. Whatever may be the causal factors, from now on the tooth continues to grow out at a regular rate, through the development of new cells at the basal end of the formative organs, these cells in turn giving rise to the hard parts of the tooth. Within a few days after eruption, the use of the tooth involves the process of attrition by which, in spite of the regular rate of growth, the exposed length is kept nearly constant for any age.
 
 
 
It is generally agreed that, by reason of the protoplasmic processes which extend into enamel and dentine from ameloblasts and odontoblasts respectively, these cells must be carried along with the tooth as it moves. Thus, as there is constantly a regeneration of these cells at the basal end of the tooth, there must be an opposite process of some nature by which these cells are eventually lost at the apical end, when carried thither by the outward progress of the tooth. First we may follow the history of the ameloblasts in this locality. Before eruption, the enamel-organ is continuous with the stratified epithelium forming the sheath around the gingival margin, and this relation continues at and after eruption. As the tooth moves forward during eruption the ameloblasts must move along with it and, when those at the anterior end approach the gingival margin, they must either be held there, or be carried out on the enamel until detached. On examining longitudinal sections at 12 days (fig. 22) it is seen that the ameloblasts, as they approach the gingival margin, become shorter and shorter, until, beneath the thickened sheath of epithelium
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 81
 
 
 
forming the gingival margin they acquire a flattened form. As a continuation of these flattened cells next the tooth is seen, extending out into the space between the erupted tip of the tooth and the epithelial gingival margin, a thin layer of tissue, which must be looked upon as the portion of the enamelorgan which has been carried out during eruption. At later stages this same appearance occurs — a thin layer of flattened cells continuous with the enamel-organ lying in the space between the tooth and the epithelium of the gingival border. It may be that some of the cells are added to the epithelium of the gingival margin, but the majority appear to be continually carried out, and eventually detached.
 
 
 
The mesenchymal tissues of the pulp at the anterior end are little afl'ected by the mere act of eruption and not until some days later when attrition begins, do we see definite changes. At eruption the anterior conical extremity of the tooth is formed of osteodentine, containing within its matrix the remains of scattered cells and blood-vessels. Immediately posterior to this begins the true fine-tubed dentine with a central pulp-chamber. The cells at the anterior end of the pulp-chamber are irregularly arranged, but following backwards one soon sees the odontoblasts in parallel arrangement at the periphery of the chamber. At 10 days, when the apex of the tooth has pierced the epithelium and is easily seen from the outside, the measurements of the upper and lower teeth are 7 and 11 mm, respectively. At 12 days, they have increased to 7.5 and 11.8 mm.
 
 
 
CHANGES IN APEX OF TOOTH BY USE
 
 
 
Already at 12 days, when one examines the exposed ends of the teeth, they show little pits, which have been caused by the pressure of the opposing teeth. At 14 days, the ends are flattened, and at 16 days, because of the increased obliquity of this flattened surface due to the wearing away of the lingual side of the dentine, they are acquiring a cutting edge. The length of these occlusal surfaces continues to increase so that by 19 or 21 days (fig. 24), they have nearly the appearance typically
 
 
 
 
 
 
 
82 W. H. F. ADDISON AND J, L. APPLETON, JR.
 
 
 
seen in the fully developed teeth. The osteodentine of the tip of the tooth is softer than is true dentine, for when the young tooth is dried this end shrivels and darkens in color. This cap of osteodentine on the end of the tooth may be useful, as suggested by Sachse ('94), because of its softness, in allowing the early formation of the functional occlusal surface. When this soft substance begins to wear away the tissues of the pulp would soon become exposed were there not a provision for the filling in of the apex of the pulp-chamber. This is effected by the formation of an irregular hard matrix, which may also be called osteodentine, within the extremity of the pulp-chamber. As
 
 
 
 
 
 
 
 
 
Fig. 24 Isolated upper and lower incisors of several ages of young albino rats. The pointed shape just before eruption is seen at 7 days. At 12 days, there is yet very slight change in the apices. At 21 days the occlusal surfaces are concave, and at 26 days they have nearly the typical mature appearance. X 2.
 
 
 
the outer surface of the tooth wears away, this formation is constantly taking place a short distance from the occlusal -surface.
 
 
 
Thus in examining a longitudinal section of the tooth at an age when the process of attrition has begun, and the typical occlusal surface has been formed (e.g., 26 days, fig. 23), we find this form of secondary dentine or osteodentine filling in the distal extremity of the pulp-chamber. As one approaches the anterior end of the pulp-chamber, the pulp becomes more and more restricted and the blood-vessels appear congested. Proceeding distally, the irregular matrix formation is seen between the cells and blood-vessels and finally near the occlusal worn surface is a granular mass of osteodentine with no circulating blood in it, but spaces are still seen containing the remains of the pulp elements. Here the living elements have disappeared,
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 83
 
 
 
but by staining (e.g., with acid fuchsin), the remains of these may be made out in less calcified spots in the matrix. Evidently the odontoblasts and other tissues of the pulp which move with the dentine, become more and more compressed at the narrowing apex of the pulp-chamber, and finally there is this irregular deposit of secondary dentine between them, which serves to obliterate the pulp-chamber. As the tooth moves out, this process is constantly going on, just in advance of the occlusal surface, and keeps pace with the process of attrition.
 
 
 
It is interesting to note the rate at which the teeth are increased in length during their formative period and prior to attrition.
 
 
 
Up per Lower mm. mm.
 
 
 
1 day old 2.3 3
 
 
 
4 days old 3.6 5
 
 
 
7 days old 5 7-8
 
 
 
10 days old 7 11
 
 
 
Average growth 0.52 nun. and 0.88 mm per day
 
 
 
As will be seen later, this exceeds the rate at which the mature tooth continues to grow out.
 
 
 
DESCRIPTION OF MATURE TOOTH AND TOOTH-FORMING ORGANS, IN FIVE-MONTH ANIMAL
 
 
 
In the mature tooth, the general relations are shown in figure 25, made from a photograph of a decalcified section of the upper tooth of a 5-month animal. The regular curved outline is seen, with the greater proportion of the length imbedded within the jaw, and only a small part projecting. The formative end lies witliin an investment of bone belonging to the maxilla. At this end the dentine is very thin and the pulp greatest in amount. As one goes forward, the dentine increases regularly in thickness while the pulp-chamber becomes smaller and smaller. The vacuolated appearance at the anterior end of the chamber is due to shrinkage of the pulp tissue during fixation. The enamel has been lost in the process of decalcification except over the basal third. Numerous blood-vessels are seen within the pulp.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
???A**%f Ik ■ .ij JT'l?^ ^epithelial papillae
 
 
 
ik' ■'•**. imeloblasts
 
 
 
liter layer of enamel ner layer of enamel
 
 
 
 
 
 
 
oentine
 
 
 
 
 
 
 
'« '^!i4l^'»,^ .^. Ipodontoblasts
 
 
 
 
 
 
 
pulp
 
 
 
 
 
 
 
26
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig. 25 Longitudinal section of upper incisor of a 5-month albino rat. The letter a shows where the next illustration (fig. 26) is taken. X 6.
 
 
 
Fig. 26 Small portion of the preceding, more highly magnified, to show the enamel-organ and the enamel, and the odontoblasts and dentine. X 135.
 
 
 
84
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 85
 
 
 
The enamel-organ is continuous over the convex labial surface of the imbedded portion of the tooth but is restricted to the most posterior region of the other surfaces, extending only 1 mm. forward from the basal margin. The enamel-organ differs in its structure in three regions of the labial side, and may be described separately in these three parts: (1) at the basal formative end, (2) near the gingival margin and (3) in the long intervening region. In (1) the enamel-organ is being constantly regenerated by the addition and growth of new cells. In (2) the enamel-organ is undergoing a retrograde process, while (3) represents the region where the enamel-organ is at its highest functional development, although its activity in increasing the thickness of the enamel is restricted, as noted before, to the basal third or less in the upper and to the basal half in the lower tooth.
 
 
 
Considering first the region (3), as shown in figure 26, the enamel-organ is conspicuous by reason of its tall ameloblasts and the high, narrow papillae. The enamel-organ is described in three layers — inner, middle and outer. The inner layer consists of the ameloblasts, which measure about 40 /x in height, with nuclei situated towards the outer end of the cells. The middle is composed of two strata (a) stratum intermedium, and (b) enamel pulp. The stratum intermedium is formed of 1 or 2 rows of fairly regular cuboida cells resting upon the outer ends of the ameloblasts, but the enamel pulp is not now recognizable as a distinct layer and exists principally within the papillae. The outer layer of the enamel-organ consisted originally of a single layer of cells, but these are no longer regular in form or arrangement. Together with the remains of the enamel pulp, the outer layer forms the papillary elevations, 60 to 70 M in height. These papillae are surrounded by an abundant capillary blood supply for the nourishment of the cells engaged in the formation of the enamel, and the purpose of the elevations is apparently to increase the surface area through which absorption may take place from the blood-stream.
 
 
 
The enamel is in two layers (fig. 26), the rods while traversing the inner layer being very distinct, and inclining towards
 
 
 
 
 
 
 
86 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
the apex of the tooth at an angle of from 50 to 54° with the dentine surface. The continuations of these rods in the outer laj^er are not so distinctly seen, but the inclination, as made out in thin sections, is still greater towards the apex, forming an angle of from 20 to 25° with the plane of the surface of the dentine. As noted before in the study of enamel, the rods in the inner layer, when observed in cross-sections of the tooth, decussate at an angle of from 70 to 90°, but when they reach the outer layer all run parallel. The fact that the rods run in these various directions seems incontrovertible, but in the light of our present knowledge of enamel formation it is difficult to understand how this condition is arrived at. If each ameloblast is responsible for an enamel-rod, then it follows that because the alternate layers of rods are oblique to one another, the ameloblasts responsible for these series of rods must have changed their relative positions during the process of formation of these rods. No such phenomenon has been observed, or even suggested. The other possibility is that the matrix of the rods is formed in a regular manner, but that afterwards, before calcification is complete, the rods become re-arranged owing to pressure strains.
 
 
 
The plane of cUrection of the rods is suggestive of the importance of the enamel-organ in the persistent growth. For always the general plane of the rods, as they leave the enameldentine junction, is towards the outer end of the tooth, as if the ameloblasts, while engaged in enamel-formation were always held back by the enamel, in which their processes were imbedded.
 
 
 
The basal formative end of the enamel-organ (region 1) in the adult animal corresponds very closely in structure with what has already been described for earlier ages, e.g., 4 days. This is the region where the enamel-organ is constantly being renewed, and it retains the same embryonal character at all stages of development.
 
 
 
At the anterior end where the enamel-organ is continuous with the surface epithelium (region 2), a gradual transition oc
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH
 
 
 
 
 
 
 
87
 
 
 
 
 
 
 
curs between the typical enamel-organ and the stratified squamous epithelium (fig, 27). As one follows the innermost layer of the enamel-organ forward, the cells become shorter, until they are cubical and finally flattened in shape. Here the other layers also lose their regular arrangement, and form, with the preceding, a thin layer of stratified cells. This layer can be followed directly into contact with the epithelium of the gingivus.
 
 
 
 
 
 
 
 
 
Fig. 27 Longitudinal section of the apex of the tooth of a 5-month albino rat, showing at a the position of the outward prolongation of the remains of the enamel-organ, and at h the more granular osteodentine filling in the apex of the pulp chamber. X 10.
 
 
 
 
 
 
 
The cells, however, do not lose their identity in the surface epithelium but remain separate as a thin layer lying against the enamel (fig. 27, a). This thin layer of epithelium, therefore, represents the ultimate fate of the enamel-organ after it has completed its functional activity. It is being continually pushed out and its most anterior part must be continually being lost.
 
 
 
 
 
 
 
88 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
RATE OF GROWTH OF THE INCISOR TEETH
 
 
 
Two methods were used for determining the rate of growth of the incisor teeth (a) cutting off one or more teeth at the gingival margin and (b) making marks upon the enamel. The results here given are based on the latter method, as giving more nearlj^ the normal rate of growth. By means of a dental engine, the animal always having been anesthetized, a fine transverse notch was made on the enamel of the incisors a short distance from the gingival margin. The interval between this mark and the tip of the tooth was then measured. At the end of about a week the distance between these two limits was again taken, and the difference between the two measurements showed the amount of wearing away. Two series of experiments were made by this method upon adult animals. In the first series six animals were used and in the second four animals, and measurements were made for several consecutive weeks. The longest period that one individual was studied was six weeks.
 
 
 
The two series gave very similar results. The lower tooth was always found to grow more rapidly than the upper. The upper tooth averaged 0.31 to 0.32 mm. per day, or 2.2 mm. per week, while the lower tooth averaged 0.4 mm. per day, or 2.8 mm. per week. No doubt there are many variations of the rate of growth under different circumstances, so that these figures must be taken as representing the average rate under one particular set of conditions. The food of these animals was the mixed diet now in use in the rat colony of The Wistar Institute. A short series of trials was made with animals kept in a large glass jar and given only soft food. In these animals the rate of wearing away corresponded very closely to that seen in animals which have also hard food and have the opportunity of gnawing. In these, therefore, the interaction of the opposing teeth must have caused the attrition.
 
 
 
For assistance with these experiments we wish to thank Dr. Stotsenburg, who greatly aided us in carrying out our observations.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 89
 
 
 
It is interesting to compare these results with those obtained on the rabbit — the only other rodent which has been carefully studied in respect to the growth of its incisors. MacGillavry ('75), using a young adult rabbit, made marks upon its lower incisors 2.5 mm. and 3 mm. from the tip. After five to seven days the marks had disappeared. Evidently the rate of growth was about 2.5 to 3 mm. per week. Noe ('02) used a rabbit which happened to possess overgrown teeth. The animal accidentally broke off the lower incisors in the bars of its cage, and Noe made observations upon the rate of their growing out. This he found to be .615 mm. per day, or 4.3 mm. per week. This is larger than MacGillavry's results and may have been due to the unopposed growth and to the other abnormal conditions which may have been present in the formative organs.
 
 
 
Using MacGillavry's figures for comparison, it would seem that the lower teeth of the albino rat and of the rabbit grow out at about the same rate.
 
 
 
OVERGROWTH OF INCISORS
 
 
 
Examples of overgrowth of the incisors of rodents, especially in rabbits and hares, which were hunted as food, must have been observed from early times. In the older literature, they are referred to principally as curiosities, which have excited the interest of whoever has found them. later the causes of the malformations were also considered. Thus Jenyns ('29), to cite only one observer, found several examples in wild rabbits, and has given a good illustration of the curved aspect of the teeth. He also clearly states the several causes which, in his opinion, may give rise to the condition. In addition to the one usually accepted at his time — accidental breaking off of one tooth — he considered also as causes (a) too soft food, (b) morbid or too rapid secretion of the osseous matter of the teeth, and (c) dislocation of one of the condyles.
 
 
 
Wiedersheim ('02-'03) has reported a case occurring in a rat, where he found an associated assymmetry of the cranium. He is in doubt as to which was cause and which was effect — the overgrowth of the teeth or the assymmetry of the cranium.
 
 
 
 
 
 
 
90 W, H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
 
 
 
 
 
 
Fig. 28 Cranium of albino rat, showing the overgrown upper incisors recurving to the left side. The left incisor passes to the outer side of the skull, while the apex of the right incisor has penetrated the bone of the maxilla in the region of the basal end of the left incisor. X 1.
 
 
 
Fig. 29 Cranium of the same albino rat shown in the preceding figure, viewed from the right side. It shows the overgrown lower incisors recurving to the right side, and the cavity which the right incisor has worn in the palate bone.
 
 
 
Figures 28 and 29 show a skull obtained some years ago from the rat colony of The Wistar Institute by Dr. Stotsenburg, and prepared in the Histological Laboratory by Miss E. F. Brooks. The upper teeth curved to the left side of the head and the lower to the right side. As seen in figure 29, the right lower has penetrated through the bone of the palate into the
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 91
 
 
 
nasal chamber, while the right upper (fig. 28) has recurved and grown into the maxilla.
 
 
 
In The Wistar Institute rat colony, at the time when the animals were fed on bread and milk, frequent examples of this and similar conditions were found, but now under a more varied mixed diet they practically never occur.
 
 
 
Beretta ('13) has recently made an analysis of these abnormalities and has classified them in three groups.
 
 
 
(1) Overgrowth of the upper and lower incisors through lack of an opposing tooth.
 
 
 
(2) Overgrowth of the incisors of the upper and lower jaws through deviation of the jaws.
 
 
 
(3) Prognathism of the lower jaw, and as a result, overgrowth of the incisor of the lower jaw.
 
 
 
In the present instance, diet seemed to be the controlling factor, probably by reason of its influence on the hardness of the bone of the alveoli from which the teeth grew out.
 
 
 
SUMMARY
 
 
 
The rate of growth of the upper and lower incisor teeth of Mus norvegicus albinus, in the mature animal, averages 2.2 and 2.8 mm. per week, or 12.5 cm. and 14.5 cm. per year, respectively.
 
 
 
Growth is due primarily to the proliferation and growth of cells at the basal end of the enamel-organ, where new enamelforming cells arise, and at the basal end of the dental papilla where new dentine-forming cells develop.
 
 
 
The enamel-organ of the adult forms a narrow circular band around the basal end of the tooth, and extends forward from this on the labial side only. It coincides in its lateral boundaries with the enamel, and extends along the entire imbedded portion of the tooth. Anteriorly, it comes in contact with the epithelium of the gingival margin, and is carried out continually as a narrow band of cells lying on the enamel, between the latter and the gingival epithelial tissue.
 
 
 
The first indication of the anlage of the incisors appears in 14-day-old fetuses. In fetuses, 21 daj^s of age (just before
 
 
 
JOURNAL OF MORPHOLOGY, VOL. 26, NO. 1
 
 
 
 
 
 
 
92 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
birth), enamel and dentine formation is beginning. In animals 1 day old the upper and lower teeth measure 2.3 and 3 nrni. At 8 to 10 days the teeth erupt, and at 10 days measure 7 and 11 mm. respectively. This period is therefore characterized by the rapid elongation of the teeth.
 
 
 
The process of attrition begins within a few days after eruption, so that by 19 or 21 days of age, the typical occlusal surface is formed. Up to the time of eruption the anterior end or apex of the tooth is immediately under the oral epithelium, while the basal or growing end is continually progressing posteriorly. After eruption, the basal end becomes nearly stationary in position, while the whole tooth structure is continually moving forward. The extra-gingival length of the tooth is kept constant, however, by the attrition of the occlusal surface, either through use in gnawing or by the action of the opposing teeth.
 
 
 
The histogenesis of the enamel-organ is practically completed by the 4th day after birth, although it does not attain its final relations to the tooth as a whole, until after eruption. In the 18-day fetus the enamel-organ is similar in all parts, and the cells of the inner layer measure the same, both lingually and labially. From this period forwards, however, the labial portion continues to progress towards its fully differentiated functional structure, while the lingual portion retrogresses, until at 4 days after birth the latter is disrupted, by the ingrowth of the surrounding connective tissue. Contrasting the cells of the inner layer — the potential ameloblasts — on the labial and lingual sides, thej^ are practically the same in the 18-day fetus, but at 19 days they are found to measure 24 and 20 m respectively. In the 21-day fetus, they measure 30 to 34 and 12 fx, and 1 day after birth the true ameloblasts on the labial side have increased to 40 n, while the non-functional cells of the lingual side are only 10 fx in height. At 4 days, the latter cease to form a continuous layer, by reason of the dispersion of the cells by the surrounding connective tissue, except at the basal formative region.
 
 
 
Characteristic of the permanently-growing enamel-organ are the epithelial papillae, formed by the elevations of the outer
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 93
 
 
 
layer of the enamel-organ, and the cells of the enamel pulp. Between these elevations are numerous capillaries which insure a rich blood supply to the enamel-forming cells.
 
 
 
There are three layers in the functional enamel-organ — inner, middle and outer. The inner is constituted of the tall ameloblasts, and the middle is made up of two divisions, (a) stratum intermedium and (b) enamel pulp. The latter unites with the single layer of cuboidal cells which compose the outer layer, to form the epithelial papillae (fig. 26).
 
 
 
The apex of the primitive tooth is formed of a variety of secondary dentine — 'osteo-dentine' of Tomes — which is softer than true dentine, and differs in its structural arrangement (fig. 20). After eruption, this terminal portion of osteodentine is soon worn away by attrition, and the typical occlusal surface is developed, as seen at 19 or 21 days. At 21 and 23 days the first two molars erupt in both upper and lower jaws, and from now on the animal is able to secure food for itself, and if necessary can maintain an independent existence.
 
 
 
As the tooth continues to be worn away there is a provision for the continual filling in of the apex of the pulp-chamber by the formation of what may also be called osteodentine. This is a form of secondary dentine, containing, when first formed, cells and blood-vessels. This is always at a little distance, about 1 mm., from the occlusal surface, but as any part of the tooth, in its outward progression, approaches the occlusal surface, the soft elements disappear within the osteodentine, and the latter forms a hard continuous surface with the adjoining true dentine. The position of this osteodentine is marked as a line on the occlusal surface of the teeth (fig. 5).
 
 
 
Prior to eruption there develops around the apex of the tooth, as it lies in contact with the surface epithelium, a thickened ring of stratified epithelium. This ring of tissue is pierced by the apex of the tooth at eruption, and would seem to have the function of serving as a resistant margin for the soft tissues, and of preventing other tissues being carried along with the erupting tooth.
 
 
 
 
 
 
 
94 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
The length of the teeth varies with the size of the cranium (table 1) so that the persistent growth is not only sufficient to offset the continual attrition, but also serves to keep the length of the teeth in a definite relation to the length of the skull, as the latter increases in size.
 
 
 
The lower tooth is always longer than the upper, and this difference manifests itself even in the anlagen of these structures in the 19-day fetus (figs. 11 and 12).
 
 
 
The contour of the enamel, as seen in cross-sections, is characteristically different in the upper and lower teeth (fig. 4).
 
 
 
The enamel of the tooth is composed of two layers which are different in appearance. The enamel rods run in two sets which decussate with each other in the inner or plexiform layer, but they change their direction as they continue into the outer layer, so that in it they are all parallel. In longitudinal sections, the general direction of the rods (fig. 26), is to incline towards the apex of the tooth, as they run from the enamel-dentine boundary to the outer surface of the enamel.
 
 
 
In conclusion, we wish to thank Professor Piersol for generous assistance in many ways, and Professor Donaldson for his constant interest in the study. We also wish to acknowledge the kind assistance of Mr. E. F. Faber in the preparation of the drawings.
 
 
 
 
 
 
 
STRUCTURE AND GROWTH OF INCISOR TEETH 95
 
 
 
LITERATURE CITED
 
 
 
Adloff, p. 1898 Zur Entwicklungsgeschichte des Nagetiergebisses. Jena. Zeitschr. fiir Naturwissenschaft, Bd. 32, ss. 347-410.
 
 
 
Beretta, a. 1913 La normala dentatura dei roditori in rapporto alle anomalie dentali in questi osservate. La Stomatologia, t. 10. Abstract in Deutsche Monatsschrift fiir Zahnheilkunde, April, s. 287.
 
 
 
VON Brunn, a. 1887 Ueber die Ausdehnung des Schmelzorganes und seine Bedeutung fiir die Zahnbildung. Arch. f. mikr. Anat.. Bd. 29, ss. 367-383.
 
 
 
BuRCKHARDT, R. 1906 In Hertwig's Handbuch der Entwickelungslehre der Wirbeltiere, Bd. 2, Teil 1, Kapitel 4, ss. 349-456. "Die Entwickelungsgeschichte der Verknockerungen des Integuments und der Mundhohle der Wirbeltiere."
 
 
 
Cope, E. D. 1888 The mechanical causes of the origin of the dentition of the Rodentia. Amer. Nat., vol. 22, pp. 3 11.
 
 
 
Donaldson, H. H. 1912 The history and zoological position of the albino rat. Proceed. Acad. Nat. Sci., Philadelphia.
 
 
 
Freund, p. 1892 Beitriige zur Entwicklungsgeschichte der Zahnanlagen bei Nagethieren. Arch. f. mikr. Anat., Bd. 39, ss. 525-5c6.
 
 
 
Fritsch, C. 1914 Untersuchungen iiber den Bau und die Innervierung des Dentins. Arch, fur mikr. Anat., Bd. 84, ss. 307-20.
 
 
 
Hatai, S. 1907 On the zoological position of the albino rat. Biol. Bull., vol. 12, pp. 266-273.
 
 
 
Jenyns, L. 1829 Observations on a preternatural growth of the incisor teeth occasionally observed in certain of the Mammalia Rodentia. Loudon's Magazine Nat. Hist., London, vol. 2, pp. 134-137.
 
 
 
Jolyet et Chaker 1875 De I'acte de ronger, etudie chez les rats. Comptes Rendus et Memoires de la Soc. de Biol., pp. 73-74.
 
 
 
Lowe, L. 1881 Beitrage zur Kenntniss des Zahnes und seiner Befestigungsweise im Kiefer. Arch. f. mikr. Anat., Bd. 19, ss. 703-719.
 
 
 
MacGillavry, T. H. 1875 Les dents incisives du Mus decumanus. Arch. Neerl. Sc. exact, et nat., Haarlem.
 
 
 
Meyerheim, M. 1898 Beitrage zur Kenntnis der Entwicklung der Schneideziihne bei Mus decumanus. Dissertation. Leipzig.
 
 
 
Mummery, J. H. 1912 On the distribution of the nerves of the dental pulp. Philos. Trans. Roy. Soc, London, vol. 202, B., pp. 337-349.
 
 
 
NoE, J. 1902 Vitesse de croissance des incisives chez les Leporides. Comptes Rendus, hebd. des Seances et Memoires de la Soc. de Biol., pp. 531-532.
 
 
 
OuDET, J. E. 1823 Experiences sur I'accroissement continue et la reproduction des dents chez les lapins. Jour, de Physiol. Exper. et Patholog., Tomes 3 et 4.
 
 
 
Owen, R. 1840-45 Odontography. London.
 
 
 
 
 
 
 
96 W. H. F. ADDISON AND J. L. APPLETON, JR.
 
 
 
Reichert, E. T., and Brown, A. P. 1910 The crystallography of hemoglobin. Pub. Carnegie Inst, of Washington.
 
 
 
Retzius, a. 1838 Bemerkungen iiber den inneren Bau der Zahne mit besonderer Rlicksicht auf den in Zahn vorkommenden Rohrenbau. Mliller's Archiv.
 
 
 
Roetter, F. 1889 TJeber Entwicklung und Wachstum der Schneidezahne bei jNIus musculus. Morphol. Jahrb., Bd. 15, ss. 457-477.
 
 
 
Ryder, J. A. 1877 The significance of the diameters of the incisors in Rodentia. Proc. Acad. Nat. Sci., Philadelphia, vol. 29, pp. 314-318.
 
 
 
1878 On the mechanical genesis of tooth-forms. Proc. Acad. Nat. Sci., Philadelphia, vol. 30, pp. 45-80.
 
 
 
Sachse, B. 1894 Entwicklung der Schneidezahne bei Mus musculus. Dissertation. Leipzig.
 
 
 
Stach, J. 1910 Die Ontogenie der Schneidezahne bei Lepus cuniculus. Extrait. Bui. d'Acad. Sc, Cracovie.
 
 
 
Tomes, C. S. 1914 A manual of dental anatomy; human and comparative. 7th Ed. Edited by H. W. Marett-Tims and A. Hopewell-Smith.
 
 
 
Tomes, J. 1850 Structure of the dental tissues of the order Rodentia. Phil. Trans. Royal Society of London, pp. 529-567.
 
 
 
Tullberg, T. 1898-99 Ueber das System der Nagethiere. Nova Acta Reg. Soc. Sc. Upsaliensis, Series 3, ss. 1-514.
 
 
 
Weber, M. 1904 Die Saugetiere.
 
 
 
Wiedersheim, R. 1902-03 Ein abnormes Rattengebiss. Anat. Anz., Bd. 22, ss. 569-573.
 
 
 
Williams, J. L. 1896 The formation and structure of dental enamel. Dental Cosmos, vol. 38.
 
 
 
Woodward, M. J. 1894 On the milk dentition of the Rodentia with a description of a vestigial milk incisor in the mouse (Alus musculus). Anat. Anz., Bd. 9, ss. 619-631.
 
 
 
 
 
 
 
A PECULIAR STRUCTURE IN THE ELECTROPLAX OF THE STARGAZER, ASTROSCOPUS GUTTATUS
 
 
 
JAMES G. HUGHES, JR.
 
 
 
From the Histological Laboratory of Princeton University, U. S. A.
 
 
 
THREE FIGURES
 
 
 
The purpose of this paper is to determine the function and composition of the pecuhar pointed fibers and long pointed rods lying in the electric layer of the electroplaxes of the stargazer, Astroscopus guttatus.
 
 
 
Before proceeding with a discussion of these rods, a brief description of the electric organ of this fish (according to Dahlgren)^ will be given.
 
 
 
The electric apparatus is composed of two organs, which form two vertical columns roughly oval in horizontal section, and placed behind and somewhat under each eye. Each organ extends from the peculiar bare spot on the top of the head down to the tissues which form the roof of the oval cavity; and is composed of about 200 thin layers of electric tissue, which extend horizontally all the way across the organ. These layers of tissue are flat, and alwaj^s at the same distance from one another. Each layer contains about 20 electroplaxes, the outlines of which present a very irregular or scalloped appearance. The electric tissue in which the electroplaxes are imbedded is in appearance a jelly-like or mucous-like tissue, usually known as electric connective tissue, and which I have shown in the course of my work to be of the same composition as white fibrous connective tissue. The nerve and blood supply runs in the above tissue. The general form of a vertical section of an electroplax is shown by figure 1, which is a drawing of part of a section of a single electroplax.
 
 
 
Each electroplax is composed of three principal layers, a nervous or electric layer which forms the upper surface, a
 
 
 
1 Anat. Anz., Bd. 29, S. 387, 1906.
 
 
 
97
 
 
 
 
 
 
 
98 JAMES G. HUGHES, JR.
 
 
 
middle layer, and a lower or nutritive layer which along with the middle layer is evaginated into a large number of long papillae. All three layers are deeply marked with a dense series of fine striations, which are peculiar to the electroplaxes of several other fishes. The upper or electric surface is flat and smooth and receives the nerve endings. The current of electricity runs downward through the organ which produces it, and thus the nerve endings in accordance with Pacini's law are found on the negative pole of the electroplax.
 
 
 
Proceeding directly to the subject of this paper, we may say that one of the most interesting of the points noted in the electroplaxes, when properly fixed and stained with iron hematoxylin, is a series of rod-like or thread-like objects running horizontally in the electric layer, among, above and below the nuclei and without any apparent connection with them (figs. 1 and 2).^ These rods are of various sizes and shapes, and in form are said to resemble the classic thunderbolts seen in the hand of representations of Jove. They usually taper slowly and branch extensively at one or both ends. Some of these branches sometimes seem to be mere lines, while others are wide and heavily pointed; at their other ends the rods are usually rounded; this latter appearance may be due, however, to the cut ends of the rods, for as noted above they sometimes branch at both ends. Some are short and heavy in appearance while others are long and thread-like. Peculiar looping, twisting, or knot-like bends are sometimes found at points on the longer rods. The outlines and contour of these rods are always smooth. Their size may vary from thick or thin rods of over 300 /x in length down to small ones that do not exceed 1 m- In those electroplaxes where the rods are few they sometimes lie parallel and point in a definite parallel direction, while in others where the rods are very numerous they do not seem to have any definite arrangement. In this latter condition the rods present a very wavy appearance. Their form may be seen in figures 1 and 2, which are drawings of the electric layer of an electroplax when
 
 
 
^ All the figures are drawings of sections of electroplaxes of Astroscopus guttatus.
 
 
 
 
 
 
 
 
 
 
 
 
 
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JAMES G. HUGHES, JR.
 
 
 
 
 
 
 
 
 
Fig. 2 Horizontal section through an electroplax. Only the electric layer is seen; a, an extensively branching rod; h, fine branches of the above rod; c, a large characteristic loop in a rod; d, a cut end of a rod; e, nuclei of the electric layer. X 1200.
 
 
 
 
 
 
 
stained with iron hematoxylin. These drawings show the electric layer in which the rods are found in horizontal section (fig. 2) and in vertical section (fig. 1).
 
 
 
The purpose, function, and chemical composition of these rods have been previously unknown to histologists. In order to determine anything in respect to their function or purpose, a knowledge as to the class of organic substance to which they belong, whether muscle, connective tissue, nervous, or chitinous,
 
 
 
 
 
 
 
ELECTROPLAX OF ASTROSCOPUS 101
 
 
 
 
and also a rough knowledge of their chemical composition is imperative. The contour and form of these rods as they appear under the microscope resemble both smooth muscle fibers and fibers of elastic connective tissue. The belief that the function of these rods was somewhat of the nature of support for the delicate substance of the electroplax, and the fact that their form resembled connective tissue fibers led the writer to take for one of his first hypotheses, that they were of some form of connective tissue, and to perform accordingly the following series of experiments. As the most logical and best way for determining the kind of connective tissue, if any, of which the rods might be composed, a number of stains used by other investigators to identify similar substances were applied and the results noted. Controls were used on known tissues.
 
and also a rough knowledge of their chemical composition is imperative. The contour and form of these rods as they appear under the microscope resemble both smooth muscle fibers and fibers of elastic connective tissue. The belief that the function of these rods was somewhat of the nature of support for the delicate substance of the electroplax, and the fact that their form resembled connective tissue fibers led the writer to take for one of his first hypotheses, that they were of some form of connective tissue, and to perform accordingly the following series of experiments. As the most logical and best way for determining the kind of connective tissue, if any, of which the rods might be composed, a number of stains used by other investigators to identify similar substances were applied and the results noted. Controls were used on known tissues.
  

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Template:J Morphol. Volumes

JOURNAL OF MORPHOLOGY

Founded by C. O. Whitman


EDITED BY J. S. KINGSLEY

University of Illinois Urbana, Ill.

with the collaboration of Gary N. Calkins Edwin G. Conklin C. E. McClung

Columbia University Princeton University University of Pennsylvania

W. M. Wheeler William Patten

Bus.'iey Institution, Harvard University Dartmouth College


VOLUME 26 1915

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA

COMPOSED AND PRINTED AT THE WAVERLY PRESS

By the Williams & Wilkins Company

Baltimore, JId., U. S. A.

Contents

1915

No. 1. MARCH

Pauline H. Dederer. Oogenesis in Philosamia cynthia. Sixty-four figures (six plates) 1

William H. F. Addison and J. L. Appleton, Jr. The structure and growth of the incisor teeth of the albino rat. Twenty-nine figures 43

James G. Hughes, Jr. A peculiar structure in the electroplax of the stargazer, Astroscopus guttatus. Three figures 97

W. Rees Bremner Robertson. Chromosome studies. III. Inequalities and deficiencies in homologous chromosomes : their bearing upon synapsis and the loss of unit characters. Fourteen figures (three plates) 109

No. 2. JUNE

George W. Tannreuther. The embryology of Bdellodrilus philadelphicus. Twenty-six text figures and eight plates 143

E. E. Just. The morphology of normal fertilization in Platynereis megalops Three plates (thirty figures) 217

Wm. a. Kepner and J. R. Cash. Ciliated pits of Stpnostoma. Four figures 235

Huber GC. The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day. (1915) J Morphol. 26: 247-358.

G. Carl Huber. The development of the albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; end of the first to the end of the ninth day. Thirty-two figures 247

Huber GC. The development of the albino rat, Mus norvegicus albinus. II. Abnormal ova: end of the first to the end of the ninth day. (1915) J Morphol. 26: 359-391.

G. Carl Huber. The development of the albino rat, Mus norvegicus albinus. II. Abnormal ova: end of the first to the end of the ninth day. Ten figures. 359

Marianna van Heraverden. Comment on Miss Beckwith's paper on "The genesis of the plasma-structure in Hydractinia cchinata" and reply by Miss Beckwith

No. 3. SEPTEMBER

E. A. Baumgartner. The development of the hypophysis in SqualusAcanthias. Forty-three figures ,391

J. Frank Daniel. The anatomy of Heterodontus francisci. II. The endoskeleton. Thirty-one figures (eight plates) 447

Robert W. Hegner. Studies on germ cells. IV. Protoplasmic differentiation in the oocytes of certain Hymenoptera. Ninety-seven figures (thirteen plates) 495

No. 4. DECEMBER

Edward Phelps Allis, Jr. The homologies of the hyomandibula of the gnathostome fishes. One figure 503

James L. Kellogg. Ciliary mechanisms of lamellibranchs with descriptions of anatomy. Seventy-two figures 625

A. T. Evans. The morphology of the frontal appendage of the male in the Phyllopod crustacean Thamnocephalus platyurus Packard. Nine figures. 703

Oogenesis In Philosamia Cynthia

Pauline H. Dederer,

From the Zoological Laboratory, Columbia University

Sixty-Four Figures (Six Plates)

Contents

Introduction 1

Material and technique 2

Maturation divisions 3

1. Observations 3

a. Chromosomes in the embryo 3

b. First maturation division 3

c. Second maturation division 6

d. Fertilization 7

2 . Conclusions and comparisons 8

Early oogenesis 10

1 . Observations 10

a. Growth of the ovary: General description 10

b. Early stages in the development of the ovary 11

c. Development of nurse cells 13

d. Development of egg cells 17

e. Degenerating cells in the ovary 20

f. Abnormal nuclei in the early ovary 20

2 . Conclusions and comparisons 20

3 . Literature on the early development of eggs and nurse cells 22

Summary 25

Literature cited 27

INTRODUCTION

From the standpoint of sex production the Lepidoptera are of especial interest as compared with other insects, because the experimental evidence of Doncaster ('06, '08) and Raynor ('06), Punnett and Bateson ('08) seems to demand the assumption that there are two kinds of eggs in the moth. The absence of visible dimorphism in the spermatozoa of the Lepidoptera also lends probability to this hypothesis. In other groups of insects studied, the spermatozoa are often dimorphic. The eggs have been assumed to be all ahke, and this condition has been demonstrated by Morrill ('09) for certain coreid Hemiptera, by Morgan ('09) for phylloxerans, by Stevens ('06 a, '09) and von Baehr ('08, '09) for aphids.

In studying the history of the male germ cells in the moth Philosamia cynthia (Dederer '07), the spermatocytes were found to contain exactly similar groups of chromosomes. The same facts had been determined by Stevens ('06 b) and Cook ('10) for various other Saturniidae. Doncaster ('12) believes that in Pieris brassicae there is no dimorphism, either in the male or female germ cells. Recently, however. Seller ('13) has decribed two kinds of eggs in a lepidopteran. These two papers will be discussed later. The present work was undertaken with special reference to the question of dimorphism in the eggs. I wish to express my indebtedness to Prof. E. B. Wilson for valuable advice and criticism during the course of the investigation.

MATERIAL AND TECHNIQUE

Carnoy's aceto-alcohol-chloroform mixture, saturated with sublimate, was used almost exclusively, since it readily penetrated the tough chorion of the eggs, which were left in the fluid from two to four hours. Flemming's and Bouin's fluids were used, but proved very unsatisfactory.- The eggs were then transferred to iodized 95 per cent alcohol, and the chorion was removed with needles. After dehydration in absolute alcohol, the eggs were placed in a mixture of alcohol and chloroform followed by pure chloroform for ten minutes. Immersion in melted paraffine for fifteen minutes was sufficient for perfect infiltration. The stains used were iron hematoxylin, and in a few cases safranin.

The maturation spindle remains in first metaphase until the entrance of the sperm. The following tabulation gives roughly the maturation stages at different intervals after the eggs are laid:

Eggs just laid to ^ hour laid first anaphase

Eggs laid 1 to 1| hours second metaphase

Eggs laid 1| hours second telophase

Eggs laid 2 to 2j hours fusion of pronuclei


In the study of the early stages of oogenesis, both caterpillars and pupae were used. The ovaries in the early pupal stage lie enveloped in the fat bodies just beneath the dorsal wall of the abdomen in the fifth segment. Upon removing the wall, the ovaries were located and transferred immediately to the fixing fluid. Flemming's, Bouin's and Carnoy's fluids were used, the latter being the only one good for all stages. The stains employed were safranin and iron hematoxylin.

MATURATION DIVISIONS 1. OBSERVATIONS

a. Chromosomes in the embryo

The number of chromosomes in the spermatogonia is 26 (Dederer '07), all rounded bodies, approximately equal in size. For determining the somatic number, sections were made of eggs several hours after fertilization. Ten counts from three different lots of eggs showed clearly 26 chromosomes. In polar view of metaphase (fig. 1) they appear as slightly elongated, often bipartite, bodies of comparatively slight difference in size. Owing to their similarity it is impossible to attempt any arrangement of the chromosomes in pairs, as has been done in some animals.

h. First maturation division

The mature eggs are oval bodies about 1.5 mm. long, each invested in a tough white chorion, which is flatter and broader at the animal pole. The flattened area appears to be due to the contact of the nurse cells in this region, while in the egg tubes.

Figure 32 is from a longitudinal section through the animal pole of an egg, showing a spindle with chromosomes in anaphase, within a dense granular mass, whose long protoplasmic processes reach out into the more reticular portion of the cytoplasm. This latter region is free from yolk, and is cone-shaped in form, the apex pointing inward, and extending as a sort of narrow vacuolated core, into the center of the egg. The remainder of the egg is filled with large yolk spheres. At the periphery of the egg appears a thin layer of dark granular protoplasm.

In earlier eggs, before or at the time of laying, a clear pale vitelline membrane may be seen beyond this. The polar bodies are formed in the dense granular layer, very near the middle of the anterior end, just within the cone-shaped area.

The earliest nuclear stage obtained after the growth of the egg, is the late prophase, found in eggs which had not been laid (fig. 3). The chromosomes, 13 in number, lie enclosed within the nuclear membrane, near the surface of the egg, in the same position as the first maturation spindle. The chromosomes are smooth, elliptical or dumb-bell shaped bodies, almost equal in size. Later the nuclear wall breaks down, the spindle fibers appear and the chromosomes become arranged upon them preparatory to division (figs. 4-5). When first placed upon the spindle, the chromosomes do not all show a dyad form, but later a median constriction appears in each one. The spindle lies obliquely to the surface of the egg. The spindle fibers can rarely be traced to a point of convergence, and no centrosomes nor asters appear. Various cytoplasmic bodies lie near or attached to the spindle (fig. 4), but they are not constant in size or number, and often cannot be detected. They are present only during the metaphase.

In figures 6 and 7 are shown two first division groups. There is but slight difference in the size of the chromosomes, and each one appears to be separated into two equal parts. On account of their small size they were at first interpreted as chroraosomes of the second division, but a further study showed that this was not the case. There was no trace of a first polar body, nor of sperm within the egg. Moreover, the eggs had not been laid, but were taken from a moth which had just begun laying. Figures 4 and 5 and numerous other undoubted first metaphase stages, with larger chromosomes, were obtained from the same lot of eggs. Restaining and extraction had practically no effect in altering the size difference which remains unexplained. In twelve cases I found chromosome groups similar in size to those shown in figures 6 and 7. These figures seem to indicate that the chromosomes divide equally in the first division but a definite statement is unwarranted, for the chromosomes are so small that a shght size difference might easily escape detection; moreover the variability in size in different groups, as seen in figures 5 and 6 would render any deductions in this respect extremely hazardous.

As the chromosomes approach the ends of the spindle, the fibers thicken enormously in the middle, forming a deeply staining cell plate, which in side view, gives the appearance of a band encircling the spindle (fig. 8). Henking ('92) described similar bodies in Pieris, which he considered as waste achromatic substance. With long extraction the cell plate appears very faint, while the chromosomes remain dark. Figure 9 shows another anaphase, in which 13 chromosomes may be counted at each pole. No lagging chromosomes were observed. In a late anaphase (fig. 10) another size peculiarity is observed, each chromosome being after division approximately as large as those of the metaphase stage. The irregularity in the form of the chromosomes in late anaphase was characteristic of this stage, and was equally apparent with either dark or light staining, although in the latter case the chromosomes appeared slightly smaller. Figure 11 is an oblique polar view of a similar stage; on account of the plane of the section the spindle cannot be seen. In figures 12 to 14 are shown polar views of spindles, the groups lettered a in each case being those entering the first polar body. Four chromosomes are in the center of each group, surrounded by a ring of nine. The polar body groups sometimes appear slightly smoother in outline than the egg groups (fig. 12), smaller, and bipartite in preparation for a second division. In attempting to compare chromosomes in the polar body with those in a similar position in the egg group, it is impossible to obtain any evidence either for or against an equal division of chromosomes. The variability is extreme, within both egg and polar groups, and in many cases it is very difficult to be sure of the actual chromosome outlines.

During the formation of the first polar body the spindle fibers elongate considerably, and the granular cytoplasm forms a conspicuous projection on the surface of the egg (fig. 33). There is apparently no first telophase, for no loss of contour or massing of the chromosomes was observed between the late anaphase and the second metaphase.

c. The second maturation division

In the second division two spindles appear, as shown in figure 34. Upon one are arranged the chromosomes of the first polar body; on the other, those of the second polar division. The old spindle fibers have disappeared, and the cell plate has assumed the form of irregular deep-staining masses. At a corresponding stage in the oogenesis of Bombyx mori, Henking ('92) figured a cell plate in a similar position, but it differed from this in being a single disc-shaped body or 'thelyid', which stained very faintly. The later constriction of the first polar body in P. cynthia, as in Bombyx, does not involve the cell plate but passes between it and the outer group of chromosomes.

In figure 15 A, B, drawn from adjacent sections, are shown 13 approximately equal chromosomes, arranged upon the two spindles, preparatory to a second division. It will be observed that the groups in the egg B and the first polar body A are at this time similar in the form and size of the chromosomes. The remnants of the cell plate are composed of deep-staining bodies, so large and definite as to give the appearance of chromosomes, but they are very irregular in form, and vary in size from large masses to very small granules. Figure 16 is another view of a similar stage. In figure 17, a polar view from three succeeding sections, the cell plate is composed of 15 large bodies, and numerous granules. One chromosome is missing from the egg group, B,

For various reasons second anaphase stages were extremely diflficult to find. A few cases, however, were obtained, which seem fairly clear. In figure 19 is shown a spindle in which 13 chromosomes are seen at one pole, 11 at the other. Thi^ latter group, which is incomplete, enters the second polar body. The 13 chromosomes of the egg nucleus are very small rounded bodies nearly equal in size. There is nothing to indicate a peculiarity in behavior of any of the chromosomes. In figure 20 polar views of two groups in anaphase are shown; here 13 approximately similar chromosomes appear in each. In figure 21, a polar view of an egg group, 13 chromosomes may be counted. Figures 23 and 24 are two oblique sections through spindles in anaphase. The groups in each case have been slightly displaced. There are 13 chromosomes in each group. These examples, while not numerous, are sufficient to show that the second polar body receives a group of chromosomes similar in number to those remaining in the egg. The small size of the chromosomes, and the lack of early anaphase stages, make it impossible, as in the case of the first division, to draw any conclusions as to the equal or unequal division of the chromosomes.

The first polar body was frequently observed in anaphase, during the stage figured above. After the second anaphase the egg chromosomes show a tendency to fusion (fig. 25) and it is impossible to distinguish separate chromosomes at either pole.

d. Fertilization

In sections through eggs in the first metaphase stage, several spermatozoa may be seen within the vitelline membrane, but only occasionally within the egg. In late anaphase, sections show that the spermatozoon has penetrated into the egg and is enveloped in a dark granular island of cytoplasm. Numerous eggs were found containing two or three spermatozoa. Thus in P. cynthia, as in many other insects, polyspermy appears to be normal. Shortly after entering the egg, the sperm appears as a long tapering rod. Later, it has the form of an oval, deepstaining vesicle surrounded by a clear area, and in contact with the female pronucleus, which lies nearer the surface of the egg. Subsequently the male pronucleus becomes spherical, the clear area disappearing. The chromatin in both nuclei is in the form of irregular flocculent masses, at first darker in the male pronucleus. Later it has the same staining capacity in both, so that it is impossible to distinguish male and female except by position.

At a later period, it is possible to count the chromosomes in each nucleus. In figure 35 the pronuclei lie near the surface of the egg where the second polar body appears. The surface cytoplasm merges with the remnants of the vitelline membrane, in which the polar body lies. Figure 27 is the same section enlarged. The pronuclear walls appear broken at the region of contact, or are so thin as to be invisible. Nine chromosomes are seen in the first section of the outer nucleus, 11 in the first section of the inner; they differ s ightly in size, and some are noticeably dyad in form. To the right of these are drawn portions of the nuclei from a succeeding section, showing 4 more chromosomes in the outer nucleus, 2 more n the inner, making 13 in each. No nucleoli are present. Within the polar body figured here remains of spindle fibers and a nuclear membrane are seen. Here too, 13 chromosomes appear. This is probably the second polar body, for the first becomes very vague after the second anaphase.

In figure 28, from an egg similar to the one described, nine or more chromosomes may be counted in the second polar body. The first polar body has apparently divided, the chromosomes in each appearing as vague granular areas. It seems probable that all three degenerate shortly after the fusion of the germ nuclei. There is no evidence that they remain included within the egg.

2. CONCLUSIONS AND COMPARISONS

The evidence obtained from the foregoing study indicates that in Philosamia cynthia the 13 chromosomes seen in the late prophase of the egg all divide in both maturation divisions. The male and female pronuclei at the time of their union each contain 13 chromosomes, giving the somatic number 26, which is found in the nuclei of the blastoderm. It appears to be certain that all of the eggs contain the same number of chromosomes, but the evidence for either the presence or absence of an XY-pair is not conclusive, on account of the variability in the size of the chromosomes in the metaphase and anaphase plates. In the early oogenesis, to be described later, there is no indication of the presence of a heterochromosome, either of equal or unequal parts, and from this we might suspect its absence in later stages. Although from the totality of the evidence, it appears probable that there is no difference in the chromosome groups, the matter will have to be left an open question.

Doncaster ('12) found that in Pieris brassicae both the male and female germ cells contain an equally paired heterochromosome which constitutes a chromatin nucleus during the growth period. He believed that in Abraxas a similar condition probably prevailed, and concluded that the chromosomes here do not provide any visible basis for the sex-limited transmission of characters." More recently, however ('13) he has found some females of Abraxas with 56 chromosomes, some w'th 55, and he beheves that there s a possibility of two kinds of eggs in this form.

Until the past year, the only recorded case of nuclear dimorphism in eggs (exclusive of parthenogenetic and sexual eggs) was that of the sea-urchin described by Baltzer ('09), in which the female appeared to be the heterogametic sex. Tennant, ('12) however, discovered that in other forms the male is heterogametic. Baltzer has recently ('13) announced that the results described in his former paper are erroneous, and he is convinced that the male is the heterogametic sex. This solves the apparent contradiction within the echinoderm group, the females being homozygous for sex in all cases described.

The latest case of heterogamy in the female is that recently described by Seller ('13) for the lepidopteran Phragmatobia fuliginosa. In the spermatocyte divisions, 27 small chromosomes are present, and a large one, which, though lagging somewhat, divides equally in both maturation divisions. In the first metaphase plate of the egg, 27 small chromosomes and a large chromosome, slightly segmented or lobed appear. After the first division, at one pole of the spindle are seen 27 small chromosomes and a large one; at the other pole, 28 small chromosomes and a large one. It is a matter of chance whether the polar body or the egg nucleus receives the extra chromosome. Seller interprets the extra small chromosome as a lobe of the large autosome which has separated from it during division, since in anaphase a small chromatin mass lies near one end of the large chromosome as if detached from it. Second divisions were not observed but Seller believes they are probably equational. He suggests the tentative interpretation that the extra small chromosome is the X chromosome. Unfortunately, only polar views of the first division are given, and these only of late anaphase, so it is impossible to determine how the extra chromosome arises. It is possible that the separation of this chromosome (described by Seller) inay be merely a temporary condition, followed by a union with the large one at the second metaphase, thus giving similar groups of chromosomes in all the oocytes. In view of the fact that in echinoderm eggs an apparently clear case of dimorphism has been found to be incorrect, it seems particularly necessary "to scrutinize carefully any evidence along this line.

EARLY OOGENESIS

A study of the early oogenesis of P. cynthia was undertaken in order to determine the origin of the haploid groups of chromosomes which enter the first polar metaphase. By analogy with spermatogenesis, pairing of the chromosomes in the egg should occur before the growth period. Although the material is unfavorable for the study of oogenesis as a whole, a seriation of stages was obtained, and several points of interest were observed in regard to the tlifferentiation of primitive ovarian -cells into eggs and nurse cells, and their later relation to each other.

1. OBSERVATIONS

a. Growth of the ovary: General description

The earliest ovaries obtained were from larvae fixed the latter part of August, a few days before the spinning of the cocoon. They are pear-shaped bodies, about 1 mm. in length, slightly smaller than a mature egg. Figure 29 is a lengthwise section through a larval ovary. The oval mass of connective tissue surrounds four egg strings which take a complicated course within the capsule. The strings open into a single slightlyexpanded chamber at the surface of the ovary, from which the oviduct arises. The earliest eggs are found near the opposite end of the ovary. Oogonial stages to very early eggs were found in this and similar ovaries. Figure 30 is of a January ovary, showing an increase in size and the growth of the egg strings. Two strings are broken away from the oviduct, but their points of attachment may be seen. The stages in this ovary ranged from a few spiremes to well-developed eggs, each with its five nurse cells contained in a separate chamber in the string. The ovaries of early June were practically identical in size with those of January. AH the cells by this time have differentiated into eggs and nurse cells. In early July, the ovaries are markedly different. Figure 31 shows portions of three egg strings from a pupa about three days before the time of emergence. The ovary now consists entirely of egg strings with a decidedly beaded appearance due to the growth of the eggs.

b. Early stages in the development of the ovary

A description is given below of the stages in the development of the eggs and nurse cells from the oogonia to the first metaphase of the egg.

, Stage a {fig. 37 a): The oogonial region containing cells in various stages of final oogonial divisions and in rest before these divisions. A polar view (fig. 2) of a metaphase plate shows 26 chromosomes. In anaphase stages no lagging chromosomes were observed, nor differential divisions, such as have been described in several insects (Buchner '09, Gtinthert '10).

Stage b {fig. 36): Post-oogonial nuclei. Here the chromatin assumes the form of deep-staining bodies with ragged and irregular outhnes. No constant number can be counted. The cells are connected by dense protoplasmic strands or tubes, which appear to originate from the spindle remains of the oogonial divisions. Gtinthert ('10) figured similar connections between eggs and nurse cells in the oogonia of Dytiscus. In figures 41 and 42 are shown two tubes with their branches appearing to terminate in rounded knobs, which are merely the upturned ends of the branches. The largest number of branches observed was 6 and this is probably the correct number as sections of later ovaries show that each egg cell is connected with five nurse cells.

Stage c (figs. 38 and 39). The chromosomes are transformed into smaller irregular fragments which later assume the form of pale delicate threads. There is no trace of the uncoiling, of convoluted threads from the chromatin masses to form the leptotene stage, as described by Davis ('08) and Wilson ('12) in the spermatogenesis of insects.

Stage d (fig. 40) •' The presynaptic leptotene. The threads now appear more definite and convoluted. A few irregular clumps of chromatin may be seen, but fewer detached fragments than before. Several free ends of the spireme are visible, but it is impossible to approximate the number of threads.

Stage e (figs. 37 b and 43): The synaptic stage or synizesis. A study of this period gives most unsatisfactory results. The contraction figure seems to follow immediately upon the leptotene stage. The spireme forms a deep-staining mass closely and intricately coiled. In some animals the synaptic knot shows two kinds of threads, thick and thin, indicating a possible parasynapsis. In P. cynthia all parts show the same diameter throughout.

Stage f (figs. 37 c and 44) •' Postsynaptic spireme. The threads now begin to spread out through the nuclear cavity. A few free ends are visible. In other cases the spireme might be interpreted as continuous. It stains deeply as before, and is of the same thickness throughout. Several writers have stated that the nuclei are not enclosed at this time, the cells forming a syncytium. In many sections of this material, cell boundaries were not to be seen, but in other cases, particularly when Flemming's fluid was used, they could be traced without any difficulty. The tubes connecting the cells stain very lightly at this stage, being only occasionally visible.

From this point on*, a gradual differentiation occurs between eggs and nurse cells, so that it is convenient to treat the two separately. The further nuclear changes in the nurse cells will be considered first.


c. Development of nurse cells

A condition shown in figure 45 succeeds that of the preceding figure. The spireme is spread out through the nuclear cavity, and appears to consist of about 13 segments, most of them looped, but without any indication of polarization. A small plasmosome is present. Somewhat later (fig. 46), 13 definite segments may be counted, and the loops show a tendency to straighten out into long rods. The threads are very definite, with fairly smooth outhne, and appear very slightly thicker than when first opening out. The hajDloid number was counted in at least twenty nuclei of this period. The plasmosome is slightly larger than before, and has no chromatin associated with it, nor is there any orientation of the segments with respect to it. Toward the latter part of this stage, the nucleus increases in size, and the chromatin segments gradually become thicker and more deeply-staining, . giving rise to the condition shown in figure 47. Some of the segments are in the form of curved rods, others are sharply bent. The plasmosome is larger at this time, and frequently vacuolated.

The condition of the thick thi-eads in figure 47 is similar in general appearance to the pachytene stage of other animals, but it is probably not equivalent in its origin, since the threads are formed, not by doubling, but by a gradual widening of the thinner threads. This stage represents the pachytene period only in the sense that it is subsequent to synapsis, and gives rise to the diplotene stage.

Stage g (figure 48): The diplotene stage. A longitudinal split now appears for the first time in all the chroniosomes, and shows very clearly in cells which lie directly in contact with those of the preceding period which show no split. They are differentiated from them also in length of the chromosomes, for the split threads are considerably shorter. Doncaster ('12) describes in Abraxas the double thi'ead as arising probably by a bending over of the chromosome, with a separation later at the bend, but this is certainly not the case in P. cynthia. There is no clue whatever to the relation of these double threads to the chromosomes of the oogonia, since the spht appears de novo, and also since there is no direct evidence that the chromatin threads have conjugated in synapsis. Unfortunately, therefore, P. cynthia cannot be added to the list of forms in which either parasynapsis or telosynapsis has been observed. The impression gained from a study of the material is that reduction is accomplished by a simple segmentation of a continuous spireme into the haploid number of threads.

Following upon figure 48, a progressive shortening of the segments occurs, the chromosomes often appearing extremely ragged. In a few cases, the halves- show a divergence at one point as if beginning to separate (fig. 49) . Very rarely the threads open in the middle while remaining united at the ends, thus forming a ring, but these forms are probably accidental and due to the fact that the chromosomes are soon to disintegrate. The later condition of this stage is shown in figure 50. The threads have by this time assumed a rod-like appearance. The lengthwise split is still apparent, but the chromosomes are extremely ragged and irregular in outline. Thirteen rods are present. At about this stage in Abraxas, Doncaster ('12) found the egg cells differentiated. In P. cynthia, as will be shown, the period is somewhat earlier.

Stage h (figs. 37 d and 51). In figure 51 almost all of the rods have shortened to bipartite chromosomes, the halves being rather widely separated from each other. Frequently a constriction in each half gives a tetrad form to the chromosome. In this figure, two are still rod-like, as in the preceding stage. The large plasmosome is frequently vacuolated. In figure 52, a later condition, the chromosomes are more irregular and stain less deeply. A few of the tetrads are broken into four separate flocculent pieces; others into irregular fragments. Some chromosomes remain bipartite as before. Within the cytoplasm a dark rounded mass indicates the end of the strand or tube which connects this cell with others.

With further growth of the cell, the nucleus increases in size, and the chromatin fragments multiply considerably. Giardina ('01), Debaisieux ('09) and Gunthert ('10) have figured in the Dytiscidae a markedly regular division of tetrads, each part giving rise to a whole tetrad, this process being repeated several times. In P. cynthia there is no evidence of any order in the fragmentation, for there is the greatest irregularity in the size and shape of the pieces.

The period of fragmentation marks the first broad phase in the history of the nurse cells. It is interesting to note that the cells have passed through a cycle of changes as if for maturation divisions, since they show the reduced number of chromosomes. These are destined, however, only for disintegration.

Stages j to I. At the beginning of this phase, the eggs and nurse cells are practically similar in size (fig. 37 e). The egg cell increases steadily in size during the growth period, the nurse cells, although increasing for a time, do not keep pace with the growth of the egg, and become relatively smaller as development proceeds.

Stage j (figs. 37 e and 53): At the close of fragmentation, numerous small dark granules lie within the nucleus near the periphery, together with a variable number of larger round bodies, which, although stained very deeply in some sections, are very pale in others, and appear to be of the nature of plasmosomes. The cell contents appear shghtly granular, or reticular, with a very darkly granular, flask-shaped area extending from the nuclei toward one end of the cell, appearing to perforate the cell wall in the form of a curved tube which enters the egg cell. Marshall ('07), Giinthert ('10) and others describe similar areas, but the tubular portion is not apparent. A later stage is shown in figure 54. The nuclear wall is less easily seen on the side nearest the tubes, for the granules are thickest at this point, and lie close to the dark granular cytoplasm. In addition to this mass of granules, the nucleus contains much smaller masses scattered near the periphery, and several small plasmosomes. A thin dark band of cytoplasm often encircles the nucleus, merging with the flask-shaped portion. Beyond this the cytoplasm appears reticular. Figure 55 A shows another cell in which the nuclear cavity is indented in two regions, giving a somewhat dumb-bell shape. During these changes in the shape of the nucleus, the plasmosomes are extremely variable. Figure 55 B is a plasmosome from a similar nucleus, very irregular in form and encrusted with chromatin granules. In some cases the granules adhere in such numbers as practically to obscure the plasmosome.

Figure 56 is typical of older nurse cells. The nuclear wall appears to be thrown into a number of folds, beset with chromatin granules, which frequently obscure the wall. The nuclear cavity contains as before, clumps of granules and plasmosomes. The cytoplasm immediately surrounding the nucleus has become much broader, forming a conspicuous dark ring which merges into the flask-shaped region. The reticular portion of the cytoplasm is smaller in extent. Frequently at this stage or later, there appear very pale delicate cytoplasmic lines in the flaskshaped region, converging down into the tubular portion, probably indicating a transfer of material into the egg cell. In figure 56 two nurse tubes are shown, the egg cell into which they open not being indicated. The tubes appear longer than in the early stages, and are irregularly constricted in places, often apparently forming a series of rings lying upon each other. There appears to be a thin homogeneous membrane forming a distinct wall to the tube; this is not a continuation of the cell wall, but is formed at the edge of the flask-shaped cytoplasm, and passes through the cell wall (fig. 54). In Eacles imperialis and Telea polyphemus a similar condition was observed, although the tubes here are not so piominent.

In figure 57 a later stage of the nurse cells is shown, drawn to the same scale as figure 56. Here the nurse cells are considerably larger than before, yet smaller than the egg. The flaskshaped region, circular granular region and nuclear cavity appear as previously indicated. The plasmosomes are covered with granules, and single strands of more prominent granules partly line and extend, down into the circular region. Only two nurse cells are figured here. The total number for each egg is five, which can be readily determined by following through a series of transverse sections. Gross ('03) also found five in other Lepidoptera. In figure 57 and other similar sections, the follicle cells are arranged in a layer around the groups of eggs and nurse cells, first definitely formed at the periphery of the egg string, then growing in at the base of the egg cell. Later they grow in between the egg and its nurse cells, separating them except in the region of the tubes.

Stage I. At the end of the growth period, when the nurse cells have become very small, no definite tubes are seen, the cytoplasm opening broadly into the egg. Later the follicle cells form a continuous layer over the egg, and the nurse cells may be seen as small degenerated masses of cytoplasm which eventually disappear.

d. Development of egg cells

The further history of the egg cells, beginning with their differentiation from the nurse cells in Stage f is given below.

Stage f: Postsynaptic spireme. Among the nurse cells of this stage (figs. 44-46) a few cells may be observed in every section, in which the spireme appears to be thicker than in the surrounding cells, more continuous and more closely convoluted^ as in figure 58. The nucleus is somewhat larger, and is very frequently distinguished by a pale yellowish tinge. Characteristic of this stage is the large plasmosome, frequently surrounded by a darker rim. The cytoplasm is also slightly greater in amount. A large number of sections were examined, and these differences appeared fairly constant. Careful study of cells in the earlier contracted spireme failed to reveal any criterion by which the egg cells might be identified at this time.

After the nurse cells are well differentiated, the spireme of the egg cell appears much less convoluted (fig. 59 A), spreading out through the nuclear cavity, which has increased considerably in size. In figure 59 B is shown a portion of the spireme which was not included in the first section. It is not possible to determine accurately if the spireme is continuous, but it is my belief that this is the case. During this period, one or two large plasmosomes may appear, and frequently two smaller bodies, probably of the same nature. The entire nucleus has the yellow tinge noted in the earliest stage of its differentiation.


The cells next to be described are taken from sections of ovaries fixed in January, later than the preceding sections. Figure 57, already referred to, shows a portion of an egg string just beyond its point of emergence from the ovary proper. Figure 60 is a nucleus from a similar egg. The spireme is typical for the eggs at this period; it is still convoluted as before, with no trace of a longitudinal split. The plasmosome varies considerably in form, consisting usually of a dark spherical portion, and a light portion, sometimes lobed and vacuolated. In this figure the plasmosome gives the appearance of breaking through the nuclear membrane, and in another egg near by a similar body was observed lying in the reticular cytoplasm at a little distance from the nucleus. A few other cases were observed on the same slide. The material appeared to be unusually well fixed, but as other ovaries failed to show a similar condition, this is probably not a normal occurrence.

It is con\'enient at this point to note more definitely the relation between the eggs and nurse cells during this period of growth. In figure 57 two nurse cells are seen connected with the egg, the flask-shaped region with its faint converging lines of protoplasm is confluent through the nurse tubes with a dark granular layer which surrounds the egg nucleus, broadening out into a conspicuous mass on the farther side of the nucleus. Here it sends long processes radiating out into the reticular portion of the cytoplasm. In most sections this finely granular region has a yellowish tinge, like the nucleus, markedly diff'erent from the reticular region of the cells. The mass frequently contains small vacuoles and deep-staining granules and is similar in appearance to the so-called yolk nuclei in various eggs; it seems probable that in P. cynthia the mass is of the same nature.

Paulcke ('00), Gross ('03), and others, have described whole nurse cells entering the egg during the growth period. This would be impossible in the moth, on account of the small diameter of the nurse tubes.

Stage g: Disappearance of the spireme in the later growth period. The next stages figured are sections from the ovary shown in figure 31, from a moth fixed a few days before the time of emergence. In a few of the youngest eggs in this material, the spireme is still vaguely discernible (fig. 61) in the form of a pale network of irregular threads joined together, not the coiled spireme of earlier eggs. Several dark bodies of irregular size and shape are characteristic of this period. In figure 62 — a slightly older nucleus in the same string — all traces of the spireme have disappeared. The nuclear cavity contains a pale body with a large vacuole, and numerous smaller rounded masses, which frequently stain veiy deeply. These are probably all plasmosomes. There is extreme variability in respect to their number, size and appearance, some being apparently homogeneous, others filled with vacuoles. Figure 63 is a nucleus of about the same age as figure 62.

As the eggs increase in size, the nuclei appear to have at one side a darker region, frequently crescentic (fig. 64), which seems to be connected with a dark granular protoplasmic strand running down into the cytoplasmic region of the egg, now coneshaped, as in the mature egg. The nucleus is party surrounded by yolk spheres, lying in faintly granular cytoplasm. The crescentic region merges gradually into the lighter granular portion of the nucleus, and suggests merely a condensation of the nucleoplasm here. Over twenty-five nuclei of this stage were examined, after varying degrees of extraction. In many cases the contents of the darker region were visible, and all showed the same condition of darker granules merging into lighter ones. No plasmosomes were to be seen, nor any trace of chromatin. The nuclei lie near the periphery of the eggs in the cytoplasmic region near the nurse cells, which at this time are reduced to shrunken remnants. Later the nuclear wall seems to fade out at the side nearest the periphery, and several bipartite rod-like chromosomes may be seen within the nucleus. At a slightly later period, the chromosomes, now shorter and more dumb-bell-shaped, appear to lie in a rounded area in which no distinct nuclear boundary is discernible. In the latest prophase (fig. 3) the nuclear boundary reappears, very faint, and very much smaller than the former nuclear area.

e. Degenerating cells in the ovary

Groups of degenerating cells are to be found in almost all of the ovaries examined, occurring chiefly in the region of the tetrads. Similar cells were also noted in the spireme region, but very rarely in the egg strings. These cells have the form of clear vesicles which contain one or more deep-staining spheres of chromatin material. A number of writers have noted this condition both in oogenesis and in spermatogenesis.

No cases of amitosis were observed in any of the germ cells. The nurse cells do not divide in any manner after the last oogonial divisions, nor do the egg cells, until the maturation divisions.

/. Abnormal nuclei in the early ovary

Certain abnormal conditions were obser\'ed in nurse cells of Stage g. In two ovaries, several cells showed, instead of 13 bipartite rods, from 15 to 19 rods. These differed further in the fact that no longitudinal split was evident. As it happened that these two ovaries were the first ones examined, the problem was very puzzling, for it appeared to indicate that the chromosomes were not of the reduced number. Normal nuclei, however, were found in the same material, and sections of about 40 other ovaries failed to show any abnormal cells. Doncaster ('12) mentions a somewhat similar abnormality, in which one cell showed the diploid, instead of the haploid number of chromatic threads.

2. CONCLUSIONS AND COMPARISONS

Differential divisions in the oogonia, which have been described for the Dytiscidae, are not found in P. c^ynthia. The germ cells all appear similar in size until the post-synaptic spireme stage, agreeing in this respect with Pieris and Abraxas (Griinberg '03, Doncaster '12), the bee (Paulcke '00), and the di^.gon-fly (McGill '06, Marshall '07). Doncaster finds a differentiation appearing a little later than in P. cynthia, when the chromatin threads shorten to form bipartite chromosomes in the nurse cells. The egg spireme is not continuous, but is composed of the haploid number of interlaced threads, which have not yet contracted. Marshall ('07) found a still later dilTerentiation in Platyphylax, a neuropteran, in which the tetrad stage is common to both kinds of cells, but the egg cell is larger, and the tetrads persist longer before disintegration.

The haploid number of chromatin segments is present in the nurse cells of P. cynthia, as in Pieris (Doncaster '12), indicating a preparation for division in these cells whose function is only nutritive. A number of writers, including Griinberg ('03), Gross ('03), Marshall ('07), and Woltereck ('98), figure tetrads in the nurse cells of various animals, but do not state whether the haploid number is present. They agree, however, that differentiation of eggs and nurse cells occurs after synapsis, which would imply that nurse cells as well as eggs must have undergone pseudo-reduction.

A transfer of material takes place from the nurse cells and the egg through the connecting tubes which in P. cynthia have very prominent walls. A markedly similar condition was observed by Giinthert ('10) in Dytiscus, where converging bundles of fibrils appear, beset with chromidia or chromatin granules which enter the egg. In this case there is no definite wall to the tubes, Griinberg ('03j states that in Pieris the egg sends a large blunt process up between the nearest nurse cells. Evidently there is considerable variation in the relation of eggs and nurse cells within the Lepidoptera, for in P. cynthia it is the nurse cells which send processes into the egg.

The history of the egg nucleus seems to show that the chromosomes lose their visible identity during the growth period. I am convinced of the accuracy of the results in this particular, on account of the very careful study given to this stage. More than half the nuclei from one individual were examined, and only in the very earliest eggs were traces of spiremes to be found. I examined also egg strings of other moths. In Clisiocampa the spireme persists relatively longer, being found in large eggs. As in P. cynthia, it becomes gradually fainter and more broken the older the eggs become, and finally disappears altogether.


Throughout the growth period the nucleus contains many small non-chromatic bodies, in a pale flocculent nucleoplasm, but no trace of a spireme. Sections of Rothschildia jorulla eggs and Actias luna showed an essentially similar condition.

The literature dealing with the condition of the chromosomes during the growth period contains a number of diverse results. In several groups of vertebrates and invertebrates the persistence of the chromosomes has been demonstrated by Grifhn ('99), Stevens ('04), Dublin ('05), Marshall f'07, '10), Ruckert ('02), Born ('94), Schockaert ('02), Winiwarter and Saintmont ('08), King ('08), and others. Deton ('09) found only a pale reticulum in the egg of Thysanozoon; nevertheless he believes, with Gregoire ('09) that, whatever the appearance, the chromosomes persist autonomously up to the maturation divisions. On the other hand, evidence that the chromosomes disappear as such during the growth period, is given by the work of Carnoy and Le Brun ('99), Hacker ('95), Woltereck ('98), Bonnevie ('06), Popoff ('07), Goldschmidt ('08), and Schleip ('09). The latter found an interesting condition in Cypris; in one form the chromosomes may be traced throughout the growth period, in another they disappear. With the second group Philosamia cynthia is to be included, as the facts observed inchcate a gradual disappearance of the spireme during the growth of the egg.

3. LITERATURE ON THE EARLY DEVELOPMENT OF EGGS AND

NURSE CELLS

In this list are included only a few of the papers dealing with the various early stages in the growth of the eggs and nurse cells in insects.

Lepidoptera. Doncaster ('12) describes the early oogenesis in Pieris brassicae and Abraxas grossulariata. In Pieris, after the oogonial divisions, when 30 chromosomes are seen in the equatorial plate, the nucleus enlarges and forms a reticulum, followed suddenly by the synizesis stage, in which a chromatin nucleolus appears. In the ensuing stage, a broken spireme of 14 separate threads is seen, the fifteenth element being represented by the double chromatin nucleolus, which is interpreted as an equally paired heterochromosome. When the threads shorten to chromosomes, this is indistinguishable from the others. In Abraxas these stages are similar, A distinction is noted here between eggs and nurse cells. In the former "the bivalent threads persist to the latest stage observed — possibly till the prophase of the polar divisions;" in the latter, the bivalent threads shorten into loops to form chromosomes. In Bombyx and Pieris, as described by Griinberg ('03), the germ cells are at first all alike, with nucleolus and granules in the nucleus. The next zone in the ovary shows spiremes, in which stage synapsis occurs. Cell boundaries are not figured here. This is followed by a differentiation zone, in which the egg nucleus is distinguished by a nucleolus and threads, the nurse cells by tetrads. Details of their origin are not given. Fragmentation of the tetrads is described, and the arrangement of nurse cells near the egg, followed by a transfer of granular material to the egg cell.

Neuroptera. The observations of Marshall ('07) on the ovary of Platyphylax are meagre as regards chromatin changes. From synapsis, beaded threads appear, showing a lengthwise split. These threads give rise to tetrads, which fragment. In cells destined to form eggs, the tetrads disappear later and the nuclei are slightly larger. The further history of the eggs and nurse cells is not given.

Hy7nenoptera. In the early ovary of the bee, Paulcke ('00) observed that the eggs and nurse cells are at first similar. Beyond the synapsis stage, the nurse cells differentiate, the chromatin fragmenting and the nucleus increasing in size. Later the cytoplasm of the egg may be seen projecting into the region of the nurse cells, which are very numerous. He believed that nurse cells entire might be taken into the egg, and that amitosis occurred.

Hemiptera. In a study of the early ovary of Protenor, Foot and Strobell ('11) distinguish three zones in the ovary: Zone A consists of nuclei with numerous granules; zone B of larger nuclei with granules and a nucleolus, arising by growth from A;

zone C contains very small nuclei similar to A, arising chiefly from the cells of zone B by amitosis, and giving rise to the ova. They believe amitosis plays an important role. No cell bomidaries appear in these zones. In young ova, leptotene threads -are seen, followed by a stage of broken spireme threads. These gradually disappear, and reappear later to form chromosomes. The figures given are chiefly photographs and do not adequately illustrate the points mentioned in their paper. The importance of amitosis has been questioned b}^ Gross ('01), who concluded from his studies on Hemiptera that nuclei which divide amitotically never divide again by mitosis.

Payne ('12) also discusses the origin of the ova in Gelastocoris, another hemipteran. He finds the same three kinds of cells, although their arrangement in zones is very indefinite. He is convinced that the eggs are not derived from the small nuclei of zone C, but from certain larger cells of zone B, which he finds in synapsis, and that there is no break in the continuity of the cells from oogonia to ova. This appears to be a more reasonable interpretation of the development of the eggs, and accords more closely with the conditions observed in other insects.

Coleoptera. Debaisieux ('09) has described the early oogenesis in Dytiscus marginalis, amplifying Giardina's work on the same form. Debaisieux discovered a synaptic and a diplotene stage between the zone of differentiation of eggs and nurse cells and the growth zone. In the latter, the chromatin of the nurse cells gives rise to tetrads which fragment, the chromatin of the egg cell remaining in the diplotene stage as before. His main conclusions are, (1) that the 'chromatic mass', which Giardina believed to be derived from certain chromosomes is not true chromatin, but a condensation of the reticulum left in the nucleus after the chromosomes of the last oogonial division are formed; (2) that the chromosomes persist autonomously up to the maturation divisions. In Giinthert's paper ('10) the chief point of interest is the description of difi'erential mitosis in the oogonia. When a cell divides, a 'chromatic mass' and the spherical remains of the spindle pass into one cell undivided. This becomes the egg cell, which again divides differentially as before. At the end of the fourth differential mitosis, there are 15 nurse cells and one egg cell, which enters the resting stage. The spindle remains of the nurse cells join that of the egg, forming a protoplasmic bridge between them. Gtinthert believes that differential divisions occur in many animals; and that the 'accessory body' described by Buchner ('09) in Gryllus is merely a 'chromatic mass' indicating a differential mitosis. The origin of nurse cells from the egg by a process of budding, as described by Will, is probably to be interpreted in the same way. In the later nurse cells of Dytiscus Gtinthert finds tetrads which subdivide regularly several times, freeing thousands of granules in the nucleus. When the nuclear wall breaks down, they migrate into the cytoplasm, where they increase by division, eventualh^ entering the egg.

Orthoptera. Buchner ('09) figures a leptotene stage in Gryllus after the oogonial divisions, followed by a diplotene stage during which the 'accessory' is much vacuolated. The probable significance of this body has been referred to above. After the diplotene stage the threads shorten into rods and tetrads. No further development of the egg is given.

SUMMARY

1. In Philosamia cynthia the 13 bivalent chromosomes of the late prophase all divide in both maturation divisions.

2. The male and female pronuclei at the time of their union each contain 13 chromosomes, making the somatic number 26, which is found in the nuclei of the blastoderm.

3. On account of the great variations in the size of the chromosomes in the metaphase and anaphase plates, there is no conclusive evidence for either the presence or the absence of an X Y-pair of chromosomes.

4. In the oogonia no differential divisions occur. The germ cells all appear similar through the presynaptic and synizesis stages.

5. In the post-synaptic spireme stage, the nuclei of the future nurse cells show the haploid number of threads, indicating a preparation for division, although the chromosomes are destined only for disintegration. In the egg cell the spireme is probably continuous. A plasmosome is present in both cases.

6. During the growth period, the chromosomes of the nurse cells fragment into numerous granules. The nuclear wall becomes much infolded, and is lined with the granules.

7. A transfer of material takes place from the nurse cells to the egg, through connecting tubes derived from the spindle remains of the final oogonial divisions. The egg cell increases in size at the expense of the nurse cells.

8. Amitosis does not occur among the germ cells. Degeneration of cells is common in the region of differentiation.

9. The egg nucleus remains in the spireme stage throughout the greater part of the growth period. There is no indication of a segmentation into the haploid number of threads.

10. Shortly before emergence, a few of the youngest cells in the ovary show a faint disintegrating sj^ireme. In most of the cells no trace of chromatin is present. This indicates that the chromosomes lose their visible identity during the growth period. It is impossible to demonstrate the form which the chromatin assumes during this period of its diffusion.

11. In the oldest cells of a late ovary the chromosomes reappear in the form of 13 short rods or dumb-bell-shaped bodies characteristic of the early metaphase groups of chromosomes.

March, 1914.

LITERATURE CITED

VON Baehr, W. B. 1908 Ueber die Bildung dcr Sexualzellen bei Aphididae. Zool. Anz., Bd. 33.

1909 Die Oogenese bei einigen viviparen Aphididen imd die Si)ermatogenese von Aphis saliceti, u.s.w. Arch. f. Zellf., Bd. 3. no. 2.

Baltzer, F. 1909 Die chromosomen von Strongylocentrotus lividus und Ecdiinus microtuberculatus. Arch. f. Zellf., Bd. 2.

BoNNEviE, K. 1906 Beobachtungen an den Keimzellen von Enteroxenos cstergreni. Jenaische Zeit., Bd. 41.

Born, G. 1894 Die Struktur des Keimblaschens im Ovarialci von Triton taeniatus. Arch. f. mikr. Anat., Bd. 43.

BucHNER, P. 1909 Das accessorische Chromosom in Spermatogenese und Ovogenese der Orthopteren, u.s.w., Arch. f. Zellf., Bd. 3, no. 3.

Carnoy, J. B., AND Lebrun, H. 1899 La vesicule germinative et les globules polaires chez les batraciens. III. La Cellule, torn. 16.

Cook, M. H. 1910 Spermatogenesis in Lepidoptera. Proc. Acad. Nat. Sci., Philadelphia, April.

Davis, H. S. 1908 Spermatogenesis in Acrididae and Locustidae. Bull. Mus. Comp. Zool. Hai"vard, vol. 52, no. 2.

Debaisieux, p. 1909 Les debuts de I'ovogenese dans le Dytiscus marginalis. La Cellule, tom. 25.

Dederer, p. H. 1907 Spermatogenesis in Philosamia cynthia. Biol. Hull, vol. 13, no. 2.

Deton, W. 1909 L'etape synaptique dans le Thysanozoon brocchii. La Cellule, tom. 25.

Donc'aster, L. 1908 Sex inheritance in the moth Abraxas grossulariata. Royal Soc. Evol. Comm., rept. 4.

1912 The chromosomes in the oogenesis and spermatogenesis of Pieris brassicae, and in the oogenesis of Abraxas grossulariata. Journ. Genetics, vol. 2, no. 3.

. 1913 On an inherited tendency to produce purely female families in Abraxas grossulariata, and its relation to an abnormal cliromosome nximber. Journ. Genetics, vol. 3, no. 1.

Doncaster, L., and Raynor, G. E. 1906 Breeding experiments with Lepidoptera. Proc. Zool. Soc, London, vol. 1.

Dublin, L. 1905 The history of the germ cells in Pedicellina americana. Ann. New York Acad. Sci., vol. 16.

Foot, K., and Strobell, E. C. 1911 Amitosis in the ovary of Protenor belfragei and a study of the chromatin nucleolus. Arch. f. Zellf., Bd. 7, no. 2.

GiARDiNA, A. 1901 Origine dell' oocite e delle cellule nutrici nel Dytiscus. Internat. Monatschr. f. Anat. u. Phys., Bd. 18.

GoLDSCHMiDT, R. 1908 Uebei" das Verhalten des Chromatins bei der Eireifimg und Befruchtung des Dicrocoelium lanceatum. Arch. f. Zellf., Bd. 1.

Gregoire, V. 1909 Les phenomenes de I'etape synaptique. La Cellule, torn. 25.

Griffin, B. B. 1899 Studies on the maturation, fertilization and cleavage of Thalassema and Zirphaea. Jour. Morph., vol. 15.

Gross, J. 1901 Untersuchungen liber das Ovarium der Hemipteren, zugleich ein Beitrag zur Amitosenfrage. Zeit. f. wiss. Zool., Bd. 69.

1903 Untersuchungen fiber die Histologic des Insectenovariums, Zool. Jahrb. Bd. 18.

Grunbero, K. 1903 Keim- und Nahrzellen in den Hoden und Ovarien der Lepidopteren. Zeit. f. wiss. Zool., Bd. 74.

GtixTHERT, T. 1910 Die Eibildung der Dytisciden. Zool. Jahrb., Bd. 30.

Hacker, V. 1895 Die Vorstadien der Eireifung. Arch. f. mikr. Anat., Bd. 45.

Henking, H. 1892 Untersuchungen iiber die ersten Enlwicklungsvorgange in den Eiern der Insekten, III. Zeit. f. wiss. Zool., Bd. 54.

King, H. D. 1908 The oogenesis of Bufo lentiginosus. Jour. Morph., vol. 19.

McGiLL, C. 1906 The behavior of the nucleoli during the oogenesis of the di-agon fly, with especial reference to synapsis. Zool. Jahrb., Bd. 23.

Marechal, J. 1907 L'ovogenese des selaciens et dc quelciues autres chordates. La Cellule, torn. 24.

Marechal, J., et de Saedeleer, A. 1910 Le premier developpement de I'ovocyte chcz les Raj ides. La Cellule, torn. 26.

Marshall, W. S. 1907 Cellular elements of the ovary of Platyphylax designatus Walk. Zeit. f. wiss. Zool., Bd. 86.

Morgan, T. H. 1909 A biological and cytological study of sex determination in Phylloxerans and Aphids. Jour. Lxp. Zool., vol. 7, no. 2.

Morrill, C. V. 1910 The chromosomes in the oogenesis, fertilization and cleavage of Coreid Hemiptera. Biol. Bull., vol. 19, no. 2.

Paulcke, W. 1900 Ueber die Differenzierung der Zellelemente im Ovarium der Bienen-konigin. Zool. Jahrb., Bd. 14.

Payne, F. 1912 L A further study of the chromosomes of the Reduviidae. IL The nucleolus in the young oocytes and origin of the ova in Gelastocoris. Jour. Morph., vol. 23, no. 2.

PopoFF, M. 1907 Eibildung bei Paludina vivipara und Chromidien bei Paludina und Helix. Arch. f. mikr. Anat., Bd. 60.

Punnett, R. C, and Bateson, W. 1908 The heredity of sex. Science, N. S., vol. 27, no. 698.


RucKERT, J. 1892 Zur Entwickelungsgeschichte des Ovarialeies bei Selachiorn. Anat. Anz., Bd. 7.

ScHLEip, W. 1909 Vergleichende Untersuchung der Eireifung, u.s.w. Arch, f. Zellforsch., Bd. 2.

ScHOCKAERT, R. 1902 L'ovogcuesc chez le Thysanozoon brocchi, II. La Cellule, torn. 20.

Seller, J. 1913 Das Verhalten der Geschlechtschromosomen bei Lepidopteren. Zool. Anz., Bd. 41, no. 6.

Stevens, N. M. 1904 Further studies on the ovogenesis of Sagitta. Zool. Jahrb., Bd. 21.

1906 a Studies on the germ-cells of Aphids. Carnegie Institution. Pub. Xo. 51.

1906 b Studies on spermatogenesis. II. A comparative study of the heterochromosomes in certain species of Coleoptera, Hemiptera, and Lepidoptera, with especial reference to sex determination. Carnegie Institution Pub. No. 36.

1909 An unpaired heterochromosome in the Aphids. Jour. I^xj). Zool., vol. 6.

Tennant, D. H. 1912 a The behavior of the chromosomes in cross-fertilized echinoid eggs. Jour. Morph., vol. 23.

1912 b Studies in cytology. I. A further study of the chromosomes of Toxopneustes variegatus. II. The behavior of the chromosomes in Arbacia-Toxopneustes crosses. Jour. Exp. Zool., vol. 12.

Wilson, E. B. 1912 Studies on chromosomes. VIII. Observations on the maturation-phenomena in certain Hemiptera and other forms, with considerations on synapsis and reduction. Jour. Exp. Zool., vol. 13, no. 3.

VON Winiwarter, H., et Saintmont, G. 1908 Nouvelles recherches sur I'ovogcnese de I'ovaire des mammiferes (chat). Arch, de Biol., tom. 24.

WoLTERECK, R. 1898 Zur Bilclung und Entwicklung des Ostracocleneies. Zeit. f. wiss. Zool., Bd. 64.


All the. figures (Philosamia cynthia) were drawn with the camera lucida. The enlargement is 2100 diameters, unless otherwise specified.

PLATE 1

EXPLANATION OF FIGURES

1 Metaphase from cell of embryo, showing 26 chromosomes.

2 Oogonial metapliase, showing 26 chromosomes.

3 Prophase of first oocyte division, showing 13 chromosomes.

4 to 7 Metaphasesof first oocyte division, side view, showing 13 chromosomes.

8 Anaphase of first oocyte division, showing cell plate and chromosomes.

9 Same; 13 chromosomes at each pole.

10 and 11 Late first anaphase, oblique polar view. The upper groups of chromosomes enter the first polar body.

12 to 14 Three sister anaphase groups of the first division; a, chromosomes of first polar body; 6 chromosomes remaining in the egg.


PLATE 2

EXPLANATION OF FIGT'RES .

15 Metaphase of second oocyte division, side view, from serial sections, showing cell plate between .4, 13 chromosomes of first polar boch', and B, 13 chromosomes in the egg.

16 Same, without cell plate.

17 Same, with cell plate; polar view.

18, 19, 22, 26 Anaphases of second oocA^te division, all incomplete except lower group in figure 19.

20 Sister anaphase groups of second division, showing a, 13 chromosomes of second polar body; h, 13 chromosomes in the egg.

21 Second anaphase group of 13 chromosomes in the egg.

23 and 24 Sister anaphase groups of second division; oblique polar view, the groups slightly displaced.

25 Telophase of second division; incomplete.


PLATE 3

EXPLANATION OF FIGURES

27 Copulation of the pronuclei, from serial sections, showing 13 chromosomes in each pronucleus B, C, and 13 in second polar body, A.

28 Polar bodies from egg of similar stage; the first one has divided, and shows shadowy chromosome outlines.

29 Ovary from a larva, longitudinal section. X 16.

30 Ovary from a pupa, fixed in January; total, X 16.

31 Same, fixed in July; total, X 16.


PLATE 4

EXPLANATION OF FIGURES

32 and 33 Anaphases of first oocyte division. X 700.

34 Metaphase of second oocyte division. X 700.

35 Copulation of pronuclei; same egg as figure 27. X 700.

36 Post-oogonial nuclei with chromatin masses. The cells are connected by protoplasmic strands or tubes.

37 Longitudinal section through portion of an egg string of a larval ovary. X 400. a. Stage a, oogonial region; b, Stage e, synizesis; c, Stage f, post-synaptic spireme; d, Stage h, dyad or tetrad chromosomes in nurse cells; e, eggs and nurse cells well differentiated.

38 and 39 Stage c; the chromatin masses are transformed into small irregular fragments which later assume a thread-like form.

40 Stage d; presynaptic leptotene.

41 and 42 Two groups of protoplasmic tubes with branches.


PLATE 5

EXPLANATION OF FIGURES

43 Stage e; synizesis.

44 to 47 Stage f ; post-sjoiaptic spireme of nurse cells. 48 and 49 Stage g; diplotene stage; 13 split rods.

50 to 52 Stage h; chromosomes begin to fragment.

53 and 54 Stage j ; young nurse cells, showing tubes entering egg cell. X 700.

55 A, nucleus of nurse cell with plasmosomes and chromatin granules; X 700. B, plasmosome enlarged.

56 Older nurse cells, with tubes. X 400.


PLATE 6

EXPLANATION OF FIGUKES

57 Portion of an egg string, showing an egg and two nurse cells. X 400.

58 and 59 Stage f; early and later post-synaptic stages of egg cell; spireme probably continuous.

60 Nucleus from an egg cell similar to the one shown in figure 57. X 700.

61 Stage g; spireme disappearing in the later-growth period; plasmosomes of varying size and form.

62 Slightly older nucleus; all traces of the spireme have disappeared.

63 Same. X 850.

64 Nucleus from a nearly mature egg, showing dark crescentic region. The nuclear cavity is filled with granules. X 400.



The Structure And Growth Of The Incisor Teeth Of The Albino Rat

William H. F. Addison And J. L. Appleton, Jr.

From the Anatomical Laboratory of the University of Pennsylvania and The Wistar Institute of Anatomy, Philadelphia

TWENTY-NINE FIGURES

CONTENTS

Introduction 43

Historical survey 44

Material and methods 46

Dentition of adult animal 46

Minute description of the incisors 52

Microscopic structure of enamel and dentine 55

Development of the incisors 59

Detailed description of development up to time of eruption 61

Eruption of the teeth 78

Changes in apex of tooth by use 81

Description of mature tooth and tooth-forming organs, in 5-month animal. . 83

Rate of growth of incisor teeth 88

Overgrowth of incisor teeth 89

Summary 91

Literature cited 95

INTRODUCTION

The incisor teeth of the Rodentia have long been regarded by the zoologist as having a high value for the understanding of many of the characteristics of this order. For instance, in 1888, Cope wrote "nearly all the peculiarities of the rodent dental system and manner of mastication are the mechanical consequences of an increase in length of the incisor teeth." Tullberg ('98-'99) gives the taxonomic position of the genus Mus, proceeding from the more general to the more specific grouping: Rodentia, Simplicidentati, Sciurognathi, Myomorphi, Myoidei, Muriformes, Myodontes, Muridae, Murini, Mus. A consideration of these terms merely from an etymological view suggests the importance of the teeth and jaws in the classification of the gnawing animals. The observations here recorded are based on the study of the celhilar processes involved in the formation, eruption and growth of the incisor teeth in a single rodent form — Mus norvegicus albinus. An additional interest was lent to the work by the fact of the increasing use of this animal for laboratory purposes, which makes it desirable to learn the time-relations of its life-processes, as a basis of comparison in various forms of experimental studies. Although the rodent incisors have been the object of much study, few observers have carried out their observations through the complete life-history, including developmental stages and adult structure, in one form of animal and this it has been our aim to do.

HISTORICAL SURVEY

Oudet ('23) proved the phenomenon of permanent growth in the incisor teeth of rodents by cutting off the teeth at the gingival margin and observing that they were regenerated. Retzius ('37) and others noted the overgrowth of these teeth in cases of malocclusion. MacGillavry ('76) observed the rate of growth of the incisors of a rabbit by making marks on the teeth and noting the gradual advance and disappearance of these marks, as the teeth grew out and were worn away.

Questions which have called forth much study and controversy are (1) does the rodent incisor belong to the milk or to the permanent dentition; and (2) which of the three incisors of the typical mammalian dental formula does it represent. Without exception, all who have studied the first question agree that the large rodent incisor belongs to the second or permanent dentition. These same studies show that abortive milk incisors occur in a varying degree in the several families of the Rodentia; and that they are slightly, if at all, represented in the Muridae. As to the second question. Cope on palaeontological evidence decided that the large rodent incisor was h. Adloff ('98) on embryological evidence confirmed this view. Freund ('92), Woodward ('94) and Stach ('10) beheved it to be I,. Weber ('04) has given a resume and extended bibliography of this work, up to the date of his writing.


The histology of the incisor was briefly described by Owen ('40-'45) and more completely studied by J. Tomes ('50). The latter found a considerable diversity of arrangement of the enamel prisms in the different families of the order, so that in many cases he was able correctly to refer a tooth to a particular family by a simple inspection of thin sections of its enamel. Von Brunn ('87) showed that at eruption the tip of the incisor of the albino rat is free from enamel, and Sachse ('94) confirmed this on Mus musculus. J. L. Williams ('96), in a comparative study of the formation of enamel, gives a number of good illustrations of the structure of the enamel and enamel-organ of the rat, prepared from microphotographs.

Ryder ('78) and Cope ('88), in harmony with their views on the "Origin of the Fittest," described the form and position of the rodent incisor as manifestations of a most efficient mechanical system; and studied the various effects on skull topography, necessitated by adaptation to this system.

The enamel organ of the albino rat was studied by von Brunn ('87) who described in some detail the differences in structure between its functional labial portion and its non-functional lingual side. He also described the early continuity of the lingual side of the enamel-organ and its later penetration by the surrounding connective tissue. Roetter ('89), studying Mus musculus, denied von Brunn's position in regard to the invasion of the lingual side of the enamel-organ by connective tissue, and Sachse ('94), also using Mus musculus, agreed with Roetter and described the continuity of the lingual portion as persisting through life.

The development of the rodent incisor has been studied especially by Roetter ('89), Sachse ('96) and Meyerheim ('98). Burckhardt ('06), in his description of the development of the persistently growing rodent incisor in O. Hertwig's Handbuch der Entwickelungslehre has followed chiefly Sachse's work upon Mus musculus. In both Weber ('04) and Hertwig ('06) are extensive bibliographies and in these may be found all references not fully given in our appended list of literature cited.

MATERIAL AND METHODS

The albino rat is a variety of Mus norvegicus, the common gray rat (Donaldson '12). This has been shown by similarity of skull measurements (Hatai '07) and of hemoglobin crystals (Reichert and Brown '10) and also by the fact that the two interbreed freely.

The material used was obtained from the rat colony of The Wistar Institute. Serial sections in paraffin or in paraffincelloidin were made of decalcified heads of fetuses taken at daily intervals from the 16th day onwards until birth, and of jaws of animals newly-born and at short intervals until one month, and of several older stages. Serial sections of fetuses younger than 16 days were examined in the collection of The Wistar Institute. Ground sections were made of the isolated teeth, and the petrifaction method of imbedding in Canada balsam was used to prepare the teeth and adjacent soft parts in situ. Also a series of prepared crania, some entire and some disarticulated, was made at selected ages, varying from birth to old age. The 'gold dust' method of Davison, as tested out for different ages at The Wistar Institute was used for the preparation of the former, and maceration in tap water for the latter. Schultze's clearing method was found useful in studying the early periods of calcification.

DENTITION OF ADULT ANIMAL

The dental formula of the albino rat is I ., C ^, P „, M ' .

There is only one set of teeth, and hence the dentition is monophyodont. The time of eruption of the various teeth extends over a period of 3^ weeks. The incisors are the first to appear, viz., at 8 to 10 days after bhth. The first and second molars erupt at about the 19th and 21st days respectively, and it is after this period that the young animals may be weaned and are able to maintain an independent existence, as far as food is concerned. The third molars are delayed until 2 weeks later and do not appear until about the 35th day.


Fig. 1 Cranium of a 5-month albino rat. X 2.

Fig. 2 Cranium of a 5-month albino rat, with the bony alveoli dissected away to show the entire length of the incisor teeth. X 2.

The incisors are permanently-growing (or rootless) teeth, while the molars have a definite limited period of development and acquire roots. A wide diastema separates the incisors from the molars as may be seen by reference to figm-e 1. The incisors are strongly curved and Owen ('40-'45) has described the lower incisor as being the smaller segment of a larger circle, and the upper incisor as the larger segment of a smaller circle. In the lower incisor of the albino rat this statement needs a slight modification. For while the curvature of the upper incisor is in one plane only, the lower incisor is a portion of a flattened spiral, possessing a curve in three planes. The upper incisor is a segment of a true circle (at 5 months about 210°) and in cases of overgrowth it has often been known to complete the cu'cle. In the case of the lower incisor, however, when we project it on the sagittal, frontal or coronal planes, it gives in each case a curve. It was the very evident curved projection seen on the sagittal plane to which Owen referred. Considering only this view, the lower incisor of a 5-month animal forms a segment of about four-fifths of a semicircle (140-145°).

TABLE 1 23 1 41 ! 10 15 , 5 ; 8 10

DAYS j DAYS I WEEKS WEEKS MONTHS MONTHS MONTHS

imii. ! m7n. : mm. \ mm. mm. mm. mm.

Xaso-occipital length 29 .7 32 .5 39 40 43 44 46 .5

Interzvgomatic 13.7 14 14.5 14.6 15. -> 15.1 15.5

Uppe/diastema 7.4 9.5 10 11.4 12 .3 12 .5 13

Upper incisor— total length 12.8 15 18.3 20.3 23.3 23.7 26.2

Upper incisor — extra-alveolar

length ! 5.1 i 5.5 j 7 8.4 8.7 9 9 3

Lower diastema ' 4.6 I 5 I 5.6 6 6.7 7 6.8

Lower incisor— totallength 18.1 21.7 25.5 26.4 29.4 29.9 31.3

Lower incisor — extra-alveolar

length 6.5: 7 I 10.5 11.4 11.6 12 12.4


Measurements of the incisors and skulls of animals of different ages, were made as shown in table 1.

The teeth were measm-ed along their convex surfaces by means of silk thread wet with water, and applied to the object to be measured. The thread was then cut with scissors at the end of the object, straightened on paper and measured to tenths of millimeters.

A consideration of table 1, shows in a definite way the peculiarities characteristic of the dentition, not only of the rat but of rodents in general. As is well known, these are the great development of the incisors, the wide diastema, and the consequent posterior position of the molar teeth as related to the rest of the skull. Cope ('88) wrote that he considered "the increase in the length of these teeth has been due to their continued use, as beUeved by Ryder." The effects of this increased elongation upon surrounding parts he described under several different headings, but reference will be made here only to one, viz., upon the shape of the glenoid cavity. "A peculiarity of the masticating apparatus is the lack of a postglenoid process, and the consequent freedom of the lower jaw to slide backward and forward in mastication. Appropriately to this motion, the condyle of the mandible is extended antero-posteriorly and the glenoid ca^dty is a longitudinal instead of a transverse groove."


Fig. 3 Thimble-shaped portion of the maxiUa bone, in which the basal end of the upper incisor is located. X 2.

The lower incisors are longer and more slender than the upper and extend far back in the mandible, beneath the lower molars, to near the sigmoid notch. The upper incisors are contained within the premaxilla and maxilla, the basal end occupying a thin-walled, thimble-shaped recess of bone (fig. 3) to be seen best in the disarticulated skull, and which is attached at only one limited region to the rest of the maxilla. In both upper and lower teeth, the intra-alveolar portions are longer than the extra-alveolar. When one compares the extra-alveolar lengths of the upper and lower teeth of the mature animal, the latter are always greater, and, as may be seen by reference to table 1, the difference in lengths becomes greater with increased age and size.

In both upper and lower incisors the bone is so contoured around their imbedded portions that their course may be easilj^ recognized. The basal end or foraminal apex of the lower incisor forms on the outer aspect of the mandible a marked rounded projection, directed upwards and backwards beneath the coronoid process, and sometimes extending sUghtly posteriorly beneath the sigmoid notch. Almost directly opposite this projection on the mesial aspect of the mandible is the inferior dental foramen. This projection marks the position of the growing end of the formative organs of the incisor in the adult. In the new-born anunal it is not present, nor at the end of the first month. By the age of 2| months it may be recognized, and thereafter it increases in prominence and constitutes a very evident feature of the bone. This region of the growing end of the tooth is protected b}'^ the zygomatic arch, and also by the overlying muscles.

The course of the upper incisor may also be readily followed in the prepared skull. Laterally it is covered with a thin rounded layer of bone. Mesially it forms an elevated, distinct ridge projecting markedly into the nasal fossa. In the adult the position of its basal or growing end is not so prominent as that of the lower incisor. As these incisor teeth are an indispensable part of the rodents' existence their importance demands protection from traumatism which might injure their growing pulp. Here in the upper incisors, this protection is afforded by a flange of the maxilla running parallel to the lateral wall of the cranium, as shown in figure 1 , as well as being encased in a separate thimble-shaped recess of bone (fig. 3), beneath, and separated by a narrow interval from, the outer layer of the maxilla. These details are in harmony with Cope's idea ('88) of the influence of the incisors in moulding the general topography of the rodent skull.

The diastema in the upper jaw is always longer than in the lower (fig. 1). By reference to table 1 it may be seen that in the mature animal the upper is nearly twice as long as the lower, but that in the younger stages the difference is not so great. The upper hair-covered lips are infolded into the diastema, dividing the oral cavity into an anterior and posterior compartment. This arrangement probably prevents the debris and splinters of gnawing from entering the main oral cavity.


The mandibular symphysis is formed of fibrous tissue and allows independent rotation of either ramus with its contained tooth. This lateral movement of the lower incisors appears to be under the control of the will of the animal. According to the observations of Jolyet and Chaker (75) this mobility has a definite purpose in mastication. They observed a rapid alter



Fig. 4 Cross-sections of the (a) upper and (b) lower incisor teeth of a 5-month albino rat, taken near the alveolar margins. These show the arrangement of the enamel and the dentine, and the difference in contour of the enamel in the upper and lower teeth. The mesial surface cf each tooth is towards the right side. X 15.

nate separation and approximation of the tips of the lower incisors in the act of attempting to bite into a match or other slender object offered to the animal. At the same time the upper incisors were held stationary.

Mention may be made here of a point of variation among the Rodentia in the relation of the angle of the lower jaw to the sheath of bone around the lower incisor. In the Myomorphi and Sciuromorphi the angle arises from the lower surface of the incisive sheath, while in Hystrix the angle arises entirely on the outer side.

Ryder (77) suggested a classification of rodents based on the shape of their incisors as seen in cross-section. In some genera the diameter of the teeth is less from side to side, than in the antero-posterior direction, while in others the reverse condition is found. The present form belongs to the former group, as is shown in figure 4. From the consideration of many rodents, Ryder deduced the general principle, that where the incisors are thicker in the antero-posterior direction, the gnawing habit is greatly developed.

MINUTE DESCRIPTION OF THE INCISORS

Enamel and dentine make up the hard tooth substance, enclosing the pulp. Owen, in his "Odontography" ('40-'45, p. 399) said that there existed a general investment of cementum over the whole tooth structure. J. Tomes ('50, p. 533) was not able to agree entirely but said that in most, if not in all, incisors of rodents cementum could be seen investing the posterior surface. In the rat, it is not apparent that there is any cementum at all. The enamel is usually colored with a pigment which is yellowish in the young but becomes orange-colored with age, and is usually more pronounced in the upper than in the lower incisors. At 13 days, there is as yet no color, but at 21 days a slight tinge of yellow is perceptible in the uppers, but none in the lowers. At 25 days the uppers are distinctly yellow, and the lowers have now acquired a slight color. At 38 days, these colors have intensified, the uppers having more pigment than the lowers; and in the mature animal the same relation continues, the uppers being orange-colored and the lowers yellow. The enamel is found principally on the labial side, and this accounts for the shape of the occlusal surface. For, the enamel being harder than the dentine, the latter is more easily worn away by the action of the opposing tooth, and the more resistant enamel remains as the cutting edge or point. The shape of the incisal end of the upper and lower teeth is different, being chisellike (scalpriform) in the upper, and more rounded and narrower in the lower. The incisal line is also usually different in the upper and lower teeth. In the former, it is often sHghtly concave from side to side, while in the latter it is convex (fig. 5). As is shown in figures 1 and 5 the occlusal surface is an elongated concave area on the lingual aspect of the teeth, and in the living animal extends practically to the gingival margin. Due to the difference in the curve of the upper and lower teeth, the occlusal surface of the lower teeth is always longer than that of the upper, and in the mature animal it is usually found to be nearly twice as long.



Fig. 5 Labial and lingual aspects of the extra-alveolar portions of the (a) upper and (b) lower incisors of a 5-month albino rat, showing the occlusal surfaces and incisal edges of the teeth, and the outline of the bony alveolar margins. X 2.


It follows that because these teeth are constantly growing, the occlusal surfaces are constantly being worn away. As we shall see, when discussing the growth of the teeth, the elongated temporo-mandibular articulation is important, in allowing the teeth to have either the position pictured in figure 1 or to have the opposite relation, with the lower teeth outside of the upper. Thus the very important factor in the animal's economy — the proper regulation of the length of the opposing incisors — is controlled by their own inter-action.


The pulp-chamber has the characteristic shape found in all permanently growing teeth, as is well seen, for instance, in the elephant's incisor. Its cross-area is greatest at the basal end of the tooth, and gradually diminishes anteriorly. The pulpchamber is found to extend in the tooth beyond the line of the gingivus, and very nearly to the occlusal surface. The shape


Fig. 6 Upper incisor of a o-month albino rat (X o) and cross-sections of it at different points (X 8), to show the relative cross-area of the dentine and of the pulp chamber at these regions. The dotted line indicates the position of the margin of the alveolus.


in cross-section of the pulp-chamber at different levels may be seen by reference to figure 6. The position of the filled-in pulpchamber is usually well marked on the occlusal surfaces as a line (fig. 5). In weathered specimens of rats' teeth from recent geological formations this last-formed part which fills in the pulpchamber at the end of the tooth, is usually found to be lacking, and is evidently not of the same hardness as the surrounding parts of the tooth.


MICROSCOPIC STRUCTURE OF ENAMEL AND DENTINE

Sections of enamel show two layers; an outer thin and an inner thicker layer, as noted by Owen ('40-'45, p. 399). The enamel rods run in different directions in the two layers as fully described by J. Tomes in 1850. In the inner layer the enamel rods appear to run in two sets, obliquely to one another, while in the outer layer the rods are all parallel. The outer layer has also been called the fibrous layer, and in its superficial part is situated the yellow or orange pigment which gives the color to the enamel.

Figures 7 and 8 show the arrangement of the enamel rods in the two la3^ers. In the inner or plexiform layer, when examined in cross section, the alternating series of enamel rods decussate, forming an angle varying between 70 and 90°. In longitudinal sections (fig. 26) these rods are slightly S-shaped, running outwards from the enamel-dentine surface at an angle of 50 to 54°, and inclining towards the anterior end of the tooth. Figure 8 is from a ground-section in which the enamel was broken during the process of preparation, and the broken edge shows distinctly the two sets of rods running at nearly right angles to each other. Under high magnification the rods are slightly notched.

In cross-sections of the outer fibrous layer, the rods are parallel and form in the mid-line of the tooth an angle of 90° with the outer surface. As one proceeds away from the mid-line of the tooth, whether mesially or laterally, the general tendency of the long axis of the rods as they pass from the dentine junction to the periphery, is to incline in the direction away from the mid-line of the tooth. The ameloblasts usually form an obtuse angle with the rods of the outer layer and seldom coincide in direction with them (fig. 7). In longitudinal sections the rods of the outer layer are not usually so distinctly seen as in cross-sections. In favorable longitudinal sections, however, they are seen to run quite obliquely, inclining towards the apex of the tooth, and forming an angle of 20 to 25° with the plane of the enamel-dentine junction. The pigment, as will be seen



Fig. 7 Portion of cross-section of lower incisor with enamel-organ, prepared by the petrifaction method, showing the decussation of the enamel-rods in the inner or plexiform layer and their jiarallel arrangement in the outer or fibrous layer. X 350.

Fig. 8 Small piece of enamel, showing the rods of the inner or plexiform layer running in two directions nearly at right angles to one another. X 350.


below, is confined to the outermost part of the fibrous layer. There appears to be no Nasmyth's membrane over the enamel, which means that there has been a complete transformation of the enamel matrix into enamel rods. The pigment extends about two-thirds of the total length of the upper tooth, and about one-half of the total length of the lower tooth, and hence it follows that the deposition of enamel is completed within the basal third of the upper and the basal half of the lower tooth. By examining cross-sections of the tooth at different regions (fig. 6) it would seem that the full thickness of the enamel is attained within even a smaller area at the basal end of the tooth.

The arrangement of the enamel over the labial aspect of the upper and lower teeth is shown in figure 4, drawn from crosssections of the teeth of a 5-month animal. In both teeth the sections were made just posterior to the alveolar border. In both upper and lower teeth the enamel is thickest over the labial aspect, and is continued over the adjacent mesial and lateral surfaces. In both, the enamel is continued farther on the lateral than on the mesial surfaces, and relatively farther on the lateral surface in the lower than in the upper tooth. In the upper tooth the enamel has a flattened external surface labially, while in the lower it has a rounded contour. In the upper there is a distinct labio-mesial and a labio-lateral angle, the enamel being somewhat thicker at the former. In the lower there is a labio-mesial angle, though less prominent than in the upper, and the labio-lateral angle is practically absent.

In a 5-month animal the thickness of the enamel and its constituent layers was measured in the mid-line of the teeth, as follows:

Upper Lower

Total thickness 100-110 lJO-150

Outer fibrous layer 30-40 20-30

Pigmented portion of outer fibrous layer 8-10-12 6-8

Inner plexiforni layer 70 120-125

It will be observed, however, in figure 4 that the enamel is not thickest in the mid-line of the upper tooth, but at the lateral and mesial angles. While the enamel of the upper tooth measures only 100 to llO^t in the mid-line, it measures 160 to 180/x at the region of these angles, and is, therefore, thicker here than the enamel of the lower tooth. The increased thickness at the angles is principally in the inner plexiform layer, the other layer being increased only slightly or not at all. The outer fibrous layer is distinctly thicker in the uppers and has a slightly wider band of pigment in it superficially. This, no doubt, is the basis of the more deeply pigmented appearance of the labial surface of the upper as compared with the lower teeth.

The dentine, unlike the enamel, grows continually thicker as one passes towards the outer end of the tooth. At the basal, growing, end it begins as an extremely thin layer. The thickness at different points is seen in figure 6. As the dentine increases in thickness, the pulp-chamber is in consequence proportionately reduced. At the distal end there is no longer any pulp-chamber and the site of its previous position has been filled in by the formation of a kind of secondary dentine. C. Tomes ('14) notes that "in some rodents the final closure of the axial tract takes place almost by a continuance of the formation of normal fine-tubed dentine, with very little secondary dentine of different structure, while in others there is a large area of dentine with vascular tracts in it." In the rat there is relatively little of this secondary dentine. It is laid down in irregular trabeculae, with the pulp tissue, including bloodvessels, at first within it. At the exposed surface, however, it forms a continuous granular mass with apparently no soft tissues in it (fig. 27). The ordinary dentine of the tooth is quite typical in structure, with numerous parallel dentinal tubules, each having many fine lateral branches. The tubules are slightly sinuous, and the lateral branches anastomose with those of neighboring tubules. Sometimes a tubule sends off at an acute angle a branch nearly equal in diameter to the continuation of the main tubule. This is usually in the dentine not covered by enamel. Where these large branches come off the diameter of the tubule is greater than elsewhere, measuring nearly 2^. Elsewhere the diameter varies from 1 to l.T/z. Slight differences may be seen between the tubules (a) in the dentine covered by enamel, and (b) in the dentine free from enamel. The tubules of the anterior region (a) of the dentine, covered by enamel, are more regularly parallel and have finer lateral branches than elsewhere. They also seem to taper slightly as one follows them towards the enamel. In the dentine not covered by enamel (b) the tubules are more sinuous and irregular, the irregularities marking the position of origin of the larger lateral branches. In all parts at the periphery of the dentine the tubules end in a great number of very fine anastomosing arching branches. As a consequence of the smaller diameter of the little tubules here, a narrow zone at the periphery of the dentine has usually a more homogeneous appearance than has the remainder. Towards the anterior end of the tooth, in the vicinity of the pulp-chamber, are vascular channels in the form of loops within the dentine. The tubules must necessarily take a curved course around these vascular channels, and thus the position of the vessels is more easily seen.

In the dentinal tubules Mummery ('12), Fritsch ('14) and others have demonstrated not only the processes of the odontoblasts, but also fine non-medullated nerve fibers. As to why the exposed dentine on the lingual aspect of the teeth is insensitive, there are no definite observations to decide. A contributing factor may be the compression which the pulp tissues undergo at the anterior end of the pulp-chamber, leading to the physiological cutting off of the nerve supply to the dentinal tubules.

DEVELOPMENT OF THE INCISORS

The times of the early stages of development of the incisors were seen as follows:

14-day fetus — slight thickening of oral epithelium

15-day fetus — distinct thickening and growth inwards of oral epithelium

16-day fetus — dental ledge and beginning of flask-shaped enamel organ

17-day fetus — dental papilla with crescentic enamel organ capping it

19-day fetus — both ameloblasts and odontoblasts differentiated

new-born animal — enamel and dentine formation begun

8 to 10 days — eruption of the tooth

Throughout life growth continues, and in the adult animal is on the average 2.2 mm. per week in the upper and 2.8 mm. per week in the lower incisor.


The structures to be described here, as in the development of the crowns of all teeth, are the enamel-organ with the ameloblasts, and the dental papilla (which becomes the pulp-substance) with the odontoblasts. There are two factors, however, which alter the usual history of the development of these structures, and especially of the enamel-organ. First, in permanently growing teeth of which these are examples, all these structures continue functional throughout life, so that the enamelorgan is also a persistent structure. The other factor and one correlated to some extent with the first, is that the enamel is formed on one side of the tooth only, and here only does the enamel-organ develop to its most highly differentiated functional condition.

The history of the development and growth of the tooth may be conveniently considered in two stages (1) pre-eruptive, and (2) post-eruptive. The pre-eruptive stage extends from the 14th or 15th day of fetal life until eruption of the tooth takes place between the 8th and 10th post-natal days. Until near the time of birth there is no formation of enamel and dentine, but from birth onwards these substances are laid down rapidly, so that at eruption, the teeth have their characteristic elongated narrow form. This pre-eruptive stage is characterized by the rapid elongation of the tooth-forming organs, and by the teeth attaining very similar relations to the other structures of the jaw which the imbedded portions of the erupted teeth possess. Thus, the anlage of the lower incisor appears under the oral epithelium in the anterior region of the mandible, and grows continually backwards, until its growing end reaches the region beneath the developing molars. At this time the growing end presumably reaches a region which, by reason of its increasing calcification, offers resistance to further progress. The result of the ever-continuing mitotic division and cell growth at the basal end, is the pushing of the whole tooth and its formative organs, in the opposite direction, and the consequent eruption of the tooth. During the latter half of this pre-eruptive stage, the anterior tip of the developing tooth structure is immediately beneath the oral epithelium, and remains at a fixed point, while the posterior end is continually growing backwards and changing its relations. At eruption this condition changes, and the posterior extremity becomes practically a fixed point from which the whole tooth moves forward. That there is, however, a gradual change in the position of the posterior end of the tooth may be seen in figure 9. As the jaw grows, the entire tooth not only grows to keep the same general relative position, to surrounding structures, but it may be seen that the growing end progresses gradually posteriorly. In the full-grown animal this end occupies a distinct outpushing of the bone (fig. 1).

During the post-eruptive period, which continues throughout life, this outward growth is continued at a regular rate, and at the same rate the outer end has to be worn away. This wearing-away process would soon result in the pulp becoming exposed were not the occlusal end of the pulp-chamber also being continually filled in. As may be seen from figure 6 the dentine continues to increase in thickness until near the end of the tooth. This means that the odontoblasts continue their regular functional activity until near the end of the tooth. However, the final fiUing-in of the pulp-chamber to form a continuous hard occlusal surface is accomplished by the deposition of a hard matrix between the pulp elements and by the probable calcification of the latter. The result is, that as the tooth is worn away, the soft pulp never becomes exposed. Although the pulp reaches very near to the end of the tooth, a hard substance always fills in the end of the pulp-chamber, and so protects the pulp beneath.

DETAILED DESCRIPTION OF DEVELOPMENT UP TO THE TIME OF ERUPTION

The anlage of the enamel-organ of each incisor arises as an epithelial ingrowth, distinct and separate from that for the molars.

In frontal sections of the 14-day fetus, there are slight diffuse thickenings of the oral epithelium in the four positions, which represent the sites of the future tooth-formations.


Fig. 9 Series of mandibles of the albino rat at ages \arying from birth to ten months, viewed from the lateral aspect. These show the changing relation of the basal end of the incisor to the rest of the mandible during this period.


At 15 days these thickenings have become more definite, and in the lower jaws especially have begun to push into the underlying mesenchyme, and may be described as the dental ledges or dental laminae.

At 16 days the ingrowths have continued to increase as broad masses of cells, pushing deeper into the underlying mesenchyme, and in the lower jaws the enamel organs may be distinguished as expanded structures, each connected by a slightly narrower mass of cells with the oral epithelium. In the upper jaws the differentiation of the enamel-organs from the remainder of the epithelial ingrowth is not so marked.

At 17 daj^s (fig. 10) the dental papillae are beginning, and the enamel-organs in both upper and lower jaws have a crescentic outline. In the enamel-organs there is already an indication of the differentiation into three layers. As seen in sagittal sections, the papillae develop on the posterior side of the enamelorgans, thus foreshadowing the axis of growth of the toothforming organs in the antero-posterior direction.

Eighteen-day fetus

Series of frontal sections of 18-day lower jaws, show that the enamel-organs are growing over the dental papillae more rapidly on the labial and lingual surfaces than elsewhere, and extend more posteriorly on these surfaces. There are thus two projections of the posterior margin of each enamel-organ as already noted by Meyerheim ('98). The labial process is broad and thin and extends more posteriorly than the lingual process, which is somewhat narrower and thicker. One may her-e remark, therefore, an early difference between the labial and lingual part of the enamel-organ. Other differences which will soon appear have not yet developed. Thus, the inner layer of the enamel-organ is made up of columnar elements which are still similar in all parts, both labially and lingually. In the dental papilla no columnar odontoblasts are yet seen.

The enamel-organ remains connected with the surface epithelium by a broad band of epithelial cells. In the lower jaw, immediately laterad to the line of j miction of this stalk of the enamel-organ to the surface epitheliimi, the lip furrow is a depression, the plane of which is continued into the underlying mesenchyme by an ingrowth of surface epithelium several layers of cells in thickness. It is by the subsequent splitting of this epithelial layer into two, that the separation of the lip will be effected.

Nineteen-day fetus

At 19 days, the enamel-organ in the upper jaw (fig. 11) is crescentic in outline in sagittal section, and in the lower jaw (fig. 12) is more elongated and conical in shape. At this age odontoblasts are first seen as columnar cells on the labial aspect of the mesodermal papilla. Three layers are recognizable in the enamel-organ, but the middle layer (enamel pulp), as has been also described by Sachse ('94) for the mouse, is extremely thin, and therefore is not present in the great quantity tj^pically seen in the development of rooted teeth. It appears as a more lightly stained zone between the inner and outer layers, and is thickest at the basal end of the enamel-organ as shown in figures 11 and 12. It averages about 20^i in thickness and is made up of stellate cells loosely arranged. Already there is an indication of a compact arrangement of the two or three rows of cells next the inner layer of the enamel-organ, which will result in the so-called stratum intermedium seen at later ages. This middle layer is also slightly more abundant at the anterior end in the region where the enamel-organ is continuous with the stalk which joins it to the oral epithelium.

At this age the enamel-organs in the lower jaws have a greater total length than those in the upper, and especially in the lower

Fig. 10 Longitudinal section of upper jaw of 17-day fetus, showing tooth anlage of incisor, with the enamel-organ longer labially than lingually when measured from the point of junction of the stalk of the enamel-organ. The dental papilla is on the posterior aspect of the enamel-organ. X 70.

Fig. 11 Longitudinal section of upper incisor anlage of 19-day fetus of albino rat, showing the crescentic outline of the enamel-organ, its greater length labially than lingually, and its thickened basal margin. X 70.

Fig. 12 Longitudinal section of lower incisor anlage of 19-day fetus of albino rat, showing the conical outline of the enamel-organ and its greater length than in the upper jaw^ at the same age. X 70.


jaws distinct differences may be made out between the oral and labial sides of the tooth-forming organs. These differences are:

(1) The enamel-organ is longer labially than on the oral side.

(2) The staining of the inner layer of the enamel-organ on the labial side is more intense, and here the cells are slightly longer than in other parts of the enamel-organ, measuring 24/x in length and assuming the typical appearance of ameloblasts. Measurements show the similar cells on the lingual side to be about 20m in length. It is also to be noted at this age that the site of the most advanced cells which are differentiating to become ameloblasts is not at the apex of the enamel-organ, as is the case in the development of rooted teeth. For as one follows the cells of the labial side of the enamel-organ from the apex towards the base, while at the apex the cells are columnar they become longer as one goes posteriorly, and then towards the base of the enamel-organ diminish again. So that the site of most advanced differentiation here is a short distance posterior to the apex on the labial side. This is true also of the developing odontoblasts which are longest opposite the tallest ameloblasts.

(3) The outer layer of the enamel-organ on the labial side is becoming slightly wavy in outline, and this denotes the beginning of the papillae, which form such a characteristic part of the mature functional enamel-organ (fig. 26).

(4) The odontoblasts are seen only on the labial side of the dental papilla.

Mitoses are abundant in all parts of the developing tissues.

Twenty-one-day fetus

Thus the anlage of the rodent incisor begins in the usual way, and for a short time continues along the typical mammalian course. From 19 days onward, however, the differences which have already begun, become more distinct and definite. At 21 days (end of gestation) the enamel-organ has become more definitely differentiated into a labial and a lingual region. Of the three constituent layers, the inner especially is strikingly different in these two parts. On the labial side at the anterior end, the organ has advanced to the condition where functional activity is beginning, while the oral side has remained stationary, or has actually retrogressed. Thus in the innermost layer on the labial side of the lower incisor, where the ameloblasts have begun to form enamel, these cells measure 30 to 34ju in length, while the non-functional cells on the oral side of the innermost layer are low columnar or cubical in shape and measure only 12 /j. in length (fig. 13). Comparison of these measurements with those at 19 days shows that the cells of the inner layer of the labial side of the enamel-organ have advanced in length from 24/i to 30 : or 34m, while the cells on the lingual side have decreased from 20 to 12ju. There is, therefore, a primary tendency for the cells of the inner layer to develop equally in all parts, but very soon the nonenamel-forming cells of the lingual side begin to retrogress, while the functional cells of the labial side continue to grow. This constitutes another point of contrast with the development of the crowns of rooted teeth. For here in the 21 -day fetus, when the enamel and dentine formation has just begun, these substances are thickest, not over the apex of the tooth-forming organs, as in the usual method, but at a short distance posterior to this point, on the labial surface. Thus, not only are the odontoblasts and the ameloblasts first differentiated on the labial side, posterior to the apex, but at this region enamel and dentine formation is also evidently first begun.

Over the apex of the dental papilla there is apparently a very thin outline of dentine deposited, but within this, in the tissues of the apex of the dental papilla, there is also beginning an irregular formation of a hard matrix. Between the cells of the pulp, trabeculae of a bone-like material are appearing. As development proceeds this substance increases until the final result is, as seen in figure 20, that the primary apex of the tooth has a bone-like structure, consisting of cells imbedded in lacunae within a dense matrix. This has been called by Tomes ('04) 'osteo-dentine.'

A similar difference between the labial and oral sides is noted in the cells on the margin of the dental papilla, which are becoming odontoblasts. In the basal half of the papilla (fig. 14), odontoblasts occur only on the labial side opposite the tall ameloblasts, the peripheral cells of the other sides being still irregular or cuboidal in shape. Farther forwards the odontoblasts are found also on the lateral and mesial surfaces of the dental papilla, but not on the lingual. In the apical one-fourth of the dental papilla odontoblasts occur all round, measuring 20 to 24/x in length, and are engaged in the formation of dentine (fig. 13). The dentine is thickest on the labial side.

In the region where enamel and dentine formation has begun no mitoses were seen in the formative ameloblastic and odontoblastic cells, but posteriorly, where the deposition of enamel and dentine has not yet commenced, many mitoses occur in the layers of developing ameloblasts and odontoblasts, as well as elsewhere. The nearer one approaches the basal margin of the enamel-organ the more numerous are the mitoses and it is apparent that it is principally in this region that growth by addition of new cells is taking place.

One day old

At the end of the first day of post-natal life, there has been great progress in the enamel and dentine formation, and the narrow, pointed outline of the tooth has been already laid down. In the upper jaw the teeth measure about 2.3 mm. in length and in the lower jaw about 3 mm. Definite changes in its relation to the oral epithelium have occurred also at the anterior end of the tooth. The original epithelial stalk connecting the enamelorgan with the oral epithelium has increased in size and the end

Fig. 13 Cross-section of developing lower incisor of 21-day fetus of albino rat, nearer the anterior extremity of the tooth than figure 14. Shows the greater thickness of the labial side of the enamel organ, as compared with that of the other sides, and shows odontoblasts around the entire periphery of the pulp. Enamel and dentine formation has begun. X 110.

Fig. 14 Cross-section of developing lower incisor of 21-day fetus of albino rat, posterior to the region shown in figure 13. No enamel or dentine yet formed at this point. Odontoblasts highest on the labial aspect, decreasing in height laterally but not yet differentiated as columnar elements on the lingual side. Enamel-organ thickest on the labial side. X 110.


of the tooth has apparently advanced somewhat into it. So this thick stratified layer of epithelium forms a close-fitting investment about the tooth apex, and is continuous posteriorly with the remainder of the enamel-organ. But in this epithelial cap there are no ameloblasts and consequently there can be no enamel over the osteodentine which forms the tip of the primitive tooth. This substance forming the tip of the unerupted tooth is a form of secondary dentine with its cells located in the lacunae of the matrix. Passing backwards, one comes to the ordinary dentine containing the vascular pulp with odontoblasts situated at the periphery of the pulp-chamber in a regular manner.

As the odontoblasts were first differentiated labially, and dentine formation began there before on the other side, the dentine of the labial side is thicker than on the lingual side. Thus at a point about the middle of the entire tooth structure, the dentine measured 54/^ labially and 20/x orally (fig. 15). Between the odontoblasts are numerous fine capillary loops. At this region may also be seen the characteristic structure of the enamel-organ (fig. 15). This extends all around the tooth, but is much thicker on the labial side than elsewhere. This difference in thickness is seen in all the constituent layers. In the inner layer, the tall ameloblasts of the labial surface measure iOfj., while the similarly situated cells on the other surfaces are cubical and measure only 10^. Comparing these with the previous stage described, it is seen that the cells on the labial surface have increased and those on the other surfaces have decreased. Of the middle layer on the labial side, the stratum intermedium is a distinct line of cuboidal cells, one to two rows in thickness, lying behind the ameloblasts. The other constituent — the original enamel pulp — is small in amount and is principally within the elevations of the outer layer, which form the beginning of the epithelial papillae. The cells of the outer layer, somewhat irregular in shape with round nuclei, are in a single row. Between the developing papillae (called by Sachse StLitzpapillen) are numerous capillary blood-vessels. On the other surfaces, practically nothing remains of the middle layer, although the outer layer still persists as a layer of flattened cells. Thus lingually the enamel-organ is represented by only two rows of cells — one representing the inner, the other the outer layer of the enamel-organ.



Fig. 15 Cross-section of developing lower incisor of 1-day albino rat, showing the great development of the ameloblasts on the labial side, and the thinness of the enamel-organ elsewhere. The space between the ameloblasts and the dentine is an artefect, and was formerly partly filled by the enamel, which has disappeared in the pi'ocess of decalcification. In the layer of odontoblasts are seen the nuclei of the endothelial cells of the walls of capillaries. X 110.


Two days old

Figure 16 shows a longitudinal section of a 2-day upper incisor. The epithelial enamel-organ is continuous over the whole tooth, but only shows its specialized functioning structure on the labial side. On the lingual side it is still intact and consists only of two rows of cuboidal or flattened epithelial cells. On the labial side, along the region where enamel has been formed (fig. 17) the


Fig. 16 Longitudinal section of upper incisor of 2-day albino rat showing the enamel-organ continuous over the labial surface and terminating posteriorly in the thickened margin. X 18.

Fig. 17 Small portion of preceding figure more highly magnified, to show the structure of the enamel-organ and the odontoblasts, a, outermost layer of enamel-organ and epithelial papillae; b, enamel pulp; c, stratum intermedium; d, layer of ameloblasts; e, layer of dentine; f, layer of odontoblasts. X 175.


ameloblasts measure about 40^. These are backed by two rows of darkly staining flattened cells composing the stratum intermedium. Next to these is the looser arrangement of stellate cells, comparable to the enamel pulp of ordinary tooth development, but with much smaller spaces between the cells. This tissue is covered by the layer of cells constituting the outer layer of the enamel-organ, and the two together constitute the epithelial papillae. At the summit of each of these papillae the cells of the outer layer are grouped in a more compact manner. With higher magnification processes can be seen running from the ameloblasts into the developing enamel — the so-called enamel processes of Tomes.

At the basal formative part of the enamel-organ the three original layers show distinctly. At the thickened basal margin of the enamel-organ, around its entire circumference, is a mass of rapidly dividing cells. As seen in figure 16 this thickened margin is more noticeable on the labial side. Its peripheral zone as seen in longitudinal sections is deeply staining and its cells, more or less columnar in shape, are compacted together. The interior, of more lightly stained appearance, is composed of oval or elongated cells, irregularly parallel, but more loosely arranged than the cells of the periphery. This region constitutes the site of origin of the cells of the ever-forming enamelorgan. From this pass forward the outer and inner layers, and between them, in larger quantity than is found more anteriorly, the tissue of the middle layer. This for a short distance is all enamel pulp and shows no differentiated layer of stratum intermedium.

In this formative region on the labial side, the inner layer consists of columnar cells, the future ameloblasts, in which many mitoses are seen. While the outer layer consists of cells which are columnar near the margin, a short distance anterior to this (150/x) they change shape, first to cubical, then to flattened cuboidal. Between the two layers are cells representing the enamel pulp. At this region there are no papillae, although numerous blood-vessels are seen alongside the outer layer of the enamel-organ. About 0.5 nmi. from the basal end this outer layer of the enamel-organ becomes sinuous, and low papillae are being formed.

On the lingual side, the structure of the basal end of the enamelorgan is similar, but somewhat simpler. Thus there are three layers at and near the basal margin, but soon, proceeding anteriorly, these become reduced to two by the disappearance of the middle layer. The lingual side then consists of two rows of cuboidal or flattened cells, one constituting the outer and the other the inner layer of the enamel-organ in this situation.

The dental papilla is made up of closely packed small stellate cells, with rounded nuclei. The mesenchymal cells which lie against the basal margin of the enamel-organ are rounded or irregular in shape, but within a short distance (0.5 mm.) anterior to this margin, the peripheral cells become first cubical and then columnar in shape. Where they are beginning to form dentine they measure 30m in length. From the odontoblasts processes enter the dentinal tubules of the dentine. The outer surfaces of the odontoblasts from which these processes arise show a distinct cuticular margin. Between the odontoblasts at short intervals capillaries form loops around the cells. These are evidently for the purpose of insuring an ample blood supply to these functionally active cells.

Four days old

By 4 days of age there has been continued growth, and deposition of enamel and dentine. The upper incisor measures 3.6 mm. in length and the lower 5 mm. The position of the apex of the tooth is in close relation to the oral epithelium (fig. 18). A thickened mass of epithelium, partly a derivative of the original stalk of the enamel-organ, and partly an ingrowth from the

Fig. 18 Longitudinal section of upper incisor of 4-day albino rat, showing the increased curvature of the outline of the tooth and the relation of the apex of the tooth to the ingrowth of the oral epithelium. X 16.

Fig. 19 Longitudinal section through basal end of labial side of enamelorgan of 4-day albino rat showing the region of the thickened margin, a, margin composed of mass of proliferating cells; b, region where three layers are seen; c, region where stratum intermedium becomes differentiated from rest of middle layer. Anterior to the region of this figure the epithelial papillae appear and the ameloblasts begin to form enamel. X 80.


surface epithelium surrounding the tip of the tooth, is a preparation for the eruption of the tooth, and will serve as a resistant ling of tissue through which the tooth will be pushed at eruption. It may be looked upon as a protective device, to prevent adjacent tissues from being carried out by the erupting tooth.

The typical enamel-organ seen on the labial side does not cover the apex, for the tall columnar cells give place here, first to cubical and then to flat squamous epithelial cells, which form but a part of the thick mass of stratified epithelium, constituting the epithelial sheath over the end of the dentine. The other layers of the functioning enamel-organ also lose their identity at the region where the ameloblasts cease to have their characteristic elongated form. As maintained by von Brunn ('87) and Sachse ('94), there is no enamel apparent over the dentine at the apex of the tooth.

The cells representing the enamel-organ on the lingual side can be traced forward for a short distance as a two-layered stratum. These cells are flattened, with oval nuclei. Beyond this point only a single regular row of cells is apparent, and about half way along the length of the tooth-structure, even this ceases to be definite, and apparently here the mesenchymal cells of the peridental tissues have grown between and scattered these cells. As a result of this activity of the mesenchymal cells in this region, the enamel-organ now ceases to exist as a complete conical investment of the tooth. Approaching the apex of the tooth on the lingual side, one finds the prolongation of the epithelial sheath as a thin layer of flattened cells which thickens as it passes forwards into the epithelial sheath.

The basal formative end of the enamel-organ consists of a thickened band of tissue, as shown in figure 18, and under higher magnification in figure 19. This end is thicker on the labial side than elsewhere and it curves inwards, as seen in longitudinal sections, thus considerably diminishing the diameter of the entrance to the pulp-chamber. The extremity of this mass of tissue (fig. 19, a), constitutes a common origin for the several layers of the enamel-organ and contains many dividing cells. A short distance (0.1 to 0.2 mm.) from the extremity (fig. 19, b)


the cells form three layers, inner, middle and outer. The inner and outer layers, made up of columnar elements, stain more darkly than the middle layer, and the inner is thicker than the outer. The middle layer consists of elongated cells with oval nuclei, arranged for the most part with theii* long axes parallel to the surface of the enamel-organ. Frequent mitoses are also to be seen here, especially in the inner layer.

In the region about 0.6 mm. anterior to this (fig. 19, c), where enamel formation has not yet begun, the innermost layer shows a single row of distinct tall columnar cells, the ameloblasts. The middle layer now shows two subdivisions (a) two or three layers of compacted flattened cells lying against the ameloblasts, and composing the stratum intermedium, and (b) a somewhat thicker stratum, lightly staining, of more loosely arranged cells, constituting the enamel pulp. The outermost layer is a single row of cubical cells, which form a straight continuous surface for the enamel-organ. Beyond this layer and in contact with it are numerous small blood-vessels. Passing still farther forwards, the outermost layer becomes more sinuous in outline, and blood-vessels occupy the depressions between the elevations. This arrangement shows the beginning formation of the typical epithelial papillae.

Seven days old

At 7 days the tip of the tooth is in the oral epithelium (fig. 20), and ready for eruption, being separated from the outside by only a thin layer of superficial cornified epithelium. The epithelial tissues immediately about the apex of the tooth show the appearance of pressure atrophy. The cell boundaries are more indistinct than elsewhere, the tissue takes the acid stain deeply, and there is increased granularity — evidently degenerative effects due to the pressure of the advancing tooth.

In the upper jaw, the basal end of the tooth in its backward growth has reached the region of the maxilla, into which it continues to grow, pushing before it a little pocket of thin bone. The average length of the upper teeth at this age is 5 mm., and of the lower teeth, 7 to 8 mm. Their pointed apices, and their comparatively slight curvature are shown in figure 24.



Fig. 20 Osteodentine of apex of tooth of 7-day albino rat imbedded in the surface epithelium, showing cells in the lacunae in the matrix. X 175.

ERUPTION OF TEETH

Eight to ten days

During the process of eruption (fig. 21), the tooth and its formative organs gradually move forward as a whole, and the apex of the dentine forming the anterior end of the tooth pierces the surface epithelium. This procedure is accompanied by new changes in the tooth-forming organs. For while the same process of cell-division continues at the basal end of the dental papilla and enamel-organ, these structures are subjected to

Fig. 21 Longitudinal section of the upper tooth of an 8-day albino rat, showing the apex of the tooth piercing the surface epithelium. X 10.

Fig. 22 Longitudinal section of the upper tooth of a 12-day albino rat, showing the increased size and curvature of the tooth, the basal end directed more towards the palatal surface and the progression of the apex of the tooth through the epithelium. X 10.

Fig. 23 Longitudinal section of the upper incisor of a 26-day albino rat, showing the well-established occlusal surface, the approximation of the basal end towards the palatal surface, abundant blood-vessels in the pulp, and the position of the granular osteodentine filling in apex of the pulp chamber. X 10.

new conditions at the erupting end of the tooth. Before detailing these changes, it may be advisable to state, in a general way, the changing circumstances attendant upon eruption. Up to this time the anterior end of the tooth has been nearly stationary, but there has been continued growth backward of the posterior extremity. At this time the rate of progression foi-ward is greatly increased, and the rate of progression backward much reduced. As suggested before, the process of eruption may depend largely upon the fact of increasing calcification in the bones, rendering them more resistant to the backward growth of the developing tooth. Whatever may be the causal factors, from now on the tooth continues to grow out at a regular rate, through the development of new cells at the basal end of the formative organs, these cells in turn giving rise to the hard parts of the tooth. Within a few days after eruption, the use of the tooth involves the process of attrition by which, in spite of the regular rate of growth, the exposed length is kept nearly constant for any age.

It is generally agreed that, by reason of the protoplasmic processes which extend into enamel and dentine from ameloblasts and odontoblasts respectively, these cells must be carried along with the tooth as it moves. Thus, as there is constantly a regeneration of these cells at the basal end of the tooth, there must be an opposite process of some nature by which these cells are eventually lost at the apical end, when carried thither by the outward progress of the tooth. First we may follow the history of the ameloblasts in this locality. Before eruption, the enamel-organ is continuous with the stratified epithelium forming the sheath around the gingival margin, and this relation continues at and after eruption. As the tooth moves forward during eruption the ameloblasts must move along with it and, when those at the anterior end approach the gingival margin, they must either be held there, or be carried out on the enamel until detached. On examining longitudinal sections at 12 days (fig. 22) it is seen that the ameloblasts, as they approach the gingival margin, become shorter and shorter, until, beneath the thickened sheath of epithelium forming the gingival margin they acquire a flattened form. As a continuation of these flattened cells next the tooth is seen, extending out into the space between the erupted tip of the tooth and the epithelial gingival margin, a thin layer of tissue, which must be looked upon as the portion of the enamelorgan which has been carried out during eruption. At later stages this same appearance occurs — a thin layer of flattened cells continuous with the enamel-organ lying in the space between the tooth and the epithelium of the gingival border. It may be that some of the cells are added to the epithelium of the gingival margin, but the majority appear to be continually carried out, and eventually detached.

The mesenchymal tissues of the pulp at the anterior end are little afl'ected by the mere act of eruption and not until some days later when attrition begins, do we see definite changes. At eruption the anterior conical extremity of the tooth is formed of osteodentine, containing within its matrix the remains of scattered cells and blood-vessels. Immediately posterior to this begins the true fine-tubed dentine with a central pulp-chamber. The cells at the anterior end of the pulp-chamber are irregularly arranged, but following backwards one soon sees the odontoblasts in parallel arrangement at the periphery of the chamber. At 10 days, when the apex of the tooth has pierced the epithelium and is easily seen from the outside, the measurements of the upper and lower teeth are 7 and 11 mm, respectively. At 12 days, they have increased to 7.5 and 11.8 mm.

CHANGES IN APEX OF TOOTH BY USE

Already at 12 days, when one examines the exposed ends of the teeth, they show little pits, which have been caused by the pressure of the opposing teeth. At 14 days, the ends are flattened, and at 16 days, because of the increased obliquity of this flattened surface due to the wearing away of the lingual side of the dentine, they are acquiring a cutting edge. The length of these occlusal surfaces continues to increase so that by 19 or 21 days (fig. 24), they have nearly the appearance typically seen in the fully developed teeth. The osteodentine of the tip of the tooth is softer than is true dentine, for when the young tooth is dried this end shrivels and darkens in color. This cap of osteodentine on the end of the tooth may be useful, as suggested by Sachse ('94), because of its softness, in allowing the early formation of the functional occlusal surface. When this soft substance begins to wear away the tissues of the pulp would soon become exposed were there not a provision for the filling in of the apex of the pulp-chamber. This is effected by the formation of an irregular hard matrix, which may also be called osteodentine, within the extremity of the pulp-chamber. As



Fig. 24 Isolated upper and lower incisors of several ages of young albino rats. The pointed shape just before eruption is seen at 7 days. At 12 days, there is yet very slight change in the apices. At 21 days the occlusal surfaces are concave, and at 26 days they have nearly the typical mature appearance. X 2.

the outer surface of the tooth wears away, this formation is constantly taking place a short distance from the occlusal -surface.

Thus in examining a longitudinal section of the tooth at an age when the process of attrition has begun, and the typical occlusal surface has been formed (e.g., 26 days, fig. 23), we find this form of secondary dentine or osteodentine filling in the distal extremity of the pulp-chamber. As one approaches the anterior end of the pulp-chamber, the pulp becomes more and more restricted and the blood-vessels appear congested. Proceeding distally, the irregular matrix formation is seen between the cells and blood-vessels and finally near the occlusal worn surface is a granular mass of osteodentine with no circulating blood in it, but spaces are still seen containing the remains of the pulp elements. Here the living elements have disappeared, but by staining (e.g., with acid fuchsin), the remains of these may be made out in less calcified spots in the matrix. Evidently the odontoblasts and other tissues of the pulp which move with the dentine, become more and more compressed at the narrowing apex of the pulp-chamber, and finally there is this irregular deposit of secondary dentine between them, which serves to obliterate the pulp-chamber. As the tooth moves out, this process is constantly going on, just in advance of the occlusal surface, and keeps pace with the process of attrition.

It is interesting to note the rate at which the teeth are increased in length during their formative period and prior to attrition.

Up per Lower mm. mm.

1 day old 2.3 3

4 days old 3.6 5

7 days old 5 7-8

10 days old 7 11

Average growth 0.52 nun. and 0.88 mm per day

As will be seen later, this exceeds the rate at which the mature tooth continues to grow out.

DESCRIPTION OF MATURE TOOTH AND TOOTH-FORMING ORGANS, IN FIVE-MONTH ANIMAL

In the mature tooth, the general relations are shown in figure 25, made from a photograph of a decalcified section of the upper tooth of a 5-month animal. The regular curved outline is seen, with the greater proportion of the length imbedded within the jaw, and only a small part projecting. The formative end lies witliin an investment of bone belonging to the maxilla. At this end the dentine is very thin and the pulp greatest in amount. As one goes forward, the dentine increases regularly in thickness while the pulp-chamber becomes smaller and smaller. The vacuolated appearance at the anterior end of the chamber is due to shrinkage of the pulp tissue during fixation. The enamel has been lost in the process of decalcification except over the basal third. Numerous blood-vessels are seen within the pulp.


Fig. 25 Longitudinal section of upper incisor of a 5-month albino rat. The letter a shows where the next illustration (fig. 26) is taken. X 6.

Fig. 26 Small portion of the preceding, more highly magnified, to show the enamel-organ and the enamel, and the odontoblasts and dentine. X 135.

The enamel-organ is continuous over the convex labial surface of the imbedded portion of the tooth but is restricted to the most posterior region of the other surfaces, extending only 1 mm. forward from the basal margin. The enamel-organ differs in its structure in three regions of the labial side, and may be described separately in these three parts: (1) at the basal formative end, (2) near the gingival margin and (3) in the long intervening region. In (1) the enamel-organ is being constantly regenerated by the addition and growth of new cells. In (2) the enamel-organ is undergoing a retrograde process, while (3) represents the region where the enamel-organ is at its highest functional development, although its activity in increasing the thickness of the enamel is restricted, as noted before, to the basal third or less in the upper and to the basal half in the lower tooth.

Considering first the region (3), as shown in figure 26, the enamel-organ is conspicuous by reason of its tall ameloblasts and the high, narrow papillae. The enamel-organ is described in three layers — inner, middle and outer. The inner layer consists of the ameloblasts, which measure about 40 /x in height, with nuclei situated towards the outer end of the cells. The middle is composed of two strata (a) stratum intermedium, and (b) enamel pulp. The stratum intermedium is formed of 1 or 2 rows of fairly regular cuboida cells resting upon the outer ends of the ameloblasts, but the enamel pulp is not now recognizable as a distinct layer and exists principally within the papillae. The outer layer of the enamel-organ consisted originally of a single layer of cells, but these are no longer regular in form or arrangement. Together with the remains of the enamel pulp, the outer layer forms the papillary elevations, 60 to 70 M in height. These papillae are surrounded by an abundant capillary blood supply for the nourishment of the cells engaged in the formation of the enamel, and the purpose of the elevations is apparently to increase the surface area through which absorption may take place from the blood-stream.

The enamel is in two layers (fig. 26), the rods while traversing the inner layer being very distinct, and inclining towards the apex of the tooth at an angle of from 50 to 54° with the dentine surface. The continuations of these rods in the outer layer are not so distinctly seen, but the inclination, as made out in thin sections, is still greater towards the apex, forming an angle of from 20 to 25° with the plane of the surface of the dentine. As noted before in the study of enamel, the rods in the inner layer, when observed in cross-sections of the tooth, decussate at an angle of from 70 to 90°, but when they reach the outer layer all run parallel. The fact that the rods run in these various directions seems incontrovertible, but in the light of our present knowledge of enamel formation it is difficult to understand how this condition is arrived at. If each ameloblast is responsible for an enamel-rod, then it follows that because the alternate layers of rods are oblique to one another, the ameloblasts responsible for these series of rods must have changed their relative positions during the process of formation of these rods. No such phenomenon has been observed, or even suggested. The other possibility is that the matrix of the rods is formed in a regular manner, but that afterwards, before calcification is complete, the rods become re-arranged owing to pressure strains.

The plane of cUrection of the rods is suggestive of the importance of the enamel-organ in the persistent growth. For always the general plane of the rods, as they leave the enameldentine junction, is towards the outer end of the tooth, as if the ameloblasts, while engaged in enamel-formation were always held back by the enamel, in which their processes were imbedded.

The basal formative end of the enamel-organ (region 1) in the adult animal corresponds very closely in structure with what has already been described for earlier ages, e.g., 4 days. This is the region where the enamel-organ is constantly being renewed, and it retains the same embryonal character at all stages of development.

At the anterior end where the enamel-organ is continuous with the surface epithelium (region 2), a gradual transition occurs between the typical enamel-organ and the stratified squamous epithelium (fig, 27). As one follows the innermost layer of the enamel-organ forward, the cells become shorter, until they are cubical and finally flattened in shape. Here the other layers also lose their regular arrangement, and form, with the preceding, a thin layer of stratified cells. This layer can be followed directly into contact with the epithelium of the gingivus.



Fig. 27 Longitudinal section of the apex of the tooth of a 5-month albino rat, showing at a the position of the outward prolongation of the remains of the enamel-organ, and at h the more granular osteodentine filling in the apex of the pulp chamber. X 10.


The cells, however, do not lose their identity in the surface epithelium but remain separate as a thin layer lying against the enamel (fig. 27, a). This thin layer of epithelium, therefore, represents the ultimate fate of the enamel-organ after it has completed its functional activity. It is being continually pushed out and its most anterior part must be continually being lost.


RATE OF GROWTH OF THE INCISOR TEETH

Two methods were used for determining the rate of growth of the incisor teeth (a) cutting off one or more teeth at the gingival margin and (b) making marks upon the enamel. The results here given are based on the latter method, as giving more nearlj^ the normal rate of growth. By means of a dental engine, the animal always having been anesthetized, a fine transverse notch was made on the enamel of the incisors a short distance from the gingival margin. The interval between this mark and the tip of the tooth was then measured. At the end of about a week the distance between these two limits was again taken, and the difference between the two measurements showed the amount of wearing away. Two series of experiments were made by this method upon adult animals. In the first series six animals were used and in the second four animals, and measurements were made for several consecutive weeks. The longest period that one individual was studied was six weeks.

The two series gave very similar results. The lower tooth was always found to grow more rapidly than the upper. The upper tooth averaged 0.31 to 0.32 mm. per day, or 2.2 mm. per week, while the lower tooth averaged 0.4 mm. per day, or 2.8 mm. per week. No doubt there are many variations of the rate of growth under different circumstances, so that these figures must be taken as representing the average rate under one particular set of conditions. The food of these animals was the mixed diet now in use in the rat colony of The Wistar Institute. A short series of trials was made with animals kept in a large glass jar and given only soft food. In these animals the rate of wearing away corresponded very closely to that seen in animals which have also hard food and have the opportunity of gnawing. In these, therefore, the interaction of the opposing teeth must have caused the attrition.

For assistance with these experiments we wish to thank Dr. Stotsenburg, who greatly aided us in carrying out our observations.


It is interesting to compare these results with those obtained on the rabbit — the only other rodent which has been carefully studied in respect to the growth of its incisors. MacGillavry ('75), using a young adult rabbit, made marks upon its lower incisors 2.5 mm. and 3 mm. from the tip. After five to seven days the marks had disappeared. Evidently the rate of growth was about 2.5 to 3 mm. per week. Noe ('02) used a rabbit which happened to possess overgrown teeth. The animal accidentally broke off the lower incisors in the bars of its cage, and Noe made observations upon the rate of their growing out. This he found to be .615 mm. per day, or 4.3 mm. per week. This is larger than MacGillavry's results and may have been due to the unopposed growth and to the other abnormal conditions which may have been present in the formative organs.

Using MacGillavry's figures for comparison, it would seem that the lower teeth of the albino rat and of the rabbit grow out at about the same rate.

OVERGROWTH OF INCISORS

Examples of overgrowth of the incisors of rodents, especially in rabbits and hares, which were hunted as food, must have been observed from early times. In the older literature, they are referred to principally as curiosities, which have excited the interest of whoever has found them. later the causes of the malformations were also considered. Thus Jenyns ('29), to cite only one observer, found several examples in wild rabbits, and has given a good illustration of the curved aspect of the teeth. He also clearly states the several causes which, in his opinion, may give rise to the condition. In addition to the one usually accepted at his time — accidental breaking off of one tooth — he considered also as causes (a) too soft food, (b) morbid or too rapid secretion of the osseous matter of the teeth, and (c) dislocation of one of the condyles.

Wiedersheim ('02-'03) has reported a case occurring in a rat, where he found an associated assymmetry of the cranium. He is in doubt as to which was cause and which was effect — the overgrowth of the teeth or the assymmetry of the cranium.


Fig. 28 Cranium of albino rat, showing the overgrown upper incisors recurving to the left side. The left incisor passes to the outer side of the skull, while the apex of the right incisor has penetrated the bone of the maxilla in the region of the basal end of the left incisor. X 1.

Fig. 29 Cranium of the same albino rat shown in the preceding figure, viewed from the right side. It shows the overgrown lower incisors recurving to the right side, and the cavity which the right incisor has worn in the palate bone.

Figures 28 and 29 show a skull obtained some years ago from the rat colony of The Wistar Institute by Dr. Stotsenburg, and prepared in the Histological Laboratory by Miss E. F. Brooks. The upper teeth curved to the left side of the head and the lower to the right side. As seen in figure 29, the right lower has penetrated through the bone of the palate into the nasal chamber, while the right upper (fig. 28) has recurved and grown into the maxilla.

In The Wistar Institute rat colony, at the time when the animals were fed on bread and milk, frequent examples of this and similar conditions were found, but now under a more varied mixed diet they practically never occur.

Beretta ('13) has recently made an analysis of these abnormalities and has classified them in three groups.

(1) Overgrowth of the upper and lower incisors through lack of an opposing tooth.

(2) Overgrowth of the incisors of the upper and lower jaws through deviation of the jaws.

(3) Prognathism of the lower jaw, and as a result, overgrowth of the incisor of the lower jaw.

In the present instance, diet seemed to be the controlling factor, probably by reason of its influence on the hardness of the bone of the alveoli from which the teeth grew out.

SUMMARY

The rate of growth of the upper and lower incisor teeth of Mus norvegicus albinus, in the mature animal, averages 2.2 and 2.8 mm. per week, or 12.5 cm. and 14.5 cm. per year, respectively.

Growth is due primarily to the proliferation and growth of cells at the basal end of the enamel-organ, where new enamelforming cells arise, and at the basal end of the dental papilla where new dentine-forming cells develop.

The enamel-organ of the adult forms a narrow circular band around the basal end of the tooth, and extends forward from this on the labial side only. It coincides in its lateral boundaries with the enamel, and extends along the entire imbedded portion of the tooth. Anteriorly, it comes in contact with the epithelium of the gingival margin, and is carried out continually as a narrow band of cells lying on the enamel, between the latter and the gingival epithelial tissue.

The first indication of the anlage of the incisors appears in 14-day-old fetuses. In fetuses, 21 daj^s of age (just before birth), enamel and dentine formation is beginning. In animals 1 day old the upper and lower teeth measure 2.3 and 3 nrni. At 8 to 10 days the teeth erupt, and at 10 days measure 7 and 11 mm. respectively. This period is therefore characterized by the rapid elongation of the teeth.

The process of attrition begins within a few days after eruption, so that by 19 or 21 days of age, the typical occlusal surface is formed. Up to the time of eruption the anterior end or apex of the tooth is immediately under the oral epithelium, while the basal or growing end is continually progressing posteriorly. After eruption, the basal end becomes nearly stationary in position, while the whole tooth structure is continually moving forward. The extra-gingival length of the tooth is kept constant, however, by the attrition of the occlusal surface, either through use in gnawing or by the action of the opposing teeth.

The histogenesis of the enamel-organ is practically completed by the 4th day after birth, although it does not attain its final relations to the tooth as a whole, until after eruption. In the 18-day fetus the enamel-organ is similar in all parts, and the cells of the inner layer measure the same, both lingually and labially. From this period forwards, however, the labial portion continues to progress towards its fully differentiated functional structure, while the lingual portion retrogresses, until at 4 days after birth the latter is disrupted, by the ingrowth of the surrounding connective tissue. Contrasting the cells of the inner layer — the potential ameloblasts — on the labial and lingual sides, thej^ are practically the same in the 18-day fetus, but at 19 days they are found to measure 24 and 20 m respectively. In the 21-day fetus, they measure 30 to 34 and 12 fx, and 1 day after birth the true ameloblasts on the labial side have increased to 40 n, while the non-functional cells of the lingual side are only 10 fx in height. At 4 days, the latter cease to form a continuous layer, by reason of the dispersion of the cells by the surrounding connective tissue, except at the basal formative region.

Characteristic of the permanently-growing enamel-organ are the epithelial papillae, formed by the elevations of the outer layer of the enamel-organ, and the cells of the enamel pulp. Between these elevations are numerous capillaries which insure a rich blood supply to the enamel-forming cells.

There are three layers in the functional enamel-organ — inner, middle and outer. The inner is constituted of the tall ameloblasts, and the middle is made up of two divisions, (a) stratum intermedium and (b) enamel pulp. The latter unites with the single layer of cuboidal cells which compose the outer layer, to form the epithelial papillae (fig. 26).

The apex of the primitive tooth is formed of a variety of secondary dentine — 'osteo-dentine' of Tomes — which is softer than true dentine, and differs in its structural arrangement (fig. 20). After eruption, this terminal portion of osteodentine is soon worn away by attrition, and the typical occlusal surface is developed, as seen at 19 or 21 days. At 21 and 23 days the first two molars erupt in both upper and lower jaws, and from now on the animal is able to secure food for itself, and if necessary can maintain an independent existence.

As the tooth continues to be worn away there is a provision for the continual filling in of the apex of the pulp-chamber by the formation of what may also be called osteodentine. This is a form of secondary dentine, containing, when first formed, cells and blood-vessels. This is always at a little distance, about 1 mm., from the occlusal surface, but as any part of the tooth, in its outward progression, approaches the occlusal surface, the soft elements disappear within the osteodentine, and the latter forms a hard continuous surface with the adjoining true dentine. The position of this osteodentine is marked as a line on the occlusal surface of the teeth (fig. 5).

Prior to eruption there develops around the apex of the tooth, as it lies in contact with the surface epithelium, a thickened ring of stratified epithelium. This ring of tissue is pierced by the apex of the tooth at eruption, and would seem to have the function of serving as a resistant margin for the soft tissues, and of preventing other tissues being carried along with the erupting tooth.


The length of the teeth varies with the size of the cranium (table 1) so that the persistent growth is not only sufficient to offset the continual attrition, but also serves to keep the length of the teeth in a definite relation to the length of the skull, as the latter increases in size.

The lower tooth is always longer than the upper, and this difference manifests itself even in the anlagen of these structures in the 19-day fetus (figs. 11 and 12).

The contour of the enamel, as seen in cross-sections, is characteristically different in the upper and lower teeth (fig. 4).

The enamel of the tooth is composed of two layers which are different in appearance. The enamel rods run in two sets which decussate with each other in the inner or plexiform layer, but they change their direction as they continue into the outer layer, so that in it they are all parallel. In longitudinal sections, the general direction of the rods (fig. 26), is to incline towards the apex of the tooth, as they run from the enamel-dentine boundary to the outer surface of the enamel.

In conclusion, we wish to thank Professor Piersol for generous assistance in many ways, and Professor Donaldson for his constant interest in the study. We also wish to acknowledge the kind assistance of Mr. E. F. Faber in the preparation of the drawings.


LITERATURE CITED

Adloff, p. 1898 Zur Entwicklungsgeschichte des Nagetiergebisses. Jena. Zeitschr. fiir Naturwissenschaft, Bd. 32, ss. 347-410.

Beretta, a. 1913 La normala dentatura dei roditori in rapporto alle anomalie dentali in questi osservate. La Stomatologia, t. 10. Abstract in Deutsche Monatsschrift fiir Zahnheilkunde, April, s. 287.

VON Brunn, a. 1887 Ueber die Ausdehnung des Schmelzorganes und seine Bedeutung fiir die Zahnbildung. Arch. f. mikr. Anat.. Bd. 29, ss. 367-383.

BuRCKHARDT, R. 1906 In Hertwig's Handbuch der Entwickelungslehre der Wirbeltiere, Bd. 2, Teil 1, Kapitel 4, ss. 349-456. "Die Entwickelungsgeschichte der Verknockerungen des Integuments und der Mundhohle der Wirbeltiere."

Cope, E. D. 1888 The mechanical causes of the origin of the dentition of the Rodentia. Amer. Nat., vol. 22, pp. 3 11.

Donaldson, H. H. 1912 The history and zoological position of the albino rat. Proceed. Acad. Nat. Sci., Philadelphia.

Freund, p. 1892 Beitriige zur Entwicklungsgeschichte der Zahnanlagen bei Nagethieren. Arch. f. mikr. Anat., Bd. 39, ss. 525-5c6.

Fritsch, C. 1914 Untersuchungen iiber den Bau und die Innervierung des Dentins. Arch, fur mikr. Anat., Bd. 84, ss. 307-20.

Hatai, S. 1907 On the zoological position of the albino rat. Biol. Bull., vol. 12, pp. 266-273.

Jenyns, L. 1829 Observations on a preternatural growth of the incisor teeth occasionally observed in certain of the Mammalia Rodentia. Loudon's Magazine Nat. Hist., London, vol. 2, pp. 134-137.

Jolyet et Chaker 1875 De I'acte de ronger, etudie chez les rats. Comptes Rendus et Memoires de la Soc. de Biol., pp. 73-74.

Lowe, L. 1881 Beitrage zur Kenntniss des Zahnes und seiner Befestigungsweise im Kiefer. Arch. f. mikr. Anat., Bd. 19, ss. 703-719.

MacGillavry, T. H. 1875 Les dents incisives du Mus decumanus. Arch. Neerl. Sc. exact, et nat., Haarlem.

Meyerheim, M. 1898 Beitrage zur Kenntnis der Entwicklung der Schneideziihne bei Mus decumanus. Dissertation. Leipzig.

Mummery, J. H. 1912 On the distribution of the nerves of the dental pulp. Philos. Trans. Roy. Soc, London, vol. 202, B., pp. 337-349.

NoE, J. 1902 Vitesse de croissance des incisives chez les Leporides. Comptes Rendus, hebd. des Seances et Memoires de la Soc. de Biol., pp. 531-532.

OuDET, J. E. 1823 Experiences sur I'accroissement continue et la reproduction des dents chez les lapins. Jour, de Physiol. Exper. et Patholog., Tomes 3 et 4.

Owen, R. 1840-45 Odontography. London.

Reichert, E. T., and Brown, A. P. 1910 The crystallography of hemoglobin. Pub. Carnegie Inst, of Washington.

Retzius, a. 1838 Bemerkungen iiber den inneren Bau der Zahne mit besonderer Rlicksicht auf den in Zahn vorkommenden Rohrenbau. Mliller's Archiv.

Roetter, F. 1889 TJeber Entwicklung und Wachstum der Schneidezahne bei jNIus musculus. Morphol. Jahrb., Bd. 15, ss. 457-477.

Ryder, J. A. 1877 The significance of the diameters of the incisors in Rodentia. Proc. Acad. Nat. Sci., Philadelphia, vol. 29, pp. 314-318.

1878 On the mechanical genesis of tooth-forms. Proc. Acad. Nat. Sci., Philadelphia, vol. 30, pp. 45-80.

Sachse, B. 1894 Entwicklung der Schneidezahne bei Mus musculus. Dissertation. Leipzig.

Stach, J. 1910 Die Ontogenie der Schneidezahne bei Lepus cuniculus. Extrait. Bui. d'Acad. Sc, Cracovie.

Tomes, C. S. 1914 A manual of dental anatomy; human and comparative. 7th Ed. Edited by H. W. Marett-Tims and A. Hopewell-Smith.

Tomes, J. 1850 Structure of the dental tissues of the order Rodentia. Phil. Trans. Royal Society of London, pp. 529-567.

Tullberg, T. 1898-99 Ueber das System der Nagethiere. Nova Acta Reg. Soc. Sc. Upsaliensis, Series 3, ss. 1-514.

Weber, M. 1904 Die Saugetiere.

Wiedersheim, R. 1902-03 Ein abnormes Rattengebiss. Anat. Anz., Bd. 22, ss. 569-573.

Williams, J. L. 1896 The formation and structure of dental enamel. Dental Cosmos, vol. 38.

Woodward, M. J. 1894 On the milk dentition of the Rodentia with a description of a vestigial milk incisor in the mouse (Alus musculus). Anat. Anz., Bd. 9, ss. 619-631.


A Peculiar Structure In The Electroplax Of The Stargazer, Astroscopus Guttatus

James G. Hughes, Jr.

From the Histological Laboratory of Princeton University, U. S. A.

THREE FIGURES

The purpose of this paper is to determine the function and composition of the pecuhar pointed fibers and long pointed rods lying in the electric layer of the electroplaxes of the stargazer, Astroscopus guttatus.

Before proceeding with a discussion of these rods, a brief description of the electric organ of this fish (according to Dahlgren)^ will be given.

The electric apparatus is composed of two organs, which form two vertical columns roughly oval in horizontal section, and placed behind and somewhat under each eye. Each organ extends from the peculiar bare spot on the top of the head down to the tissues which form the roof of the oval cavity; and is composed of about 200 thin layers of electric tissue, which extend horizontally all the way across the organ. These layers of tissue are flat, and alwaj^s at the same distance from one another. Each layer contains about 20 electroplaxes, the outlines of which present a very irregular or scalloped appearance. The electric tissue in which the electroplaxes are imbedded is in appearance a jelly-like or mucous-like tissue, usually known as electric connective tissue, and which I have shown in the course of my work to be of the same composition as white fibrous connective tissue. The nerve and blood supply runs in the above tissue. The general form of a vertical section of an electroplax is shown by figure 1, which is a drawing of part of a section of a single electroplax.


1 Anat. Anz., Bd. 29, S. 387, 1906.


Each electroplax is composed of three principal layers, a nervous or electric layer which forms the upper surface, a middle layer, and a lower or nutritive layer which along with the middle layer is evaginated into a large number of long papillae. All three layers are deeply marked with a dense series of fine striations, which are peculiar to the electroplaxes of several other fishes. The upper or electric surface is flat and smooth and receives the nerve endings. The current of electricity runs downward through the organ which produces it, and thus the nerve endings in accordance with Pacini's law are found on the negative pole of the electroplax.

Proceeding directly to the subject of this paper, we may say that one of the most interesting of the points noted in the electroplaxes, when properly fixed and stained with iron hematoxylin, is a series of rod-like or thread-like objects running horizontally in the electric layer, among, above and below the nuclei and without any apparent connection with them (figs. 1 and 2).^ These rods are of various sizes and shapes, and in form are said to resemble the classic thunderbolts seen in the hand of representations of Jove. They usually taper slowly and branch extensively at one or both ends. Some of these branches sometimes seem to be mere lines, while others are wide and heavily pointed; at their other ends the rods are usually rounded; this latter appearance may be due, however, to the cut ends of the rods, for as noted above they sometimes branch at both ends. Some are short and heavy in appearance while others are long and thread-like. Peculiar looping, twisting, or knot-like bends are sometimes found at points on the longer rods. The outlines and contour of these rods are always smooth. Their size may vary from thick or thin rods of over 300 /x in length down to small ones that do not exceed 1 m- In those electroplaxes where the rods are few they sometimes lie parallel and point in a definite parallel direction, while in others where the rods are very numerous they do not seem to have any definite arrangement. In this latter condition the rods present a very wavy appearance. Their form may be seen in figures 1 and 2, which are drawings of the electric layer of an electroplax when

^ All the figures are drawings of sections of electroplaxes of Astroscopus guttatus.


Fig. 2 Horizontal section through an electroplax. Only the electric layer is seen; a, an extensively branching rod; h, fine branches of the above rod; c, a large characteristic loop in a rod; d, a cut end of a rod; e, nuclei of the electric layer. X 1200.


stained with iron hematoxylin. These drawings show the electric layer in which the rods are found in horizontal section (fig. 2) and in vertical section (fig. 1).

The purpose, function, and chemical composition of these rods have been previously unknown to histologists. In order to determine anything in respect to their function or purpose, a knowledge as to the class of organic substance to which they belong, whether muscle, connective tissue, nervous, or chitinous, and also a rough knowledge of their chemical composition is imperative. The contour and form of these rods as they appear under the microscope resemble both smooth muscle fibers and fibers of elastic connective tissue. The belief that the function of these rods was somewhat of the nature of support for the delicate substance of the electroplax, and the fact that their form resembled connective tissue fibers led the writer to take for one of his first hypotheses, that they were of some form of connective tissue, and to perform accordingly the following series of experiments. As the most logical and best way for determining the kind of connective tissue, if any, of which the rods might be composed, a number of stains used by other investigators to identify similar substances were applied and the results noted. Controls were used on known tissues.

Before taking up the connective tissue stains, however, a description of the results from the iron hematoxylin staining is now noteworthy; the material used being fixed in pure corrosive sublimate.

The jelly connective tissue stained a very light gray. The nutritive, striated, and electric layers stained a much darker gray. The nuclei in all the layers stained somewhat black. The pointed fibers, and rods found in the electric layer stained a deep black, thus being clearly differentiated from the surrounding cytoplasm. In some electroplaxes they were very numerous, while in others the number was rather small.

The connective tissue stains applied as follows:

(1) Mallory's connective tissue stain, using the modification given in Lewis' Text-book of histology." White fibrous connective tissue should stain blue in this medium.

Paraffin sections of the electroplaxes fixed in corrosive sublimate were stained for 10 to 12 minutes in a 1 per cent aqueous solution of acid fuchsin. They were then transferred directly to a stain consisting of 0.05 grain of aniline blue (soluble in water) and 0.2 grain of orange G dissolved in 100 cc. of a 110 per cent aqueous solution of phospho-molybdic acid. In this they remained from 2 to 3 minutes. They were then rinsed in distilled water, dehydrated rapidly, cleared and mounted.


102 JAMES G. HUGHES, JR.

A description of an electroplax as seen under the 2 mm. oil immersion lens is as follows: The white fibrous or electric connective tissue stained a light blue or purple. The electric nutritive and middle layers or the electroplax proper stained a reddish purple, and the nuclei as a whole in all of the layers, stained somewhat lighter than their surrounding cytoplasm.

The peculiar rods and fibers stained a deep red, and were thus clearly differentiated from the other elements of the electroplax. They were very numerous and as noted above their outlines were always smooth. Blood corpuscles lying in the jelly connective tissue, of which there were only a few, stained a brilliant red, much the same as the rods.

The white fibrous or jelly electric connective tissue (between the electroplaxes) , as noted above, stained a light blue. The pointed fibers and rods stained a deep red. This would indicate therefore, that these rods are not composed of white fibrous connective tissue. It would seem also that they are not muscle for they stained a different shade of color from the rest of the electroplaxes, which I have found in the course of my work to stain much the same as muscle.

(2) Vcn Gieson's connective tissue stain, in which white fibrous connective tissue should stain red:

Paraffin sections of the electroplaxes fixed in corrosive sublimate were stained for 4 to 5 minutes in a 1 per cent aqueous solution of hematoxylin. They were rinsed in distilled water and transferred to a stain consisting of a saturated aqueous solution of picric acid containing 20 per cent acid-fuchsin. They remained in here 15 to 20 minutes and were then rinsed, cleared, and mounted. The white fibrous connective tissue layer stained pink. The three layers of the electroplax stained brown. The nuclei and rods stained the same color, that is, brown; and, while the nuclei could be seen with difficulty, the rods were scarcely visible owing probably to their similar refractive index. The fact therefore that the rods did not stain the same color as the white fibrous connective tissue, which stained pink, indicates again that they are not composed of white fibrous connective tissue. By Van Gieson's stain, therefore, I have


ELECTROPLAX OF ASTROSCOPUS 103

confirmed the evidence as presented by Mallory's stain in respect to the composition of the rods; that is, they are not white fibrous connective tissue.

I now undertook to apply stains which were tests for elastic fibers, and the first under this head is Weigert's resorcin-fuchsin stain in which connective tissue according to Weigert and other writers stains dark blue. The stain was made up as follow^s:

One per cent of basic fuchsin and 2 per cent of resorcin were dissolved in water; 50 cc. of the solution were raised to the boiling point, and 25 cc. of liquor ferri sesquichlorate P.G. were added and the whole boiled with stirring from 3 to 5 minutes; a precipitate was formed. After cooling, the liquid was filtered, and the precipitate which remained on the filter was boiled with 50 cc. of 95 per cent alcohol. It was then allowed to cool, filtered and the filtrate made up to 50 cc. with alcohol, and 1 cc. of hydrochloric acid added.

Paraffin sections of the electroplaxes fixed in corrosive sublimate were stained for 6 hours in the above alcoholic solution of the precipitate. They were then washed in 95 per cent alcohol, dehydrated quickly, cleared and mounted. A description of the results are as follows: The electroplax proper, with its three layers, did not take the stain at all. The jelly electric tissue around the electroplax stained a brilliant blue. The rods contained in the electric layer of the electroplax were invisible. The fact therefore that the rods did not take the stain at all shows they are not composed of elastic connective tissue. The control used in this case for elastic tissue was a section of the ligamentum nuchae of a horse, which, when put in the stain for exactly the same time as the electroplax, came out a deep blue. The ligamentum nuchae was chosen as a control since it is, perhaps, the best known and best representative of elastic tissue found in the animal kingdom.

Not desiring to rely solely on the above stain to prove that the rods are not composed of elastic tissue, the electroplaxes were treated with the digesting fluid, pepsin, and the results noted. Vertical sections of electroplaxes, having been freed from paraffin, were put for 3 minutes in a very weak solution


104 JAMES G. HUGHES, JR.

of pepsin in 0.2 per cent HCL (the concentration being 0.5 gram of commercial pepsin to 100 cc. of 0.2 per cent aqueous HCL). The electroplaxes were then treated with the following stains: (1) Iron hematoxylin. The result was that all the rods and part of the nuclei, and less dense portions of the electroplax had been digested. (2) Mallory's connective tissue stain was applied, the electroplaxes being stained according to the directions given in the first part of the paper. The results were the same as with the iron hematoxylin, namely, the less dense portions of the electroplaxes and the rods had disappeared. The fact therefore that these rods were disgested in 3 minutes by a weak solution of pepsin discredits absolutely the hypothesis that the rods are composed of elastic connective tissue, for elastic fibers are digested only very slowly by pepsin, the time required being several hours; the control in this case being the same as that noted above — the ligamentum nuchae of a horse.

Having shown that the rods are not composed of connective tissue of any sort, the next most logical hypothesis was that they were of some form of keratin, chitin, or chondrin, and the following test was performed accordingly.

Vertical sections of the electroplaxes of Astroscopus, after the paraffin had been removed, were put for 24 hours in a 72 per cent solution of hydrochloric acid. The acid was then washed out and the sections stained with iron hematoxylin and then examined with the microscope. The rods could not be found, but hollow spaces corresponding to the shapes of the rods were found in the electric layer of the electroplax. Figure 3, a drawing of a slide after treatment with hydrochloric acid, shows these spaces clearly. The spaces are seen to be a little wider and larger than the rods, showing that the rods must have swelled to a certain extent before being dissolved by the acid. This latter statement was also confirmed by treating the rods with the acid for only a short time, and then examining with the microscope; the result being that the rods had swelled to a considerable amount. The other elements of the electroplax, nuclei, etc., in their general form remained intact. There


ELECTROPLAX OF ASTROSCOPUS 105






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Fig. 3 Oblique section through an electrophix treated with 73 per cent hydrochloric acid. The portion of the electric layer shows the electric nuclei, and also the spaces which were occupied by the rods before they were dissolved. The spaces have a greater diameter than that of the rods because the rods swelled before being dissolved. X 1200.

fore this indicates clearly that the rods are not composed of chitin or keratin, for these substances are not attacked by hydrochloric acid of the above strength. To strengthen this statement we have only to quote the results from the pepsin treatment noted above, in which the rods were digested in 3 minutes. They could not therefore have been composed of chitin or keratin, for according to Encycl. mikr. Technik, these substances are not attacked at all by pepsin. From the abo\'e facts therefore we may with a good degree of certainty conclude that the rods are not composed of any kind of chitinous or keratinous substance. Having found that the rods do not consist of any kind of connective tissue or of chitin or keratin the only reasonable hypothesis left was that they were some form of muscle fiber, not having however the same chemical com


106 JAMES G. HUGHES, JR.

position as the ordinary striated fiber, from which the electroplax is derived. Accordingly the following stains were apphed :

(1) Van Gieson's picro-nigrosine, which stains muscle a yellowish-green and connective tissue blue, mixed as follows: To 45 cc. of saturated aqueous solution of picric acid, 5 cc. of 1 per cent aqueous solution of nigrosine were added, the whole mixed thoroughly. Paraffin sections of the electroplaxes (vertical) fixed in pure corrosive sublimate were stained for 12 hours in this mixture; then washed in picric alcohol, dehydrated rapidly, cleared and mounted. A description of an electroplax is as follows: The jelly electric tissue stained blue. The electroplax proper with its three layers stained a yellowishgreen. The rods were just visible, and were stained the same color and to the same degree as the electroplax. The fact therefore that the rods stained the same color as the electroplax and as muscle shows that they are very probably composed of some muscle-like substance. It may be noted here that the electroplax proper always stains about the same as voluntary muscle, a control consisting of voluntary muscle having proved the truth of this statement several times. The reason for this is apparent when we recognize the fact that the electroplax is derived from a striated muscle fiber. This stain also confirms the results of Weigert's resorcin-fuchsin stain that the rods are not composed of any kind of connective tissue, and also indicates decidedly that the jelly electric tissue is white fibrous connective tissue. The controls used were the adductor muscle from an oyster, the ligamentum nuchae of a horse, and the white fibrous connective tissue in the umbilical cord of a sheep, all being stained exactly the same period of time as the electroplax.

(2) Van Gieson's picro-fuchsin, which stains muscle yellow and connective tissue red, was mixed as follows: To a saturated solution of picric acid were added a few drops of a saturated aqueous solution of acid fuchsin, until the mixture became a deep red. The stain was then ready for use. (It may be noted that, if too much acid fuchsin be added, muscle as well as connective tissue will stain red).


ELECTROPLAX OF ASTROSCOPUS 107

Paraffin sections of the electroplaxes (vertical) fixed in pure corrosive sublimate were stained for 6 hours in the above mixture. The results were as follows: The jelly connective tissue stained red, the electroplax proper with its three layers stained yellow. The rods were scarcely visible, staining the same color (yellow) as the electroplax. The fact that the rods stained yellow would indicate again that they are composed of muscle tissue, and this in connection with the picro-nigrosine stain in which the rods stained the same color as muscle gives us strong ground for beheving that the rods are composed of muscle tissue; probably involuntary muscle fibers as the rods are not striated. The fact also that the jelly electric tissue stained red shows again that it must be of the same composition as ordinary white fibrous connective tissue. The controls were the same as those for Van Gieson's picro-nigrosine.

It may be objected that the rods did not stain the same color as the electroplax proper with Mallory's connective tissue stain. To explain this we may say that the rods stained a brilhant red, which is the color that muscle tissue should take in the above stain. The fact that only the electroplax stained a reddish-purple shows that the rods are more strictly composed of a muscle substance than the electroplax itself.

The only tissue now left of which the rods might be composed is nervous; that is, the rods might be nerve endings of some kind, which they resemble a little. In order to test this Paton's silver nitrate stain for demonstrating nerve fibers and endings was apphed and the results carefully noted. The stain and fixation were as follows:

The electric organ was fixed in 10 per cent formalin solution, neutrahzed with magnesium carbonate. It was then cut in small strips about 4 mm. thick. These were washed with running tap water for 12 hours and then in three changes of distilled water for about 30 minutes. The tissue was then put in 1 per cent silver nitrate solution for 6 days in the dark. The tissue became a reddish-brown in color. (It may be noted here that if the tissue becomes a yellowish-brown the stain has not been applied correctly, and it is advisable to throw the whole speci JOURNAL OP MORPHOLOGY, VOL. 26, NO. 1


108 JAMES G. HUGHES, JR.

men away.) It was then placed in a freshly prepared solution of silver nitrate, made as follows:

To 20 cc. of 1 per cent silver nitrate solution, two drops of a 40 per cent solution of caustic soda were added. A gray precipitate was formed. Twenty to thirty drops of strong ammonia were then added — just enough to dissolve the precipitate. The tissue was allowed to remain in this for 45 minutes; then washed in distilled water containing a few drops of glacial acetic acid (25 drops of acid to 100 cc. of water). It was left in this for about 10 to 15 minutes, until the reddish-brown color had changed to yellowish-brown. It was then washed in distilled water, and placed for 12 hours in 1 per cent hydroquinone containing 5 per cent neutral formal. It was again washed with distilled water, dehydrated, and imbedded in paraffin through chloroform. The tissue was then cut and sections mounted with balsam.

A description of a vertical section of an electroplax so stained is as follows: The electroplax proper stained a very light brown. The nuclei, staining a little deeper, could be clearly seen. The electric connective tissue did not stain at all, and was not even visible. The nerve endings, which were very abundant, stained dark brown or black, thus being clearly differentiated from the rest of the electroplax. The rods could not be found. If present, they must have stained light brown, the same color as the electroplax, for they certainly were not present as nerve endings, which as I noted above, stained a dark brown or black. This conclusion therefore is evident, that the rods are not nerve endings of any kind.

The fact therefore that these fibers and rods have been shown to be composed of a muscle-like substance brings up a new kind of muscle fiber to the attention of histologists. These fibers have not been shown to be contractile, are non-striated, dense and exist without any apparent connection with each other. In view of the above facts we cannot give to these elements of the electroplax any other function except that of support, and this will consequently have to suffice until some other investigator presents a better interpretation.


CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT

HARVARD COLLEGE. NO. 256.


CHROMOSOME STUDIES

iit. inequalities and deficiencies in homologous chromosomes: their bearing upon synapsis

AND the loss of UNIT CHARACTERS W. REES BREMNER ROBERTSON

FOURTEEN FIGURES (THREE PLATES)

In November, 1910, while preparing a larger paper upon the problem of synapsis in the germ cells of certain grasshoppers belonging to the subfamily Tettigidae (Robertson '15), I found some individuals in which there occurred a pair of unequal homologous chromosomes. I made a series of drawings of these pairs at the time, intending to incorporate them later into my larger paper. That paper is not yet completed, owing to the duties of teaching during the last year at Kansas University. The importance of these inequalities in members of a chromosome pair, however, seems to warrant publishing the results in brief form. In this account I wish to point especially to a possible relation between these unequal chromosomes and the behavior of certain unit characters in breeding; also to the bearing which the permanency of such chromosomes has upon the problem of parasynapsis.

In the Tettigidae, a subfamily of the short-horn grasshopper family Acrididae, I have found, for all the species of at least four different genera which I have examined, the number of chromosomes to be uniformly 14 in the female (figs. 1, 11) and 13 in the male (figs. 4, 9). I have also found all to have, with limited variation, among the autosome (ordinary chromosome) series, two extremely long pairs of chromosomes, the 7's and 6's (figs. 1, 3, 4), two intermediate pairs, the 4's and 3's (figs. 1-3) or o's and 4's (figs. 9, 10), and two very small pairs, the 2's and I's (figs. 1-3, Tettigidea) or the 3's and 2's (figs. 9-10, Acridium).

109


110 W. E. B. ROBERTSON

The sex chromosome — single in the male and paired in the female — may rank in size as No. 5 (Tettigidea), No. 3 (Paratettix) or No. 1 (Acridium), depending upon the genus, but this variation between different genera does not seem to be accompanied by any very considerable difference in the relative sizes of the ordinary chromosomes.

Of the constancy of these size relations, I am very certain, having examined a large number of individuals in each species. Being certain of the size relations, I was very ready to recognize any abnormal variations in this respect when they appeared. I found such variations in two genera: two cases in Tettigidea parvipennis, only one of which is given (figs. 4-8), and two in Acridium granulatus (figs. 11-13).

1. A DEFICIENT NO. 4 CHROMOSOME IN TETTIGIDEA PARVIPENNIS

To understand the abnormal chromosomes we must first examine the normal ones. Figures 1 to 3 are of cells from normal individuals. Figure 1 is from the wall of an egg tube. It shows 14 chromosomes, the number characteristic of the female. The chromosomes are numbered and paired according to size. Two of the chromosomes (nos. 1 and 2) have been drawn at one side for convenience. This figure is also typical for male 2 x cells, with this exception, that the sex-chromosome. No. 5, is always unpaked in the male. Figures 2 and 3 are lateral views of the first maturation division in the male germ cells. These show the members of pairs about to separate from each other. The members are still in contact at the distal ends in most cases. The No. 7 pair in figure 2 has formed a cross and is still paired through a greater extent than in the other pairs. The sex chromosome ranks fifth in size in Tettigidea and may be seen passing undivided to one pole in these cells. Among the autosomes (ordinary chromosomes) of figures 2 and 3 there can be seen a very slight difference in size between the I's and 2's, a considerable difference between the 3's and 4's, and a much greater difference between the 6's and 7's. We are concerned at present with the relative sizes of the 3's and 4's only.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 111

Since the diameters of the chromosomes are uniform throughout the series in any given cell, the relative sizes can be learned best by measurement of lengths. My measurements have been made from drawings outlined by means of a camera lucida, the image being projected at the level of the base of the microscope. A 2 mm. Leitzoil-immersion objective and an 18 X Zeiss compensating ocular were used for outlining. I feel reasonably certain that my drawings show approximately the correct relative sizes of the chromosomes in each cell.

The average length (doubled) of the No. 3 chromosomes in the figures ( X 2600) of six cells from two normal individuals of Tettigidea parvipennis is 15 mm. The average length of the No. 4 chromosomes under the same conditions in the same six cells is 17.05 mm. The ratio of the No. 3 to the No. 4 chromosomes is therefore as 1 to 1.14. This may be taken as approximately the normal size relation of the No. 3 and No. 4 chromosomes; that is, No. 4 is about one-seventh longer than No. 3.

In figures 4 to 8 are cells from a male individual of the same species, in which one member of the No. 4 pair of chromosomes (4 — ) is abnormally small. The average length of the No. 3 tetrad is here 13.1 mm. This is shorter than in the preceding case of six cells, but in this instance all chromosomes are affected similarly, the reduction in size being due probably to the cells having been in slightly different stages when killed. This does not affect the relative lengths of the chromosomes, however. The average (doubled) length of the larger (no. 4) diad (figs. 5-8) in the same cells is 15.5 mm. The ratio therefore of the No. 3 tetrad in these cells to the larger diad of the abnormal No. 4 tetrad, or to a No. 4 tetrad made up of two such diads, would be as 1 to 1.18; i.e., the ratio of the No. 3 chromosomes to the normal member of the No. 4 pair here is as 1 to 1.18. This is not far from the normal ratio, 1.14, and the difference is quite within the range of the probable error due to inaccurate measurement, etc. But the average (doubled) length of the smaller diad of the abnormal No. 4 tetrad is 12.7 mm., instead of 15.5 mm. The ratio therefore of the No. 3 tetrad to a tetrad made up of two such No. 4 diads would be as 1 to 0.97; i.e., the ratio of the


112 W. R. B. ROBERTSON

No. 3 chromosomes to the smaller No. 4 chromosome is as 1 to 0.97. This shows the smaller No. 4 diad to be even smaller than the No. 3 chromosomes. I believe that the larger diad of this abnormal No. 4 tetrad is the normal No. 4 chromosome, since its ratio, 1.18, is so near to the normal ratio, 1.14, and that the smaller diad is abnormal and has lost a part of its distal end ; (my reason for thinking the distal end deficient will appear later) . Its ratio, 0.97, instead of 1.14, gives it a shortage of 0.17, or nearly one-sixth of 1.14. It seems therefore to have lost one-sixth of its normal length.

The size of this deficient No. 4 chromosome is constant for this individual. All division figures which occurred in lateral view showed the members of this unequal pair in practically the same relative sizes. Not only the germ cells but also somatic cells (fat body) exhibited the same proportions. The fact that the size ratio is constant in all germ cells and even in body cells seems to point to its germinal origin; i.e., it must have been present in the fertilized egg from which this animal developed.

There is no constant relation between the members of this unequal pair of chromosomes and the sex chromosome, as the cells of the maturation division show (figs. 5, 6, 8), for the sex chromosome passes as often to the pole which receives the small No. 4 chromosome as to the pole which receives the large member.

Another very noticeable evidence of the abnormality and defectiveness of this No. 4 chromosome is shown in its manner of contact with its larger normal mate in the first maturation spindle (figs. 5, 6, 7 c). In these three figures its attachment is not strictly terminal, as it should be. This, it seems to me, is evidence of a deficiency at the distal end of this chromosome. This will be better understood if I describe briefly the method of chromosome pairing in the Tettigidae (Robertson '15). During the period of synapsis, which occurs in the early part of the growth period of the first spermatocyte, homologous chromosomes pair side to side (parasynapsis) , as shown in figures Ai, A2 (pi. 3) . After this period of synapsis, during the growth period which follows, a separation of the pairing chromosomes takes


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 113

place. This separation begins at the proximal ends of the pair and' gradually moves along toward the distal ends (figs. A3 to A5) . By the proximal end I mean that from which the spindle fiber springs and that which travels in advance toward the pole. The opposite, blunt end is the distal end. The proximal ends of the pair diverge, each through an angle of 90°; i.e., until they are 180° apart (figs. A3 to A5). The proximal ends now point in opposite directions, and the pair, thus attached at their distal ends, form a rod. In this condition the pair (tetrad) enters the first maturation spindle (fig. A5).

The defective tetrad, like the others, has gone through this process. But a portion of the distal end of one of the chromosomes being gone, the chromosome cannot conjugate properly with its mate and so has a tendency to slip to one side when the tetrad reaches this stage, as figures 5 to 7, and E2 to E5 show. The normal conjugant (diad) finds at its distal end no corresponding portion in the defective mate with which to pair. The chromosomes were probably paired normally at the proximal ends and in all parts which are present, leaving the distal part of the larger normal member extending beyond the shortened distal end of the defective mate (fig. Eo) . In 'the process of separation which follows conjugation the defective chromosome (diad) had its shortened distal end rotating on the side of the distal end of the longer, normal mate. When the diads had rotated apart 180° this shortened end was as a result out of line with the end of its longer mate (figs. E2 to E5). This is the condition in which the defective chromosome of figures 5, 6 and 7 c is seen.

2. AN UNEQUAL PAIR OF ANOTHER TYPE OCCURRING IN ACRIDIUM GRANULATUS

While working out the chromosomes in dividing ovarian follicle cells of the female individuals of Acridium granulatus, I found one animal which showed among its 14 chromosomes five long members (fig. 11) instead of four (the two 6's and the two 7's). This puzzled me in my pairing of the chromosomes. The material was laid aside for the time. A few weeks later I found a male individual which showed in its first maturation divisions


114 W. R. B. ROBERTSON

the same five long chromosomes. My problem was solved at once when I saw that this extra long chromosome paired in maturation with one of the ordinary chromosomes of the complex, the No. 1 (figs. 12-13).

This unequal pair appears to be of a type different from the deficient tetrad just described, and so must be treated separately. It is of primary importance in its bearing on questions of synapsis and of maturation division. Only two animals having it were found, a male and a female. These, and most others of this species worked upon, were collected from a spot 75 feet square near Waverly, Massachusetts, and are probably related individuals.

Figures 9 and 10 show cells from normal individuals exhibiting the typical condition of the chromosomes in Acridium granulatus. There are two long pairs (6's and 7's), two intermediate pairs (5's and 4's), two short pairs (I's and 3's), and the sex chromosome, which in size ranks No. 2 in this genus. A small, faintly staining, fragmentary body is shown (dotted) in figure 9. It is not present in all cells, and is probably a nucleolar structm-e of some sort. The normal chromosomes paired for the first maturation division may be seen in figure 10. Their size relations are clearly shown there: two large pairs (6, 7), two intermediate pairs (4, 5) and two small pairs (1, 3), as well as the small 'accessory' chromosome (2).

Figures 11 to 13 are of the abnormal male and female. In figure 11 (female) the five, instead of four, long chromosomes are clearly shown. The fifth long chromosome (1) appears odd because it has no equal chromosome with which to pair. The first maturation division in the male (figs. 12-13), however, shows it pairing with the small (no. 1) chromosome. In the male cells, moreover, it shows a constriction at a region about as far from its distal end as the length of the small (no. 1) chromosome with which it is paired.

The other chromosomes are quite normal in these animals (figs. 11-13). In the female (fig. 11) there are the two long pairs (7's and 6's), the two intermediate pairs (5's and 4's), the short pair (3's), the 2's (which are the sex chromosomes and are


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 115

paired in the female), one normal No. 1 chromosome, and the abnormal (long) No. 1. In the male cells (figs. 12-13) there are two large pairs (7 and 6), two intermediate pairs (5 and 4), one small pair (3), and the sex chromosome (2) unpaired; the other small tetrad is represented by one small No. 1 chromosome paired with the abnormal long chromosome. It is evident from these two figures that this long structure is connected with one of the small (No. 1) chromosomes.

The long abnormal diad bears no constant relation to the sex chromosome in its distribution to the second spermatocyte cells, as figures 12 and 13 show. A large number of dividing cells examined showed that in the reduction division it passed as often to the pole which did not receive the sex chromosome as to that which did. I feel certain, therefore, that this chromosome bears no relation to the sex-determining chromosome.

2a. Discussion

What is the origin of the long chromosome?

The partially constricted off distal portion of this long body (figs. 12-13) is probably a No. 1 chromosome and in synapsis it was possibly paired side to side with the opposite normal No. 1, leaving the remainder of the long chromosome, the proximal end, to extend beyond it (figs. Bi and B2). In the Tettigidae, as I have said above, side-to-side pairing in synapsis occurs during the bouquet stage. Following this period the chromosomes of a pair separate from each other, beginning at the proximal ends. Remaining in contact at the distal ends, they continue to rotate apart until the pair appears upon the first maturation spindle as a long rod frequently constricted in the middle (figs. 10, 12, 13 and figs. Ai to A5). That is exactly what has occurred in the case of this abnormal tetrad, but to the end of one of the No. 1 members is attached this extra 1^ portion, which increases the length of this member to two and one-half times its normal dimension (figs. Bi to B4). Unfortunately no satisfactory prophase stages are present to show the behavior of this element in synapsis, but, judging from the constriction


116 W. R. B. ROBERTSON

at one end and knowing the behavior of the unequal tetrads of other individuals (figs, 4-8), I believe that here we probably have the result of an unequal division of some preceding generation where a No. 1 tetrad failed to divide at the proper place, in the middle between the two members, but instead so divided as to give a ^ portion of a No. 1 pair to one pole and a halfportion to the other pole (Ci and C2). As a result of this, it might be imagined that there would be some generations of individuals with a sesqui-valent (l|-valent) No. 1 chromosome present pairing with the normal No. 1 (figs. D], D2). The normal No. 1 might pair with the normal part, or with the fragment. In order to get a 2^-valent No. 1 chromosome the parts of which are largely oriented in one direction it will now be necessary to suppose that, at some reduction division of the germ cells in succeeding generations of animals, a normal No. 1 chromosome became attached during the synapsis period to this sesqui-valent No. 1 chromosome. It might do this by becoming fused with, or by failing to separate from, the fragmentary (cut off) end of the sesqui-valent member after some method as is indicated in figures Di to De, giving as a result a 2|-valent chromosome (fig. De), such as we have in figures 10 to 13. All portions of this chromosome, except the sesqui-valent fragment in the middle, would then be oriented in one direction (see arrows in figs. Ci, Co, and Di to De). My reason for thinking that at least the terminal parts, which make up the greater part of the long chromosome are oriented in the same direction, is based upon the fact that I always see the chromosome arranged on the spindle in perfectly normal position, pointing directly toward the pole to which it is about to go, and upon the fact that the mantle fiber of this chromosome always springs from the longer (1^) portion of the chromosome (figs. 12, 13, and Bi to B5). The chromosome always travels with the larger, sesqui-valent portion in advance (figs. 12, 13, B4 and B5).

In this animal evidently the normal No. 1 chromosome paired during synapsis with the distal No. 1 portion of the 2^-valent chromosome (figs. 12, 13, and Bi to B5). It should pair also


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 117

with the sesqui-valent portion, but I have seen no evidence that this occurs. Possibly the sesqui-valent portion has become so non-functional that it fails properly to attract the normal No, 1 chromosome in the synapsis period.

This explanation is the best I can propose, in the light I now have, to account for the origin of this long chromosome. The facts are that it is approximately two and one-half times the length of a normal No. 1 chromosome, all or most of its parts are evidently oriented in one direction, and a constriction near the distal end marks off a portion which evidently pairs in synapsis in the normal manner with the No. 1 chromosome and separates from this chromosome in the normal manner in the first maturation division (figs. 12, 13, and Bi to B5).

An essential fact is that we have here cases of an abnormally large chromosome, which is constant in size in individuals of both sexes. Its relative size is the same in all the dividing germ cells found in the male, and hkewise in the somatic cells (follicle) of the female. I believe that it is a permanent structure, so far as these two individual animals are concerned.

A second important fact is that this abnormally large chromosome alternates with a normal chromosome and it may or may not be present in either sex. Theoretically, we may have, depending on the presence or absence of this chromosome, three sorts of male individuals and three sorts of female individuals; those containing two normal No. 1 chromosomes, those containing one normal and one abnormal No. 1 chromosome and those containing two abnormal No. I's. I have found the first two cases in both sexes. The latter case has not yet been found in either sex. We have therefore a basis for a Mendelian ratio. The presence of one long and one short chromosome might be considered the cell condition of the heterozygote, or hybrid; the presence of two short chromosomes or of two long ones would give the homozygotes, recessive and dominant, if such relations may be imagined in so far as these chromosomes are concerned.


118 W. R. B. ROBERTSON

3. ON SYNAPSIS AND REDUCTION

In their behavior these unequal tetrads throw a good deal of hght on the nature of the synapsis of chromosomes and their separation from each other in the reduction division. In regard to the beginning of this process, the individuals furnishing these abnormal chromosomes do not have much to show, as I have said above. This beginning, however, I determined from normal spermatogenesis, and have worked it out in my second paper.

I have found the pairing process in the Tettigidae to start as a parasynapsis and to end in a telosynapsis (end-to-end pairing). In the synizesis period following the last spermatogonial division, the members of each pair of chromosomes, six pairs in all, having assumed the thread condition, are seen to arrange themselves in side-by-side fashion. The number of threads is at first twelve, in addition to the accessory chromosome. The twelve threads are seen to become six double and finally six single threads. During this period the sex chromosome usually lies at one side of the nucleus. This condition of pairing continues into the later growth period, when the members of each pair move apart, the separation beginning first at the proximal ends (figs. Ai to A4), the chromosomes of a pair remaining attached to each other at the distal ends (figs. A4, A5). Each rotates through 90° and the two then form a rod (fig. A5), which may show, by a slight constriction, the point of contact of the two members. In this condition the paired chromosomes (tetrads) enter upon the first maturation division.

In the behavior of these unequal pairs I feel perfectly certain that the same thing must have- taken place, for T see no reason to think that these abnormal pairs should behave differently from the normal chromosomes during this process. Their behavior on the maturation spindle and in the anaphase following is in every way similar to that of the normal chromosomes, and we have every reason to believe that a parasynapsis ending in a telosynapsis has taken place. The unequal pairs appear on the maturation spindle in the telosj^napsis condition.

Attention, first of all, should be called to the permanency of size of these abnormal pairs, especially of the abnormal members


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 119

of each paii-. In the first type of inequality which I described (figs. 4-8), all dividing cells examined showed the members of the No. 4 pair unequal and constantly of the two respective sizes. The normal No. 4 chromosome was always of the size normal for No. 4 chromosomes and its deficient (No. 4) mate was hkewise of a constant size but uniformly one-sixth less than the normal No. 4 in all cells, both somatic and germinal. The constancy of this relative inequality was very clearly shown, especially in those figures of the first spermatocyte division which appeared in lateral view (figs. 5-8) . A similar uniformity of sizes I found in the unequal pair of the second type (figs. 12, 13) in both male and female animals. Abundant evidence of the relative sizes was given, especially in the tetrads of the first spermatocyte divisions. Every cell showed the No. 1 portion of the abnormal tetrad to be of the normal No. 1 size (figs. 10-13) and the longer 2|-valent portion to be likewise of a constant size. It was not only of uniform size in all the cells of the male individual, both somatic and germ cells, but in another individual, a female, it was also found to be of the same length (fig. 11).

These facts indicate that in the latter case this abnormally long chromosome must have been handed on as such, not only through many generations of cell division, but also through many generations of individual animals. In the former case the deficient No. 4 chromosome — which was of practically the same size, not only in a large number of spermatocyte cells, the germ cells, but also in the body cells — indicates again that such an abnormal chromosome maintains its identity from generation to generation, from fertilized egg to fertilized egg.

The permanency of these abnormal chromosomes has an important bearing upon one problem of synapsis; upon the question as to whether or not a complete side-to-side fusion of homologous chromosomes takes place during this period. I have attempted to illustrate my ideas by diagrams (figs. Fi to F5, Gi to Ge, and Hi to He). If we suppose that a fusion of the chromosomes takes place, we should expect to find in the case of the unequal No. 4 pair something like figures Fi to F3 occurring. The shortened No. 4, pairing with that portion of the normal


120 W. R. B. ROBERTSON

No, 4 to which it corresponds, or with which it is homologous, and leaving the distal end of the normal No. 4 projecting beyond (fig. Fi), would, on fusion with it, form a single cylindrical body having one end, the distal, smaller in diameter than the remainder of the cylinder. If a splitting occur at the end of synapsis in a plane formed regardless of the old plane of fusion, having merely for its object the splitting of this single fused body into symmetrical halves, there would result two daughter chromosomes of equal size but with smaller diameter at the distal end than throughout the remainder of the chromosome (figs. Fs to F5). There would then be no such uniform inequality of the No. 4 chromosomes preserved in the first spermatocyte divisions of this animal as we have found. ' Instead of having a normal sized 'No. 4 and a (say) five-sixth-sized No. 4 in every cell after the first maturation division, therp would be two No. 4 chromosomes of equal length but shorter than the normal (fig. F5). On the contrary every first spermatocyte metaphase and anaphase (figs. 5-7) shows one normal sized No. 4 chromosome of the same fixed relative size and likewise one deficient No. 4 chromosome of a definitely fixed relative size.

If we turn to the abnormally long tetrad (figs. 12, 13), we have even more definite proof that fusion longitudinally and splitting along a new plane in all probability does not occur. The normal No. 1 chromosome evidently has paired with the distal portion of the long No. 1 chromosome in a manner similar to the diagram shown in figures Bi and B2. If in such pairing it fuse completely with its longer mate, its would form a cylinder again, with one end of larger diameter, the opposite of smaller diameter (figs. Gi to G4). If, on the splitting of the fused thread, the new split be formed, not along the old plane or fusion, but upon any plane giving two equal daughter threads, we would get two long chromosomes of the same length with diameters large at the distal end instead of a long chromosome and short chromosome, of the same relative lengths and equal diameter, with which we started. Again, if the new split appeared in the fused chromosome in a plane at right angles to that of fusion (figs. Hi to He), we should


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 121,

get a similar result, which is also contrary to the facts in the case. The chromosomes, when they come out of the pairing process, are evidently of the same size they were on entering the process. The abnormal sized 2^-valent No. 1 chromosome is of the same size in all the germ cells after the process as in the germ cells of the same individual before the process, and even in the body cells of another individual (fig. 11). The same may be said of its normal mate. No tags or projections were seen in any case to indicate that a new longitudinal plane of division had occurred.

In both types of unequal tetrads we have very strong evidence that homologous chromosomes, on entering the side-to-side pairing process of synapsis, remain as distinct individuals, retain their identity throughout the period and come out of it with at least the same size they had on entering it. Each pairing chromosome maintains its distinct individuality during this period.

This is opposed to the ideas of Jannsens ('09) and Morgan ('11) as expressed in the theory of the 'chiasma type.' In their theory they assume that homologous chromosomes in parasynapsis twist about each other and fuse. On splitting, a plane passes down the fused body, regardless of the previous spiral fusionplane, resulting in two daughter chromosomes which may not be identical with the two chromosomes which entered the process. Each new one may contain parts of both original chromosomes. If such had been the case, the separation or formation of a short and a long chromosome out of the fused chromosome (Bo to B5), with such regularity of size, etc., as we have shown, could not have occurred. Or, supposing that spiral twisting and fusion had occurred and that splitting again was limited to the larger end of the fused chromosome (figs. G3 to G4), we should expect that the shorter member resulting (figs. B2 to B5) would at least show fragments of the side of the long chromosome attached to its proximal point in those cases where the splitting plane did not coincide with the old plane of fusion between the short and long chromosomes. No such attached fragments were found. All short or normal No. 1 chromosomes in the metaphase and anaphase of the first spermatocyte divisions were of uniform length


122 W. R. B. ROBERTSON

and showed that they retained the same size and were as free from terminally attached fragments as they were on entering the pairing process.

In regard to the question of pre- and post-reduction, I have evidence here that the first maturation division is the reduction division so far as regards three of the seven pairs of chromosomes of the Tettigidae subfamily of grasshoppers. These pairs are the Ts, the 4's and the sex chromosomes. The abnormally long No. 1 chromosome is seen in Tettigidea separating from its normal mate (figs. 12, 13), the deficient No. 4 is seen separating from its normal mate (figs. 5-8), and the unpaired sex chromosome (paired in the female, figs. 1, 11) is seen passing over whole to one of the poles in the male cells in smiilar manner, just as if it had had a mate from which to part at this division, where such parting is being carried out by the members of the ordinary chromosome pairs (figs. 2, 3, 5, 6, 8, 10, 12, 13). The other chromosome pairs (the 2's, 3's, 6's, and 7's) behave similarly to the I's and 4's in every respect during the synapsis, prophase, metaphase, and anaphase periods of the first spermatocyte divisions. This leads one to think that probably the first spermatocyte division is the reduction division for all pairs of chromosomes, in the Tettigidae subfamily of grasshoppers at least.

4. THE DEFICIENT NO. 4 CHROMOSOME AND THE LOSS OF UNIT CHARACTERS

The most important of these unequal tetrads are those in which one member of the pair is of less than the normal size (figs. 4-8) . The importance lies in this, that such a deficiency may be the result of the dropping of a' part of a chromosome and in this way may furnish the basis in the germ cell for the loss of unit factors in heredity.

In animal and plant breeding there are many unit characters which might be considered to have resulted from the loss of something from the germ plasm, whether this something be an enzyme of some sort or a substance upon which an enzyme might act.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 123

It has been shown by Castle ('11) and others that in mice, rabbits, and guinea-pigs the ordinary gray color of the wild rodent is not a simple character, but on the contrary, is very complex. It depends — for example, in mice — upon the simultaneous presence of at least seven or eight color factors. The complete dropping of any one of these from the germ plasm modifies the color of the animal.

The factor most commonly dropped is that for the production of color, the animal — whether potentially gray, black, or brown, etc. — being an albino, actually without color. The next factor very commonly dropjDed is that for barring of the fur. This does not show unless black or brown pigment is present in the hair. The barring of the hair is due to the fact that some factor prevents the development of black and brown pigment granules in a jDortion of the individual hair immediately below the tip, leaving a black tip and a dark base with a light yellow band between them. When this barring factor is dropped, we get a black or a brown animal instead of a gray. The next most commonly dropped factor is that for the production of black, giving cinnainon or brown animals. By dropping the factor for self color, spotting results, spotting of two sorts, white upon ai^olored coat, or yellow upon a black, brown, or gray coat,^giving in the extreme cases white with black or brown eyes ^nd yellows with ])lack or brown eyes. By dropping the dark-^ed character we obtain pink-eyed animals with a scarcity of pigment in the fur. By dropping the intensity-of-pigmentation factor we get diluteness of pigment, giving dilute-pigmented gray, black ('blue'), brown ('cream'), or yellow animals. When all of these factors are present we have the wild gray type. By the dropping of any one of them entirely the color may be modified accordingly.

It is evident that these animals lack something. The dropping of the black pigment, for example, gives animals which are cinnamon or brown. No black can be produced in the race until this factor is again added to the mixture. It is entirely absent. In this case black cannot be considered a latent character which has been restricted in some way from attaining its development, as is the case in albinos of gray or black animals;

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these on being crossed with brown, or any animal that contains the color factor, will give gray or black, etc., showing the gray or black characters to have been present in substance though not developed. If the black be absent, nothing can restore it except the addition of black again. There is evidently a complete absence of the black pigment material from the fur of the animal.

In the varieties of domestic plants many similar cases may be given. But before mentioning the color varieties I wish to call attention to the cross between the 'cupid' and the 'bush' variety of sweet pea as one of the best examples. The cupid is a dwarf plant whose internodes are very short, making the stem correspondingly short, about 9 to 10 inches. It has the prostrate habit, that of lying on the ground, due to the diverging habit of the branches. The 'bush' variety produces branches which do not diverge but grow upright, making a tall bush growing 42 to 48 inches in height. These varieties, on being crossed, produce in the Fi generation plants which show a reversion to the habit and size of the wild sweet pea of Sicily (Punnett '11). They have the long internodes and the long stem of the bush variety combined with the prone, prostrate habit of the cupid variety. By inbreeding the Fi individuals the Fo show the tall 'bush' variety, the tall procumbent, the short procumbent or original 'cupid,' and the short 'cupid' bush-like variety. The factors concerned are the long internodes versus the short, and the procumbent versus the erect habit. The two varieties evidently owe their origin to the dropping of one or the other of these factors from the germ plasm of the wild type. The bringing of them together in the hybrid supplies the lack in both races (the 'cupid' and the 'bush' varieties), the result being the wild type.

In flower color of sweet peas we have a >similar case. Most white sweet peas breed true to white, but there are two varieties of white which on being crossed produce a purple colored variety, like the wild Sicilian species. On being inbred, the F2 generation shows nine of the colored variety and seven of the white. Of the whites some breed true, some give a 3.1 ratio, i.e., three whites


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 125

to one colored, while some give all colored again. This shows that there are two factors concerned. The presence of both is needed to produce the colored flowers, the absence of either one giving white. It is the r2 result of an ordinary dihybrid Mendelian ratio, in which there are nine cases where both factors are present and colored results, three cases where only one factor is present and white results, three cases where the other factor is present and w^hite results, and one case where neither factor is present and a white which breeds true results.

The colored individuals in the F2 generation are found to belong to six classes depending upon the presence or absence of a purple factor, a light-wing factor giving a bi-colored flower, and a factor for intensity of pigmentation, in the absence of which a diiutely colored flower results. The wild type is intensely colored, and bi-colored, i.e., purple with blue wings. By dropping the purple a red bi-colored or uniformly colored flower is obtained in both the dilute and intense series, by dropping the bi-color factor uniformly purple or red are obtained, by dropping the intensity factor both varieties of the red are obtained in dilute form, and, finally, by dropping either the color-producing base or the color developer we get any of these color varieties in the albino form. The wild purple Sicilian species contains all these factors. By the dropping of these factors one by one and by inbreeding, all the color varieties of our domestic sweet peas have been obtained.

Now, it seems to me that it would be quite possible to account for phenomena of this kind in plants and animals as the result of an unequal division of the chromosomes in the reduction division, similar to what I have found evidence of in Tettigidea parvipennis. If but one member of the pair of chromosomes showed the deficiency it might not give a result in the organism. When both chromosomes of the pair show such a deficiency, a condition w^hich would result only when inbreeding occurs, then such a deficiency might be shown in the somaplasm by some such defect as albinism, blackness, dwarfishness, etc. It is true that such a chromosome would evidently be an abnormality of a deficient kind, but are not all such traits as albinism in plants


126 W. R. B. ROBERTSON

and animals, melanism in rodents, retarded mental development, feeblemindedness in man, dwarfisms, etc., abnormal deviations from the type of the species which may be classed as deficient characters?

The amount which may be dropped from one chromosome, such as I have shown in Tettigidea, may be considered too much to allow of the existence of the organism in the homozygous condition, i.e., where both members of the pair lack it. It is conceivable that a limit in the amount that may be absent might occur and that, going beyond that limit, the homozygous condition might be lethal, as in yellow mice. Here the homozygote always dies, or may never be formed, as has been shown by the results obtained from breeding yellows together. The litters are three-quarters the normal size and the young are found to be two-thirds yellow and one-third gray or other colors, never breeding true to yellow. This indicates that the yellow parents were heterozygotes and that the one-fourth pure yellows, which we would expect in F2, have never been formed. Baur ('07) found, on crossing two varieties of snapdragon — the green leaved with the golden leaved — that the offspring were 50 per cent green and 50 per cent golden leaved. The greens bred true but the golden variety produced 25 per cent greens, 50 per cent golden and 25 per cent that were almost white. The latter died at the end of germination, when the food in the seed was used up, since they possessed no chlorophyll. The golden variety was variegated with green patches (chlorophyll-containing cells) and so could manufacture its own starch}^ food.

The yellow condition in mice and in the snapdragon may be due to a greatly deficient chromosome, so greatly deficient that a zygote having two such deficient chromosomes might have too great a deficiency to be able to develop, or, after having developed, may lack some substance, such as the chlorophyll in the snapdragon, necessary for carrying on one of the vital life processes. In addition, this lack might be so great that it would over-ride the normal chromosome in the heterozygous zygote, giving a yellow mouse instead of a gray, black, or brown, as the case may be; or in the snapdragon a golden variety instead of a green


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 127

variety. That something of this nature has occurred, we are inclined to believe from the fact that the golden variety is simply a plant which lacks chlorophyll in small variegated patches over its surface, whose color is therefore due to this condition. In the cases here given possibly there is a law at work correlating the amount of material that may be lacking from a chromosome with the recessiveness or dominance of the trait resulting in the organism. A defective chromosome may continue to give a trait which is recessive to the normal condition until the defectiveness of the chromosome reaches that point where its deficiency becomes so great that the homozygous zygote cannot develop. At that point the defect becomes dominant to the normal condition and individuals can exist only in the heterozygous or normal condition.

It is strange that yellow should be dominant in mice while in most other species of domestic animals it is recessive. This may be due to the position of the yellow determinant along the chromosome. In most species it may be thought of as lying near the end of the chromosome and accordingly could be dropped very easily, causing little disturbance and giving a recessive trait. In the mouse it may be conceived of as lying farther from the end of the chromosome. On dropping enough of the chromosome to cause yellow, a greater disturbance would be created and the defective trait resulting would accordingly be dominant to the normal trait. I make this as a suggestion merely.

The chances for such abnormal divisions are limited by the number of pairs of chromosomes in the species and by the varying amounts which may be dropped from each chromosome in each case. In guinea-pigs the number of pairs is twentyeight (Stevens '11). The chances for abnormal divisions in guinea-pigs are therefore large. Where the number of chromosomes is small the chances are smaller. The amount that may be cut off from each individual chromosome might vary enough to give several varieties due to the variation in this respect in one pair of chromosomes. The same might be said of each of the twenty-eight pairs in guinea-pigs. Any of these varying conditions in a single pair of chromosomes might combine with


128 W. R. B. ROBERTSON

all possible combinations of similar variations in the twentyseven other pairs. Such might be the basis of all of our variations in guinea-pig inheritance.

It is noticeable in any animal or plant which becomes domesticated that very soon there appear whites, blacks, browns, spotted, yellows and others of the color varieties common to domesticated species. The same may be said of other characters of the species, size for instance. On domestication inbreeding occurs. This gives rise to homozygous strains, which maj^ be isolated. In the wild state inbreeding is not so prevalent. Promiscuous mixing occurs. A summing up of the characters results and all normals, which are usually dominant, are present and show. The weaker recessive characters, if present, are covered up. They exist in the single or heterozygous condition and so do not show. If they exist in the homozygous condition they may show, as in albinos, but the organisms may be killed off by natural selection. Under domestication man preserves these recessives.

The loss of parts of chromosomes may explain very easily the appearance of such phenomena in the domestication of species. The fact that there appear always the same or nearly the same color varieties in each species may be due to this, that theiichromosomes are more or less similarly organized, that there are approximately the same number and in many respects the same individual chromosomes to be dealt with in each species, and finally, that these chromosome pairs are subject to the same vicissitudes of fortune in division at the maturation period in each species. True, there are minor variations, in chromosomes as we pass from species to species. There are also minor variations in color inheritance as we pass from species to species. But these variations are small when we think of the similarities. All species of rodents show grays, all show albinos, all show blacks, all show some form of brown, and yellow^s. The method of inheritance of the yellows, for instance, might vary from species to species, but they are yellow, nevertheless.

The same spontaneous variations, such as albinism, probably occur not once but over and over again in the same species in various parts of the world entirely independently of each other.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 129

If the basis of albinism lie in an abnormal reduction division of a certain pair of chromosomes, we should expect it to do just that sort of thing. So long as the same number of pairs of chromosomes occurs in a species, so long will the same variations continue to occur. In this way we shall continue to have white animals produced anew by 'mutation' — blacks, yellows, spotted, and all the varieties not only of color but in respect to other properties of the body as well. In the same way we might always expect to have produced a certain percentage of defective human beings, such as the classes of feeble-minded, imbeciles, epileptics, etc., each of which seems to be due, in many cases at least, to something lacking in the germ plasm (Davenport '11). Germinal variations of this kind, it seems to me, might be at the basis of De Vries' 'eversporting varieties,' which gave such abnormalities as striped flowers, five-leaved clovers and monstrosities of various sorts, such as pistoUody, twisted and flattened stems, etc. Again, it seems possible that germinal variations of this sort might lie at the basis of those of De Vries' mutations which he distinguished as retrogressive in character; i.e., which were characterized by the dropping out of some character from the parent species. They might lie at the basis of some mutants which he considered progressive, but which showed some retrogressive traits, such as the brittleness of the stem in Oenothera rubrinervis. My reasons for supposing this are as follows: De Vries found his 'eversporting varieties' producing their abnormal individuals continually. He found his parent species, Oenothera lamarckiana, continually throwing off the same mutants in certain proportions. No single parent plant proved ever to be wholly destitute of mutability." In his parent species, lamarckiana, he has probably a constant fundamental number of chromosomes to deal with. He has a reduction division taking place every time a germ cell is formed. He has the same possibility of abnormal, unequal, divisions of tetrads at the time of this division, giving a deficient homologous chromosome. He has self fertilization (inbreeding), which would tend to bring such defective chromosomes together. He has frequent cases of sterility in the inbred offspring of his mutants,.


130 W. R. B. ROBERTSON

as one would expect in instances where a vital factor had been dropped. He has mutations which, with one or possibly two exceptions, are of a retrogressive nature; i.e., lacking something necessary which was present in the parent spe.^ies. The gigas variety was due evidently to a doubling of the number of chromosomes (Gates '09). It seemed to lack nothing, but the other mutants seemed all to lack traits, some more useful, some less useful, which were present in the parent species. These phenomena, it seems to me, point to something for their basis like the abnormal variations in reduction divisions, such as I have described in Tettigidea parvipennis.

It is interesting to compare the number of mutations De Vries obtained from lamarckiana with the number of chromosomes. His number of chromosomes was 14, seven pairs. He obtained seven mutations from his plants. One of these, gigas, was evidently due to a doubling of the number of chromosomes. It, however, was not a defective mutant, and so may be left out of account here. The other mutants seemed to have something lacking, and there were six of them. Of these scintillans seems to have been heterozygous, producing on inbreeding, lamarckiana and scintillans. It also produced, in a small percentage, some of the mutants most frequently produced by lamarckiana. Possibly scintillans had one over-deficient chromosome, such as I have postulated for yellow mice or the golden snapdragon. Many sterile pollen grains were found. Possibly the cause of sterility lies here. On this hypothesis, the fact that scintillans can produce oblonga, lata, and nannella is not surprising. The other chromosome pairs are just as liable to accidents in germ cells of scintillans as of lamarckiana. Lamarckianas may be produced by scintillans; why may not the mutants of lamarckiana be produced also? Each of the other five mutations might be based respectively upon deficiencies in one of the remaining five pairs of chromosomes. Since these remaining mutants breed true in each case, it would be supposable that, in order to show, they must be in the homozygous condition. Thus we may possibly account for the five remaining mutants. This hypothesis, it seems to me, is worthy of consideration here. I


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 131

see many reasons to suppose that De Vries was dealing, in part of his mutants at least, with something similar to what T have described in this paper as deficient homologous chromosomes.

Deficient chromosomes, such as I have found paired with their normal sized mates in Tettigidea parvipennis, it seems to me, furnish a sufficient explanation for the loss of unit factors from the germ plasm. Looked at in the light we now have of the behavior of unit characters which belong to this 'loss' group, the hypothesis appears very probable. It seems to me that Professor Morgan and his students, who are working upon Drosophila, should also take into consideration the possibility of such deficient chromosomes. It is a good working hypothesis, and I am going ahead with breeding experiments upon this species, Tettigidea parvipennis, in the hope of getting some results. As to what connections there may be between these deficient chromosomes and the greater problem — the origin of new characters — it is difficult to imagine. This matter had better be left until we have more knowledge of the behavior of these chromosomes.

The observational work upon which this paper is based was done at Harvard University under the direction of Prof. E. L. Mark. The writing was completed at Kansas University. I wish to express here my gratitude to Dr. Mark for the help and criticism he has so kindly given to me from time to time.


132 W. II. B. ROBERTSON

ADDENDUM

After this paper was worked out, a treatise by Miss Carothers ('13) appeared, describing unequal tetrads in three species of the 23-chromosome grasshoppers, Brachystola magna, Arphia simplex, and Dissosteira Carolina. I wish to consider her paper briefly here.

In the twenty specimens of the three species examined she finds the members of one of the three pairs of small chromosomes always unequal in size. The unequal pair occurs in spermatogonial, in first spermatocyte and (separated) in second spermatocyte cells. The members of the unequal pair agree with those I have found, in that they become separated from each other during the first and not during the second maturation division. This is evidence again that the first maturation division is the reducing division. In their passage to the second spermatocytes these unequal meml^ers (diads) agree with those of my material in that they are distributed to these cells irrespective of the presence or absence of the sex chromosome. In this respect our unequal pairs differ from those of Gryllotalpa, described by Payne ('12), where the longer chromosome was always accompanied b}^ the sex chromosome in the anaphase of the reduction division. Payne may have been dealing with a group of sex chromosomes similar to what he has already worked out in another order of insects ('09).

Miss Carothers' material differs from mine, however, in that she finds the unequal pair present in every animal. I cannot agree with her in this respect, in Acridium or Tettigidea, as my drawings have shown. Possibly further search will show that the unequal pair is not always present in the species upon which she has worked. If this should be the case, the hypothesis of selective fertilization which she advances would be unnecessary. So far as Tettigidea and Acridium are concerned, it is not necessary to postulate selective fertilization.

In thinking that the great number of combinations of chromosomes possible is sufficient to account for all variations, I fear Miss Carothers may be mistaken, (iates ('09) has shown that in


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 133

Pisum the number of pairs of chromosomes (seven) is not large enough to account for the number of pairs of allelomorphic characters which beha^'e independently of each other in breeding experiments, if we assume that the basis of each member of one allelomorphic pair must be permanently located in one member of a single pair of chromosomes:

In Pisum eleven or more pairs of allelomorphs have been observed and the reduced number of chromosomes is only seven; which shows that in this case at least, several characters must reside in one chromosome. The characters must then be confined to separate particles or corpuscles of the chromosomes, and an interchange of homologous particles according to chance during maturation would give the Mendelian combinations.

I am not quite willing to believe that the basis of an allelomorph may slip from one chromosome to another. Yet it is very evident, so far as I can see, that the number of chromosome pairs behaving independently of each other is too small to allow them to be the basis for the number of allelomorphic pairs of characters behaving likewise independently of each other. Possibly some of these extra pairs of allelomorphs may be accounted for by the deficient chromosome hypothesis which I have advanced, or possibly by the 'chiasma' theory of Morgan, though I have evidence against the latter in these unequal chromosomes and in the V-chaped chromosomes of Chorthippus and Jamaicana (Robertson '15).

Additional instances of unequal chromosomes, so far as I have been able to find in the literature, have been reported by Baumgartner (Science '11), Hartman ('13), and, I have been informed, by Voinov ('12). The last mentioned paper is published in a European journal to which I have been unable to get access. Baumgartner reported an unequal pair in Gryllotalpa, but he gave no drawings and no description. Since that time, Payne ('12) has shown that the unequal pair in Gryllotalpa is related to the sex chromosome, the larger member of the pair going with it in the reduction division.

A short time ago Mr. F. A. Hartman called my attention to the fact that he had already described unequal divisions of some


134 W. R. B. ROBERTSON

of the small chromosome tetrads in the first spermatocyte cells of Schistocerca in his paper (March '13) on Variations in the size of chromosomes. " His paper deals chiefly with variations in size of chromosomes due to, what he believes to be, their unequal growth in the cell. Thinking that the whole paper was devoted to 'variation due to unequal growth,' I overlooked the latter part in which he illustrates and describes briefly a few cases of what he considers unequal division in the first spermatocyte. One of these cases (his fig. 86) may possibly be due to faulty conditions of sectioning. The other cases (his figs. 83, 85 and 87 to 91) are probably variations similar to those Carothers has since described in Brachystola, Arphia, etc. That "size variations may be due to unequal growth" I am inclined to doubt, but Hartman is to be given credit for recognizing the importance of variation in chromosome size in its relation to 'variation' in 'animals,' since his work was done while teaching in a high school away from any university contact and especially since he was entirely ignorant of the Mendelian laws and their relation to variation. Had he known of these and the related work, he would likely have come to the same conclusions that I have.


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES 135

BIBLIOGRAPHY

Baur, E. 1907 Untersuchungen fiber die Erblichkeitsverhaltnisse einer nur in Bastardform lebensfiihigen Sippe von Antirrhinum majiis. Bericht Deutsch. Bot. Ges., Bd. 25, pp. 442-454.

Carothers, E. Eleanor 1913 The Mendelian ratio in relation to certain Orthopteran chromosomes. Jour. Morph., vol. 24, no. 4, December, pp. 487-504.

Castle, W. E. 1911 Heredity. D. Appleton & Company; 184 pp.

Davenport, C. B. 1911 Heredity in relation to eugenics. Holt & Company; 298 pp.

DeVries, H. 1904 Species and varieties. Open Court Publishing Company; 847 pp.

Gates, R. R. 1909 a The stature and chromosomes of Oenothera gigas De Vries. Archiv fiir Zellf., Band 3, Heft 4, pp. 525-552.

1909 b Cytological basis of Mendelism. Bot. Gaz., vol. 47, pp. 79 81.

Hartman, F. a. 1913 Variations in size of chromosomes. Biol. Bull., vol. 24, no. 4, March, pp. 223-238, 4 pis.

JanNsens, F. a. 1909 La theorie de la chiasmatypc. La Cellule, tome 25, fasc. 2, pp. 387-411, 2 pis.

LuTZ, Anne M. 1907 A preliminary note on the chromosomes of Oenothera lamarckiana and one of its mutants, O. gigas. Science, N. S., vol. 20, pp. 151-152.

Morgan, T. H. 1911 Random segregation versus coupling in iNlendelian inheritance. Science, N. S., vol. 34, no. 873, p. 384.

Payne, F. 1912 The chromosomes of Gryllotalpa borealis Burm. Archiv fiir Zellf., Band 9, Heft 1, pp. 141-148.

Punnett, R. C. 1911 Mendelism. Third edition. INIacmillan Comjiany; 192 ])p.

Robertson, W. R. B. 1915 Chromosome studies. I and II. (Not j-et pubJishcd.)

Stevens, N. M. 1911 Heterochi'omosomes in the guinea-pig. Biol. Bull., vol. 21, no. 3, pp. 155-167.

VoiNOv, D. X. 1912 La spermatogcnese chez Gryllotalpa vulgaris, C. R. Soc. Biol. Paris, tome 72, pp. 621-623. (This reference is taken from Carothers' ('13) Bibliography).


EXPLAXATIOX OF PLATES

The drawings of plates 1 and 2 were outlined with an Abbe camera lucida at a nuignification of 3900 diameters obtained with a Leitz 2 mm. oil-immersion objective and a Zeiss X 18 compensating ocTilar. with draw-tube set at 150 mm. and drawing made at the level of the base of the microscope. In the process of rei)roduction, they have been reduced one-third, and therefore appear at a magnification of 2600 diameters. The numerals affixed to the chromosomes indicate their relative sizes, the smallest being numbered '1."

PLATE 1

EXPLANATION OF FIGURES

' 1 to S Tettigidea parvipennis

1 Chromosomes of an oogonial cell; female.

2 First spermatocyte; tetrad No. 4 is normal; No. 5 is the sex-chroniosoine; male.

3 Chromosomes of a first spermatocyte of a third animal (male) showing a normal No. 4 tetrad; No. 5 and No. 2 seen somewhat foreshortened.

4 to 8 From a fourth animal, male.

4 Chromosomes of a spermatogonium; all chrome somes are s})lit and in luetaphase; one of the chromosomes of pair No. 4 is deficient.

5 First spermatocyte; the deficient No. 4 (4 — ) separating from its mate (4).

6 First sijermatocyte; deficient No. 4 ('4 — ) in abnormal, oblifjue contact with its mate (4).

7 Deficient No. 4 tetrads taken from three other first spermatocyte di\iding cells in the same animal, showing uniform size l)ut variation in manner of contact of the conjugating chromosomes.


13(3


rNEQUALITIES IN HOMOLOGOUS CHROMOSOMES

W. R. B. EOBERTSOX


PLATE 1




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PLATE 2


EXPLANATION OF FIGURES


8 Deficient chromosome (4 — ) in the anaphase of the first spermatocyte.

9 to 13 Acridium granulatmn. 9, 10 From two normal males.

9 Spermatogonium; No. 1 chromosomes normal.

10 First spermatocyte, No. 1 tetrad normal.

11 Follicle cell of female. One abnormally long (2^-valent) No. 1 chromosome. 12, 13 From abnormal male.

12 First spermatocyte, the 2^-valent, abnormal, No. 1 (1) separating from its normal mate (1) ; going with the sex chromosome.

13 First spermatocyte, the 2i-valent No. 1 chromosome not going with the sex chromosome.


138


INKQUALITIE8 IN HOMOLOGOUS CHROMOSOMKS

\V. R. B. ROBERTSON


PLATE 2



139


JOUBNAI, OF MORPHOLOGV, VOL. 26, NO. 1


PLATE 3

EXPLANATION OF FIGURKS

Ai to He, schematic

Ai to Ae Illustrating pairing in synapsis and separation at first maturation division of homologous chromosomes in Tettigidae. Arrows indicate direction of orientation of the chromosomes; lines and dots indicate respectivelj' maternal and paternal origin of the pairing chromosomes.

Bi to B5 Illustrating manner in which the 2|-valent abnormal No. 1 chromosome pairs with and separates from its normal mate; arrows indicate orientation of parts.

Ci to De A supposed method of origin for the 2|-valent No. 1 chromosome.

Ci Co Accidental unequal division in the reduction mitosis, giving a sesquivalent No. 1 chromosome; arrows indicate orientation of parts.

Di Pairing in synapsis of the sesqui-valent No. 1 chromosome of Co with a normal No. 1.

D2 D3 Separation in late synapsis.

D3 to Do Imagined revolution of the normal No. 1 about the fragmentary end of the sesqui-valent No. 1, giving a 2|-valent No. 1 chromosome having the two end portions oriented in the same direction.

De Carried over whole in a reduction division giving a 2§-valent No. 1 chromosome. The distal No. 1 portion corresponds to that part of the long chromosome in Bi to Bo, which pairs with the normal No. 1.

El to E5 Evident method of pairing of the deficient No. 4 of figures 4 to 8. Normal No. 4 projects beyond its deficient mate (fig. E2) at its distal end. In separation the deficient mate evidently has rotated on the side of this projecting end, and has come into position on the metaphase spindle out of line with its normal mate; compare figures 5, 6, 7 b, and 7 c.

Fi to F5 Showing result expected if in parasynapsis on the pairing of the deficient No. 4 with its normal No. 4 mate there occurred a complete fusion followed by a splitting of the fused body into two symmetrical parts. The chromosomes would be alike in size in the anaphase of reduction (contrary to fact).

Gi to Ge Showing expected result if the same as above took place in the pairing of the 2|-valent No. 1 with its normal No. 1 mate. There would be two long chromosomes of equal length having clubshaped distal and slender proximal ends, the short No. 1 getting a portion of the long No. 1 (contrary to fact).

Hi to He Showing the result if the 2^-valent and 1-valent chromosomes paired and separated in a plane at right angles to the above split (figs. Gi to Ge), the plane of splitting not agreeing with the plane of fusion of the short and long chromosome (fig. Go). Two equal chromosomes would result each composed similarly of like parts of both (contrary to fact).


140


INEQUALITIES IN HOMOLOGOUS CHROMOSOMES

\V. R. B. ROBERTSO>f


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141


THE EMBRYOLOGY OF BDELLODRILUS PHILADELPHICUS

GEORGE W. TANNREUTHER

Zoological Laboratory, University of Missouri

TWENTY-SIX TEXT FIGURES AND EIGHT PLATES

CONTENTS

Introduction 144,

Natural history 146

Brief outline of development 147

Cleavage 149

1 . Designation of cleavage cells 149

2 . Types of cleavage 149

a. Oblique period of cleavage 149

b. Transitional period of cleavage 161

c. Bilateral period of cleavage 163

3 . The first somatoblast 1 63

4. The second somatoblast 169

5 . The entoblast 170

General history of the germ bands 172

1 . Inner stratum of the germ bands 173

2 . Middle stratum of the germ bands 175

3 . Outer stratum of the germ bands 176

The ectoderm and its products 177

1 . The nervous system 177

2. The excretory system 184

Growth " 186

A comparative study of different forms 187

1 . The first somatoblast 190

2 . The second somatoblast 193

3 . Variations in the method of mesodermal formation 199

a. Ecto-mesoblast 199

b. Coelo-mesoblast 199

4. Variations in the source of entoderm 205

General adaptation and interpretation of cleavage 207

1 . In the cleavage of Bdellodrilus 208

2 . In the cleavage of other forms 212

General summary 213

Literature cited 215

143

JODRNAT. OF MORPHOLOGY, VOL. 20, NT. 2 JUNE, 1915


144 GEORGE W. TANNREUTHER

INTRODUCTION

The Discodrilidae are, in many ways, extremely favorable for the study of annelid development. The material can be readily obtained at almost any season of the year. The development of any one egg can be followed from the time of fertilization to its complete development. The smallness of the eggs and the chitinous-like cocoon are the most objectionable features to contend with. Notwithstanding these facts, this group of annelids has been almost completely neglected by investigators in the study of cell lineage.

Moore, in his paper on The anatomy of an American Discodrilid (Bdellodrilus illuminatus)," refers incidently to his 'embryological studies' in the course of his investigations, but has published no account of the Discodrilid development.

Salensky, in his paper on "The development of Branchiobdella an European Discodrilid, parasitic on Astacus fluvitalis," gives an account of the cleavage, axial relation, origin of the germ layers and the formation of the adult structures. But the points of chief importance are so inadequately described and imperfectly worked out, that his results have no special significance in the study of Discodrilid development.

The development of Bdellodrilus philadelphicus, one of the Discodrilidae, has, so far as I can learn, never been worked out. It is in many respects a very important form, not only from the standpoint of development, but from its adult anatomical structure as well.

The more important points in the following paper may be briefly summarized as follows:

1. The cleavage is unequal and regular, but may be variable in some eggs. A very small cleavage cavity is present. The gastrula is formed by the epibolic process. The blastopore occupies a very small area on the ventral surface. Its point of closure corresponds to the median ventral side of the adult worm.

2. The early cleavage planes are definitely related to the future organs of the adult worm; i.e., every cleavage foreshadows


EMBRYOLOGY OF BDELLODRILUS 145

the position of some definite future formation. The large macromere D after the formation of d^ divides very unequally, the smaller cell becomes the entomere D and the larger cell becomes the mesomere d^ (M). The entire mesoblast is derived from the large cell M after its equal cleavage. The primary mesoblasts M, M, or meso-teloblasts, are completely grown over by the derivatives of X and the cleavage cells of the third generation of ectomeres. The descendants of the primary mesoblasts are differentiated into two distinct groups of cells. The first group becomes the dorsal mesoderm of the adult worm. The second the mesodermal germ bands, becomes the ventral and lateral mesoderm. The cells of the first group remain undifferentiated until late development. The latter becomes differentiated into muscle tissue much earlier than the former.

3. When completely formed, the germ bands consist of three distinct strata of cells: (a) An outer stratum, ectoblast from one to two cells thick, which is produced by the three generations of ectomeres. This layer persists as the definitive hypodermis and secretes the cuticle; (b) A middle stratum, which gives rise to the nervous system and the nephridia; (c) An inner stratum, mesoblast which gives rise to all of the mesodermal elements, blood vessels, septa, reproductive organs, etc.

4. The middle stratum is composed of eight distinct longitudinal rows of cells, which at first lie at the surface and form part of the general ectoderm (ectoblast), but afterwards become completely covered over by the ectoderm. There are four rows in each germ band, terminating at the posterior end in a large cell or teloblast. The inner or ventral neural row on each side gives rise to the corresponding half of the nervous system. The three remaining rows of cells (nephridial) on either side, give rise to the nephridia. The outer nephro-teloblast often proliferates but few cells.

5. The brain or cephalic ganglia take their origin from the extreme anterior ends of the neural rows and are distinctly independent of the ectoderm.

6. The cleavage of the entomeres A, B, C, and D is continued to the end without delay. The entire digestive tract,


146 GEORGE W. TANNREUTHER

with the exception of the very short stomodaeum and proctodaeum is derived from the entomeres. The proctodaeum is on the dorsal side of the tenth segment. The stomodaeum is formed at the apical pole. The embryo is completely tm'ned on itself, i.e., the extreme anterior and posterior ends are in immediate contact. The outer or curved surface, becomes the ventral side of the future adult worm.

NATURAL HISTORY

The Discodrilids occupy rather a unique position in the annelid group. They resemble the Hirudinea in their parasitism, in their general shape, in the presence of an anterior and posterior sucker and in the existence of chitinous jaws. The last character is not found in any other oligochaet, but occurs in a large number of leeches. These facts, perhaps not important in themselves, are indications of a very close relationship between the Discodrilids and the Hirudinea, a group which they approach, not merely in such habits as the formation of the cocoon in which the eggs are enclosed, but in many other points of internal and external structures. The fundamental differences between the two groups are not numerous and are not of such importance as has been assigned them by different writers. The Discodrilids are classified as a distinct family of the Ohgochaeta.

Bdellodrilus philadelphicus occurs very abundantly on Cambarus virilis, especially in the early spring and summer months. A few may persist throughout the entire winter in their natural habitat.

For convenience, the animal may be divided into three distinct regions; the head (pharynx), the body proper, and the posterior sucker. The head is much broader than the anterior body segments. The head is composed of four distinct annuli, which perhaps represent distinct segments. The first or peristomal annulus is divided into very mobile dorsal and ventral lobes or lips, which exhibit slight median emarginations, but are otherwise entire. It has sensory hairs and papillae, which are common in this family. The fourth annulus is very narrow.


EMBRYOLOGY OF BDELLODRILUS 147

The middle two appear as muscular rings. The chitinous jaws are triangular, the dorsal with a single tooth, the ventral jaw with a pair of smaller teeth. No lateral mucous glands which are very common in some of the species are present.

The body proper consists of eight strongly bi-annulate somites or rings. The anterior somites are longer and broader than the posterior. When contracted, the minor annuli of the somites are telescoped within the major annuli. The fifth, sixth, and seventh somites are sexual. The first, second, third, fourth and eighth somites are nephridial. The spermatheca is broad, thin walled, and nearly cylindrical. The penis is carried to the exterior by the eversible bursa, into which its projecting end is received. There is a conspicuous prostate in addition to the large glandular sperm sac. These parasitic forms remain attached to the ventral surface of the host throughout their entire life history. The eggs are deposited on the ventral surface of the host, more abundantly where the water is kept in constant motion by the movement of the appendages. Each egg is enclosed in a distinct separate stalked cocoon. The base of the stalk is firmly attached to the host. The deposition of eggs occurs during the entire year, if the parasites be kept in aquaria at room temperature. In their natural habitat eggs are not deposited during the severe winter months.

BRIEF OUTLINE OF DEVELOPMENT

The cleavage of the ovum takes place with considerable precision and regularity. Especially is one impressed with this striking phenomenon, after following the cleavage of many ova. The only perceptible variations being (a) slight differences in the time at which the individual cells divide; (b) slight variations in the size of the same cells in different ova. The rate of cleavage varies somewhat with temperature. Occasionally all the cleavage cells of an individual egg are nearly equal and it is impossible to orient the embryo before the germ bands begin their formation. This, however, is an exception, rather than a usual occurrence.


148 GEORGE W. TANNREUTHER

As development progresses the variations between individual embryos become less apparent and as far as can be recognized, do not affect the final result.

The history of the cleavage is distinguished by three well marked periods, namely: oblique, transitional, and bilateral. In the first period, which extends to the twenty-four-cell stage, the germ layers are differentiated, and the parent cells, which give rise to the future organs are definitely marked out.

The first cleavage is nearly transverse to the median longitudinal axis of the adult worm. The second cleavage plane occurs at an angle of forty-five degrees to the first. The third cleavage plane is horizontal and separates the four ectomeres above from the four macromeres below.

Three generations of four ectomeres each are successively separated, from the macromeres A, B, C and D. The first generation of ectomeres (a^, b', c^ and d^, are formed in a right handed direction. The second generation of ectomeres (a-,b^ c- and d-), are formed in a left handed direction. The third generation of ectomeres (a^*, b^, c^ and d/*), are formed, in a right handed direction. From these twelve ectomeres the entire ectoderm is formed.

The ectomere d- gives rise to all or nearly all the ectoderm of the trunk, to the nervous system and to the nephridia.

The oblique type of cleavage is maintained in the division of macromeres. At the close of the oblique period the embryo consists of twenty-four cells (text fig. 6 and fig. 36). The relation of the cleavage cells to the germ layers is as follows:

4 macromeres entpderm

1 mesomere mesoderm

„„ , fl9 ectoderm

20 ectomeres. < , ,,,. , , , , . ,.

1^ 1 (d^). .ectoderm, nervous system, nephridia

Bilateral division now occurs in some of the ectomeres, while others may continue to divide obliquely. The transitional period shows both types of cleavage. Oblique cleavage persists in some of the cells until the fiftieth or more cell stage.

In the third period, the cleavage becomes essentially bilateral


EMBRYOLOGY OF BDELLODRILUS 149

in most of the cells and the teloblasts of the right and left halves of the embryo are formed. Bilateral symmetry now becomes definitely established and the animal increases in length very rapidly.

CLEAVAGE 1. DESIGNATION OF CLEAVAGE CELLS

In the designation of the cleavage cells, for the sake of uniformity and convenience, I have for the most part adopted the system followed by Wilson in his "Cell lineage of Nereis," and Lillie in his study on "The embryology of the Unionidae." The first four cells (macromeres) are designated by the capital letters A, B, C and D. The generations of micromeres (ectomeres) by the small letters a, b, c, and d. The first index number indicates the generation to which the ectomere belongs. Thus a\ b^" or c^'i'- or d^'^ all belong to the first generation; c^, b',d2'* belong to the second generation, etc. A, B, C and D correspond to the vegetal pole; a, b, c and d to the apical pole. When a cell divides the products receive the designation of the parent


cell with the addition of a further index number; thus b


2 ,1


lb2.


Exceptions to this rule are made only in the case of special cells, which, for convenience, receive special designations: thus d^ of the second generation of ectomeres becomes the 'first somatoblast' and is designated by (X), and its small derivatives by x^, x2, x^ etc.; d^ the 'second somatoblast' is designated by (M).

2. TYPES OF CLEAVAGE

a. The oblique period of cleavage: one to twenty-four cells

First cleavage: The first cleavage occurs about five to ten hours after the deposition of the egg. The time varies somewhat with external conditions. The plane of division passes through the area where the polar bodies are formed (fig. 1) and divides the egg into two very unequal parts, AB and CD (text fig. 1 and fig. 2). The smaller of the two cells AB is anterior, and


150


GEORGE W. TANNREUTHER




EMBRYOLOGY OF BDELLODRILUS


151



1 b


2 '^



Fig. 1 Two-cell stage from apical pole view. Fig. 2 Early three-cell stage from apical pole Fig. 3 Four-cell stage from apical pole view. Fig. 4 Four-cell stage from vegetal pole.

a-h, median longitudinal plane of future adult; 1-1, first cleavage plane; 2-2 second cleavage plane; a, anterior; h, posterior; r, right side; I, left side.

the larger cell CD is posterior. The cleavage at first is very deep and the cells are rounded, but soon they begin to press against each other and flatten at their point of contact. Before the second cleavage begins the egg assumes its original elliptical shape and the point of contact externally, between the two cells is represented by a mere line or shallow groove. No actual fusion of the two cells ever takes place; sections always show a distinct line of separation between them.

The deutoplasm is equally distributed in both cells. The cytoplasm surrounding the nucleus contains very little yolk


152 GEORGE W. TANNREUTHER

material. This makes it possible, not only to recognize the position of the nucleus, but to be able to make out the exact position of the cleavage spindle in the living egg.

Second cleavage: The second cleavage is meridional and takes place at an angle of forty-five degrees to the median plane of the future adult. The two cells divide at different times (occasionally both cells divide simultaneously). These two cleavages taken together represent the second cleavage in other annelids. CD divides first into two very unequal parts (text fig. 2 and figs. 3, 78). The division of AB is nearly equal (figs. 5 and 6). The largest cell, D, is posterior. B is anterior, inclined a little to the right. C is right (text figs. 3-4) and A is left with reference to the median axis of the future worm. The large cell D has a tendency always to divide first. The exact formation of the four macromeres must be carefully worked out, and correctly understood, since their position largely determines the orientation of the future organs.

For descriptive conveniences the region of the first generation of ectomeres will be considered as the upper or apical pole and the point directly opposite, as the lower or vegetal pole. The centers of the upper and lower poles of the dividing ovum, coincide with the median longitudinal plane of the adult worm. The poles however may be shifted somewhat anteriorly or posteriorly, with reference to the macromeres, more especially to D in the formation of d-. When viewed from the upper pole A and C are in contact, while B and D are separated. But when viewed from the ventral pole A and C are separated and B and D are in contact (figs. 6-7). This extensive cross furrow found at the vegetal pole is also present in forms like Nereis, Clepsine and Crepidula; while in those forms like Unio, in which the greatest mass of the four macromeres is concerned in the formation of ectoderm instead of endoderm, the cross furrow is greatest at the animal pole. These cross furrows ('Brechungslinie' of Rauber) have no special significance in the development of Bdellodrilus, as the cleavage of the macromeres is carried to the end, immediately after the three generations of ectomeres are formed. In those forms like Nereis, where the cross furrow


DESCRIPTION OF PLATES

All drawings were made with a camera lucida under a magnification of about 125 diameters. All whole amount drawings, with one or two exceptions, were made from the living egg. The variation in size of the surface views is due to a difference in the size of the eggs. The sections were not uniformly magnified. Stippling has been adopted for the sake of clearness.


REFERENCE LETTERS

a., anterior mi. a., minor annulus

bl., blastopore mj.a., major annulus

c, ciliated cells mo., mouth

C.C., cleavage cavity p., posterior

C.I., cerebral lobes pb^~', polar bodies

coe., coelom ph., pharynx

e.cp., egg capsule pr., proctodaeum

ec, ectoderm so.mes., somatic mesoderm

en., entoderm sp.mes., splanchnic mesoderm

gn., ganglia st., stomodaeum

7nes., mesoderm v., ventral

A, left macromere

B, anterior macromere

C, right macromere

D, posterior macromere

aS b^, cS dS a'S etc., first generation of ectomeres a^ b^, c^, d^, a^-^', etc., second generation of ectomeres a^, b^, c', d^, a^'\ etc., third generation of ectomeres a"*, b*, c"*, d, etc., fourth generation of micromeres X = d^ first somatoblast X, X, right and left proteloblasts X('), neuroblast Z(2), X(3), X(4), nephroblasts M = d'* second somatoblast , 7n, secondary mesoblast x^-x', small derivatives from X N, posterior end of nephric rows Nc, posterior end of neural rows nc, neural rows np, nephric rows


153


PLATE 1

EXPLANATION OF FIGURES

1 Surface view of an unsegmented ovum, to show the polar bodies and the cleavage nucleus.

2 Two-cell stage from the upper pole.

3 Two-cell stage; cell CD dividing.

4 Three-cell stage from the upper pole; division of CD is complete.

5 Same stage as preceding; cell AB dividing.

6 Four-cell stage from the upper pole; the cleavage spindle for the first ectomere forming.

7 Four-cell stage, ventral view.

8 Four-cell stage viewed from the left side.

9 Same as the preceding, viewed from the right side.

10 Four-cell stage from the upper pole, showing the formation of the first ectomere.

11 Five-cell stage from the upper pole, d^ formed.

12 Stage showing the cleavage spindle of the second ectomere.

13 Six-cell stage from the upper pole; cleavage spindles for third and fourth ectomere forming.

14 Eight-cell stage from the upper pole; the macromeres are considerably flattened.

15 Same as the preceding, from the ventral pole; similar to the same view of the four-cell stage.

16 Eight-cell stage from the left side.

17 Same as the last showing the behavior of the macromere D in the formation of the first somatoblast.

18 Stage a little later than the preceding.

19 Nine-cell stage from the left side after the formation of the first somatoblast.

20 Xine-cell stage turned a little to the left, so that all the cells are visible.


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EMBRYOI.OGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 1



Kline, del


156 GEORGE W. TANNREUTHER

persists until late development, it serves as an unmistakable point of orientation.

Figures 8 and 9 show the four-cell stage from the left and right sides. The dorsal ventral axis of A, B and C is about the same as that of D, but immediately after the formation of the first generation of ectomeres, the cells A, B and C shorten and become more rounded (f g. 16). In later stages of development these cells often become very much flattened and cause the developing embryo to appear unusually large, when viewed from the upper or lower poles.

Third cleavage (eight-cell stage): In the formation of the first generation of ectomeres (d^, c^, b^ and a^, each of the four macromeres divide obliquely. The ectomere end of the cleavage spindle is uppermost. The macromere D divides first; d^ is budded off from D towards the upper pole, in the direction of the hands of a watch (dexiotropic), (figs. 10-11). We have thus a fivecell stage. Each of the macromeres C, B and A next bud off a small cell towards the upper pole. These are not formed simultaneously, but in the invariable order g\ h^ and a\ Thus there occurs successively, a six, a seven and an eight-cell stage (figs. 11-14). In figure 13, an upper pole view, D and C have divided and A and B are preparing to divide. In both A and B the asters of the ectomere end of the spindles are visible. The position of the opposite end of the spindles are indicated by circles. This figure shows the oblique nature of the cleavage spindles. The spindle in A points to the space between A and B. The spindle in B points to the space between B and C. In figure 14, an eight-cell stage, the exact relation of the ectomeres and macromeres are shown as they normally appear from the apical pole. The position of the first generation of ectomeres is obvious. They suggest a possible rotation, after their formation, through an angle of about forty-five degrees in the direction of the hands of a watch. If actual rotation did occur there would be no difficulty in explaining their final position. But the fact that the cleavage spindles are oblique and the position of the completely divided nucleus can be definitely determined, before there is any indication of the cytoplasmic division of the


EMBRYOLOGY OF BDELLODRILUS 157

parent cell in the formation of the daughter cells, suggest that the apparent rotation process is not mechanical or even produced by pressure of the macromeres. The formation of d^ in figure 10 shows how the process takes place; d^ is budded off obliquely from the macromere D over the inner posterior edge of A and becomes partly imbedded in A. Its final position is determined by the direction of the cleavage spindle. This characteristic method in the formation of the ectomeres is quite a prevalent one. It occurs not only in the eggs of annelids, but in those of the molluscs and polyclads as well.

Fourth cleavage: A nine-cell stage is reached in Bdellodrilus by the division of the macromere D in an oblique direction. Figure 16, an eight-cell stage viewed from the left side, shows the position of the macromeres A, B and C with reference to the macromere D, before the formation of the ectomere d^. The large macromere D contains about two-thirds of the volume of the dividing ovum. In preparation for the formation of d^, D elongates in an obhque direction at an angle of about forty-five degrees to the horizontal plane of the developing embryo. The ventral anterior portion of D shifts forward beneath A, B and C (fig. 17). After the formation of d-, D takes a position directly beneath the first generation of ectomeres, and completely covers the inner ends of A, B and C (figs. 19-20). In some instances D is shifted more anterior and completely covers the ventral surface of the other macromeres (fig. 20) ; but in most cases, as in the nine-cell stage, D occupies the region of the ventral pole, directly beneath the first generation of ectomeres (figs. 22-23). The formation of d^ is shown in figures 16 to 19. The division is equal in most cases. When unequal, d'^ is the larger cell. In figure 20, a nine-cell stage turned to the observers left so that all the cells are visible, A and B are preparing for the formation of a- and b-. In most the succeeding stages d-, the 'first somatoblast' will be designated by the capital letter X. It is also the first cell of the second generation of ectomeres. The formation of a-, b' and c- is shown in figures 20, 21, 22 and 24 in side and top views respectively. The second generation of ectomeres, with the exception of d^ (X), is about the same size as those of the


PLATE 2

EXPLANATION OF FIGURES

21 Nine-cell stage, viewed from the right side.

22 Same stage as the preceding, from the upper pole; spindles for the second generation of ectomere are forming.

23 The same stage from the ventral pole.

24 Twelve-cell stage from the upper pole; spindles for the third generation of ectomeres are forming.

25 Thirteen-cell stage from the left side; the cell d^ nearly formed.

26 Same stage as the preceding from the right side; the embryo is considerably elongated.

27 Fourteen-cell stage from the right side; X dividing to form x^.

28 Same stage as the preceding, ventral view.

29 Fifteen-cell stage from the upper pole; c' budded off symmetrical with d^.

30 Fifteen-cell stage from the left side; upper pole turned considerably to the left.

31 Same stage as the preceding from the right side; the small cell x^ is drawn out between c^ and X.

32 Fifteen-cell stage, ventral view, as a transparent object, with the position of all the cells indicated; drawn from a prepared specimen, cleared in xylol.


158


EMBRYOLOGY OF BDELLODRILUS


PLATE 2


■G. W. TANNREUTHER





C-/



. A R




. c


H


d'


>.::&,:■;?




jill^d'




-p^


3 ■■"• -^'


X


3/


Kline and Tannreuther,


deL



-;r'-~ ^


y^


vX


-D




e' 32



159


JOUBNAL OF MORPHOLOGY, VOL. 26, NO. 2


160 GEORGE W. TANNREUTHER

first. The order of their formation is d- (X), c-, b-, a^, the same as the first generation. Figure 24 illustrates the twelve-cell stage from the upper pole. The cells are somewhat flattened. The macromere D is located a little to the left of the median longitudinal plane, while X is symmetrical with reference to the median axis of the future worm.

The cleavage spindles of the third generation of ectomeres form in an oblique direction. The thirteen-cell stage is reached by the formation of d. The manner in which d is formed, is rather unique when we take into consideration the size and position of D with reference to the other macromeres A, B and C (figs. 23-25). It is budded off from the outer surface of D and takes up a position symmetrical with c^ The fourteencell stage is reached, by the formation of x^ (fig. 27), it is budded off from the median right side of X. Its final position is between X and C. Figure 28 shows the same stage as the preceding in ventral view, turned a little to the observer's right.

Immediately after the formation of x^ the macromere C buds off c', thus making a distinct fifteen-cell stage (fig. 29). The order of formation of the third generation of ectomeres is the same as the first and. second. Figures 29, 30, 31 and 32 show the fifteencell stage in dorsal, left, right and ventral views respectively. In figure 33, a'^ and b^ are formed and. the first generation of ectomeres are preparing to divide; d' and c^ divide first; at the same time x^ is budded off from X, symmetrical with x^, between D and d'^ thus making a twenty-cell stage (fig. 34). Figure 35 is a side view of a twenty-two-cell stage after a^ and b^ have divided. This figure represents an anterior posterior elongation of the embryo, which is a very common occurrence. The cells, taken as a mass, are very plastic and may assume different shapes. This peculiarity is only secondary and has no special significance. The cells become more spherical before division and flatten out somewhat after the division is complete.

The twenty-three-cell stage is reached by the formation of x^ from the upper posterior side of X between c^ and d^ (fig. 36).

The division of the first generation of ectomeres is unequal and. radial rather than oblique. From the twenty- to the thirty


EMBRYOLOGY OF BDELLODRILUS 161

cell stage several types of cleavage are present; oblique, radial and bilateral. This period of variable cleavage will be designated as the transitional period.

b. The transitional period of cleavage: twenty- to thirty-cell stage

After the formation of x'^ there is a short inactive period and in many of the developing embryos, the cleavage furrows become very indistinct. Cleavage is again initiated by the formation of d^ from the large macromere D. The cleavage is oblique and very unequal (text fig. 5 and fig. 85).

The smaller cell is almost completely hidden when first formed. It is budded off directly between A and B, near the ventral anterior surface (fig. 37). The smaller cell persists as D (entomere) and the larger cell d^ or M becomes the 'second somatoblast.' After the formation of M the entire endoderm is contained within the entomeres A, B, C and D (figs. 37-38).

The germ layers are now distinctly separated and the embryo at this stage of development is composed of twenty-four cells (text figs. 6-7). Nereis at the same period of differentiation, shows thirty-eight cells. Unio (Lillie) at the time of the separation of the germ layers contains thirty-two cells. This difference is due, in case of Bdellodrilus to the lagging of division in the cells of the upper pole.

The composition of the embryo at the twenty-four-cell stage is as follows:

Entomeres A, B, C, D 4

Ectomeres of first generation 8

Ectomeres of second generation 4

Ectomeres of third generation 4

Mesoblast M 1

First somatoblast derivatives 3

24

Many of the cells during the transitional period have a definite shape and if separated from the cell complex, they could be readily recognized. The embryo at this stage of development is somewhat spherical (figs. 36-38). Immediately after


162


GEORGE W. TANNREUTHER

D



Fig. 5 Twenty-four-cell stage in ventral view, to show the division of the large macromere D. The larger of the two cells d^ (M) becomes the 'second somatoblast.' The smaller cell D, becomes one of the four entomeres; D is scarcely visible from the exterior.

Fig. 6 Same as the preceding, in an apical pole view.

Fig. 7 Twenty-four-eell stage, ventral view, shows the bilateral division of M. D after the bilateral division of M becomes more distinct from surface view.

Fig. 8 Horizontal section of an early embryo to show spindles in the formation of x^ from either proteloblast X, X.

Fig. 9 Horizontal section little later than preceding, to show the small cell x^-x^; apical pole view.


EMBRYOLOGY OF BDELLODRILUS 163

the establishment of the germ layers, the bilateral division of the 'first and second somatoblasts' occurs. The bilateral division of the 'second somatoblast' usually precedes that of the first; occasionally they divide simultaneously.

c. The bilateral period of cleavage: twenty-five-cell stage

The first bilateral cleavages occur in the first and second somatoblasts (text fig. 7 and figs. 37-43). The small superficial cells of the lower pole are derived from the second and third generation of ectomeres and from the derivatives of X. The arrangement of these cells with reference to the blastopore is shown in figure 42. The entomeres A, B, C and D are partly grown over by the other cells and the open space becomes the blastopore. It is bounded anteriorly and laterally by small cells from the second and third quartettes, and posteriorly by the primary mesoblasts M,M. Its hinder lip, which is formed by the primary mesoblasts, lies anterior to the center of the lower pole. The closure of the blastopore takes place by a convergence of the cells from all sides. The principal growth of cells is from in front backwards, formed by the derivatives of the second and third generation of ectomeres (figs. 42, 43, 51). The entomeres now divide very rapidly and the cells soon become smaller than those of the ectomeres, which grow over them (figs. 43, 51).

3. THE FIRST SOMATOBLAST

The history of the 'first somatoblast' in Bdellodrilus is of considerable interest when considered from the standpoint of its origin and its derivatives. When first formed from the posterior macromere D, it contains one- third of the entire bulk of the developing embryo. As already described, it first buds off the small cell x^ on the right, x- symmetrically on the left and a third cell, x^, on the median posterior upper side. These three small cells are symmetrical with reference to the median longitudinal axis. The fourth cleavage divides the somato


PLATE 3

EXPLANATION OF FIGURES

33 Seventeen-cell stage from the upper pole; spindles in the first generation of ectomeres forming.

34 Twenty-cell stage from the upper pole; cells c^~\ cU~' and x- just formed.

35 Twenty-one-cell stage from the right side; b^~\ new cell; this embryo is unusually elongated.

36 Twenty-three-cell stage from the upper pole; d'~' and x^, two new cells formed; embryo considerably flattened.

37 Twenty-four-cell stage from the ventral side; the unecjual division of D has just occurred; D partially visible; the cleavage spindle of M forming.

38 Twenty-five-cell stage, ventral view; division of M complete and the spindle for the first bilateral division of X is forming.

39 Twenty-five-cell stage from the left side.

40 Twenty-five-cell stage from the upper pole; embryo is nearly spherical.

41 Twenty-six-cell stage, ventral view.

42 Same as the preceding, ventro-anterior view.

43 Twenty-nine-cell stage, same aspect as preceding; a"*, b"* and c* are the three new cells formed.

44 Forty-two-cell stage from the upper pole; increase in number of cells due to the rapid division of the ectomeres.


164


EMBRYOLOGY OF BDELLODRILUS

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PLATE 3



42 X'

Kline and Tannreuther, del.


43 X/


165


PLATE 4

EXPLANATION OF FIGURES

45 Sixty-three-cell stage from the upper pole.

46 Same stage as the preceding, from the ventral pole; very few of the ectomeres are visible.

47 Seventy-cell-stage, ventral view. Increase in number due to division of small cells. Consult table of cleavage. First nearly equal division of the proteloblasts, X, X. This division separates the neural and nephridial elements.

48 Seventy-two-cell stage, ventral view.

49 Little later than the preceding stage, to show x^ and x".

50 Embryo, ventral view, to show the first division of the nephroblasts; transverse axis greater than the longitudinal.

51 Embryo, ventral view, turned anteriorly to show the blastopore.

52 Embryo to show the lengthening of the anterios-posterior axis. The small cells, x", x", are good points to mark the orientation of the different figures. All figures on plate are similarly orientiated with reference to the right and left sides of the embryo.

53 Stage a little later than the preceding.

54 Embryo from upper pole, to show derivatives of x^ between the nephroblasts.

55 Embryo with upper surface turned posteriorly, to show the rapid division of the ectoblast cells.

56 and 57 Show that either nephroblast X^-^) or X(') may divide, to form the three nephroblasts on either side.


166


EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 4



X-X

Kline and Tannreuther, del


167


168 GEORGE W. TANNEEUTHER

blast into two equal parts, right and left (figs. 40-41). These two cells, for convenience in description, will be called the posterior right and left proteloblasts. At the fifth division each proteloblast buds off a small cell, x^, external to x^ and x- respectively (fig. 44). At the sixth division each of the proteloblasts buds forth a small cell, x^ on either side of x, beneath the derivatives of c^ and d^ (text fig. 8). At the seventh division each of the proteloblasts buds off a small cell, x" on the ventroanterior edge at the junction of the two cells (fig. 46).

At the eighth division each proteloblast, on either side of the median plane of the embryo, divided into two equal parts (figs. 47, 48, 93). The four cells formed become the posterior teloblasts, X<i^ and X*, right and left of the median axis (fig. 47). X'l' without any further division, becomes the neuroblast on either side, and X'^' becomes the nephroblast on either side of the median axis (figs. 47-48). Next X'-', right and left, divides equally, giving X<2* and X^' (figs. 50, 52). Next either X'-" or X*^' divides equally. If X*^ divide, which is the common occurrence, we get X'3' and X'^'. But if X<-' divide instead of X^^\ the final result is the same. In either case, we get four teloblasts on each side (one neuroblast and three nephroblasts) as shown in figures 56 and 57. Next X'-^ on either side divides very unequally and gives rise to x^ on the anterior ventral outer surface (fig. 49).

The progeny of the 'first somatoblast,' when the teloblasts are completely formed, is twenty cells. Table 2 shows the derivatives of the 'first somatoblast.'

TABLE 2

x'x'x*x^V' x'x'x'x'


1/ /^


X'


(') x^


^^^'^ i Son'iQloblast \<Xx)*

^ Proteloblast ProteloWest


EMBRYOLOGY OF BDELLODRILUS 169

4. THE SECOND SOMATOBLAST

Immediately after the formation of X from the posterior macromere D, d^ is budded off from D (figs. 24-25). Next D divides very unequally in an oblique direction and gives rise to d^ (M) the 'second somatoblast,' as previously described. Figures 37 and 85 show the position of D and M after the cleavage of the macromere D. The twenty-five-cell stage is reached by the bilateral division of M (figs. 37-38). The cells M, M at first are a little to the left of the median plane, but later in course of development they become symmetrical to the longitudinal axis of the adult worm.

Soon after the bilateral division of M, the 'second somatoblast,' each cell M,M right and left buds off five or more small cells directly beneath the first generation of ectomeres (figs. 86-87). It is impossible to detect these cells except by means of sections, hence the uncertainty as to their exact number. They are characterized by their large nuclei with homogeneous staining chromatin and they contain but little yolk material. These small cells divide once or twice soon after their formation from the primary mesoblasts and then remain inactive until late embryological development, at least until after the germ bands are completely formed and the embryo has undergone considerable differentiation (as the formation of the lumen of the digestive system, etc.).

These undifferentiated mesodermal cells occupy the region which becomes the central dorsal side of the embryo, at the point where the developing worm is completely turned on itself (figs. 92, 98-99). The history of these cells can be readily followed through their different stages of development, so that there can be no question as to their exact origin and history. When the embryo begins to straighten, the progeny of these small cells extend toward either end and form the splanchnic and somatic mesoderm on the dorsal side of the worm. This secondary mesoderm later becomes continuous with the primary mesoderm, which forms directly from the mesoblasts M,M.


170 GEORGE W. TANNREUTHER

5. THE ENTOBLAST

The formation of the entoblast in Bdellodrilus represents an unusual type of development among the annelid worms. The macromeres A, B, C and D, after the formation of d\ give rise to the entire entoderm, D is greatly reduced after the formation of d^ The position of the entomeres is shown in figures 40 and 42. In figure 40 A, B and C appear rather large, because of the flattened condition of the cells. In figure 42 (from the ventral pole) the cells are rounded and appear more normal. The position of the entomeres and their boundary cells are distinctly shown. This figure shows more clearly the bulk of the entoderm, when compared with the mass of the entire egg. In figure 43 (a twenty-nine-cell stage) A, B and C have divided nearly equally. This division is considered by some investigators as the formation of the fourth generation of micromeres; d^ of the D quadrant has formed earlier. Figures 44 and 45 (apical pole views) show the upper outer edge of the entodermal cells. In figure 46 (the same stage as preceding from the ventral pole) a very small part of the entodermal and ectodermal cells are visible. This figure shows the prominence of the four large cells, which later form the ten teloblasts. These four large cells, from their position, resemble the four large entomeres, which are so prominent in many other forms. These cells (X, X, M, M), according to Selensky, share equally in the formation of the germ layers, i.e., ectoderm, endoderm and mesoderm are produced by each of them.

In forms like Clepsine, Crepidula and others, at a similar or later stage of development, the entomeres are very prominent and the ectomeres with the first and second somatoblasts, form a cap of cells on their upper surface. In Bdellodrilus the conditions are different. The ectomeres and the entodermal cells form a cap of cells on the upper anterior surface of X, X, M and M. This difference is due to the prominence of the first and second somatoblasts, which constitute the greatest bulk of the embryo. At about the seventy-cell stage the ectodermal and endodermal cells are nearly uniform in size (figs. 47-49). In


EMBRYOLOGY OF BDELLODRILUS 171

figure 51 (a little later stage) the blastopore is nearly closed. This early closure of the blastopore in Bdellodrilus, is due largely, to the ventro-anterior shifting of the macromere D over A, B and C in the formation of the somatoblasts (figs. 18, 85).

The closure of the blastopore, in some of the annelids, occurs at a very late stage of development. In Clepsine the teloblasts give rise to rows of cells, which pass anteriorly around the entomeres A, B and C beneath the edge of the blastodisc or cap of cells. The blastodisc with these rows of cell cover about half of the entomeres. By the downward growth of the blasdodisc and the concrescence of the germ bands, the closure of the blastopore is completed. The closure of the blastopore in Clepsine occurs on the ventral side, nearer the anterior end. In Bdellodrilus, the germ bands are not formed until later and take no part in the closure of the blastopore. Text figures 10 to 13 show the position of the ectoderm, entoderm, and the first and second somatoblasts, at different stages in the closure of the blastopore. The region of closure is similar to that of Clepsine.

At the time of the formation of the secondary mesoblast just beneath the first generation of ectomeres, the entire entoderm is situated in the anterior half of the embryo. But soon after the formation of the m cells (text fig. 12 and fig. 86), the entodermal cells by a rapid proliferation extend posteriorly between the m cells and the primary mesoblast. During the formation of the primary mesoblast, the meso-teloblasts themselves are carried posteriorly, ahead of the entoderm. The entoderm, thus becomes situated between the m cells or secondary mesoderm above and the mesoblast bands or primary mesoblast below. The entoderm in reality never reaches the posterior limit of the meso-teloblasts, as shown in figures 98 and 99.

The interior of the developing embryo, now consists of a solid mass of small entodermal cleavage cells (figs. 95-99), heavily laden with yolk. These cells are readily distinguished from the surrounding mesodermal cells, by their deeper cytoplasmic stain. Figure 99 (a vertical longitudinal section near the median plane) shows the position of the entodermal cells in the embryo.


172 GEORGE W. TANNREUTHER

As the embryo elongates, the entodermal cells increase in number. This process of growth is continued until the digestive tract is completely formed. Figure 99 shows the anterior and posterior limits of the digestive tract, which is formed from the four entomeres. The anterior end shows a distinct lumen, while the posterior end is yet a solid mass of cells. The entire digestive tract, except the insignificant stomodaeum and proctodaeum, is entodermal in origin. The proctodaeum is not formed until the time of hatching. It occurs on the dorsal side of the tenth segment. The embryo is completely turned on itself (fig. 99) and brings the anterior and posterior ends of the digestive tract in close proximity. The differentiation of the digestive tract begins anteriorly and progresses posteriorly. As growth continues, the outer cells of the entodermal mass form an epithelial layer. At first the cells are somewhat flattened, but soon take a columnar position, and form the columnar epithelium of the digestive tract. The cells within soon lose their staining properties, break up and. serve as food for the developing embryo.

The digestive tract in its course of development, passes through the following stages: the first stage is represented by the four entomeres A, B, C and. D; the second, stage by a solid mass of cleavage cells (the cell boundaries are often very indistinct) within the center of the embryo; the third stage by an elongation of the entodermal mass as the larva lengthens, and. by the establishment of a lumen; the fourth stage by a thin layer of flattened epithelium and. later a columnar epithelium; the fifth stage, the cells within the epithelial layer serve as food ; and sixth the formation of the stomodaeum and. proctodaeum.

GENERAL HISTORY OF THE GERM BANDS

The term 'germ bands' has been variously interpreted by different investigators on cell lineage. The term germ bands, or the German equivalent 'Keim Steifen,' is usually restricted to the strata derived, from the teloblasts, the ectoblastic layer being excluded. It is held by others that the germ bands of annelids are purely mesoblastic.


EMBEYOLOGY OF BDELLODRILUS 173

Balfour, Hatschek, Goette, Kowalevsky and many others made use of the term 'mesoblastic bands' as the equivalent of the germ bands. In Hirudinea, according to Whitman, the germ bands are composed, of three distinct layers; the ectoblastic, mesoblastic and the neuroblastic elements. Wilson gave the same interpretation in his studies on The embryology of Lumbricus." In Bdellodrilus the term 'germ bands' includes the three strata of cells as in Hirudinea and Lumbricus.

1. INNER STRATUM OF THE GERM BANDS

After the formation of the teloblasts, five on either side of the median axis (one neuroblast, one mesoblast and three nephroblasts), the mesoblasts or meso-teloblasts are the first to begin the formation of the germ bands by a forward proliferation of cells near the posterior hp of the blastopore (text figs. 12, 13, 17). The plane of division, is nearly at right angles to the formation of cells in the secondary mesoblast (text figs. 10, 13). The cells of the mesoblast bands are considerably smaller than the teloblasts from which they originate. They grow forward between the entoderm and the ectoderm and finally meet at the anterior end of the larva. As these bands grow forward they become several cells broad, but seldom more than two cells deep. Their differentiation begins anteriorly and progresses backward. The first cells of the mesoblast bands, when formed, are on the surface, but soon become covered by the ectodermal cells. As the mesoblast band, extends forward below and around the entoderm, it forces its way to the extreme anterior end of the embryo beneath the ectoderm. It finally encloses the digestive tract on the ventrcl and lateral sides and becomes continuous with the secondary mesoblast on the dorsal side. The two mesoblast bands fuse first at the anterior end along the median ventral side and subsequently with the dorsal secondary mesoderm. In figure 99 (a longitudinal section) the mesoblast is differentiated into splanchnic and somatic layers, with the coelom between. The longitudinal muscles become differentiated before the circular. At the extreme posterior end the


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Vertical section to the left of the median plane.

Vertical section of a ninety-cell stage.

Vertical section of an embryo at the time of closing of the blasto


Figs. 10-13 Diagrammatic figures to show the ventro-posterior extension of the ectomeres, in the closure of the blastopore.

Fig. 10 Thirty-three cell stage, taken a little to the right of the median plane.

Fig. 11

Fig. 12

Fig. 13 pore. The mesoblast bands have just begun.

The heavy stippling represent endoderm; the light stippling mesoderm, and the unstippled the ectoderm or ectomeres. m, secondary mesoblast; M, mesoblasts; hi, blastopore; hlc, point where the blastopore closes; A', derivatives of the 'first somatoblast;' mcs, mesoblast bands.

meso-teloblasts are represented by an undifferentiated mass of cells, which later give rise to the musculature of the last three segments of the worm, and are directly concerned in the formation and movements of the posterior sucker.


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175


2. MIDDLE STRATUM OF THE GERM BANDS

The middle stratum of the germ bands can readily be distinguished while the embryo is still nearly spherical. Upon close examination it is seen that the ectoblast cells are arranged into four distinct rows, on either side of the median ventral axis (figs. 65-66). Each row terminates posteriorly in a large cell or teloblast.

Text figures 15 and 17 and figure 58 show the early formation of these rows of cells. Sections of these various stages show that



Fig. 14 Surface view from upper pole, to show the position of the ten teloblasts. The meso-teloblasts or mesoblasts have budded off eight or ten cells in the formation of the mesoblast bands. Their position is indicated by dotted outline. The broken outline represents the region of the entoderm. The position of the ectoderm is indicated by a continuous line.

Fig. 15 Third horizontal section from the top, passing through four of the large nephroblasts. The spindles represent the beginning of the first division in the formation of the middle germ band. The anterior end and the right side of the section are a little below the horizontal plane.

Fig. 16 Seventh horizontal section from the top. It passes through the upper portion of the entoderm and the secondary mesoblast (m).


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these rows of cells form a part of the general ectoderm, being partly covered here and there by adjoining cells. In later stages of development, these rows of cells become completely covered as they gradually sink beneath the surface, and thus come to lie between the mesoblast and the ectoderm or ectoblast.



Fig. 17 Twelfth section from the top, to show the anterior extension of the mesoblast bands below and around the entoderm.

Fig. 18 Nineteenth section from the top, to show the lower side of the mesoblast. The section passes below the entoderm. The spindles represent the beginning of the first division of the neuroblasts to form the neural rows.

Heavy stippling represents entoderm; light stippling mesoderm and the unstippled portion the ectoderm. The sections of figures 15-18 were eight micra thick. There were 21 sections in all. a, anterior, and p, posterior, represent the respective ends of the cleavage cells, but not the future ends of the embryo; r, right; I, left; ent, entoderm and ect, ecoderm.


3, OUTER STRATUM OF THE GERM BANDS

This stratum forms the definitive ectoderm and needs no further description at this point of development.

The embryo now elgongates very rapidly, and the general shape of the adult worm becomes recognizable. The teloblasts become less and less distinct, until finally the cell rows terminate posteriorly in a group of small cells. The mesoteloblasts extend farther posteriorly than the neuro-teloblasts. New cells are always formed from the anterior surface of the teloblasts. There can be no question as to the origin of the germ bands from corresponding teloblasts, as their formation


EMBRYOLOGY OF BDELLODRILUS 177

can be followed step by step. The mesoblastic and neuroblastic portions of the germ bands can be traced to the anterior end of the embryo. The meso-teloblasts are the last to disappear. They are distinct until after the formation of the stomodaeum and its connection with the pharynx. The concrescence of the germ bands begins anteriorly and progresses posteriorly.

THE ECTODERM AND ITS PRODUCTS

The three generations of ectomeres are given entirely to the formation of the ectoderm, which later becomes differentiated into the definitive hypodermis, with its glands, the cuticle and the anterior and posterior ends of the digestive tract. The ectoderm includes, in addition to the above, all of the teloblasts, except the two larger and deeper ones which represent the mesoblasts. The reason for regarding the eight teloblasts and their derivatives as a part of the general ectoderm, is on account of their origin and position. In position, they are superficial at first and can not be distinguished from the general ectoderm, except by their arrangement in rows. Small cells are budded off from the teloblasts, which form the trunk ectoderm. In Clepsine these teloblasts are at first superficial at the posterior end of the embryo. In Lumbricus they are found directly in the general ectoderm, and beyond question form a part of it.

1. THE NERVOUS SYSTEM

The nerve chain in Bdellodrilus first appears as a double row of cells, nearly uniform in size. Each row of cells originates from a single cell, the neuroblast. The neuroblasts, when first formed, are widely separated, but symmetrical to the median axis of the body. Figures 47 and 48 (in ventral view) show their position when first formed by an equal division of the proteloblasts X and X. They take up their position on either side of the mesoblasts (figs. 47, 48, 93). When first formed the neuroblasts are turned somewhat anteriorly as shown in the horizontal section of figure 93. This movement of the cells to their final position, independent of the former position of the cleavage


178 GEORGE W. TANNREUTHER

spindle, is a common occurrence in Bdellodrilus. In some instances it is necessary to employ sections, in order to determine the origin of cells. The transverse axis of the embryo at this stage is often greater than the longitudinal (figs. 49-50). This condition persists for a brief period only, during the formation of the teloblasts. As the embryo increases in length the neuroblasts are carried more and more posteriorly (figs. 56-57).

In order to get a better understanding of the origin and orientation of the neuroblasts — X^^^ right and X* left — with reference to the other teloblasts, the figures of plate 4 are so arranged that the left side of the developing embryo corresponds to the reader's left. In figure 45 the upper pole is turned a little posteriorly, to show the upper outer edges of the entodermal cells. Figure 47 (from ventral pole) shows some of the ectodermal cells. The remaining figures are either turned forward or backward on their transverse axes. The ectomeres x** and x*^ right and left serve as good points for orientation (figs. 46-53) . After the formation of the teloblasts, bilateral symmetry is fully established. The meso-teloblasts, however in some instances, are still a little to the left of the median axis. This variation in the symmetry of the mesoblast does not in any way change the end result. In the early history of the germ band formation the teloblasts X^^^ and X^^^ are slightly separated, while X^^' and X^-^ are widely separated from the corresponding teloblast on the opposite side (figs. 56, 58). The neuroblasts and the nephroblasts begin their proliferation of cells to form the germ bands, about the same time (fig. 58). At this stage of development, the exact orientation of the embryo is distinct. Since the embryo is completely turned on itself, the further use of the terms, apical and ventral poles, is significant only as being convenient in description. The mouth, as stated above, is formed in the center of the apical pole and the anus in close proximity on the dorsal side of the tenth segment. Figure 59 (upper pole view) shows the complete curvature of the embryo. The heavily shaded portion represents approximately, the boundary between the anterior and posterior ends. This figure shows that the teloblasts are coming more and more in a straight line. Since the two ends of


EMBRYOLOGY OF BDELLODRILUS 179

the embryo are in immediate contact, it is impossible, except by longitudinal sections, to determine the exact point of separation. The ectoderm of the anterior end of the embryo, which is derived from the three generations of ectomeres is continuous with the ectoderm derived from the 'first somatoblast.'

The separation of the two ends of the embryo becomes recognizable in the early formation of the germ bands, as shown in figures 59 and 60. The posterior and ventral shifting of the neuroblasts (figs. 58-60) continues until all of the teloblasts are in a direct line. The small cells between the teloblasts are derived from the first somatoblast. In viewing the embryo from the upper pole (which now corresponds more to the anterior and posterior ends of the future animal) the germ bands extend laterally, downward and forward, being curved somewhat posteriorly as they pass from the upper to the lower pole (fig. 59). The meso-teloblasts in figure 58 are still visible from the exterior. In figure 59 they are almost grown over, while in figure 60 they are completely covered. This is due to the posterior shifting of the neuroblasts and the growth of the ectomeres from above and below. In an embryo viewed from the right side (fig. 61, a little older than fig. 60), the position of the neural and nephric rows of the germ band are shown. As the rows extend anteriorly they are more difficult to distinguish from the ectoderm. The neural rows alone can be followed to the extreme anterior end. The posterior end of the embryo is widely blunt, while the anterior end is more rounded. The heavily shaded portion represents the point of separation between the two ends.

Figure 62 represents the same embryo from the upper pole, with the ends of the embryo rotated or turned a little posteriorly. In figures 63 and 64 (from right and left sides respectively) the embryo is more elongated and the point of separation between the two ends is more distinct. The neuroblasts are lagging in their posterior extension. Their position is median ventro-posterior, as shown in figure 65. Their concrescence is not yet complete at the posterior end. In the following stages of development the cells of the neural and nephric rows divide


PLATE 5

EXPLANATION OF FIGURES

58 Embryo from upper pole, tilted a little to the right. The position of the ten teloblasts are shown; the small cells between the teloblasts on the surface are derived from x® and x^ on either side.

59 Same view as the preceding; the neuroblasts have migrated a little posteriorly and are approaching each other.

60 The ecto-teloblasts are nearly in a direct line; the germ bands have begun to form; the two primary mesoblasts M, M are no longer visible from the exterior; the transverse heavily shaded joortion shows the approximate point of separation between the two ends.

61 Embryo viewed from the left side; the posterior end is extremely blunt.

62 Same embryo as preceding, from the upper pole (upper pole corresponds to the anterior and posterior ends). Shows very strikingly the close proximity of the two ends.

63-65 Represent the same embryo from the right, left and ventral sides respectively. The ectoderm which partially covers the germ bands is not shown.

66 Embryo from upper pole; bilateral symmetry is well marked; the teloblasts are considerably reduced by the time they come in contact with their fellows on the opposite side.


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EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 5



6^ )(

Tannreuther, del.


181


PLATE 6

EXPLANATION OF FIGURES

67 Embryo turned slightly to the left to show the anterior and the posterior ends; the embryo at this stage begins to rotate within the egg membrane.

68 The same as the preceding from the ventral pole, turned a little to the left.

69 Embryo viewed from the right side; condition before the posterior end becomes drawn out or pointed.

70 Embryo from the upper pole; shows compressed condition of the two ends; at this stage the embryo rotates very rapidly.

71 Embryo viewed from the right side; the teloblasts are partially visible at the posterior end; the tapering of the posterior end is well marked.

72 Embryo to show the overlapping of the ends ; indications of segments are visible anteriorly; the stomadaeum is distinct.

73 Unusual condition, where the two ends remain in immediate contact until after the form of the worm is distinct; this occurs in eggs with an unusually large cocoon.

74-6 Different stages in the final growth of the embryo.

77 Condition of embryo at the time of emergence from the egg.


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G. W. TANNREUTHER


PLATE 6



75

Kline and Tannreuther, del


183


184 GEORGE W. TANNREUTHER

very rapidly and gradually become covered by the ectodermal cells as they sink beneath the surface (figs. 66-72), and form the middle stratum of the germ bands. In a nearly median longitudinal section (fig. 99), the relation of the parts are shown. The neural plate at different points shows the formation of ganglia. The anterior end of the section passes through the exact median plane and does not show any gangha. The neuroblasts still persist at the posterior end.

The entire nervous system arises as two simple longitudinal rows of cells, and each row is produced by the continued proliferation of cells from a single cell, 'the neuroblast.' This proof is established by the study of surface preparations in connection with sections taken in different planes through the developing neural elements. The neural rows can be followed to the extreme anterior end, where they pass up around the pharynx and give rise to the cerebral ganglia on either side (fig. 96) by a thickening of the anterior extremity of the corresponding neural rows. There are exactly four rows of cells in the middle stratum of each germ band. The outline of the rows can be easily seen in surface views of the living embryo (figs. 63-66). They are more distinctly marked at the posterior ends, and become less distinct anteriorly, which is due to the more advanced development; that is, each row becomes double, then triple, etc. (figs. 68-69) and at the same time, its boundary lines become less distinct.

2. THE EXCRETORY SYSTEM •

After the bilateral division of the 'first somatoblast,' each proteloblast contains the neural and nephric elements of their respective sides. According to Whitman, these two cells are called the neuro-nephroblasts. But when each proteloblast X, X divides equally the neural and nephric elements become separated, X*^^ neural and X*-' nephridial (figs. 47-48). The nephroblast X'-' on either side next buds off a very small cell x^, which becomes ectodermal (fig. 49) . Immediately after the formation of this small cell, X'-' divides nearly equally, and forms


EMBRYOLOGY OF BDELLODRILUS 185

X'-' andX*^' on either side (figs. 50-54). Both cells are nephridial. This fact perhaps is made more suggestive by the behavior of X*"' and X'^\ Either of these cells may divide equally, but never both in the same embryo. In either case we have three teloblasts derived from the nephroblast X'^* on either side. The cells of the nephridial rows are somewhat smaller and narrower than those of the neural rows. In some cases the outer nephridial row of cells is very short. In other embryos it is composed of but one or two cells and its presence is hard to verify, suggesting a possible disappearance in the group. As stated above, the nephridia arise in connection with a continuous nephric cord of ectoblastic origin, which forms a part of the middle stratum of the germ band and lies along side of the neural row. Each nephric cord terminates at the posterior end in three teloblasts. Thus the entire nephric cord of cells is formed by the continued division of the 'nephroblasts,' which agree precisely with the neuroblasts in structure, action and mode of origin. The nephric cord at first is composed of three rows of cells posteriorly, but passing forward the rows are no longer definitely separated and. the nephric cord or plate consists of an irregular series of cells which extend anteriorly to the posterior end of the pharynx. The formation of the nephridia progresses from in front backwards and keeps pace with the formation of new segments in the embryo. The beginnings of a pair of nephridia are found in each of the main segments. Only two pairs of nephridia are retained in the adult worm. The details of the formation of these segmental organs have not been worked out.

Berg considers the entire nephridia in Criodrilus as mesodermal in origin; Whitman held the extreme opposite view, that the entire nephridium was ectodermal in origin; while Wilson regarded the nephridia as being part mesodermal and part ectodermal in origin. In Bdellodrilus the nephridia are ectodermal. The anterior pair occurs in the first, second, third and fourth body segments. The left nephridia of the anterior pair, extends from the first to the third segments inclusive, while the right extends from the second to the fourth segments inclusive. Both


186 GEORGE W. TANNREUTHER

have a common opening on the dorsal side of the third segment. The funnel of the left occurs in the second and. that of the right in the third segment. The posterior pair is found in the eighth segment. Each nephridium has a separate opening to the exterior on the dorsal side of the eighth segment.

GROWTH

The developing embryo does not increase appreciably in bulk until after the teloblasts are formed. Up to this period it is merely a division of the egg content into the various cell complexes. Even at this stage the increase in the long axis of the embryo is brought about by a decrease in the transverse diameter. Figures 50 and 55 show the transverse axis greater than the longitudinal, while in figure 56 and 57 the longitudinal axis is greater, due more to a change in shape than to growth. The egg content is very plastic and when removed from the cocoon the egg membrane, in most cases, is not of sufficient tenacity to retain the embryo intact. The ten teloblasts are shown in figures 56 and 57.

The first increase in length is due to the formation of the mesoblastic portion of the germ bands (text figs. 17-18). The neuroblastic and nephroblastic portions of the germ bands begin simultaneously after the meso-teloblasts have formed eight or ten cells (text figs. 15, 18 and fig. 58). Figures 58-71 show the various stages in the formation of the germ bands. Figure 71 is about the last stage when the germ bands can be detected externally. A longitudinal section of figure 71 near the median axis shows a differentiation of the germ bands into their incipient organs (fig. 99). From this point of development, growth is very rapid, and the embryo begins to rotate on its transverse axis. The movement is produced by the action of cilia, which occur on the large ectodermal cells in the median ventral half of the anterior end of the embryo (figs. 96-99). These ciha disappear before hatching, but the cells from which they are produced persist as a part of the ectoderm. The anterior and posterior ends are no longer in immediate contact, as in figure 71, but begin to overlap. The ends of the embryo


EMBKYOLOGY OF BDELLODRILUS 187

now take the position within the egg membrane of the least resistance to their further growth. Figure 74 shows the overlapping of the ends. The stomodaeum is completely formed and the annuh of the pharynx are visible. Figure 73 shows an unusual condition in the position of the ends. At this stage of development the animal often turns on its longitudinal axis, largely on account of the action of the muscles, and, instead of the convex side being ventral, it now becomes dorsal. This rotation on its longitudinal axis has no significance, as has been thought by previous investigators, in the later stages of development. The animal is extremely plastic and may assume any position or shape, as shown in figures 74 and 76. Figures 77 shows the completely developed animal at the time of emergence from the cocoon. The number of the segments is distinct. This peculiar form of growth within the cocoon is merely adaptive. Occasionally, when the cocoon is of an unusual size, the developing worm is less bent on itself.

A COMPARATIVE STUDY OF DIFFERENT FORMS

In following the cleavage cells of annelids, molluscs and polyclades, one is impressed with the striking resemblances in their different stages of development. If this marked similarity alone were a sufficient criterion for a basis of classification, some of the most widely separated forms, when considered from the standpoint of their early development, would be grouped as closely related species. How can such resemblances in development be explained in animals which are so unlike in their late stages of growth? Are they merely the result of such mechanical principles as surface tension, alternation of cleavage, and pressure, or is the nature and structure of the protoplasm the common cause? According to Driesch, 'the striking similarity' between the types of cleavage in annelids, molluscs and polyclades does not appear startling and is easy to understand, since cleavage is of no systematic worth. However, the more recent investigators on cell lineage, according to Heath, look upon the early cleavage stages as something more than a mere


188 GEORGE W. TANNREUTHER

manifestation of simple mechanical forces. Rather are the blastomeres the expression of the active intrinsic forces, which control development from the earliest stages unto the end. Gravity, surface tension, cohesion and pressure no doubt are effective, but not to the extent that they become the controlling or coordinating agents in development. The early cleavages are as important as those occurring in later life, and may even be considered more so. Also the long continued resemblances which exist in the development of these different forms from the earliest segmentation of the eggs are as fundamental and deep seated as are the homologies existing in the adults."

The number of these resemblances in the annelids and molluscs is surprisingly great. In all forms accurately studied, the first three generations of ectomeres give rise to the entire ectoderm. The mesoblast arises at the fourth division of the posterior macromere D. The remaining members of this quartette and the macromeres produce the entoderm. The division and position of the cells up to the twenty-four or thirty-cell stage are identical in many different species. Beyond this point Wilson believes a divergence between the two classes ensues, and that development proceeds upon two entirely different lines. However, subsequent investigators have shown that the supposed differences are more superficial, and that the points of resemblances become more numerous and extend throughout longer periods of development. Lillie ('95) showed that points of resemblance existed in the lamellibranchs and the annelids, and that in both classes there is an essential similarity between the development of the 'first somatoblast.' In annelids this structure develops to a greater extent than in Unio, but the two have many points in common.

Mead ('97) and Conklin ('97) showed that the rosette series had the same origin and position in aimeUds and molluscs, and that in both it probably gave rise to the apical sense organ. According to Conklin, it also gave rise to the cerebral ganglia, while Mead considered this particular point doubtful. Furthermore, Mead in his annelid studies demonstrated that the same cells in five different annelids gave rise to the entoderm; that


EMBRYOLOGY OF BDELLODRILUS 189

the head kidney in Amphritrite and Nereis developed from the same cells. Conklin further states that the axial relation of all the blastomeres, with the possible exception of the macromeres, are the same in both the annelids and molluscs, and that the larval mesoblast in Crepidula and Unio arises from the same group of ectodermal cells.

Heath ('99) found that the prototroch in anneUds and molluscs was homologous, and that the twenty-two to twenty-five cells concerned have exactly the same origin, direction of cleavage, and destiny. Also that the remainder of the first quartette, forming the head vesicle with its rosette series and moUuscan cross cells or intermediate girdle cells, has in all probability, the same fate in both. He found many other resemblances and concludes :

Thus it is seen that not only in the origin and position of the various quartettes do resemblances appear, but that the early cleavage of these are in many cases cell for cell the same. In later stages close cell homologies cease, but the relation of the cell groups and their development in giving rise to larval or adult structures follow along much the same path. After passing these facts in review and considering the various structures in detail and modifications which they undergo, one fact presents itself with greatest clearness, that between Ischnochiton and the annelids the resemblances are moref undamental and closer than are the differences.

For a more direct comparative study of Bdellodrilus with the annelids and molluscs, special references will be made to Clepsine (Hirudinea), Dinophilus (Polychaete) , and Unio (Lamellibranch). In all these forms the first and second cleavages are meridional and divide the eggs into four unequal macromeres (text figs. 19-22). In Dinophilus C and D are approximately posterior and A and B are anterior. In the other three forms B is anterior, D posterior, C right and A left. In each case D is the largest cell; A, B and C are nearly equal; B is usually the smallest when variation occurs. The eight-cell stage has the same structure, and in all probability arises in the same manner in the four forms, the only apparent difference being the much greater relative size of the ectomeres in Dinophilus than in the three remaining forms. The first cleavage plane in Bdellodrilus


190


GEORGE W. TANNREUTHER




Fig. 19 Four-cell stage of Unio, upper pole (after Lillie).

Fig. 20 Four-cell stage of Bdellodrilus, upper pole.

Fig. 21 Four-cell stage of Dinophilus, upper pole (after Nelson).

Fig. 22 Four-cell stage of Clepsine, upper pole (after Whitman).

occurs at nearly right angles, while in Unio and Clepsine it is inclined at an angle of about forty-five degrees to the sagittal plane of the future adult. In Dinophilus the direction of the first cleavage is in doubt. The second cleavage plane in Unio, Clepsine and Bdellodrilus occurs at an angle of about fortyfive degrees to the sagittal axis. The origin of the ectoderm, the entoderm and the mesoderm is approximately the same in each form.

1. THE FIRST SOMATOBLAST

The first somatoblast in each instance is derived from the large posterior macromere D (text figs. 23-26). The cell d(X) is extremely large and occupies a median posterior position. In Clepsine (Whitman) d- (X) is called the 'neuro-nephroblast.'


EMBRYOLOGY OF BDELLODRILUS 191

It divides into two, four and finally eight large cells called the teloblasts; the middle stratum of the germ bands is derived from them. These eight teloblasts are arranged into two groups of four cells each. Each group, which later is composed of four rows of cells, produces the middle stratum of the germ band on the corresponding side. The inner row of each band lies ultimately near the median ventral plane and gives rise to the corresponding half of the nervous system. The adjoining rows — 'nephroblasts' — give rise to the nephridia. The derivatives of the outer row are still in doubt, but probably take part in the formation of the ectoderm.

In Dinophilus (Nelson) d^ (X) is formed by a laeotropic division of the macromere D (text fig. 25) ; D is much smaller than X. Immediately after the formation of X, x^ is budded off to the right at a low level. Next x^ is budded off to the left at a higher level than x^; x^ is next formed by a dexiotropic division from the dorsal side, a little to the left. Next X divides equally and produces X and X, right and left. These two large cells correspond to the proteloblasts in Bdellodrilus. Finally X on either side divides equally, and produces the two teloblasts on each side of the median plane. These four cells, according to Nelson, correspond to the posterior teloblasts of Nereis. They also correspond to the neuroblasts and nephroblasts of Bdellodrilus. The division of X in Dinophilis and Nereis differs no more than do the corresponding divisions in Nereis and other annelids (Amphitrite, etc.). At the time of the closure of the blastopore in Dinophilus, the descendants of X are distributed dorsally and laterally to the posterior stem cells. In Neries the main bulk of the descendants of X lay on the vegetal side of the stem cells.

In Unio (Lillie) the 'first somatoblast' X is formed by an unequal division of D (text fig. 24) in a median posterior position; xi is budded off from X, just behind C on the vegetal pole; next x^ is budded off from X symmetrically with x^ on the right side, just posterior to d^; next x^ is formed from X towards the apical pole, posterior to d'-^; then x^ is budded off from X anteriorly, towards the vegetal pole. This division of X does

JOURNAL OF MORPHOLOGY, VOL. 26, NO. 2


192


GEORGE W. TANNREUTHER




Fig. 23 Nine-cell stage of Clepsine, upper pole (after Whitman).

Fig. 24 Nine-cell stage of Unio from behind (after Lillie).

Fig. 25 Nine-cell stage of Dinophilus, left side (after Nelson).

Fig. 26 Nine-cell stage of Bdellodrilus, left side.

not occur in this manner in Dinophilus, Bdellodrilus or even in Nereis. The fourth division in the above three forms is equal and bilateral, while in Unio the fifth cleavage of X is the first bilateral division and forms X, X right and left. Next X, X on either side divides nearly equally and gives rise to the shell gland. These four cells might be regarded as the posterior teloblasts, which occur in other forms, as in Nereis and Dinophilus.

In Bdellodrilus X is formed by an equal division of the macromere D (text fig. 26), and takes a median posterior position First xi is budded off from X to the right, posterior to C. Then x2 is budded off to the left, symmetrical with x^ and posterior to d^ Next x^ is formed from the median dorsal anterior edge of X, between d^ and c^ Now the first bilateral division of X takes place and forms the proteloblasts X, X, right and left. Each of the proteloblasts bud off x^ x^ and x" respectively. At the


EMBRYOLOGY OF BDELLODRILUS 193

next division each proteloblast divides nearly equally, and gives rise to X^i\ neuroblast, and X*^^, nephroblast, on each side of the median axis of the embryo. Next each nephroblast divides nearly equally and produces X'-'^ and X'^\ Now a very interesting thing happens; either X^^) or X^^^ divides (but never both in the same egg) and produces the three nephroblasts on each side, which are designated as X^^)^ x^^' and X^^*'. In Clepsine only two of these teloblasts are concerned in the formation of the nephridia. The lateral teloblasts, as stated above, are probably ectodermal.

These four forms unquestionably show that there is a marked similarity in the cleavage of the 'first somatoblast,' not only in widely different individuals in the same group, but in individuals of widely separated groups. This comparison could be extended to other groups or forms, but the above will suffice for our purpose.

2. THE SECOND SOMATOBLAST

In Clepsine, the 'second somatoblast' has rather a unique origin. It is formed at about the twelve-cell stage. D, after the formation of X, becomes directly the 'second somatoblast' or M. (These cells are differently designated by Whitman; D is represented by x, X by x^ and the mesoblasts by x and xy.) M divides nearly equally and produces the right and left mesoblasts, from which the inner stratum of the germ bands is formed.

In Bdellodrilus, M is formed by a very unequal division of the macromere D, at the twenty-four-cell stage (fig. 85). The larger cell or M is formed in front of X. It is inclined a little to the left of the median axis. The first division of M is equal, producing the mesoblasts, one on either side. These primary mesoblasts now bud off a number of small cells, directly beneath d^ and c^ (figs. 86-87). It is very difficult to make out the exact number of these small cells, since they are not visible externally. There are at least twelve formed, six on either side from each mesoblast. After this small group of secondary mesodermal cells are formed, the mesoblasts M, M, give rise to


PLATE 7

EXPLANATION OP FIGURES

78 Two-cell stage, horizontal section; CD dividing.

79 Four-cell stage; horizontal section taken above the center of the egg.

80 Same as the preceding, with plane of section below center.

81 Nine-cell stage, parasagittal section, to right of median plane.

82 Horizontal section of a nine-cell stage, taken four sections from the top. Taken from embryo composed of 21 sections, each eight micra in thickness.

83 Same as the preceding; sixth section from top.

84 Same as figures 82 and 83; fifteenth section from top.

85 Parasagittal section of a twenty-four-cell stage; plane of section little to left of median axis. This figure shows the unequal division of the macromere D in the formation of the second somatoblast.

86 Thirty-three-cell stage. Parasagittal section to left of the median plane. Shows the formation of the secondary mesodermal cells (m cells) from the primary mesoblasts.

87 About the same stages as the preceding, to show the distribution of the yolk in different cells.

88 Horizontal section of an eighteen-cell stage, fourth section from top. Series composed of 20 sections, each eight micra in thickness.

89 Same as the preceding; seventh section from top.

90 Taken from series same as figure 88; eighth section from top.


194


EMBRYOLOGY OF BDELLODRILUS

G. W. TANNREUTHER


PLATE 7



88

Tannreuther, del


195


PLATE 8

EXPLANATION OF FIGURES

91 Taken from the same series as figures 88 to 90; eighth section from the top.

92 Horizontal section of an eighteen-cell stage; sixth section from the top; shows the nem-oblasts and nephroblasts.

93 Same as the preceding; tenth section from top; this figure shows the persistence of the cleavage spindles after the cell membranes are distinct.

94 Same as 92 and 93; fourteenth section from top; taken from a series of 20 section each eight micra thick. This figure shows the upper side of the macromere D wedged in between the other cells.

95 Transverse section of an embryo represented by figure 64; section taken at plane 2 — 2, or at a region corresponding to plane 2 — 2, of figure 98.

96 Transverse section of stage corresponding to figure 63; section taken at plane 2 — 2 ; figures 95 and 96 shows germ bands only partially covered by the ectoderm.

97 Transverse section of embryo represented in figure 69; section taken at level marked by line 2 — 2; here the germ bands are completely covered by the ectoderm.

98 Longitudinal section, near median line, of stage represented by figure 69.

99 Longitudinal section of an embryo represented bj' figure 71 ; plane of section near median line.


196


EMBRYOLOGY OF BDELLODRTLUS

G. W. TANNREUTHER


PLATE 8



98 P ^^WM

Tannreuther, del.


197


198 GEOKGE W. TANNREUTHER

the inner stratum of the germ bands. The plane of division in the formation of the primary mesoblast is at right angles to that of the secondary.

In Dinophilus, M is formed at the twenty-nine-cell stage, by an unequal division of D. M is much larger than D, as in Bdellodrilus, and is in front and below X, slightly to the left of the median plane. The division of M is now delayed until the seventy-two-cell stage, when two small cells are budded off anteriorly towards the vegetal pole, close to the line of junction of the two mesoblasts. At the next division two small cells are budded off, one on either side of the first pair. The following cleavages are teloblastic and produce the mesoblastic bands. The mesoblasts do not move into the cleavage cavity as in many other annelids, but remain on the surface until covered by the ectoderm.

In Unio, at the thirty-two-cell stage, M is formed by a very unequal division of the macromere D. The first division of M is equal and bilateral. Their position is immediately behind the entomeres. The next division of the two mesoblasts is very unequal, two small cells, m, m, are budded off at the posterior lip of the blastopore. Later the mesoblasts are included within the segmentation cavity, where they take up their final position behind the archenteron and give rise to the definitive mesoblastic germ bands with lateral teloblasts.

From the forms compared above it is very evident that there is a remarkable similarity with Bdellodrilus, not only in the early cleavage stages, but in the establishment of the germ bands as well. Thus cells having the same origin and lineage have the same final result in a wide series of forms (d"* the mesoblasts). Again, cells of unlike origin have a different fate (first and second somatoblasts) ; or cells of a different origin may have the same fate (d^ of annelids and the second and third generation of ectomeres in polyclades) . Then cells of the same origin may have a different fate (a-- in Unio and Bdellodrilus). These contradictions, however, are far less striking than the resemblances. The 'first somatoblast' in each of the above four forms gives rise to the ventral plate and all or nearly all of the trunk ectoderm, while


EMBRYOLOGY OF BDELLODRILUS 199

the 'second somatoblast' produces the definitive mesodermal elements of the adult animal.

3. VARIATIONS IN THE METHOD OF MESODERMAL FORMATION

All annelids and molluscs which have been carefully studied show that the ectoderm arises from the three generations of ectomeres, the mesoderm from M and the entoderm from the remaining cells. There are, however, a few minor variations in forms like Clepsine, Crepidula and Nereis. In polyclades the mesoderm is directly associated with the ectomeres. The second and third generations of ectomeres, as in Discocoehs, produce the mesoblast, and the macromeres the entoblast. In molluscs and annelids the mesoderm is more closely associated with the macromere D. There is considerable variation in the cleavage of D in the formation of the 'second somatoblast;' in some forms D is given over entirely to the mesoderm; in other forms it shares equally, or in still others it contributes but little to the mesoderm.

Forms in which the mesoblast has two sources

(a) Ecto-mesoblast and (b) coelo-mesoblast. In Thalassema, Torrey distinguished between ecto-mesoblast, from the ectoderm and coelo-mesoblast from M. He states that the coelomesoblast is present in two bands, each consisting of five sub-equal cells. These are closely applied to the body wall and lie in the usual position on each side of the neural rows, but are more widely separated than in most anneUds. The ecto-mesoblast on the other hand, derived from the first and second quartette of ectomeres, is present in great abundance and many of the cells have already undergone considerable differentiation in the formation of the muscles. He further states that the two mesoblast cells, M, M, are the last to sink in at gastrulation, instead of the first, as in the case where development is more direct (Nereis and Amphitrite). The two coelo-mesoblast bands of five cells each are shown to have the same origin and early history as the mesoblast bands in other annelids. The coelo-mesoblast, which is meagerly developed in the trochophore, is clearly correlated with


200 GEORGE W. TANNREUTHER

the long duration of its free swimming and almost stationary larval existence. In all forms where there is a tropchophore stage of long duration, as is the case in all annelids with equal cleavage, the two coelo-mesoblast cells do not, in the early stages at least, bud like teloblasts. This is tue in Hydroides, some species of Polygordius, in Thalassema and Podarke.

Many of the annelids and molluscs show that the so-called ecto-mesoblast (designated, as larval mesoblast or as mesenchyme by some authors) arises from certain ectodermal cleavage cells of the second or third quartette of ectomeres and. is entirely independent of the coelo-mesoblast. In Thalassema (Torrey) ten large ectodermal cells sink in. from the ectoderm and. give rise to all the mesenchyme. Three of these cells are from the a, c and d quadrants of the third, quartette and seven from the first quartette of ectomeres. The most important source of mesenchyme in Thalassema is from the three cells of the third quartette (3d.-"-\ 3c- •^■-•\ and Sa---). The first two sink into the cleavage cavity, just before gastrulation and at first lie close to the two coelo-mesoblast cells. They soon migrate laterally and bud off simultaneously small cells toward, the M cells. They divide like teloblasts, but in the reverse order to the ordinary direction. So close is the connection of these cells with the coelo-mesoblast that one would be certainly led to think that they formed a part of these bands, unless their cytogeny had been carefully followed. Similar conditions are described by Treadwell in Podarke obscura. The progeny of these two cells forrn almost the entire mesenchyme of the post throchal region and become differentiated for the most part into muscles of the digestive tract. The progeny of the other ectodermal cell migrates to the mid-ventral line. The ecto-mesoblast cells of the first quartette sink into the primary body cavity later than those of the third; their exact cell lineage has not been traced, but probably give rise to gut musculature. This mesoblast has commonly been considered as purely larval and transitory. In some instances it is possible to determine its exact origin, but in many others merely the general region from which it arises.


EMBRYOLOGY OF BDELLODRILUS 201

During the last thirty years embryologists have differed in their conception of the origin of the mesoderm and of its phylogenetic significance. Hatschek ('78) was among the first to distinguish between mesenchyme and mesoderm, but held, after studying the embryology of Polygordius, Echiurus and Eupomatus, that these two morphologically different mesoblasts, arise from a common foundation. This same view was later put forth by the Hertwigs ('81) in their 'Coelomtheorie,' which, according to Meyer, has formed the foundation of all later work on mesoderm. Roule ('89 and '94), Burger ('91 and '94), Fraipont ('88), Hacker ('95), and others, have described the mesoblast as having a single origin. On the other hand, those who have studied the embryology of annelids and molluscs, consider the origin of the mesenchyme distinct from that of the mesoblast or coelo-mesoblast. This later view was first described by Kleinenberg ('78 and '86), and later by Whitman ('87), by Berg ('90), by Schimkewitsch ('94), by Meyer ('01), by Torrey ('03), and others.

A larval mesoblast was first described by Lillie ('95) in Unio. It arises asymmetrically from the derivatives of a- - and later migrates into the segmentation cavity, where it divides equall}^ and. becomes symmetrically arranged on either side of the midline. The derivatives of these two cells become metamorphosed into 'myocytes' and larval adductor muscles, which are functional only during larval life.

Treadwell ('97) regards both mesenchyme and mesoderm as morphologically the same tissue, the apparent difference in their mode of origin being of no significance. And, further, Wilson regards the larval mesoblast (ecto-mesoblast, because of its origin from the ectoderm) as a distinct tissue from that of the definitive mesoblast or ento-mesoblast, and states that it is homologous with the mesenchyme of the turbellarian ancestors of the annelids, while the mesoblast from which the adult structures arise is phylogenetically younger and is represented prophetically in the ontogeny of such a form as Discocoelis (polyclade) by the peculiar lateral division of M, and states that the ecto-mesoblast and endo-mesoblast are phylogenetically of


202 GEORGE W. TANNREUTHER

different origin. This same point was previously urged by Meyer.

The condition, however, found by Wilson in Nereis and Lumbricus does not indicate a hard and fast distinction between the two kinds of mesoblast. In Nereis, cells from the anterior end of the germ bands separate early and pass forward into the segmentation cavity where they give rise to the larval musculature. This corresponds exactly in structure and function with the larval mesoblast of Unio (Lillie) and Podarke (Treadwell). In Lumbricus the origin of the mesenchyme is similar to that in Nereis. These two kinds of larval mesenchyme have also been described by Eisig ('98) as occurring in the same individual (Capitella, a polychaete annelid).

In Thalassema and Podarke the larval mesenchyme arises directly from the ectoderm, while in Nereis and Lumbricus it arises from the anterior ends of the mesoblast bands. According to Treadwell, no one has yet proven that no 'mesenchyme' arises from the germ bands in cases where a larval mesenchyme exists. If we accept Wilson's view that mesenchyme and mesoderm are different phylogenetically, we must regard the two sets of larval mesenchyme which have the same structure and function, as non-homologous, or we must regard the mesenchyme and mesoderm as morphologically the same tissue and the difference in their modes of origin as of no significance. Furthermore, Wilson has pointed out that the trochophore, as it occurs at present, is more than a mere ancestral stage, for it contains in a concentrated form the anlage of the whole future body. According to Mead, the ectoderm behind the first septum in Amphitrite arises from a group of cells which surround the proctodaeum of the young trochophore and are descended from a single cell, the 'first somatoblast.' The same is true of other trochophore forms. There is no need to assume phylogenetically a new formation of ectoderm for the body as distinct from that of the head. Neither is there any necessity to assume a distinct phylogenetic origin of the larval mesoblast from that of the mesoderm.


EMBRYOLOGY OF BDELLODRILUS 203

It is evident that in Nereis and Lumbricus, both kinds of mesoblast have the same origin, and simply shows a more complete concentration of the mesoderm than in Thalassema and Podarke, where the mesenchyme is formed direct from the ectoblast. The mesoblast cells collected at the posterior end of the trochophore, which are derived from M, represent the mesoderm of the body. It is morphologically continuous with that of the head, as in Nereis, and is concentrated at this point to provide for the elongation as new segments are formed. The difference in the concentration of the mesodermal elements, as to whether they have a single or double origin in no way interferes, as already pointed out, with the morphological unity of the tissue, and as to the source of its origin, whether from the ectoderm or from the endoderm phylogenetically, we are not able to say (Treadwell).

Meyer ('01) in his study of the phylogenetic significance of the two kinds of mesoblast, gave a view directly opposed to that expressed by Treadwell. After an exhaustive review of the whole mesodermal question, he concludes that the great mass of evidence, both embryological and anatomical, points to the conclusion that in annelids, at least, there are two entirely distinct forms of mesoblast, the ecto-mesoblast (primary mesoblast) and the coelo-mesoblast (secondary mesoblast). Of these two he considers the primary mesoblast to be phylogenetically the older, and as a rule, to be derived from the ectoderm. The coelo-mesoblast, on the other hand, is regarded as a later formation, which has originated from the gonad cells.

The formation of the ecto-mesoblast in annelids and molluscs from certain cells of the first, second, third and fourth generation of micromeres, can well be regarded as vestiges or survivals of the process which occurs in all four cells of the second and third quartettes of certain polyclads. The origin of the ecto-mesoblast from the ectoderm in annelids and molluscs, partially bridges the gap between them and the polyclads. In order to have a complete homology of the mesoderm in the polyclads, annelids and molluscs, it is necessary to find a polyclad in which there is a double origin of the mesoderm. The development of the polyclad Leptoplana (Wilson) is the nearest representative to complete the homology. In Leptoplana only a part of the four quadrants of the second quartette contributes to the entire mesoderm, the typical condition in polyclads being that all of


204


GEORGE W. TANNREUTHER


the second and third quartette is mesodermal. The behavior of d^ in the polyclad Discocoehs, is also very suggestive. Here the division of d^ is equal and gives rise to two symmetrically placed cells at the posterior end of the embryo, comparable to the primary mesoblasts found in annelids and molluscs. Some investigators have even suggested that these two posterior cells in the polyclads may give rise to the mesoblast bands in this particular group. This latter point, however, has never been verified.

Table 3 shows that the first, second, third and fourth generation of micromeres, in a series of widely separated forms, may contribute to the formation of the mesoderm.


TABLE 3



1st gen.


2d gen.


3d gen.


4th gen.


Annelids:






Thalassema


part of a, b and c quad's


none


1 cell each of

a, b and c

quadrants


d^ part mes.


Bdellodrilus


none


none


none


d^ all mes.


Molluscs:






Unio


none


a2-2 (larval)


none


d^ all mes.


Crepidula


none


aS b^, c2


none


d^ part mes.


Physa


none


none


b^, c^


d^ part mes.


Podarke


none


none


a^3-2-2-2 (,3-2-l-2. (J3-2-2-2


d^ part mes.


Polyclads :






Discocoelis


none


all mes.


all mes.

none


Leptoplana


none


part of each quadrant


none


none


In case of the ecto-mesoblast a complete series could be arranged, in which all of the cells of certain quartettes contribute to the mesoblast, to those forms in which only a small part of certain quartettes is mesoblastic. Again in case of the coelo-mesoblast we have a wide range of variation, in which all of d^ is mesodermal, to those in which only a small part of d^ is mesodermal. As far as records show, Capitella is the only annelid in which


EMBRYOLOGY OF BDELLODRILUS 205

d^ does not contribute to the coelo-mesoblast. Here then we have quite a unique series ranging from those forms where the entire mesoderm is ectodermal in origin, or where it is both ectodermal and entodermal, to those where it is entirely entodermal. From the above it is evident that the entire mesoblast of polyclads is derived from the ectomeres, and, if homologies be any significance, it would be fair to conclude that this mesoblast is represented by the ecto-mesoblast in the annelids and the molluscs.

The origin and development of the mesoblast in Bdellodrilus contributes but little to the phylogenetic significance cf the primary and secondary mesoblast. Here, beyond question, when considered from the standpoint of their origin, they are one and the same tissue. Both are formed directly from the primary mesoblasts. The secondary mesoblast cells are budded oE from the two primary mesoblasts before the germ bands begin their development. Similar conditions are found in other forms, as in Lumbricus; here, however, the secondary mesoblast is formed later directly from the anterior ends of the mesoblastic germ bands. The difference is only in the point of time in their formation. In Bdellodrilus there can be no hard and fast distinction made between the two kinds of mesoblast. Both must be considered as the same tissue phylogenetically.

4. VARIATIONS IN THE SOURCE OF THE ENTODERM

In general, as stated above, the ectoderm originates from the three generations of ectomeres, the mesoderm from d^, and the entoderm from the remaining cells. The origin of the three germ layers, however, depart somewhat from the above rule in some of the annelids and molluscs. In some species cells from the first, second and third quartettes contribute to the mesoderm; in others d^ gives rise to entoderm as well as mesoderm. In all annelids and molluscs. A, B and C, after the formation of the first three sets of ectomeres, are distinctly entodermal. The macromere D, after the formation of d^ is likewise entodermal. In some forms D is the same size as its fellows, in others


206 GEORGE W. TANNREUTHER

it is reduced until it is little more than a mere nucleus, while in others it has completely disappeared as an entomere, and is given over entirely to the formation of mesoderm.

In annehds, in a gradually decreasing series, D (Nereis) is the same size as the entoblast cells A, B and C. In Dinophilus it is about half the size of these cells. In Bdellodrilus D is little more than a mere nucleus; while in Clepsine D is given over entirely to the formation of the mesoderm. In molluscs it is a fairly common condition to find the entoblast cell D smaller than A, B and C, or even greatly reduced. In Crepidula it is very little reduced; in Unio it is more than half reduced, while in Ischnochiton, D is often little more than a mere nucleus. The second somatoblast, M, may contribute to the formation of entoderm as well as mesoderm. In forms like Crepidula M is mostly entodermal. In Fiona (Casteel) the division of M in the formation of the entoderm is very similar to that in Crepidula. In Unio two small cells are budded off from M, which lie near the entoderm, and are probably concerned in the formation of that layer.

In some of the annelids the primary mesoblasts bud off small cells directly posterior to the macromeres. This number varies; in Nereis there are six to ten, and in Aricia there are but two. In many of the other annelids and also in some of the molluscs, where their cell lineage has been traced, it is found that these small cells give rise to entoderm. There are at least sixteen to twenty species of annelids and molluscs in which similar cells have been found (small cells from the primary mesoblasts.) Diverse accounts of their behavior and fate have been given by different investigators. Table 4 shows the fate of these small cells in a few of the annelids and molluscs.

In the mollusc Alpysia, according to Carazzi, each primary mesoblast buds off four small cells. Three of these are mesoblastic and one is entoblastic. This interesting condition might be considered as a transitional form or as a connecting link between those forms in which these small cells are entirely mesodermal and those in which they are entodermal. Again we could arrange a series of annelids and molluscs in which at one extreme


EMBRYOLOGY OF BDELLODRILUS


207


TABLE 4


ENTODERM


MESODERM


NOT CERTAIN


Crepidula


Amphitrite


Dreissensia


Nereis


Arenicola


Patella


Podarke


Umbrella


Spio


Thalassema


Planorbis


Serpulorbis


Fiona


Unio?


Cyclas


Ischnochiton


Limax


Aricia


Physa fontanalis




Phj^sa hyponurum




Aplysia




the entoblast derived from M is greater in amount than the mesoderm, as found in Crepidula, and at the other extreme, where but two rudimentary cells of M are entoblastic, as in Aricia.

According to Wilson, a series of this nature may indicate a gradual elimination of the entodermal element from the macromere D of the fourth quartette, and finally its complete transformation into the mesoblast. Kovalevksy ('71) suggested that this transformation shows quite forcibly that the mesoblast pole cells are to be regarded, phylogenetically, as derivatives of the archenteron, because of their close association with the posterior entoblast cell, D.

The primary entoblasts. A, B, C and D, undergo but little change until late development in those forms which possess a larval stage, and may remain in this condition until after the trochophore is developed, or until after the blastopore is closed. In those individuals with a fetal type of development, they often remain distinct until after the germ bands are completely formed, as in Clepsine.

GENERAL ADAPTATION AND INTERPRETATION OF CLEAVAGE

The cleavage of eggs of widely separated forms exhibit unique resemblances. At certain stages of development these resemblances exceed their differences. Is the persistence of these features due to the influence of ancestral inheritance, or are they due more to the adaptive conditions of their environment, to


JOURNAL or MORPHOLOGT, VOL. 26, NO. 2


208 GEORGE W. TANNREUTHER

meet the highest need of the developing animal? It has been demonstrated, again and again, in anneUds, molluscs and even in polyclads, that homologous cells of like generations give rise to like parts in the developing embryo and the adult. The occurrence of these conditions in such widely separated forms furnishes a very interesting and important phase in the study of cell hneage. The tendency has been rather to emphasize these resemblances, than to give special stress to the exact conditions which occur in any one species in its different stages of development. It is true, however, that the general form of cleavage may be inherited from a long series of ancestors, probably from some of the Turbellarian worms. But the problem of more direct importance in any one group is, why such variation in the size, form, direction and rate of cleavage?

1. IN THE CLEAVAGE OF BDELLODRILUS

In Bdellodrilus we have a determinate type of cleavage, i.e., the fetal as well as the adult structures can be shown to have a definite or direct cell lineage, and can be traced back to the unsegmented egg. The structure of the ovum is quite homogeneous, and at the time of maturation, the egg can be definitely oriented as to the future axis of the body. Before the first cleavage is complete, the parts of the ovum which give rise to the different germ layers can be traced or ascertained with a fair degree of accuracy, i.e., definitely localized parts which • give rise to definite organs or structures.

"Adaptation in cleavage can manifest itself only in three possible ways or modes of cleavage variation, which are, as has been pointed out by Lillie, Mead, Conklin and others, the following: first differences in the rate of cleavage; second differences in the size; and third, differences in the direction of cleavage."

The general plan of cleavage in Bdellodrilus, is similar to that of other forms. The ectoderm is derived from the four basal cells, by three successive horizontally formed cleavages. The mesoderm from a fourth cleavage of the posterior macromere D and the entoderm from the remaining cells. The first cleavage


EMBRYOLOGY OF BDELLODRILUS


209


in Bdellodrilus is meridional and very unequal. In the two-cell stage the larger cell is posterior and the smaller cell anterior. The larger cell divides first and very unequally, while the smaller cell divides nearly equal (text figs. 1-3 and fig. 5). In the fourcell stage D is posterior, C right, A left and B anterior, inclined a little to the right. Thus it is very evident that the four-cell stage illustrates a difference in the rate of cleavage, a difference in the size of the cells, and a difference in the direction of the cleavage. The significance of these variations may be emphasized as follows:


a. Difference in the rate of cleavage of cells

If we compare a thirty-two-cell stage of Bdellodrilus with other forms or perhaps, better, with an ideal ovum, in which there is a uniform rate of cleavage in the formation of the cleavage cells, a uniform size and a uniform direction of cleavage, a distinct variation occurs as shown in table 5.


TABLE 5






CKEPIDULA


IDEAL OVUM


NEREIS


BDELLODRILUS


First generation of ectomeres


12 9 5 2 4

32


16 8 4


16 8 4


8


Second generation of ectomeres


11


Third generation of ectomeres


4


Mesoblast


2


Entoblast


4 i 4


7



32


32


32


It is evident that in the first generation of ectomeres Bdellodrilus departs very far from the ideal condition. The first generation contains eight cells instead of sixteen. This means that the cells have divided more slowly than in the ideal ovum. In an ideal ovum these cells form the prototroch and the entire region in front of it, with the apical plate in the center. In Bdellodrilus, this region is degenerate and no trace of the apical plate appears. This indicates an adaptive modification — eight cells instead of sixteen — due to a degenerate frontal region.

In the second generation of ectomeres, the ideal number is


210 GEORGE W. TANNREUTHER

eight, while in Bdellodrilus it is eleven. This increase above the ideal is due entirely to the rapid succeeding divisions of one cell — d^, the first somatoblast. The other cells of the quartette have not divided, while d^ has given rise to three new cells. Does the behavior of d^ suggest any significance, or is it adaptive? From d- the ectoderm of the trunk region, the nephridia and the entire nervous system is derived ; d^ is not only the largest but the most actively dividing cell of the entire embryo; hence its rate of cleavage is well adapted to its resulting formations.

The number of ectomeres in the third generation is the same as in an ideal ovum; d^ however is often formed before aor b^ of the second generation. This interesting phenomenon is due to the tendency of the basal cell, D, and its derivatives to divide more rapidly than those of A, B or C. The differences in the rate of cleavage in the first, second and third generation of ectomeres, no doubt possess prospective significance, looking forward to the definitive parts. This may fairly be called adaptation in the rate of cleavage.

In Bdellodrilus the more rapidly dividing cells do not form the first functioning parts. The cells of the first quartette are the first to function, in the production of cilia for the movement of the embryo. The variation in the rate of cleavage is not due to the varjdng conditions of the media, or the dividing ovum would be uniformly affected, as a whole. Nor is it diie to the size of the individual cells, as the largest cells divide more rapidly. At the thirty-two-cell stage the ideal ovum contains four entodermal cells, while in Bdellodrilus there are seven. Here the cleavage is carried to the end without any resting stage of the four basal cells. This is due to the fact that the larva develops very rapidly and the entodermal cells must keep pace with the rapid development in order to reach their final position, just where they are needed.

b. Variation in the size of cells

The relative sizes of the cells in the early cleavage of the eggs of Bdellodrilus are adapted to the later developing parts.


EMBRYOLOGY OF BDELLODRILUS 211

The largest cell at the four-cell stage is D. Its first division is very unequal, and the smaller cell is less than one-tenth the size of the larger. It is the first ectomere of the first generation formed. The second division, in most instances, is equal; when unequal, the largest cell passes into the upper product, and forms d^, the first somatoblast. The third division is unequal, and d^ the smaller product, is again uppermost; and finally, the fourth division is very unequal and only a small portion remains as the macromere D. The greater bulk, d^ becomes the second somatoblast. In each of the above instances the larger cells form a large part of the embryo and the adult, while the smaller cells, in every instance, form a very insignificant portion. The unequal division in each instance is evidently adaptive, for the great bulk of the material passes into the two somatoblasts and gives rise to the muscular, nervous and excretory systems.

c. Variation in the direction of cleavage

Here only some of the special cleavages will be emphasized. The first division of the second somatoblast is equal, and each part forms equal parts of the mesoderm. Next, each primary mesoblast buds off five or six small cells beneath the first quartette of ectomeres. These small cells remain quiescent for a considerable period and later give rise to the dorsal mesoderm. Immediately after these small cells are formed, the mesoblast bands are begun by a forward prohferation of cells from the anterior face of the primary mesoblasts. The plane of division iS at right angles to that of the small cells. These bands extend forward between the ectoderm and the entoderm, and at the same time the entodermal cells extend posteriorly between the mesoblast bands and the group of small cells, thus separating the primary and secondary mesoderm. Here the direction of the cleavages place the. cells where they are later used in the formation of some special part, adapted for that particular region.

The first somatoblast buds off x^ to the right, x^ to the left, and x^ median dorsal anterior. X now divides equally and


212 GEORGE W. TANNREUTHER

t

each proteloblast buds off a small cell, x"*, one to the right of x^ and the other to the left of x^. Again each proteloblast buds off a small cell, x^, on either side of x^ At the next division each buds off a small cell, x^ on the ventral anterior edge. Later, x^ is budded off from each neuroblast on the ventral side. These small cells give rise to the trunk ectoderm and the larger cells to the nephridia and nervous system. Here again the cells are formed just where they are needed; the smaller on the exterior or outer surface while the larger remain within.

2. ADAPTATION IN THE CLEAVAGE OF OTHER FORMS

In following the variation of cleavage cells in annehds and molluscs, special cells can be arranged in a complete series, from those of an almost insignificant size to an extremely large cell. In following these variations, step by step, we can not fail to be convinced that these variations are adaptive to the future needs and habits of the larva and of the adult animal.

In forms with equal cleavage, the first somatoblast gives rise to the ectoderm of the trunk region. In Polydorgus, Podarke, Hydroides, Eupomatus and others with equal cleavage, d^ is the same in size as the cells of the other quadrants. Equal cleavage has been offered by some as due to a lack of differentiation in the early stages but in such forms as Podarke with equally cleavage, very early differentiation occurs, and the prominence of these early functioning parts varies according to the size of the initial cell from which they are formed.

In forms with unequal cleavage, the first somatoblast differs in size from the remaining members of the same quartette. Beginning with Amphi trite, the relative size of d- increases successively in Chaetopterus, Arenicola, Nereis, Capitella, Aricia, Spio, Clepsine and Bdellodrilus. Those forms with equal cleavage pass through a distinct trochophore stage and are characterized by an almost equatorial prototroch, a very large exumbrella, and with a very slow trunk development. In those with unequal cleavage, especially in the second generation of ectomeres, there is a gradual decrease in the prominence of the


EMBRYOLOGY OF BDELLODRILUS 213

trochophore to its approximate or complete disappearance; on the other hand, there is a gradual acceleration in the time of the trunk development, varying according to the increase in the relative size of the first somatoblast or X. Treadwell states that the extra amount of material stored in the macromere D is in some way related to the amount of somatic and mesoblastic material needed in the future organism. This statement is true of the condition that occurs in such annehds as Bdellodrilus and Clepsine.

GENERAL SUMMARY

The undivided egg of Bdellodrilus philadelphicus is nearly oval. Its median longitudinal axis through the region of the polar bodies corresponds to the median axis of the future adult. The polar bodies occupy the region which later becomes the anterior end of the embryo.

The first cleavage plane is nearly at right angles to the median axis of the resulting individual, and divides the egg into two very unequal parts. The second cleavage occurs at an angle of about forty-five degrees to the first. It divides the smaller cell nearly equally and the larger cell very unequally; the larger cell divides first. In a four-celled embryo the large cell D is posterior, B is anterior, inclined a little to the right; A left and, C right.

The ectoderm is separated from the four macromeres by a series of three oblique cleavages. The first generation of ectomeres is formed in a dexiotropic direction. The second generation laeotropically and the third in a dexiotropic fashion.

In the fourth generation of micromeres, d^ is mesoblastic. The other cells of the fourth quartette, together with the four macromeres, form the entoderm. The cleavage of the entodermal cells is carried to the end without delay, in the formation of the digestive tract, and the interior of the embryo becomes a solid mass of entodermal cleavage cells, which later become differentiated into the epithehal portion of the alimentary canal. As the core of entodermal cells grows posteriorly,


214 GEORGE W. TANNREUTHER

it separates the primary and secondary mesoderm. The extreme ends of the digestive tract are ectodermal. Among other annehds and also in molluscs, so far as is known, the entodermal cells are not broken up into cells but enter directly into the formation of the digestive tract.

The posterior cell, d^ (X), of the second generation of ectomeres is the largest cell of the segmenting ovum. The derivatives of X are symmetrically placed with reference to the median plane of the future individual. The large cell, X, gives rise to the trunk ectoderm, the nervous and the excretory systems. The nervous system is derived from the two neuroblasts. The brain is formed from the extreme anterior end of the neural rows.

The largest cell, d* (M), of the fourth generation of micromeres, gives rise to the entire mesoderm. It is the first cell to divide in a bilaterally symmetrical manner. The primary mesoblast cells, M, M, bud off five or six small cells each, beneath the first quartette of ectomeres, which give rise to the secondary mesoderm on the dorsal side of the embryo. Immediately after these small cells are budded off, the primary mesoblasts, by a teloblastic proliferation of cells, produce the mesoblast bands.

The embryo increases but little in bulk before the germ bands are formed. The embryo as a whole, during its early stages of development, is extremely plastic and may vary considerably in its transverse and longitudinal axes. The developing embryo is completely turned on itself, and the anterior and posterior ends are in immediate contact. The outer surface is ventral and the turned in portion is dorsal. This pecuHarity of development is foreshadowed in the position taken by the early cleavage cells.

At the beginning of the germ-band formation, the embryo begins to rotate on its transverse axis. This movement is due to the action of cilia, which are produced by the ectodermal cells on the median ventro-anterior end of the embryo; the rotation alternates.

As growth continues within the cocoon, the ends of the embryo soon begin to overlap. The embryo may assume almost


EMBRYOLOGY OF BDELLODRILUS 215

any position in the cocoon during its later stages of development. The embryo is completely developed before emergence; the trochophore stage is completely suppressed; the gastrulation is of the epiboHc type.

Columbia, Mo. November 3, 1914

LITERATURE CITED

Balfour, F. M. 1893 Elements of embryology. London.

Berg, R. S. 1888 Zur Bildungsgeschichte der Excretionsorgans bei Ciido drilus. Arb. a. d. zool. Inst. VViirzburg, Bd. 7, Heft 3. BiGELOW, A. M. 1899 Notes on the first cleavage of Lepas. Zool. Bull.,

vol. 2, no. 4. BtJRGER, O. 1891 Beitrage zur Entwicklungsgeschichte der Hirudineen. Zool.

Jahrb., Bd. 4. Carazzi, D. 1900 L'embriologie dell'Aplysia limacina. Anat. Anz., Bd. 18. Casteel, B. D. 1904 The cell lineage and early development of Fiona marina,

a nudibranchiate mollusc. Proc. Ac. Nat. Sci., Philadelphia. Castle, W. E. 1896 The early embryology of Ciona intestinalis Flemming

(L). Bull. Mus. Comp. Zool. Harvard College, vol. 27, no. 7. Child, C. M. 1900 The early development of Arenicola and Sternaspis.

Arch. f. Entwick. der Organismen, Bd. 9, Heft 4. Conklin, E. G. 1897 a The embryology of Crepidula. Jour. Morph., vol. 13.

1897 b Cleavage and differentiation. Biol. Lecture, Woods Hole. EisiG, H. 1898 Zur Entwickelung der Capitelliden. Mittheil. a. d. Zool.

Stat. Neapel, Bd. 13. Fraipont, J. 1887 Le genre Polygordius. Fauna u. Flora d. Golfes von

Neapel, Monographic, tom. 24. GoTTE, A. 1882 Ueber die Entwickelung der Chaetopoden. Leipzig. Hatschek, B. 1885 Entwickelung der Trochophora von Eupomatus uncina tus. Arb. zool. Inst. Wein, Bd. 6. Heath, H. 1899 The development of Ischnochiton. Zool. Jahrb., Bd. 12,

Heft. 4. Hertig, O. und R. 1881 Die Coelomtheorie. Jenaische Zeitschr., Bd. 14. Kleinenberg, N. 1879 The development of the earthworm. Quart. Jour.

Micr. Sci., vol. 19. Kowalevsky, a. 1871 Embryologische Studien an Wiirmern und Arthropoden.

Mem. de I'Acad. Imp. de Sc. de St. Petersbourg, tom. 16, no. 12. Lang, A. 1884 Die Polycladen des Golfes von Neapel. Flora und Fauna

des Golfes von Neapel, Bd. 6. LiLLiE, F. R. 1895 The embryology of Unionidae. Jour. Morph., vol. 10.

1899 Adaptation in cleavage. Biol. Lecture Woods Hole.

1901 The organization of the egg of Unio. Jour. Morph., vol. 17. Mead, A. D. 1897 The early development of marine annelids. Jour. Morph.,

vol. 13.


216 GEORGE W. TANNREUTHER

Meyer, E. 1890 Die Abstammung der Anneliden. Biol. Centralb., Bd. 7. Moore, J. P. 1895 The anatomy of Bdellodrilus illuminatus, an American

Discodrillid. Jour. Morph., vol. 10. Nelson, A. J. 1904 The early development of Dinophilus. Proc. Ac. Nat.

Sci., Philadelphia. RouLE, L. 1899 Etudes sur le developpement des annelides et en particulier

d'un Oligochete limicole marin (Enchytraeoides marioni). Ann.

Sci. Nat., (7), tome 7. Salensky, W. 1885 Developpement de Branchiobdella. Arch, de Biol.,

tom. 6, fasc. 1, p. 1. ScHiMKEWiTSCH, W. 1895 Zur Kentniss des Baues und des Entwickelung des

Dinophilus vom weissen Meere. Zeit. wiss. Zool., Bd. 59. ToRREY, J. C. 1903 The early embryology of Thalassema mellita (Conn).

Ann. New York Acad. Sci., vol. 24, no. 3. Treadwell, a. L. 1898 Equal and unequal cleavage in annelids. 'Biol.

Lecture Woods Hole.

1901 The cytogeny of Podarke obscura. Jour. Morph., vol. 17. Whitman, C. O. 1878 The embryology of Clepsine. Quart. Jour. Micr. Sci.,

vol. 18.

1887 The germ layers in Clepsine. Jour. Morph., vol. 1. Wilson, E. B. 1887 The germ bands of Lumbricus. Jour. Morph., vol. 1.

1889 The embryology of the earthworm. Jour. Morph., vol. 3.

1891 The origin of the mesoblast bands in annelids. Jour. Morph., vol. 4.

1892 The cell lineage of Nereis. Jour. Morph., vol. 6. WiERZEJSKi, A. 1905 Embryologie von Physa fontinalis. Zeit. f. wiss. Zool.,

Bd. 62.


THE MORPHOLOGY OF NORMAL FERTILIZATION IN PLATYNEREIS MEGALOPS

E. E. JUST

THREE PLATES (THIRTY FIGURES)

1. INTRODUCTION

In a previous paper on Platynereis megalops, which described the egg-laying habits, it was stated that insemination takes place in the body cavity of the female and, further, that the eggs will not fertilize when inseminated in sea water. The present paper is a description of the normal fertilization process in Platynereis. An experimental analysis of fertilization in Platynereis appears elsewhere (Just, '15).

2. NORMAL FERTILIZATION OF PLATYNEREIS

The living egg. The egg of Platynereis is compressed and irregular in shape while in the body cavity. Those eggs which happen to be uninseminated when laid gradually round out in sea-water as almost perfect spheres equatorially, but with a rather shorter polar axis. The large, centrally placed, germinal vesicle is slightly elongated in the polar direction. The largest eggs, fully rounded out, measure 180 to 200 fx. They are almost perfectly transparent, have an equatorial ring of oil drops, and a well marked transparent exoplasm or cortical layer of protoplasm with very faint granules forming a delicate mesh. In short, the living egg closely resembles that of Nereis; it is larger (but of. Wilson, '92) not so deeply pigmented, and lacks the characteristic yolk spheres of the Nereis egg.

A. Fertilization in the living egg

We may consider the fertilization of the egg under the following heads: (1) insemination, (2) penetration of the sperm, and (3) copulation of the germ nuclei.

217


218 E. E. JUST

1. Insemination. In Platynereis insemination normally takes place in the body cavity ( Just, '14). The eggs, when laid, have the sperm attached within a thin hull of jelly, the secretion of the cortical layer. If the worms be allowed to deposit eggs in India ink ground up in sea-water it can be proved satisfactorily that a hull of jelly, as in Nereis, envelops inseminated eggs. This jelly, absent in uninseminated eggs, is formed from the exoplasm of the egg as the result of stimulation through sperm attachment. In sea-water the zone between India ink particles and the vitelline membrane gradually widens, not so much because of the slow diffusion of the jelly from the egg, as because of the swelling of the extruded jelly.

Insemination in some way brings about oviposition. The presence of the sperm in the female is a stimulus to egg laying; as in Nereis (see Lillie and Just) the presence of the sperm in the sea-water brings about the shedding of the eggs. The first result of the attachment of the sperm to the egg is jelly formation through cortical secretion, with the consequent formation of the perivitelline space; and this process must begin in the body cavity, since eggs have a thin jelly investment when laid. As in Nereis the vitelline membrane is preformed; the sperm does not cause 'membrane formation.'

For twenty to thirty minutes after oviposition, the sperm remains external to the egg. During this time profound changes take place in the egg, many of which doubtless are to be interpreted as changes incident to maturation, the mechanism of which is released with the breakdown of the' cortical substance and the consequent formation of the perivitelline space. These changes: breakdown of the germinal vesicle, formation of the spindle, polar body formation, and cytoplasmic movements, are easily followed in the living egg.

2. Penetration. In Nereis a striking phenomenon of sperm attachment is the fertilization cone (Lillie, '11, '12). In Platynereis no sharply defined cone is found. There are, however, cytoplasmic disturbances at the point of sperm entry. In polyspermic eggs the cytoplasm may form a low blunt protrusion, with as many as five spermatozoa attached to it, but this is not


FERTILIZATION IN PLATYNEREIS MEGALOPS 219

a cone. Twenty-five minutes after oviposition a slender strand of protoplasm may be discerned across the perivitelline space and beneath the point of sperm entry; even this does not seem to be constant, but appears to be formed only in the animal hemisphere. This protoplasmic strand lies, first, in a radius of the egg, but gradually bends so that it now lies almost tangential to the egg. It is found after sperm entry.

After thirty minutes the spermatozoon is engulfed. Often the formation of the sperm aster is discernible, the middlepiece and tail remaining outside. The maturation asters are always visible in the living egg. Mathews ('06) has called attention to the difTerence between the structure of the asters of living eggs and of fixed material. The difference is certainly striking in Platynereis. Instead of the short stiff astral fibres of chrom-osmic material or the long slender ones of mercuric fixation, one sees in the living egg beautiful broad rays sweeping through the cytoplasm.

3. Copulation of the germ nuclei. About fifty minutes after egg-laying, the germ nuclei copulate, the cleavage asters form, and at sixty minutes the egg divides unequally. The egg at this time exhibits a stratification of protoplasmic stuffs. During maturation the cytoplasmic currents shift the materials. The equatorially placed oil drops, about eighteen in number, gradually become massed at the vegetative pole, the coarser (yolk) granules lie above these; at the clearer animal pole are the male and female pronuclei. Beneath the polar bodies the cytoplasm is most transparent. The asters are very distinct. One cannot get an adequate picture of these structures from sections. In the living egg they are incomparably clear; large broad rays which bear little resemblance to the short stiff fibres seen in the sections.

The penetration path of the spermatozoon may often be followed, the copulation path always followed. The spermatozoon enters at any point of the egg and through this, as in Nereis (Just, '12), the first cleavage plane passes along the copulation path of the germ nuclei.


220 E. E. JUST

B. Observations on the sectioned egg

Observations of the phenomena of fertiHzation in the Hving egg were supplemented with a study of sectioned material.

Technique. Eggs were fixed in Meves' fluid for thirty minutes, one hour, or twelve hours. Aceto-osmie-bichromate mixtures (Mathews, 1 '99; Bensley) ; Bouin's fluid, modified by the addition of an equal volume of water; and Gilson's mercuric-nitric mixture were likewise used. Although very destructive to the yolk and oil, the modified Bouin proved helpful in the study of certain details in connection with sperm penetration.

The difficulties of fixation, which are great in this egg, as in Nereis, may in large measure be overcome by the subsequent treatment. The following methods were used after fixation with Meves :

(a) Clearing with double distilled anihn oil from 80 per cent alcohol.

(b) Clearing in cedar oil from 95 per cent alcohol.

(c) Clearing in cedar oil from 95 per cent alcohol after treatment with glycerine (eggs put in 70 per cent alcohol plus an equal amount of glycerine).

(d) Clearing in xylol from 95 per cent alcohol or from absolute alcohol.

In all cases xylol was used before imbedding in paraffin or in paraffin with some admixture of Johnston's rubber-asphalt mass. It was found that avoidance of absolute alcohol left the eggs less brittle and therefore less refractory in cutting. By far the most natural contours of both the Platyfiereis and the Nereis eggs are preserved through the use of aniline oil after 80 per cent — a clearing agent that I have used successfully for several years. Staining was with iron hematoxylin alone. Sections were cut four micra thick.

Spermatozoa, after fixation, were studied for the most part unstained after the methods of Koltzoff, de Meyer, etc. The iodine mixture recommended by Mayer for Volvox proved in ^ From the legend of Mathews' figures it appears that he used aceto-osmicbichromate mixtures.


FERTILIZATION IN PLATYNEREIS MEGALOPS 221

valuable. For permanent preparations Bensley's staining mixtures were used.

1. Stages previous to the penetration of the sperm. The egg of Platynereis rivals in structure the beauty of the Nereis egg. A section of an uninseminated egg (fig. 1) teased out of the. female directly into Meves' fluid gives many of the details. The cytoplasm is sharply marked off into two regions: the exoplasm made up of clear cortex and zone of oil and yolk and the deeply staining endoplasm.

The outer portion of the exoplasm is a mesh of pale blue delicate fibrils, the alveoli of the cortical jelly. The outer limits of this cortical layer — slightly more dense than the deeper portions — is studded with black granules immediately below the vitelline membrane. The inner border of the cortex arises from a zone of closely-packed, deep-staining bodies, from which apparently the walls of the cortical alveoli project. Below this inner border is the region of oil drops which lies in the equatorial zone, among spherules which prove, from their later behavior, to be yolk spheres, although even in the best preparations, the fine granules of which they are composed tend to shrink from their spherical walls (c/. LiUie, '11; figure of Nereis egg fixed in Fleming) . These yolk spheres are evenly crowded against the deeply stained basal ♦ area of the cortex. Around the germinal vesicle and closely applied to it is the endoplasmic mass, made up of fine granules which take the stain very tenaciously. Its outer limits are uneven, encroaching on the area of oil drops and yolk spheres as blunt projections.

Scattered throughout the germinal vesicle, as in Nereis, are the chromosomes — fourteen tetrads. These lie among many black granules of varying size. Although an attempt has been made to study their number, distribution, etc., and to ascertain any constant characters, nothing now can be said further of them. These granules tend to be spherical and to grade down to minute bodies.

The whole egg, therefore, exhibits a granular structure, both living and fixed, as Mathews some time since ('06) for echinoderm eggs and more recently Kite for some other eggs have shown.


222 E. E. JUST

Lillie ('06), too, in a most elaborate study on the egg of Chaetopterus, has determined the granular structure of the cytoplasm. Vacuoles found in mercuric-nitric or picro-acetic preparations are filled with yolk or oil in Meves' preparations or in the living Platynereis egg (c/. Wilson, '98, on the cytoplasmic structure of eggs, including that of Nereis).

The egg of Platynereis, as compared with that of Nereis fixed with the same methods, does not show so clearly the radial striation in the cortical layer or the homogeneous yolk spheres.

Ten minutes after laying the germinal vesicle is breaking down and maturation asters, formed outside its wall, are pushing into its substance. The deepest of the cortical alveoli are often still unemptied; the whole process of jelly extrusion can easily be followed from its beginning in inseminated eggs. On one or between two of the apices of the wavy vitelline membrane the spermatozoon is found attached by its perforatorium. Sperm head, middle-piece, and tail are readily distinguished (fig. 2).

Fifteen minutes after laying, the cortical jelly has been wholly extruded (fig. 3) and the first maturation spindle formed, with the chromosomes in late prophase. The endoplasm, with the extra-chromatin substance of the germinal vesicle, imbeds the spindle. In toto mounts of the egg at this stage, as is true of the Nereis egg, give no view of the spindle. One sees only a deeply stained core of substance which incloses the spindle. The egg is irregular in shape and the vitelline membrane is closely applied.

The spermatozoon is visible on the membrane (figs. 3 and 4) above a group of granules similar to those more thinly scattered throughout the periphery of the egg. These granules are markedly like those described by Meves and are doubtless 'mitochondria;' but in Platynereis they cannot possibly have the significance that Meves ascribes to them in the eggs of various forms. The granules appear massed beneath the point of sperm entry, but these masses assume no definite form. I have purposely figured those that give the nearest approach to cone formation (figs. 3, 4, and 5). A slender strand of cytoplasm may extend toward the membrane just below the perforatorium.


FERTILIZATION IN PLATYNEREIS MEGALOPS 223

The granules in the region may appear as a disc, but never as a retracted cone, as in Nereis. The cortical breakdown has released the close application of the yolk spheres to the inner cortical margin; they are now irregularly spaced and among them lies the granular cytoplasm.

The figures (2 to 5) also give good pictures of the spermatozoa. They are much like the living spermatozoon. The head is almost spherical, the perforatorium a large blunt cap; the middle-piece and tail are often clearly defined.

2. Penetration of the spermatozoon. The penetration of the sperm head begins at twenty to twenty-six minutes after laying (c/. Nereis, forty-five minutes after insemination). The first maturation spindle, in the metaphase, is oriented in the polar plane of the egg; the inner endoplasmic mass which incloses the spindle is, at this stage, triangular in section; the outer aster of the spindle is near the apex of the triangle. The base of the triangle is less blunt than in previous stages and reaches farther outward along a radius of the egg. The various stages of penetration are shown in figures 6 to 18. The sperm substance enters the egg as a slender black thread, which gradually increases in size at its inner end. The sperm head, in my preparations, is usually homogeneously black, but often the external bulb is not so dark; or lighter areas appear along the entering thread; (particularly figs. 10, 11, 12, and 14). Often, especially in sections stained for twelve hours only, in stages just after the attachment of the perforatorium to the cytoplasm, the head appears, not as a homogeneous chromatin mass, but as a slightly differentiated body. One gains, therefore, the impression that the spermatozoon flows into the egg (c/. Koltzoff and Lillie, who, with different methods, find Nereid spermatozoa extremely ductile) .

Cytoplasmic changes due to sperm entry are clearly marked during the later stages of penetration ; striae appear in the cytoplasm around the entering spermatozoon, the area stains more deeply, and a projection from the endoplasmic mass reaches out toward the point of sperm entry (see figs. 14 to 17) (c/. on these points. Foot, Gardiner, Vedjovsky, Jenkinson, and Lillie, '12).


224 E. E. JUST

As the head is drawn into the egg, the inner bulb turns with its growth. Finally, the portion forming the external bulb is engulfed. The middle piece and tail, as in Nereis, never enter the egg (fig. 17). They may often be found in sections outside the membrane after penetration of the sperm head (see figures).

I have never found the spermatozoon in the Nereis or in the Platynereis egg at the time or in the form figured by Wilson ('96 and '00).

Does the sperm head rotate? I could not positively determine the rotation of t