Paper - Histochemical horizons in human embryos - Stage 14

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Mckay DG. Adams EC. Hertig AT. and Danziger S. Histochemical horizones in human embryos. II. 6 And 7 millimeter embryos-Streeter horizon XIV. (1956) Anat. Rec.126(4): 433-63 PMID 13403206

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This historic 1955 paper by Mckay and co-authors describes human Carnegie horizon (stage) 14 embryos. Currently only a brief abstract is included on this page.


See also by this author: Mckay DG. Adams EC. Hertig AT. and Danziger S. Histochemical horizons in human embryos. I. Five millimeter embryo, Streeter horizon XIII. (1955) Anat. Rec. 122(2): 125-51. PMID 13238850
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Histochemical Horizons in Human Embryos II. 6 and 7 Millimeter Embryos — Streeter Horizon XIV

Donald G. Mckay, Eleanor C. Adams, Arthur T. Hertig and Sara Danziger


Department of Pathology, Harvard Medical School, Boston, Massachusetts, and the Pathology Laboratories, The Free Hospital for Women, Broolcline, Massachusetts and the Boston Lying-in Hospital, Boston, M assaohusetts

Eighteen Figures

  • This investigation was supported by research grants C-1471 and C-2451 (c) from the National Cancer Institute, of the National Institute of Health, Public Health Service, by the Institutional Grant to Harvard Medical School by the American Cancer Society, and the Carnegie Institution of Washington, Department of Embryology.

Introduction

As a result of a histochemical study of a 5mm human embryo (Streeter horizon XIII) certain physiological implications related to embryogenesis were derived (McKay, Adams, Hertig and Danziger, ’55). Alkaline phosphatase activity was concentrated in the membranes separating intercellular fluids of major difference, i.e. in placental trophoblast which separates maternal plasma from chorionic fluid; in the dorsal mesoblast of the liver which separates chorionic fluid from embryonic venous plasma; and in arteriolar capillary endothelium which separates embryonic arterial plasma from embryonic intercellular fluid. This finding suggested that phosphate ions are concentrated at these interfaces. In relation to the trophoblast, biochemical support for this hypothesis was obtained from the observation of a high phosphate content of chorionic fluid and fluid from the vesicles of hydatidiform moles (McKay, Roby, Hertig, Richardson, ’55a, ’55b).


Alkaline phosphatase activity was found to be greatest in the most rapidly growing tissues of this embryo, including neural tube, limb bud epithelium, gastrointestinal tract, dorsal portion of the liver and the trophoblast. Since these are regions of most active nucleoprotein synthesis, it was suggested that phosphate ions released in these tissues are largely utilized in the synthesis of nucleoproteins.


Endothelium lining blood Vessels was found to be nonhomogeneous, indicating a varied metabolic function in Veins, arteries and capillaries.


The liver and yolk sac appear to function as a “preporta ” unit, and the chemical constituents or metabolic activities in which the early liver is deficient are supplied by the yolk sac. In later stages of development, as the yolk sac involutes, the liver assumes these metabolic functions.


These observations apply also to the three embryos in horizon XIV which are described in this report. One 6mm and two 7 mm human embryos were available for study and the histochemical reactions observed in this group serve to confirm the observations on the 5 mm embryo.


Nevertheless, in spite of the similarities between horizons XIII and XIV, some differences exist, and these will be described. Also, because of the method of sectioning, the anlagen of a few organs such as the anterior hypophysis, thyroid, pancreas, metanephric bud and gall bladder, which were not found in the 5mm embryo, were observed in the embryos of Horizon XIV. In general, the major significance of embryos of this stage of development lies in the fact that this is the period of establishment of organs and organ systems.

Streeter horizon XIV (Streeter, ’51).

For the purposes of orientation and classification, the following summary is presented of the characteristics of the group into which these embryos fit.

“In summary, age group XIV covers the time of the invagination of the optic lens, and various stages of the process are seen, from a shallow pocket to a vesicle still open to the surface by a narrow pore. The ear vesicle has a well defined endolymphatic appendage which can be seen in transparent specimens. The mandibular and hyoid bars have acquired ascendency over the third or g1ossopharyngeal bar. The latter is partially concealed in the receding sinus cervicalis. The arm buds have become elongated and curve toward the body, but one cannot yet speak of a hand plate. The leg buds are finlike. The greatest length of the embryos is usually somewhere between 6.0 and 7.0 mm. The ovulation age of this group has an estimated range of between 28 and 30 days.”

Materials and Methods

Three embryos in our collection fall within the developmental limits of horizon XIV: Fetus 33, 6mm (FHW S-531910); Fetus 29, 7 mm (NWH S-53-288, a tubal pregnancy); and Fetus 31, 7 mm (FHW S-53-593). All were fixed in cold acetone, paraffin embedded, serially sectioned, and reacted or stained histochemically by the techniques reported previously (McKay, Hertig, Adams and Danziger, ’53). Recently Burton’s (’54) modification of the acid phosphatase azo-dye method has been utilized in addition to the previously reported azo-dye methods. It employs a substrate concentration of 0.05% and a Diazo Blue concentration of 0.2% in an acetate buffer at pH 5.8 with incubation at 37° C. for two hours.

  • Fetus 29, 7 mm (NWH S-53-288, a tubal pregnancy)<ref> We wish to thank Dr. David Skinner, Pathologist at the Newton-Wellesley Hospital, for this specimen.


Table 1 is a composite report of the results of identical techniques on each of the three embryos. Both the localization pattern and the relative intensity pattern of enzyme activity and the other substances studied showed excellent agreement between the three embryos. In a few instances Where there were minor differences, the results tabulated are based on agreement between two of the three embryos. Although the enzyme localization pattern was identical in the three embryos, one 7 mm specimen (Fetus 31) had a slightly reduced enzyme activity when compared with the other two, although its own relative intensity pattern (i.e. relative intensity per structure) was in good agreement with the other two embryos.


The difference in the results obtained by the two acid phosphatase techniques deserves comment. When the Burton method was used with a pH of 5.8 and an incubation time of two hours, very few tissues showed evidence of acid phosphatase activity. When the modified Seligman method was used with a pH of 5.0 and overnight incubation, almost every tissue showed apparent acid phosphatase activity. (With both techniques the liver, trophoblast, yolk sac, mesonephros and macrophages revealed the greatest intensity of staining.) Three explanations can be offered: (1) the pH of 5.8 in the Burton method is too high for the demonstration of maximal acid phosphatase activity; (2) the overnight incubation at pH 5.0 caused diffusion of the enzyme and/or of the product of hydrolysis causing false positive localizations; or (3) incubating the tissues overnight in the modified Seligman technique allows autolysis of the tissues which activates acid phosphatase. Gustafson (’54) has pointed out the fact that acid phosphatase is activated by allowing tissues to stand for long periods of time in vitro. The use of a third acid phosphatase method involving a post incubation coupling technique devised by Rutenburg and Seligman (’55) revealed virtually no acid phosphatase activity in any of these embryos.

With the digested PAS reaction in horizon XIII only the most intensely stained areas were reported as glycoprotein positive. In horizon XIV all tissues or cells with any visible pink staining material have been reported as positive.

No calcium was detected histochemically in any of the three embryos.

Description

Because of the general similarity between horizons XIII and XIV in the 4 specimens thus far examined, only those tissues which were seen for the first time in horizon XIV or which showed a reproducible difference will be described. The table, a composite summary of three embryos, may be refered to for a complete description of this horizon.

Epidermis

The histochemical reactions in the skin appear the same as in horizon XIII except that it has been additionally noted that the developing olfactory area is thickened and the cells in this region contain large amounts of glycogen, ribonucleoprotein and alkaline phosphatase activity. The olfactory area thus resembles other areas of thickened epidermis including the tips of the limb buds and the pharyngeal region.

Connective tissue

The connective tissues of the mesonephros, the sclerotome and around the eye no longer contain glycogen, which constitutes a change from the preceding horizon. The connective tissue immediately surrounding the lung buds has also become altered to resemble the mesoblast of the limb buds with abundant ribonucleoprotein but no glycogen or alkaline phosphatase activity. With these .exceptions the connective tissues of these embryos present the same histochemical pattern as in horizon XIII. (It was erroneously stated in the text of our previous report that the mesoblast of the limb buds contained glycogen and was enzymatically active. Actually the mesoblast of the limb buds of both horizon XIII and XIV does not contain glycogenand is free ofalkaline phosphatase activity.)

Nervous system

The dorsal and lateral portions of the brain and spinal cord exhibit abundant alkaline phosphatase activity. The neuroblasts contain large amounts of ribonucleoprotein but are free of glycogen. In contrast, there is a ventral streak of cells, beginning in the medullary region and running the length of the spinal cord which contains glycogen and is free of alkaline phosphatase activity. In the ventral portion of the brain, rostral to the medulla, the zone showing no alkaline phosphatase activity broadens laterally. Rathke’s pouch is lined by epithelium with the same histochemical reactions as the neighboring pharyngeal epithelium. The spinal ganglion cells contain abundant cytoplasmic ribonucleoprotein and moderate alkaline phosphatase activity, but do not exhibit non-specific esterase activity. The histochemistry of the lens and retina of the eye have not perceptibly changed except for the appearance of a hyaline material (primitive vitreous humor) between them which gives the reaction for glycoprotein. The cells lining the otic vesicle have acquired glycogen and glycoprotein. In the preceding horizon these were free of glycogen and were indistinguishable from the neuroblasts of the central nervous system.


Table 1

Table 1 - Embryos 33, 2.9, 31, composite description of horizon XIV
Glycogen Glycoprotein Ribonucleoprotein Alpha Naphthyl Alkaline Phosphatase pH 9.4 Acid Phosphatase pH 5.0 Acid Phosphatase pH 5.8 Non-specific Esterase pH 7.8
EPIDERMIS
A. General (thin)
B. Limb buds and pharyngeal region (thick)
C. Olfactory pit
D. Basement membrane
CONNECTIVE TISSUE
A. Notochord Cells
Basement membrane
B. Mesoblast (limb buds)


C. Mesenchyme Lung Body stalk a. near chorion b. at ventral body wall

Ventral body wall Root of mesentery Gut

Mesonephric ridge Pharynx

Surrounding eye Surrounding ear Surrounding thyroid Surrounding notoehord

D. Macrophages

E. Somites Dermatome Myotome Sclerotome


++ ++

+ +I

I

+ + +++++I+II++I I+I

GLYCOPROTEIN

+ +++ I+I I

+++++++++++I I++

RI'BONUGIaE0PROTEIN

+

++ ++

+ +I+

+ +++++++++++I +++


— (periderm +) +

++ + ++ +

+|+ + III I

III+++++++++ +++ %‘ "U E 9 J, I+++++++IIII I+I


IIIIIIIIIII+ III

5-NUcm:o'rmAsl pH 7.5

II

I

I

IIIIIIIIIIII III

NON srlcmlc

ISTIRASE pH 7.8

III I

||IIIIIIIII+ III 439

Rathke ’s pouch Brain Ventral

€95

Dorsal

C. Spinal cord Ventral Dorsal

D. Ganglia

E. Nerves

F. Eye Lens Retina

Gr. Otic vesicle H. Sympathetic nerve I. Limiting membrane

GUT TRACT AND DERIVATIVES

A. Pharynx Epithelium Basement membrane Thyroid Thyroglossal duet Esophagus Traehea Lung buds Stomach Pancreas Gall bladder

Liver Coelomic mesoblast Hepatic cells Sinus endothelium Kuptfer cells

K. Mid gut L. Hind gut M. Cloaca

+

+ (medulla. only)

+

+

++ ++]+ +l+ I +I+

not identified ~ +

prominent ventrally

>k

7++++++ +Hw+++ + ++ +++++++-H+1+++

+ + + +++} +++

+ +1 ++++-1‘-+++ +++} +++

+ (surface)

+

I+++ I++ I

+| ++++++++ +I+I +++

+ + + ++++ +++ I

+ +| +++-l-+++-+ +++++++

not identified

I

H++ll 440

TABLE 1 (continued) Embryos 33, 29, 31, composite description of horizon XIV

BLOOD VASCULAR system

A. Heart muscle Auricle Ventricle Bulbus cordis Heart endothelium Auricle Ventricle Bulbus cordis Myoendocardial cushion Heart valve:

B. Blood vessels

Aorta . a. endothelium b. wall Small arteries Umbilical artery a. within embryo b. in body stalk Vitelline artery a. within embryo b. in yolk stalk Cardinal veins Venous plexi (pharynx, cloacal region) Umbilical veins 3.. Within embryo b. in body stalk Vitelline vein a. within embryo b. in yolk stalk

GLYGOGEN

++ +++ +++++ +++ ++ HI I

1+

ll

GLY00PROTEIN

+++ +++++ +++ ++ +++ I

++ ++

RIBONUCLEOPROTEIN

+|+ +++:+ +++ ++ +++ + ++ ++

ALPHA NAPHTHYL ALKALINE PHOSPHATASE pH 9 .4

+1! II+Il +I+ +I ++1 I

+1

ll

ACID PHOSPHATASE pH 5.0

+++ +++|+ +++ ++ ++[ I

ACID PHOSPHATASE pflm

++l lllll Ill 1|

5-NUCLEOTIDASE pH 7.5

III ++lll III 1+

11+ + [+ ++

NONSPECIfiC EBTERASI pH 7.8

III lllll lllll

lll

ll PEIMITIVE (mam cnnns

Mnsonxsznes A. Corpuscle B. Secretory tubules C. Mesonephric duct

Mmuumrnmc PRIMORDIA A. Epithelium‘ B. Connective tissue

Connomo EPITHELIUM

A. Visceral (gut, abdominal) B. Parietal (abdominal)

C. Pleural

D. Hepatic

E. Mesonephric

BLOOD CELLS A. Erythroblasts B. Normoblasts

C. Polymorphonuclear leukocytes

44

H You: SAG A. Epithelium B. Endothelium

ALLANTOIS

PLACENTA A. Syncytium B. Cytotrophoblast C. Connective tissue D. Capillaries of stroma

AMNION


Basement membrane is + in these structures I

+++ ++ +I I

I +

I

I

++ + +++++

+ +++ ++ +++++ II

+++ ++++ I

+ ++I

+I+ -I-I

++ +'++++

I

+++ ++ +4-+++ ++ I

+++

++++ +

+

++

++I

++I II

IIIII II

++I!

I+Il


Gastrointestinal tract and derivatives. The cells lining the gastrointestinal tract continue to exhibit glycogen, ribonucleoprotein, glycoprotein and alkaline phosphatase activity. The thyroid and thyroglossal duct contain large amounts of glycogen and ribonucleoprotein and a moderate alkaline phosphatase activity.

A few changes have taken place in the liver. The sinus endothelium, Kupffer cells and the hepatic cells now show non-specific esterase activity. five—nucleotidase activity appears in the cytoplasm of the hepatic cells as well as in the sinus endothelium. The pancreas and gall bladder anlagen contain glycogen, glycoprotein, ribonucleoprotein and alkaline phosphatase activity. The gall bladder epithelium is outstanding because of its greater alkaline phosphatase activity.

Blood vascular system. The heart muscle is essentially unchanged except for a virtual disappearance of ribonucleoprotein from the cytoplasm of the cells of the ventricle and the appearance of a small amount of alkaline phosphatase activity in a few cells of the auricles. The endothelium lining the auricle and ventricle no longer show alkaline phosphatase activity. The interstitial ground substance of the myoendocardial cushion is characterized only by the presence of glycoprotein. However, the connective tissue cells within this structure contain glycogen and ribonucleoprotein.

The endothelium of the aorta and arteries presents the same histochemical pattern as in horizon XIII. The endothelium of the vitelline artery differs from the systemic arterial endothelium since no glycogen is present in the vitelline vessels. The umbical artery in the body stalk is free of alkaline phosphatase activity yet this same vessel within the embryo contains the enzyme. five—nucleotidase activity is present in the umbilical artery in the body stalk. This is the only artery in these embryos exhibiting 5-nucleotidase activity. In contrast to arterial endothelium, the majority of the veins do not contain glycogen. The one exception to this rule is the portion of the umbical vein in the body stalk. The vitelline veins present the same histochemical pattern as the cardinal veins.

Mesonephros. The developing corpuscles and the secretory tubules contain glycoprotein, ribonucleoprotein, acid phosphatase, non-specific esterase and alkaline phosphatase activity. These structures are free of glycogen. The mesonephric duct on the other hand contains glycogen, glycoprotein, and ribonucleoprotein, but no alkaline phosphatase or non-specific esterase.

Metcmcphros. The epithelium of the metanephric primordia contains glycogen, glycoprotein, ribonucleoprotein and alkaline phosphatase. The connective tissue around the metanephric buds is free of glycogen but contains glycop-rotein, ribonucleoprotein and alkaline phosphatase.

Coelomic epithelium. A few changes appear to have taken place in certain portions of the coelomic epithelium. Although patchy in distribution, all areas now exhibit some alkaline phosphatase activity. Glycogen is no longer found in the pleural region but occurs in the gut region.

Blood cells. The red cell series gives no reaction with the several methods used, except for acid phosphatase (pH 5.0), and a few nucleated red cells with ferric iron granules in the cytoplasm. The granulocytes, however, contain glycogen and non-specific esterase activity.

Yolk sac. The histochemical reactions of the endoderm are unchanged. The endothelium of the vessels of the yolk sac no longer contain glycogen or 5-nucleotidase activity.

Allantois. The epithelium of this structure» presents the same histochemical pattern as the cloaca.

Ohorion. The only change observable in the trophoblast is the appearance of non-specific esterase in the cytotrophoblast. The amnion contains only glycogen and glycoprotein. A few ferric iron granules were found in the stroma of the villi and in the syncytial cytoplasm.


Interpretation

Liver and yolk sac

As in the previous horizon, the liver cells contain no glycogen; a finding consistent with the biochemical determinations of Villee (’53) on somewhat older embryos. The inability of the liver cells to store glycogen at this stage of development may be related to the fact that they are incapable of producing glucose. Villee has further observed that the early placenta is capable of glucose formation and has suggested that this organ rather than the liver controls the glucose content of the early embryonic circulation. Anatomical and histochemical considerations suggest that the yolk sac also may play a role in the regulation of blood glucose early in embryonic life. The vascular drainage of the yolk sac leads back through the liver to the sinus Venosus thus forming an early model of the portal circulation. The cells of the yolk sac endoderm are filled with glycogen and may be supplying glucose to the fetal circulation through the liver.

One of the earliest evidences of a shift in function from the yolk sac to the liver can be observed in the 5—nucleotidase activity of these structures. In horizon XIII this enzyme was found in the sinus endothelium of the liver and in the endothelium of the yolk sac vessels. In horizon XIV 5-nucleotidase is no longer present in the yolk sac vessels; is still present in the vessels of the yolk stalk and liver endothelium; but is now found for the first time in the cytoplasm of the liver cells. N on—specific esterase is another enzyme that shows a beginning shift from yolk sac to liver. Although it is still very active in yolk sac epithelium, it makes its first appearance in the liver cells in this horizon.

The histochemical observations on the human yolk sac epithelium may be compared to those of Wislocki, Deane and Dempsey (’46) in the rodent yolk sac. This epithelium in man and the rodent contains abundant cytoplasmic ribonucleoprotein, glycogen, glycoprotein and acid and alkaline phosphatase activity. In contrast, the rodent yolk sac epithelium contains abundant iron, whereas the human yolk sac epithelium, in the horizons thus far examined, is free of iron. In this early developmental period there appears to be an interesting difference in iron metabolism between rodent and man. Iron is present in the epithelial cells of the uterine glands of rodents and is secreted into the uterine lumen. It is then absorbed by the yolk sac of several rodent species. In the human, however, neither the endometrial epithelium, the gland secretions nor yolk sac endoderm contain histochemically demonstrable iron. This diflerence in iron metabolism may be a functiontal expression of the fundamental anatomic difierence between the yolk sacs of rodent and man. (It should be noted that in subsequent horizons a few granules of iron appear in a few cells of the yolk sac endoderm.)

Central nervous system

The central nervous system at this stage of development is of interest because of the longitudinal streak of cells in the ventral groove that differ so markedly from the remainder of the cells of the brain and spinal cord. They are unique since they contain glycogen but are free of alkaline phosphatase activity. The physiological significance of this observation is not apparent but it is worth noting that these cells were the first to invaginate in the process of formation of the neural tube, and are the cells that lie nearest to the notochord. The importance of the notochord in influencing the shape of the neural tube has been indicated experimentally. Lehmann (’35) has shown that the slit-shape of the normal neural tube depends on the presence of the notochord. Weiss (’55) states:

“The effect may be credited to a vertical system of fibers, spanning the thickness of the plate along a median strip coextensive with the notochord and apparently attached to it, Which holds the midline firmly anchored as a hinge about Which the flanks of the plate fold up.”

In the histochemical preparations no fibers were seen, but the different metabolic properties of these ventral midline cells indicates their special nature and their location suggests a relationship to notochord.

Studies of alkaline phosphatase activity in the central nervous systems of the chick and mouse embryos (Moog, ’43; Chiquoine, ’54) reveal many similarities to the human. In general, the brain and spinal cord of all these species in the early developmental stages exhibit intense alkaline phosphatase activity. The early nerve bundles are similarly intensely reactive and this enzyme is found in the dorsal ganglia of all these species. It has not been detected in the cells of the roof of the 4th ventricle in any. A ventral streak of cells free of alkaline phosphatase similar to that in the human embryos was observed in the chick on the third day (Moog) and in the mouse on the 8th day (Chiquoine).

Limb buds

In considering the possible forces responsible for initiating early limb bud development, Streeter has considered it unlikely that spinal nerves, migrant cells from the somites or proliferation of cells from the coelomic tract should be responsible. This leaves but two sources for the pioneer cells of the limbs, i.e. the apical ectoderm and the mesoblast of the lateral body wall. Experimental studies by Zwilling (’55) on chick embryos have demonstrated the importance of the apical ectoderm on limb bud development. Removal of the apical ectoderm resulted in development of partial limbs, While reimplantation of apical ectoderm onto denuded limb buds restored normal development. Transplantation of wing bud ectoderm to the leg bud mesoderm resulted in the development of a leg. Transplantation of leg bud ectoderm onto wing bud mesoderm caused the development of a. wing. He concluded that in this experimental situation the presence of the apical ectoderm is a specific requisite for limb outgrowth but has no influence on the determination of limb type. HISTOCHEMISTRY or 6-7 MILLIMETER EMBRYOS 447


Zwilling noted the relative absence of morphological distinction between the apical ectoderm and the remainder of the ectoderm in spite of the difference in biological potential of these two tissues. Histochemically in the human the apical ectoderm can be readily distinguished from the remaining ectoderm because of its greater metabolic activity. The presence of large amounts of glycogen, ribonucleoprotein and alkaline phosphatase activity may be directly related to the growth promoting effects of this specialized ectoderm. Alkaline phosphatase may supply by diffusion an increased number of phosphate ions and the glycogen an increased concentration of carbohydrates to the underlying mesoblast at the apex of the limb buds which might favor the growth and multiplication of connective tissue cells at the apex and in this manner cause lengthening of the limb buds. The accumulation of phosphorus in the region of the limb bud has been illustrated by Dent and Hunt (’54). Using P32 as a tracer these authors found high concentrations of phosphorus in developing limb buds of amphibia and in the limb bud region in stages of development prior to the actual appearance of the bud. They further stated that phosphorus tends to become localized in those regions in which growth and/or differentiation were progressing most rapidly.


The thickened ectoderm in the nasal placode and in the pharyngeal region are histochemically similar to that of the apex of the limb buds and may play a similar role in the difl"erentiation of the underlying connective tissue structures of the nose and pharynx. It may be that other, more specific, growth promoting substances are released by the thickened ectoderm in these special areas.

Acid and alkaline phosphatase

In horizon XIV as well as in the preceding horizon, acid phosphatase appears in greatest concentration in the liver, yolk sac epithelium, mesonephros and in the syncytial trophoblast. In these locations the enzyme appears to be distributed evenly throughout the cytoplasm. This is quite in contrast to alkaline phosphatase which is found in the same tissues but on the surface of cells rather than inside them. This may be indicative of important functional differences between acid and alkaline phosphatase, the former an “intracellular” enzyme, and the latter a “surface” enzyme.

Summary

This report contains a histochemical description of three human embryos belonging to Streeter horizon XIV. Procedures for the histochemical demonstration of glycogen, glycoprotein, cytoplasmic ribonucleoprotein, alpha naphthyl alkaline phosphatase, acid phosphatase, 5-nucleotidase, nonspecific esterase, calcium and iron were employed. Interpretations of the possible physiological significance of these observations have been presented.

It is suggested that:

  1. The high concentration of alkaline phosphatase and glycogen in the apical ectoderm of the limb bud may supply by diffusion an increased concentration of phosphate and carbohydrate to the underlying mesoblast at the apex of the limb buds. This might favor the growth and multiplication of adjacent connective tissue cells and in this manner cause lengthening of the limb buds.
  2. Because of the large amount of glycogen in the yolk sac endoderm and its virtual absence from the livercells, the yolk sac may be an important structure in supplying glucose to the embryonic circulation during the first weeks of embryonic life. Because of the absence of stainable iron in the yolk sac, the endometrial glands and their secretions in the human, the acquisition of iron by the human embryo appears to be via a different pathway from that in the rodent, in which the yolk sac epithelium appears to absorb iron from the secretions of the endometrial glands.
  3. In general alkaline phosphatase appears as a “surface” enzyme and acid phosphatase as an “intracellular” enzyme in these early embryos.


Literature Cited

BURTON, J. F. 1954 Histochemical demonstration of acid phosphatase by an improved azo-dye method. J. Histochem. and Cytochem., 2: 88-94.

CHIQUOINE, A. D. 1954 Distribution of alkaline phosphomonesterase in the central nervous system of the mouse embryo. J. Comp. Neur., 100: 415-440.

DENT, J. N., AND E. L. HUNT 1954 Radiotracer techniques in embryological research. J. Cell. and Comp. Physiol., 43: 77-96.

GUSTAFSON, T. 1954 Enzymatic aspects of embryonic difierentiation. International Review of Cytology V01. III. Ed. by G. H. Bourne and J. F. Danielli. Academic Press. Inc., N. Y., 277-327.

LEHMANN, F. E. 1935 Die Entwicklung von Riickenmark Spinalganglien und Wirbelanlagen in chordalosen Korperregionen von Tritonlarven. Rev. Suisse Zool., 42: 405-415.

Mckay DG. Adams EC. Hertig AT. and Danziger S. Histochemical horizons in human embryos. I. Five millimeter embryo, Streeter horizon XIII. (1955) Anat. Rec. 122(2): 125-51. PMID 13238850

MCKAY, D. G., C. C. Rosy, A. T. Hmrrre AND M. V. RICHARDSON 1955 (a) Studies of the function of early human trophoblast. I. Observations on the chemical composition of the fluid of hydatidiform moles. Am. J. Obs. and Gyn., 69: 722-734.

1955 (b) Studies on the function of early human trophoblast. II. Preliminary observations on certain chemical constituents of chorionic and early amniotic fluid. Ibid., 69: 735-741.

MOOG, F. 1943 The distribution of phosphatase in the spinal cord of chick embryos of one to eight days incubation. Proc. Nat’l. Acad. Sci., 29: 176-183.

RUTENBURG, A. M., AND A. M. SELIGMAN 1955 The histochemical demonstration of acid phosphatase by a post—incubation coupling technique. The J our. of Histochem. and Cytochem., 3: 455-470.

Streeter GL. Developmental horizons in human embryos. Age groups XI to XXIII. (1951) Carnegie Institution of Washington, Washington, D. C.

VILLEE, C. A. 1953 Regulation of blood glucose in the human fetus. J. Applied Physiol., 5: 437-444.

WEISS, P. A. 1955 Nervous system (neurogenesis). Analysis of development. Ed. by B. H. Willier, P. A. Weiss, V. Hamburger, W. B. Saunders Co., Philadelphia, 1955, p. 371.

WISLOCKI, G. B., H. W. DEANE AND E. W. DEMPSEY 1946 The histochemistry of the rodent’s placenta. Am. J. Anat., 78: 281-346.

ZWLLING, E. 1955 Ectoderm-mesoderm relationship in the development of the chick embryo limb bud. J. Exp. Zool., 128: 423-438.

Plates

Plate 1

Mckay1956 plate01.jpg

Cross section through an entire embryo at the level of the pancreas and liver. Note alkaline phosphatase activity in the spinal cord except at the ventral region, in the dorsal area of the somites, in the spinal ganglia and in the thickened epidermis at the distal tips of the limb buds. Enzyme activity is also seen in the corpusoles and secretory tubules of the mesonephros (but not in the mesonephric duct), in the mescnchyme of the mesonephric ridge and of the mesentery, in the lumens of the pancreatic glands, in the gut epithelium, in the coelomic epithelium and sinus endothelium of the dorsal area of the liver, and in the endothelium of the dorsal aorta, small arteries of the limb bud and the umbilical veins in the lateral body walls. Fetus 33, 6mm, X 80 (green filter).

Plate 2

Eye and olfactory placode. Alkaline phosphatase activity in optic evagination and retina of eye, in the thickened epidermis of the olfactory placode, and in the cndothelium of small blood vessels. Note absence of this enzyme in the indentation of the lens. Fetus 29, 7 mm, X 50 (green filter).

Yolk sac and stalk. Alkaline phosphatase activity in the endoderm of the yolk sac and yolk stalk and in endothelium of the vitelline artery. Note absence of activity in endothelium of yolk sac and in vitelline veins. Fetus 33, 6mm, X 70 (green filter).

Yolk sac and yolk stalk. Ribonucleoprotcin in cytoplasm of endodcrm of yolk stalk and yolk sac. Fetus 33, 6mm, X 70 (green filter).

Yolk sac and yolk stalk. Ribonuclease digestion of section adjacent to figure 4. Ribonucleoprotein has been removed from endoderrn. Fetus 33, 6mm, X 70 (green filter).

Pancreas, gut and liver. Alkaline phosphatase activity in the lumina of developing pancreas and of the gut, in the rnesenchyme of the mesentery, and in the coelomic epithelium and sinus endothelium of the dorsal region of the liver. Fetus 33, 6mm, X 90 (green filter).

Gut and gall bladder. Alkaline phosphatase activity is marked in the cytoplasm of the gall bladder epithelium. Note reaction also in mesenchyme of mesentery and in the endothelium of the umbilical veins seen laterally.

Fetus 33, 6mm, X 90 (green filter).

Tail region. Alkaline phosphatase activity ill the spinal cord at the left and in the lumen of the tail gut at the right. Note also a faint reaction in the somites and in the mesenchyme, but absence of alkaline phosphatase activity in the endothelium of the venous plexi and notochord. Fetus 33,

6 mm, X 100 (green filter).

Tail region. five-nucleotidase activity is seen in the endothclium of the venous plexi (haematoxylin counterstain of nuclei). Fetus 33, 6mm, X 100 (green filter).


Plate 3

10 Myencephalon and otic vesicles. Alkaline phosphatase activity is found in patches in the epithelium of the otic vesicles, in the neuroblasts of the developing thalamus and in the cndothelium of the blood vessels in the connective tissue around the brain. Fetus 29, 7mm, X '75 (green filter).


Plate 4

Liver (left), and heart (right). Glycogen deposits are seen in the muscle of the wall of the auricle (below), the ventricle (above) and the bulbus

cordis (right). Glycogen is also seen in the epidermis, but is not found in the liver cells at this stage. Fetus 29, 7 mm, X 75 (green filter).


Plate 5

Spinal cord, somitcs, ganglia, dorsal aorta, mesonephric ridge, and mesentery of the gut. Alkaline phosphatase activity is concentrated in the dorsal portion of the central nervous system, in the mesonephric tubules (but not in the duct) and in the primitive germ cells seen ill the mesentery and the developing gonadal folds. Enzyme activity is also present in the dorsal region of the sornites, in the ganglia, in the dorsal aorta, and in the connective tissue of the mesonephric ridge. Fetus 29, 7 mm, X 75 (green filter).

Pharynx with notochord above and thyroid, bulbus cordis and heart below. Note heavy glycogen deposits in the notochord, the pharyngeal epithelium, the epidermis, the thyroid and in the heart muscle. Glycogen is also present in the loose mesenchyrne above the pharynx and in the arterial endothelium. Fetus 29, 7mm, X 75 (green filter).


Plate 6

14 Gut, liver and heart. Alkaline phosphatase is seen in the coelomie epithelium and the sinus endothelium in the dorsal region of the liver, in the thick epidermis covering the distal tips of the limb bud (seen at the sides), and in the capillary endothelium in the lateral regions of the connective tissue between the liver and the heart. Fetus 29, 7 mm, X 75 (green filter).

1:3 Gut and liver. Acid phosphatase is seen in the cytoplasm of the liver cells. Fetus 29, 7 mm, X 150 (green filter).

Plate 7

Ventral tip of spinal cord, nerves and somites. Alkaline phosphatase activity is found in nerves and intersegmental arteries. Note absence of activity in the neuroblasts at the ventral tip of the spinal cord. Fetus 29, 7mm, x 75 (green filter).

Somites. Note heavy glycogen deposit in the myotomes and in the epidermis. Fetus 29, 7mm, X 75 (green filter).

Tongue and pharynx at left, trachea at center, esophagus at right. Alkaline phosphatase activity is seen in the pharyngeal epithelium, the connective tissue of the pharynx (except for the bone anlage below the tongue) and in the epithelium of the trachea and the esophagus. Fetus 29, 7 mm, X 75 (green filter).



Cite this page: Hill, M.A. (2019, December 16) Embryology Paper - Histochemical horizons in human embryos - Stage 14. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_Histochemical_horizons_in_human_embryos_-_Stage_14

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