Development of Olfactory Organ in Rana nigromaculata

Since Goette’s classical paper on the development of the toad (Goette, 1875) there have appeared many accounts of the ontogeny of the olfactory organ of Amphibia. The most thorOugh-going work has been that of Hinsberg (1901), who followed very closely the early developmental steps but not the later ones. In the developmental history of the amphibian nasal organ two points in particular have arrested the attention of students of comparative embryology and caused a good deal of controversy. One is the existence of the oro-nasal groove, the other is the location of the choanal opening in the oral cavity. Kurepina (1931) claimed that there is present in the amphibian larva an oro-nasal groove, comparable to the nasal groove in embryos of the Amniota, while Hinsberg (1901), Kawagae (1933), and Schneider (1935) were unable to find such a groove. Kurepina also found that the choana opens into the ectodermal part of the oral cavity as it does in all Amniota. In this he was supported by Watanabe (1936). On the other hand, Goette (1875), Hinsberg (1901), and others (Fahrenholz, 1925; Kawagae, 1933; Schneider, 1935) contended that the choanal opening is in the endoderm. However, neither side has given conclusive evidence.

Rana nigromaculata Hallowell is a common frog in North China. Its olfactory organ has not been investigated. The aim of the present paper is to make a thorough and complete study of the development of that organ and to see if fresh light can be thrown on those controversial points mentioned above.

The present investigation was conducted in the Biology Department, the National University of Peking, Peiping. When the wo rk was nearly completed, it was interrupted by the Japanese invasion and was finally completed in Kunming.

Rana nigromaculata is very abundant in Peiping. One can get its eggs and tadpoles in ponds, pools, and rice fields from April to July. A complete series of stages, ranging from larvae 2 mm. long to young frogs was collected. Identification was based on descriptions in Boring, Liu, and Chou (1932, pp. 3949). The body-length was measured and recorded. One-half of the larvae of the same stage was preserved in 10 per cent, formalin for gross observation and comparison with sections. The other half was fixed in Spuler’s fluid (Muller’s solution, 70 c.c., saturated sublimate solution, 30 c.c., concentrated formalin, 10 c.c., and glacial acetic acid, 2 c.c.) or in a mixture of acetic acid and sublimate (saturated sublimate solution, 35 c.c. and glacial acetic acid, 20 c.c.). Each fixative was prepared immediately before use. The fixed material was imbedded in paraffin. Both cross and sagittal serial sections were cut 5-9μ. in thickness and stained with Delafield’s haematoxylin or a combination of Delafield’s haematoxylin and eosin.

It was necessary to employ reconstructions. For this purpose I tried to use the blotting-paper method described in Gage (1936, pp. 511-20) instead of the wax-plate method. But I had trouble in cutting. I then tried to infiltrate a piece of blotting paper with beeswax which made the paper much firmer. The cutting was very easily done with the knife used by Chinese wood-carvers. The method is as follows:

A sheet of blotting paper (48×61 cm.) was cut into three strips and immersed in a bath of melted beeswax, to 100 parts of which 5 parts of paraffin were added. After two minutes they were taken out of the bath and both sides of the paper were quickly wiped with a piece of cloth in order to make the waxed paper even and smooth. Every strip of the waxed paper was cut again into five. To determine the thickness of the waxed paper, every piece was measured in 8 different places with a micrometer caliper. The mean of the 120 readings (15×8) was taken to be the correct thickness. The most suitable thickness for cutting a complicated model was 0·4 to 0·5 mm. Thinner pieces would be, of course, easier to cut, but involved more work. Paper thicker than 0·5 mm. was employed only for making simple and large models. For sections cut 8μ. thick, paper 0·4 mm. in thickness was just right for magnifying them 50 × ; two thicknesses of paper for 100×.

Serial drawings were made on the waxed blotting paper with a fine needle under an Edinger drawing machine or a ‘Panphot’ (manufactured by Leitz). It was found easier, however, to make sketches on a piece of blotting paper with a sharp pencil before infiltration with wax. Further treatment is just the same as in the wax-plate method.

Models constructed with wax-infiltrated paper are very strong. This turned out to be very fortunate. Since the Sino-Japanese war, the models together with other materials had to be hurriedly shipped South. On the way they were subjected to rough handling and subtropical heat. If the models had been made of wax, they would have been hopelessly ruined. My models arrived, however, in perfectly good condition.

The course of development of the olfactory cavities may be conveniently divided into four stages.

From the Inception of the Olfactory Anlage to the Formation of the Olfactory Placode (length of larva, 2·5-5 mm.).—When the neural groove just closes to form a tube, the ectoderm of the head of the larva shows clearly the surface and the sensory layer (Goette, 1875; Corning, 1899). The larva at this stage is about 2·5 mm. long. The anlage of the olfactory placode arises as a pair of thickenings from the sensory layer of the ectoderm at the top of the head and in front of the optic vesicle. When strongly magnified, the external surface of this anlage can be barely made out as a slight rounded depression (Text-fig. 6). A larva of about 2×8 mm. in length shows the primitive placode definitely depressed to form the olfactory pit, which can be already seen under 24 × magnification (Text-fig. 1). Hinsberg (1901) working on Ban a fuse a stated that the olfactory pit appears only when the larva reaches 5 mm. in length. Innigromaculatait appears then much earlier. No further changes could be observed at this stage except the increase in dimension of the placode, and that with the enlargement of the head at the brain region the placode which first appeared dorsally is displaced ventrally. Hinsberg (1901) discussed at length the question, whether the surface layer of ectoderm takes part in the formation of olfactory placode and concludes that in fusca the placode is derived solely from the sensory layer. My findings in Rana nigromaculata completely support those of Hinsberg.

In the head of the larva of Rana nigromaculata there is also a forehead streak, the ‘Stimstreifen’ of Hinsberg (1901). It will be dealt with in a separate paper, for it has nothing to do with the development of the olfactory organ.

Fresh specimens, serial sections and models of the head in the first and later stages were examined, but no trace of the oro-nasal groove of Kurepina (1931) was found.

(a) Formation of the Nasal Lamina (length of larva, 5-9·5 mm.).—The olfactory pit remains a shallow depression as long as the lens is still attached to the sensory layer of the ectoderm. The formation of the nasal lumina synchronizes with the separation of the lens from the sensory

(Text-figs. 6-12 drawn with the aid of a camera lucida and magnified 86 ×.)

Antlos, anterior lower sac; Cavinf, cavum inferius; Cavmed, cavum medium; Cavprinc, cavum principale; Cho, choana; Cle, clefts; Cons, constriction; Dim, diencephalon; Dori, dorsal lumen; Det, ectoderm; Ent, endoderm; Entrcan, entrance canal; Epi, epiphysis; Ethch, ethmoidal part of chondrocranium; Extnar, external naris; Hypo, hypophysis; Inf, infundibulum of nasal cavity; Inf’, infundibulum of brain; Latap, lateral appendix; Latgl, lateral nasal gland; Latgr, lateral groove; Latp, lateral portion; Llid, lower eyelid; Llip, lower lip; Medgl, medial nasal gland; Medp, medial portion; Mes, mesencephalon; Met, metencephalon; Midl, middle lumen; Nar, nasal rim; Nasduct, naso-lacrymal duct; Nasduct’, external opening of naso-lacrymal duct; Noto, notochord; Olfp, olfactory pit; Olfpl, olfactory placode; Opines, optic vesicle; Orca, oral cavity; Or ep, oral epithelium; Palep, palate epithelium; Phagl, pharyngeal gland; Pham, pharyngeal membrane; Phar, pharynx; Postlos, posterior lower sac; Postpro, posterior prolongation; Pricho, primitive choana; Proj, projection; Pros, prosencephalon; Recalar, recessus alaris; Reclat, recessus lateralis; Recmed, recessus medialis; Recsac, recessus saccj ormis; Serilay, sensory layer; Sic, skin; Surlay, surface layer; Suplatgr, super-lateral groove; Uplip, upper lip; Ups, upper sac; Venl, ventral lumen.

layer. The nasal pit extends inward and upward to form a tube, the enlarged end of which forms the dorsal lumen (Text-fig. 7).

After the formation of the dorsal lumen, the neighbouring cells behind it are immediately differentiated to form a blind sac, which is called the lateral appendix. At the time of its formation it lies just at the dorso-lateral region of the placode (Text-fig. 8). Later, with the more rapid expansion of the placode to the dorsal and medial side, the lateral appendix is gradually shifted latero-posteriorly and a marked constriction appears between it and the placode (Text-figs. 10 and 18). The part of the placode posterior to the lateral appendix is again differentiated into two portions, a thin lateral one and a thick medial one. Between them there originate several minute clefts (Text-fig. 8). These are at first discontinuous but later communicate with one another to form the ventral lumen posterior to the olfactory pit (Text-fig. 10). Contemporaneous with the appearance of the clefts, the olfactory pit begins to extend inward and gives rise to another lumen, the middle lumen, which is immediately anterior to the dorsal lumen (Text-fig. 9) and runs ventro-posteriorly. Its medial wall is derived from the olfactory placode, while its lateral wall is from inward extension of the skin (Text-fig. 10). It communicates with the dorsal lumen from the very beginning (the larva at this time is 7 mm. in length) and with the ventral one when the clefts run together to form a whole lumen (the length of the larva now is 8·5 mm.). Between the middle and ventral lumen there is a small inward projection, which is particularly plain in nigromaculata (Text-figs. 10 and 14). This projection serves at this time as a boundary between the middle and ventral lumen. Its subsequent development will be described later.

(b) Formation of the Primitive Choana (length of larva 5·5-9·5 mm.).—The formation of the primitive choana in the oral cavity is closely related to the formation and rupture of the pharyngeal membrane. The latter is formed by fusion of the intruded ectoderm and the protruded endoderm. As the brain expands, the forehead is thrust forward and downward, so that the pharyngeal membrane is then shifted backward (Text-fig. 11). At each side of the pharyngeal membrane the two embryonic layers meet to form the lateral walls of the primitive oral cavity. The method of fusion of these two layers is somewhat peculiar. The endoderm protrudes above, while the ectoderm intrudes below (Text-fig. 13). As the result of this kind of fusion, the meeting line of the two layers is not vertical but runs obliquely from the dorso-anterior towards the ventro-posterior (Text-figs. 11 and 14). Therefore, in the roof of the oral cavity the endoderm extends much more anteriorly than it does in the floor (Text-figs. 12 and 14). By the difference in microscopic structure one can easily distinguish the two layers. The ectoderm in the oral cavity is thicker and in a given area contains many more cells, the nuclei of which are crowded together. The endoderm is a thin layer; its cells are not so crowded and contain many yolk-granules. This layer takes the stain less deeply (Text-fig. 12).

The olfactory placode in the 5-5 mm. larva shows a small prolongation at its most posterior end. This prolongation extends ventro-posteriorly towards the oral cavity (Text-fig. 13). The tip of it is later thrust into the endodermal part of the oral cavity at the point where the lateral wall meets the roof (Textfigs. 12 and 14). This occurs just after the rupture of the pharyngeal membrane. At this stage, the ventral nasal lumen is still represented by the discontinuous clefts (p. 66). After these clefts have run together to form the ventral lumen, it extends into the prolongation and opens at its distal end into the oral cavity. This distal opening is the primitive choana. Since the tip of the prolongation grows into the portion of the oral cavity lined by endoderm, the choana opens definitely into the endoderm.

The choana possesses medial extension on the palate in the form of a transverse groove (Text-fig. 16). The roof of this groove is derived from the posterior prolongation of the olfactory placode, while the walls flanking it consist of the mixture of the placodal and endodermal tissues.

Formation of Blind Sacs and Changes of the External Naris and the Primitive Choana (length of larva, 9·5-50 mm.).—Up to the end of the second stage the olfactory placode with its himina is more or less straight (Text-fig. 17). This simple structure undergoes profound changes to become a very complicated organ.

The embryonic olfactory organ as it appears at the end of the third stage may be divided according to natural demarcations into three blind sacs. Using the designations of Hinsberg (1901) the three sacs are called the upper sac, the anterior lower sac, and the posterior lower sac (Text-figs. 21 and 22). The upper sac includes the external naris and contains the middle nasal lumen. At first, it is the largest sac (Text-fig. 17). The anterior lower sac is a small one situated ventro-anteriorly to the upper sac. The posterior lower sac is under the upper sac. It contains the ventral lumen and includes the choana. The three sacs arise in the following manner.

The part of the olfactory placode around the middle nasal lumen enlarges and the lumen expands along with it, thus bringing about the formation of the upper sac. Contemporaneous with the coming into being of the upper sac, the placodal region around the ventral lumen together with the posterior prolongation and the primitive choana (into which the ventral lumen opens) enlarges and the whole complex now becomes the posterior lower sac. At the beginning it is not marked off from the upper sac (Text-fig. 17).

It will be recalled that between the middle and ventral lumen there is an inward projection (p. 66). As the upper and posterior lower sacs are being formed, this inward projection also enlarges. When viewed from the outside of the olfactory organ at this stage, this inward projection is a small depression (Textfig. 17). Following the expansion of the upper and the posterior lower sacs this depression increases both in length and in depth. This structure I designate as the lateral groove. It marks off the upper sac on the outer side from the posterior lower sac (Text-fig. 18). In the medial region the upper sac projects out and hangs over the latter, though on that side there is no definite groove to mark them off (Text-fig. 18).

The anterior lower sac is simply an evaginated part of the ventro-anterior region of the upper sac (Text-fig. 17). It protrades forward under the upper sac. In the early stage, this sac looks somewhat like a flattened egg.

After the formation of the three blind sacs, the olfactory organ undergoes the following changes.

The head as a whole increases more rapidly in size than the olfactory organ. As a consequence there is now intercalated between the external naris and the middle lumen (which were previously in close contact) a canal formed by the invaginated skin inside the naris. This canal is the entrance canal (Textfig. 17). The surrounding skin of the external naris is raised to form a rim around it (Text-fig. 17). This rim exists up to the time when the larva is 18 mm. long; then it begins to disappear.

The larva of 25 mm. in length shows no trace of such a rim around the nasal opening.

After the disappearance of the rim the entrance canal dilates (Text-fig. 18). The upper part of it extends dorso-Iaterally and constitutes a small sac (Text-fig. 20, x) which in the next developmental stage gives rise to several small structures (see p. 83). The lower part extends in the opposite direction—to the ventro-medial—to form another blind sac which is thrust between the upper and anterior lower sac and becomes the cavum medium in adult (Text-fig. 20). Concomitant with the above changes the external naris is shifting sideways so that the two nasal openings which were close together become at the end of this stage wide apart (Text-fig. 21).

The lateral appendix, as described above (p. 66), is the earliest developed blind sac. It increases very rapidly at the beginning and reaches the highest development at the end of the second stage. It then remains stationary. Upon the development of the upper sac it is gradually shifted to the lateral and posterior (Text-fig. 18). At this time it starts also to degenerate, becoming increasingly smaller (Text-figs. 19 and 20) until it is obliterated by the end of the metamorphosis. It is remarkable that the lateral appendix, which differentiates very early and, for a time, is the only blind sac in the early larval olfactory organ, should completely disappear in the course of further development. Watanabe (1936) is of the opini’ n that it may be a sense organ for the larval life, but he gives no evidence to support his conjecture.

At the beginning of the disappearance of the lateral appendix the posterior part of the upper sac which is heretofore continuous with the posterior lower sac (Text-fig. 18) becomes elevated and marked off from the lower one (Text-figs. 19, 20, and 21).

Just above the lateral groove and under the lateral appendix there arises now another groove which extends anteriorly upto the proximal end of the anterior lower sac (Text-figs. 19 and 20). I call it the super-lateral groove, as it is above that structure. The part of the lateral wall of the upper sac between these two grooves is now separated from the rest of the wall and becomes a ledge overhanging the lateral wall of the posterior lower sac. It becomes the recessus lateralis in the adult (Textfigs. 19 and 20).

The anterior lower sac now increases and extends more anteriorly than in other directions, while the upper sac extends in all directions but very little anteriorly. The result is that the former sac lies far to the front at the end of this stage (Text-figs. 20, 21, and 22). In the larval stage its communication is always with the middle lumen of the upper sac. In the adult it becomes connected with the recessus lateralis, which is a derivative of the upper sac. These changes will be described in the fourth stage.

As pointed out in a preceding paragraph, the posterior lower sac is not fully separated from the upper sac until the end of the present stage. It expands rapidly, so that at the end of this stage it outstrips the upper sac in size and becomes the largest sac of the time. The expansive growth is far greater at the posterior part, and its form at the end of this stage is roughly a triangular pyramid with its apex directed anteriorly (Textfig. 22).

With the expansion of the posterior lower sac, the primitive choana also enlarges until the narrow groove becomes quite a large irregular opening (Text-fig. 22). The projecting side walls of the former groove persist for a time, but, towards the beginning of the fourth stage, completely disappear so that the edge of the choana is now perfectly smooth (Text-fig. 25).

From the Beginning of the Metamorphosis to the Adult (from larva of 50 mm. in length to adult frog).—When the tadpole reaches 50 mm. in length, its tail begins to be resorbed. There occurs a great change in the nasal cavity. It now undergoes simplification in outline and, at first, noticeable shrinkage in size. This decrease in size is understandable when we compare the head of the tadpole just before metamorphosis with the young frog. The head of the tadpole is much larger and has a rounded outline about the mouth (Text-fig. 2 A). After metamorphosis the head has shrunken in all dimensions and has a triangular outline (Text-fig. 2 B). The change in the size of the head is graphically represented in the Text-fig. 3.

The shrinkage of the head is evidently related to the emergence of the animal from water, which is probably accompanied by dehydration. The connective tissue in the head was very loose in the larva, but becomes compact in the young frog. The simultaneous decrease in the size of the olfactory organ may also be due to the loss of water from its tissues. This point cannot be established histologically, however, since measurements of the cells of the organ before and after metamorphosis do not reveal any shrinkage in their size.

The entire nasal cavity appears also to be shifted forward after metamorphosis and the nostrils are now at the very tip of the head (Text-fig. 2 A and B). This is brought about by the fact that the upper lip now completely folds under as the mouth widens.

The shape of the olfactory organ at the end of the last stage is very irregular and the three sacs are distinct from one another (Text-figs. 21 and 22). During metamorphosis the furrows separating the upper and posterior lower sacs along the medial and posterior region (Text-fig. 21) become smoothed out as a result of distension of the sacs. The elevated posterior part of the upper sac and the lateral appendix lose their identity and the whole of the upper sac and the main portion of the posterior lower sac merge into one large cavity, designated as the chief cavity or cavum principale in the mature organ (Text figs. 24 and 25). It is flattened dorso-ventrally, shifts medially, and also extends far anteriorly over the anterior lower sac (Text-fig. 25).

The super-lateral groove separating the former upper sac and the posterior lower sac at their lateral parts remains, how-ever. The whole lateral wall of the posterior lower sac extends lateralwards carrying the ledge along with it, forms a side extension of the cavum principale, and is designated as recessus lateralis (Text-figs. 24 and 26).

The anterior lower sac lies now completely under the cavum principale. The main part of it together with the foremost extension of the recessus lateralis, which projects until it is level with the anterior lower sac, constitutes the cavum inferius of the mature organ (Text-figs. 23 and 25), while the medioposterior portion of the sac is called recessus medialis (Textfigs. 25 and 27).

The small sac derived from the upper part of the entrance canal, and situated under the external naris (Text-fig. 20, x) expands to form the anlagen of the recessus alaris, infundibulum and recessus sacciformis (Text-fig. 23). The cavum medium (p. 78) is then shifted to the lateral under the infundibulum (Text-fig. 23) instead of its original position. The external naris enlarges and along with the cavum principale extends forward (Text-figs. 24 and 26).

The olfactory organ increases in size along with the growth of the head. In a later stage it is still further flattened out dorso-ventrally. The cavum principale at its hind portion stretches medially and the recessus lateralis extends further laterally. The choana becomes greatly appressed (Text-fig. 27). It is now relatively large, but in the mature organ its relative size to that of the nasal cavity is much smaller (Tsui, 1985, fig-6).

From this point on, the structure of the nasal cavity undergoes no important change. In the adult stage, the olfactory organ of Rana nigromaculata is essentially the same as that of fusca and esculenta (Tsui, 1935). Itis, therefore,unnecessary to carry the description farther.

Extra Blind Sac s.—Extra blind sacs have been occasionally found in the nasal cavity. In three cases such sacs were observed—all in the stage when the cartilaginous nasal capsule is already formed. In one specimen an invaginated sac is present in the cavum principale (Text-fig. 4). In the other two, one or two small folds occur in the medio-anterior part of that cavity (Text-fig. 5). All these extra pouches are symmetrically present in both nasal cavities. The formation of these unusual structures is perhaps due to the fact that the cells in certain regions of the olfactory epithelium increase abnormally. On account of the presence of the cartilaginous nasal capsule, the epithelium cannot extend freely and, as a result, a blind sac is formed. An undetermined blind sac was reported in fusca (Tsui, 1935). It was probably formed in the same way.

1. Naso-lacrymal Ducts.—The development of the naso-lacrymal duct is very sudden. It arises in the dorso-lateral part of the cavum principals behind the external naris (Textfig. 24). Due to the formation of the recessus sacciformis it is pushed farther laterally and is connected with the latero-posterior end of the cavum medium (Text-fig. 26). It lengthens posteriorly, the distal end of the duct bifurcates, and the two branches open into the lower eyelid (Text-fig. 26).

According to Born (1876), this duct in the amphibians he examined is first solid and hollows out to form a duct. Transverse serial sections were made of the duct of the present species. It was found that the proximal end of this duct is at first solid. The duct lengthens very rapidly and when it reaches the eyelid, it is already hollow along most of its length. Finally the proximal end also becomes hollow. The formation of the lumen seems to begin from the distal end.

2. Lateral Nasal Gland.—At the same level as, and medially to, the naso-lacrymal duct there arises the lateral nasal gland. It is a small diverticulum (Text-figs. 24 and 26). Soon it increases in size, and branches laterally towards the naso-lacrymal duct to become a compound tubulo-acinous gland. When mature, it occupies a great surface between the cavum principale and recessus lateralis and covers the anterior part of the naso-lacrymal duct (Tsui, 1935, fig. 7).

8. Medial Nasal Gland.—At the beginning of the third developmental stage of the olfactory organ, the cells of the olfactory epithelium in the medio-posterior angle of the anterior lower sac differentiate gradually into a tiny diverticulum composed of simple epithelium. This is the medial nasal gland (Text-fig. 17). It grows forward and chiefly along the medial edge of the anterior lower sac until it is on a level with the distal end of the latter (Text-fig. 18). The structure is quite simple, containing only a few branched tubules. The proximal tubule becomes the short duct of this gland, which opens at the place of its origin. The distal tubules then branch farther and extend now chiefly medially and posteriorly, but very little dorsally, ventrally, or anteriorly. As the anterior lower sac later extends more anteriorly and medially, the gland assumes relatively a more posterior position (Text-fig. 22). Later it ramifies profusely to become a small branched tubular gland. It now grows mainly backwards and laterally. Still later the cartilaginous nasal capsule is formed and closely encases the olfactory organ including this gland, restricting its-growth along the medial and posterior region. Consequently the further growth of the gland is chiefly towards the front (Text-figs. 25 and 27).

It may be pointed out here that during metamorphosis when the cavity shrinks in size (p. 82), this gland also undergoes considerable shrinkage. A detailed cytological investigation of this gland and the lateral nasal gland will be presented in a separate paper.

4. Glands of Bowman.—Glands of Bowman are imbedded in the epithelium of the cavum principale. They do not appear until the larva is 46 mm. long. The mouth part of the gland arises earlier than the body. Moreover, the glands do not arise simultaneously in the whole cavum principale, but appear in the posterior part of it earlier than in the anterior.

5. Pharyngeal Gland.—The pharyngeal gland appears just at the beginning of the fourth stage behind the lateral nasal gland along the groove separating cavum principale and recessus lateralis (called the super-lateral groove). It extends posteriorly and runs over the roof of the choana and down the medial wall of it (Text-figs. 24 and 26).

Careful as Hinsberg’s work (1901) was, he left a big gap in the ontogeny of the nasal organ. The changes described in the fourth stage were completely overlooked by that author. The shrinkage of the organ during metamorphosis is interesting and, as far as I know, has not been reported by previous writers.

With regard to the question whether the primitive choana opens into the endodermal or ectodermal part of the oral cavity, it is sufficient to discuss the investigations of two authors, Kurepina (1931) and Watanabe (1936), who paid special attention to this point.

Kurepina made reconstructions from sagittal sections. Such sections show plainly a lateral constriction on either side of the oral cavity. Kurepina took this constriction to be the vestige of the pharyngeal membrane. Since this membrane marks the meeting place of the two embryonic layers, Kurepina concludes that the choanal opening, which is anterior to the constriction, must be ectodermal. In Rana nigromaculata such a pair of lateral constrictions is also present (Text-fig. 15). My reconstructed model of the head of nigromaculata at this stage shows structures identical with that illustrated by Kurepina (cf. Text-fig. 15 with Kurepina’s fig. 15, p. 26). But the lateral constriction in nigromaculata does not represent a vestige of the pharyngeal membrane. It is a constriction near the front end of the primitive pharynx. The endoderm on the upper part of the oral cavity stretches far beyond it and the pharyngeal membrane is formed in front of this pair of lateral constrictions (Text-figs. 12, 13, and 14). At the time of the formation of the primitive choana this membrane has ruptured and left no vestige to serve as a guide. Microscopic observations show, however, that the tissue into which the choana opens is definitely endodermal. Evidently Kurepina made nd careful microscopic observation of his sections, but relied solely on his models which could not reveal detailed histological structure. One has reason to suspect that what is true of Rana nigromaculata also obtains in Pelobates fuscus, which he figured, and perhaps in other amphibians he studied.

Watanabe (1986) also believes that the choana opens into the ectoderm, but gives no clear evidence to support his contention. He made his studies on cross-sections only. From my experience such sections are unsuitable for the elucidation of this point. To ascertain whether the choana opens into the ectoderm or endoderm, it is desirable that these two layers be well represented in a given section so that they can be easily compared and one is certain which layer he has under view. For such studies, sagittal sections should be employed. In a cross-section generally only one or the other of these embryonic layers is present, or, when both are included, they are represented by fragments which do not lend themselves to such comparative studies. I have cross-sections which closely resemble the figures in Watanabe’s paper but which proved worthless for determining the nature of the choanal opening. It is only after one has familiarized oneself with the character of these two layers by studying sagittal sections that one can tell them apart in cross-sections.