Development of Olfactory Organ inSana nigromaeulata .

By

Ghi-Lan Tsui,Depa r tmen t of Biology, t he Nat ional Universi ty of Yunnan , "Kunming,

Yunnan , China.

Wi th 27 Text-figs.

C O N T E N T S . PAGE

INTRODUCTION . 6 1

M A T E R I A L AND T E C H N I Q U E . . . . . . . 62

D E V E L O P M E N T A L A N A T O M Y . . . . . . . . 63

D I S C U S S I O N 88

SUMMARY 89

I N T R O D U C T I O N .

SINCE Goette's classical paper on the development of thetoad (Goette, 1875) there have appeared many accounts of theontogeny of the olfactory organ of Amphibia. The mostthor6ugh-going work has been that of Hinsberg (1901), whofollowed very closely the early developmental steps but not thelater ones. In the developmental history of the amphibiannasal organ two points in particular have arrested the attentionof students of comparative embryology and caused a good dealof controversy. One is the existence of the oro-nasal groove,the other is the location of the choanal opening in the oralcavity. Kurepina (1931) claimed that there is present in theamphibian larva an oro-nasal groove, comparable to the nasalgroove in embryos of the Amniota, while Hinsberg (1901),Kawagae (1933), and Schneider (1935) were unable to find sucha groove. Kurepina also found that the choana opens into theectodermal part of the oral cavity as it does in all Amniota.In this he was supported by Watanabe (1936). On the otherhand, Goette (1875), Hinsberg (1901), and others (Pahrenholz,1925; Kawagae, 1933; Schneider, 1935) contended that the

62 CHI-LAN TSUI

choanal opening is in the endoderm. However, neither side hasgiven conclusive evidence.

B a n a n i g r o m a c u l a t a Hallowell is a common frog inNorth China. Its olfactory organ has not been investigated.The aim of the present paper is to make a thorough and com-plete study of the development of that organ and to see if freshlight can be thrown on those controversial points mentionedabove.

The present investigation was conducted in the BiologyDepartment, the National University of Peking, Peiping. Whenthe work was nearly completed, it was interrupted by theJapanese invasion and was finally completed in Kunming.

MATERIAL AND TECHNIQUE.

E a n a n i g r o m a c u l a t a is very abundant in Peiping. Onecan get its eggs and tadpoles in ponds, pools, and rice fields fromApril to July. A complete series of stages, ranging from larvae2 mm. long to young frogs was collected. Identification wasbased on descriptions in Boring, Liu, and Chou (1932, pp. 39-49). The body-length was measured and recorded. One-halfof 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 e.c, concentratedformalin, 10 c.c, and glacial acetic acid, 2 c.c.) or in a mixtureof acetic acid and sublimate (saturated sublimate solution,35 c.c. and glacial acetic acid, 20 c.c). Each fixative wasprepared immediately before use. The fixed material was im-bedded in paraffin. Both cross and sagittal serial sections werecut 5-9/x. in thickness and stained with Delaneld's haematoxylinor a combination of Delafield's haematoxylin and eosin.

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

BANA NrGBOMACULATA 68

A sheet of blotting paper (48x61 cm.) was cut into threestrips and immersed in a bath of melted beeswax, to 100 partsof which 5 parts of paraffin were added. After two minutesthey were taken out of the bath and both sides of the paper werequickly wiped with a piece of cloth in order to make the waxedpaper even and smooth. Every strip of the waxed paper wascut again into five. To determine the thickness of the waxedpaper, every piece was measured in 8 different places with amicrometer caliper. The mean of the 120 readings (15x8) wastaken to be the correct thickness. The most suitable thicknessfor cutting a complicated model was 04 to 0-5 mm. Thinnerpieces would be, of course, easier to cut, but involved morework. Paper thicker than 0-5 mm. was employed only formaking simple and large models. For sections cut 8/z thick,paper 0-4 mm. in thickness was just right for magnifying them50 x ; two thicknesses of paper for lOOx.

Serial drawings were made on the waxed blotting paper witha fine needle under an Edinger drawing machine or a 'Panphot'(manufactured by Leitz). It was found easier, however, to makesketches on a piece of blotting paper with a sharp pencil beforeinfiltration with wax. Further treatment is just the same as inthe wax-plate method.

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

DEVELOPMENTAL ANATOMY.

A. Olfactory Cavi t ies .

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

1. F i r s t S tage .From the Incep t ion of the Olfactory Anlage to

64 CHI-LAN TSUI

the Fo rma t ion of the Olfac tory P lacode (lengthof l a rva , 2*5-5 mm.).—When the neural groove just closesto form a tube, the ectoderm of the head of the larva showsclearly the surface and the sensory layer (Goette, 1875; Corning,1899). The larva at this stage is about 2*5 mm. long. The anlageof the olfactory placode arises as a pair of thickenings from thesensory layer of the ectoderm at the top of the head and infront of the optic vesicle. When strongly magnified, the externalsurface of this anlage can be barely made out as a slightrounded depression (Text-fig. 6). A larva of about 2-8 mm. inlength shows the primitive placode definitely depressed to formthe olfactory pit, which can be already seen under 24 x magni-fication (Text-fig. 1). Hinsberg (1901) working on Ban afuse a stated that the olfactory pit appears only when thelarva reaches 5 mm. in length. Inn igromacula ta i t appearsthen much earlier. No further changes could be observed atthis stage except the increase in dimension of the placode, andthat with the enlargement of the head at the brain region theplaeode which first appeared dorsally is displaced ventrally.Hinsberg (1901) discussed at length the question, whether thesurface layer of ectoderm takes part in the formation of olfac-tory placode and concludes that in f usea the placode is derivedsolely from the sensory layer. My findings in Ran a n igro-macu la t a completely support those of Hinsberg.

In the head of the larva of E a n a n ig romacu l a t a thereis also a forehead streak, the ' Stirnstreifen' of Hinsberg (1901).It will be dealt with in a separate paper, for it has nothing to dowith the development of the olfactory organ.

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

2. Second S tage .(a) Fo rma t ion of the Nasal Lumina (length of

l a r v a , 5-9-5 mm.).—The olfactory pit remains a shallowdepression as long as the lens is still attached to the sensorylayer of the ectoderm. The formation of the nasal luminasynchronizes with the separation of the lens from the sensory

H

Maao.-

TEXT-FIG. 1.

Lateral view of the larva (2-8 mm. long), x 30. lstga, first gillarch; Hyoa, hyoid arch; Mana, mandibular arch; Olfp, olfactorypit; 2ndga, second gill arch; Sue, sucker.

ABBREVIATIONS.

(Text-figs. 6-12 drawn with the aid of a camera lucida andmagnified 86x.)

Antlos, anterior lower sac; Cavinf, oavum inferius; Cavmed, cavuinmedium; Cavprinc, cavum principale; Cho, choana; Cle, clefts;Cons, constriction; Dien, diencephalon; Borl, dorsal lumen;Ect, ectoderm; Ent, endoderm; Entrain, 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, lateralnasal gland; Latgr, lateral groove; Latp, lateral portion; Llid,lower eyelid; Llip, lower lip; Medgl, medial nasal gland; Medp,medial portion; Mes, mesencephalon; Met, nietencephalon; Midl,middle lumen; Nar, nasal rim; Nasduct, naso-lacrymal duct;Nasduct', external opening of naso-lacrymal duct; Noto, noto-chord; Olfp, olfactory pit; Olfpl, olfactory placode; Optves, opticvesicle; Orca, oral cavity; Orep, oral epithelium; Palep, palateepithelium; Phagl, pharyngeal gland; Pham, pharyngeal mem-brane; PJiar, pharynx; Postlos, posterior lower sac; Postpro,posterior prolongation; Pricho, primitive choana; Proj, pro-jection; Pros, prosencephalon; Recalar, recessus alaris; Reclat,recessus lateralis; Recmed, recessus medialis; Recsac, recessussaccuormis; Serilay, sensory layer; 8k, skin; Surlay, surface layer;Suplatgr, super-lateral groove; Uplip, upper lip; Vps, upper sac;Venl, ventral lumen.

NO. 345 F

66 CHI-LAN TSUI

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 neighbouringcells behind it are immediately differentiated to form a blindsac, which is called the lateral appendix. At the time of itsformation it lies just at the dorso-lateral region of the placode(Text-fig. 8). Later, with the more rapid expansion of the

Jostril \ /\gS^ NoSlTil

A BTEXT-JIG. 2.

Comparative dimensions of the head before (A) and after (B)the metamorphosis, x 3.

placode to the dorsal and medial side, the lateral appendix isgradually shifted latero-posteriorly and a marked constrictionappears between it and the plaeode (Text-figs. 10 and 18). Thepart of the placode posterior to the lateral appendix is againdifferentiated into two portions, a thin lateral one and a thickmedial one. Between them there originate several minute clefts(Text-fig. 8). These are at first discontinuous but later com-municate with one another to form the ventral lumen posteriorto the olfactory pit (Text-fig. 10). Contemporaneous with theappearance of the clefts, the olfactory pit begins to extend in-ward and gives rise to another lumen, the middle lumen, whichis immediately anterior to the dorsal lumen (Text-fig. 9) andruns ventro-posteriorly. Its medial wall is derived from theolfactory placode, while its lateral wall is from inward extensionof the skin (Text-fig. 10). It communicates with the dorsallumen from the very beginning (the larva at this time is 7 mm.in length) and with the ventral one when the clefts run togetherto 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

RANA NIGROMACULATA 67

projection, which is particularly plain in n igromacula ta(Text-figs. 10 and 14). This projection serves at this time as aboundary between the middle and ventral lumen. Its subsequentdevelopment will be described later.

(b) F o r m a t i o n of the P r i m i t i v e Choana (lengthof l a r v a 5-S-9-5 mm.).—The formation of the primitivechoana in the oral cavity is closely related to the formation and

50 45 40 35 3o 25 20 15 20 25 W

Length of the body in mm.d'nthetadpole stoje the tail is Included^

TEXT-FIG. 3.

Graphic representation of the changes in the dimensions of the headin relation to metamorphosis. The arrows mark the beginningand end of metamorphosis.

rupture of the pharyngeal membrane. The latter is formed byfusion of the intruded ectoderm and the protruded endoderm.As the brain expands, the forehead is thrust forward and down-ward, so that the pharyngeal membrane is then shifted back-ward (Text-fig. 11). At each side of the pharyngeal membranethe two embryonic layers meet to form the lateral walls of theprimitive oral cavity. The method of fusion of these two layersis somewhat peculiar. The endoderm protrudes above, whilethe ectoderm intrudes below (Text-fig. 13). As the result ofthis kind of fusion, the meeting line of the two layers is notvertical but runs obliquely from the dorso-anterior towards the

68 CHI-IiAN TSUI

ventro-posterior (Text-figs. 11 and 14). Therefore, in the roofof the oral cavity the endoderm extends much more anteriorlythan it does in the floor (Text-figs. 12 and 14). By the differencein microscopic structure one can easily distinguish the two

The ectoderm in the oral cavity is thicker and in a given

/lect.rifis.

PYOc.max.post.

axil.

TEXT-FIG. 4.

Camera lucida outline of cross-section of the head through themiddle region of nasal cavity shoiving an extra blind sac. X 38.Bowgl, Bowman's gland; Gartdbl, cartilago obliqua; Gutgl,cutaneous gland; Extrs, extra blind sac; Maxil, maxilla; Olfep,olfactory epithelium; Promaxpost, processus maxillaris posterior;Septnas, septum nasi; Solnas, solum nasi; Tectnas, tectum nasi.For other abbreviations, see list of abbreviations.

area contains many more cells, the nuclei of which are crowdedtogether. The endoderm is a thin layer; its cells are not socrowded and contain many yolk-granules. This layer takes thestain less deeply (Text-fig. 12).

The olfactory placode in the 5-5 mm. larva shows a small

RANA NIGROMACULATA 69

prolongation at its most posterior end. This prolongationextends ventro-posteriorly towards the oral cavity (Text-fig. 13).The tip of it is later thrust into the endodermal part of the oralcavity at the point where the lateral wail meets the roof (Text-figs. 12 and 14). This occurs just after the rupture of thepharyngeal membrane. At this stage, the ventral nasal lumen

Tect.nas. Sept nas-

EKW.S.

TEXT-ITG. 5.

Camera luoida outline of cross-section of another head at the levelof the external naris showing three extra blind sacs. x 3 5 . Labelslike those used in Text-fig. 4.

is still represented by the discontinuous clefts (p. 66). Afterthese clefts have run together to form the ventral lumen, itextends into the prolongation and opens at its distal end intothe oral cavity. This distal opening is the primitive choana.Since the tip of the prolongation grows into the portion of theoral cavity lined by endoderm, the choana opens definitelyinto the endoderm.

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

3. Third S tage .Fo rma t ion of Blind Sacs and Changes of the

External Naris and the P r i m i t i v e Choana (length

70 CHI-LAN TSUI

TEXT-FIG. 6.

Cross-section of a portion of the head through the olfactoryplacode (length of larva, 2-5 mm.).

Pros.

TEXT-FIG. 7.

The same, through the olfactory pit and dorsal lumen of theolfactory placode (length of larva, 7 mm.).

RANA NXGROMACULATA 71

of l a rva , 9-5-50 mm.).—Up to the end of the second stagethe olfactory placode with its lumina is more or less straight(Text-fig. 17). This simple structure undergoes profoundchanges to become a very complicated organ.

The embryonic olfactory organ as it appears at the end of thethird stage may be divided according to natural demarcations

Pros.

Opt.ve$

The same, through lateral appendix of the olfactory placode(length of larva, 8-5 mm.).

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 uppersac includes the external naris and contains the middle nasallumen. At first, it is the largest sac (Text-fig. 17). The anteriorlower sac is a small one situated ventro-anteriorly to the uppersac. The posterior lower sac is under the upper sac. It containsthe ventral lumen and includes the choana. The three sacsarise in the following manner.

72 CHI-LAN TSUI

. 9.The same, through the communication of dorsal and middlelumen of the olfactory placode (length of larva, 8-5 mm.).

TEXT-FIG. 10.

The same, through the communication of dorsal, middle, and centrallumen of the olfactory placode (length of larva, 9-5 mm.).

BANA NIGROMACULATA 73

The part of the olfactory placode around the middle nasallumen enlarges and the lumen expands along with it, thusbringing about the formation of the upper sac. Contem-poraneous with the coming into being of the upper sac, theplacodal region around the ventral lumen together with the

Mas.

Met

Dten-

Pros.

Ect

Pha.m.

TBXT-EIG. 11.

Sagittal section through the pharyngeal membrane (length oflarva, 5-5 mm.).

posterior prolongation and the primitive choana (into which theventral lumen opens) enlarges and the whole complex nowbecomes the posterior lower sac. At the beginning it is notmarked off from the upper sac (Text-fig. 17).

It will be recalled that between the middle and ventral lumenthere is an inward projection (p. 66). As the upper and pos-terior lower sacs are being formed, this inward projection alsoenlarges. When viewed from the outside of the olfactory organ

74 CHI-LAN TSUI

at this stage, this inward projection is a small depression (Text-fig. 17). Following the expansion of the upper and the posteriorlower saes this depression increases both in length and in depth.This structure I designate as the lateral groove. It marks off

Pros,

Postpra

TEXT-FIG. 12.

The same, through the posterior prolongation of the olfactoryplacode. (The posterior prolongation is thrust into the ento-dennal part of the oral cavity. The entodermal tissue behind theprolongation gives a tangential view of the upper corner of theoral cavity, and thus appears as if it were a thick transection.Length of larva, 7-5 mm.)

the upper sac on the outer side from the posterior lower sac(Text-fig. 18). In the medial region the upper sac projects outand hangs over the latter, though on that side there is nodefinite groove to mark them off (Text-fig. 18).

The anterior lower sac is simply an evaginated part of theventro-anterior region of the upper sac (Text-fig. 17). It pro-

EANA NIGHOMACUIJATA 75

trades forward under the upper sac. In the early stage, thissac looks somewhat like a flattened egg.

SI.

TEST-FIG. 13.

Lateral view of a model (made from the same specimen a section ofwhich is illustrated in Text-fig. 11) showing the olfactory organand the lateral wall of the primitive oral cavity, x 35.

TEXT-FIG. 14.

Lateral view of a model showing the olfactory organ and the lateralwall of the oral cavity. The oblique meeting-line of the ectodermand entoderm is very clear (length of larva, 8-5 mm.), x 35.

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

The head as a whole increases more rapidly in size than the

76 CHI-LAN TSUI

olfactory organ. As a consequence there is now intercalatedbetween the external naris and the middle lumen (which werepreviously in close contact) a canal formed by the invaginated

TEXT-ITS. 15.

Medial view of a model (made from the same specimen a section ofwhich is illustrated in Text-fig. 12) showing the olfactory organ,oral cavity, and the entodermal constriction of the latter, x 35.

TEXT-EEG. 16.

Antero-medial view of a model showing the olfactory organ and theprimitive choana in the roof of the oral cavity (length of larva,9-5 mm.), x 35.

skin inside the naris. This canal is the entrance canal (Text-fig. 17). The surrounding skin of the external naris is raised toform a rim around it (Text-fig. 17). This rim exists up to thetime when the larva is 18 mm. long; then it begins to disappear.

RANA NIGROMACULATA 77

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

After the disappearance of the rim the entrance canal dilates(Text-fig. 18). The upper part of it extends dorso-laterally and

TEXT-FIG. 17.

Anterior view of a model showing the beginning of the differentiationof the olfactory organ into three embryonic blind sacs (length oflarva, 11 mm.). x25.

xtnor.Entrcao.

TEST-FIG. 18.

Dorso-lateral view of a model showing the formation of thelateral groove (length of larva, 22 mm.). x25.

constitutes a small sac (Text-fig. 20, x) which in the nextdevelopmental stage gives rise to several small structures (seep. 83). The lower part extends in the opposite direction—to theventro-medial—to form another blind sac which is thrust

78 CHI-LAN TSUI

N: Lst.fiB

TEXT-FIG. 19.

Latero-posterior view of a model showing the formation of thesuper-lateral groove and recessus lateralis. At this stage theupper sac is clearly demarcated from the posterior lower sac(length of larva, 41 mm.). x25.

Extnff

Psstkr

TEST-FIG. 20.

Lateral view of a model made from the olfactory organ of a specimenjust before metamorphosis (length of larva, 50 mm.), x 25.

between the upper and anterior lower sac and becomes thecavum medium in adult (Text-fig. 20). Concomitant with the

KANA NIGROMACULATA 79

above changes the external naris is shifting sideways so thatthe two nasal openings which were close together become atthe end of this stage wide apart (Text-fig. 21).

The lateral appendix, as described above (p. 66), is theearliest developed blind sac. It increases very rapidly at thebeginning and reaches the highest development at the end of

Medgl:

TEXT-FIG. 21.

Dorsal view of the same model.

the second stage. It then remains stationary. Upon thedevelopment of the upper sac it is gradually shifted to the lateraland posterior (Text-fig. 18). At this time it starts also todegenerate, becoming increasingly smaller (Text-figs. 19 and20) until it is obliterated by the end of the metamorphosis. Itis remarkable that the lateral appendix, which differentiatesvery early and, for a time, is the only blind sac in the earlylarval olfactory organ, should completely disappear in thecourse of further development. Watanabe (1936) is of theopinir n that it may be a sense organ for the larval life, but hegives no evidence to support his conjecture.

80 CHI-LAN TSUI

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

Just above the lateral groove and under the lateral appendixthere arises now another groove which extends anteriorly up

•Pestles.

TEXT-FIG. 22.

Ventral view of the same model.

to the proximal end of the anterior lower sac (Text-figs. 19 and20). I call it the super-lateral groove, as it is above that struc-ture. The part of the lateral wall of the upper sac betweenthese two grooves is now separated from the rest of the walland becomes a ledge overhanging the lateral wall of the posteriorlower sac. It becomes the recessus lateralis in the adult (Text-figs. 19 and 20).

The anterior lower sac now increases and extends moreanteriorly than in other directions, while the upper sac extendsin all directions but very little anteriorly. The result is thatthe former sac lies far to the front at the end of this stage

BANA NIGROMACUI/ATA 81

(Text-figs. 20, 21, and 22). In the larval stage its communica-tion is always with the middle lumen of the upper sac. In theadult it becomes connected with the recessus lateralis, which isa derivative of the upper sac. These changes will be describedin the fourth stage.

As pointed out in a preceding paragraph, the posterior lower

TEXT-FIG. 23.

Anterior view of a model made from the olfactory organ of aspecimen during metamorphosis (length of larva, 38 mm.—tailhas been resorbed). x 25.

sac is not fully separated from the upper sac until the end ofthe present stage. It expands rapidly, so that at the end of thisstage it outstrips the upper sac in size and becomes the largestsac of the time. The expansive growth is far greater at theposterior part, and its form at the end of this stage is roughlya triangular pyramid with its apex directed anteriorly (Text-fig. 22).

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

NO. 345 G

82 CHI-LAN TSUI

4. F o u r t h S t a g e .P rom the Beginn ing of the Metamorphos i s to

t h e Adul t (from l a r v a of 50 mm. in l eng th to a d u l tfrog),—When the tadpole reaches 50 mm. in length, its tailbegins to be resorbed. There occurs a great change in the nasalcavity. It now undergoes simplification in outline and, at first,noticeable shrinkage in size. This decrease in size is under-standable when we compare the head of the tadpole just beforemetamorphosis with the young frog. The head of the tadpoleis much larger and has a rounded outline about the mouth(Text-fig. 2 A). After metamorphosis the head has shrunken inall dimensions and has a triangular outline (Text-fig. 2 B). Thechange in the size of the head is graphically represented in theTest-fig. 3.

The shrinkage of the head is evidently related to the emer-gence of the animal from water, which is probably accompaniedby dehydration. The connective tissue in the head was veryloose in the larva, but becomes compact in the young frog. Thesimultaneous decrease in the size of the olfactory organ mayalso be due to the loss of water from its tissues. This pointcannot be established histologically, however, since measure-ments of the cells of the organ before and after metamorphosisdo not reveal any shrinkage in their size.

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

The shape of the olfactory organ at the end of the last stageis very irregular and the three sacs are distinct from oneanother (Text-figs. 21 and 22). During metamorphosis thefurrows separating the upper and posterior lower sacs along themedial and posterior region (Text-fig. 21) become smoothed outas a, result of distension of the sacs. The elevated posterior partof the upper sac and the lateral appendix lose their identityand the whole of the upper sac and the main portion of theposterior lower sac merge into one large cavity, designated asthe chief cavity or cavum principale in the mature organ (Text-

RANA NIGROMACULATA

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 sacand the posterior lower sac at their lateral parts remains, how-

TEXT-FIG. 24.

Dorsal view of the same model.

ever. The whole lateral wall of the posterior lower sac extendslateralwards carrying the ledge along with it, forms a sideextension of the cavum principale, and is designated as recessuslateralis (Text-figs. 24 and 26).

The anterior lower sac lies now completely under the cavumprineipale. The main part of it together with the foremostextension of the recessus lateralis, which projects until it islevel with the anterior lower sac, constitutes the eavum inferiusof the mature organ (Text-figs. 23 and 25), while the medio-posterior portion of the sac is called recessus medialis (Text-figs. 25 and 27).

The small sac derived from the upper part of the entrancecanal, and situated under the external naris (Text-fig. 20, x)expands to form the anlagen of the recessus alaris, infundibulum

84 CHI-LAN TSUI

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 narisenlarges and along with the cavum principale extends forward(Text-figs. 24 and 26).

The olfactory organ increases in size along with the growth

Re&altr.

TEXT-FIG. 25.

Ventral view of the same model.

of the head. In a later stage it is still further flattened outdorso-ventrally. The cavum principale at its hind portionstretches medially and the recessus lateralis extends furtherlaterally. The choana becomes greatly appressed (Text-fig. 27).It is now relatively large, but in the mature organ its relativesize to that of the nasal cavity is much smaller (Tsui, 1935,fig. 6).

From this point on, the structure of the nasal cavity undergoesno important change. In the adult stage, the olfactory organof E a n a n i g r o m a c u l a t a is essentially the same as that offusca and esculenta (Tsui, 1935). Itis, therefore,unnecessaryto carry the description farther.

E x t r a Blind Sac s.—Extra blind sacs have been occasion-ally found in the nasal cavity. In three cases such sacs were

RANA NIGROMACULATA 85

observed—all in the stage when the cartilaginous nasal capsuleis already formed. In one specimen an invaginated sac ispresent in the cavum principale (Text-fig. 4). In the other two,one or two small folds occur in the medio-anterior part of thatcavity (Text-fig. 5). All these extra pouches are symmetricallypresent in both nasal cavities. The formation of these unusualstructures is perhaps due to the fact that the cells in certain

Cav.inf.

Cav.mtd.

'av.imt

Phoji

TEXT-FIG. 26.

Dorsal view of a model made from the olfactory organ of a youngfrog (length of the body, 20 mm.), x 25.

regions of the olfactory epithelium increase abnormally. Onaccount of the presence of the cartilaginous nasal capsule, theepithelium cannot extend freely and, as a result, a blind sacis formed. An undetermined blind sac was reported in fusca(Tsui, 1935). It was probably formed in the same way.

B. Accessory Glands and Naso-lacrymal Ducts.1. Naso-lacrymal Ducts.—The development of the

naso-lacrymal duct is very sudden. It arises in the dorso-lateral

86 CHI-LAN TSUI

part of the cavum prineipale behind the external naris (Text-fig. 24). Due to the formation of the recessus saeciformis it ispushed farther laterally and is connected with the latero-posterior end of the cavum medium (Text-fig. 26). It lengthensposteriorly, the distal end of the duct bifurcates, and the twobranches open into the lower eyelid (Text-fig. 26).

According to Born (1876), this duct in the amphibians he

TEXT-FIG. 27.

Ventral view of the same model.

examined is first solid and hollows out to form a duct. Trans-verse serial sections were made of the duct of the presentspecies. It was found that the proximal end of this duct is atfirst solid. The duct lengthens very rapidly and when it reachesthe eyelid, it is already hollow along most of its length. Finallythe proximal end also becomes hollow. The formation of thelumen seems to begin from the distal end.

2. L a t e r a l Nasa l Gland.—At the same level as, andmedially to, the naso-laerymal duct there arises the lateralnasal gland. It is a small diverticulum (Text-figs. 24 and 26).Soon it increases in size, and branches laterally towards the naso-

RANA NIGROMACULATA 87

lacrymal duct to become a compound tubulo-acinous gland.When mature, it occupies a great surface between the cavumprincipale and recessus lateralis and covers the anterior partof the naso-lacrymal duct (Tsui, 1935, fig. 7).

3. Medial Nasal Gland.—At the beginning of the thirddevelopmental stage of the olfactory organ, the cells of theolfactory epithelium in the medio-posterior angle of the anteriorlower sac differentiate gradually into a tiny diverticulum com-posed of simple epithelium. This is the medial nasal gland(Text-fig. 17). It grows forward and chiefly along the medialedge of the anterior lower sac until it is on a level with the distalend of the latter (Text-fig. 18). The structure is quite simple,containing only a few branched tubules. The proximal tubulebecomes the short duct of this gland, which opens at the placeof its origin. The distal tubules then branch farther and extendnow chiefly medially and posteriorly, but very little dorsally,ventrally, or anteriorly. As the anterior lower sac later extendsmore anteriorly and medially, the gland assumes relatively amore posterior position (Text-fig. 22). Later it ramifies pro-fusely to become a small branched tubular gland. It nowgrows mainly backwards and laterally. Still later the carti-laginous nasal capsule is formed and closely encases the ol-factory organ including this gland, restricting its"growth alongthe medial and posterior region. Consequently the furthergrowth of the gland is chiefly towards the front (Text-figs. 25and 27).

It may be pointed out here that during metamorphosis whenthe cavity shrinks in size (p. 82), this gland also under-goes considerable shrinkage. A detailed cytological investiga-tion of this gland and the lateral nasal gland will be presentedin a separate paper.

4. Glands of Bowman.—Glands of Bowman are im-bedded in the epithelium of the cavum principale. They do notappear until the larva is 46 mm. long. The mouth part of thegland arises earlier than the body. Moreover, the glands do notarise simultaneously in the whole cavum principale, but appearin the posterior part of it earlier than in the anterior.

5. P h a r y n g e a l Gland.—The pharyngeal gland appears

88 CHI-LAN TSUI

just at the beginning of the fourth stage behind the lateralnasal gland along the groove separating cavum principale andrecessus lateralis (called the super-lateral groove). It extendsposteriorly and runs over the roof of the choana and down themedial wall of it (Text-figs. 24 and 26).

DISCUSSION.

Careful as Hinsberg's work (1901) was, he left a big gap in theontogeny of the nasal organ. The changes described in thefourth stage were completely overlooked by that author. Theshrinkage of the organ during metamorphosis is interestingand, as far as I know, has not been reported by previous writers.

With regard to the question whether the primitive choanaopens into the endodermal or ectodermal part of the oralcavity, it is sufficient to discuss the investigations of two authors,Kurepina (1931) and Watanabe (1936), who paid special atten-tion to this point.

Kurepina made reconstructions from sagittal sections. Suchsections show plainly a lateral constriction on either side of theoral cavity. Kurepina took this constriction to be the vestigeof the pharyngeal membrane. Since this membrane marks themeeting place of the two embryonic layers, Kurepina concludesthat the choanal opening, which is anterior to the constriction,must be ectodermal. In Eana n ig romacu la t a such a pairof lateral constrictions is also present (Text-fig. 15). My recon-structed model of the head of nigromaculata at this stageshows structures identical with that illustrated by Kurepina (cf.Text-fig. 15 with Kurepina's fig. 15, p. 26). But the lateralconstriction in nigromaculata does not represent a vestige ofthe pharyngeal membrane. It is a constriction near the frontend of the primitive pharynx. The endoderm on the upper partof the oral cavity stretches far beyond it and the pharyngealmembrane is formed in front of this pair of lateral constrictions(Text-figs. 12, 13, and 14). At the time of the formation of theprimitive choana this membrane has ruptured and left novestige to serve as a guide. Microscopic observations show,however, that the tissue into which the choana opens is definitelyendodermal. Evidently Kurepina made no careful microscopic

RANA NIGROMACULATA 89

observation of his sections, but relied solely on his models whichcould not reveal detailed histological structure. One has reasonto suspect that what is true of E a n a n i g r o m a c u l a t a alsoobtains in P e l o b a t e s fuscus , which he figured, and per-haps in other amphibians he studied.

Watanabe (1936) also believes that the choana opens intothe ectoderm, but gives no clear evidence to support his con-tention. He made his studies on cross-sections only. From myexperience such sections are unsuitable for the elucidation ofthis point. To ascertain whether the choana opens into theectoderm or endoderm, it is desirable that these two layers bewell represented in a given section so that they can be easilycompared and one is certain which layer he has under view.For such studies, sagittal sections should be employed. In across-section generally only one or the other of these embryoniclayers is present, or, when both are included, they are repre-sented by fragments which do not lend themselves to suchcomparative studies. I have cross-sections which closelyresemble the figures in Watanabe's paper but which provedworthless for determining the nature of the choanal opening.It is only after one has familiarized oneself with the characterof these two layers by studying sagittal sections that one cantell them apart in cross-sections.

SUMMARY.

1. The anlage of the olfactory placode arises from thesensory layer of the ectoderm.

2. There is no oro-nasal groove between the olfactory placodeand stomodaeum.

3. The primitive choana opens into the endodermal part ofthe oral cavity.

4. The lateral appendix is the earliest formed blind sac. Itdegenerates and disappears at the end of the third stage.

5. There are three blind sacs in the larval stage: the upper,anterior lower, and posterior lower sac. The first and thirdsacs together become the cavum principale and recessuslateralis, while the second becomes the cayum inferius andrecessus medialis in the adult stage.

90 CHI-LAN TSUI

6. The nasal cavities undergo considerable shrinkage duringmetamorphosis.

7. Extra blind sacs may be formed in the later larval stage.8. The naso-lacrymal duct arises in the dorso-lateral part of

the cavum principale and extends to the lower eyelid. Theprocess of hollowing out appears to begin from the distal end.

9. There are four kinds of glands in the nasal cavity—medialnasal, lateral nasal, Bowman's, and pharyngeal gland. The firstone arises earlier than the other three.

LITERATURE CITED.

Boring, Liu, and Chou, 1932.—'Handbook of North China. Amphibia andreptiles.' Peking, China.

Born, G., 1876.—"Nasenhohlen und Trannennasengang der Amphibien",'Morphol. Jahrb.', 2.

Corning, H. K., 1899.—"Entwicklungsvorgange am Kopf der Anuren",ibid., 27.

Fahrenholz, C , 1925.—"Entwicklung des Gesichtes und der Nase bei derGeburtshelferkrote", ibid., 54.

Gage, S. H., 1936.—'The Microscope.' Ithaca, New York.Goette, A., 1875.—'Entwicklungsgeschichte der Unke.' Leipzig.Hinsberg, V., 1901.—"Entwicklung der Nasenh6hle bei Amphibien",

'Arch. f. mickrosk. Anat.', 58.Kawagae, I., 1933.—"Entwicklungsgesch. des Geruchsorgans von Megalo-

batrachus japonicus", 'Folia Anat. jap.', 10.Kurepina, M., 1931.—-"Entwickl. des Geruchsorgans der Amphibien",

'Zool. Jahrb., Abt. Anat. u. Ontog.', 54.Schneider, P. P., 1935.—"Primitive Entwicklung der Nase in der Eeihe

der Wirbeltiere", 'Zeitschr. f. Anat. u. Entw.', 104.Tsui, C. L., 1935.—"Histolog. Untersuch. der Nasen-driisen des Frosches",

'Zool. Jahrb., Abt. f. Anat. u. Ontog.', 60.Watanabe, St., 1936.—"Entwiekl. des Geruchsorgans von Rhacophorus

schlegelli", 'Zeitschr. f. Anat. u. Entw.', 105.