Book - Introduction to Vertebrate Embryology 1935-4

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Shumway W. Introduction to Vertebrate Embryology. (1935) John Wiley & Sons, New York

Shumway (1935): Preface - Contents | Part I. Introduction | Part II. Early Embryology | Part III. Organogeny | Part IV. Anatomy of Vertebrate Embryos | Part V. Embryological Technique
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Introduction to Vertebrate Embryology (1935)

Part IV Anatomy Of Vertebrate Embryos

Chapter XI The Anatomy Of Frog Embryos

In earlier chapters we have discussed the fertilization of the frog’s egg (page 57), its cleavage (pages 97, 103), and germ-layer formation (pages 109, 118), and have observed that while the germ layers are being laid down the process is complicated by the early localization of some of the organ systems, notably the sensorynervous complex (page 129). In this account of later organogeny, three stages of development seem especially significant: first, an early embryo of about 3 mm. body length in which the visceral grooves are apparent, a stage attained in Rana pipiens about the second day after the eggs are laid; second, the newly hatched larva of about 6 mm. with external gills developing, about two weeks old; third, a young “ tadpole ” stage of about 11 mm. with the opercula covering the internal gills, about the age of one month.

These stages are easily identified even though the lengths and ages can be given only approximately, for the rate of development is greatly influenced by the prevailing temperature, and the size of the tadpole is determined largely by external factors, such as the amount of food available.

The student must bear in mind that the sections illustrated in this and the two chapters following are for the sole purpose of giving him starting points from which he 1s expected to study all the sections in the series furnished him. He will probably never encounter sections exactly like those selected for these illustrations, but he will discover sections very like them from which he can commence his own observations.

A. THE EARLY EMBRYO (3 MM.)

External form. — This stage corresponds approximately to the embryo of 33 mm. described by Marshall. The head region, through its more rapid growth, has become easily distinguishable from the trunk, which bulges ventrally on account of the large

amount of contained yolk, and a well-marked tail bud is present. 275 276 THE ANATOMY OF FROG EMBRYOS

The neural folds have fused throughout their length, and enclosed the blastopore. In the head the stomodeum appears as an antero-posterior slit on the anterior ventral surface, and is enclosed by ridges identifiable as the maxillary processes and mandibular arches. On either side and slightly ventral to the stomodeum, are the primordia of the sucker or oral gland. At the dorso-lateral margins the olfactory placodes have begun to evaginate. Lateral bulges on either side of the head are due to the developing optic vesicles. The ear is now in the otic vesicle stage. The gill region shows five visceral grooves. Immediately behind the last arch, a swelling is caused by the developing pronephros. Dorsally, slight furrows indicate the boundaries of thirteen soEpiphysie mites. Beneath the tail Optic vesicle bud, the proctodeum Prosencephalon has united with the

Oral gland hind-gut to form the Visceral pouch eloacal aperture.

Fore gut Endodermal derivaay Liver tives. - The anterior portion of the gastrocoel is now a large fore-gut with a thin-walled lining. From this, on a . either side, the begin eurenteric . : canal nings of three visceral Fig. 182. —3 mm. frog embryo, viewed from right pouches can be seen.

side as a transparent object. X15.

From the fore-gut a narrow evagination grows backward into the floor of the mid-gut as the primordium of the liver. The mid-gut is distinguishable by its relatively narrow lumen and thick yolk-laden floor. The small but thin-walled hind-gut opens above into the neurenteric canal by which it is connected with the neurocoel, and opens ventrally to the exterior by way of the proctodeum. An axial rod, the hypochord, is found beneath the notochord. It originates from the roof of the gastrocoel and disappears soon after hatching.

Mesodermal derivatives. — The notochord is large and vacuolated and enclosed by two sheaths. The somites have now attained their maximum number (13) in the trunk, but are not



Mesencephalon

Otic vesicle Rhombencephalon

Somite I Notochord + THE EARLY EMBRYO 277

yet distinguishable in the tail region. The intermediate mesoderm, after a temporary division into nephrotomes, is now reunited into a nephrotomal band in which spaces have appeared opposite the second, third, and fourth somites, indicative of the pronephric tubules which are to develop. A thickening along the



Mesencephalon PD, Prosencephalon


Neurenteric canal

Fia. 1838. —3 mm. frog embryo. Sagittal section: 50.

nephrotomal band immediately below the ventro-lateral margins of the somites is the primordium of the pronephric duct. Immediately below the floor of the fore-gut, the lateral mesoderm has separated into dorsal splanchnic and ventral somatic layers, while the contained space is the beginning of the pericardial cavity, the only region of the coelom yet apparent.

Ectodermal derivatives. — The epidermis at this stage is ciliated. The neurocoel, as has been remarked above, is con278 , THE ANATOMY OF FROG EMBRYOS

nected with the hind-gut by the neurenteric canal. At the anterior end, the brain is distinguishable by its relatively larger lumen and by the cranial flexure over the anterior end of the notochord. The divisions between the three primary vesicles are not marked by the constrictions characteristic of many vertebrates, but are distinguished by the following points of reference: the prosencephalon extends to a Optic line projected from a thickvesicle ening on the floor known as the tuberculum posterius to a point just in front of a similar thickening on the Hypophysis — dorsal wall; the mesencephOral gland = aon, from the boundary of the prosencephalon to a line Fia. 184. —3 mm. frog embryo. Transverse connecting the tuberculum section through optic vesicle. 50. anda point just behind the dorsal thickening; the rhombencephalon merges imperceptibly into the spinal cord. From the prosencephalon, the optic vesicles extend on either side and cause the external bulges already noted. From the ventral side of the prosencephalon, a depression, the infundibulum, extends towards the hypophysis, which in the frog grows inward as a solid wedge of ectodermal cells anterior to the stomodeum. Dorsally, the epiphysis appears as a median evagination.



Fore-gut

B. THE LARVA AT HATCHING (6 MM.)

External form. — Although the larva, if it may be so called, has emerged from the protecting membranes of egg jelly, the mouth has not yet opened and for several days the yolk is still the sole source of food. The head region is still easily distinguishable from the trunk, while the tail has increased greatly in length and has become bilaterally compressed. In the head, the stomodeal pit has deepened at the anterior end, and the maxillary processes and mandibular arches are more sharply sculptured. The invagination of the nasal (olfactory) placodes has THE LARVA AT HATCHING 279


Fig. 185.—3 mm. frog embryo. Transverse section through otic (auditory) vesicle. 50.


Fig. 186. —8 mm. frog embryo. Transverse section through mid-gut and liver. X50. 280 THE ANATOMY OF FROG EMBRYOS


Fig. 187. — 3 mm. frog embryo. Frontal section through optic stalks, liver, and hind-gut. 50. THE LARVA AT HATCHING 281

continued to the point where they may be called pits, connected to the anterior margins of the stomodeal pit by oro-nasal grooves. The bulge of the eye is still prominent. The primordia of the oral glands have fused to form a U-shaped sucker ventral and posterior to the stomodeum. The visceral grooves are still separated from the visceral pouches by closing membranes, while on the third and fourth arches external gills have appeared. Behind them the pronephric elevation is well marked, and continues backward as a slight ridge marking the pronephric duct. Intersomitic grooves are still apparent. On the ventral side at the base of the tail is the cloacal aperture.

Prosencephalon




Optic cup

Mesencephalon é

Otic vesicle a

Heart Rhombencephalon S External gills Pronephros i a— Liver


Yolk

Myotomes

A Fig. 188. — 6 mm. frog larva (just hatched). Transparent preparation, viewed from right side. X15.

Endodermal derivatives. — On either side of the fore-gut are to be seen five visceral pouches, although they would hardly be recognized as such since they are so compressed. A groove on the ventral side of the pharyngeal cavity is the primordium of the thyroid gland. At this stage, also, the dorsal epithelial 282 THE ANATOMY OF FROG EMBRYOS

bodies of the first two visceral pouches (hyomandibular and first branchial) may be distinguished. The liver diverticulum has increased in length. The hind-gut has lost its connection with the neurocoel through the occlusion of the neurenteric canal, but now receives the posterior ends of the pronephric ducts.

Mesodermal derivatives. — The notochord has grown back into the tail. The somites have now become differentiated into the myotomes, dermatomes, and sclerotomes, while from the myotomes muscle cells have been formed. The pronephros is now established. There are three pronephric tubules, each opening into the coelom by means of a ciliated nephrostome. Opposite these, a mass of capillaries, connected with the dorsal aorta, forms the so-called glomus, equivalent to the separate glomeruli of other vertebrates. The pronephric tubules grow backward into the pronephric ducts, which have acquired lumina. At the time of hatching, the primordia of the heart have fused to form a tube, twisted slightly and almost S-shaped, suspended in the pericardial cavity by a dorsal mesocardium. ‘Two regions may be distinguished, the posterior atrium and anterior ventricle. From the ventricle leads the bulbus, arising from the fusion of paired primordia. This connects with the dorsal aorta, also the result of fusion, by means of aortic arches in the third and fourth visceral arches (vestiges of the first and second aortic arches have already appeared and disappeared). At a slightly later stage, loops from these arches will grow out into the external gills to form a branchial circulation. The anterior ends of the dorsal aortae are prolonged to form the internal carotids, while the posterior ends unite directly above the heart, and just after uniting give off the glomi on either side. Both the somatic and splanchnic venous systems are represented at this stage. Two vitelline veins unite to enter the heart at the sinus venosus. The cardinal veins at this time are represented by irregular lacunar spaces in the head and near the pronephros.

Ectodermal derivatives.— The epidermis is still ciliated. From the prosencephalon the thin-walled cerebral vesicle has appeared. The epiphysis is well marked, and the infundibulum is in contact with the hypophysis. At this time the primordia of cerebrospinal nerves may be distinguished. In the spinal nerves, dorsal roots arise from the ganglia produced by the segTHE LARVA AT HATCHING


283 Infundibulum Epiphysi Mesencephalan. piphysis Prosencephalon Rhombencephalon Fore gut Oral gland

Heart

Notochord

Liver

Sittegretee SEN Bengawere ee

ee

pear = a 2

os

a ‘5-3 eo Re Od SA eGo

a Hoe Raa pide Pina SRL

eC PRES ee

BJ

eS Fs “

i) ce ‘ ret OKs Ve on

a

A)

a

Yolk

Ps ae oe

Fig. 189. — 6 mm. frog larva.

Sagittal section, anterior portion. 50. 284 THE ANATOMY OF FROG EMBRYOS



Prosencephalon

Optic cup Lens Optic . ZH stalk FF 5 —_ Notochord

Fore-gut

Stomodeum

Fig. 190. — 6 mm. frog larva. Transverse section through optic cup. 50.

Otic vesicle

cavity

Fig. 191. — 6 mm. frog larva. Transverse section through otic vesicle. 50. THE LARVA AT HATCHING 285

mentation of the neural crest while the ventral roots arise from neuroblasts in the spinal cord. In the head, four ganglia arise and with each is associated a placode of nervous ectoderm. From the first ganglion and placode, the trigeminal (V) nerve arises. The second combination gives rise to the facial (VII) and acoustic (VIII) cranial nerves, while the remainder of this placode invaginates to form the otic vesicle. The third ganglion and placode produce the glossopharyngeal (IX) cranial nerve, and the

Pronephric tubules


Fig. 192. — 6 mm. frog larva. Transverse section through pronephros. 50.

fourth gives rise to the vagus (X). The fourth placode grows back as far as the tail, giving off as it goes small groups of cells which later become the lateral line organs of the trunk. Those of the head arise from the second and third placodes. At this time, also, ganglion cells are migrating toward the dorsal aorta to aggregate as the ganglia of the autonomic nervous system. The eye is well advanced in development, as the optic vesicles have invaginated to form the optic cup and the lens placode has separated from the epidermis and acquired a cavity. The ear is in the otic vesicle stage with an endolymphatic duct. The nose is still represented by the nasal pits. From the prolongation of the fourth placode referred to above, the lateral line system is in process of formation. 286 THE ANATOMY OF FROG EMBRYOS




Visceral 1 pouch

I Visceral arch

Pronephric tubules

Segmental muscles

Fig. 193. —6 mm. frog larva. Frontal section through nasal pit and visceral

pouches. 450. THE YOUNG TADPOLE 287

C. THE YOUNG TADPOLE (11 MM.)

External form. — The head and trunk are now fused into a common ovoid mass, sharply distinguished from the long bilaterally compressed tail. The mouth is open and equipped with horny raspers, while the oral gland is reduced to two vestiges on the ventral side of the head. On the dorsal surface, the large eyes, now functional, protrude slightly. Anterior to these are the external openings of the nasal tubes (external nares). The external gills, which were developing at hatching, have now degenerated and been replaced by internal gills concealed from view by the opercula. On the left side, the opercular aperture serves as a means of egress for the water from which the gills obtain their oxygen. The tail, now two-thirds the length of the tadpole, has a dorsal and a ventral fin. Close to the margin of the latter, at the base of the tail, is the cloacal opening.

Endodermal derivatives. — The mouth has been formed by the breaking through of the oral membrane. From the pharynx, all the visceral pouches except the hyomandibular and the vestigial sixth pouch open to the exterior as visceral clefts; and demibranchs have arisen on the anterior and posterior margins of the third, fourth, and fifth visceral arches and on the anterior margin of the sixth. These are the internal gills which hang down into the opercular cavity. The epithelial bodies from the hyomandibular pouch have disappeared. Those from the second pouch form the thymus gland, while similar buds arise from the third and fourth but presently disappear. The ventral epithelial bodies of the second pouch are said to give rise to the carotid gland, and those of the third and fourth to “ parathyroids.” The fifth pouch never gains communication with the exterior but gives rise to the ultimobranchial bodies. The thyroid is now separated from the pharynx. In the tadpole the pulmonary organs consist of a pair of thin-walled sacs, the lungs, arising from a laryngeal cavity connected with the pharynx by a narrow opening, the glottis. Posterior to the pharynx comes the esophagus, which was occluded just before the opening of the mouth but now possesses a narrow lumen opening into the stomach, which is not greatly dilated. The vesicle, which formerly represented the liver, persists as the gall bladder and common bile duct, rela288 THE ANATOMY OF FROG EMBRYOS


Internal gills

Intestine

Fig. 194. — 11 mm. frog larva.1_ Transparent preparation viewed from right side. X15.

1 Figs. 194-198 inclusive are from preparations loaned me by Dr. A. R. Cahn. In earlier editions they were labelled 9 mm., as measured after preservation. THE YOUNG TADPOLE 289


Infundibulum

Stomach Notochord Intestine Dorsal aorta Yolk Muscles of tail

ie Fig. 195. — 11 mm. frog larva. Sagittal section, anterior part. 40. 290 THE ANATOMY OF FROG EMBRYOS

tively small in comparison with the great glandular mass of the liver. Although the pancreas arose from paired primordia of the duodenum, these have now shifted their position so that their ducts open into the common bile duct. The intestine is extremely long and coiled into a double spiral. It terminates in a slightly dilated rectum, opening into the cloacal cavity which also receives the pronephric ducts and opens to the exterior by the cloacal aperture.

Mesodermal derivatives. — The notochord has elongated toward the posterior end, accompanying the growth of the tail. The two most anterior somites have disappeared, leaving eleven in the trunk region and a much larger and variable number in


Fig. 196. — 11 mm. frog larva. Transverse section, through eye. X40.

the tail. In the tail the myotomes have given rise to the dorsal and ventral musculature. The pronephros has become larger and more complicated through the branching of the pronephric tubules. The coelom consists of a pericardial cavity containing the heart, whose myocardia have disappeared, and an abdominal cavity in which the gut is suspended by the dorsal mesentery. These cavities are still continuous up to the time of metamorphosis. In the heart the sinus venosus is now a large transverse sac; the atrium is partially divided by the interatrial septum; the ventricle has thick muscular walls; and the short bulbus opens into the ventral aorta which is divided into proximal and distal portions by a pair of valves. The ventral aorta is divided into THE YOUNG TADPOLE 291

four afferent branchial arteries, the ventral portions of aortic arches III-VI. From these the blood passes through the internal gills by means of capillaries and is conveyed to four efferent branchial arteries, the dorsal portions of the aortic arches referred to above, which in turn lead to the dorsal aortae. The carotid arteries are connected in front of and behind the infundibulum by commissural vessels, and continue forward as the anterior cerebral arteries. From the anterior commissure the basilars run backward and the anterior palatines forward. The pharyngeal

Myelencephalon


Otic vesicle


Horizontal

Fig. 197. — 11 mm. frog larva. Transverse section through ear. X40.

artery, running forward from the dorsal aorta, at a point just posterior to the anterior commissure, represents the dorsal portion of the mandibular arch; the lingual artery arises independently and unites with the first efferent branchial. From the efferent branchial arteries of the sixth arch, the pulmonary arteries grow backward to the lungs. The vitelline veins have been broken up, by their inclusion in the developing liver, into hepatic veins, opening into the sinus venosus, and hepatic-portal veins from the intestine. The anterior cardinal veins are formed by the union of the superior jugular and facial veins and empty into the common cardinals. From the ventral side of the head the inferior jugulars drain into the common cardinals. The posterior somatic veins are the posterior cartlinals, which return the blood from the 292 THE ANATOMY OF FROG EMBRYOS

region of the pronephros into the common cardinals. The lymphatic vessels of the tadpole have arisen from the confluence of numerous, small intercellular spaces in the mesenchyme. Ectodermal derivatives. — The epidermis is no longer ciliated. The cerebral vesicle is now subdivided into right and left portions, while immediately behind this is the choroid plexus of the diencephalon. The pineal gland is connected with the diencephalon by:a small stalk; the pituitary gland has lost all connection with the exterior. In the mesencephalon the optic

Neural tube



Notochord



Intestine

Fig. 198. — 11 mm. frog larva. Transverse section through pronephros. X40.

lobes are just apparent. The metencephalon is distinguishable by the thickness of its walls as compared with the choroid plexus of the myelencephalon. All cranial nerves and spinal nerves are now established. The eye now contains all elements necessary for functioning; rods and cones of the sensory layer connect with the neurons of the optic nerve; pigment is deposited in the pigment layer; the choroid and sclerotic layers have been formed from mesenchyme; the lens is transparent, as is the cornea formed from the ectoderm. The otocyst is partially divided by a dorsal partition into an outer saccule and inner utricle. The nasal pits have grown backward as solid rods which by now have acquired lumina and will soon open into the

pharynx. REFERENCES 293

Diencephalon

Ear

Notochord

Pronephros


Wall of Intestine

Fig. 199. — 11 mm. frog larva. Trontal section through nose, eye, and ear. 40.

REFERENCES

Huxley, J. S., and de Beer, G. R. 1934. The Elements of Experimental Embryology, Chap. 2.

Jenkinson, J. W. 1913. Vertebrate Embryology, Chap. 7.

Kellicott, W. E. 1913. Chordate Development.

Marshall, A. M. 1898. Vertebrate Embryology, Chap. 3.

McEwen, R.S. 1931. Vertebrate Embryology, 2nd Ed., Part 2.

Morgan, T. H. 1897. The Development of the Frog’s Egg.

Zeigler, H. E. 1902. Lehrbuch der vergleichenden Entwickelungsgeschichte der niederen Wirbeltiere. . CHAPTER XII THE ANATOMY OF CHICK EMBRYOS

The traditional stages in the development of the chick Gallus domesticus) for laboratory practice are those at the end of each of the first three days of incubation. So many important changes take place during the second day, however, that it is advisable to study an additional stage intermediate between twenty-four and forty-eight hours in age. The chick of thirty-three hours is selected because the form of the embryo is not yet affected by torsion or flexure, and the headfold of the amnion has not yet slipped over the head of the chick.

As it is a well-known fact that, in these first few days of incubation, embryos of the same age have attained varying degrees of development, the length of the embryo has been proposed as a mark of identification. The flexures of the body, however, make this standard impracticable, and the remaining alternative is to select the specific development of some particular structure as a basis of arrangement. For this purpose the number of somites, suggested by Lillie, is admirable. Still, it must be remembered that on account of the effect of temperature upon the rate of development, the number of somites is not correlated exactly with the number of hours of incubation, as may be seen from the following table.



TABLE 12 Duval Keibel Lillie Patten About 24 hours Fig. 76 Vig. 9, 9A Vig. 59 Fig. 36

(24 hrs. 6S) (24 hrs. 7-88) (25 hrs. 7S) (27 hrs. 8S)


About 33 hours Fig. 93 Fig. 10, 10A Fig. 68 Fig. 39 (33 hrs. 168) (32 hrs. 9 S) (33 hrs. 128) (33 hrs. 128)


About 48 hours Fig. 109 Fig. 16, 16A Fig. 93 Fig. (48 hrs. 27-28S)| (52 hrs. 278) a8 hrs. 278) (55 hrs. 2 S)


About 72 hours Fig. 115 Fig. 18, 18A g. 117 g. 63 (68 hrs. 37S) {(67 hrs. 35-37 S) (ak 18s, 35S) ah ee 368)






° 294 TWENTY-FOUR HOURS 295

A. THE TWENTY-FOUR HOUR STAGE

At the end of the first day of incubation, the chick embryo has completed the period of cleavage (pages 98, 105) and germ-layer formation (pages 111, 121), and is in the early stages of organogeny.

Anterior neuropore

Head fold Proamnionrn t hy








Amnio te Anteriorcardiac ntestinal portal vesicle Neural fold

Neural groove

4th Somite

Area & pellucida Primitive knot

Primitive streak

Area vasculosa

Fie. 200. — 24 hour chick embryo. Cleared preparation from dorsal side. X25.

External form. — The embryo, 3.3 mm. in length, lies along the axial line of the slipper-shaped area pellucida which in turn is surrounded by the crescent-shaped area vasculosa, whose anterior horns, separated by the proamnion, reach about to the level of 296 THE ANATOMY OF CHICK EMBRYOS

tip of the head. At the anterior end, the head fold of the embryo is lifted above the proamnion from which it is separated by the subcephalic pocket. In the head fold is contained the fore-gut,

0.59 mm. in length, which opens at its

posterior end into the yolk cavity by

means of the anterior intestinal portal.

On either margin of the portal the pri mordia of the vitelline veins are to be

recognized in thick bands of splanchnic mesoderm. The neural plate has already given rise to the neural folds which extend back as far as the first somite. They have united just posterior to the region where the optic vesicles are _ to appear and thus have given rise to a neural tube 0.3 mm. in length, which is widely open in front and behind as the anterior and posterior neuropores, respectively. Behind the head fold the axial mesoderm is segmented into six somites. Between the neural folds the notochord can be recognized as a faint line which joins, at its posterior end, the

primitive streak, now reduced to 0.83

mm. in length.

Endodermal derivatives. — The only differentiation which has taken place in the endoderm consists of the establishment of the fore-gut by means of the folding off of the head from the proamnion. As this process continues the fore gut will be lengthened at the expense of

Fia. 201. — 24 hour chick em- the widely open mid-gut, and the an ean Sagittal section. terior intestinal portal: will progress steadily backward.

Mesodermal derivatives. — The mesoderm proper does not extend into the head, but a loose aggregate of mesenchyme derived from it is present. Posterior to the head the axial mesoderm is divided into six somites. Transverse sections show that TWENTY-FOUR HOURS 297

Epidermis , , Brain




Mesenchyme

Splanchnic mesoderm

Proamnion


Somatic mesoderm Fore-gut


Ectoderm Endoderm.

Fig. 202. — 24 hour chick embryo. Transverse section through brain region. The neural folds have met but are not yet fused together. X50.


Axial mesoderm Notochord ge ie See SO ete Ry oe ayer Vitelline vein Amnio-cardiac Splanchnopleure vesicle

Fig. 203. — 24 hour chick embryo. Transverse section through region of intestinal portal. X50.

Neural groove

| Somite IY Ectoderm

Exocoel





PO Blood island nom i ram) Canes.

Notochord So Endoderm ~

Fig. 204. — 24 hour chick embryo. Transverse section through fourth somite. X50.

Intermediate mesoderm

Primitive groove

Endoderm

HH, BY Fig. 205. — 24 hour chick embryo. Transverse section through primitive streak. x50. 298 THE ANATOMY OF CHICK EMBRYOS

each has a minute cavity, or myocoel. The intermediate mesoderm does not divide into nephrotomes as in the frog. The lateral mesoderm is divided into the somatic and splanchnic layers. In the latter, numerous blood islands appear and give the characteristic mottled appearance to the area vasculosa. The coelom of the embryo is continuous with that of the extra-embryonic regions, or exocoel. In the region on either side of the head, between the proamnion and the intestinal portal, the coelom is distended into an amniocardiac vesicle, so called because the somatopleure will contribute to the head fold of the amnion, while the splanchnic mesoderm will give rise to the primordia of the heart, and the cavities of the vesicles will unite to form the pericardial cavity. The notochord, from its point of origin, the primitive streak, extends forward into the head.

Ectodermal derivatives. — The ectoderm at this stage consists of the elongate neural plate, with its groove and folds which are already in process of fusion, and the epidermis or non-nervous ectoderm.

B. THE THIRTY-THREE HOUR STAGE

External form. — In the chick embryo, after thirty-three hours’ incubation, the length has increased to 4.3 mm. There is a slight bending of the head downward over the end of the notochord, foreshadowing the cranial flexure. The area vasculosa, in which the blood islands are being converted into capillaries, now has grown in toward the embryo, so that the area pellucida persists only around the head and tail regions. The anterior horns of the area vasculosa have met in front, completely inclosing the proamnion. The head has increased in length not only by actual forward growth but also by the backward extension of the lateral margins of the head fold, so that the enclosed foregut is now 1 mm. long. The vitelline veins are prominent at the margins of the intestinal portal and continue on the ventral side of the fore-gut to meet at the posterior end of the heart, which is now a single tube, slightly bent toward the right. The neural folds are fused as far back as the eleventh somite, where the posterior neuropore is now known as the rhomboidal sinus. The anterior neuropore is about to close, and in the head the neural tube shows three regions of dilation which represent the THIRTY-THREE HOURS 299

Head fold |. of amnion



4 neuropore Prosencephalon

Optic

7 vesicle Mesencephalon Foregut Rhombencephalon & Heart

- Vitelline vein

Somite 6

Sinus rhomboidialig

Primitive streak

Fig. 206. — 33 hour chick embryo. Cleared preparation from dorsal view. X25. 300 THE ANATOMY OF CHICK EMBRYOS

fore-brain, mid-brain, and hind-brain, respectively. The sides of the fore-brain are evaginating to produce the optic vesicles.

Head fold . of amnion

Prosencephalon— Subcephalic F pocket Mesencephalon Fore-gut Pericardial cavity


Rhombencephalon# 6.4. /"

Notochord

Fig. 207. — 33 hour chick embryo.

Sagittal section.

are ~ Anterior

intestinal portal

i Primitive cm, streak

aN

X25.





In the hind-brain, five neuromeres can be identified. Twelve somites may be counted. The notochord extends forward to the fore-brain from the primitive streak which is now reduced to 0.3 mm.

Endodermal derivatives. — The anterior end of the fore-gut is in contact ventrally with the stomodeum separated only by the oral plate, composed of ectoderm and endoderm. At the sides, the walls of the fore-gut are fused to the ectoderm at points where the first visceral pouches (hyomandibular) will be located.

Mesodermal derivatives. — The somites now number twelve, and myocoels are still apparent. The mesomere is still unsegmented, but pronephric tubules have appeared in the region corresponding to somites 5-12. The four posterior tubules are growing back to form the pronephric duct. In the splanchnic mesoderm the blood islands are being converted into capillaries. The vitelline veins are prominent and continue forward into the heart, of which the endo -cardium and myocardium are dis tinct. The heart is supported by the dorsal mesocardium, the ventral mesocardium having disappeared. The primordial tubes, from the

fusion of which the heart arose, continue forward as the ventral aortae which bend around the pharynx (first aortic arches) and THIRTY-THREE HOURS 301

continue backward along the dorsal surface of the pharynx as the dorsal aortae. At the level of the primitive streak they are lost in a capillary nexus which foreshadows the vitelline arteries. From a point immediately in front of the optic vesicle, the anterior cardinals course backward on either side of the neural tube, bending down ventrally to enter the heart with the vitelline veins. The notochord is slightly bent at the anterior end.

Ectodermal derivatives. The ncural folds now extend to the eleventh somite and have fused throughout the length of the head. The anterior neuropore is almost closed. The three

Prosencephalon

Epidermis Mesenchyme

Optic vesicle

=» _

ts wa P Exocoel é OOF


Splanchnopleure Sub-cephalic pocket

Fia. 208. — 33 hour chick embryo. Transverse section through optic vesicles. X50.

dilations which represent the prosencephalon, mesencephalon, and rhombencephalon are distinct. From the prosencephalon the two optic vesicles extend to the ectoderm of the sides of the head. Five neuromeres may be identified in the rhombencephalon. It has been asserted that in earlier stages three neuromeres may be identified in the prosencephalon and two in the mesencephalon, while the first of the five noted above has resulted from the fusion of two original neuromeres destined to give rise to the metencephalon. At about this time a shallow depression in the floor of the prosencephalon, just in front of the tip of the notochord, marks the appearance of the infundibulum. The auditory placodes may sometimes be seen in sections as thickenings at the level of the constriction separating the last two neuromeres on either side. 302 THE ANATOMY OF CHICK EMBRYOS

Rhombencephalon






Notochord Fore-gut Otic ( auditory) placode Somatopleure TET Dorsal aorta Dorsal [Bi soy 7S uateral sulcus mesocardium B Feria

5 Way, Oe

ee ae ox ed fhe & wou ee se Os a lamest ~ Endocardium Splanchnopleure Fig. 209. — 33 hour chick embryo. Transverse section through otic placodes.

x50.

Spinal cord




Dorsal aorta Somite

Intermediate mesoderm Exocoel

Se

Vitelline vein

Fie. 210. — 33 hour chick embryo. Transverse section through vitelline veins. x50.

Spinal cord

Neural crest Somite



Intermediate mesoderm Somatic layer

NAPRY

Fig. 211. — 33 hour chick embryo. Transverse section through sixth somite. 50. FORTY-EIGHT HOURS . 803

Cc. THE FORTY-EIGHT HOUR STAGE — External form. — The chick at the end of the second day of incubation has usually attained a length of 7 mm., but the form

of the body has been altered profoundly. As the head has been lifted away from the blastoderm, it has increased greatly in size,









Ww ir ot y h h, Mesencephalon ‘Rhombencephalon

Otic vesicle Optic cup

Lens vesicle Visceral cleft I Prosencephalon

0 i

Sinus venosus— Vitelline vein—

Atrium Bulbus arteriosus Ventricle

Amniotic fold Somite XIV

Vitelline artery

Neural tube

Tail fold

aed cent Fia. 212.—48 hour chick embryo. Transparent preparation from dorsal view (head from right side). X15.

and the cranial flexure, which was just appearing in the thirtythree hour chick, has become so pronounced that the anterior end of the head is directed backwards. With this growth and flexure the head is twisted normally to the right, until it lies on one side, a phenomenon known as torsion. At forty-eight hours, this torsion involves the chick as far back as the seventeenth somite. The posterior end of the chick lies in its original position, and at the extreme caudal end a tail fold is being formed. In the Fig. 213. — 48 hour chick embryo. (304)

frontal section due to torsion.

x50.

Head in sagittal section, somite region in

Stomodaeal plate Telencephalon.


Diencephalon

Myelencephalon


Metencephalon




Mesencephalon FORTY-EIGHT HOURS 305

area vasculosa the capillaries have formed attachments with the vitelline arteries and veins, and at the border of this area is a circular vessel, the sinus terminalis. The fore-gut is now 1.4 mm. in length, and the first of the three visceral pouches now communicates to the exterior following the rupture of the closing plate which separated it from the corresponding visceral groove. The second and third visceral grooves are apparent, but their closing plates are still unperforated. In the visceral arches the first three aortic arches are apparent, arising from the ventral aorta. The heart is now twisted so that the ventricular loop is upper Anterior cardinal vein Dorsal aorta

Otic pit





eB 5

Chorion



Yolk sac tent Notochord eet Blood island Visceral groove I a Pigment layer

Visceral pouch I Hypophysis Sensory layer


Fia. 214. — 48 hour chick embryo. Transverse section through otic pit and optic cup. 650.

most. The vitelline veins are large and conspicuous, as are the vitelline arteries which leave the body at the level of the twentysecond somites. The neural tube is completely closed. In the head the five definitive regions of the brain are outlined, the prosencephalon having given rise to the telencephalon and diencephalon, and the rhombencephalon to the metencephalon and myelencephalon. The eye is now in the optic cup stage, and the invagination of the optic vesicle continues down the stalk to form the choroid fissure. The lens is in the form of a pit which has almost attained the vesicle stage. The ear is represented by an otic pit which, owing to the cervical flexure, is about on a level with the eye. There are twenty-seven somites at this stage. The primitive streak is found only in the tail fold. At this time 306 THE ANATOMY OF CHICK EMBRYOS

the head fold of the amnion has grown back over the chick as far as the sixteenth somite.

Endodermal derivatives. — The stomodeum, an ectodermal invagination from the ventral surface of the head fold, has formed the oral membrane by contact with the fore-gut a little back of its most anterior point. Hence there is a blind pocket in front of the oral plate, known as the preoral gut. Three visceral pouches are present, the first of which opens into the corresponding visceral furrow following the rupture of its closing membrane. The primordium of the thyroid is represented by a ventral depression in the floor of the pharynx at the level of the second visceral pouches. The primordia of the lungs (sometimes difficult to distinguish) extend to the level of the sinus venosus. The liver arises at the level of the anterior intestinal portal from two evaginations of the endoderm, one below and one above the meatus venosus. ‘The mid-gut now has two shifting boundaries, the anterior intestinal portal and the posterior intestinal portal. The latter is barely apparent as the opening of a shallow endodermal pocket or hind-gut in the tail fold.

Mesodermal derivatives.—-The somites, twenty-seven in number, show a varying degree of specialization, with the most advanced at the anterior end. In these two regions can be distinguished: a loose aggregate of cells at the median ventral angle (the sclerotome); and a cap of epithelial cells at the lateral dorsal angle. The cells of this cap nearest the epidermis will form the dermatome, while those nearest the neural tube will form the myotome.

The pronephric tubules in the more anterior somites have disappeared and mesonephric tubules are appearing in the mesomere posterior to the thirteenth somite. The pronephric (now the mesonephric) duct has acquired a lumen but has not yet attained its complete backward growth.

The heart is still tubular, but the ventricular limb of the cardiac loop has grown back and over the atrial limb so that the ventricular region is now caudal and dorsal with relation to the atrial region. Three aortic arches are present as a rule, but infrequently the third has not developed. From the first aortic arch a network of capillaries extends into the head. From these the carotid arteries will be formed. The dorsal aortae have fused FORTY-FIGHT HOURS 307

from a point back of the sixth somite as far as the level of the fifteenth somite. The vitelline arteries leave the dorsal aortae at the level of the twenty-second somite but the aortae continue

Common cardinal Bulbus vein arteriosus Chorion

I.- poem 2s Cra, /

Dorsal aorta Fore-gut er , bAY

Notochord




Spinal cord

,

as

Epidermis 5 J

Amnion

“ Somiter Coelom

‘Dorsal mesocardium Fia. 215. — 48 hour chick embryo. ‘Transverse section through heart. X50.

backward as the caudal arteries to the last somite. The vitelline veins are fused at their point of entrance into the heart as the sinus venosus. The anterior cardinals are prominent and extend from a capillary plexus in the head back toward the heart, where they

Somite

Notochord | Dorsal aorta





Vitelline vein,

Choria

- Armnion— Gi N Coelom —

Posterior cardinal vein

Ventricle


Meatus Mid-gut: *

venosus

Fig. 216. — 48 hour chick embryo. Transverse section through liver. 50.

are joined by the posterior cardinals and proceed as the common cardinals to: enter the heart in the angles between the sinus venosus and the vitelline veins. The posterior cardinals may be traced back to the last somite. The heart of the chick commenced 308 THE ANATOMY OF CHICK EMBRYOS

beating at the forty-fourth hour of incubation, so that the course of the blood is through the ventral aorta to the aortic arches and thence to the dorsal aorta. From the first aortic arch a network of capillaries supplies the head with blood (which is returned by way of the anterior cardinals). The main current of the stream passes down the dorsal aortae to the point where these fuse to form the median dorsal aorta. From the dorsal aorta, the somites are supplied by capillaries, which will later become the intersegmental arteries. This blood is returned through the posterior cardinals. Leaving the dorsal aorta by way of the vitelline arteries, the blood passes through the capillaries of the area vasculosa to the sinus terminalis, and thence to the capillary drainage of the vitclline veins which return it to the heart.

The notochord is bent, not only at its tip (cranial flexure) but also at the point where the myelencephalon merges with the spinal cord (cervical flexure).

Ectodermal derivatives. — The brain now has acquired its five definitive vesicles. The telencephalon is enlarged but shows

Amniotic raphe




— Posterior cardinal vein

Mesonephric tubules

Beast

Lateral sulcus Fig. 217. —48 hour chick embryo. Transverse section through mesonephros. 50.

no particular differentiation. From the diencephalon project the constricted optic stalks which bear the optic cups with their inner sensory layer and outer pigmented layer. (The pigment will not arise until later.) The invagination by which the cups were formed continues down the stalk as the choroid groove. On the ventral surface of the diencephalon the infundibulum has deepened. Growing in toward it from the stomodeum is an ectodermal invagination, the hypophysis, which will fuse with the infundibulum to form the pituitary gland. The lens of the eye SEVENTY-TWO HOURS 309

is in the pit stage, resulting from the invagination of a sensory placode. When the process is complete, the lens will be a vesicle completely withdrawn beneath the surface of the ectoderm, as will the otic vesicle, the primordium of the inner ear. Along the rhombencephalon and cord, the neural crest is to be seen as a narrow band of cells on each dorso-lateral angle.

Myelencephalon


Otic vesicle %, Metencephalon

he 4 te

Visceral cleft I * q Mesencephalon wy Choroid fissure Optic cup Atrium and lens

Diencephalon

Nasal pit - Epiphysis Telencephalon Ventricle Anterior limb bud 3 Somite 26 Vitelline 2 artery Vitelline ; . . : Posterior vem limb bud

Fig. 218. — 72 hour chick embryo. Transparent preparation from dorsal view, head seen from right side. X15.

D. THE SEVENTY-TWO HOUR STAGE External form. — At the end of the third day of incubation, the total length of the embryo is 9.5 mm., but the curvature of the body is so great, on account of the cranial and cervical flexures in addition to the newly developed caudal flexure, that the greatest length, from neck to tail, is 7 mm. Torsion involves the 310 THE ANATOMY OF CHICK EMBRYOS

body as far back as the vitelline arteries and will become complete during the fourth day. Anterior and posterior limb buds are now apparent at the levels of somites 17-19 and 26-32 respectively. The tail is curved forward. The fore-gut is still 1.4 mm. in length but has undergone further differentiation, indicated externally by the fact that the first three visceral clefts are open while the fourth is still interrupted by its closing plate. In the branchial arches four aortic arches may be seen. The telencephalon has given rise to the primordia of the cerebral hemispheres, and from the roof of the diencephalon, a small evagination represents the epiphysis or primordium of the pineal gland. The eye and ear, which were formerly in the same transverse section, are now nearly in an antero-posterior relationship. The olfactory pits have made their appearance in the head. The semilunar (fifth cranial nerve), geniculo-acoustic (seventh and eighth), and petrosal (ninth) ganglia may be seen. There are approximately thirty-five somites. The primitive streak has disappeared. The amnion is completed by the fusion of head and tail folds. The allantois, a small sac-like evagination, protrudes ventrally between the posterior limb buds.

Endodermal derivatives. — At the end of the third day the oral aperture has been formed by the rupture of the oral membrane separating the stomodeum and the fore-gut. Immediately anterior to this opening the preoral gut persists. The fore-gut is still the same length as in the chick of forty-cight hours, but is more complex in structure. The thyroid gland, which appeared during the second day, has now become differentiated into the distal dilation which will give rise to the gland proper and the thyroglossal duct. The first three visceral pouches are open to the exterior, but the epithelial buds destined to give rise to the thymus and parathyroids are not yet apparent. The fourth visceral pouch is still separated from the corresponding groove by the closing plate. The laryngeo-tracheal groove has developed in the floor of the pharynx just posterior to the fourth visceral pouches. At its posterior end the dorsal margins of this groove have closed together to form the primordium of the trachea which is thus set free from the esophagus above. The trachea is bifurcated at the posterior end, thus giving rise to the two bronchial buds which are the primordia of the lungs. SEVENTY-TWO HOURS 311

The esophagus, which is relatively narrow, is followed by a dilation which is to become the stomach. Posterior to this, the primordium of the liver may be seen as an evagination from the








Aortic arches

Dorsal.

aorta Myelencephalon

Metencephalon . Roy fandibatam

Sinus

venosus Isthmus

Atrium Spinal cord , Notochord ++—%

>Amnion Mesencephalon

Diencephalon

Telencephalon Epiphysis Mesonephros

Notochord

Spinal cord—

Fiq. 219. — 72 hour chick embryo. Sagittal section. X25.

ventral floor of the duodenal region of the gut. The dorsal pancreas arises from the duodenal region just dorsal to the liver at the end of the third day. The ventral primordia will not appear for another day. 312 THE ANATOMY OF CHICK EMBRYOS



halon

fe


Fig. 220. — 72 hour chick embryo. ‘Transverse section through otic vesicle. X25.

L esicle Dorsal aorta Optic ome

Aortic arches

d cup Sensory layer ii


Pharynx yyy

Visceral arches Fia. 221. — 72 hour chick embryo. Transverse section through optic cup. X25.









/ Esophagus Primary ‘Common cardinal Bulbus arteriosus “ . : i Chorion Amnion bronchus yon polite eee! oa 3 A ang ots + ~ Somite AfSead- m,

\ rst; Epidermis ip

3

‘Yolk sac



Pleural groove Sinus Atrium Nasal pit “venosus Pericardial cavity Fig. 222. — 72 hour chick embryo. Transverse section through heart and lung. X25.

‘ SEVENTY-TWO HOURS 313

The mid-gut region is gradually lessened by the advancing sulci which are cutting off the body of the embryo from the yolk. This region opens into the yolk stalk which is still quite wide.

The hind-gut contained in the tail fold has not yet acquired its cloacal aperture nor has the proctodeum appeared. The floor of the hind-gut between the tail bud and the posterior intestinal portal evaginates to give rise to the allantoic primordium.

Mesodermal derivatives. — The somites, typically thirty-five in number, still show a varying degree of differentiation which is carried to its furthest point in the more anterior somites. The dermatome is now a thin sheet of cells along the dorso-lateral

Posterior Dorsal cardinal} Dorsal Li _Amnion aorta vein mesentery iver






ao

Spinal cord

Notochord

Coelom

Allantoic vein


Ventral mesentery

Meatus venosus Fig. 223. — 72 hour chick embryo. Transverse section through liver. X25.

angle of the embryo, with the myotome parallel and internal; the sclerotome in these anterior segments is a large and loose aggregate of cells investing the neural tube, notochord, and aortae.

The pronephric tubules have degenerated to a considerable extent, but the nephrostomes opening into the coelom may persist. The mesonephric tubules are now in process of development, with those in the more anterior segments most highly differentiated. The tubules between the thirteenth and _ thirtieth somites have progressed from the vesicle stage characteristic of those behind the twentieth somite, and some have acquired a lumen and joined the pronephric duct which henceforward is known as the mesonephric duct. A few of the more anterior tubules develop nephrostomes, but these soon disappear. 314 THE ANATOMY OF CHICK EMBRYOS

Behind the twentieth somite, as far back as the thirtieth, only vesicles are formed. The mesonephric ducts have grown back and united with the cloaca.

The heart now shows a constriction between the atrial and ventricular region. Four aortic arches are developed, of which

Amniotic raphe

Dermatome, Sclerotome \ Spinal cord


Mesonephric Vitelline tubule artery

Lateral sulcus Dorsal aorta

Fig. 224. — 72 hour chick embryo. Transverse section through vitelline arteries leaving body. X25.

the first is becoming smaller, and somctimes has disappeared at this stage. The internal carotid arteries are now well developed, growing forward into the head from the point of union between the first arches and the dorsal aortae. From the ventral end of the first aortic arch the external carotid takes its origin. The

Chorion Mesonephric . Amnion duct Somite Dorsal ao


Fig. 225. 72 hour chick embryo. Transverse section through allantois. 25.

pulmonary is sometimes apparent as a posterior prolongation of the ventral aorta at the point where the fifth arches will appear during the next twenty-four hours. The intersegmental arteries are now apparent as dorsal diverticula from the aorta between each pair of somites. The vitelline veins have fused for a short distance behind the sinus, thus giving rise to the meatus venosus. REFERENCES 315

The anterior cardinal vein now possesses many branches from the head, among which are three intersegmental veins. The posterior cardinal has continued its backward growth dorsal to the mesonephric duct as far as the thirty-third somite. It receives the intersegmental veins of this region. Where the posterior cardinals unite with the common cardinals, a capillary network indicates the beginnings of the allantoic veins.

Ectodermal derivatives. —-'The brain at the end of the third day has its five definitive vesicles even more sharply demarcated. From the telencephalon two lateral vesicles have evaginated to form the primordia of the cerebral hemispheres. In the diencephalon the epiphysis has appeared as a dorsal evagination. On the floor of this vesicle the infundibulum is almost in contact with the hypophysis. The mesencephalon is separated from the metencephalon by a deep constriction known as the isthmus. Along the sides of the myelencephalon may be distinguished the following cerebral ganglia: the semilunar of the fifth cranial nerve; the acoustico-facialis which will later separate into the geniculate ganglion of the seventh and the acoustic of the eighth; and the petrosal ganglion of the ninth. The eye has increased in size, and the lens is now free from the epidermal ectoderm. The ear, too, is in the vesicle stage and possesses a short endolymphatic duct, which has lost its connection with the epidermis. On the third day the primordium of the nose is represented by two olfactory pits anterior to the mouth.

REFERENCES

Arey, L. B. 1934. Developmental Anatomy, 3rd Ed., Chap. 18.

Duval, M. 1889. Atlas d’embryologie.

Keibel and Abraham. 1900. Normaltafeln II, des Huhnes (Gallus domesticus). Lillie, F. R. 1919. The Development of the Chick, 2nd Ed.

McEwen, R. 8S. 1931. Vertebrate Embryology, 2nd Ed., Part 4.

Patten, B. M. 1929. The Early Embryology of the Chick, 3rd Ed. CHAPTER XIII THE ANATOMY OF THE 10 MM. PIG EMBRYO

Pig embryos of 10 to 12 mm. body length are particularly instructive for laboratory work in mammalian embryology as they

Myelencephalon Metencephalon

Ear,



Mesencephalon

Trachea +

Anterior __| a —Body stalk

limb bud Roots of spinal nerves Posterior

Fig. 226. — 10 mm. pig embryo. Transparent preparation from right side. X11.

are sufficiently large for the study of external structures and yet small enough to afford serial sections for a detailed study of the anatomy. The primordia of practically all the organ systems, excepting the skeleton and musculature, are present. In comparing the accounts given by different authors of this particular stage, it should be remembered that a large amount of shrinkage

takes place during the preparation of fresh sections, so that, as 316 ENDODERMAL DERIVATIVES 317

pointed out by Patten, an embryo of 12 mm. will not measure more than 93} mm. when prepared for sectioning. The account which follows corresponds in general to the pig (Sus scrofa) of 10 mm. described by Keibel, of 12 mm. (Minot), 10 mm. (Prentiss) and 9.4 mm. (Patten), but is not so advanced as the 13.5 mm. pig (Boyden).

External form. — The pig embryo at this stage is relatively ‘more advanced than the chick of seventy-two hours. The body is sharply flexed, owing to the presence of the cranial, cervical, dorsal, and caudal flexures. In the head region the olfactory pits are well developed and are connected by the naso-lachrymal groove to a depression which surrounds the bulging eyeball. The five divisions of the brain are apparent through the relatively thin overlying epidermis. Four visceral grooves can be seen, the first of which, or hyomandibular, is the primordium of the external auditory meatus. The third and fourth grooves are compressed by the cervical flexure into a deeper depression known as the cervical sinus. A frontal view of the head shows the oral cavity bounded above by the frontal process in the middle, the maxillary processes at the side, while the lower jaw is represented by the mandibular arch.

In the trunk region, the buds of the pectoral and pelvic appendages are large but show no further differentiation. The contours of the somites, now forty-four in number, are apparent along the back, and ventral to these can be seen the outlines of the heart, liver, and mesonephros. In some specimens there appears between the limb buds a thickened ridge from which the mammary glands develop and which is therefore known as the milk line. | The umbilical cord projects from the ventral side of the embryo. Between this and the base of the slender tail is a small protuberance, the genital tubercle, or primordium of the external genitalia.

Endodermal derivatives. — The preoral gut still persists anterior to the oral aperture. Ventral to this, and seen best in sagittal section, is the long and slender hypophysis, now in contact with the infundibulum of the diencephalon. Both the hypophysis and infundibulum, it should be remembered, are of ectodermal origin. The pharynx is dorso-ventrally compressed, and from its floor the tongue is arising. Four visceral pouches 318 THE ANATOMY OF THE 10 MM. PIG EMBRYO

are present, corresponding to the visceral grooves already noted. These do not unite to become visceral clefts but remain separated by their closing membranes. Between the second and third

Metencephalon Myelencephalon



Pharynx


Posterior vena cava





//_ . Ductus venosus

i Liver Duct of ventral pancreas

Spinal { nw ; artery fDuodenum___Vitelline vein on—__ Body Dorsal stalk pancreas i ses mbilical SSS ) rte Vitelline 4 7 SN d Af ty (ant. mesenteric)’ oN i R artery cl Notochord oaca Metanephros Aorta

Mesonephros

Fia. 227.— 10 mm. pig embryo. Sagittal section. 164.

pouches the thyroid gland appears. From the level of the fourth pouch a short laryngeal groove is prolonged into the trachea which has given rise to the bronchial buds, three in number. Two of these, the primary bronchi, have arisen by the bifurcation of ENDODERMAL DERIVATIVES 319

the trachea; the third or apical bud, which will give rise to the eparterial bronchus, develops anterior to the right primary bronchus. The esophagus is relatively long and narrow and, just posterior to the level of the lung buds, passes into the stomach which is dilated and shows a slight dorsal curvature. Posterior to the stomach the duodenal glands, liver, and pancreas are well developed. The liver, now a large glandular mass traversed by




W- Nerve XI

Nerve X and jugular F Ganglion IX ganglion (superior)

Otic vesicle

~Ganglion VOI Myelencephalon ¥} (acoustic) Ganglion Y~}(semilunar) P Pog IT a1 Basilar Fee eof artery Nerve III Internal

carotid artery

Mesencephalon


Fig. 228. — 10 mm. pig embryo. Transverse section through otic vesicles. 163}.

the capillaries of the hepato-portal veins, retains its original connection with the duodenum as the common bile duct from the distal end of which the gall bladder is forming. Both dorsal and ventral primordia of the pancreas are present, the duct of the latter arising from the common bile duct. The long and slender intestine extends into the umbilical cord as the intestinal loop, to which the yolk stalk is still attached. Just posterior to this, a slight enlargement may sometimes be observed which indicates the boundary between the large and small intestine. The hind-gut is dividing into a dorsal rectum and ventral urogenital 320 THE ANATOMY OF THE 10 MM. PIG EMBRYO

sinus, prolonged into the allantoic stalk. The sinus and rectum unite in a common cloaca which has not yet established connection with the proctodeum. Immediately posterior to the cloacal plate, a small blind pocket represents the postcloacal gut.

Spinal cord.


it Dorsal root co Spinal ganglion

re Ventral root Dorsal ramus








Notochord

Anterior cardinal vein Ganglion X Aortic ( nodosum ) arch OT Radix aortae Visceral

Visceral arch

a Hypophysis Anterior cardinal vein

Sensory layer Pigment layer

Diencephalon

Fig. 229.10 mm. pig embryo. Transverse section through optic cup. 164.

Mesodermal derivatives. — The notochord extends from the vicinity of the floor of the mesencephalon into the tail, following the flexures of the body.

The somites have long since become differentiated into the myotome, dermatome, and sclerotome. In the tail region, the sclerotomes are separated into the cranial and caudal arcualia from which the vertebrae will originate.

In the pig of 10 mm., the pronephric stage has been passed; the mesonephros is at the height of its development, forming a great “Wolffian” body with a complicated network of interwoven tubules; while the mesonephric duct (originally the pronephric duct) may be recognized along the ventral margin. Emerging MESODERMAL DERIVATIVES 321

from the mesonephros, each duct enters the urogenital sinus at the same level as the allantoic stalk. From each duct a narrow stalk runs dorsally and forward as the metanephric duct, or ureter, which at its distal end is enlarged to form the pelvis of the metanephros. Around the pelvis the posterior portion of the nephrotomal band will produce the secretory tubules of the definitive kidney at a later stage. On the median ventral margin of each




Notochord

Dorsal aorta

Oesophagus Anterior

ardinal vein

Left atrium

Telencephalon

Fig. 230. — 10 mm. pig embryo. Transverse section through nasal (olfactory) pit. X 163.

mesonephros are slight swellings which will later become the genital ridges, primordia of the gonads. The coelom is partially divided into the pericardial and abdominal cavities by the septum transversum. The mesenteries of the principal viscera are in evidence. The liver is still suspended in the ventral mesentery. A dorsal mesocardium is present.

The heart of the 10 mm. pig has the four main chambers established, although not yet completely separated into right and left halves. The sinus venosus now enters the right atrium through 322 THE ANATOMY OF THE 10 MM. PIG EMBRYO

a slit guarded by the valves of the sinus. The right and left atria are partially separated by the interatrial septum in which can be seen an opening, the foramen ovale. The atrio-ventricular canal leading to the ventricle is partially separated into right and left halves by the endocardial cushion. The ventricle is partially divided by the interventricular septum. From the ventral aorta three aortic arches curve around the pharynx to unite with the dorsal aorta. These are the third, fourth, and sixth aortic arches; the first and second have degenerated, while the fifth

Spinal cord .




Ganglion Notochord

Anterior limb bud

Common Dorsal aorta cardinal vein Eparterial Oesophagus bronchus Trachea Valves of sinus Left atrium Right atrium Left Right \ ventricle ventricle

Fia. 231. — 10 mm. pigembryo. Transverse section through sinus venosus. 16}.

seldom appears as a separate structure. The pulmonary arteries are growing back from the sixth aortic arches.

As prolongations of the original paired ventral and dorsal aortae, the external and internal carotid arteries, respectively, run forward into the head. The internal carotid arteries are united at the level of the isthmus between the mesencephalon and the metencephalon with the basilar artery, which serves to unite them with the vertebral arteries, arising from the anastomosis of intersegmental arteries in the cervical region. At the 10 mm. stage the vertebral arteries have lost their intersegmental connections with the aorta except at the posterior end, where the MESODERMAL DERIVATIVES 323







Anterior limb bud:

Coelom

Posterior vena cava

Right

atrium { i Left Right i ventricle ventricle

Notochord

Dorsal aorta

Glomerulus

ketee . g ‘ Posterior Vea me AN Stomach vena cava i

by \ v

Septum 4 Pericardial

ransversum — cavity a

Fig. 233. — 10 mm. pig embryo. Transverse section through stomach. X16}. 324 THE ANATOMY OF THE 10 MM. PIG EMBRYO

seventh cervical intersegmental artery persists and grows out into the pectoral limb bud to form the subclavian artery. Near the point of origin of the subclavian, the dorsal aortae are fused and run back as a single median aorta into the tail. Dorsally, branches are given off from the aorta as intersegmental arteries of the trunk. Laterally, many small branches supply the glomeruli of the mesonephros. Ventrally, the dorsal aorta gives off the coeliac artery and anterior mesenteric arteries to the gut.



Ganglion Notochord

Left umbilical vein

Fig. 234. — 10 mm. pig embryo. Transverse section through gall bladder. 163.

Two large umbilical (allantoic) arteries run from the dorsal aorta into the umbilical cord. The aorta continues into the tail as a relatively slender vessel, the caudal artery.

The vitelline veins are much smaller than in the chick of seventy-two hours, for the yolk sac from which they drew their blood is nearly degenerated. In the pig at this stage they drain the gut area and cross into the liver where they become the portal vein. Within the liver they are broken up into capillaries which emerge as the hepatic veins to the sinus venosus. Of the somatic MESODERMAL DERIVATIVES 325

veins, the anterior cardinals are still prominent and are joined by an extensive series of head veins. In the cervical region the anterior cardinals receive the dorsal intersegmental veins as well as the external jugular from the mandible. As the anterior cardinals enter the common cardinal veins, they are joined by the posterior cardinals, which have already lost part of their drainage

Spinal cord oO .







Notochord Posterior cardinal vein Posterior vena

Left vitelline (portal) vein Left mbilical vein Left vitelline artery

duct

umbilical artery

Fused umbilical veins

Fig. 235. 10 mm. pig embryo. Transverse section through umbilical stalk in region of intestinal loop. X16}.

area to the subcardinal veins passing through the ventral portions of the mesonephroi. Numerous small venous channels serve to connect the subcardinals and postcardinals during this period. The posterior caval vein has already made its appearance as a direct connection from the subcardinals to the liver. The umbilical (allantoic) veins proceeding from the allantois toward the heart are fused together in the umbilical cord. In the body they 326 THE ANATOMY OF THE 10 MM. PIG EMBRYO

pass through the liver, within which they are, like the vitelline veins, broken up into capillaries. The left umbilical maintains a broad channel through the liver. This vessel, now known as the ductus venosus, connects the umbilical with the posterior caval vein.



Posterior Mesonephric

limb bud duct Umbilical artery Metanephric duct

Caudal artery

Notochord Spinal cord

Fig. 236. — 10 mm. pig embryo. Transverse section through metanephric duct and posterior limb buds. X16}.

Ectodermal derivatives. — The epidermal derivatives of the ectoderm have already been enumerated in the description of external form. There remain for consideration the nervous system and sense organs. ‘The five definitive vesicles of the brain are well marked. From the telencephalon arise the two lateral cerebral vesicles. This division of the brain is separated from the diencephalon by two points of reference, the optic recess in the floor, and the velum transversum in the roof. From the diencephalon spring the optic stalks, leading to the optic cups, and the infundibulum, now in contact with the hypophysis as mentioned above. The posterior boundary of the diencephalon is indicated by the tuberculum posterius arising from the brain floor. The epiphysis seldom appears at this stage. The mesencephalon, with the third cranial nerve arising from its floor, is ECTODERMAL DERIVATIVES 327

demarcated at its posterior end by the deep constriction of the isthmus. The metencephalon is distinguished from the myelencephalon by its thicker roof. From the isthmus the fourth cranial nerve runs forward laterally over the sides of the brain to the mass of mesoderm surrounding the eyeball, from which the









Basilar artery

Anterior cardinal

vein Internal carotid artery Thymus Olfactory pit | Visceral 8rd Aortic arch

4th Aortic arch 6th Aortic arch. Sinus

venosus

Right atrium

pouch

Ductus venosus


[J Subcardinal

  1. /J anastomosis,

Notochord

Fig. 237. — 10 mm. pig embryo. Frontal section through aortic arches and ductus venosus. X16}.

eyeball muscles will be formed. Conspicuous at the anterior ventro-lateral margin of the metencephalon is the large semilunar ganglion of the fifth cranial nerve. From the floor of the myelencephalon, the sixth cranial nerve emerges to run forward toward the eye. Immediately following this, the geniculate ganglion of the seventh and the acoustic ganglion of the eighth are in close 328 THE ANATOMY OF THE 10 MM. PIG EMBRYO

connection. The ninth cranial nerve has two ganglia, the dorsal superior ganglion and ventral petrosal, while the tenth similarly possesses a dorsal jugular and ventral nodose ganglion. The eleventh cranial nerve possesses at this stage a small ganglion (of Froriep) which disappears in the adult. The last of the cranial nerves, the twelfth, arises from the floor of the myelencephalon by a number of small roots and without a ganglion. In the region of the spinal cord the segmental nerves arise from the cord by two roots, of which the dorsal is associated with a spinal ganglion. The trunk is very short and soon divides into three main branches. The dorsal and ventral rami run to these respective regions of the body wall, while the third, or communicating ramus, unites the spinal nerve with a ganglion of the sympathetic chain. The sympathetic ganglia may be recognized as small masses of cells dorsal to the aorta.

The nose is represented by the olfactory pits. The eye is in the optic cup stage with a well-marked choroid fissure and groove, while the lens is completely separated from the outer ectoderm and is in the vesicle stage. Of the various regions of the ear, all the primordia are now established. The otic vesicle with its endolymphatic duct, representing the inner ear, is in close juxtaposition to the first visceral pouch (hyomandibular) which will give rise to the auditory tube and chamber of the middle ear; the external auditory meatus, or outer ear, will arise from the first or hyomandibular groove.

REFERENCES

Arey, L. B. 1934. Developmental Anatomy, 3rd Ed., Chap. 19.

“Boyden, E. A. 1933. A Laboratory Atlas of the Pig Embryo.

Keibel, I’. 1897. Normaltafeln, I, des Schweines (Sus scrofa domesticus).

Lewis, F. T. 1902. The gross anatomy of a 12 mm. pig, Am. Jour. Anat., Vol. 2, pp. 211-226.

‘Minot, C.S. 1911. A Laboratory Textbook of Embryology, 2nd Ed.

Patten, B. M. 1931. The Embryology of the Pig, 2nd Ed.

Wallin, E. 1917. A teaching model of a 10 mm. pig embryo, Anat. Rec., Vol. 5, pp. 17-45.

Part V Microscopical Technique

Chapter XIV Preparation Of Embryological Material

A method much employed in the study of comparative embryology is that of cutting a preserved egg or embryo into a series of extremely thin slices, and arranging these in order upon a glass slide, so that they may be examined under the microscope. The older embryologists, however, were limited to the study of entire embryos and of minute dissections. These methods are still of great value in supplementing the study of serial sections, for it is a difficult mental exercise to translate sections into terms of the whole embryo. The single section, especially, is meaningless except when interpreted as a part of the complete series. It is very helpful, therefore, when facilities permit, for each student to prepare for himself a whole mount and a series of sections through one of the embryos he is to study.

A. COLLECTION AND REARING OF EMBRYOS

Although preserved embryos of the more important laboratory types may be obtained from the biological supply houses, it is often desirable to collect and rear live embryos.

THE FROG. — There are some sixty species of tailless Amphibia within the continental limits of the United States. Although the capture of adults in a pond where eggs are found is strong circumstantial evidence as to the species of the eggs, even this evidence is often lacking, so that the ability to identify the eggs or larvae from their own characteristics is highly desirable. A key to the eggs and larvae of some of the common Eastern frogs and toads is found in Wright’s ‘‘ Life History of the Anura of Ithaca, N. Y.” For the Pacific slope fauna, see Storer, “A Synopsis of the Amphibia of California.”’ The eggs of the salamander, Ambystoma, are laid at the same time and in the same localities as those of the early frogs, but may be distinguished from them by the greater proportion of jelly to the eggs in the

mass of spawn. 331 332 PREPARATION OF EMBRYOLOGICAL MATERIAL

Experiments dealing with the effect of pituitary hormones have led to the discovery that one of these hormones will induce ovulation in the female frog, and the drive to amplexus in the male, out of the breeding season. Rugh! (1934) has described in detail a technique for inducing ovulation and bringing about artificial fertilization which has been since used in several laboratories, including the author’s, with complete success.

The rate of development of the frog’s egg depends upon the temperature of the water. In the laboratory, the eggs will hatch in about one week after laying, at the ordinary room temperature. The egg masses should be kept in clean glass containers with at least ten times as much water. The water should not be changed until after hatching, when the larvae should be transferred to fresh water with aquatic plants. After the assumption of the tadpole form, they should be fed small pieces of finely ground meat. Metamorphosis may be hastened by feeding fresh or desiccated thyroid tissue.

Artificial fertilization is the best method of obtaining the earliest stages of development. The testes and vasa deferentia of the male are teased out in a watch glass of water. The eggs from the distal portions of the oviducts are placed in this water for five minutes and then removed to glass containers with not more than four inches of water.

THE CHICK. — In collecting hens’ eggs for incubation, it is a truism that they must be fresh and fertile. The best results are obtained from trap-nested eggs in the spring semester. The egg is normally laid in the gastrula stage (Chapter II), but in those cases where the egg does not reach the distal end of the oviduct by 4 P.M., it is retained till the following morning and undergoes further development. After laying, the egg cools and development ceases until incubation is commenced. The fertilized egg is viable for five weeks at a temperature of 8°-10° C. The time of hatching, as in the frog’s egg, is dependent upon the temperature. The minimum temperature at which development will take place is about 25° C.; the optimum is 37° C., at which temperature the egg will hatch in twenty-one days; the maximum temperature is about 41° C. In incubating eggs, care must be

1R. Rugh. Induced Ovulation and Artificial Fertilization in the Frog, Biol. Bull. 66, 22-29. PRESERVATION OF MATERIAL 333

taken to keep the air in the incubator moist and to rotate the eggs once a day.

Instructive demonstrations may be made by opening the shell and shell membranes under aseptic conditions and removing a bit of the albumen. <A window of celloidin placed over the opening and carefully sealed will permit of observations on the development of the embryo for several days. An alternative method is that of opening the egg and placing the contents in a sterilized small stender dish. A glass ring is placed on the yolk to keep it beneath the surface of the albumen, and the dish is covered and placed in the incubator. If this operation is carried on under aseptic conditions, development will continue for two or three days.

THE PIG. — The early stages of development in any mammal are valuable. The larger embryos are visible as protuberances on the inner side of the uterine tubes. The tube should be slit open and the embryos exposed by cutting open the embryonic membranes which surround them. Smaller stages are obtained by washing out the contents of the tube with normal salt solution or preserving it entire.

Pig embryos may be obtained in quantities from any good-sized packing house. As many as eighteen may be found in a single female, but the average number is eight. The period of gestation in the pig is 121 days. Pig embryos of 10 mm. body length are the most useful in the elementary course. Later stages are of value in the detailed study of organogeny.

B. PRESERVATION OF MATERIAL

The preliminary preparation of material for microscopical work involves three distinct operations: killing, fixing, and preservation. In practice, two or three of these operations are performed by a single reagent known as a “ fixing fluid.” Such a reagent should kill the embryo so rapidly that it will undergo the minimum of post-mortem changes; it should preserve the structures of the embryo with as life-like an appearance as possible; and it should harden the soft parts so that they may undergo the later processes of technique without loss of form or structure. Some fixing fluids, such as alcohol or formalin, may be used indefinitely as preservatives, but the majority are used for a particular optimum period, and then washed out and replaced by alcohol. 334 PREPARATION OF EMBRYOLOGICAL MATERIAL

THE FROG. — The frog’s egg, before hatching, is best fixed by Smith’s fluid.

Potassium bichromate.......... cece eee eee eee 0.5 gram Glacial acetic aCid. . 6... ce cece cece cece eee eee 2.5 ce. Formalin. 2.0... ccc ccc cece cee cece cence eeaee 10.0 ce.

Distilled water... 2... . cece eee eee eee eee 75.0 cc.

1. Cut the egg masses into small pieces of about twenty-five eggs each, and submerge them in a dish of Smith’s fluid for twenty-four hours. A quantity equal to ten times the volume of the eggs should be used.

2. Rinse the eggs in water and wash with a 5 per cent aqueous solution of formalin until no more free color comes out. The eggs may be kept indefinitely in this fluid. If it is desired to remove the egg membranes, proceed as follows:

3. Wash in water for twenty-four hours, changing the water several times.

4. Place the eggs in eau de Javelle, diluted with three time its volume of water, and shake gently from time to time during a period of 15 to 30 minutes until the membranes are almost dissolved and will shake off.

5. Rinse in water and run through 50 per cent and 70 per cent alcohol, an hour to a day each, and preserve in 80 per cent alcohol.

After hatching, larvae are best fixed in Bouin’s fluid.

Picric acid, saturated aqueous solution................ 75 cc. Formalin. 2.0... cece cece eee een eeeeees 25 cc. Glacial acetic acid... .. ec cee cece eeeeees 5 ce.

1. Larvae are left in this fluid from one to eighteen hours, according to size.

2. After rinsing in 50 per cent alcohol, wash in 70 per cent alcohol, to which has been added a few drops of lithium carbonate, saturated aqueous solution, until the yellow color is extracted, and preserve in 80 per cent alcohol.

THE CHICK. — The chick embryo must be removed from the shell, albumen, and yolk before fixation. As the early stages are more difficult to handle, it is advisable to practice this operation on embryos of seventy-two hours’ incubation and then work backward toward the stages of the first day. THE CHICK 335

1. Place the egg in a dish 3 inches high and 6 inches in diameter, two-thirds full of normal saline solution, warmed to 40° C.

2. Crack the shell at the broad end with the flat of the scalpel, and pick away the pieces of shell until an opening slightly larger than a half dollar has been made. Remove the outer and inner shell membranes. Invert egg beneath the surface of the salt solution and allow the contents to flow out. The blastoderm, containing the embryo, will rotate until it is uppermost. With fine-pointed scissors, cut rapidly a circle of blastoderm, about the size of a quarter, with the embryo at the center. With blunted forceps, pull the blastoderm and adherent vitelline membrane away from the yolk and albumen, waving it gently beneath the surface of the salt solution to remove all yolk.

3. Submerge a syracuse watch glass in the salt solution and float the embryo into this. Remove the watch glass carefully from the large dish and examine the embryo with a dissecting lens. If the vitelline membrane has not yet separated from the blastoderm, it should be removed at this time with fine-pointed forceps and needles. Make sure that the embryo lies dorsal side up, as it did when the egg was opened.

4, Slide a cover glass under the embryo, and remove all salt solution: with a pipette, taking care that the embryo lies in the center of the cover glass. Lift the cover glass by one corner so that the overhanging edges of the blastoderm fold under, and place it in a dry watch glass on a piece of thin absorbent tissue paper and add fixing fluid at once. While the embryo is becoming attached to the cover glass, remove the yolk, albumen, and pieces of shell from the dish of salt solution to a slop jar, reheat the salt solution to 40° C., and prepare another embryo. Three embryos of each stage are to be prepared.

5. After five minutes, drop the cover glass, embryo side up, into a small stender dish of Bouin’s fluid and leave from two to four hours.

6. Rinse in 50 per cent alcohol, wash for two days in 70 per cent alcohol to which lithium carbonate has been added or until the yellow color is extracted from the embryo, and preserve in 80 per cent alcohol. 336° PREPARATION OF EMBRYOLOGICAL MATERIAL

THE PIG. — Embryos of 6 mm. body length and over are easily located in the uterine wall. Slit open the uterus and remove the embryo with fine-pointed forceps and a horn spoon, taking pains not to rupture the membranes. Place at once in Bouin’s fluid. Embryos of 10 mm. body length should be fixed for four hours. Rinsing and preserving are done as for the frog or chick. Larger embryos should have the body cavity slit open to admit the fixing fluid. Fetal pigs of 6 inches or more should be injected through the umbilical artery with formalin (20 per cent aqueous solution). This solution is also injected into the body cavity and cranium, after which the fetus is submerged in the same medium for a week and preserved in 6 per cent formalin.

“°C. WHOLE MOUNTS

It is very helpful to have some embryos mounted entire for comparison with the serial sections. In making these whole mounts, the embryos are stained, cléared, and mounted, i.e., transferred to a final medium for preservation and examination on the slide beneath a cover glass.

THE FROG. — Frog eggs and embryos may be mounted as opaque objects with the natural pigmentation, or they may be cleared and stained as transparent mounts.

Opaque mounts. —

1. Prepare a saturated aqueous solution of thymol. Filter the solution, and add gelatin until saturated. Remove the supernatant liquid.

2. Liquefy the gelatin by immersing a small quantity, in a test tube, in a dish of hot water. Fill a hollow-ground depression slide with gelatin and allow to cool.

3. With a hot needle, melt a small hole in the gelatin, sufficiently large to hold the embryo. Place the embryo in the desired position and hold it in place until the gelatin has cooled.

4, Add a drop of gelatin just warm enough to be liquid and cover with a cover glass which has been slightly warmed. When the gelatin has cooled, any surplus may be removed from the edges of the cover glass with a toothpick wrapped in moist cotton. In order to prevent the later formation of bubbles, the edges of the cover glass should be painted with gold size or Valspar.

Free-hand sections and dissections are admirably mounted by THE CHICK 337

this method, but great care must be exercised to prevent the formation of air bubbles through cracks in the gold size.

Transparent stained mounts. —

1. Bleach the embryo, until white, in hydrogen peroxide. About one week is required for this purpose. Embryos that have been preserved in 80 per cent alcohol should first be passed through 70 and 50 per cent alcohol to water, an hour or more in each fluid. Embryos in formalin must be rinsed in water for one hour.

2. Stain in dilute borax carmine four days or more.

Borax, 4 per cent aqueous solution. .................. 100 ce. Carmine. . 0.0.2... ccc cece eee eeneee 1 gr. Boil until dissolved and add alcohol, 70 per cent....... 100 ce.

To dilute, take 5 cc. of the borax carmine and 95 ce. of 35 per cent alcohol and add a crystal of thymol.

3. If overstained, remove the surplus color with hydrochloric acid (1 per cent solution in 70 per cent alcohol) after passing through water and 50 per cent alcohol, an hour each.

4, Run up through 80, 95, and 100 per cent alcohol, an hour each, and place in xylene (xylol) until transparent.

5. Prepare a mounting diagram by drawing an outline of a slide on a piece of cardboard and in this laying off an outline of the cover glass to be used. Place a clean slide on the diagram, and, just’ inside the right and left margins of the cover-glass outline, attach a thin strip of celluloid, 15/1000 of an inch in thickness, by means of a drop of acetone. Greater thicknesses may be obtained by attaching other strips as necessary. When these supports are dry, place a few drops of Canada balsam, dissolved in xylene, between the supports, place the embryo in position, and lower a clean cover glass gently. Try to avoid the formation of air bubbles. If these appear later they may be removed by a needle which has been heated or dipped in xylene. A little fresh balsam may be run into the cavity.

THE CHICK. — Total mounts may be stained either with the borax carmine or with Conklin’s modification of Delafield’s hematoxylin. Delafield’s hematoxylin, which gives a blue color to the embryo, is made as follows: 338 PREPARATION OF EMBRYOLOGICAL MATERIAL

Hematoxylin (16 per cent solution in 100 per cent al COMO]... cc ee een eee nes 25 ce. Ammonia alum (saturated aqueous solution).......... 400 ce. Hydrogen peroxide, neutralized. . 0... 6. cee eee ee eee 25 cc. Glycerin. 20... cc cece eet ee eee eens 100 ce. Alcohol methyl... 0.0.0... ccc cence nens 100 ce.

Conklin’s modification consists of diluting the stain with four times the volume of distilled water and adding to each 100 ce. of the dilute stain 1 ec. of picrosulphuric acid, prepared by adding 2 cc. of sulphuric acid to 98 cc. of picric acid (saturated aqueous solution).

1. Run the embryo from the 80 per cent alcohol down to water through changes of 70 and 50 per cent alcohol, an hour each.

2. Stain in borax carmine, undiluted, over night, or in hematoxylin from one to three hours. Either stain may be diluted still further and the staining period prolonged. In the author’s laboratory the schedule demands a four-day staining period and the borax carmine is diluted 5 x, the hematoxylin 20 x.

3. Destain, if necessary, in acid alcohol until the desired color is obtained. Embryos stained with hematoxylin will turn red in the acid alcohol, and the blue color must be restored by washing them in running water or, after washing in neutral 70 per cent alcohol, placing them in alkaline alcohol (1 per cent ammonia in 80 per cent alcohol).

4. Run up the alcohols, 80, 95, and 100 per cent, half an hour each. Pour off half the 100 per cent alcohol and add an equal amount of xylene. When the diffusion currents disappear, transfer to pure xylene and leave until the embryo is transparent. In rainy weather, or when 100 per cent alcohol cannot be obtained, phenol-xylene (phenol crystals, 25 gr. and xylene 75 cc.) may be substituted.

5. Remove the embryo from the cover glass (if it has not already detached itself) and trim the surrounding blastoderm to the form of an oblong or circle. Arrange a clean slide on the mounting diagram, as described for the frog, attach celluloid support, and mount the embryo in Canada balsam with the same side uppermost as when the egg was opened. Put the slide away where it may lie flat and free from dust until the balsam has hardened. This will take at least a week, after which the slide EMBEDDING IN PARAFFIN 339

may be cautiously cleaned and studied. The process may be hastened by drying the slide in the paraffin oven.

THE PIG. — Embryos up to 10 mm. body length may be prepared as whole mounts by staining in dilute borax carmine, destaining until only a trace of color persists, and mounting in Canada balsam. The time spent in each alcohol should be at least an hour for the larger embryos.

D. SERIAL SECTIONS

In the preparation of serial sections of an embryo, the fixed material is (1) embedded in a suitable matrix and (2) sliced into extremely thin sections, which are (3) mounted in serial order upon slides. The embryo may be stained before or after sectioning.

Embedding. — There are two principal methods of embedding, in paraffin or in celloidin. For especially delicate objects, the best results are obtained by a combination of these methods, the embryo being first impregnated with celloidin in order to avoid the shrinkage (about 10 per cent) caused by paraffin embedding, and the block of celloidin then immersed in paraffin so that ribbons of serial sections may be cut.

Embedding in paraffin. — In preparing the first few embryos for sectioning, it is advisable to stain, dehydrate, dealcoholize, and clear as if for a total mount. Later, the staining may be omitted until after the sections are affixed to the slide.

1. After clearing in xylene, which should be done in a warm place, for example, the low-temperature oven at about 40° C., pour off half the xylene and add an equal amount of paraffin chips. In the author’s laboratory a paraffin of about 55° melting point, obtained by mixing commercial paraffin with parawax, is used. The parawax, unfortunately, varies in melting point, so that the formula is empirical. The embryo may be left in this xylene paraffin for two days.

2. If the mixture has hardened it should again be melted in the low-temperature oven. Fill a clean stender dish with melted paraffin, transfer the embryo to this, and place in the high-temperature oven at about 56° C. (or one degree above the melting point of the paraffin used) for not more than two hours. The xylene paraffin should be thrown in the slop jar. Take care not 340 PREPARATION OF EMBRYOLOGICAL MATERIAL

to get any xylene in the high-temperature oven or paraffin used for the final embedding.

3. Smear the interior of a small watch glass with a 10 per cent aqueous solution of glycerin (or vaseline), and fill with fresh melted paraffin. Transfer the embryo to this, making any necessary adjustments in position with a heated needle. Place the embryo dorsal side up, and note the position of the head. Cool the surface of the paraffin by blowing on it gently until it is congealed. Then plunge it immediately into a dish of cold water or waste alcohol and leave it there for five minutes. Mark the block for identification. Objects may be left in paraffin indefinitely.

4. On removing the block of paraffin from its container, examine for the following flaws:

a. Air bubbles, if they are not near the embryo, may be removed with a hot needle. Otherwise it is better to trim the block close to the embryo, put it into melted paraffin, and re-embed.

b. Milky streaks are due to the presence of xylene. These will crumble during sectioning, so that it is best to re-embed if they occur near the embryo.

c. If the paraffin has “ fallen ” in the center, it is because the surface was cooled too long before the block was immersed in the water. If any part of the embryo is exposed, it must be reembedded.

Sectioning after paraffin embedding. — Before sectioning your first embryo, be sure you understand the mechanism of the microtome (there are many varieties, of which the rotary type is best adapted to beginning students), and have practised the technique on a block of paraffin. There are three standard planes of sectioning corresponding to the axes of the body (Fig. 238). Transverse sections are obtained by cutting the cephalic end of the body first, with the knife entering the left side. Sagittal sections are made by cutting the right side first, with the knife entering the ventral surface. Frontal sections are made by commencing at the ventral surface, the knife entering the left side. It is best to begin with transverse sections.

1. Attach the paraffin block to the object-carrier of the microtome in the proper manner to obtain the type of section desired. This is done by heating the surface of the carrier until it will just SECTIONING AFTER PARAFFIN EMBEDDING 341

melt paraffin, pressing the block against it in the desired orientation, and lowering into a dish of cold water. A little melted paraffin may be poured around the base of the block and this again cooled to secure additional support.

2. Place the object-carrier in the microtome and, after orienting the block with respect to the knife, trim it so that the end of the block is a perfect rectangle with one of the longer sides parallel to the knife edge. If one of the angles is cut off slightly there will be a series of indentations in the ribbon which will assist in orienting the sections on the slide.

3. If microtome knives are not available, place a new safetyrazor blade (Autostrop type) in the holder provided, allowing the



Transverse Sagittal

Fig. 238. — Diagram to show method of orienting embryo with reference to microtome knife according to type of section desired.

edge to project between a sixteenth and an eighth of an inch. Screw the holder in the knife-carrier so that the edge of the blade is tilted inward about 10° from the perpendicular.

4. Set the regulator for 20 microns (thousandths of a millimeter). .

5. Run the feed screw as far back as it runs freely; do not force it.

6. Advance the knife-carrier until the edge of the blade just clears the block.

7. Release safety catch and turn the wheel steadily until the knife begins to cut the block. Cut slowly, making necessary adjustments to the block and knife until you are cutting a perfectly straight ribbon without wrinkles or splits. The principal causes of trouble and their remedies are as follows: 342 PREPARATION OF EMBRYOLOGICAL MATERIAL

a. The ribbon curls to right or left. This happens because (1) the block is thicker on the side away from which the ribbon curls, or (2) the knife is duller on the side toward which the ribbon curls. Remedy: (1) trim the sides of the block parallel; (2) shift the knife to one side.

b. The sections curl and the ribbon is not continuous. This is due to (1) too much tilt of the knife, (2) too hard a grade of paraffin, or (3) too cold a room. Remedy: (1) lessen tilt of knife; (2) re-embed in softer paraffin; (3) move microtome to warmer place, light an electric light or micro-bunsen burner near microtome, or cut thinner sections.

c. The ribbon wrinkles badly. This is caused by (1) too little tilt to the knife, (2) too soft a grade of paraffin, (3) too warm a room, or (4) a dull or dirty knife. Remedy: (1) increase the tilt of the knife; (2) re-embed in harder paraffin; (8) move to a cooler room, or cool the knife and block by dropping alcohol on them and blowing vigorously, or cut thicker sections; (4) clean knife edge with cloth moistened in xylene or shift to a new place on the knife.

d. The ribbon splits lengthwise. This is due to (1) a nick in the knife, (2) a bubble in the paraffin, or (3) dirt on the knife edge or side of the block. Remedy: (1) shift to new cutting edge; (2) paint surface with thin celloidin; (3) clean knife edge and block.

e. The sections refuse to ribbon; they fly apart or cling to the knife or the block. This is due to the electrification of the sections caused by unfavorable atmospheric conditions. Many remedies have been suggested; the best is to ground the microtome to a water pipe. Usually it is advisable to wait for more favorable conditions.

8. Remove the ribbon in 6 inch lengths with a camel’s hair brush and arrange these in order, shiny side down, in a cardboard box cover. Avoid air currents of all kinds. The ribbons may be put away in a dust-free place if the room is not too warm. It is better to affix them to slides as soon as possible.

Affixing paraffin sections to the slide. — 1. Prepare a mounting diagram by laying off the outline of a slide as before, but enclose in this the outline of a long cover glass (25 by 50 mm. approximately) and leave space for a label on the right-hand side.

2. Clean a slide thoroughly by washing with acid alcohol EMBEDDING IN CELLOIDIN 343

followed by distilled water. Place this over the mounting diagram and brush over the surface above the outline of the cover glass with the following dilute solution of egg albumen:

Egg albumen, beaten and skimmed.................. 50 ce. Glycerin... 0... cece cece eee eee neeeeeeees 50 ce. Filter and add Thymol.............. 0. ccc cee ee ee ees a crystal Dilute 2 drops of this to distilled water............... 25 ce.

3. Cut the ribbon into lengths about 2 per cent shorter than the length of the cover glass. Using the wet brush from which most of the albumen solution has been squeezed, pick up these lengths and arrange them on the albumenized slide so that the sections will follow each other like the words on a printed page. The shiny side of the ribbon should be next to the slide. Great care should be taken to lower the ribbon slowly so as to prevent the formation of air bubbles beneath it.

4. Carefully warm the slides on a warming plate or a piece of plate glass, previously heated in the paraffin oven, until the sections are expanded and perfectly smooth. If bubbles appear beneath the ribbon, prick them with a hot needle while the ribbon is still soft and hot. Drain off the surplus water, carefully realign the sections, mark the slides with a glass-marking crayon, and set them away in the low-temperature oven to dry, at least two days. They may be kept indefinitely in this condition if not exposed to dust. .

Embedding in celloidin. — This method is preferred by some technicians as no heat is used in the process and the shrinkage is less than that resulting from the paraffin method. However, thin sections are not so easy to obtain and the sections must be handled individually.

1. Embryos are dehydrated as for the paraffin method. Leave in absolute alcohol one day.

2. Absolute alcohol and ether, equal parts, one day.

3. Thin celloidin, three days to one week.

Alcohol, 100 per cent. ... 0... eee eee eee eee eee 100 ce

Ether... 0.2... cece ccc eee cence eee eeeeee 100 ce

Celloidin. £0... ccc ccc cece eee e eee e eee eaes 5 gr. 4, Thick celloidin, two days to two weeks.

Alcohol, 100 per cent... 1... 0.0... ccc cece eee 100 ce

BO 6) a 100 ce 344 PREPARATION OF EMBRYOLOGICAL MATERIAL

5. Remove the embryo to a small watch glass and pour thick celloidin over it. Cover lightly, or place under a bell jar until the celloidin is hard enough to cut with a scalpel.

6. Dip a block of vulcanized fiber in thick celloidin. Cut out a block of celloidin containing the embryo from the watch glass and, after moistening the end by which it is to be attached in ether alcohol, press it firmly against the prepared fiber block.

7. Pour a little chloroform into a stender dish, add the block and embryo, cover tightly, and allow the celloidin to harden in the fumes for thirty minutes.

8. Fill the stender dish with chloroform and cover. Leave for thirty minutes.

9. Pour off half the chloroform and add an equal amount of cedar oil. Leave for one hour.

10. Transfer to pure cedar oil where it may remain indefinitely.

Sectioning after celloidin embedding. — Celloidin sections are usually cut with some form of sliding microtome. Be sure to study the mechanism and cut a piece of hardened celloidin before proceeding further.

1. Set the knife with a little more tilt than would be used for paraffin, and obliquely to the object so that at least half the cutting edge will be drawn through the block.

2. Orient the block upon the object-holder so that the desired type of sections may be obtained. The long side of the block should be parallel to the edge of the knife.

3. Cut sections 20 » or more in thickness, using a steady drawing cut. Mount sections as they are cut.

Affixing celloidin sections to the slide. — This is best done as the sections are cut.

1. Using the mounting diagram as before, rub on a thin film of undiluted albumen solution to cover the areas of the cover glass. Rub in well with the ball of the finger.

2. Arrange the sections in order on this area. When this is filled, lay a cigarette paper over the sections and press gently with another slide. The slides may be kept in a dust-free container.

Double embedding in celloidin and paraffin. — This process, although tedious, combines the best points of the two methods already given. AFTER STAINING IN BULK 345

1. Embed in celloidin according to the method above, omitting step 6.

2. Trim the celloidin block close to the embryo and wash out the cedar oil with xylene, three changes in two hours.

3. Embed in paraffin as described above, commencing at step 2.

4. Section according to the method given for paraffin.

5. Affix to the slide according to the method given for paraffin sections.

Staining serial sections. — When the embryo has been stained before sectioning, it is only necessary to remove the paraffin (or celloidin), replace with Canada balsam, and cover, if the stain proves to be satisfactory. Sometimes, however, it is advisable to strengthen or weaken the stain or to add a contrasting dye.

After staining in bulk. —

1. Paraffin sections on the slide should be put in a Coplin staining jar of xylene and left until the paraffin is dissolved, up to fifteen minutes.

2. Transfer to a mixture of xylene and 100 per cent alcohol, equal parts, five minutes.

3. Transfer to 100 per cent alcohol, five minutes.

4, Examine slide rapidly under microscope after wiping the back of the slide.

a. If the stain is satisfactory:

5a. Absolute alcohol and xylene, five minutes. 6a. Xylene, ten minutes. 7a. Mount in balsam under cover glass.

b. If the stain is too intense: 5b. Ninety-five and 85 per cent alcohol, one minute each. 6b. Acid 70 per cent alcohol, until stain is correct. 7b. Sections stained in hematoxylin should have the blue color restored in alkaline 85 per cent alcohol.

8b. Eighty-five, 95, and 100 per cent, one minute each. 9b. Absolute alcohol and xylene, five minutes.

10b. Xylene, ten minutes.

11b. Mount in balsam.

c. If the stain is too light: 5c. Ninety-five, 85, 70, and 50 per cent alcohol, one minute each, 346 PREPARATION OF EMBRYOLOGICAL MATERIAL

6c. Stain until desired effect is secured. 7c. Distilled water, five minutes. 8c. Fifty, 70, 85, 95, 100 per cent alcohol, one minute each. 9c. Absolute alcohol and xylene, five minutes. 10c. Xylene, ten minutes. llc. Mount in balsam.

Celloidin sections on the slide should be exposed to the fumes of the aleohol-ether mixture for half a minute, dried for one minute, and placed in a staining jar of 95 per cent aleohol. All other operations may be carried on as above except that phenol-xylene should be substituted for 100 per cent alcohol.

Counterstaining after staining in bulk. — In order to differentiate the parts of the embryo more sharply, it is often desirable to add a second stain contrasting with the first. The stains that have been employed in the previous exercises are nuclear dyes; that is, when extracting by acid alcohol, the color will persist in the nucleus after it has been washed out of the cytoplasm. The second stains affect the cytoplasm and should contrast in color with the nuclear stain employed. After borax carmine, a 0.5 per cent solution of anilin (Lyons) blue in 95 per cent alcohol is employed; after hematoxylin, a similar solution of cosin should be used.

1. Proceed as in the preceding section as far as 60.

2. Destain in acid alcohol until the color persists only in the nuclei.

3. Restore the blue color to hematoxylin-stained sections in alkaline 80 per cent alcohol.

4, Eighty and 95 per cent alcohol, one minute each.

5. Counterstain lightly, dipping the slide into the solution repeatedly until a light color persists in the sections, one-half to one minute.

6. Rinse in 95 per cent alcohol, dehydrate with 100 per cent alcohol, followed by xylene-absolute, clear in xylene, and mount.

Staining with Delafield and eosin on the slide. — Follow directions given for sections stained in bulk (where stain is too light), as far as step 6c, and follow with directions for counterstaining as given above.

Staining with Heidenhain’s hematoxylin. — This is one of the most important embryological stains. OPPEL’S POLYCHROMATIC STAIN 347

1. Remove the paraffin from the sections and run down the alcohols to distilled water.

2. Four per cent aqueous solution of iron alum, one hour to over night.

3. Rinse in distilled water and place in 0.5 per cent aqueous solution of hematoxylin, same time as in the iron alum.

4. Rinse in distilled water and return to the iron alum until sections are a pale gray. Check from time to time by rinsing in distilled water and examining under microscope to see that the desired structures are still visible.

5. When sufficiently destained, wash in running water for twenty minutes, or in distilled water, with frequent changes, for two hours.

6. Run up the alcohols, clear, and mount.

Fuchsin and picro-indigo-carmine. — This polychromatic stain is especially fine for organogeny.

1. Remove the paraffin and run down the alcohols to distilled water.

2. Stain in basic fuchsin, saturated aqueous solution, twenty minutes.

3. Rinse in distilled water and place in picro-indigo-carmine for five minutes.

Picric acid, saturated aqueous solution................ 50 ce. Indigo-carmine, saturated aqucous solution............ 50 ce.

4. Pass rapidly through 70, 95, and absolute alcohol into xylene-alcohol. The green dye is extracted most rapidly by the 70 per cent alcohol, the red by the absolute. Only experience will teach the right time allowance for each alcohol.

5. Clear in xylene and mount.

Oppel’s polychromatic stain. — This gives beautiful effects with older embryos and larvae.

1. Fix in Bouin.

2. Stain in bulk with undiluted borax-carmine, one to two days. Destain for the same period.

Embed, preferably by the double method. Cut sections, 15-20 u.

Run down the alcohols to water.

Stain in picro-indigo-carmine, 14 minutes. Stain in picro-fuchsin, one minute.

NES OUR 348 PREPARATION OF EMBRYOLOGICAL MATERIAL

Picric acid, saturated aqueous solution................ 50 ce. Acid fuchsin, saturated aqueous solution.............. 50 ce. 8. Wash in distilled water, changed repeatedly, five minutes. 9. Ninety-five per cent alcohol, two minutes. 10. Phenol-xylene, xylene, and mount.

E. TECHNICAL RECORDS

Not the least important part of technique is the keeping of exact records covering every technical operation. For each embryo there should be a card, giving the following data: Kind of embryo and stage of development. Method of fixation, time and date. Bulk staining, time and date. Method of embedding, time and date. Plane and thickness of sections, and date. Slide staining, time and date. Method of mounting, and date. Name of preparator.

PN OOP Wh Ee

F. OUTLINE OF TECHNICAL PROCEDURE FOR CHICK EMBRYOS

1. Remove embryo from egg in warm normal salt solution.

2. Fix for two hours in Bouin’s fluid.

3. Wash in 70 per cent alcohol (plus lithium carbonate), at least one change, for two days.

4. Pass through 50 per cent alcohol and water, one hour each.

5. Stain in dilute borax-carmine or Delafield’s alum-hematoxylin, four days.

6. Destain in acid 70 per cent alcohol until desired effect is obtained.

7. Wash in neutral 85 per cent alcohol. (The hematoxylinstained specimen is transferred to alkaline 85 per cent alcohol until blue color is restored.) Two days.

8. Dehydrate and clear: 95 per cent, 100 per cent alcohol, absolute alcohol-xylene, xylene, twenty minutes each.

Mount in Canada balsam OR

9. Prepare for embedding by pouring off half the xylene and adding an equal amount of paraffin chips. Keep in warm place up to four days. REFERENCES 349

10. Continue by transferring embryo to melted paraffin and place in paraffin oven for an hour and a half. 11. Embed in fresh paraffin and cool in water. Make blocks. 12. Cut transverse sections 20 u in thickness on microtome. 13. Prepare clean albumenized slide, float sections on this in order, warm until sections are expanded, remove surplus water. Dry for at least two days. 14. Remove paraffin with xylene, and A. Mount in balsam, or B. Run down alcohols to 70 per cent and destain. Run up the alcohols, through absolute alcohol and xylene and xylene, mount in balsam, or C. Run down alcohols to water and restain, dehydrate, clear and mount, or D. To 95 per cent and counterstain for one minute. Dehydrate, clear, and mount.

REFERENCES

Baker, J. R. 1933. Cytological Technique.

Ballentyne, F. M. 1928. An Introduction to the Technique of Section Cutting.

Carleton, H. M. 1926. Histological Technique.

Gage, S. H. 1925. The Microscope, 14th Ed.

Guyer, M. F. 1917. Animal Micrology, 2nd Ed.

Lee, A. B. 1929. The Microtomist’s Vade-Mecum, 9th Ed.

McClung, Ch. 1929. Handbook of Microscopical Technique.

Oppel, A. 1914. Embryologisches Practikum und Entwicklungslehre.

Rugh, R. 1934. Induced Ovulation and Artificial Fertilization in the Frog. Biol. Bull. 66, 22-29.

Shumway, W. 1926. Fuchsin and Picro-indigo-carmine, a Polychromatic Stain for Vertebrate Organogeny. Stain Technology I, 1. CHAPTER XV STUDY OF EMBRYOLOGICAL PREPARATIONS

During the carly stages of development, embryos are too small to be studied with the unaided eye. Some observations, to be sure, may be made with the dissecting lens, but most embryological work requires the use of the compound microscope. Although the student may be familiar with the use of the microscope from the elementary course in biology, he should nevertheless review this subject before proceeding further. In addition, he should at this time familiarize himself with the simpler methods of measuring objects with the aid of the microscope, as embryological drawings require a strict accuracy as to proportions. A great convenience in embryological work is the camera lucida or some other device by means of which accurate outlines may be traced. Finally, we must consider the methods by which the embryo may be reconstructed in magnified form from serial sections, thus returning, in a sense, to the point where the study of embryological technique was begun.

A. THE USE OF THE MICROSCOPE

Nomenclature of the microscope. — The separate parts of the microscope (Fig. 239) may be grouped into two systems, the mechanical parts, and the optical parts. The principal mechanical parts are the base, from which arises the pillar, attached to which is the arm, which may be inclined at the joint. Attached to the arm, just above the joint, is the stage, upon which the slide is placed for examination, and beneath this, the movable sub-stage equipment, consisting of a condenser-sleeve, and one or two iris-diaphragms, by means of which the amount of light to be used is regulated. At the base of the arm is the mirror, a silvered double mirror, with a plane surface on one side and a concave surface on the other. At the upper end of the arm are two screws, the coarse and fine adjustments, by means of which

the barrel of the microscope may be raised or lowered either 300 THE OBJECTIVES 351

rapidly or very slowly. The barrel is composed of the bodytube, connected to the arm by a rack and pinion, in the upper end of which is enclosed an inner tube, the draw-tube, on which is a graduated scale of millimeters representing the tube length exclusive of the revolving nose-piece at the lower end. The optical parts of the microscope are systems of lenses, the condenser, placed in the condensersleeve, the objectives, attached to the revolving nose-piece, and the oculars, one of which is placed at the upper end of the drawtube.

The condenser. — This is a system of lenses which increases the amount of illumination O thrown upon the object, and is BJECTIVE required only with the higherpower objectives.

The objectives. — These are systems of lenses which produce an enlarged and inverted image of the object under proper conditions. Objectives were formerly marked by arbitrary letters V1¢. 239.— Diagram showing parts of or numbers, with the lowest-pow- ae compound microscope. (From er objectives beginning the series. age.)

To-day they are usually indicated by the equivalent focal length (E. F.), that is, the focal length of a simple lens at 250 mm. or 10 inches, or else by the actual magnification (x) at 160 mm. (Leitz microscopes, 170 mm.). In some of the older microscopes the tube lengths indicated on the draw-tube were calibrated without including the length of the revolving nose-piece, then an accessory part. When setting up these instruments the length of the nose-piece (Leitz, 18 mm.) must be deducted and the drawtube set at the reduced length (Leitz, 152 mm.). The most useful objectives for general embryological purposes are the 25-mm. or 6 X, which will hereafter be spoken of as the lowerpower objective; the 16-mm. or 10 X, which will be called the



Coarse Adjustment

4yBuayz-aqny 352 STUDY OF EMBRYOLOGICAL PREPARATIONS

medium-power objective; and the 4-mm. or 40 x, known as the high-power objective. For the study of the germ cells, an oilimmersion objective, of which the front lens must be in contact with the cover glass by means of a drop of cedar oil, is necessary. The most generally used immersion objective is that of 1.9 mm. E. F. or approximately 95 xX.

Oculars. — These are systems of lenses which magnify the real image formed by the objective. Like objectives, these were, in the past, usually numbered or lettered, beginning with that of the lowest power, but now are marked with the E. F. at 250 mm. or the actual magnification at 160 mm. (Leitz oculars, 170 mm.). The most useful oculars are the 50-mm. (5 X) or low-power ocular, and the 25-mm. (10 x) or high-power ocular. When used with the objectives given above, a range of magnification from 30 xX to 450 * may be obtained. A method of obtaining the exact magnification will be described in connection with the directions for reconstruction given below.

The use of the microscope. —

1. Place the microscope squarely in front of you with the pillar toward you and the stage horizontal.

2. Place the low-power ocular in the draw-tube, and adjust this to a length of 160 mm. (170 mm. for Leitz instruments) as indicated on the millimeter scale. Swing the low-power objective into position. Place the mirror bar in the median line and adjust the mirror to secure an even illumination. Use the plane side of the mirror. The concave side is employed only when the condenser is not in use.

3. Place the slide on the stage so that the object to be examined is in the center of the stage aperture, and fasten it down with the spring clips provided. With the coarse adjustment, lower the body-tube until the objective nearly touches the cover glass. Then, with the eye at the ocular, slowly raise the body-tube until the object comes into plain view. With the fine adjustment, raise and lower the body-tube a little at a time until the point at which the smallest details show clearly is discovered. This is the focal point.

4, When using the low-power and medium-power objectives, the condenser should be lowered until the illumination is evenly distributed. With the high-power objective, the condenser should MICROMETRY 353

be raised almost to the level of the stage. The iris diaphragm should be open sufficiently to illuminate about three-quarters of the aperture of the objective. In other words, it is more widely open for the low-power objective than for the high-power objective.

5. If a greater magnification is desired, change to the highpower ocular, which will double the magnification. If this is not sufficient, return to the low-power ocular and swing the mediumpower objective into position, and so on. On most modern instruments, the objectives are par-focal; that is to say, the lengths of the objectives are such that when another objective is swung into place the object will still be visible. If, however, the object is not in focus, it is best to lower the body-tube until the new objective almost touches the cover glass, and focus up until the object comes into view. If the oil-immersion objective is to be used, lower the condenser and place a drop of oil on its upper surface; then raise it until it touches the bottom of the slide. Place another drop immediately over the object on the cover glass and lower the body-tube with great care until the front lens of the objective touches the oil. Focus by means of the fine adjustment only. .

6. All optical parts of the microscope must be cleaned with lens-paper. After the oil-immersion objective has been used, the front lens, condenser, and slide should be wiped with a bit of lens-paper dipped in xylene and then dried with a fresh piece. Never separate any of the optical parts. The microscope should be lifted by the pillar unless a special grip is provided to the arm. The microscope should be kept in the case when not in use. One of the oculars should be left in the draw-tube at all times to prevent dust getting on the upper lenses of the objectives. Beginners should try to avoid the error of closing the eye that is not in use. Practice will enable the microscopist to work with both eyes open and even to alternate the right and left eye at the ocular.

Micrometry. — The unit of measurement in microscopy is the micron (x). It is the one-thousandth part of a millimeter. Measurement of microscopic objects is performed with the aid of micrometers, of which there are two types, the stage micrometer and the ocular micrometer. The former is a glass slide, in the center of which, under a cover glass, is a line, usually 2 mm. long, 354 STUDY OF EMBRYOLOGICAL PREPARATIONS

divided into 200 equal parts, each of which, therefore, is equivalent to 10 wu. The ocular micrometer is a glass disc, placed in an ocular at the level of the ocular diaphragm, on which is engraved a scale, with arbitrary subdivisions. Some oculars are furnished with a draw-tube so that the upper lens of the system may be focused more sharply upon the scale. The value of the divisions indicated on the scale varies according to the amount of magnification of the real image, and so must be obtained for each objective independently, according to the following method:

1. Arrange the microscope as before, taking particular care to secure the proper tube-length.

2. Focus the eye-lens on the ocular micrometer scale by means of the ocular draw-tube. Focus the objective on the stage micrometer.

3. Make the lines of the stage micrometer parallel with those of the ocular micrometer, and determine the value of the divisions of the ocular micrometer in terms of those of the stage micrometer. Thus, if it requires 10 spaces of the ocular micrometer, and the latter is equal to 0.1 mm., then the value of a single space of the ocular micrometer for that particular objective and at that particular tube-length is 0.01 mm. or 10 ». Determine the value of the ocular micrometer for each objective in the same way.

B. EMBRYOLOGICAL DRAWINGS

Free-hand drawings of microscopic objects can only approximate an accurate representation. However, great pains should be taken to secure at least accurate proportions, neat and cleancut lines, and complete labels. Accurate outlines can be secured by the aid of the camera lucida, various types of projection apparatus, or microphotography.

Equipment. — The student will need a hard lead pencil (4H), a medium pencil (HB), and blue, red, and yellow colored pencils, an eraser, and bond paper to fit the note-book cover used in earlier courses.

Free-hand drawing. —

1. Lay off the space to be occupied by the drawing, by placing four dots at the corners. Rule in two lines, intersecting at the center of this space. These will represent the dorso-ventral ABBE CAMERA LUCIDA 355

and the dextro-sinistral axes, if the drawing is to be of a transverse section.

2. Measure the corresponding axes of the sections by means of the ocular micrometer, multiply by the desired magnification of the drawing, and lay off these magnified measurements on the cross lines already drawn. The following magnifications are recommended: for the twenty-four hour chick, 100 x; for the thirty-three hour chick, 75 x; for the forty-eight hour chick, 50 xX; for the seventy-two hour chick, 30 x; for the 10 mm. pig, 20 x.

3. Draw in a careful outline of the section and of the internal structures, paying particular attention to the proportions, which should be measured with the ocular micrometer and laid off on the axes at the proper magnification.

4, On one side of the dorso-ventral axis, all structures should be colored with the crayons in accordance with the following scheme: ectoderm, blue; mesoderm, red; and endoderm, yellow.

5. Label all structures represented in the section, using broken lines at right angles to the long axis of the paper to connect the label with the structure indicated.

6. Identify the drawing fully, by means of a serial number, the species, and stage of development, the number given to the series, slide, and section, the type of sections, and the amount of magnification. Example: No. 23, Chick, 48 hours, Series 1102, Slide 2, section 28, transverse section 50 x. If a drawing has already been made of the total embryo or a total mount, indicate on this, by means of a heavy ruled line, the position of the section just drawn, and number this line with the serial number of the section.

Abbé camera lucida. — This is an attachment which reflects the light from the drawing board, by means of a mirror, to a silvered prism, whence the light is reflected to the eye, superimposed on the image of the object which is transmitted through a small hole in the silvered surface of the prism directly above the ocular of the microscope (Fig. 240).

1. Attach the camera to the draw-tube of the microscope in such a way that the mirror projects to the right, and the opening in the prism lies above the center of the ocular.

2. Extend the mirror arm to its greatest length and set the 356 STUDY OF EMBRYOLOGICAL PREPARATIONS

mirror at an angle of 45°. The mirror arm must be parallel to the drawing board.

3. Try various combinations of objectives and oculars until an image of the desired magnification appears on the paper. Magnifications intermediate to those obtainable in this way may be secured by varying the tube-length or by raising or lowering the drawing board. If the stage of the microscope interferes with the drawing, the mirror should be set at an angle of 40° or 35° and the drawing board tilted toward the microscope at an angle of 10° or 20°, respectively, by means of wooden images. If the image is stronger than the reflection of the pencil point, a smoked glass may be placed beneath the prism, or the aperture of the iris diaphragm may be reduced. If the reflection of the pencil is stronger than the image, smoked glass may be placed . at the side of the prism or the amount Fig. 240. — Diagram showing prin- Of light falling on the paper reduced

ciple of the Abbé camera lucida. by means of a screen.

Path of image seen in microscope 4, Draw in the outlines of the sec shown in broken lines, that on, .

drawing paper shown in unbroken tions and the larger internal struc lines. (From Gage.) tures. The details may be added

free-hand.

5. Remove the slide and substitute a stage micrometer. Trace in part of the scale by means of which both the magnification of the drawing and the absolute size of the object may be computed readily.

Projection apparatus. — Where many drawings are to be made, as in the case of reconstructions, some form of apparatus by means of which the image of the section may be projected directly upon the paper is very helpful. There are many types of projection apparatus, directions for the use of which may be obtained with the instruments. THE GRAPHIC METHOD (OF HIS) 357

Microphotography. — The photography of minute objects with the aid of the microscope is of great assistance in embryology. However, the methods are so difficult, the apparatus so complex, expensive, and delicate, and the process requires so much technical knowledge and skill, that microphotography has been considered a field too advanced for the beginning student, although a method described by Headland seems to overcome these difficulties to a large extent. In recent years the motion-picture camera has been adapted for use with the microscope, and excellent results have already been obtained.

C. RECONSTRUCTION

After an embryo has been sectioned, it is sometimes necessary to reconstruct some part of it from the sections. There are two important methods: graphic reconstruction, in which a geometric projection of a sagittal section, for example, might be made from transverse sections; and plastic reconstruction, in which magnified replicas of each section are made of wax and piled together so as to make an enlarged model of the object to be studied. A complete series of sections of uniform thickness and accurate orientation is required for either type of reconstruction, and an outline drawing of the embryo before sectioning is of great assistance.

The graphic method (of His).— This method can best be described by giving practical directions for a particular problem, e.g., to prepare a geometrical sagittal projection 20 x of the neural tube of a 10 mm. pig embryo from a series of transverse sections 20 pz in thickness.

1. From the lateral view of the embryo drawn before sectioning, make an outline drawing 20 x.

2. Draw a median line corresponding to the cephalo-caudal axis, the length of which, in this case, should be 200 mm.

3. Count the number of sections in the series, in this case, 500.

4. Locate the position of each transverse section which you have drawn on the median line of the outline. Thus if the most anterior section drawn was the fiftieth of the series of 500 sections, it would be located at a point 1/10 of the total length of the axis (200 »), or 20 mm. from the cephalic end.

5. Theoretically, each section is at right angles to the median 358 STUDY OF EMBRYOLOGICAL PREPARATIONS

line, but this angle may be greater or less as a result of variations in technique. Study each drawing of a cross section in connection with the ‘drawing of the total embryo and determine the angle made by that section with the cephalo-caudal axis of the embryo. Draw in, at each point located on the median line, a cross line at the proper angle so determined. These lines represent the dorso-ventral axes of the transverse sections. Their lengths should correspond with those of the dorso-ventral axes of the drawings of the transverse sections previously made at the same magnification, 20 x.

6. Plot in on each section-plane line (dorso-ventral axis) the dorsal and ventral boundaries of the neural tube as determined from measurements of the drawings already made. Interpolate by direct measurement and magnification of these points on intervening sections.

7. Sketch in the contours of the neural tube by connecting up the points which have just been plotted. Compare the drawing with a sagittal section of an embryo in the same stage of development.

Plastic reconstruction. — This method also will be indicated by practical directions for the reconstruction of a particular organ, in this case, a model 50 x of the heart of a 10 mm. pig, from a series of transverse section, 20 u in thickness.

1. Prepare a number of wax plates of the proper thickness. In this case, if every section is to be reconstructed, the thickness of the plates must be 50 X 20 u,orlmm. Nearly as good results can be obtained by reconstructing every second section and making the plates twice as thick. The wax is prepared according to the following formula:

Beeswax... 0... ccc cc cece eee eee e eee eeeeee 6 parts Paraffin, 56° C. mp... ccc eee cceeec eee eeeeeee 4 parts Rosin, white lump. ...... 0. cee eee cece ee eee eee 2 parts

Mix and melt.

Pour 130 grams of this wax into a pan with an inside measurement of 500 X 280 mm., into which boiling water has been poured to a depth of 15 mm. This amount of wax will make a plate 1 mm. in thickness. Bubbles in the wax may be removed by playing the flame of a bunsen burner over the surface as it is cooling. As the surface hardens, cut the edges free from the sides of PLASTIC RECONSTRUCTION 359

the pan. When the wax has set put is still malleable, roll up the plate and remove it to a soapstone slab, where it is unrolled and allowed to cool.

2. With the help of a camera lucida or projection apparatus, prepare outlines 50 x of the heart in all the sections in which it isfound. Number the drawings consecutively and note the serial number of the sections drawn, so that it will be possible to check the drawings later if necessary. Note also whether the right and left sides of the drawing actually correspond with the right and left sides of the embryo or whether this condition is reversed. This is very important, as a mistake at this point would render the reconstruction valueless.

3. Transfer the drawings to the wax plates by means of carbon paper. Place the wax plates on a sheet of glass, and cut out the parts to be preserved with a sharp scalpel, leaving bridges of wax to connect the parts which would otherwise be separated. These bridges are best made in the form of arches bending towards the outside of the section.

4. Pile the sections in order, taking care to avoid the reversal of right and left sides, and to get an accurate fit. It is best to group the sections in piles of ten. A steady pressure of the hand will be sufficient to cause the sections to adhere to each other. The bridges may be cut away and stout pieces of wire substituted. Heat the wire at each end and press into position. After the wire is set, the wax bridges are cut away and the edges of the piece smoothed with a heated scalpel or aluminum modeling tool.

5. When all the sections have been combined in groups of ten, these groups should be united and the completed model smoothed in the same way. Such models may be painted or dissected, and mounted on wooden supports as desired. They are quite permanent if not exposed to high temperatures. Plaster of Paris molds and casts may be made from them in the customary manner.

REFERENCES

Belling, J. 1930. The Use of the Microscope. Cage, S. H. 1932. The Microscope, 15th Ed. Guyer, M. F. 1917. Animal Micrology, 2nd Ed: 360 STUDY OF EMBRYOLOGICAL PREPARATIONS

Headland, C. I. 1924. A Simple and Rapid Photomicrograph for Embryological Sections. Anatomical Record, XVII, 2.

Lee, A. B. 1929. The Microtomist’s Vade-Mecum, 9th Ed.

Mueller, J. F. 1935. A Manual of Drawing for Science Students.

Norman, J. R. 1923. Methods and Technique of Reconstruction. Journal of the Royal Microscopical Society.