Paper - The earliest stages of development of the blood-vessels and of the heart in ferret embryos 2

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Wang CC. The earliest stages of development of the blood-vessels and of the heart in ferret embryos. (1918) J Anat. 52(2): 137-85. PMID 17103832

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This historic 1918 paper by Wang describes early blood-vessels and of the heart in ferret embryos.



See also earlier Wang CC. The earliest stages of development of the blood-vessels and of the heart in ferret embryos. (1917) J Anat. 52(1): 107-36. PMID 17103829



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The Earliest Stages of Development of the Blood-vessels and of the Heart in Ferret Embryos

By Chung-Ching Wang, M.D., Ch.B. (Edin.),

Acting Lecturer and Demonstrator in Anatomy, University College, London; tate Carnegie Research Fellow in Embryology, and Assistant in the Depariment of Anatomy, Edinburgh University.

(Continued from October Number.)

Stage IV.

This stage is represented by one embryo 25 mm. in length, the cranial extremity of which has been reconstructed in wax.

Description of the Plastic Reconstruction of the Heart and the Cranial Portion of a Ferret Embryo 2°5 mm. in Length with 12 Paired Somites. (F. Ap. 13/28/08.)

A plastic reconstruction of the cranial portion of this embryo has been made, which was exhibited, along with several other reconstructions of older specimens, before the Anatomical Section of the International Medical Congress at its meetings held in London in August 1913.

Technique.

The mother was killed with chloroform and the abdomen opened immediately. The whole uterus was removed and the embryo fixed en bloc with warm Zenker’s fluid. It was afterwards washed in running water and transferred to industrial spirit coloured with iodine in the usual manner.

The embryo, with about half of the uterine wall attached to it, was stained in bulk with Meyer's acid hemalum, and counterstained with eosine. After subsequent treatment in the usual way, the specimen was embedded in paraffin, trimmed and provided with guiding-lines. The embryo, which was exposed to view after the dorsal half of the uterine wall was removed, was carefully measured before being fixed and stained, and was found to be 2°5 mm. in length, age being 14 days old ‘approximately. It was then cut in serial sections with a thickness of 10 microns, and yielded 240 perfect sections, there being, therefore, a slight but uniform shrinkage of ‘1 mm. The plane of section was almost directly transverse to the long axis of the embryo in caudo-cranial succession.

On microscopical examination the resulting sections are found to be in excellent quality, and present a perfect histological picture with frequent mitotic figures and a normal condition of the general contour of the epithelial linings of the various organs, vessels, and body spaces—all pointing to the specimen being normal.

Tracings of every section of the cranial end of the embryo were drawn with the aid of the projector apparatus at a magnification of 100 diameters, and these, in turn, were made into wax plates of 1 mm. in thickness. When the plates were cut out and methodically adjusted into position with careful manipulation of the guiding-lines, and in their numerical order, it was found that they superimposed one another most accurately, and that the structures faithfully took up their relative anatomical positions. After the model of the embryo had thus been built up, the original guiding-lines were dispensed with, but, to minimise any error that might arise in the subsequent division of the reconstruction into detachable blocks to expose to view the deeper organs, new guiding-lines were made on the surface of the reconstruction. - The plates were then solidified and slightly smoothed, and the structures painted over with different colours to represent the various organs. The model is now to be seen at the Anatomical Depart- ment, Edinburgh University.


Such plastic reconstructions, generally known as. Born’s reconstruction, as has often been pointed out, are not to be considered as absolutely free from error even with the most accurate manipulation of the wax plates provided with the most reliable guiding-lines ; for it has been noticed that the variations of temperature in the room at the time when the model is under the process of reconstruction, or even after the reconstruction is completed, materially alter, though in a small degree, the consistency of the wax employed, and therefore affect the model; but the results obtained with the Born’s method are such as cannot be so conveniently produced by any other known method.


General Description.

It should be pointed out that the embryo under consideration came from the same uterus as the one of 13 somites described by Yeates (’15), to which references will be made hereafter.


The embryo is, in certain ways, similar to the human embryo described by Alexander Low (’08), which measures 2°6 mm. in length and exhibits 13-14 somites. Three visceral pouches can be distinguished, as in the case of Yeates’ (15) specimen, but there are only two external clefts in the specimen (figs. 21 and 25). In the latter respect the ferret embryo may be regarded as being in a somewhat younger stage of development than the human embryo described by Low, which shows three visceral clefts besides possessing an S-shaped heart tube. The brain flexure of the ferret embryo at this stage corresponds, in a measure, more to the ferret embryo of 13 somites described by Yeates and-to the human embryo of 13 somites described by Ivan E. Wallin (13) than to the one described by Low.

As in all of the embryos above quoted, but resembling more closely to the 4-mm. human embryo described by Bremer (05-06), the medullary tube opens to the exterior at its cranial and caudal extremities, the former (figs. 22, 25, and 27) with a slit-like but bent aperture over a distance of 28 sections, and the latter with an opening of equal length. This feature harmonises with the statement made in Keibel’s Normentafeln, that in both the pig and the rabbit embryos the closure of the medullary tube is completed only after the formation of the head and neck bends. It may be mentioned here that the bucco-pharyngeal membrane is present (figs. 22 and 27), and that the rudiment of the nephric body is first seen opposite the 7th somite, and thereafter it is not separated from the paraxial and lateral mesoderm.

Somites.

Twelve pairs of well-formed somites are present. They were determined by careful counting under the microscope. The first formed somite is distinctly caudal to the otic plate by a distance of 2mm. As the ganglia are not yet well developed, it is futile to attempt, at this stage of develop- ment, to allot the different somites to their respective regions. Each somite has a uniform thick wall three or four cells in depth, enclosing a cavity (myoccel) which looks very distinct in the more caudal somites, but less so in those situated more cranially. Many of the nuclei, which lie near the coelom of the somite, show definite signs of mitosis. The more cranially placed somites (fig. 23) are distinctly triangular in shape on cross section, with the apices pointing ventro-medially and the bases dorso-laterally, while the more caudal ones (tig. 24) are more or less quadrangular in outline.

Allantois.

The allantois may. be described as having the appearance of a small ‘diverticulum on the caudal end of the entodermal sac. Its caudal extremity



Fig. 21. — Dorsal view of plastic reconstruction of ferret embryo 2°5 mm. x 75.


Fig. 22. — Ventral view of plastic reconstruction of ferret embryo 2°5 mm. x 75.


is, however, bifid, as noted by Yeates of London (11); the bifidity includes both the entodermal and mesodermal components This mode of development of the allantoic cavity seems to occupy an intermediate position between that of the typical mammal and that of the: lizard.


Fig. 23.—Transverse section through embryo. x 100.


Nervous System.

The medullary tube, as already pointed out, is closed except at its cranial and caudal ends, where the cranial and caudal neuroporic apertures are present. The mesencephalon is flexed upon itself, in such a way as to bring the prosencephalon almost to a plane parallel with the long axis of


Fig. 24. — Transverse section through embryo. x 100.

the rhombencephalon, which merges, without any definite line of demarca- tion, into the spinal medullary tube (fig. 25). The pontine and cervical flexures are definitely absent in this specimen. The otic vesicle is repre- sented only by a thickened plate of epithelium which shows no indication of invagination, and is situated ‘2 mm. cranial to the first pair of somites

142 Dr C. C. Wang (vide swpra). There is yet no lens thiekening to be detected. The primary optic vesicle on each side is, however, well represented by. a slight bulge of the lateral wall of the anterior portion of the prosencephalon of the corre- sponding side, much in the same way as in the specimen described by Yeates (715). .

When the whole series of the sections is examined under the high power of the microscope, the notochord is found to be free throughout its

Neuropore.—



Bulbus. s-—- Ist visceral pouch.

—~ Dorsal aorta.

Ventricle. Medulla.

Atrium 2nd visceral pouch.

Mesodermic tissue.

ws 8rd visceral pouch. Sinus venosus.

— Vitelline vein.

Fie. 25.—Lateral view of plastic reconstruction of ferret embryo 2°5 mm. x 100.

whole course, except at the cranial part,-where it is still connected with the gut wall from a point opposite the first somite to the bucco-pharyngeal membrane, and at the caudal end, where it is connected with the entoderm from the level of the 7th somite to the end of the body of the embryo. The notochordal canal at this stage of development, as described by Mall (12), Eternod (95, 99), and Grosser (13), is just disc@rnible under the high power (fig. 26). In the ferret embryo of 13 somites, it is to be noted no lumen could be made out by Yeates (’15) in either the cranial or the caudal parts of.the notochord. In places the cells of the notochord are arranged in two

‘lateral masses which suggest the presence of bilateral symmetry (fig. 23). The cells of the chorda are comparatively large, oval, and clear. Development of Blood-vessels and Heart in Ferret Embryos 143

Pericardium:

The reconstruction shows that at this stage of development the peri- cardial cavity is closed except at the dorsal part of its caudal extremity, where it communicates, on each side, with the pleuro-pericardial canals which are situated dorso-medial to the vitelline veins (figs. 22 and 30). The pericardium, measuring 875, in width and 350, in length, is reflected on to the two endothelial tubes on its dorsal aspect, forming the dorsal meso- cardium, but ventrally there is not even the vestige of the ventral

e Medullary tube. Somite. | Ceelom. Ectoderm. '


Vitelline vein. | Vitelline vein. Entoderm. Notochord.

Dorsal aorta.

Fic. 26.—Transverse section through embryo. x 100.

mesocardium which, in birds and amphibians, is so conspicuously seen at this stage of development.

Heart.

With the ventral portion of the pericardium removed (fig. 22) it is obvious that the ventral aspect of the heart rudiment is separated in the caudal but larger portion of its extent into a right and left segment by a cranio-caudal sulcus. On each half there are also several. transverse sulci, but the significance of these various sulci is rather obscure, as the segments marked out by them do not correspond, in any- way, with the primary divisions of the heart tube, although at first sight it appears that the left segment is apparently atrial, and the right, ventricular. The fact that this appearance is deceptive becomes at once obvious when a portion of the muscular wall of the heart is removed (fig. 27); for it is then seen that within the lumen of the muscular tube, and separated from its wall by a more or less thick mass of loose mesodermic tissue, lie two endothelial tubes which are quite separate from one another in the whole of their extent (figs. 27 and 28a-d). Each endothelial tube can be followed caudally to the 144 Dr C. C. Wang

septum transversum, where it is continuous with the corresponding vitelline vein immediately cranial to the umbilical orifice (figs. 27 and 28a), and cranially it is continuous with the first cephalo-aortic arch, which runs dorsally through the mandibular arch round the cranial border of the first visceral pouch (figs. 25, 27, and 29). In the reconstruction (fig. 27) the muscular coat of the heart rudiment










Neuropore. Prosencephalon.

ist aortic arch.

Bucco-pharyngeal

membrane. Mesodermic tissue. ee 6s Bulbus cordis. > ; Ist visceral pouch. Ventricle.

Right heart tube.

Mexesermie Tans: 2nd visceral pouch. Epicardium. ee 4 Acereei,

Mesodermic tissue. 3rd visceral pouch. Pericardium.


Arrow in pleuro- oe pericardial canal. Umbilical orifice.

Sinus venosus. Vitelline vein.

Fic, 27.—Ventral view of ferret embryo 2°5 mm. with muscular wall of heart partially removed. x 100.

has been removed entirely on the left side but only partly on the right side. In this way the left.endothelial tube is, therefore, fully exposed to view on its ventral and lateral aspects, and it exhibits clearly a very definite indication of separation into divisions which appear to indicate, in caudo-cranial succession, the positions of the sinus venosus, the sino-atrial canal, the atrium, the atrio-ventricular canal, the ventricle and the bulbus — cordis (fig. 27).

The dilatation (figs. 25 and 27) which represents the sinus venosus Development of Blood-vessels and Heart in Ferret Embryes——145

occupies the most caudal portion of the tube and is partly embedded in the substance of the septum transversum and partly projects into the

Vena capitis

ihe Fore-gut. medialis.

Dorsal aorta.

-


Vitelline vein.

Pericardial cavity.

Left endothelial tube.

Fie, 28a.—Transverse section through heart region of 2°5 mm. ferretembryo. x 100.

Dorsal Venacapitis Medullary Fore- aorta. medialis. tube. gut.

Pericardial cavity.

Dorsal meso- cardium.


Right endo- Left endo- thelial tube. thelial tube.

Fic. 280.—Transverse section through heart region of 2°6-mm. ferret embryo. x 100,

pericardial cavity. At its caudal end near its dorsal aspect it receives the corresponding vitelline vein, which turns abruptly from the transverse

to a caudo-cranial direction before it empties itself into the sinus venosus (fig. 27). Dr C. C. Wang

The constriction which indicates the. position of the sino-atrial canal is distinetly marked laterally and dorsally (fig. 27). It is less conspicuous

Vena capitis medialis. i

146

Dorsal aorta.

Fore-gut.

Pericardial cavity.

Dorsal meso- cardium, ©


Right endothelial { b Left endothelial

tube. tube. x 100.

Fic. 28c.—Transverse section through heart region of 2°5-mm. ferret embryo.

Vena capitis medialis. Dorsal aorta.



1st visceral _ pouch. Medullary tube.

Fore-gut.

Right endo- & thetial tube ©


, i —_ “Left endo- Cut surface of Dorsal thelial tube. cranial pericar- —_mego-

dial reflection. cardium.

Fig. 28d.—Showing cranial pericardial reflection. x 100.

ventrally, and is altogether absent on the medial side of the tube. The dilatation (figs. 25 and 27) which takes the place of the atrium is com- paratively small, and the constriction (fig. 27) which indicates the position Development of Blood-vessels and Heart in Ferret Embryos 147

of the atrio-ventricular canal can easily be recognised on the medial and ventral aspects of the tube and slightly on the dorsal, but there is nothing to suggest a constriction on the lateral aspect in this situation (fig. 25).

The ventricle which is situated at the most cranial end of the éndo- thelial tube is bent upon itself (fig. 27), and the most dependent point of the tube is the most ventral part of the ventricular portion (figs. 25 and 30). It is divided by a shallow depression, clearly recognisable on the lateral aspect (fig. 27), into a caudal and a cranial limb; the latter is separated by a somewhat oblique constriction from the bulbus cordis, which is bulged laterally and then turns cranially to be continuous with

1st visceral pouch. Fore-gut. Medullary tube.

Dorsal aorta. eae oes 1st aortic arch.

Right endo- » thelial tube. 1


/- 228 Cut surface of cranial pericardial reflection.


Fra. 29.—Showing first aortic arch. x 100

the ventral aorta immediately below the caudal extremity of the ventral part of the first visceral pouch.

The right endothelial tube, which has not been fully exposed in the: reconstruction, appears .to present similar dilatations and constrictions, the outlines of which can be followed, to a certain extent, through the muscular covering, but, since in the model it is still covered in parts by muscular substance, the exact details cannot be worked out at present, and therefore no positive statement regarding it can be put forward. It is, however, more dorsally situated than the left tube (fig. 30); moreover, the whole heart is bent slightly towards the right side in the ventricular region (fig. 27).

- The heart at this stage of development is attached to the dorsal wall ‘of the pericardium (figs. 28a-d). There is positively no trace of a ventral mesocardium in the ferret embryo (a point already alluded to, but to be 148 Dr C. C. Wang

fully discussed later), and the dorso-ventral length of the dorsal meso- cardium is extremely short. The heart is applied closely to the ventral aspect of the pharynx in the region of the second visceral pouch.

In this communication it is not proposed to enter in detail into the development of the blood-vessels beyond the region of the heart, but, in passing, it is perhaps of interest to note that the ventral aorta com- municates from the dorsal end of the bulbus cordis and runs cranially ventral to the first visceral pouch, then turns dorsally round the cranial border of the pharynx to form the first cephalo-aortic arch, and, finally,

Somite.

Meduiasy tau, | | Dorsal aorta. Pleuro-pericardial canal.

Right vitelline vein. '







Dorsal ENA be

pee ats ; EN Left vitelline vein. aorta. Foes ee

Arrow in pleuro-pericardial canal.

Mesodermic tissue. ~

Left sinus venosus. — 5 ~ Cut surface of myocardium.

—~ Right endothelial tube partially exposed.

Left ventricle. —

Fie. 30.—Caudal view of plastic reconstruction of ferret embryo 2°5 mm, x 100.

it pursues a course caudally along the dorsal wall of the fore-gut as the dorsal aorta (figs. 21 and 25). It is a relatively wide vessel, its calibre throughout being distinctly greater than that of the bulbus cordis.

Immediately dorsal to the dorsal aorta there is situated on each side of the embryo a series of apparently isolated sections of a minute blood- vessel (figs. 28a-d). These capillaries lie close against the medullary tube. This vessel appears to be the vena capitis medialis of Grosser (’95), which Miss Parker (’15) also has found to be present in her Stage III. Perameles nasuta 7°5 mm.

Intersegmental offshoots from the dorsal aortze in the region of the caudal somites are described by Miss Parker in Perameles nasuta, but no such offshoots from the dorsal aortee were present in the ferret specimen under consideration. In the caudal region of the embryo, the Development of Blood-vessels and Heart in Ferret Embryos 149

two dorsal aortze become continuous with the vitelline arteries, which spread themselves out in a plexiform manner on the wall of the yolk- sac (fig. 24). This stage of development of the dorsal aorta in the ferret agrees, in some ways, with the 1:3-mm. human embryo described by Eternod (95, ’99).

The vitelline vein (fig. 27), which opens into the caudal end of the sinus venosus, so far as it lies on the embryonic region, runs at first transversely towards the median plane in the substance of the septum transversum immediately cranial to the umbilical orifice. As it approaches the median plane it changes its course suddenly, making a sharp bend upon itself cranially, and terminates, as has already been indicated, in the dorsal extremity of the caudal part of the corresponding sinus venosus. It is applied so closely to the margin of the umbilical orifice that its caudal border causes a distinct bulging of the boundary of the orifice (fig. 27).

For the sake of comparison, and for the purpose of bringing out the chief differences, as far as the development of the heart is concerned, between this specimen and that of Yeates (’15), the description of the endothelial tubes of the latter given by Yeates might be quoted: “As the endothelial tubes course through the primitive cavity of the heart (the primitive cavity of the heart means the muscular tube—the myoepi- cardium) they are separated by a variable but distinct interval from the myoepicardium The myoepicardial tube has previously been stated to present constrictions at each extremity, at the sinuatrial junction and at the atrio-ventricular canal. The endothelial tubes present corresponding constrictions. In the region of these constrictions the endothelial tubes are relatively close to the primitive myoepicardium, whilst they gradually recede from the heart wall as the middle of each of the three primitive cavities of the muscular heart is approached. In other words, the muscular cavity is more expanded, and the endothelial is more tubular between the constrictions. ‘The endothelial tubes are in contact medianly in the cranial two-thirds of the atrium, in the ventricle and in the region of the bulbus. In the atrio-ventricular canal they are not only in contact but have fused and are partially absorbed, so that their cavities communicate across the median plane. On the other hand, in the region of the sinus venosus and of the truncus, the tubes are free and separate from each other. The portions of the tubes which are in contact within the atrium are con- nected by delicate endothelial strands with the inner aspect of the ventral wall of the atrium along the crest of the irregular ridge, which has been spoken of as the remains of the primitive cardiac septum. The ventri- cular limbs of the endothelial tubes are subdivided into ventricle, bulbus 150 © Dr Cc. C. Wang

cordis, and truncus arteriosus by two faintly marked constrictions. The endothelial heart, therefore, consists of not only sinus venosus, atrium, and ventricle, as in the muscular heart, but also of bulbus cordis and truncus arteriosus.” .

It will be noticed, then, that Yeates’ specimen is decidedly in a more advanced stage of development, since the two endothelial tubes have partially fused in the region of the atrio-ventricular canal, much in the same way as the Stage V. specimen, to be immediately described.

Subdivisions of both of .the endothelial tubes into sinus venosus, atrium, ventricle, bulbus cordis, and truncus arteriosus have been observed also by Yeates in his specimen; but, unlike those of the Stage IV. - 25-mm. ferret embryo, these subdivisions correspond in the main in positions to those exhibited in the muscular (myoepicardial) wall of the heart tube. One point which is clear is that the endothelial tubes differentiate into their various subdivisions more completely than the muscular tube.

It should be pointed out that the second aortic arch in Yeates’ specimen has already made its appearance.

It is to be noted that, as far as the external appearance of the heart is concerned, the ferret embryo at this stage of development shows certain prominent features which are in many respects identical with those of the dog of a similar stage of development, and which have been investigated by Bonnet (01). In his paper it has not been possible to find a com- prehensive description of the development of the heart of the dog. In fig. vi. (Anat. Hefte, 1901, Bd. xvi.) Bonnet depicted a dog embryo 57 mm. long with 10 somites. There the heart is represented by two endothelial tubes which are distinctly separated from one another and are both bent towards the right side in the ventricular region; each endothelial tube has, for its caudal continuity, the corresponding vitelline vein, which runs latero-medially and at the same time cranially towards the sinus venosus - There seems to be no distinctive demarcation between the sinus venosus and the atrium, but between the atrium and the ventricle there is a constriction both on the lateral side and the medial side. The ventricle looks very much dilated in the figure, so much so that, when viewed from its ventral aspect, the whole heart appears to be formed by the distended. ventricle, with the bulbus cordis and atrium attached to it cranially and caudally respectively as mere appendages. The bulbus cordis is clearly constricted off from the ventricle, and its calibre is barely one-third of that of the ventricle. Development of Blood-vessels and Heart in Ferret Embryos 151

Stace V.

The material for this stage consists of one embryo which measures 3°14 mm. in length.

Description of the Graphic Reconstruction of the Heart of a Ferret Embryo 3°14 mm. in Length with 13-14 Somites. (F. 15d. (e).)

The general development of this embryo is so similar to that of the Stage IV. specimen as to merit no separate description.

The specimen is slightly older than the Stage IV. ferret embryo. As in the case of the other embryos described, the state of preservation of this embryo is perfect. In length it undoubtedly exceeds the Stage IV. specimen, but when other measurements are taken, the fact is revealed that the embryo in question is relatively a small one. The pericardial cavity measures 400 in its cranio-caudal diameter and 840u from side to side. :

No plastic reconstruction of this embryo has been made. The graphic reconstruction of the heart, however, shows that the two endothelial tubes have united in part of their extent (figs. 31 and 32). The fused portion, extending through some sixteen sections of 10u each, appears to be the ventricular part and lies more to the right side (figs. 31 and 32). Cranially the fused ventricle divides into two vessels, each of which represents the bulbus cordis (figs. 31 and 32) and becomes continuous with its correspond- ing dorsal aorta by looping round the cranial end of the pharynx, thus constituting the first cephalo-aortic arch (fig. 31). The paired atria run into the fused ventricle cranially, and each receives its sinus venosus caudally (fig. 34).

The vitelline veins are very much in the same stage of development as those observed in Stage IV. (compare figs. 27 and 31). The right vein pursues a more transverse course latero-medially, and terminates at its corresponding sinus venosus. In this, as in the preceding specimens, there is no trace of a ventral mesocardium. The dorsal mesocardium is, however, present in this specimen (figs. 32, 33, and 34).

It may be observed that at this stage of development the ferret heart, though resembling very closely the heart of the Stage V. Perameles obesula (19, viii., 03) described by Miss Parker, yet differs from it in many respects. In both cases the two endothelial tubes have partly fused. In the ferret the fusion occurs in the ventricular region (fig. 31). In the Perameles obesula, according to Miss Parker, the bulbus is the only portion of the heart in which the endothelial tubes have actually fused _ at this stage. oO 152 Dr C. C. Wang

AeA.


Fig. 31.—Graphic reconstruction of the heart of ferret embryo 8°14 mm., ventral view. 100

AOo.A., aortic arch; D. Ao., dorsal aorta; B., bulbus cordis; A., atrium; 9.v., sinus venosus ; _ P., pericardial reflection ; V.v., vitelline vein ; V., ventrieles (fused).

Medullary tube.





Dorsal aorta.

Dorsal mesocardium.

Fused ventricular portion of the two endothelial

tubes. ~ Pericardial cavity.


Fic. 32.—Transverse section through fused ventricular region of 3°14-mm. ferret embryo. x 100. : Development of Blood-vessels and Heart in Ferret Embryos 153

Asymmetry of the two endothelial tubes has also been noted in Parameles embryos. Miss Parker observes that whilst the left heart tube

Medullary tube.



Dorsal aorta. ——~


Fore-gut.=——_%

Dorsal mesocardium.

Left bulbus cordis.

Right bulbus cordis.

Pericardial cavity.

Fig. 33.—Transverse section through bulbus cordis. x 100.

i rta, Dorsal mesocardium. Dorsal a0 ta

Fore-gut. X Medullary tube. /

/

Pleuro-peri- , cardial canal.

Right vitelline vein.

@

Caudal reflection of pericardium Left sinus at the level of septum transversum. venosus.

Fic. 34.—Transverse section through sinus venosus. x 100.

is practically straight, the right tube shows well-marked curvatures. In

the ferret it has been found that the two tubes, prior to fusion, appear to

have been shifted as a whole towards the right side (fig. 27), and that they

remain in this position even after partial fusion has taken place (fig. 31). VOL. LII. (THIRD SER. VOL. XIII.)—JAN. 1918. 11 154 Dr C. C. Wang

Two pairs of aortic arches are found arising from the fused bulbus in the Perameles embryo, but only one pair of vessels can be recognised in the ferret embryo at this stage, and these come from the yet unfused bulbus (fig. 31). In both instances the ventricular portion is the most dependent part of the endothelial tubes.

Stage VI. Macropus ruficollis 52 mm. of Miss Parker is distinctly older and is in a more advanced stage of development than the Stage V. ferret embryo, but it is interesting to note that in Macropus the right and left heart tubes are fused except im the region of the sinus venosus, where they remain separate. Three pairs of aortic arches are described. The fused heart has already begun to acquire the S-shaped curvature, so that the bulbus arteriosus lies dorsal to the cephalic extremity of the ventricle. The bulbus arteriosus is continued into a short median ventral aorta which bifurcates to form the first pair of aortic arches. The second and third pairs of aortic arches arise from the median ventral aorta immediately caudal to its bifurcation. The atrial limb of the S is carried into position dorsal to the ventricle. On the other hand, in the ferret of 3°14 mm. the two heart tubes are only fused in the middle parts of their extents.

DISCUSSION. Origin of Blood Cells and Vessels.

The transformation of the blood-cells into red and white corpuscles. lies outside the scope of the present communication, in which the develop- mental relationships which the blocd-cells have in common with’ the vascular endothelium will alone be considered.

In the paragraph dealing with the development of the extra-embryonic vascular rudiments in mammals, it has been pointed out that in embryos of the higher vertebrates the earliest vascular rudiments have invariably been described by most authors as appearing, at first, in the form of localised cell cords (“angioblast” of His). Thus far all observations on this point incline to support the work of His (’00).

Further, in the literature of the past twenty-five years there are numerous descriptions and illustrations of the origin of blood-cells from the vascular linings. In 1892 Schmidt described the transformation of individual endothelial cells into white and red blood corpuscles. In

support of Schmidt’s view, Maximow (’09) states that the endothelial cells

and blood-cells are closely related and arise from a common stem-cell in the blood islands, and may continue to do so from such a cell during later development.

The most damaging evidence against Maximow’s view is to be found Development of Blood-vessels and Heart in Ferret Embryos 155

in the recent work of Stockard (’15), who, after having conducted a series of experiments on Fundulus, comes to the conclusion that endothelial lining of vessels is utterly incapable of giving rise to any form of blood- cells, and that vascular endothelium arises in loco in many parts of the embryonic body in which blood-cell rudiments are not present.

If Stockard’s view is correct, it necessarily follows that, even if the two groups of cells have a common origin, they are not interchangeable nor can one replace the other: nevertheless it is the common opinion, based upon the results of many investigators, that the angioblast cells produce both blood-cells and endothelium, but there is no agreement as to whether the angioblast cells are derived from mesoderm or from entoderm. Nearly all investigators in this field of work have assumed that blood cells and vessels have a common origin which some attribute to the mesoderm and others to the entoderm.

The facts now to-be recorded do not support the view of a common angioblastic origin of both endothelium and blood-cells, and in this communication the term angioblast will be restricted to the progenitors of the blood-cells. _

It has been pointed out that in the ferret embryos, Stage I. (a) and Stage II. (6) (figs. 1, 156, 17a and 6) it is possible to identify mitotic division in the entodermal cells in the neighbourhood of angioblastic clusters, and there is no evidence to show that, in the ferret, endothelial cells are capable of giving rise to blood-cells. In figs. 2a, b, 8, 4, 5, and 15a it is to be observed that angioblast cells are in abundance on the yolk-sac. These are frequently adherent to the entodermal cells, which, if not in direct protoplasmic continuity with the blood-cells, are, in many cases, in close contact with them. It is to be further noted that where the apposition of these cells is intimate, it is impossible to distinguish the angioblast cells from many of the entodermal cells, the nuclei, size, and shape of the two kinds of cells having a close resemblance. On the other hand, a great dissimilarity exists between the blood-cells and the neighbouring mesodermal cells, for in the former the cells are, without exception, spheroidal in shape, their nuclei are large, staining more deeply, and the protoplasm is comparatively small in amount; whilst in the latter, the cells are usually spindle-shaped, their nuclei have mostly differentiated and taken on a lighter stain (figs. 3, 4, 5, and 15a). Further- more, in the ferret embryos angioblast cells have been demonstrated in regions of the yolk-sac where invasion of the mesoderm has not yet taken place. The above facts appear to indicate that, in the ferret at least, if not in all the other mammals, the origin of angioblast cells from the entoderm is highly probable. 156 Dr C. C. Wang

With regard to the yenetic origin of the vascular endothelium, numerous investigators have recorded wandering mesenchymal cells upon the yolk- sac. Stockard (15) claims that in Fundulus these wandering mesenchymal cells ultimately give rise to four different kinds of cells—the endothelial cells, the black chromatophores, the brown chromatophores, and the blood- cells.

Another current view is that after the so-called “angioblast ” has made its appearance, the vascular endothelium arises from the cells of the blood islands by a rearrangement of the peripheral cells of the blood islands to form lining endothelium and the central ones to remain as blood-cells, and that further extension of the endothelium is brought about by buddings of the endothelium which appear, at first, as solid cords but later become hollow. There is so far no evidence to show that the peripheral cells of the angioblast group in the ferret are capable of being transformed into endothelial cells. It is to be noted that in the ferret, endothelial cells, whether in the form of solid cords or grouped together with a lumen, are invariably spindle-shaped from the very beginning.

Ziegler (’87), as will be remembered, maintains that the system of blood-vessels and that of the lymphatic vessels are produced from the remnants of the blastocoel which remain behind as vessels, lacuns, or interstices. Felix (’97), however, inclines to the belief that the circulatory system is, from a developmental point of view, closely related with the ccelom. ,

In connection with this question Stockard (’15) states: “The vessels arising from independent mesenchymal cells in the space of the blastoccel in the teleost yolk-sac entirely overthrow any notion that vessels arise ontogenetically as portions of the ccelomic epithelium. The vascular lumen is originally continuous with the primary body cavity, the segmenta- tion cavity, and never with the secondary body cavity or ccelomic cavity.”

It is clear that these authors agree, at least, that the origin of the endothelium is from the mesoderm. In the ferret it is possible to demon- strate that endothelial cells take their origin from the splanchnic layer of the mesoderm. In some of the sections of the ferret embryo of Stage II. (a) (figs. 10a-d) there are indications to support the view of Felix (’97) that portions of the ccelomic space surrounded by mesoderm may be cut off to form vascular endothelium and to lie between the mesoderm and entoderm.

The opinion that endothelium develops independently of blood-cells is furthered strengthened by the observations of Stockard (15), who finds that vascular endothelium arises in loco in many parts of the embryonic body of the Fundulus, in which blood-cell rudiments are not present, and that independent blood islands, having no connection with the intermediate Development of Blood-vessels and Heart in Ferret Embryos 157

cell-mass, are found on the yolk-sac, and even in extremely young embryos blood islands may appear on the ventral yolk surface at a great distance away from the intermediate cell-mass. He maintains, also, that early blood islands are invariably destitute of endothelial walls. Though favour- ing the view that vascular endothelium and blood-cells are independent of each other in their mode of development, Stockard firmly believes that both types of cells are derived from wandering mesenchymal cells. This author summarises his statement by saying: “The differences among the four types (endothelial cells, black chromatophores, brown chromatophores, and blood-cells) produced are from the standpoint of our present knowledge in all probability due to the potential differences among the apparently similar mesenchymal cells from which they arose. The four types includ- ing endothelial cells and erythrocytes we must consider from an embryo- logical standpoint as arising from different mesenchymal anlagen.”

It will be observed then that Stockard, whilst admitting, on the one hand, (a) that endothelial cells are quite different from blood-cells in shape, in position, and in the period of migration; (b) that the former develop independently of the latter; and (c) that blood-cells when first formed are devoid of endothelial surroundings, claims, on the other hand, that both types of cells have a common parent trunk—the wandering mesenchymal cells. In the Fundulus this is perhaps true, but the evidence produced by the ferret shows the conditions are not the same.

In the ferret there are indications to show that vascular endothelium is mesodermic in origin and that blood-cells are, at all events in the first instance, derived solely, by proliferation, from entodermal cells. Such being the case, it is obvious that the sources of origin of the blood-cells and vascular endothelium are distinct, and that these two different vascular rudiments cannot be considered to have a common origin.

If the biphyletic origin of blood-cells and vascular endothelium is to be accepted, two more points still remain to be solved, namely, how, when, and where the first blood-cells enter the circulation. This has been variously described not only in embryos of different species but probably even among embryos of the same species. Ziegler thinks, however, that just beyond the lateral plates in the plasma-filled spaces of the yolk-sac which lie between the periblast and ectoderm, the first blood-cells project into the circulation. Stockard describes that in Fundulus embryos the earliest blood-cell formation occurs in the yolk-sac blood islands. The cells in these islands continue to divide until they become surrounded by endo- thelium. As to how these blood-cells are provided with endothelial covering, Stockard makes the following statement: “A growing vascular tip may be observed at certain stages to come in contact with a group of 158 Dr C. C. Wang

erythroblasts, or actually a blood island unsurrounded by vascular endo- thelium. The tip of the vessel seems to disorganise to some extent, and its cellular elements slowly surround the group of corpuscles which are later taken into the circulation as the current becomes established in the includ- ing vessel.”

Unfortunately the ferret embryos, at present worked upon, provide no definite evidence on this point, but it is quite clear that angioblast cells are formed outside the embryonic area, and that blood-vessels are formed inside the embryonic area, and are at first devoid of blood corpuscles. Moreover, it has been pointed out that there is no evidence in the very young embryos dealt with that any blood corpuscles are formed by division of or budding from the endothelial walls of the independently formed blood - vessels. It would appear therefore that the earliest blood - cells probably enter the embryo from the periphery.

Intra-Embryonic Blood- Vessels.

In the review of the literature on the subject of the origin of the intra- embryonic blood-vessels, it has already been indicated that the problem has proved to be one of the most difficult in the development of the vertebrate animals. Numerous conflicting views have been advanced regarding the precise mode of the origin of the intra-embryonic blood- vessels. Thus His (’00) and Hertwig (’92) have associated themselves with the theory that the early blood-vessels in the body of the embryo are formed by a budding or ingrowth of the endothelial lining of the vessels from the extra-embryonic vascular area, and Sobotta (02) supports the belief that vessels in the embryo develop in situ, and those on the wall of the yolk-sac are secondary as a result of an outgrowth from the intra- embryonic blood-vessels. Rabl (’86), on the other hand, pointed out the possibility of the vessels of, at least, the cranial region, if not the whole vascular system of the embryo, having been formed by the extension of the paired heart rudiments when these are developed. Recently Riickert and Mollier (06) maintain that the embryonic vascular system, or at least a part of it, arises in situ from the mesoderm of the embryo. Felix (97) states _ that in birds, the aorta and certain veniplexuses all arise in loco.

The question is therefore still open, and each view invites further criticisms or support. In birds the caudal portion of the dorsal aorta is, according to Vialleton (’92), His (00), and Evans (09), formed from the medial margin of the vitelline plexus which has grown into the embryo in the manner already indicated in the beginning of this communication. In the ferret, Stage II. (a), 1:97 mm., it has been found that the caudal portion of the dorsal aorta has established its communication with the Development of Blood-vessels and Heart in Ferret Embtygs_)=


vitelline plexus. Precisely how the caudal end of the dorsal aorta in the ferret is developed, no definite statement can be made, but as far as evidence goes, it is probable that this part of the dorsal aorta arises much in the same way as described by Vialleton (’92) and His (’00).

For the development of the cranial portion of the dorsal aorta, on the other hand, various opposite views are held. His (00) attributes it to the result of a further growth of the same extra-embryonic vitelline plexus which forms the caudal part of the aorta, but which is reduced to a capil- lary chain growing cranially, eventually turning ventrally over the blind end of the fore-gut and fusing with the cranial portion of the heart tubes. In support of this theory Lewis (04) affirms that all intra-embryonic blood- vessels of rabbits are apparently derived as offshoots from the extra- embryonic network of vessels in the splanchnopleure of the yolk-sac, the vitelline plexus ending medially in the embryo in the form of two vessels —the dorsal aorte. Quite recently Bremer (’12) states that in the rabbit embryo of 5 somites, the dorsal aorta, the first aortic arch, the conus arteriosus, and the lateral heart are all parts of an original network of angio- blastic cords derived from the extra-embryonic plexus of blood-vessels.

Riickert and Mollier (06) maintain that the cranial portion of the aorta is developed in situ from the mesodermic cells of the lateral plate of the mesoderm of the cranial region of the embryo. In support of the autoch- thyonic origin of the cranial portion of the dorsal aorta, the work of Huntington (10, 14) and M‘Clure (’10, 12) may be cited. Recently this view is further strengthened by the results of the experiments of Miller and M‘Whorter (’14) on the origin of blood-vessels in the chick embryo. Further support is to be found in the more recent experimental evidence presented by Reagen (15), which shows the origin im loco of vessels in isolated parts of chick embryos, and by Stockard (15), which claims beyond doubt that in Fundulus embryos the. heart endothelium and aorta arise in loco within the embryo, and here there are no vessels, nor even meso- derm, present on the yolk-sac in the cranial portion.

Fig. 6a represents the graphic reconstruction of the vascular system of the cranial portion of the Stage II. (a) ferret embryo. In this specimen the heart rudiment is represented merely by a transverse blood channel which lies across the median plane and unites the cranial ends of the two vitelline veins. The pleuro-pericardial cavity, together with the pleuro-pericardial canals, has already been described as having the shape of an inverted U-shaped canal which lies dorsal to the vitelline vein and the heart rudiment. Two rudimentary dorsal aorte can be made out in this specimen. They run caudo-cranially one on each side of the medullary groove. They are still more or less plexiform in character, and they terminate blindly 160 Dr C. C. Wang

at their cranial extremities. The absence of the first aortic arch, which is so conspicuously seen in the next stage, deserves particular notice. It is clear that at this stage of development in the ferret, the heart rudiment and the caudal part of the dorsal aorta are present, but the connection, 2. the cranial dorsal aorta, the first aortic arch, and the conus arteriosus, between the heart and the caudal dorsal aorta, is still wanting (fig. 6a). A stage further in the development of the cranial portion of the dorsal aorta is illustrated by the Stage III. embryo (fig. 18). Here the dorsal aorta is seen to have established its connection with the heart rudiment through the first aortic arch. But exactly how this connection takes place, there is no evidence from which to form any definite conclusion. It is as yet impossible to decide whether the first aortic arch and the conus arteriosus when developed, as seen in the specimen just referred to, should be attributed to the result of a cranialward growth from the dorsal aorta, or as the direct outcome of an extension of the heart rudiment growing round the cranial end of the fore-gut to join the dorsal aorta and to constitute the conus arteriosus and the first aortic arch. All that can be said is that probably coinciding with the formation of the head fold the two dorsal aortz are carried, pari passu, cranialward over the cranial end of the fore-gut, and possibly, as the result of a further growth from the blind ends of the aorte towards the heart rudiment, these structures establish their communications with the heart. If this contention re- presents precisely what really takes place in the ferret embryo, the conus arteriosus and the first aortic arch must be considered as being the result of a further growth from the dorsal aorta. But if the other theory is to be accepted, that is, that the development of the conus arteriosus and the first arch is due to an extension of the heart growing round the fore-gut, then the development of these parts of the vascular system does not conform with the statement made by Bremer (’12) relating to the early development of the blood-vessels in the rabbit embryo of 5 somites. ‘This investigator asserts that the dorsal aorta, the first aortic arch, the conus arteriosus, and the lateral heart are all parts of an original network of angioblast cords derived from the extra-embryonic plexus of blood-vessels. Another mode of origin which cannot, however, be overlooked, is that, after the heart rudiment and the dorsal aorta have been laid down, the remaining parts of the main vascular system of the cranial region of the embryo may develop im situ from the mesoderm. The view that parts of the intra-embryonic vascular system arise im situ cannot be ignored, for there is an overwhelming accumulation of conclusive evidence to indicate that the formation of the intra-embryonic blood-vessels is much more extensive and important than has hitherto been supposed. Development of Blood-vessels and Heart in Ferret Embryos 161

From whichever point of view the development of the dorsal aorta, the first arch, the conus arteriosus, and the heart is to be looked upon, the fact remains that these structures do not develop simultaneously. This is clearly shown in fig. la, in which the heart rudiment and the dorsal aorta at this stage of development are all represented, yet there is nothing to indicate or to represent the future first aortic arch and the conus arteriosus.

It is certain that, in the ferret, mesoderm is laid down in the whole of the embryonic area before any blood-vessels appear in the area. It is also certain that some of the splanchnic mesoderm cells arrange themselves into an anastomosing plexus of larger and smaller strands. One of the larger strands lies lateral to the notochord on each side and ultimately is trans- formed into the dorsal aorta, whilst previously a strand along the anterior border of the area is transformed into the channel of communication between the vitelline vein of opposite sides.

The dorsal aorta and the transverse communication between the vitelline veins are at first quite separate from one another, except that they are both connected with the surrounding splanchnic mesoderm.

As the head fold develops the dorsal aorta and the transverse channel between the vitelline veins become united and the first aortic arches are formed. How the union occurs is not clearly shown by the specimens, for intermediate stages are wanting. It might be by growth caudalwards from the vitelline veins, or cranialwards from the aorte, but it is more probable that arches of communication are formed in situ from the mesoderm, and that the whole process of blood-vessel formation in the embryo is one of transformation of cords of splanchnic mesodermal cells into tubes. The transformation takes place first in the anterior and posterior ends of the embryonic area, but extends more rapidly forwards than backwards, hence the dorsal aorte grow from behind forwards and the last formed parts of the great vessels are the aortic arches.

Development of the Human Vascular System.

Some points in the early development of the human vascular system may now be discussed, although I have only had the opportunity of examining two very young specimens. According to Evans (’12), it is certain that in man, long before any vascular rudiments are found in the body of the embryo, and at a time before any mesodermic somites are ° formed, typical vascular rudiments are detected irregularly scattered, at .first, over the surface of the ventral pole of the yolk-sac only, but on account of its comparatively small size the vascularisation of the whole surface of the yolk-sac is soon completed.

It is generally believed that, as in other vertebrates already studied, 162 | Dr C. C. Wang

these vascular rudiments make their appearance as nodular swellings of that part of the wall of the yolk-sac known as the area vasculosa, and are cell clumps lying between the mesoderm and the entoderm. It is claimed also that very shortly after their appearance, the peripheral cells of these cell clumps arrange themselves to form endothelium while the central ones remain as blood-cells.

In young human embryos it has been possible to demonstrate that, at a

period before any vascular rudiments on the yolk-sac proper can be distinguished, there develop in the belly-stalk and chorion of the embryo indisputable blood-vessels which appear, at first, as strands of spindle cells possessing a lumen. This has been described by Fetzer (10), and also observed by Graf Spee (96) in the embryo Von Herff of :37 mm. Others (Jung (’07) and Herzog (’09)) have called attention to the aggregations of endothelial cells in the belly-stalk. True blood islands in the belly-stalk near the allantois have been described also by Grosser (13) and Debeyre (12). Frassi (08) also is in favour of the view that well-formed angio- blastic cords can be detected on the ventral surface of the yolk-sac and in the belly-stalk and chorion. ' [have examined very carefully the whole series of sections of “A very early human ovum embedded in the uterus” described by Johnstone (14), and have found that true vascular endothelium in the form of isolated cords is present in the ventral pole of both of the twin vesicles near their attachments to the blastocysts. Dr Johnstone, however, considers these endothelial cords as merely localised thickenings which he was unable to denote definitely as the precursors of vessels.

Judged in the light of observations made by other investigators, it is not unreasonable to look upon what have been considered as the “mere localised thickenings” by Dr Johnstone as in reality true vascular endothelial cords. The reason for this belief is the fact that, in position and in their general characters, the cell cords or “thickenings” in question bear a close resemblance to those which have been described in other early human embryos as endothelium by Fetzer, Graf Spee, Jung, Frassi, and others.

Recently Bremer (14) has stated that, in human embryos, the earliest blood-vessels appear separately in the yolk-sac and in the belly-stalk in the form of multiple rudiments which are for the greater part funnel-shaped invaginations of the surface of the mesoderm. By a partial fusion of the walls of an ingrowth, a portion of the ccelom, bordered by mesoderm, may. be cut off as a separate cavity, lying deep within the substance of the belly-stalk. This investigator, therefore, believes that the endothelium arises either by delamination from the walls of such a detached portion. of Development of Blood-vessels and Heart in Ferret Embryos 163

the ccelom, or by direct extension, in the form of an angioblastic cord, from the mesothelial ingrowth.

Most authors believe that the early development of the vascular rudi- ments in the belly-stalk and chorion in human embryos happens before the yolk-sac proper exhibits any vascular elements. That this should be the case is due to the fact that in human embryos the vitelline circulation is of secondary importance. The belly-stalk and chorion, on the other hand, constitute the primary connection between the embryo and the placenta, and are therefore the first to be vascularised. This deviation from the ordinary type of development is but one of the remarkable series of variations with which man is distinguished from his fellow-creatures.

All facts, therefore, tend to point that in man the first vascular endothelium is laid down in the belly-stalk and chorion. This furnishes an additional evidence in favour of the biphyletic origin of blood-cells and blood-vessels. The next question to be considered is whether the vascular- isation of the yolk-sac proper is to be regarded as the extension of a further growth from the vascular rudiments in the belly-stalk and chorion, or whether the process arises im situ by separate vascular rudiments. Bremer (14) thinks that the vascularisation of the yolk-sac proper is a separate manifestation, but it is quite possible that the first vascular endothelium of the yolk-sac is the result of an extension from the endothelium of the belly-stalk and chorion. Unfortunately, Johnstone’s specimen gives no definite indication either of the origin of the cell cords or of their extension. ‘

Development of the Heart and Pericardiwm.

In the literature dealing with the development of the heart and peri- cardium, much has been written regarding the developmental processes of these structures in mammals, but in man much is required yet before a comprehensive knowledge of the developmental phenomena can be obtained. It is still a speculation if the earliest rudiment of the human heart is ” essentially similar to that of the mammalia. Tandler (’12) inclines to the belief that they are similar. It is not to be expected, at present, that the comparison of the development of the human heart with that of the mammalia will throw much light upon the subject until the various developmental processes of the different members of the mammalian embryos are first understood.

In mammals, as has been stated previously, the first rudiment of the heart is the appearance of a number of cells—the angioblast of His, which are distinguishable in embryos of 2-3 primitive somites. These vascular cells appear between the entoderm and mesoderm in the cranial region of 164 Dr C. C. Wang

the embryo on each side not far from the median plane. They are responsible for the development of the endothelial heart tubes only, the remaining structures of the wall of the heart being derived from that part of the visceral ccelomic wall which has been designated by Mollier (06) the heart-plate. It is generally believed that the first appearance of the vascular cells of the mammalian heart is bilateral and is located on the ventral aspect of the pleuro-pericardial canals. Hensen (°76) is credited as being the first who observed this bilateral origin of the heart. A fusion of the two endothelial tubes next takes place, and the paired heart rudiments are therefore transformed into an unpaired heart tube, but the precise mode of this transformation is, up to the present, sub judice.

As far as the bilateral origin of the heart rudiments is concerned there seems to be still a little doubt. In all vertebrates that have been investi- gated all writers incline to believe that the heart rudiments originate in two lateral parts, but precisely how these two parts are brought together to form an unpaired heart is much a disputed point. ,

As already pointed out (vide swpra), some believe (a) that in mammals, as in birds, the two endothelial tubes, out of which the heart is formed, appear at a time when the lateral folds which are said to form the ventral wall of the throat are only just visible; (b) that, on the formation of the lateral folds of the splanchnic walls, the two halves of the heart, enclosed within the hitherto symmetrical and laterally placed pleuro-pericardial cavities, become carried medially and ventrally until they fuse on the ventral aspect of the fore-gut; and (c) that the heart is therefore provided, at least for a time, with a ventral and a dorsal mesocardium.

Professor Wilson of Sydney (’14), writing in favour of the presence of a ventral mesocardium in human embryos, asserts that the human heart, like the amphibian, has, at a certain period of development, a ventral mesocardium. This author bases his conclusion on the result of the ex- amination of a series of sections of a human embryo measuring 1:78 mm. catalogued H3 in his series, which was aborted and: is admittedly in an _ indifferent if not bad state of preservation. In addition it is stated that the specimen was cut “in a plane intended to be transverse to the long axis of the embryo, but which turned out to be distinctly oblique.” The obliquity appears to have taken place in two planes instead of one, namely, a ventro-dorsal and a lateral. No reconstruction of the heart of the embryo has been made to verify his statement, and the numbér of somites has been hesitatingly determined to be three pairs.

In fig. vi. of Wilson’s paper an incomplete “septum” in the peri- cardial cavity has been named by him the “septum proprium interperi- cardiacum.” This he looks upon as evidence of the bilateral origin of Development of Blood-vessels and Heart in Ferret Embryos 165

the pericardial cavity, which, according to his observation, ends on each side “blindly without establishing any communication with other ccelomic cavity such as occurs later, eg. in Mall’s embryo, No. 391.” In the same figure the heart appears to have an attachment to the ventral wall of the pericardium which presumably Wilson calls the ventral mesocardium.

It is quite possible, as far as the figure shows, that the so-called septum proprium interpericardiacum is merely a fold of the pericardial wall which has never, at any time, formed a true septum. The statement made regarding the blind termination of the pericardial cavity on both sides of the embryo is contrary to all known facts established by other observers on this structure in mammals. The narrow communication between the pericardial cavity and the ccelomic cavity has probably been overlooked by Wilson in his specimen on account of the poor histological details of the sections, or the pleuro-pericardial canal might have been obliterated in such a way as to render the communication unrecognisable. If the ob- servation of Wilson is the true interpretation of the condition of the peri- cardium at this stage of development, then the balance of evidence in favour of the primary communication of the pericardial cavity with the general ccelomic cavity in mammals is totally upset. But the unsatisfactory condition of the sections, from a histological point of view, unfortunately affords ground to doubt the accuracy of the observation, otherwise this embryo opens a new field for further investigations into the true nature of the pericardial cavity at this particular stage of development in the human embryo. The connection between the heart tube and the ventral wall of the pericardial cavity in Wilson’s specimen can be feasibly explained in the following way. If the obliquity of the plane of section is such that it passes through the septum transversum as well as the heart tube situated immediately cranial to it, it will be seen that what appears to be the ventral mesocardium is really a part of the septum transversum. More- over, it is quite possible that what Wilson considers to be the two endo- thelial tubes, may after all turn out to be the two vitelline veins passing through the septum transversum to reach the sinus venosus cranially, and that the specimen he has been dealing with might have been pathological.

I was fortunate enough to have at my disposal also the second young human embryo which has been described by Dr Johnstone (14) in his “ Contribution to the Study of the Early Human Ovum.” When the sections came to my hand, it was found that the series was unfortunately not entirely complete; consequently a reconstruction of the heart of this in- teresting stage had to be abandoned. Nevertheless the series serves one particular purpose very well, namely, that it shows that this specimen has been cut in a plane not dissimilar to that of Wilson’s embryo, and that the 166 . Dr C. C. Wang

heart tube in Dr Johnstone’s specimen: bears a close resemblance to that of Professor Wilson’s specimen, in so far as the anatomical relationships of the heart are concerned.

Though no description of the heart of the embryo has been made by Dr Johnstone in his paper, a few very interesting points can be. detected by an examination of the serial sections. At first glance the section of Johnstone’s specimen, from which fig. 35 is taken, appears to indicate that the heart has a ventral mesocardium connecting the ventral part of the heart tube with the ventral wall of the pericardium. But on closer examination, what seems to be ventral mesocardium is found to be really

Fore-gut. Medulla. Dorsal aorta.

Dorsal mesocardium.

Pericardial cavity. Endotnelial tube.

Mesodermic tissue.


Vitelline Septum Muscular heart tube. vein. transversum.

Fie. 35. -Human embryo. x 100.

part of the septum transversum, for when this structure is traced forwards and backwards in the series, it is found that the vitelline veins traverse it to join the sinus venosus. Fig. 36 shows that the vitelline vein passes through the septum transversum to reach the heart. Cranially to this there is nothing to indicate a ventral mesocardium, the cavity of the pericardium extending without interruption from side to side (fig. 37) Professor Robinson has very kindly looked through the ‘sections of Dr Johnstone’s specimen with me and has confirmed the above statement.

In the chick, on the other hand, a ventral mesocardium is recognisable, but this is due, as Robinson (’02) points out, to the relatively late penetra- tion of the pleuro-pericardial canals into the mesoderm in the cranial region. The pleuro-pericardial canals do not extend round and unite in front of Development of Blood-vessels and Heart in Ferret Embryos 167

the medullary plate in early stages, but only at a later stage do they penetrate into the floor of the fore-gut after the mesoderm has been

Mesodermic tissue.

Muscular heart tube.


Endothelial tube.


Vitelline vein. . . Septum transversum.

Fic. 36.—Septum transversum in fig. 36 magnified. x 500.

Medulla.

Dorsal aorta.

Fore-gut. Pericardial cavity.

Dorsal mesocardium.

Pericardial cavity. =

Muscular heart tube. be Endothelial heart tube.


Mesodermic tissue.

Fic. 37.—Human embryo. x 100.

formed. The lateral cavities therefore do not at once become continuous, but remain separated from each other by a double layer of mesoderm which constitutes the ventral mesocardium. 168 7 Dr C. C. Wang

According to Robinson (’02) the pericardial mesoderm appears in the pericardial portion of the embryonic area, and it is there completely differentiated into somatic and splanchnic layers before the head bend is developed; there is, therefore, a single pericardial cavity to begin with, which extends from side to side along the cranial boundary of the embryonic area. As the head bend develops, the single pericardial cavity is reversed, and it is carried into the ventral wall of the fore-gut, where it forms a U-shaped tube which communicates at each end with the general

‘ecelom. The he&rt rudiments are formed in the splanchnic layer of the peri- cardial mesoderm ; therefore, after the reversal of the area, they lie in the dorsal wall of the pericardial cavity attached only by a dorsal mesocardium to the ventral wall of the fore-gut, but they are never, at any time, con- nected with the ventral wall of the pericardium by a ventral mesocardium.

Rouviere (04), whilst agreeing with Robinson as to the absence of the ventral mesocardium, describes the formation of the lateral pleuro-peri- cardial canals which grow cranially round the cranial end of the brain- plate and fuse to form a continuous channel. Miss Parker (’15), in her investigation into the early stages in the development of Marsupials, modifies Rouviére’s opinion, saying “that while the initiation of head-fold formation is in all probability due to the forward growth of the brain-plate, there occurs also an active backward growth of the anterior intestinal portal (umbilical orifice). This process is associated with the rapid expansion of the pericardium which occurs at this period of development, and which brings about the backward and inward growth of the layer of splanchno- pleure limiting the pericardium.”

Against the view that a backward growth occurs in the cranial margin of the umbilical orifice is the contention of Robinson (’02) that “the orifice (of the umbilicus) is not reduced in size during the early stages of develop- ment by the convergence of its margins towards a central point. This being the case, no tucking off of the embryo from the surface of the ovum can occur; on the contrary, what does occur is almost the exact opposite of such a process, for the margin of the area remains asa relatively slow- growing region, whilst the embryonic and extra-embryonic portions of the wall of the ovum rapidly increase in extent. Under these circumstances, it follows that the margin of the embryonic area will soon appear as a ring between the upper or embryonic, and the lower or extra-embryonic parts of the ovum, both of which have expanded beyond it in all directions.”

The evidence afforded by the ferret material I have described shows that there is no ventral mesocardium in the ferret, and all the indications it gives are strongly opposed to the idea that any part of the gut closure is affected by the fusion of the lateral folds. Development of Blood-vessels and Heart in Ferret Embryos 169

In the Stage IT. (a) and (6), in which the head fold of the ferret embryo has not yet appeared (figs. 6a and 6, lla and 6), the pleuro-pericardial cavity is present in the median plane, and immediately caudal and ventral to this the two vitelline veins communicate with one another across the

- median plane. There is no evidence that they were even separate from one another, but the union may be called the primary union of the heart rudiments ; it should be specially noted that the term “ primary ” proposed signifies that the right and left parts separate from one another and are again fused by a “secondary” union. To this point reference will be made subsequently in connection with the discussion of the formation of the unpaired heart rudiment from the two separate endothelial tubes.

At Stage II. of development the separation of the right and left parts of the rudimentary heart has commenced, and the pleuro-pericardial cavity - and the heart rudiments are all represented before there is any indication of the formation of the fore-gut or the head fold (fig. 6a). It should also be noticed that when the heart rudiments and the pleuro-pericardial cavity first make their appearance, the former invariably lie ventral to the latter (figs. 6b and 8). In the subsequent stages of development (Stage III. and onwards) a change takes place in their position, with the result that the heart rudiments occupy a plane dorsal to the pleuro-pericardial cavity, and are connected with the ventral aspect of the fore-gut only by the reflection of the splanchnic wall of the pleuro-pericardial cavity. The reversion of positions of the heart and the pleuro-pericardial cavity can only be explained, as suggested by Professor Robinson (02), by a forward growth of the head fold to form the fore-gut with the cranial margin of the umbilical orifice remaining stationary. With the development of the head fold the heart rudiments suffer a rotation round a transverse axis over the pleuro-pericardial cavity. Consequently when the two heart rudiments which became separated are again. brought together to form one endo- thelial tube, it is connected with the ventral surface of the fore-gut only by a dorsal mesocardium, which is formed by the reflection of the splanchnic layer of the pleuro-pericardial cavity over the lateral and ventral surfaces of the two heart tubes; the portion of the splanchnic wall of the pleuro- pericardial cavity in which the endothelial tube lies is in the meantime pushed ventrally, and thus the dorsal mesocardium is elongated. There is no indication of a ventral mesocardium.

Since the time of Hensen (’76), the first appearance of the heart rudi- ments has been considered to be bilateral, that is to say, the heart rudiments develop as two separate endothelial tubes, one on each side of the embryo not far from the median plane and on the ventral aspect of the pleuro-

pericardial canals. Recently the bilateral endothelial tubes have been VOL. LI. (THIRD SER. VOL. XIII.)—JAN. 1918. 12 170 Dr C. C. Wang

traced to the stage of angioblastic cords, which have also been described as lying between the mesoderm and the entoderm. Spaces soon make their appearance in the vascular masses, and when these coalesce, two endothelial tubes are thus formed, one on each side of the embryo, ventral to the pleuro-pericardial channels. In the ferret the coalescence of the vascular endothelium is to -produce one endothelial tube (the “primary” union of the heart rudiments) lying across the median plane caudo-ventral to the pleuro-pericardial cavity (Stage II. (a), figs. 6a and 6b). This occurs, it should be remembered, at a time before the formation of the fore-gut has made its appearance. When this “primary” endothelial tube is traced laterally, it is found to communicate with the two vitelline veins (fig. 6a). A search through the literature on the early stages of development of the mammalian heart has failed to discover any account of this “primary ” union of the heart rudiments. Miss Parker, in the course of her investiga- tion into the early stages in the development of Marsupials, notices the early or “primary” union of the heart rudiments across the median plane in Stage II. Dasyurus viverrinus (85 mm.); its significance, however, has not been explained by her.

That the “primary” union of the heart rudiments to form a single endothelial tube is not a singular occurrence due to any abnormal or pathological conditions, and that this stage of development of the heart is an important une is proved by the fact that a similar phenomenon repeats itself in another ferret embryo (Stage IT. (6), figs. lla, b, and 12a, b, c), in which the heart and the pleuro-pericardial cavity exhibit features similar to those observed in the Stage II. (a) ferret embryo. It has to be emphasised once more that the “primary” union of the heart rudiments across the median plane as a transverse vascular channel lying caudo-ventral to the pleuro-pericardial cavity occurs at a period before any indication of the head fold or the formation of the fore-gut can be detected.

The next stage of development of the heart in the ferret embryo is represented by the 2°3-mm. embryo (Stage III.). In this specimen, which is obviously slightly older than the Stage II. (a) and (6), the medial part of the “primary” endothelial tube shows indications of splitting ‘into two lateral endothelial tubes, for two non-vascular loculi divide its central portion incompletely into a cranial and a caudal portion (fig. 18). Ap- parently the destruction of the central part of the rudimentary transverse heart (“primary” union) proceeds still further as development goes on, until it is completely separated into a right and a left half; for in the’ Stage IV. ferret embryo the heart rudiments are represented there by two separate longitudinal endothelial tubes lying side by side close together (fig. 27). It is again to be noted that the head fold and the fore-gut of Development of Blood-vessels and Heart in Ferret Embryos 171

this ferret embryo (Stage III.) have already begun to develop, since at the cranial end two vessels, one on each side of the median plane, run cranially from the heart rudiment. They arch round the cranial end of the fore-gut and form the first pair of aortic arches, which terminate dorsally in the corresponding dorsal aorta (fig. 18).

In the comparison of this stage of development of the heart in the ferret with that of other mammals of a similar stage of development, the Stage III. Perameles nasuta (1 S) of Miss Parker may-be cited; the total length of her specimen, after partial flattening under cover glass, from the anterior margin of the brain-plate to the hinder extremity of the primitive streak is, according to Miss Parker, 7°5 mm. In her specimen two endo- thelial tubes are depicted lying side by side in the pleuro-pericardial cavity. Cranially each endothelial tube becomes continuous with the ventral aorta, which runs underneath the ventral surface of the fore-gut. As the cranial extremity of the fore-gut is reached, the ventral aorta bends dorsally round the blind. end of the fore-gut and communicates with the dorsal aorta, so forming the first aortic arch. Whilst still in the pleuro- pericardial cavity the heart tube is described as giving off a lateral branch which is presumably the second aortic arch. What is most striking and interesting in Miss Parker’s specimen is, that “in the median space between the anterior ends of the endothelial heart tubes, are a number of scattered angioblast cells lying between the splanchnic mesoderm and the entoderm.” Miss Parker believes that these cells possibly represent the primordia’ of the capillaries and afford an instance of the origin of angioblast cells from the splanchnic mesoderm after the establishment of the definite endothelial heart tubes. Judging from what has been observed in the ferret embryo of Stage III, it is perhaps more correct to interpret these angioblast cells as being the remains of the once “primary” union of the heart rudiments. This “primary” union is well developed in the ferret embryo of Stage IT. (a) and (b), and also in the Stage II. Dasywrus viverrinus (85 mm. A) of Miss Parker. The explanation for the breaking up of the “ primary ” union of the heart rudiments to form two separate endothelial tubes is, however, not apparent in the light of our present knowledge of the development of the mammalian heart. It is conceivable that partly as a result of the development of the fore-gut cranialward, in the manner pointed out by Robinson (’02), and partly also due to the rotatory movement of the heart rudiments from a position ventral to the pleuro-pericardial cavity to a position dorsal to it round a transverse axis, the cross channel, that is, the “primary ” union of the heart rudiments is, at first, put on the stretch, and finally separated into two endothelial tubes lying side by side, each of which becomes continuous with its corresponding dorsal aorta through the 172 Dr C. C. Wang

conus arteriosus and the first aortic arch. In the ferret embryo of Stage III. (fig. 18) the connection between the heart and the dorsal aorta has already established itself before the heart (after its “ primary ” union) has again completely separated into two halves. In the case of the Stage II. Dasyurus of Miss. Parker, the “ primary” union of the heart rudiments is described as giving rise to the first aortic arch, which follows the antero- lateral margin of the gut almost to the median plane and there becomes continuous with the corresponding dorsal aorta. But what has been taken for the first aortic arch by Miss Parker may, after all, prove to be the plexus between the dorsal aorta and the vitelline vein, which has been observed by Bremer (’12) in the 3°4-mm. rabbit embryo.

It will be noticed, then, there is evidence to show that, at a period before the formation of the fore-gut, the first rudiment of the heart in the ferret is single and is situated in the median plane of the embryo, caudo-ventral to the pleuro-pericardial tube cavity, in the form of a transverse endo- thelial tube which is destitute of any blood-cells. Laterally it is in direct communication with the two vitelline veins, one on each side of the embryo. But exactly how this “primary” transverse heart tube is formed it is impossible to make any definite statement. The contention of His (00) does not fully account for the origin of this “primary” union of the two vitelline veins, for, according to His (00), two endothelial tubes are’ directly formed from the ingrowth of the two vitelline veins which, by a further growth, communicate with the two dorsal aorte. Possibly the “primary” union of the heart rudiments is the result of an ingrowth from the vitelline veins which, instead of linking up each with its corresponding dorsal aorta, as His would have imagined, have grown across the median plane. In this way the U-shaped vitelline system is, at least for a time, not connectéd with the longitudinal aortic system cranially. This is exactly what is seen in the ferret embryo of Stage II. (a) (fig. 6a). It is also possible, and perhaps more probable, that the “primary” union arises im situ and is afterwards joined to the two vitelline veins.

From whatever point of view the development of the heart is looked upon, it is clear that in the ferret the first rudiment of the heart appears as a cross channel situated ventro-caudally to the pleuro-pericardial cavity, and is connected only with the venous system, which is composed of the two vitelline veins laterally. The arterial system, that is, the dorsal aorte, remains, at least for a time, distinct and unconnected with the heart rudiment. No explanation can at present be offered as to why the heart rudiment, when first represented, should only be united with the venous circulation. It is evident that further light in this field of investigation is required before a solution can be obtained. Development of Blood-vessels and Heart in Ferret Embryos 173

Quite recently Professors Robinson and Gibson (’16), in their description of a reconstruction model of a horse embryo twenty-one days old, mention that “the allantoic blood-vessels consist of a number of dilated capillaries which form a coarse network on each side. Each lateral network receives two branches from the caudal end of the dorsal aorta of the same side, and it terminates, at the caudal end of the allantoic mass, in a terminal trans- verse sinus from which the umbilical veins take their origin. But, in addition to the connection with both umbilical veins through the terminal sinus, each vascular network also communicates directly with the umbilical vein of the same side.” The terminal transverse sinus at the caudal end of the allantoic mass of the horse and the transverse heart rudiment situated in the cranial end of the ferret embryo may together represent at one time a portion of an original complete ring of circulation.

The next phase of development of the heart is represented by the separation of the “primary ” heart rudiment into two distinct endothelial tubes lying closely together, one on each side of the embryo, not far from the median plane (Stage IV.). Coinciding with the development of the heart, the head fold appears, and as the formation of the fore-gut proceeds, the heart rudiment suffers a reversion with regard to its relation with the pericardium, for it is seen that when the fore-gut is formed the heart is found on the dorsal aspect of the pleuro-pericardial cavity and is attached to the ventral surface of the fore-gut.

Each tube is covered by the splanchnic wall of the pleuro-pericardial cavity on its lateral, ventral, and medial surfaces. Dorsally they are connected with the ventral aspect of the fore-gut by the reflection of the wall of the pleuro-pericardial cavity. Ventrally the pleuro-pericardial cavity passes from side to side (fig. 27). It is to be noted that the two tubes lie far apart from each other cranially and caudally where they emerge from the pleuro-pericardial cavity. The distance between them is greater caudally (fig. 26) than cranially (figs. 27 and 28d). It is obvious that at a stage further, when the two heart rudiments are brought together to form one endothelial tube, it is connected only with the ventral surface of the fore-gut by a dorsal mesocardium and no ventral mesocardium can possibly be developed.

Cranially each endothelial tube can be traced to its corresponding dorsal aorta through the first aortic arch, and caudally each tube com- municates with its corresponding vitelline vein in the region of the septum transversum (figs. 21, 25, and 27).

It is generally held that the rate of growth of the two endothelial tubes exceeds that of the pleuro-pericardial cavity. Consequently, as the result of the different rate of growth, the two tubes, instead of having a 174 Dr C. C. Wang

straight appearance, are thrown into loops before the “secondary” union takes place.

Miss Parker (’15) thinks, however, that at this stage of development the pericardium grows rapidly in length and decreases in width so that the heart tubes are brought together by longitudinal stretching of that part of the pericardial wall which lies between them. In the ferret it is clear. that this is not the case; the two endothelial tubes seem to grow more rapidly in length than the pericardium, with the result that loops — are formed with constrictions here and-there to mark out the different subdivisions of the heart (atrium, ventricle, bulbus, etc.), before any fusion of the two tubes takes place (Stage IV. fig. 27), these subdivisions appear to remain even when the two endothelial tubes have partially united (Stage V. fig. 31). The appearance of these loops in the endothelial tubes speaks against the theory of stretching advanced by Miss Parker. For, if it were true, the result of any stretching of that part of the pleuro-peri- cardial wall which lies between the medial borders of the two endothelial tubes would, in the first instance, be the undoing of the loops, or the pre- vention of their formation, before any approximation of these tubes could be effected.

In the ferret (Stage IV.) what has really happened is this, that as the result of the rapid growth, the two endothelial tubes are thrown into loops, and as the result of a further growth of the two tubes medially towards each other, the part of the pleuro-pericardial wall which lies between them is pushed ventrally. When fusion of the two endothelial tubes takes place, the unpaired heart is, therefore, attached only to the ventral wall of the fore-gut by the dorsal mesocardium. Ventrally the splanchnic wall of the pleuro-pericardial cavity passes from side to side across the ventral aspect of the fused heart tube, there being no fusion of this part of the pleuro-pericardial wall, as supposed by Miss Parker.

Asymmetry of the heart tubes, either before or after fusion takes place, has been noted in the ferret embryos (Stages IV. and V., figs. 27 and 31), as in the Stage IV. Perameles nasuta and also, in a measure, in the Stage V. Perameles obesula (10, viii., 03) of Miss Parker and likewise in the 5-7-mm. dog embryo of Bonnet (01). There seems to be a tendency for the two endothelial tubes, even before fusion, to curve and to be shifted distinctly as a whole to the right side with the concavity of the bend facing the left. In Stage V. (fig. 31) the fusion of the two endothelial tubes in the ferret takes place in the ventricular region, and the fused part lies clearly on the right side of the median plane. The fact of this occur- rence contradicts, in a very convincing manner, the theory of stretching of the pleuro-pericardial wall, for the fused portion of the two endothelial Development of Blood-vessels and Heart in Ferret Embryos 175

tubes should occupy the median plane of the embryo if the two tubes were really brought together by the uniform stretching of that part of the splanchnic wall of the pleuro-pericardial cavity which lies between them. There is no evidence to prove that the fusion of the two tubes, occurring on the right side, may be due to an uneven stretching of the dorsal wall of the pleuro-pericardial cavity.

In the Stage IV. Perameles nasuta (2 P) of Miss Parker the descrip- tion of the heart in the text does not seem to agree with her illustration— Plate 1, fig. 5. In the text it is stated: “They (the two endothelial tubes) have fused at their cephalic extremity, the fused portion extending through some eighteen sections and representing the most closely approxi- mating portions of the endothelial tubes. . . . From it is derived the bulbus (conus) arteriosus. . . . Posterior to this fused portion, the endothelial tubes lie close together but unfused for a considerable portion of their length and then diverge widely and pass into the vitelline veins.” In her illustration, on the other hand, it appears that the fused portion represents really more than the conus arteriosus. Possibly the ventricles also have fused across, because in the figure it shows that at least the cranial half, if not more, of the two endothelial tubes have fused. If this is the case, it is difficult to explain why in the next stage of development (Stage V. Perameles obesula (10, viii., 03) of Miss Parker) the bulbus is the only portion of the heart in which the endothelial tubes have actually fused, and the ventricles have become once more separated.

In the text and the figures illustrating Stage IV. Perameles nasuta (2 P) and Stage V. Perameles obesula (10, viii. 03) of Miss Parker, there are no data to be found upon which the magnification and length of the embryos in question can be gauged. But as far as one can judge from the illustrations alone, the figures of Stage V. Perameles obesulu are undoubt- edly of a higher magnification although probably taken from an embryo of a greater length than that of the Stage IV. Perameles nasuta. If it is so, an explanation is needed to account for the decrease in length of the fused portion of the two endothelial tubes seen in Stage V. It is unfortu- nate that these important data should have been omitted by Miss Parker in her paper, for their absence diminishes the value of her communication when an attempt is made to use it for the purpose of solving the points under consideration.

Hitherto the heart tube with its time-honoured S-shaped character has been the subject of much dissertation. The literature on the development of the S-shaped heart rudiment is so voluminous and confusing that only a brief summary on this subject can be given. All writers have laid great stress on the subsequent development of the fused endothelial tube which 176 Dr C. C. Wang

is now covered with its myocardial coat. It is generally believed that the next phase of development of the fused heart tube, consisting as it does of an inner endothelial tube and an outer myocardial covering, is the acqui- sition of the well-known S- “shaped form. The caudal portion of the single heart tube grows cranio-dorsally and to the left to form the atrial » limb, and the cranial portion grows caudo-ventrally and to the right to form the ventricular limb. In the Stage IV. ferret embryo model (fig. 22), it has been shown that the external configuration of the muscular wall of the heart does not necessarily furnish a true index to the condition of the development of the enclosed endothelial tubes, for it has been remarked upon that although the muscular coat of the heart rudiment may have acquired the familiar S-shaped appearance, yet the two endothelial tubes have not begun to fuse. Moreover, the segments exhibited in the muscular tube do not correspond in the least with the dilatations and constrictions of the underlying endothelial tubes. Furthermore, in the dog, it has been pointed out by Bonnet, and in Marsupials by Miss Parker, that primary divisions of the endothelial tubes into sinus venosus, atrium, and ventricle occur before they have completely fused to form a single tube. Similar divisions of the heart tubes are present also in the Stage IV. ferret embryo (vide supra). These facts therefore show that the primary divisions of the heart rudiment take place in the endothelial tubes at a time when no fusion can be detected, and that the convolutions of the S-shaped muscular coat of the heart do not necessarily represent the different segments of the underlying endothelial tubes.

In connection with the fusion of the two endothelial tubes there is one more point which requires attention. Many investigators have noted the asymmetry of the heart rudiments at this period of development. Devia- tion of the two heart tubes to the right, before and after fusion, has also been observed in the mammalian heart. In the ferret these features are present. The significance of the asymmetry and deviation of the two endothelial tubes have, so far, not been fully explained by those who have made these observations. It has been noted that the ventricular portion of the heart is the first to fuse, and is situated on the right side of the embryo (Stage V. fig. 31). The fused portion in fact represents the junc- tion between the ventricular and the atrial limbs of the future S-shaped heart tube. As fusion proceeds cranially and caudally, more parts of the heart tubes are taken in to form the two limbs of the S-shaped heart rudiment until the two tubes are completely fused. The result of this fusion is to produce a ventricular limb which is situated ventrally and to the right side and an atrial limb which is directed dorsally and to the left.

It should also be remembered that in the space between the myo- - Development of Blood-vessels and Heart in Ferret Embryos 177

cardium and the endothelium of the heart tubes there is a more or less thick mass of loose mesodermic tissue which separates not only the former from the latter but also the two endothelial tubes from one another before their fusion (Stage IV. figs. 27 and 28a, b,c). This mass of loose tissue, though playing an important role in the establishment of the sino-ventri- cular bundle (Mall (’12)) in the subsequent development of the heart, is essentially a passive element at this stage of development. It permits, on the one hand, the growing endothelial tubes to assume their various bend- ings, constrictions, and dilatations independently, and adapts itself, on the other hand, to the characteristic curling of the muscular tube into an S- shaped appearance. This point is clearly shown in the Stage IV. ferret embryo in which the two endothelial tubes have, as already noted, dif- ferentiated into their various divisions in advance of their fusion (fig. 27), . whilst the muscular wall of the heart has seemingly acquired the familiar S-shaped form (fig. 22).

It is obvious, therefore, that owing to the interposition of this more or less thick mass of mesodermic tissue, the endothelial tubes do not follow, in a faithful manner, the various curvatures and bulges of the myocardial covering of the heart, and it would be a mistake to try to determine, at this stage of development, the true nature of the two endothelial tubes by the simple examination of the condition and shape of the muscular coat, without ascertaining, at the same time, the various features of the under- lying endothelial tubes and comparing these with those exhibited by the muscular covering.

It may therefore be concluded that, at least, from the first appearance of the paired heart rudiments as two endothelial tubes to the time of their “secondary ” fusion, the myocardium has little or no common relationship with the underlying endothelium; that the two structures are quite in- dependent of each other, as far as their individual growth is concerned ; that the various constrictions and dilatations of the two endothelial tubes have a definite significance; that these constrictions and dilatations fore- shadow the site and limits of the future sinus venosus, sino-atrial canal, atrium, atrio-ventricular canal, ventricle, and bulbus cordis; and that the muscular tube comes into conformity only at a later period.

SUMMARY AND CONCLUSIONS.

A. The Blood Cells and Vessels.

Hitherto it has been the general belief that blood-cells and vascular endothelium in mammals are derived from a common origin which, accord- ing to some, is mesodermic, and others, entodermic, and that, whichever 178 Dr C. C. Wang

may be the source of origin, the vascular rudiment appears, at first, as angioblastic cells lying between the mesoderm and entoderm. It has been claimed also that the peripheral part of the angioblast soon resolves itself into an. uninterrupted network of endothelium, and the central part into clusters of blood-cells. It has been stated further. that the endothelium so formed is capable of giving rise to new blood-cells.

The facts revealed by the study of the early stages in the development of the ferret point to the conclusion that, whilst blood-cells and vascular éndothelium are closely related to each other and are found invariably between the mesoderm and entoderm, there is evidence to show that, in the ferret, the origins of these two vascular elements are separate and distinct—the blood-cells arising from the entoderm and the vascular endo- thelium from the mesoderm.

Blood-cells develop first extra-embryonically in the area vasculosa in the form of clusters of spheroidal cells which are provided with large and round nuclei and with a comparatively small amount of protoplasm. These are for the most part found adherent to the entoderm in the neigh- bourhood of their origin before they are engulfed by the endothelium, and are, in most instances, identical in structure with the entodermal cells where the contact is intimate.

On the other hand, the cells which form the endothelial rudiment are mesodermic in origin and are, without exceptions, spindle or flattened in shape. They are generally connected with one another by long slender protoplasmic processes, the result of which is to form a network of endo- thelium. This network is capable of extension by buddings which grow either into the embryo or on to the yolk-sac. The blood-cells are next engulfed by the vascular endothelium which grow round them, and in this way they are taken into the circulation.

B. The Intra-Embryonic Blood-Vessels..

The caudal portion of the dorsal aorta communicates, at a very early stage of development, with the yolk-sac circulation through the vitelline arterial plexus. Cranially the dorsal aorta stops short and remains un- connected with the heart rudiment as long as there is no head fold or fore- gut. This part of the dorsal aorta makes its appearance in association with the development of the head fold and fore-gut.

The conus arteriosus and the first aortic arch develop after the “primary” heart rudiment, the two vitelline veins, and the dorsal aortz are all represented, and at the time when the formation of the head fold and fore-gut has begun.

The vitelline veins are two in number, one on each side of the embryo. Development of Blood-vessels and Heart in Ferret Embryos 179

They lie for the greater part of their extent ventro-medial to the pleuro- pericardial canals. They grow from the wall of the yolk-sac into the cranial extremity of the embryo. They are, at an early period, united across the median plane to form the “ primary ” heart rudiment which lies between the pleuro-pericardial cavity dorso-cranially and the bucco- pharyngeal membrane caudally. A short distance caudal to the “ primary ” heart rudiment, each vitelline vein sends out offshoots medially to anastomose with the cranial end of its corresponding dorsal aorta.

C. The Pericardium and the Heart.

Before there is any indication of the head fold or the formation of the fore-gut, the pleuro-pericardial canals have grown across the median plane of the cranial end of the embryo to form the pleuro-pericardial cavity which, in relation with the “primary” heart rudiment, lies cranio-dorsal to it. As the head fold and fore-gut develop by growing cranially, a rotation of the pleuro-pericardial cavity and the heart rudiment round a transverse axis takes place, with the result that the former occupies a position ventral to the latter and the “primary” heart rudiment is now found ventral to the fore-gut.

In the subsequent stages of development the “ primary ” heart rudiment divides into two endothelial tubes, each of which is covered laterally, ventrally, and medially by the splanchnic wall of the pleuro-pericardial cavity and is attached only dorsally with the ventral aspect of the fore-gut by the reflection of the pleuro-pericardial wall. As a result of the growth of the two endothelial tubes towards the median plane, the part of the splanchnic wall of the pleuro-pericardial wall which lies between them is thereby pushed ventrally. When fusion of the two endothelial tubes occurs, the “ secondary ” heart tube is consequently attached only dorsally to the ventral surface of the fore-gut by the dorsal mesocardium. Ventrally there is no fusion of the pleuro-pericardial wall, and therefore no ventral mesocardium can possibly be developed.

At an early period when the pleuro-pericardial cavity has not yet reversed its position, and when there is no indication of the formation of the head fold or the fore-gut, the heart rudiment appears as a transverse blood channel—the “primary” heart rudiment—situated in the median plane ventro-caudal to the pleuro-pericardial cavity and cranial to the bucco-pharyngeal membrane. Laterally the “primary” single heart rudiment communicates with the two vitelline veins, one on each side of the embryo. At this stage of development the cranial extremities of the two dorsal aorte are found, for a considerable distance, caudal to the heart rudiment. 180 Dr C. C. Wang

In the subsequent development of the embryo when the head fold and the fore-gut make their appearance by growing cranially, the pleuro- pericardial cavity and the “primary” heart rudiment undergo a rotation round a transverse axis, with the result that their positions are reversed, so that the heart rudiment now lies dorsal to the pleuro-pericardial cavity.

The division of the “primary” single transverse heart rudiment into two longitudinal endothelial tubes is due to the fact that, at this period, the fore-gut grows rapidly cranially in length and ventrally in width, together with the expansion of the pleuro-pericardial cavity in all directions, so that the transverse heart rudiment which lies between these structures is at first put on the stretch, and is subsequently divided across into two endothelial tubes, a right and a left. Concurrently with the development of the head fold and the fore-gut, the two dorsal aorte establish their communications with the “primary” heart rudiment even before the latter is completely separated into two endothelial tubes.

The next phase of development of the heart is the appearance of the two separate endothelial tubes, one on each side of the median plane. Each tube is continuous cranially with the dorsal aorta and caudally with the vitelline vein. As development proceeds they tend to grow towards the median plane, and in this way the part of the splanchnic wall of the pleuro-pericardial cavity which lies between them is pushed ventrally. The two tubes remain in contact, but not fused, with each other for a time. Owing to the fact that the endothelial tubes grow more rapidly than the pleuro-pericardial cavity, the former are thrown into loops which are separated by constrictions. These dilatations and constrictions indicate in caudo-cranial succession the future sinus venosus, the sino-atrial canal, the atrium, the atrio-ventricular canal, the ventricle, and bulbus cordis. The most dependent and approximating parts of the endothelial tubes are the ventricular portions. They incline more to the right side of the embryo, and are the first to become fused and to form the “secondary ” single heart tube.

The myocardium assumes the familiar S-shaped appearance at a stage when the heart is still in the condition of two separate endothelial tubes. The segments and sulci, appearing on the surface of the myocardial tube, do not correspond, in any way, with the dilatations and constrictions of the two underlying endothelial tubes.

ACKNOWLEDGMENTS.

I take this opportunity to express my thanks to Professor Arthur Robinson for the use of the ferret embryos from his collection which he Development of Blood-vessels and Heart in Fefret Embryos 181 mm SOC. placed at my disposal. To him I am also grateful for. pelptnl advice frequently asked and readily given. Ss

For.the two early human embryos I am grateful to Dr R. W. Johnstone of the Midwifery Department, Edinburgh University, who, as stated, has kindly permitted me to make use of the serial sections.

To the Carnegie Trust my sincere thanks are due for pecuniary support which has greatly facilitated the completion of this part of my research, and a grant to defray the cost of illustrations. The earlier part of the expenses incurred whilst working at the reconstructions has been borne by a grant from the Karl of Moray’s Fund, for which I desire to put on record my gratitude.

The illustrations taken from sections of embryos and from drawings have all been photographed by Mr W. Watson of the Royal College of Physicians Laboratory, Edinburgh, under my personal supervision.

Last, but not the least, I am indebted to Mr J. T. Murray for the drawings of figs. 18, 21, 22, 25, 27, and 30, which represent the original reconstructions.


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VOL. LIl. (THIRD SER. VOL, XIII.)—JAN. 1918. 13


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