Book - A Laboratory Manual of Vertebrate Embryology (1947) Frog

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Adamstone FB. and Shumway W. A Laboratory Manual of Vertebrate Embryology. (1947) John Wiley & Sons, London.

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Pages where the terms "Historic" (textbooks, papers, people, recommendations) appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms, interpretations and recommendations may not reflect our current scientific understanding.     (More? Embryology History | Historic Embryology Papers)

A Laboratory Manual of Vertebrate Embryology

Part I Anatomy of Frog Embryos

(Jtana Tnpiervi)

STAGES

(After Shumway, 1940)

No. Name Age in Hours at 18" Cent. Length in mm.

1.

Unfertilised egg

0

1.7

2.

Fertilised egg (gray crescent)

1

“

3.

Two-cell

3.5

“

4.

Four-cell

4.5

5.

Eight-cell

5.7

"

6.

Sixteen-cell

6.5

7.

Thirty-two-cell

7.5

8.

Mid-cleavage (early blastula)

16

9.

Late cleavage (blastula)

21

1.9

10.

Dorsal lip (early gastrula)

26

“

11.

Lateral lip (mid-gastrula)

34

12.

Ventral lip (late gastrula)

42

“

13.

Neural plate (ncurula)

50

“

14.

Neural fold (early)

62

2

15.

Rotation (late neural fold)

67

2.3

16.

Neural tube

72

2.6

17.

Tail bud

84

2.9

18.

Muscular response (to mechanical stimuli)

96

4

19.

Heart beat (gill buds)

118

5

20.

Hatching (capillary circulation ‘in first gill)

140

6

21.

Mouth opening (cornea transparent)

162

7

22.

Tail fin circulation

192

8

23.

Opercular fold

216

9

24.

Right operculum

240

10

25.

Operculum complete

284

11

The Germ Cells and Fertilization

The Ovum (Stage 1)

The frog's egg is spherical with a diameter of 1.7 mm. Its upper or animal hemisphere is black; the lower or vegetal hemisphere is white. It is surrounded by a very thin envelope, called the vitelline membrane, which is tightly pressed against the egg. Outside this is a thicker and tougher layer, the chorion, which is not to be confused with the chorion formed by developing birds, reptdes, and mammals. Outside the chorion is the egg jelly, which consists of an inner and an outer layer. These egg envelopes are best observed in living eggs.

Hie nucleus, whidi lies in the animal hemisphere, can be seen in sections of ovarian or uterine eggs. This hetui^ere is niarked by the dark pigment granules lying just under the periphery. Scattered tlirou# the cytoidasm of the egg are the ovoid yolk granules.

The Spermatozoon

The male germ ceil is extremely minute, approximately 0.03 mm. in length. There is a long cylindrical head containing the nucleus and a slender tail which is twice the length of the head.

The Fertilized Egg (Stage 2)

Under natural conditions the eggs of the frog mature simultaneously in the spring, a condition which may be brought about in the laboratory by mjections of pituitary gland ^ into the body cavity.

The eggs escape from the follicles of the ovary and are collected in the oviducts. When stimulated by the embrace (amplexus) of the male, the eggs are discharged from the cloaca, while the spermatozoa of the male are simultaneously emitted as a stream of milt. Pituitaryinduced eggs may be artificially fertilized with spermatozoa removed from the sperm ducts of the male.

In living material it is possible to see at the animal pole two polar bodies, one formed before, and one after, fertilization. One can also recognize a narrow gray crescent lying between the black animal and white vegetal hemispheres on one side of the egg. This is the side opposite the one on which the cperm entered the egg. These observations are extoemely dxGEicult to make on preserved eggs.

As the eggs enter the water, the egg jelly swells and the outer layer becomes sticl^ so that all the eggs from one ovary tend to form a large mass of spawn containing about 2(K)0 eggs.

Laboratory Directions

Examine the demonstrations of eggs and sperm provided.

CLEAVAGE

First Cleavage (Stage 3). A furrow appears in tile animal hemisphere after fertilization. This furrow grows down throu^ the vegetal hemisphere, thus dividing the egg into the first two blastomeres. In fresh material it can be seen that this furrow usually passes through the gray crescent.

Fra. 1 Gerin cells of the frog (Aona ptpieru) a, Croa9-«ectto& of the ovarian egg (approximately 26 X); b, Bpemiatoeoon (apptozimately 1000 X>

Second Cleavage (Stage 4 ). The second cleavage plane also commences at the animal pde, crossing tiie first at ri§^t anftisB and dividing the egg into four blastomeres. Cktreful inspectkm of the animal hemiqihere shows that the two furrows do not meet in an exact miss. Two of the blastomoes are in dose contact, forming a 8h<»rt hmgitodinal groove known as tiie pdfir furrow.

Bee R. Bngb, "Embryoiue Material for Laboratoiy EzpsrimeBtafiim,’' Am. Ski. Ttachtr, BW.

CLEAVAGE

3

Third Cleavage (Stage 5). This stage is marked by the formation of a latitudinal furrow sli^^tly above the equator of the egg and l3ring in the animal hemisphere. It divides the egg into eight blastomeres. Careful examination shows that this furrow is not an exact circle but has irregularities where it intersects the other cleavage planes.

Fourth Cleavage (Stage 6). Two furrows appear at this stage, beginning in the animal hemisphere, and grow down through the vegetal hemisphere to produce sixteen blastomeres. These furrows may mtersect each other at the animal pole or naay be parallel to each other. Careful exammation of the e-nitwH.! hemisphere should be made.

Fifth Cleavage (Stage 7). Again two furrows are formed, one in the animal hemisphere and one in the vegetal. The one in the anunal hemisphere appears first. On the completion of the lower furrow, thirty-two blastomeres are formed. Those in the animal hemisphere are smaller and are called micromeres; those of the vegetal hemisphere are larger and are called macromeres. Prom this stage on, increasing irregularity of the cleavage planes is apparent.

Blastula (Stage 9). The fifth cleavage stage is followed by the sixth, etc., until the end of the cleavage period comes with the formation of the blastula. In this stage the pigmented area has increased at the expense of the unpigmented area. The cells in the pigmented area are so small that they are seen only with difficulty even when observed with a hand lens. If the blastula is split with a safety-razor blade through the animal pole,^ it wfll be seen that it contains a large cavity, the blastocoel. The roof of this cavity (animal hemisphere) is very thin; its floor (vegetal hemisphere) is very thick. This is because of the concentration of yolk in the macromeres of the vegetal hemisphere.

L uh orafory Dirtcfions

Biamine the stages desoibed above and make outline sketdies (about one inch in diameter) of each bk igpaos provided opposite the paragraphs above.

4

ANATOMY OF FROG EMBRYOS

GERM LAYER FORMATION

After the fonnulation of the blastula the embryo is converted into a gastrula with several germ layers. Externally the process of germ-layer formation or gastrulation is marked by the progressive

Fio. 2. Germ layer formation in the frog. Diagrammatic, a, doraal lip stage; b, lateral lip stage; c, ventral lip stage; d, neural plate stage after rotation. The smaller figures to the upper left are surface views; the larger ones to the right are sagittal sections. An, animal pole; Vcp., vegetal pole; D, dorsal; V, ventral. The small arrows indicate the direction of cell movements. The presumptive germ layers are designated after the data of Vogt and Paateels; epidermal ectoderm, white; neural plate ectoderm, cross lines; notochord and mesoderm, stipple; endoderm, small circles.

formation of the blastopore, conveniently divided into three stages; (Stage 10} the appearance of the dorsal lip; (Stage 11) the development of the lateral lips from the dorsal lip; and (Stage 12) the formation of the ventral lip by the union of the lat^eral lips.

The study of sections shows that at the dorsal lip, surface cells roll inward to form the roof of a slitlike cavity, the gastrocoel. As the dorsal lip advances over the surface of the vegetal hemisphere, it also elongates to form the lateral lips, where a similar in-rolling occurs, thus adding to the side walls of the gastrocoel. In its advance the dorsal lip moves almost completely over the vegetal hemisphere so that when the lateral lips unite to form the ventral lip, the completed lip of the blastopore forms a small circle enclosing the only white area left on the ^irface of the e^. Hiia white area which protrudes slightly above the levd of the Uastop<»e m the yolk plug.

‘ Hus may be dons by demonstration.

FORMATION OF THE NEURAL TUBE

5

The partition between the newly formed gastrocoel and the old blastocoel is either ruptured or pushed back until the blastocoel is obliterated, and the gastrocoel is the only cavity persisting. Since the heavy mass of yolk-laden macromeres is now on one side of the gastrula, the embryo rotates until the weight is at the bottom, which brings the bliustopore to the posterior end of the embryo (Fig. 2).

In the frog’s egg the layer of cells left on the surface after gastrulation is complete is the ectoderm. The layer of cells turned in at the lips of the blastopore gives rise to the notochord and mesoderm which form a temporary roof of the gastrocoel. Soon, however, endoderm cells move up beneath the chordamesoderm and transform it into a middle germ layer. The median axial strip of this middle layer is the primordium of the notochord; the rest becomes the mesoderm, which soon grows out between the ectoderm and endoderm in other regions of the embryo. In an exactly sagittal section it can be seen that the notochord forms part of the roof of the gastrocoel (Fig. 2d). During the formation of the neural tube the endoderm grows beneath the notochord from either side (Fig. 3).

Laboratory Directions

Examine total preparations of the dorsal-lip, lateral-lip, and ventral-lip (yolk-plug) stages of gastrulation. Make outline sketches in the space provided as in the last exercise. Prepare a half gastrula^ by splitting from top to bottom, bisecting the yolk plug. Observe the gastrocoel and its lining of small white cells, the endoderm. Sketch and label. Examine demonstration slides which show the position of all three germ layers.

FORMATION OF THE NEURAL TUBE (Reorulation)

Neural Plate (Stage 13). During gastrulation the ectoderm separates into a thinner outer layer, which gives rise to the epidermis, and a tiiicker inner layer, which will give rise to the neural tube, neural crest, and the sensory placodes. This is known as the nervous layer. The thickening of this layer on the dorsal side produces a flattening of the dorsal surface of the embryo, called the neural plate. At this stage the blastopore is latersdly compressed to form a dorsoventral slit.

SThJs may be ehtnm by damonstral^

6

ANATOMY OF FROG EMBRYOS

Fia. 8. Neural tube formatiou in the frog (£ana pipiens). a, transverae section through neural plate; b, transverse section through neural folds and groove; e, transverae section through neural tube. All at 60 X- Germ layers represented as in Fig. 2. X, nervous ectoderm which forms neural crest.

Neural Folds, Early (Stage 14). The embryo is now increasing in length. The margins of the neural plate are elevated as the neural folds while the shallow groove which they enclose is the neural groove. At the anterior end of the embryo extending around the neural folds on either side is a crescentic elevated area known as the sense plate. The blastopore is surrounded by the neural folds on the right and left. Sections show that the neural folds are produced by a thicke|iing of the nervous layer in that region. The lateral portion of each neural fold wiU give rise to neural crest cells. The notochord is separated from the mesoderm on each side but it is in close ccmtact with the advancing sheets of endoderm which will form the roof of the gut (Fig. 3a).

Neural Folds, I^ite (Stage 15). Ihe emlnyo is.still inereaffing in length and ciliated cells have appeared in the epidermal layer of the ectoderm. The action of these cilia causes the endDvyo to rotate within the egg jelly. The neural folds are in cohtaot with

3-MM. EMBRYO

7

each other along the inid>dor8al line but have not yet fused. Posterior to Ihe sense plate, the gill plate can be seen on either side of the neural folds. Here the visceral grooves will develop. Sections show that the neural crests are being drawn over the developing neural tube, and that the neural folds enclose a longitudinsJ canal, the neurocoel. The mesoderm nearest the notochord is segmented into blocks, or somites, of which three or four pairs may be distinguished in sagittal or frontal sections. The gut is now completely roofed by endoderm (Fig. 3h).

Neural Tube (Stage 16). The embryo is now a little more than 2.5 nun. in length, and the neural folds have fused to produce the neural tube. From the dorsal aspect the embryo is pear shaped and marked on each side by short transverse grooves enclosing the gill plate and representing the first and fifth visceral grooves. On the ventral surface at the anterior end are two swellings, the primordia of the mucous glands. Between them is a depression which is the beginning of the stomodeum. At the posterior end the blastopore is obliterated by the fused neural folds, and at the ventral end of the strip a depression, resulting from this fusion, marks the beginning of the proctodeum. Slight transverse grooves on the sides of the embryo indicate the presence of six somites. In certain sections it can be seen that the neural tube is completely separated from the epidermis and that the neural crest material lies between them.* Between the notochord and the roof of the gut lies the hypochord, a temporary structure formed from endoderm which disappears in later development (Fig. 3c).

Laboratory KrecHons

Examine the stages in neural tube formation described above and sketch each from the dorsal aspect in the space provided. Examine demonstrations of transverse sections through each stage.

3-MM. EMBRYO (Stage 17, Tail Bud)

The embryo of approximately 3 mm. body length is chosen somewhat arbitrarily as an example of an early embryo. Externally it is marked by the possession of a distinct tail bud. This has grown backward from the posterior end of the fused neural folds. The gill plate now shows four slight visceral grooves known as the hyoid, first, second, and fourth branchial grooves, respectively. The third bran^ial groove appears at a later stage. The mucous glands have enlarged considerably. In ventral view, a groove extending from the posterior end forward in each gives them the appearance of an elongated U. Between them the stomodeum may be identified. At the posterior end of the body, a short distance ventral «id anteriw to the base of the tail bud, is the proctodeum, which now opens into the hind-gut. In lateral view, a swelling in the sense plate region indicates the outgrowth of the optic vesicle. Posterior tp the gOl plate and at a level i^proximating the base of the neural folds a series of slight transverse grooves mvks ^e presence of twelve or thirteen somites. Ventral to these and just behind the gill ^ piate i$ a, awdluE^ oa^ised by the developing nephrotomal band. (These details may be seen better in

aeleig^imdstaiii^

ANATOMY OF FROG EMBRYOS

S

PHARYNGEAL DERIVATIVES AND ASSOCIATED STRUCTURES

Visceral

(pharyngeal)

pouches

EniodmMl

Visceral

(branchial)

arches

Visceral

(pharyngeal)

clefts

Aortic arches

Visceral

(branchial)

grooves

Ectodermal

—

I

Mandibular

—

I

—

I

Hyomandibular

—

I

Hyomandibular

—

I

Hyomandibular

—

II

Hyoid

—

II

—

II

—

II

—

II

—

Ill

Ist branchial

—

Ill

Carotid

—

Ill

—

HI

—

Ill

—

IV

2nd branchial

—

IV

Systemic

—

IV

—

IV

—

IV

—

V

3rd branchial

—

V

—

V

—

V

—

V

—

VI

4th branchial

—

VI

Pulmonary

—

VI

Vestigial in frog

—

VI

Lacking in frog

—

VI

Lacking in frog

The eonfnaioii srieiiig in the mind* of etudante who atteront to do eoUataral readme emnniinc the erohee and pouolua may be avoided Iqr the etlidy ol tlila table. It win be a|»parent that the difficulty eentem about the faMU^minate nee of %» oped “h nmah ial’' in fajBeet to the atebee. Soma auibeniMe the wiMdler ^ the anhea, othert lemriot iu uae to the yflideariny arahaa aa we hara doM hm We have, hotmvtr, (fv«n Oie aHmnntivo uMie in miwthww. ae tea firm no dt^toapiiaartofoieeoiiratdatioaon oareeUtacuta. Sea alio Flfa. 10 and tl.

3-MM. EMBRYO

Lahortttor^ DhecHons

Make an outline sketch of the S-mm. embryo from the left side in the space provided below. Label all structures you have identified.

The 3-mm. Frog Embryo.

Serial Sections

The internal anatomy of embryos from this stage onward is studied much better from embryos which have been cut into a continuous series of sections or slices and mounted in order on slides for jni>

Fra. 4. Block diagram of 3-mm. frog embryo {Rana pipient), to ahow position of transverse section through liver primordium in relation to the whole embryo represented as a transparent body.

croscopic obsowation. Figure 4 indicates the rdationship of a transverse section to the embryo as a whole. In order to read a series of sections you should examine each section, identifying the structures you fAnKxvB.

10

ANATOMY OF FROG EMBRYOS

ECTODEBMAL DEBIVATIVES

The epidermis is ciliated except on the ventral side, but the cilia themselves are too small to be seen with your microscope. It will be noticed with high power that the ectoderm is divided into two layers, the epidermal layer and the nervous layer. The stomodeum, between the two mucous glands, has not yet joined the fore-gut. From the dorsal region of the stomodeum a solid hypophysis grows toward the forebrain. Notice with high power the structure of the mucous glands. The cells in these glands are more qiecialized at this time than any others of the body. The proctodeum has opened into the hindgut, thus forming the cloaca.

The neural tube is now completely cut off from the exterior. The anterior end or prosencephalon is bent slightly around the anterior end of the notochord, owing to the cranial flexure. The prosencephalon extends backward on its ventral side to the pt^terior level of the optic vesicle. On the dorsal side it extends backward to the anterior end of a thickening in the roof of the brain. The mesencephalon extends backward from these boundaries to the anterior level of the notochord. The rhombencephalon begins at this point. The boundaries of the mesencephalon and rhombencephalon are not easily distinguished in transverse sections. These and the epiphysis are distinguished more easily in a sagittal section. The spinal cord has grown into the tail bud.

From the ventral halves of the lateral walls of the prosencephalon, the optic vesicles protrude outward, so producing a bulge on either side of the head of the embryo. Farther back, in the region of the rhombencephalon, the nervous layer of the ectoderm is greatly thickened and has invaginated on each side to form an otic (auditory) pit. Notice that the epidermal layer of the ectoderm is not invaginated. The nose at this time is in the form of thickenings of the nervous ectoderm, the olfactory (nasal) placodes, in sections anterior to, or at, the level of the prosencephalon.

BimOOEBMAn DEBIVATIVES

The gastrocoel is now lengthened and dilated in the region anterior to the yolk mass to form a foregut. A depression in the floor of the fore-gut extends towards the stomodeum but the two are still separated. Posteriorly a fingerlike evagination of the fore-gut extends into the yolk mass as the primordium of the liver. Frontal sections show the beginning of the visceral pouches I, II, and III growing out from each side of the fore-gut towards the corresponding visceral grooves already noticed in the ectoderm.

The mid-gut is formed from the gastrocod ovmiying the yolk mass. It has a very thick floor and relatively thin roof. Above it lies a thin rod much like the notochord but smaller. It is called the hypochord and is formed from the endodermal roof of the mid-gut, has no known function, and disappears early.

The hind-gut, posterior to the yolk mass, has united with the proctodeum to form the cloaca, opening to the exterior by the small vent already noticed.

MBSODEBMAL DEBIVATIVBS

The notochord extends forward to the base of the mesencephalon where it turns down very slightly. At the posterior end it joins a mass of relatively undifferentiated tissue which forms the core of the tail bud.

On either side of the notochord is the axial mesoderm from whidi twelve or thirteen somites have been formed. The axial mesoderm terminates in the core of the tail bud mentioned in the above pari^ graph. The intermediate mesoderm was partially divided into nephrotomes at an earlier stage. These have now fused together to form a nephrotonud bmid. This is best seen in sections out Ihrou^ the • region of the liver. The lateral mesoderm is divided into an outer dinner somatic layer and an.inn^ thicker splanchnic layer. No coelom is formed between them as yet except beneath the fore-guti it is to be seen in sections cut in the region of the otic pit. Here the (g>ace rep'resmitil the b^j^hiaJ&g bf the pericardial cavity. V

3-MM, EMBRYO

11

Laboratory DirecHoru

Study the set of sections provided you and make sketches of sections through (1) the optic vesides, (2) the otic pits, (3) the liver, and (4) the cloaca.

Section through optic vesicles.

Section through otic pits.

Section through liver.

Section through doaca.

Transverse Sections of 3-mni. Prog Embryo.

12

ANATOMY OF FROG EMBRYOS

6>MM. EMBRYO (Stage 20, Hatching)

' The embryo of Ram pipiens batches spontaneously at a length of 6 mm. The stage now to be described is marked exactly by the beginning of circulation in the capillary loops of the first external gill.

Extebnal Form

The head is marked off from the trunk by a notch behind the mucous glands which have approached each other at their posterior ends. The stomodeum, now a deeper pit, is anterior and slightly dorsal to the anterior ends of the mucous glands but has not yet opened into the fore-gut. On either side of the stomodeum and somewhat dorsal to it is a nasal pit. On the sides of the head optic bulges are still prominent. All five visceral grooves (hyomandibular and branchials 1-4) have appeared but have not opened into the corresponding visceral pouches. External gills are developing on the first and second branchial arches, and circulation can be demonstrated in the first of these.

Transverse intersomitic grooves appear on the trunk, and beneath them a slight ridge marks the region of the pronephric duct. On the ventral side the cloaca! opening is to be seen just anterior to the base of the tail.

The tail is now one-half of the body length. Intersomitic grooves continue into the tail. A prominent tail fin extends completely around the tail from the vent on the ventral side to the dorsal junction with the trunk.

Laboratory Directions

Make a drawing of the 6-mm. embiyo from the left side.

The 6-mm. Frog Embryo.

6-MM. EMBRYO

13

Rbphesentativb Sections of the 6-mm. Embryo

The sections described and illustrated are selected from a series of 275 sections cut transversely to the long axis of the embryo at a thickness of 20 microns (0.02 mm.). They are reproduced from preparations made to compensate for the reversal produced by the compound microscope. Right on the illustration is right on the embryo. The student is cautioned that because of slight variations in the developmental tempo of embryos and variations in the plane of the section he will probably never encounter sections exactly like those illustrated.

A. Section through the Epiphysis and Nasal Pit (Section 10, Fig. 5)

Because of a slight lateral curvature of the head, this section shows a slight asymmetry. The plane of the section passed* through the left side at a region that is anterior to that on the right. The nasal pit (1), therefore, is cut on the left through its openini^ to the exterior, and on the right through its posterior side wall. The groove on the ventral side is the beginning of the stomodeum (2). The brain is cut through its most anterior division, the telencephalon (3), at the level of the primordium of the pineal

Fra. 5. Rana pipieru, 6-mm. em- Fra 6. Rana pipiens, â– 6-mm. embryo.

brj’O. Transverse section through Transverse section through optic cup.

nasal pit. 50 X- 50 X.

gland, or epiphysis (4). An examination of the epidermis (5) with the high power will reveal a few larger rounded cells, projecting slightly from the surface, which bear cilia. The space between these structures (all of ectodermal origin) is fiDed with a loosely connected tissue, the mesenchyme (6), partly of mesodermal derivation and partly from the ectoderm of the neural crest. A few small cavities represent the neural capillary plexus (7) from which the blood vessels of the head are forming at this time.

B. Section through Optic Cup and Lens Vesicle (Section 22, Fig- 6)

There is still a slight asymmetry so that the section passes through the lens vesicle (1) on the left and behind it on the right. The vesicle has just detached itself from the epidermis and has a small central cavity. The optic cup has two layers, an inner, thicker sensory layer (2o) and an outer, very thin, heavily pigmented one (26). The neural tube is partially divided by a constriction into the ventral dienoephalon (3) and the more dorsal mesencephalon (4). Beneath the floor of the diencephalon

14

ANATOMY OF FROG EMBRYOS

lies a flat thick plate of ceUs, the hypophysis (5). The epidermis (6), with its ciliated cells, covers tiie surface of the embryo. On the ventral side are two larger mucous glands (7), the one on the ri^t cut through its cuplike groove.

In the ventral half of the section is the large cavity of the thin-walled pharynx (8) and beneath it a solid laterally compressed rod, the thyroid gland (9).

Between these structures lies the mesenchyme (10) containing a few blood vessels. Those located between the eye and brain are parts of the ophthalmic vein (12) which lead to the anterior cardinals (see later). Those between the brain and the pharynx are part of the cerebral arteries (11) which represent anterior extensions from the carotids (see later).

C. Section through Otic Vesicles, Heart, and External Gilla (Section 38, Fig. 7)

At this level the curvature which resulted in the aqrmmetry of the two preceding sections has now been passed. As this section passes through the two otic vesicles (1), the portion of the brain between them must be referred to the myelencephalon (3) with its thinwalled roof. A slight projection on the inner dorsal angle of each otic vesicle is the beginning of its endolymphatic duct (2).

Beneath the brain is the conspicuous notochord (4) with large vesicular cells. Beneath this in turn lies the widdy expanded pharynx (5). The lateral thickenings of its walls represent the fourth visceral pouch (6). Ventral to this in the cavity of a great bulging projection lies the heart, represented by the dorsal atrium (8) and ventral ventricle (7). It is contained in the most anterior part of the coelum, the pericardial cavity (9). Note that this cavity is lined on the outside by the somatic layer of the mesoderm (10) and on the inner side by the splanchnic layer of the mesoderm (11), which gives rise to the myocardium and visceral pericardium. The endocardium (12) separated at an earlier stage.

From either side of the embryo projects the first external gill (13) borne on the third visceral arch. This gill is now

branched and has several capillary loops (14). Circulation in this gill can be seen in the living embryo. The capillary loops

heart, fio X- beneath the pharynx. The capillary loops of the gill discharge

their blood to the first efferent branchial artery, which is dorsal to the side of the pharynx and is uniting (on the right side) with the radix aortae (16). These arteries represent the third aortic arch of vertebrates. Below and lateral to the otic vesicle is the ventral branch (inferior jugular) of the anterior cardinal vein (17). Between the otic veside and the brain may be seen the dorsal branch (superior jugular) of the anterior cardinal vein (18). The mesenchyme and epidermis are readily identified.

D. Section through Pronephros and Liver (Section 55, Fig. 8)

This section cuts through the region where the liver (1) joins the intestine (2). The brain b cut in the region of the myelencephalon (8) as shown by the thin roof. Lying beneath it is the nc^odiord (10) and just below this is a smaller rod, the hypochord (11), formed from the roof of the gut fout^unr separated from it by the dorsal aortae (6) which are h^ uniting together.

On either side of the notochord and stretching up alonpide the mydenoephalon ill a myotd^ with which the dermatome is still associated. Between the myotmne and ^ ^idspals (JtS), a above the level of the notochord, the lateral line n^e (7) may be seeh.^ T^ latest appear until much later. The intermediate mesodenn or nepSuxdoinal bimd k

6-MM. EMBRYO

15

it have appeared the three pronephric tubules (5).- One of these is cut to show the iiephrostome (3) or opening into the coelom (14). This cavity is lined on Ihe outside by the somatic layer of the mesoderm (15) and on the inside by the splanchnic layer (16). Note the slight bulges at the inner dorsal angles of the coelom caused by the outward growth of each glomus (4) from the dorsal aorta. Above and to the outer side of each glomus, the large anterior cardinal veins (12) appear. The posterior cardinal veins (13) are seen beneath the pronephric tubules. On the ventral side of the liver, enclosed by the splanchnic layer of the mesoderm, are the large vitelline veins (17). The epidermis (18) presents nothing different from other sections.

£. Section through Proctodeum (Section 159, Fig. 9)

If it were not for the presence of the proctodeum (1) this would be a typical section through the tail region. Note the tail fin (2) above and below. In the mid-region are seen the spinal cord

Fio. 9. Rarm jripicnii

' 6-mm. embryo.

Fra. 8. Rana pipienn, S-mm. embryo. Transverse section

Transverse section through liver and through tail at level

pronephros. SO X- of cloacal opening.

(3), notochord (4), and hypochord (5). On either side is one of the tail myotomes (6). Beneath the hypochord is the small caudal artery (7), and some little distance beneath this the caudal vein (8). lire tail fiin is composed chiefly of mesenchyme (9) and epidermis (10) with many ciliated cells.

Thk Study of Serial Se(3tion8

After studying the representative sections just described, the student should be ready to survey the anatomy of an embryo from a complete set of serial sections. Such a set consists of a complete aeries of slices araross the long axis of the embryo arranged in order from the tip of the head to the end of the-tail. EverythinI; which could be found by dissection, if the embryo were larger, can be identified in some part of series. Once identified the structure can be traced forward and backward until a complete mental fflcture of the organ and its relationship to others is formed. It is advisable before proobcdin^ further to identify the plane of sectioning by laying a ruler across the lateral view of the whole mAayo have ^awn earlier mid tracing lines at the levels of the representative sections just studied.

m identifying the structures mentioned in Uie following paragraphs, make a record in the bUmh q;>aoeB provided (1) the number of the slide in the set you use, (2) the

16

ANATOMY OF FROG EMBRYOS

number of the row of sections in which you first identify the structure (counting from top to bottom), and (3) the number of the section in the row (counting from left to right). A certain number of the structures ajre starred. It is recommended that, in the margin of this manual opposite the starred structure, you make a simple line sketch to show its appearance in the section you have identified.

ECTODERMAL DEBIVATIVBS

I. EPIDERMAL STRUCTURES

a. Epidermis. A single layer of ectoderm cells forming the outer covering of the body, many of them ciliated.

b. Stomodeum. A pit on the ventral anterior surface of the head separated by a plate (ectoderm on the outside, endoderm on the inside) from the fore-gut.

c. Proctodeum. A pit on the ventral surface of the trunk where it joins the tail, opening directly into the hind-gut to form the cloaca.

d. Mucous glands. Prominences on the ventral side of the head on either mde of the stomodeum and behind it. Observe deep groove in each.

n. BRAIN AND SPINAL CORD. Starting at anterior end identify:

a. Telencephalon. First portion of the brain to appear in the series.

b. Diencephalon. Commences at the point where the epiphysis joins the brain. From its sides the optic stalks emerge. Beneath, a backward groove finally separates from the diencephalon to form the infundibulum.

1. Epiphysis. Extends forward from its point of origin. Now has lost its cavity.

2. Infundibulum. Find a section where the brain appears in two parts. The ventral portion is closely applied to the hypophysis (see below).

3. Hypophysis, a derivative from the anterior end of the stomodeum. It will unite with the infundibulum to form the pituitary gland.

c. Mesencephalon. With a thick roof. Lies between optic cups and above the infundibulum.

d. Metencephalon. A thin roof. Lies between otic vesicles.

e. Myelencephalon. A thin roof. Hardly to be distinguished from metencephalon. Continues backward to spinal cord.

f. Spinal cord. Thick walls, thinner roof and floor. Continues back into tail almost to the end.

lU. SENSE ORGANS OF THE HEAD

a. Nasal (olfactory) pit. Most anterior sense organ, on ventral side of head, mark^ by thickness of epitiielium and opeiung to the exterior <mly.

6-MM. EMBRYO

17

b. 1. Optic cup. Consists of pigment layer and thick sensory layer,

joined to diencephalon by optic stalk.

2. Lem vesicle. With central cavity, located in mouth of optic

cup Md separated from epidermis.

c. Otic vesicle. Located alongside myelencephalon. A thickwalled hollow vesicle with an endolymphatic duct just forming.

IV, CRANIAL NERVES. At this Stage there are four masses of tissue resembling the tissue of the walls of the brain in the mesenchyme on either side of the metencephalon and myelencephalon. These are ganglia which will supply sensory neurones to certain cranial nerves.

a. Anterior to the otic vesicle and proceeding forward.

1. Facial-acoustic complex. Immediately in front of the otic vesicle. Supplies afferent fibers to cranial nerves VII and

VIII.

2. Trigeminal ganglion. Very large. Supplies afferent fibers to cranial nerve V.

b. Posterior to the otic vesicle and proceeding backward.

1. Glossopharyngeal ganglion. Just posterior to the otic vesi cle and ventral to it. Supplies afferent fibers to cranial nerve

IX.

2. Vagus ganglion. Some distance farther back and a little dorsal to the last. Supplies afferent fibers to cranial nerve X.

Trace the lateral line nerve backward from the vagus ganglion. ^ *

ENDODERMAL DERIVATIVES

a. Mouth. The region of the fore-gut just behind the plate separating it from the stomodeum will form the oral cavity of the mouth. This continues as pharynx.

b. Pharynx. This region with its visceral (pharyngeal) pouches is best seen in frontal section (Fig. 10). Lay your ruler across this figure and draw a line to show the position of the transverse section shown in Fig. 7.

c. Thyroid gland. Originates from the floor of the pharynx as a solid rod from the level of the second visceral pouch to the fifth.

d. Liver. The liver diverticulum noted in the 3-mm. embryo has grown forward towards the heart. The posterior ventral portion shown in Fig. 8 will become the gall bladder.

e. Intestine. From the liver backward the mid-gut becomes the intestine, with a small cavity and an exceedingly thick floor which contains the yolk.

f. Cloaca. Starting with the proctodeum trace the cloaca forward until it unites with two small longitudinal tubes, the pronephric ducts (see later).

18

ANATOMY OF FROG EMBRYOS

g. Hypochord. This is a small rod of endoderm separating from the roof of die gut and lying beneath the notochord. It extends back into the tail.

Ra. 10. Ram pipietu, 6>min. embryo. Frontal eection through head at level of viflceral pouches and arches. 50 X*

MEBODEBMAL DEBIVATIVX8

I. NOTOCHORn. Cylindrical rod with characteristic vesicular cells. Lying beneath brain and qiinal cord. Commencing behind infundibulum and ^tending well back into the tail.

II. MESENCHYME. Loose reticular embryonic connective tissue filling space between organs.

III. nERivATivEs OF THE SOMITES. The souiites extend from behind the otic vesicle well into die tail. There are thirteen somitra in the trunk and approximately twenty more in the tail. The inner ventral portion of each so^te (sderotome) is now broken down into a mesenchymal tissue investing the not^ord and neural tube from which the axial skeleton will be framed. The dermatome or outer layer (from which the dermis of the skin will develop) is still associated with the musdeforming remainder of the somite or myotome. In these myotomes musde fibers are developing.

IV. oBBivATivES OF THE INTERMEOUTB MESOOEBM. In the frog this region forms the nephrotomal band from which the kidney and gonads develop.

a. Pronepkric tubvlea. Three on each side. Well-defined wall of thick cells. Pigment concentrated at the cell margin neanat the cavity. (Communicates with the oodran by fuhnel (naph^ stome) . It is not ciliated at this time.

b. Pronepkric duct. Originates from the last pnmeplurie tiibe and craitinues in the ventral portion of the neidtrotranal band, lyaaa the pHTon^hric duct to its junction with the doaea,

V. DEBIVATIVBS OF THE tATEBAL MESOniSM

a. Somatic meeoderm. CSomSy apqilied to the cbfihdend in; trunk. â– '

6-MM. EMBRYO

19

b. Splanchnic mesoderm. Closely applied to the endoderm in the trunk region.

c. Coelom. The cavity located between the somatic and splanchnic layers of mesoderm. The anterior portion of the coelom known as the pericardial cavity surrounds the heart. The coelom is not to be found in the head or tail.

d. Mesenteries. The right and left splanchnic and somatic layers of the mesoderm unite above the intestine and heart. These regions constitute the dorsal mesentery and dorsal mesocardium, respectively.

VI. cntcuiATORY SYSTEM. The blood vessels and lymphatics of the frog embryo originate in a system or network of capillaries (plexus) associated with the organ systems and derived from splanchnic mesoderm in the trunk region or from mesenchyme in the head or tail. They are best demonstrated by injection^ preparations.

a. Heart. Locate the heart, at the level of the external gills, proceeding backward until it divides into two large vessels entering from the liver.

1. Sinus venosus. The point where the two vessels converge, forming a stubby inverted Y.

2. Atrium. The sinus continues forward and dorsal, bending to

the right, to enter the thin-walled atrium. 3. Ventricle. The heart in more anterior sections takes on the appearance of a reversed C. The lower limb bending from the atrium to the left side is the ventricle.

4. Bulbus arteriosus. Continuing forward, the lower left por tion of the reversed C becomes detached from the atrium and is now known as the bulbus.

b. Arteries.

1. Of the head.

Ventral aorta {truncus arteriosus). Following the bulbus towards the head, it immediately assumes the form of a letter T. The base represents the bulbus, the shaft the short ventral aorta, and the right and left arms at the top are the branches of the truncus which later subdivide , to form the afferent branchial arteries.

Afferent branchial arteries. Three on each side, located in the first, second, and third branchial arches (visceral arches III, IV, and V). Those of the fourth branchial arch have not yet appeared.

Efferent branchial arteries. Also three on each side, connected by capillary loops with the afferent branchials.^

Radices aortae. The efferent branchial arteries on each side unite to form an aortic root.

^ H. M. ibower, "k Resumy of tii* Dsvdopmonts of Lymphatics and Associated Blood Vessels in Anuran Amphibia by the Method of Injectkm,” Wistar lostitate, 1939.

»The aSerent aad effemst branchial arteries are the representatives of the aortic arches III, IV, and V, generally devel^SQ^j^vutebraSs embryos.

20

ANATOMY OF FROG EMBRYOS

Dorad aorta. Formed by the union of the right and left radices aortae in the region of the pronephros.

Intemd carotid arteries. These are anterior prolongations of the radices aortae.

2. 0/ the trunk.

Glomus. This is a short side projection from the point where the radices aortae are fusing, bulging out into the coelom opposite each pronephros.

3. Of the tail.

Caudal artery. The direct continuation of the dorsal aorta.

c. Veins.

1. Splanchnic.

Vitelline (omphalomesenteric) veins. These arise on the ventral side of the thick-walled gut and pass forward around the liver to join the heart at the sinus venosus.

2. Somatic (cardinal system).

Anterior cardinal veins. These collect the blood from nu- ^ merous small tributaries in the head and convey it backward as far as the pronephros, where they turn and bend sharply downward to course along the anterior surface of that organ.

Posterior cardinal veins. These vessels course forward from the region of the cloaca ventral and lateral to the dorsal aorta, passing along the ventral surface of the pronephros until they meet and join the anterior cardinal veins.

Common cardinal veins (ducts of Cuvier). These vessels, formed by the union of the anterior and posterior cardinals on each side, course downward and enter the sinus venosus at points just dorsal to and lateral of the vitelline veins.

Caudal vein. This courses through the tail just ventral to the caudal artery. It divides into two branches which unite with the posterior cardinal veins on meeting the cloaca. Further details of the smaller arteries, veins, and lymphatics are omitted. The more advanced student is referred to the textbook and its references.

11 -MM. EMBRYO (Stage 2S, Opercuhm Ccanplete)

The embryo of Rana pipiens at the length of 11 mm. has assumed the familiar tadpole sbape. The head and trunk form an ovoid compressed dorsoventrally and the tail, compressed laterally, is twice the length of the body. The mouth is open and surrounded by homy raspers or oral combs. Tlie mucous glands have degenerated but persist as two small vestiges. The large eyes protrude sli^tly. The openings of the nose, external nares, are located anterior to the eyes. The external gills are covered by op^cular folds developed from the hyoid arches (visceral arch II). This fold is fused with the ectoderm of the body on the right side, but on the left an opening, the spiracle or <^>ercular I4»ertur8, p^eists. At the base of the tail is the vent or doacal aperture.

11-MM. EMBRYO

21

Laboraiory Directions

Make a drawing of the frog embryo from the left side.

Directions for Study of Transverse Sbrul Sections

The ll-mm. tadpole is a larval stage in the development of the frog. It is a free-swimming animal carrying on all the functions of animal life except that of reproduction. It is to be expected, therefore, that its internal anatomy will be more complex than that of the embryos studied heretofore. Accordingly drawings of certain organ systems are provided, based on wax reconstructions. The student is expected to trace the organ systems through the series of transverse sections, noting in the marginal blanks the number of the slide, row, and section in which each particular structure is well represented. It is suggested that the study be commenced by examining a typical section of the tail and comparing it in detail with the tail of the 6-mm. embryo. It will usually be easier to start at the region of the cloaca and trace all structures forward.

The ll-mm. Frog Embryo.

ECTODERMAL DERIVATIVES

I. BPiDiKMis. Nd longer ciHated except on the tail. Pigmented patches represent last vestiges of mu

cous c^da.

ANATOMY OP FROG EMBRYOS

n. SPINAL CORD AND BRAIK

a. Spinal cord. Extends forward until the roof thins.

b. Myelenceptudon. The cavity widens in more anterior sections. ' The thin roof is the primordium of the choroid plexus of the

fourth ventricle.

e. Metencephalon. Distinguished with difficulty from the myelencephalon. Located between the otic vesicles. At the anterior end, the roof thickens suddenly to become the primordium of the cerebellum.

d. Mesencephalon. Just anterior to the thickened primordium of the cerebellum, two swellings form the optic lobes (corpora bigemina) of the mesencephalon. The roof of the mesencepha> Ion expands widely anterior to the optic lobes. Its cavity is the mesocoel, iter, or aqueduct.

e. Diencephalon. Roof narrower than that of mesencephalon. Cavity called diacoel. Anterior boundary marked by epiphysis.

1. Epiphysis. Projects dorsally and anteriorly from roof of diencephalon.

2. Infundibulum. Broad evagination from floor of mesencephalon. Extends posteriorly beneath mesencephalon as far as anterior end of notochord.

3. Optic chiasma. A swelling on each ride of the of diencephalon anterior to infundibulum. Marks point of entrance!, of optic nerve. .

4. Optk recess. &nall evagination from floor of dioae^liidaBL Marks anterior botindaiy.

11-MM. EMBRYO

23

f. Telencephalon. Its posterior boundary is the optic recess. Rxwf thin . IVom roof a folded vascular membrane called the anterior choroid plexus hangs down into cavity known as telocoel. Telocoel and diacoel (see above) make up third ventricle.

1. Cerebral hemispheres. Formed by dorsal anteroposterior constriction from roof of telencephalon. Cavity of each called lateral ventricle.

m. CRANIAL NIBVBS

a. Associated with nose.

(/) Ophthalmic (olfactory). Prom ventral side of each cerebral hemisphere at its anterior end and passing directly to the mesial side of each nasal tube.

b. Associated with eye and listed in order from front to rear.

(//) Optic. From optic chiasma of diencephalon lateral and posterior to each eyeball. Can be seen passing through retina.

(III) Oculomotor. Very small. Emerges from floor of diencephalon on each side, just dorsal to infundibulum.

(IV) Trochlmr. Very small. Emerges from dorsolateral angle of metencephalon, just posterior to optic vesicles.

(VI) Ahducens. Very small. Emerges from floor of myelencephalon, just anterior and ventral to root of large trigeminal nerve (see below).

Notb; lliese three nerves supply the muscles of the eyeball. They are very small and require the highest power lenses of the student microscope.

c. Associated with ear and listed in order from front to rear 1. Just anterior to ear.

(V) Trigeminal. Large nerve with ganglion. Arises from ventrolateral angle of myelencephalon. Sends branches to maxillary and mandibular processes of first visceral arch.

' 2. Between ear and myelencephalon. dTlosely associated with

each other.

(VII) Facial. Arises from side of myelencephalon. Sends branches towards trigeminal and to hyomandibular arch. Ganglion.

(VIII) Acoustic. Arises with (posterior portion) facial. Supplies saccule and utricle of ear. Ganglion.

3. Just posterior to ear. Closely associated with each other. (/X) Glossopharyngeal. Arises with vagus but sends branches to first branchial arch. Ganglion.

(X) Vagus. Arises with glossopharyngeal but sends branches to second, third, and fourth branchial arches as well as to the viscera and to the lateral line organs. Note that the lateral line nerve is developed before the lateral line organs ap^ar.

XV. SPINAL oom AND NRRVS. Best examined in region posterior to pronephros. a. Central canal. Expanded dorsal and ventral to a constricted central portion.

ANATOMY OF FROG EMBRYOS

V. SENSE ORGANS OF THE HEAD

b. Ependymal layer. Layer of cells lining central canal.

c. Mantle layer. Thicker layer of cell bodies expanding on either side. Gray matter.

d. Margined layer. Outermost layer of axons. White matter.

e. Dorsal root. Thin bundle of afferent fibers, running ventral to join dorsal root ganglion.

f. Dorsal root ganglion. Elongate thick bundle of neurones on either aide of ventral half of spinal cord.

g. Ventral root. Thin bundle of efferent fibers, leaving ventrolateral angle of spinal cord to join nerve trunk.

h. Nerve trunk. Composed of efferent fibers from ventral root and afferent fibers from ganglion.

L Dorsal ramus. Branch from trunk to dorsal muscles.

j. Ventral ramtis. Branch from trunk to ventral muscles.

k. Communicating ramus. Passes around notochord to join sympathetic ganglion.

l. Sympathetic ganglion. A small mass of ganglion cells on either side of dorsal aorta. Not a part of the spinal nerve but associated with it.

a. Ear. Now partially divided into two regions.

1. Utricle. Mesial and dorsal portion.

Semicircular canals. Two are present : anterior dorsoventral canal, and outer horizontal canal. (The third, posterior dorsoventral has not developed.)

2. Saccule.

Endolymphatic duct. Posterior to level at which anterior semicircular canal joins utricle. Between utricle and brain. Joins saccule. a

Cochlea. Heavily pigmented and ciliated area on inner ventral angle of saccule.

b. Eye.

• 1. Sensory layer of retina. Rods and cones, inner .and outer granular layers and ganglion layer can be distinguished with high magnification.

2. Pigmented layer. Still characterized by heavy pigmentation.

3. Lens. Solid, lying in mouth of optic cup.

4. Choroid. Thin vascular layer {formed from mesenchyme).

5. Sclera. Layer of mesenchyme investing choroid.

6. Cornea. Continuation of sclera between lens and dkin.

7. Eyeball muscles. Small masses of muscle between eye and brain.

c. Nose. The nasal pits have grown back to open into pharynx.

1. External nares. Open to exterior.

2. Interned nares. Open to pharynx.

3. Sensory epithelium. On side of tube nearest brain.

11-MM. EMBRYO

25

ElfDODERMAL DERIVATIVES

The endodermal tube now opens to the exterior at both ends, by means of the ectodermal stomodeum and proctodeum, respectively. The boundaries between ectoderm and endoderm at these points cannot be traced. Commence at the mouth and trace the digestive tube and its derivatives back to the cloacal opening.

a. Mouth. Extends from the oral aperture to the point where the internal nares mark the entrance of the nasal tubes.

. b. Pharynx. The tube now flattens dorsoventrally to form the

pharsmx.

c. Visceral {branchial) clefts. The pharynx opens to the exterior at four points on each side where the visceral pouches (II, III, IV, and V) have opened into the corresponding branchial grooves. Pouch VI is vestigial and does not make contact with the exterior. The clefts do not open directly to the exterior but ventrally into the opercular cavity. The external gills are now degenerated and will be replaced by internal gills.

Fio. 12. Endodermal derivatives of 11-mm. J2ana pipicnt embryo. From a wax plate reconstruction representing a cast of the digestive tract. Dorsal view.

Thyroid gland. Beneath the floor of the pharynx, and completely separated from it, the thyroid gland is now dividing at the posterior end. It is pigmented and located just anterior to the heart.

e. Thymus glands. Those from the first visceral pouch have disappeared; those from the second visceral pouch have separated from the dorsal end of the pouch. They are not easily to be identified and may be omitted.

26

ANATOMY OF FROG EMBRYOS

f. Parathyroid glands (epithdioid bodies^. Small masees of tissue formed on the ventral ends of the third and fourth visceral pouches. Their identihcation may be omitted.

g. Ultimobranchial bodies. Small pigmented bodies on the ventral side of the sixth visceral pouch, at the level of the trachea. Identification may be omitted.

h. Trachea and limgs. A slitlike dorsoventral evagination from the ventral side of the pharynx in the region of the visceral clefts. This is the laryngotracheal groove. After passing a few sections this groove separates from the oesophagus above, becomes a tube, and sends out a branch, at sharp right angles to either side. These branches are the bronchi. As each bronchus proceeds backward its walls become highly vascular except at the growing point.

i. Oesophagus. From the region of the laryngotracheal groove the oesophagus narrows rapidly and bends to the right at a level behind the liver to enter the stomach.

j. Stomach. Easily recognized by its wrinkled lining. The stomach may be traced forward and dorsally until it curves backward to join the duodenum,

k. Duodenum. From the pyloric end of the stomach, the narrow duodenum crosses over the oesophagus, swings to the left under the hind-gut to join the coiled intestine.

l. Liver. This large gland is now on the right side of the coelom. It has grown around the vitelline veins and is highly vascularized. On the dorsal side is the original liver diverticulum, now the gall bladder. From the posterior end of the gall bladder the common bile duct (ductus choledochus) proceeds through the mesentery connecting liver and stomach until it enters the duodenum on its anterior surface.

m. Pancreas. This gland is found alongside the inner curvature of the stomach. The pancreatic duct joins the common bile duct shortly before the latter enters the duodenum.

n. Intestine. Following the duodenum, the intestine increases greatly in diameter, and forms two complete spiral coils ventrally. It then ascends as a much smaller tube coiling within the larger spirals just described. On approaching the oesophagus it bends sharply backward, rapidly decreases in diameter, and slowly bends downward until it joins the proctodeum. There is no cloacal swelling. Shortly after crossing the duoden num the intestine receives the fused ends of the pronephrio ducts.

MEiSOnEaUlAL DBSBIVATIVSS

I. MJESENCHTMB. In the head region condensations of mesenchyme have laid do^ the cranium in which cartilage-forming (chondrogenous) centers have appeared. These give rise to. Ute neurocranium. (See page 31.) Ckomeetive tissue of various kinds the Uood. ftod lymph Vtotoli.' ' of the head are also formed from the head mesenchyme. In the tailfin, InescSidhyme isabo "

11-MM. EMBRYO

27

great amount. Elsewhere in the body mesenchyme fills in the spaces between organs not occupied by body cavities.

II. NOTOCHORD. Still large and conspicuous. When examined with high magnification a thin layer of mesenchyme cells (from sclerotome) may be found around it, and continuing up around the spinal cord. This is the beginning of the axial skeleton, which lags behind the cranial skeleton.

III. SOMITES. The most anterior somites are disappearing. Specimens may have eleven or twelve in the trunk, and a much larger number (thirty to thirty-five) in the tail.

a. Dermatome. This region of the somite is now broken up into mesenchymatous cells which have made their way to the ectoderm of the dorsal half of the embryo where they will form the dermis (cutis) of the skin. This layer apparently arises from somatic mesoderm in the ventral portion of the body.

b. Sclerotome. Now forms the mesenchymatous zone, from which the axial skeleton will develop.

c. Myotome. The remainder of the somite is now converted into muscle bundles lying close to the notochord. In the tail region each is divided into a dorsal and a ventral bundle.

IV. INTERMEDIATE MESODERM

a. Pronephros. Greatly enlarged owing to the coiling of its tubules. The nephrostomes are ciliated.

Fm. 13. Excretory qratem of ll-mm. Rana ptjnent embryo. From a wax plate reconstruction. Ventral view.

b, Pronephric ducts. May be traced back on either side of the dorsal aorta and dorsal to the posterior cardinals until they unite. The fused portion bends ventrally and anteriorly to join the narrow cloaca.

ANATOMY OF FROG EMBRYOS

c. Mesonephros. Has not yet commenced its development.

d. Gonad. Not yet developed. At the level of the pancreas it is possible to find with high magnifi cation primordial germ cells. They are located in the roof of the coelom ventral to the posterior cardinal veins an^i in the dorsal mesentery at its junction with the coelomic roof.

V. LATERAL MESODERM. Somatic and splanchnic layers are closely apposed to the ectoderm of the ventral body wall and the endoderm of the viscera, respectively.

a. Coelom. Three regions may be distinguished.

1. Pericardial cavity. Still connects with the abdominal cavity posterior to it.

2. Pronephric chamber. In the process of being separated from the abdominal cavity beneath it by the growth of the lungs.

3. Abdominal cavity. Does not extend behind cloaca.

' b. Mesenteries. The dorsal mesentery is very thin but its two

layers are apparent at high magnification. The ventral mesentery is represented only by the thin sheet between stomach and liver (gastrohepatic omentum).

Vl. CIRCULATORT SYSTEM, easily distinguished.

Consists of heart, arteries, veins, and lymphatics. Red blood corpuscles now

a. Heart. Beneath pharynx. Commence at anterior end.

1. Bulbus arteriosm. First section of heart encountered.

2. Truncus arteriosus. Opens dorsally from bulbus. A short vertical vessel.

3. Ventricle. Thick walled and muscular, the most ventral of the two cavities seen in the same section. Communicates with bulbus arteriosus.

4. Atria, lie dorsal to ventricle. Partially separated into right and left atria by interatrial septum. Both conununicate with ventricle through common * atrioventricular foramen. Left atrium receives the pulmonary vein (see Fig. 14) on dorsal posterior surface.

5. Sinus venosus. Most posterior region of heart. Communicates with right atrium. Receives common cardinal veins (see Fig. 14) at extreme right and left angles. Receives postcaval vein from liver (see Fig. 14) ventral to right common cardinal vein.

b. Arteries. Only the main arteries are to be identified.

1. Afferent branchial arteries. Commence at level of truncus arteriosus. This vessel divides into left and right branches. Each brandi divides into two portions. The anterior portion swings forward and laterally and then divides to form the first and second afferent branchial arteries. The second branch swings laterally and divides to form the thikl and fourth branchial arteries. Prom the first branchial arteay, the external carotid (lingaal) artery passes forward to the lotier jaw. ;

ll-MM. EMBRYO

2. Efferent branchial arteries. Locate the dorsal aorta at a level just posterior to the liver. Running forward it divides into the two * radices aortae (aortic roots), which run forward at the lateral margins of the pharynx. Where the pharynx begins to widen each root is joined by the fourth efferent branchial artery. From the fourth efferent branchial artery on each side a pulmonary artery grows out under the phar3Tix to supply its lung, and a cutaneous artery makes its way to the skin.

Donal MTU — ^

Just anterior to the point where the fourth efferent branchial joins the aortic root, the third efferent branchial can be found. Anterior to the point where the third efferent branchial artery joins it, each root moves towards the midline, dorsal to the pharynx. They are then joined by large vessels, the second and first efferent branchial arteries, respectively. Anterior to the point where the first efferent branchial artery joins it, each root continues anteriorly and dorsally on either side of the base of the brain, to become an internal carotid artery. These arteries connect by a commissure beneath the infundibulum of the brain and divide into several smaller vessels of the head.

. Dorsal aorta. FVom junction of aortic roots backward until it enters the tail as the caudal artery. Gives off numerous mtersegmental arteries in its course.

ANATOMY OF FROG EMBRYOS

4. Olomm. A vascular mass of tissue, supplied by branches from aortic roots anterior to their point of junction. Suspended in pronephric chamber median to pronephros on each side.

6. Mesenteric artery. Large vessel leaving dorsal aorta on ventral side and proceeding down dorsal mesentery to supply intestine.

c. Veins. Usually have thinner walls than those of arteries.

1. Hepatic-portal system. Formed from vitelline veins after the liver has grown around them.

Hepatic veins. Originally from liver to sinus venosus. They are now connected with the postcaval vein, a very large vessel (see below). From this stage on, the name hepatic veins is applied to the vessels inside the liver, whereas the portion from liver to sinus venosus is called the postcaval vein.

Portal vein. Branches from pancreas, stomach, and intestine unite to enter liver at posterior surface.

2. Cardinal system. The posterior cardinals now in process of transformation to postcaval.

Posterior cardinal veins. Best studied by commencing with caudal artery in tail. This divides into two vessels which join posterior cardinals at level where pronephric duct enters cloaca. They run forward ventral to dorsal aorta and mesial to pronephric ducts. In some specimens, a little further developed, these veins fuse.

Postcaval vein (posterior vena cava). At the level where the mesenteric artery leaves the dorsal aorta, this large vein enters the dorsal surface of the liver. It can be traced back to right posterior caval vein, which it joins. It runs forward through the liver to enter sinus venosus ventral to right common cardinal vein.

Note: Anterior to point where postcaval vein unites widi right common cardinal vein, the cardinal vans become much reduced and can be seen only with high magnification. They follow the pronephric duct of each side to enter the pronephric einue, a saclike structure partially enveloping each pronephros.

Common cardinal veins (ducts of Cuvier). Drain pro nephric sinus. Descend vertically across front surface of pronephroi to enter sinus venosus at posterior lateral angles.

Anterwr cardinal veins (superior fuyular). Join common cardinals at their most dorsal level. Accompany amtie roots into head. Finally take portion dorsal and lateral to internal carotid artery. .

Inferior jugular veins. Join commtm cardinals at th^ most ventral levds. Run finrwaid on each skile of het^. Accompany external tsvotid arteiiee to lowQ>J|aw.

• 11-MM. EMBRYO

31

d. Lymphatic system. Difficult to trace without assistance of injected specimens.

Spleen. Thickening in wall of dorsal mesentery alongside mesenteric artery. Represents lodgment of lymph cells which will later develop into spleen.

VII. CHONUBOCBANiUM. The plan of the embryonic skull is laid down in connective tissue under the brain, around the sense organs, and in the visceral arches. In this embryonic stage many cartilage centers, easily recognizable by the large cells with their relatively clear cytoplasm, may be identified. The point of departure is the notochord, at the level of the anterior margin of the otic capsule, which is still in the precartilage stage, but will later form a cartilaginous capsule around the inner ear.

LOWER JAW, HYOID. SKULL AND UPPER JAW

Fw. 16. Chondrocranium of ll-mm. Ram pipiens embryo. From a graphic reconstruction . Dorsal view. Neurocranimn outlined in unbroken lines; splanchnocTunium in dotted line-s. (Note: Ptergygoquadrate, although Tunned from the first visceral arcli, is outlined in unbroken lines because it is now fused with the neurocranium.

a. Neurocranium. Around brain and sense organs.

1. Parachordals. On either side of the notochord at its anterior end.

2. BasUar plate. Immediately in front of the notochord, united with the parachordal on each side.

3. Trabeculae. These bars of cartilage swing wide to right or left and then extend anteriorly on either side of basicranial fontanelle.

4. The basicranud fontanelle. This cavity is the seat of the pituitary gland.

6. Pterygoquadrate (palatoguadrate). Joins posterior end of the trabecula by its posterior ascending process, swings ventrally and to the side, and continues forward. This cartilage is formed from the maxillary portion of the first visceral arch ^ but is mentioned here because of its union with the trabecula.

" At a level in front of the eye is the vertical muscular process

ANATOMY OF FROG EMBRYOS

for the attachment of jaw muscles. At the anterior end it swings toward the median line and rejoins the trabecula by its anterior ascending process.

6. Ethmoid (intemasal) plate. Formed by the union of the trabeculae in the median line. Just anterior to the connection between trabecula and anterior ascending process of the pterygoquadrate.

7- Cornua trabecularum (trabecular horns). Two bars which diverge at the anterior end of the ethmoid plate and continue towards the anterior end of the head, accompanying the nasal tubes.

8. Labial (suprarostral) cartilages. Two short cartilages in the upper lip, one on each side of the mouth. They meet the cornua trabecularum.

b. Splanchnocranium. Formed in visceral arches. Follow from

front to rear.

1. Mental (infrarostral) cartilages. In the lower lip on either side of the mouth. Meet in the mid-line at their posterior ends.

2. Meckelian cartilages. On the dorsal side of each mental car tilage and posterior to it. These rapidly extend to the side where muscles can be seen connecting them with the muscular process of the pterygoquadrate.

3. Ceratohyal. Several sections posterior to the last appearance of the meckelian cartilage. This large cartilage is formed in the hyomandibular arch.

4. Basibranchial (copula). An unpaired cartilage between the two oeratohyals. The basihyal (anterior to the basibranchial) has not yet developed cartilage. Its position may be recognized by the U-shaped thyroid gland which lies beneath it.

5. Hypobranchitds. Posterior to each basibranchial and cerato hyal and articulating with each. A platelike cartilage formed in the third visceral arch.

6. Ceratobranchials. Extending back from the hypobranchials as four slender processes. The most lateral of these ceratobranchials, that is, the one in the third visceral arch, is the first to be noticed as one goes through the sections from front to rear. The second is in the fourth visceral, third in the fifth visceral, and fourth in the sixth visceral arch. The ceratobranchials have not yet appeared in the dorsal pcwtions of the arches.