Talk:Paper - The histology of the umbilical cord of the pig (1919)
THE HISTOLOGY OF THE UMBILICAL CORD OF THE PIG, WITH SPECIAL REFERENCE TO THE VASCULOGENIC AND HEMOPOIETIC ACTIVITY OF ITS EXTENSIVELY VASCULARIZED CONNECTIVE TISSUE
H. E. JORDAN
Department of Anatomy, University of Virginia
The umbilical cord of the pig differs markedly from the human cord in that it is extensively vascularized, its connective tissue maintains throughout the gestation period largely its original embryonal character, and vasculogenic and hemopoietic activity persist to full term.
The material upon which this investigation is chiefly based is a complete cord, 4 cm. in length, from a fetus of 16-cm. length. This fetus was secured in utero at the abattoir and preserved in 10 per cent formalin. Reckoned by length, it lacked between one and two weeks of full term. Portions of the cord from the proximal end, the middle, and the distal end, were imbedded both in celloidin and in paraffin. Serial sections were cut from the paraffin blocks and stained with hematoxylin and eosin. Some of the celloidinsections were similarly stained ; others were stained with resorcin-fuchsin and counterstained with picricacid-fuchsin, for a study of the elastic and collagen fiber content. A second specimen of a nearly full-term cord, for which I am indebted to Prof. George S. Huntington, was used for comparison. Cords of pig embryos from 9 to 21-mm. length and five full-term and three fetal (three to seven months) human cords, variously fixed and stained, were also employed for comparative study.
Though primary interest centers upon the vasculogenic and hemopoietic activity of the connective tissue, it seems desirable to preface the description and discussion of these phenomena
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with a brief description of the comparative histology of this cord. Compared with the human cord it is very short, of considerably lesser girth, and only slightly twisted. It has the same light gray, pearly appearance, and feels of about the same consistency. In transverse section it has an irregularly oval shape (fig. 1), measuring 5 by 7 mm. Its three main bloodvessels have an approximately equal caliber and thickness of wall. It contains a large open allantoic duct and remnants of the occluded yolk-stalk. The connective tissue contains many arterioles, venules, and capillaries. Only one of the post-embryonic human umbilical cords in my collection, a full-term specimen, contains any blood-vessels besides the usual umbilical arteries and vein. In this cord occurs a venule of considerable size, lying near the surface and completely filled with red bloodcorpuscles. The human umbilical cord is typically non-vascular except for an occasional capillary at the extreme proximal end. None of my sections of these eight human cords contains any vestige of the allantois. One cord contains a small, still patent yolk-stalk; three contain a double, occluded yolk-stalk remnant. One of the full-term cords of the pig also contains a double occluded yolk-stalk (figs. 1 and 3), the other only a single, small, occluded remnant in only a few sections. In one of the human cords the persistent, double, occluded yolk-stalk is enveloped by a double layer of smooth muscle, an inner longitudinal and an outer thinner circular layer.
THE UMBILICAL ARTERIES AND VEIN
In the cord of the pig the wall of the umbilical arteries only contains circularly disposed smooth-muscle cells, more compact centrally; the vein in one of the two specimens contains also scattered, longitudinally placed cells beneath the intima. The disposition of the muscle differs from that in the human cord where the arteries and the vein contain, in addition to the chief circular layer, also a thin internal longitudinal layer and scattered bundles of longitudinally arranged cells externally. With regard to the elastic tissue content also, the pig's cord differs sharply from the human cord. The arteries of the latter lack
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an internal elastic membrane, but several muscle layers beyond the tunica intima the elastic tissue forms complete fenestrated membranes through many layers; toward the periphery of these vessels the elastic tissue only occurs as scattered delicate fibrils. The vein, on the contrary, contains a very robust internal elastic membrane, while through the central half of the wall occur relatively coarse scattered fibers. In the pig's cord neither arteries nor vein contain an internal elastic membrane. The elastic fibers are practically limited to the inner half of the wall, only very delicate and widely scattered fibrils occurring peripherally. In the arteries the elastic tissue forms membranes for from three to five layers beyond the central two or three layers. Considerable variation occurs with regard both to the amount and the disposition of both the smooth muscle and the elastic tissue constituents of the wall of the umbilical vessels both in the pig's cord and the human cord.
The covering ectoderm constitutes a stratified epithelium of generally four layers of cells (fig. 2) . In certain restricted areas the epithelium only consists of three layers of cells, in others of as many as eight layers of cells. This epithelium resembles the transitional rather than the stratified squamous type. It is comparable to the thicker portions of the epidermis of the threemonth human fetus, which consists of from four to six layers of cells, including a superficial periderm. It differs from the ectoderm of the full-term human cord, which includes only two or three layers of flattened keratized cells. It differs also from the continuous abdominal ectoderm in that the latter includes about eight layers of cells, all of which, except the basal cuboidal layer, consist of greatly flattened cells. Small patches of partially fused, keratized cells occur in five different regions of the section here shown. The lowermost layer of the epithelium consists of cuboidal cells; the outermost variously of thick, rectangular, flattened or dome-shaped peridermal cells ; the intervening layers include polyhedral and stout fusiform types. In those portions
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where the epithelium consists of more than four layers, the one next the basal cuboidal layer is generally composed of more flattened cells. The basal layer seems to rest directly upon the adjacent connective tissue, without the intervention of a definite basement membrane. After picric-acid-fuchsin counterstain, however, a narrow subepithelial layer of the connective tissue stains more deeply red.
None of the cells contains mitotic figures. An occasional cell of the superficial layer contains two or even four nuclei or a nucleus in process of fission. The nuclei of the cells of the basal layer are of spheroidal shape and contain a clear, lightly staining nucleoplasm and a distinct granular reticulum. These cells are completely filled with a slightly basophilic cytoplasm. The nuclei of the several outer layers are irregular in shape and they have a homogeneous, cloudy, more deep-staining character, and the nuclear wall is generally wrinkled. The cytoplasm of the cells of the intermediate layers is aggregated next the cell wall so that the cell appears hollow. This condition is probably a fixation artifact. The nucleus almost invariably lies next the outer wall, as if moved by currents passing toward the surface of the ectoderm. The cells of the outermost layer are again completely filled with an acidophilic cytoplasm. The latter is keratized to an extent w 7 hich could resist the action of the fixation currents that caused the peripheral shrinkage and central excavation of the cells of the intermediate layers.
The yolk-stalk remnant of one of the two practically full-term specimens of the pig's cord, and of four of the specimens of human cord used for comparative study, is a double structure. In the specimen of the pig's cord the two portions are of nearly equal size (fig. 3). They are approximately circular in outline and perfectly solid. The cells of the peripheral layer are squamous or very low cuboidal. The more central cells are polyhedral «r spheroidal. The cells next the outermost layer are generally flattened and appear , fusiform in longitudinal section. About
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twelve cells occupy the diameter of each of the two elements. Most of the central cells appear hollow, the nuclei having become moved to one side, almost invariably to that side nearest the center of the structure. These cells have a superficial resemblance to fat cells. The hollow condition is probably the result of the coagulative action of the fixing fluid upon the delicate cytoplasm. A few of the cells appear keratized, and are acidophilic in staining reaction, and solid. The nuclei of the outermost cuboidal cells are deep-staining, granular, greatly elongated bodies. Those of the more central cells have a generally irregular shape with wrinkled contour and a generally non-granular homogeneous nucleoplasm. No mitotic or amitotic figures can be detected. Each member of the double structure is enveloped by a very thin inner connective-tissue theca, forming a delicate, fibrillar, nucleated basement membrane. Both are inclosed in a common, more external theca. The intervening partition only consists of the fused basement membranes.
The fact that four of the five specimens of human cords used in connection with this study also contain a double yolk-stalk indicates that this doubling is a common condition. Two explanations suggest themselves: 1) That the doubling is due to the partition of the originally single stalk by the ingrowth of a connective-tissue septum related to the regressive changes by which the stalk becomes obliterated. 2) That the 'doubling' is only apparent, it being due in section to a sharp turning of the stalk in certain regions. The latter interpretation is supported by the evidence from one of the human cords: here one of the 'halves' is cut transversely, the other half is very obliquely cut, and the two are in continuity. In other words, the condition is such as would result if the section passed obliquely through the proximal end of one of the limbs and the connecting loop of a U-shaped structure. Opposed to the latter interpretation, however, is the fact that in the specimen of the pig's cord here described a common connective-tissue sheath envelops both moieties.
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The allantoic duct (fig. 1, All.) is shown highly magnified in figure 4. It has an irregularly oval shape in cross-section. Its wall is thrown into deep folds. Within the depth of the folds the epithelium consists of a single layer of cuboidal or even squamous cells, comparable to its condition throughout in the 21-mm. fetus; over the crests the epithelium is of the stratified columnar type, consisting of from three to four layers of cells. No distinct basement membrane can be discerned other than as indicated by the deeper red color of the immediately subjacent connective tissue after picric-acid-fuchsin counterstain. The nuclei have an irregularly oval form; their wall is wrinkled, and the nucleoplasm generally lacks a distinct network or granules. A number of the cells are hollowed out centrally. The cells next the lumen have a more condensed, probably slightly keratized, broad distal border. The enveloping connective tissue is less differentiated and denser than in any other portion of the section. It resembles early embryonic connective tissue or young mesenchyma. It contains many vasofactive cells ('angioblasts'), and one large blood-island (B. I.), which will be described below.
THE CONNECTIVE TISSUE
The character of the connective tissue varies in different portions of the transverse section (compare figs. 2,3,4, and 5). In the region immediately surrounding the allantoic duct (fig. 1) it is least differentiated. Here it is compact and resembles embryonic connective tissue or young mesenchyma (fig. 4). In this region also are numerous vasofactive cells (fig. 15). Along the peripheral border of the specimen the connective tissue is somewhat more differentiated and represents an older type of mesenchyma (fig. 2). Here the cells have generally a stellate or fusiform shape and are more widely separated. In this region also the capillaries are most abundant; these for the most part lie along the radii of the section and appear to be growing towards the ectodermal covering. Here also are found abundantly initial stages in the formation of the blood channels (figs. 8, 9, and 10).
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In the area between the lower umbilical artery on the left and the vein on the right, the connective tissue is in part of the type characteristic of the human cord; that is, it is typical mucous connective tissue, but with an occasional capillary. In this region also occur small bundles of collagen fibrils. The area around the yolk-stalk contains vascular connective tissue of an intermediate type, with occasional collagen fibers (fig. 5). In the narrow space between the two umbilical arteries, extending to a point below the allantoic duct, there occur several bloodislands (figs. 6 and 7). About midway between the central umbilical blood-vessels and the periphery occur numerous arterioles and venules. Occasionally these are arranged in pairs (fig. 5). These vessels terminate in capillaries. The arterioles are enveloped by a thin layer of smooth muscle; the wall of the venule only consists of endothelium resting upon the slightly more condensed enveloping connective tissue.
Certain of these smaller blood-vessels can be traced into connection with the umbilical arteries and the vein at their proximal (fetal) end. This is true both in the case of the younger cords (of embryos from 9 to 21 mm.) and in those near full term. In the 21-mm. fetus branches from the proximal end of the umbilical vein can be seen entering the body wall as well as the connective tissue of the cord. It may be confidently assumed that all of the blood-vessels, including the numerous capillaries, connect with vessels which ultimately connect with the main umbilical vessels proximally. But not all of these vessels are properly interpreted as vasa vasorum. Undoubtedly many function thus, as is indicated by the numerous capillaries directed towards the walls of the umbilical arteries and vein; but others are equally certainly nutrient vessels for the allantoic duct, the ectodermal covering, and the general connective tissue. Nor can the assumption be properly made that all of these vessels arise by sprouting from originally direct umbilical branches. If the capillaries grew exclusively by sprouting, their tips should show mitotic figures. Mitotic figures are practically absent in these capillary terminals. On the contrary, these tips seem to fuse with the general connective tissue, the cells of which become hollowed out, arrange
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themselves in line with the capillaries and ultimately become incorporated as part of the capillary network. In the full-term condition of the cord, blood-vessels arise in a manner identical with their primary origin in the original body-stalk, and become secondarily connected with the preexisting vascular net. The connective tissue of the full-term cord maintains the primitive vasculogenic mode by which the primitive blood-vessels were formed. The full-term cord is relatively much more extensively vascularized, and it consists of connective tissue largely of a less differentiated type than the cord of the 21-mm. fetus. Since the larger blood-vessels extend to the distal end of the cord, it may be inferred that they supply also the proximal pole of the allantois.
VASCULOGENESIS AND HEMOPOIESIS
In the description of the mode of vasculogenesis illustrated in this cord, we may begin most conveniently with the stage represented by the cell of figure 8. This cell has the general characteristics of an irregular mesenchymal cell. Three vacuoles can be seen to the right of the nucleus. A later stage may be represented by the upper cell of figure 9. Here the cell is binucleated, and the originally smaller discrete vacuoles have presumably coalesced to form a single large vacuole, the precursor of the initial capillary lumen. The appearance of the lumen has effected a modification of one of the nuclei so that it begins to assume endothelial features. In figure 10 is shown a still later phase of vasculogenesis. Here, moreover, the more typical endothelial 'cell' has taken on hemoblast features and has differentiated an erythroplastid (ep.) intracellularly. Figure 11 may be conceived to represent a transection of the cell of figure 9 or 10. Cells like those of figures 8, 9, 10, and 11 are very numerous in the more peripheral regions of the cross-section. Figure 12 illustrates a binucleated cell with essentially mesenchymal features, which has differentiated an erythroplastid and a lumei^ centrally. The cell shown in figure 13 has the nuclear and cytoplasmic characteristics more of a young hemoblast. This cell represents a mesenchymal cell which has rounded up and differen
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tiated into a hemoblast, and subsequently differentiated an erythroplastid intracellularly.
The mesenchymal cells of this primitive 'mucous' connective tissue may apparently undergo any one of several types of differentiation: 1) They may separate from the mesenchymal syncytium, round up and differentiate into potential hemoblasts, which may lie freely among the undifferentiated mesenchymal cells, but apparently never in this condition directly metamorphose into erythroplastids ; but grouped into blood-islands, about which the adjacent mesenchyme differentiates into endothelium, they develop into erythroblasts (fig. 7). 2) They may become bi- or multinucleated and, as hemogenic giant-cells, differentiate erythrocytes intracellularly (figs. 6, e, and 15, /). 3) A mesenchymal cell may acquire a lumen and join with other cells to form an initial capillary, incidentally differentiating also erythroplastids intracellularly (fig. 12). Erythroplastids may originate intracellularly also in young endothelial cells (fig. 10). Hemoblasts can, therefore, apparently differentiate into erythrocytes only when inclosed by endothelium; or in the multinucleated condition, hemoblasts can differentiate intracellular erythrocytes. The latter phenomenon is essentially like that where a hemoblast is inclosed by endothelium.
Figures 6 and 7 illustrate an earlier and later stage, respectively, in the differentiation of a blood-island. In figure 6 the bloodisland is still largely a syncytium. However, endothelium can be seen forming on its surface, and several of its cells are taking on erythroblast ('megaloblast') features (a, b, and d). The cell d has developed a large vacuole at one pole; this vacuole may form part of the subsequent lumen. Cell e has differentiated an erythrocyte. Several intercellular spaces have appeared in the syncytium; these are the forerunner of the subsequent lumen, to which certain intracellular spaces may also contribute. In figure 7 the endothelium and the lumen are developed further, and the hemoblasts are mostly in the erythroblast stage and are generally separated from each other by cell membranes.
It seems desirable at this point to indicate the chief differences, nuclear and cytoplasmic, between the young mesenchymal
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cell, young endothelial cell, hemoblast, erythroblast ('megaloblast'), and the erythrocyte. The mesenchymal cell has in general a light-staining, spheroidal or oval nucleus with a delicate reticulum; its cytoplasm contains delicate fibrillae. The young endothelial cell has a similarly light-staining, but generally more elongated nucleus; and its cytoplasm is less distinctly fibrillar. The hemoblast is generally spheroidal in shape, but it may assume various forms due to its ameboid capacity; its nucleus also generally has a spheroidal shape, but it contains a more distinct and more granular network, and the cytoplasm appears homogeneous. The nucleus of the young erythroblast ('megaloblast') has a spheroidal shape, a robust chromatic membrane, a generally deeper-staining nucleoplasm; and it contains one or several small nucleoli and numerous granules scattered over its delicate reticulum. Its cytoplasm is distinctly granular, and it stains deeply in eosin. The granular condition of the cytoplasm of the erythroblast is the most distinctive mark of this cell. This cell corresponds to the megaloblast of certain writers. The erythrocyte has a considerably smaller, generally deeperstaining, spherical nucleus, and a clear cytoplasm delimited by a distinct membrane (figs. 6, e, and 15,6 ande). Theerythroplastid has in contrast a brownish-yellow color.
In figure 15 (a to i) are illustrated various vasofactive and hemogenic cells. In fact, as these figures clearly indicate, vasofactive and hemogenic activities are intimately associated. The cell a may be regarded as at the stage of a late hemoblast or a young erythroblast. The cell b is similar, but has produced an intracellular erythrocyte. Cell c has developed a lumen, and it has differentiated an inclosed erythroplastid. Cell d is essentially a young endothelial cell with vestiges of cytoplasmic hemoblast features. Cell e has become essentially a young endothelial cell with an included small erythrocyte and an erythroplastid. Cells g and i are essentially hemoblasts ('angioblasts') which have become differentiated into binucleated endothelial cells. Cells/ and h should be interpreted together; h is essentially a multinucleated hemoblast or small 'giant-cell/ one of whose nuclei is apparently undergoing amitotic division;
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/ may be regarded as a later stage in the intracellular erythrocytogenic function of h, in which two of the nuclei and their enveloping cytoplasm have differentiated into erythroplastids (ep). Cells h, f, b, and e of figure 15 and e of figure 6, when considered in common, demonstrate that erythroplastids in these vasofactive cells do not arise as such out of the cytoplasm, but under the direct influence of a nucleus of a bi- or multinucleated hemoblast (memogenic giant-cell'). The absence of free erythrocytes and erythroplastids in the regions from which these cells are taken contravenes any suggestion that the intracellular red blood-corpuscles should be interpreted in terms of phagocytosis. Figures 12 and 14 supply similar evidence. The erythroplastid of figure 12 may appear to have arisen directly from the cytoplasm of this vasofactive cell. But figure 14 shows an essentially similar cell at a slightly earlier stage of differentiation, in which the chief nucleus has liberated a small bud. About this bud an erythrocyte and a lumen may be conceived to originate in the manner shown in e of figure 15, and so lead to a condition like that of figure 12. In other words, the cells of figures 12 and 14 are essentially multinucleated hemoblasts. In this sense multinucleated hemoblasts, hemogenic giant-cells, and blood-islands ('angioblasts') are fundamentally and potentially alike ; that is, they are essentially multiple ery throblasts enveloped by a layer of potential endothelium.
From the foregoing description it will be clear that the connective tissue of the full-term umbilical cord of the pig is extensively vascularized and that it is actually for the most part still in the condition of young mesenchyma or embryonal connective tissue. The conditions are essentially similar to those described for the body-stalk of very young human embryos. The question arises whether the connective tissue of this cord is in the primitive mesenchymal condition because it is vascularized or whether it is vascularized because the connective tissue is in the condition of undifferentiated mesenchyma. Since the bloodvessels have apparently arisen to a considerable extent in situ,
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t he latter interpretation would seem to be the correct one; that is, that this cord has maintained early embryonic conditions, like that of its anlage, the body-stalk, and in consequence retained its original capacity for vasculogenesis and erythrocytogenesis. The cause of this maintainance of early embryonic vasculogenic and hemopoietic potentialities, especially singular in connection with the advanced developmental condition of the umbilical arteries and vein, and the several small areas of fully differentiated mucous connective tissue, remains for the present undertermined. It is most probably associated with the large functional allantois, but the nature of this association is not clear. The relatively highly developed character and healthy condition of the covering ectoderm may be secondary to the presence of the large number of capillaries in the subjacent connective tissue.
Though this study can throw no light on the cause of the vascularized condition of the umbilical cord of the pig, the intense hemopoietic activity of its connective tissue supplies valuable data with respect to the initial steps in vasculogenesis. This is the chief point of value in this specimen. In this connection interest centers upon the mesenchymal cell, which becomes hollowed out to form an endothelial cell and at the same time differentiates erythrocytes (figs. 6, 12, 13, and 15). This cell combines the functions of an endothelioblast and an erythroblast. The process appears to be quite similar to that first described by Ranvier 8 (74) in the mesentery of the seven-day rabbit and in the great omentum of the cat, and independently by Schaefer 10 (74) in the subcutaneous tissue of the new-born rat, and subsequently confirmed by other workers on other forms. Ranvier named the cells concerned in the process 'vasoformative cells.' According to these investigators, mature (non-nucleated) 'erythrocytes' of greatly varying sizes are formed directly within the protoplasm of connective-tissue cells (vasoformative cells) by a process involving the coalescence of scattered granules of hemoglobin into condensed globules, which then come to lie in vesicles within the cells, the precursors of the capillary lumen.
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The method of erythrocytogenesis here described for this specimen of umbilical cord of the pig, however, differs radically from that described by Ranvier and Schaefer, in that the erythroplastid in this case differentiates from a nucleated portion of a vasofactive cell (figs. 6, e; 15, b and e, and 14). That is, the erythroplastid differentiates from a typical erythroblast in the usual mode. The nucleus of the erythrocyte disappears by karyolysis (fig. 15, e). This is apparently a very rapid process, since it can be detected in only relatively few cells. If one considered only cells like those of figures 12 and 13, the process would appear to be identical with that described by Ranvier and Schaefer; but figures 15, b, e, h, and/ demonstrate the essential difference.
Certain investigators (Spulef, 12 Fuchs, 3 et al.) have expressed dissent from Ranvier's and from Schaefer's interpretation of their observations; they explain these phenomena, the occurrence of which they confirm, in terms of regressive changes and phagocytosis. They believe that the so-called Vasoformative cells' are either isolated portions of a disintegrating embryonic vascular plexus or erythrophagic connective tissue cells. It is obvious that since the vasofactive phenomena here described for the umbilical cord of the pig are fundamentally different, while superficially apparently identical with those described by Ranvier and Schaefer, the criticisms of Spuler and Fuchs have no pertinancy to this case. Moreover, the red cells involved in this process in the umbilical cord of the pig show no distinct nuclear or cytoplasmic marks of degeneration. This cord, except for the almost complete absence of mitotic figures, appears in perfectly healthy condition. No free erythrocytes are available for phagocytosis in the regions here described. There is no indication of a disintegration of blood-vessels; on the contrary, the full-term cord is relatively more extensively vascularized than the cord of the 21-mm. fetus. Finally, and most significantly, this intracellular mode of erythrocytogenesis is strictly comparable to that described for other hemopoietic organs, e.g., yolk-sac of 10-mm. pig embryo, 5 yolk-sac of mongoose embryos, 6 and red bone-marrow. 7
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The matter may be summed up with figures 15, a, b, and e, and 15, h and /. A connective-tissue cell becomes transformed into a hemoblast (erythroblast) with vasofactive capacity. This cell may become bi- or multinucleated. One or several of the nuclei with their enveloping cytoplasm may differentiate into erythrocytes. Meanwhile a lumen appears within the cell, and one or two of the original nuclei may persist as the nuclei of the peripheral cytoplasm of the differentiating cell, which now forms the endothelial wall of the initial capillary. In later stages in the yolk-sac and in the red bone-marrow generally, the peripheral 'endothelial' layer of the original 'vasofactive' cell disappears, thus freeing the intracellularly differentiated erythrocytes into the confining blood spaces. The mesenchymal cell thus appears endowed with divers hemogenic "potentialities: it may become an endothelial cell or a hemoblast (erythroblast) . The endothelial cell may secondarily differentiate into a hemoblast. These hemoblasts may differentiate into erythroblasts or, as multinucleated cells, they may differentiate both intracellular erythrocytes and a potential endothelial cell. These facts demonstrate the very close relation between mesenchyme, endothelium, and hemoblasts.
Sabin 9 records a similar vacuolization of mesenchymal 'angioblasts' in the living blastoderm of the two-day chick embryo grown in Locke's solution, by which the blood-vessel lumen forms. But these observations do not justify her conclusion that they prove "that the lumen of a blood-vessel is intracellular" (p. 200). The data supplied by the umbilical cord of the pig show that the definitive lumen of the blood-vessel derived from a blood-island is of both inter- and intracellular origin.
This brings us to the matter of the factors which determine whether the mesenchymal cell shall become an endothelial cell or a hemoblast, and relates this investigation to the discussion regarding theories of hemogenesis, that is, whether blood development proceeds according to the monophyletic or the polyphyletic mode. This much seems certain regarding this tissue: single hemoblasts, freed from the mesenchyme and wandering within its meshwork, d*o not differentiate into erythrocytes. It
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is only when such a cell becomes inclosed by endothelium that it differentiates into a red blood-cell. Thus a group of such cells may form and in consequence produce pressure upon the surrounding mesenchyme, which then becomes transformed into endothelium. Under these conditions the enveloped hemoblasts become erythrocytes (figs. 6 and 7). Such endothelium is simply an adaptive form of mesenchyme, as originally maintained by Huntington 4 and by Schulte, 11 and it may subsequently return to mesenchyme, remain as endothelium, or differentiate hemoblasts either intra- or extraluminally. Endothelium, accordingly, develops originally by two different methods, both clearly represented in the specimen under consideration: 1) By adaptation of mesenchyme about a blood island; 2) by vacuolization of vasofactive mesenchymal cells ('angioblasts').
A point of special interest concerns the fact that the nucleated periphery of a multinucleated hemoblast supplies the same favorable conditions or factors for determining erythrocytogenic differentation as an endothelial wall. This phenomenon becomes intelligible when we consider that both endothelial cells and hemoblasts are only slightly modified mesenchymal cells, and that the latter, as vasofactive cells, may become hollowed out to form the lumen of an original capillary or differentiate intracellular erythrocytes. The central fact here pertains to the obviously very minute difference between the environmental conditions or stimuli that determine whether the same cell (the potential hemoblast, 'vasoformative cell', or 'angioblast') shall become an endothelial cell or an erythroblast. This suggests that also the factors which determine whether the hemoblast shall become a leucocyte or an erythrocyte, in accordance with the monophyletic theory of blood-cell origin, are similarly relatively subtle and of minute degree. Original confinement by endothelial walls furnishes the stimulus which determines erythrocytogenesis; extra vascular differentiation leads to granulopoiesis. As shown by the recent experiments of Danchakoff, 1,2 the original poly valency of the hemoblast, however, is lost by the erythroblast, and this degree of differentiation is irreversible. An erythroblast freed from its endothelial confines and
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thrown into the surrounding mesenchyme, as in the allantoic spleen grafts of DanchakofT, will not differentiate into a leucocyte, but into an erythrocyte. A mature endothelial cell does not normally, as originally, differentiate into a hemoblast. And an extra vascular hemoblast which is in process of differentiation into a granulocyte apparently cannot redifferentiate into an erythrocyte after it has wandered into the blood-vessel lumen.
The results of this study of the umbilical cord of the pig, which maintains to full-term largely the embryonic condition of the original body-stalk, emphasize the polyvalent capacity of the mesenchymal cell and its hemoblast derivative, and supply further evidence in agreement with the monophyletic view of hemogenesis. The multinucleated hemogenic giant-cell furnishes the same essential stimuli for the differentiation of erythrocytes as does an inclosing endothelium. It is comparable to a blood-island, and produces erythrocytes intracellularly in a manner similar to that by which erythrocytes separate out of a blood-island syncytium. This tissue demonstrates also the origin of endothelium both by adaptation of mesenchyme about a blood-island and by vacuolization and fusion of vasofactive mesenchymal cells. It shows, moreover, that the lumen of the original blood-vessels includes both inter- and intracellular contributions.
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1 Danchakoff, Vera 1918 Cell potentialities and differential factors con sidered in relation to erythropoiesis. Am. Jour. Anat., vol. 24, p. 1.
2 1918 Equivalence of different hematopoietic anlages (by method of stimu lation of their stem-cells). II. Grafts of adult spleen on the allantois and response of the allantoic tissues. Am. Jour. Anat., vol. 24, p. 127.
3 Fuchs, H. 1903 Uber die sogcnannte "intracellulare" Entstehung der
roten Blutkorperchen junger und erwachsener Sauger. Anat. Hefte, Bd. 22, S. 95.
4 Huntington, G. S. 1914 The development of the mammalian jugular
lymph sac, of the tributary primitive ulnar lymphatic and of the thoracic ducts from the viewpoint of recent investigations of vertebrate lymphatic ontogeny, together with a consideration of the genetic relations of lymphatic and hemal vascular channels in embryos of Amniotes. Am. Jour. Anat., vol. 16, p. 259.
5 Jordan, H. E. 1916 The microscopic structure of the yolk-sac of the pig
embryo, with special reference to the origin of the erythrocytes. Am. Jour. Anat., vol. 19, p. 277.
6 1917 Hemopoiesis in the mongoose embryo, with special reference to the
activity of the endothelium, including that of the yolk-sac. Pub. 251 of the Carnegie Institution of Washington, p. 291.
7 1918 A contribution to the problems concerning the origin, structure,
genetic relationship and function of the giant-cells of hemopoietic and osteolytic foci. Am. Jour. Anat., vol. 24, p. 225.
8 Ranvier, L. A. 1874 De dcveloppement et de 1'accroisement des vaisseaux
sanguins. Arch, de Physiol., T. 6, p. 429.
9 Sarin, F. R. 1917 Preliminary note on the differentiation of angioblasts
and the method by which they produce blood-vessels, blood plasma and red blood cells as seen in the living chick. Anat. Rec, vol. 13, p. 199.
10 Schaefer, E. A. 1S74 The intracellular development of blood corpuscles
in mammals. Mon. Micr. Jour., vol. 11, p. 261.
11 Schulte, H. vox W. 1914 Early stages of vasculogenesis in the cat (Felis
domestica) with special reference to the mesenchymal origin of endothelium. Memoir, Wistar Inst. Anat. and Biol., no. 3, pp. 1-92.
12 Spuler, A. 1S92 Uber die "intracellulare" Entstehung roter Blutkor perchen. Arch. f. mikr. Anat., Bd. 40, S. 530.
EXPLANATION OF FIGURE
1 Photomicrograph of transverse section of umbilical cord of pig. The cord is covered with a stratified epithelium from three to eight (generally four) layers thick, resembling somewhat the transitional type. Tufts of keratized cells occur at certain points (E). To the right of the allantoic duct (All) are the umbilical arteries. The umbilical vein lies below the double remnant of the occluded yolk-stalk (Y.S.). At B.V. is one of the larger arterioles of the extensively vascularized connective tissue. In the area between the lower umbilical artery and the vein, the connective tissue resembles the mucous type; elsewhere it resembles more young mesenchyme, and contains many capillaries, arterioles and venules (A.V.), and also numerous hemoblasts and several typical bloodislands. (Photos, by W. S. Dunn, Cornell University Medical College, N. Y. City. The illustrations were made from the Columbia specimen.) X 18.
UMBILICAL CORD OF THE PIG
H. E. JORDAN
EXPLANATION OF FIGURES
2 Photomicrograph of the ectodermal covering of the cord in the region, Som., of figure 1. It includes four or five layers of cells and resembles transitional epithelium. The lowermost layer is composed of cuboidal cells; the outermost layer includes dome-shaped, rectangular, and flattened peridermal cells; the intermediate layers include spheroidal and polyhedral cells. X 300.
3 Photomicrograph of remnant of yolk-stalk (fig. 1, Y.S.). It is double and occluded, and each moiety includes about twelve cells in its diameter. Art., arteriole. X 300.
UMBILICAL CORD OF THE PIG
H. E. JORDAN
EXPLANATION OF FIGURES
4 Photomicrograph of allantoic duct. The lining epithelium is thrown into folds. In the troughs of the folds the epithelium consists of a single layer of cuboidal or flattened cells ; over the crests, of from three to four layers, constituting a stratified columnar epithelium. Art., arteriole; B. I ., 'blood-island' of erythroblastic X 235.
5 Photomicrograph of pair of blood vessels (the branching venule cut obliquely) and the surrounding mesenchyme (region A.V. of fig. 1). X 300.
UMBILICAL CORD OF THE PIG
H. E. JORDAN
ML ■ *
EXPLANATION OF FIGURES
6 Drawing of blood-island, from region just below the allantoic duct (fig. 1, All.). Peripherally the cells are becoming differentiated into an endothelium. Centrally the syncytial mass is becoming vacuolated through the appearance of intercellular spaces, and certain of the cells have entered the early erythroblast ('megaloblast') stages (a and b). One erythroblast (d) contains a large vacuole. A hemoblast (e) has differentiated an intracellular erythrocyte. A hemoblast (h) is separating from the differentiating endothelium. Between the two endothelial cells below, appears another hemoblast. Figures 6 to 9 are magnified 1500 diameters.
7 Blood-vessel in process of differentiation from the mesenchyme. This drawing is from the region to the right of the allantoic duct between the two umbilical arteries (fig. 1), and includes approximately the middle third of this entire vascular anlage. The forming lumen contains seven young erythroblasts ('megaloblasts'), separating out of an originally syncytial mass.
8 Young vasofactive cell, with generally mesenchymal features and three vacuoles, the precursors of a capillary lumen.
9 Slightly older vasofactive cell with large vacuole, the underlying nucleus assuming endothelial features.
UMBILICAL CORD OF THE PIG
H. E. JORDAN
EXPLANATION OF FIGURES
10 Vasofactive cell with three nuclei. The nucleus at the right has original mesenchymal features; the endothelial 'cell' below the lumen has assumed hemoblast features, and has differentiated an intracellular erythroplastid (ep). Figures 10 to 15 are magnified 1500 diameters.
11 Vasofactive cell differentiating a lumen. This figure corresponds to a transverse section of figure 10.
12 and 13 Vasofactive cells with an erythroplastid in the lumen.
14 Vasofactive cell in early stage of differentiation from mesenchyme. The cell has in general hemoblast features. The larger nucleus has produced a small bud at the left. From such nuclear buds and their enveloping cytoplasm develop intracellular erythroplastids.
15 (a to i) Vasofactive mesenchymal cells at various stages of differentiation: a) Typical young erythroblast ('megaloblast'). h) Hemoblast that has differentiated an erythrocyte intracellularly. c) Cell with vascular lumen and an intracellularly differentiated erythroplastid. d) Cell with lumen, having assumed endothelial features, e) Cell with lumen, containing an . erythrocyte (ec, 'normoblast') and an erythroplastid (ep.). f) Binucleated cell with two intracellularly differentiated erythroplastids (ep). g) Cell with lumen and two nuclei, both with endothelial features, h) Cell with four nuclei, one apparently in process of amitotic division. The centrally located nuclei with their enveloping cytoplasm may differentiate into erythrocytes. ?') Binucleated cell, endothelial in character, with lumen containing an erythroplastid.
IMBILICAL CORD OF THE PIG
H. E. JORDAN
Resumen por el autor, Henry Alexander Murray, Jr.
Colegio de Medicos y Cirujanos, Columbia University.
El desarrollo del asa cardiaca en el conejo, con especial mention
del surco bulbo-ventricular y el origen del
El autor ha estudiado varios embriones de conejo para determinar si la formacion del asa cardiaca en esta especie es semejante a la descrita por Schulte en el gato. Los procesos de formacion son semej antes en ambas especies en lo referente al origen del asa primaria, que se produce a consecuencia del hundimiento de la hendidura bulbo-ventricular izquierda, y la fusion de los tubos miocardicos se verifica por la intervencion de la placa cardiaca media, que esta representada temporalmente por una cresta bien distinta situada en la pared interna del miocardio. En las series de embriones de conejo, sin embargo, esta elevation se oblitera algunas veces por completo y no susministra prueba alguna sobre su intervencion en la formacion del surco inter-ventricular primitivo.