CELL JUNCTIONS IN AMPHIBIAN SKIN on October 15, 2016 Downloaded from

29 Pages·2003·3.33 MB·English

CELL JUNCTIONS IN AMPHIBIAN SKIN
MARILYN G. FARQUHAR, Ph.D., and GEORGE E. PALADE, M.D.
From the Department fo Pathology, University fo School California fo Medicine,
San Francisco, and The Rockefeller Institute, New York
ABSTRACT
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Cell junctions have been investigated in the amphibian epidermis, a stratified squamous n
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epithelium, and compared to those described previously in simple columnar epithelia of ad
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mammalian cavitary organs. d fro
In adult frogs and toads, and in larvae approaching metamorphosis, belts of membrane m h
fusion or zonulae setnedulcco of considerable depth are regularly found between adjoining cells ttp
of the outermost layer of the stratum corneum, binding the cells together into a continuous, ://rup
un(wihnent errupted such a layersheet. is Another present), set and of occlua ding third set zonules usually appears occurs in in the the outesremcoonds t cornified layer of layer the ress.org
stratum granulosum. Specialized elements described as "modified" and "composite" desmo- /jcb
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cesomes lls; ordinary are encountered desmosomes along and the maculae lateral setnedulcco and basal (i.e., aspects, spots of respectively, membrane fusion) of the are cornified found rticle-p
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in all other strata. The usual 200 A intercellular gap is generally maintained between the f/2
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cells of the stratum germinativum at the basal ends of the intercellular spaces. Hence, the inter- /1/2
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cellular spaces of the epidermis form a largely continuous network, closed to the external 3/1
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medium and open to the dermal interstitia. The situation is comparable to that found in 8
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columnar epithelia, except that the intercellular spaces are much more extensive, and an 08
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extracellular subcompartment (or two) apparently exists in the stratum corneum and between 63
the latter and the stratum granulosum. The last subcompartment is usually filled with a dense .pd
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substance, probably derived from discharged secretory granules. The tripartite junctional y g
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,snereahdacomplex charactaenrids tic desmosome) of lumen-liis nseen ing only in epithelia early larvae; (i.e., a zonula occludens in adults and in followed larvae approaching by a zonula est on
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metamorphosis, the occludiznogn ule followed is directly by a series of modified desmosomes. 3 Ja
Interpreted in the light of current physiological data, these findings suggest that the dif- n
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fusion of water, ions, and small, water-soluble molecules is impeded along the intercellular ry 2
spaces of the epidermis by zonulae setnedulcco while it is facilitated from cell to cell within the 02
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epidermis by zonulae and maculae .setnedulcco
In a previous paper (1), we surveyed the epithelia as zonula occludens (tight junction), zonula ,snereahda
lining a number of glands and cavitary organs of and macula adhaerens (desmosome). Along the
the rat and guinea pig and, in all cases investi- occluding zonule, the intercellular space is obliter-
gated, found a characteristic tripartite junctional ated by the fusion of the outer leaflets of the adja-
complex between adjacent epithelial cells. The cent cell membranes; by contrast, a distinct inter-
complexes are located at the luminal end of the cellular gap is maintained along the other elements
intercellular spaces and consist of three morpho- of the complex. Since the occluding zonules are
logically distinct junctional elements identified apparently continuous around each cell and
362
throughout the epithelium, we suggested that they sively used staining in block, before dehydration,
serve as "closing belts" which seal off the inter- in aqueous uranyl acetate solutions. 2
cellular spaces from the lumen, and we postulated Sections from all these blocks were stained for 30
that they play a passive role in the maintenance minutes with lead alone (8) or doubly stained with the
latter preceded by ~}1 hours in 5 per cent uranyl
of chemical and electrochemical potential grad-
acetate. They were examined in a Siemens Elmiskop
ients across epithelia by impeding back diffusion
I, operating at 80 kv with a double condenser. Fur-
(leaks) along the intercellular spaces. This as-
ther details of the embedding and staining pro-
sumption was tested and confirmed for large
cedures can be found elsewhere (1). For light micros-
molecules by using concentrated protein solu- copy, sections of 1 to 2 # were cut from the same
tions (hemoglobin and zymogen) as mass tracers• blocks and stained with azure II and methylene blue
The solutions filled the lumina but did not pene- (9).
trate the intercellular spaces beyond the occluding
OBSERVATIONS
zonules (1). The impermeability of these zonules
to water, ions, and small water-soluble molecules The observations presented are limited primarily
remained, however, to be demonstrated. Since no to cell junctions and associated structures (i.e.,
direct means of investigating this problem is cell membranes, intercellular spaces, etc.) of the
available, we approached it indirectly by examin- frog epidermis. Other aspects, such as differentia- D
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ing the frog epidermis, an epithelium known to be tion and keratinizafion of the epidermis and the w
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practically impermeable to certain small mole- structure of the dermis, are not treated in any loa
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cbuileosl. ogical The membrane,fr og skin fries quently an extensively used in studsiteuddi es dbertiaielf. description However, of for the theg eneral sake organizatoifo n orientation, of the a d from
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of transport functions of epithelia (2). It is known skin is included. The bulk of the observations ttp
to exhibit a unilateral osmotic response (3) and pertain to the skin of the frog; observations on ://ru
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to maintain marked electrochemical potential toad and larval skin are mentioned where they re
gradients, and both properties have been localized are appropriate or instructive. ss.o
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tisoom lupbeler meable the empoiledceulrems,i s to water, (2)t. hey If shouldi ons, occluding and be presents mall zonules watienr - atrhee GEepneiradl ermis Histology and Cytology of Frog /jcb/article-p
frog epidermis, binding together the cells close The frog skin consists of an epidermis composed df/2
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to its outer surface. The observations to be pre- of 5 to 7 layers of epithelial cells, and an underly- /1/2
sented I fully confirm these expectations. ing dermis containing blood vessels, glands, and 63/1
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MATERIALS AND METHODS 2 This is an adaptation, for animal tissue, of the 28
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Skin taken from the abdomen and back of adult frogs procedure of Kellenberger te .la (7). Specimens 8/26
anaR( pipiens and Rana )anaibsetac were the principal fixed in OsO4 or glutaraldehyde followed by OsO4 3.p
tissues studied. For comparison, more limited observa- are treated for 2 hours at room temperature with df b
tions were carried out on abdominal skin from adult 0.5 per cent uranyl acetate in Michaelis buffer. The y gu
amotsytllbmoaA(ardvsa e Pieces (Bufo( stage of skin )mutatcnupmarinus), 55), taken and from taitlasi l approaching fins pithed of ofy oung saalnaimmalasn, der metamorphosis. Xenopus or amplaur-v ae laeds sfnaiionnmarply l ified salt addiptiHves is of necessaryb y the usinas g solution a for diluenatc etate-Veronals taining, is 5.0. for Since thbe oth procedure neithbeutrfhf ee r OsO4 cwalictihuomu t can and be est on 13 Janu
tceantte d OsOta4i ls, were in either fixed for acetate-Veronal la/~ to 2 hours ora t 0 phospha° te in 1 per ugrlauntyalr aldehyde acetate. folIlofw ed the tissue by Ois sO4 fixed in in phosphatOesO 4 or ary 202
buffer, the blocks must be washed 2( to 3 changes 3
buffer (pH 7.6). Other pieces were fixed in 2 per cent
each of 51 minutes) before transfer to the staining
glutaraldehyde (6) in either 0.1 M phosphate or 0.1 M
solution in order to avoid precipitation of uranyl as
cacodylate buffer (pH 7.4) for 4 to 61 hours, fol-
uranyl phosphate. This technique is particularly
lowed by "postfixation" for 2 hours in OsO4. Araldite
effective in demonstrating the stratification of all
or Epon was used for embedding. Some specimens
cellular and intracellular membranes. A certain
were stained in block with phosphotungstic acid
(PTA) or KMnO4 (cf. ,)1 during dehydration, to en- amount of extraction seems to be incurred by the
specimens in the uranyl solution; it is more noticeable
hance the contrast of fibrillar and membranous struc-
after fixation in OsO4 in acetate-Veronal than after
tures, respectively. More recently we have exten-
the other procedures mentioned. Contrast in sections
1 Preliminary reports of these findings have already prepared from blocks processed according to this
been published (4, 5). technique can be further increased by staining.
264 ThE LANaVOJ FO CELL YGOLOIB • EMVLOV ,62 1965
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GSCe:n eral stratum Abbreviations corneum din: dense material in intercellular space /article-p
SG: stratum granulosum er: endoplasmic reticulum (rough surfaced) df/2
SS: stratum spinosum f: cytoplasmic filaments 6/1
SGe: stratum germinativum fl: fusion line /26
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B: basement membrane g: Golgi complex /13
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Co: collagen fibril i: intermediate line in desmosome 28
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D: dermis il: inner leaflet of the cell membrane 8/2
Is: intercellular space ol: outer leaflet of the cell membrane 63
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Zo: zonula ocdudens (occluding zonule) m: mitochondria df b
cm: cell membrane mc: mucous coat y g
".d desmosome n: nucleus ue
cd: composite desmosome r: ribosomes st o
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rod: modified desmosome sg: small secretion granule 13
lg: large secretion granule Ja
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ERUGIF 1 Photomicrograph showing the general organization of the abdominal skin of the frog (Rana ry 2
p/p/ens). The epidermis (between arrows 1 and )2 is a stratifiede pithelium with 1 to ~ layers of partially 0
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cornified, squamous cells in the stratum corneum (SC), 1 to 3 layers of cuboidal or polyhedral cells in
the stratum granulosum (SG) and stratum spinosum (SS), and a basal layer of cuboidal or columnar cells
in the stratum germinativum (SGe). The dermis is divided into a loose layer, the stratum spongiosum (Ssp)
(between arrows 2 and )3 which adjoins the epidermis, and a deeper more compact layer, the stratum
compactum (SCo) (arrow 3 to bottom of field). The s. spongiosum contains blood vessels (V), bodies of
glands (G), pigment cells (p), and various fibrillar and cellular connective tissue elements. The s. com-
pactum (SCo) consists of a series of undulating layers, each composed of bundles of collagen fibrils in
parallel array. From layer to layer the direction of the bundles changes by ~90 °. A discontinuous layer
of extracellular, material, which stains red with this procedure, forms a series of arches (unnumbered
arrows) between the two dermal strata.
Near the top of the epidermis at either edge of the field are two mitochondria-rich cells (M) which
have a fusiform shape and extend from the middle layers to the s. corneum. The one on the right has a
relatively pale cytoplasm.
Specimen fixed in 1 per cent OsOa in phosphate buffer (pH' ~nd embedded in Araldite. One-micron
section stained with azure II-methylene blue. X 900. "~
various fibrillar or cellular connective tissue ele- of cytoplasmic flaments become more abundant
ments (Fig. 1). The epidermis contains one or two and more tightly packed (Figs. 3 and 6). At the
outer layers of partially cornified, squamous cells same time, their rough-surfaced endoplasmic
(stratum corneum), 3 to 4 intermediate layers of reticulum becomes more voluminous and their
cuboidal or polyhedral cells (stratum granulosum Golgi complexes more elaborate, with the number
and stratum spinosum), and a basal layer of cuboidal and size of the cisternae increasing. Multivesicular
or columnar cells (stratum basale or germinatioum) bodies also increase in number, and the cells
(Fig. 1). acquire dense bodies of irregular size and shape
with a heterogeneous content. Morphologically,
Stratum germinativum
these elements represent lysosomal derivatives,
The cells of the s. germinativum (Fig. 2) 3 have i.e., residual bodies, and lytic and autolytic vac-
the usual set of subcellular components in rela- uoles (13). Moreover, they give a positive reaction
tively small quantities: a few mitochondria and for acid phosphatase in specimens fixed in glu-
rough-surfaced elements of the endoplasmic reticu- taraldehyde, incubated in Gomori medium and
lure, some free ribosomes, a relatively rudimentary examined in the electron microscope following the
Golgi complex with one or two small flattened general procedures given in reference 6. The same
cisternae and associated vesicles, and variable applies to similar bodies found in the s. corneum. D
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numbers of small (pinocytic?) vesicles found es- The frequency of all these bodies increases de- wn
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pecially near the lateral cell membranes. In addi- cidedly in the s. granulosum (Fig. 6) in which many ad
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tmiiocn, filamtehnetrse is a (forwemlelr ly developed called system tonofilaments of cytoplas- (cf. atuytpoel ytic seen in vacuoles muhivesicular contain bodies, small as vesicles, well as ofr ecog- the d from
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10)) grouped in bundles and anchored in either nizable remnants of cell components, mostly ribo- ttp
desmosomal or "basal" plates (11, 12) (Fig. 18). somes (Fig. 6, inset). This finding suggests that ://ru
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Tmheem brane basal surface which of forms these a cells continuous rests on a layer, basement 500 wtihterhe is autolytic extensive vacuoles fusion of as multivesiculacrel l differentiation bodies ress.org
to 700 A thick, at the dermal-epidermal boundary progresses. /jcb
S(Ftigrs. atum 2 and spinosum 18). and Stratum granulosum granules The cells of two of distinctthe se stratat ypes: coonnet ain type cytiso plasmic relatively /article-pd
small (100 to 150 m/z) in size; is spherical, ovoid f/2
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As the cells of the epidermis differentiate and or tear-like in shape; has a finely particulate, /1
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move from the germinal layer out to the .s spinosum moderately dense content; and has a distinct 63
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and s. granulosum, they flatten and their bundles limiting membrane. Granules of this type seem 38
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3 Fig. 2 is from the skin of the toad, but the basic to be formed within Golgi cisternae (Fig. 4). 08/2
organization is the same as in the frog. Mature granules first appear randomly distributed 63
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ERUGIF ~ Base of the epidermis in toad skin, showing portions of four large cells of the Ja
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.s germinativum, a highly indented dermal-epidermal junction, and part of the dermis ua
(D) which forms a series of micropapillae containing mostly collagen fibrils. The base- ry 2
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ment membrane (B) forms a continuous (~500 A) layer which closely parallels the basal 3
cell membranes of the germinative cells and separates dermal and epidermal elements.
The intercellular spaces (Is) between the basal cells are expanded and of complicated
geometry due to extensive interdigitation between microvilli and other processes of ad-
joining cells. The spaces are open toward the basement membrane since the apposed cell
membranes remain separated by a distinct (~00 to 400 A) gap (arrows). Note the low
frequency of the desmosomes )d( on the lateral cell aspects. The cytoplasm of the basal
cell contains prominent bundles of filaments (f) as well as mitochondria (m) and numerous
free ribosomes. The nuclei (n) show large nucleoli (no) and dense, peripheral chromatin
masses.
Specimen fixed in 1 per cent Os04 in phosphate buffer (pH 7.6) and embedded in ArM-
dire. Section doubly stained with uranyl and lead. X 14,000.
266 TR~ JOURNAL OF CELL BIOLOGY • VOLUME ~6, 1965
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M. G. FARQUHAR DNA G. E. PALADE Cell Junctions in Amphibian Skin 267
throughout the cytoplasm of cells immediately in the two layers located just beneath the s.
distal 4 to the s. germinativum; they are particularly corneum, and, in contrast to the granules of the
numerous in the cells just beneath the outermost first type, are most numerous in the outer granular
granular layer, where they accumulate along the layer. There is some suggestive evidence that they
distal front of the cell (Figs. 3 and 4); and are are formed within Golgi vacuoles (Fig. 4), but
usually absent from the outermost granular layer. ~ their ultimate fate is not altogether clear. They
In this connection, it should be mentioned that decrease in number as the cells keratinize, and
whereas the intercellular spaces in the s. germi- some, at least, are retained in such cells and finally
nativum, spinosum, and granulosum appear "empty" included and apparently destroyed in autolytic
(i.e., they are occupied by a material of low den- vacuoles.
sity), a substance similar in density and texture to
Stratum corneum
the granule content fills the intercellular spaces
between the s. granulosum and s. corneum and within Cornified elements occur only distal to the
the latter (Figs. 3, 5, and 6). The orientation of intercellular spaces filled with electron-opaque
the granules along the distal cell surface, and their material. By implication, keratinization of a cell
disappearance from the cytoplasm at a level where moving into the s. corneum begins between the
a similar material appears in the intercellular discharge of its own small granules and the dis- D
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spaces suggest that the granule content is dis- charge of the same granules from the cell moving n
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charged into, and fills the intercellular spaces. behind it into the s. granulosum. Cornification ad
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Images suggesting granule discharge by membrane occurs gradually, for all stages from partially to d fro
fusion have been occasionally encountered. fully keratinized elements can be found in this m h
membrane-limiA ted, second type of is muchc ytoplasmic larger in granule, size (300 also to la(yFeigrs.. Fu3, lly 5 tok eratinized 7), are comceplllse tely appear occupied very dense by ttp://rup
9h00i gher m~), density more anvda riable finer in texture, shape, hasa nd a occurs content pri- of cofy toplasmic cell organelles filaments, which and are constoamietni mes only included remnants in ress.org
marily in the perinuclear cytoplasm (Figs. 3 and autolytic vacuoles. Partially cornified cells have a /jcb
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4). These larger granules are found predominantly cytoplasmic matrix of a variable but generally rticle
lower density, and their organelles are more" -p
4The terms "distal" and "proximal" are used numerous and less altered (Fig. 6). Some of these df/2
tdherromuaglh out structures the text in troe lation refer ttoo thet he basepomseintito n of mem- epi- cells still have recognizable ribosomes, usually 6/1/26
brane. clumped in the remaining spaces between fila- 3/1
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5 They occasionally occur in small numbers along the ments and cell organelle remnants (Fig. 6). Ribo- 82
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distal front of these ceils. somal particles are absent or not recognizable in 08
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ERUGIF 3 Outer front of the frog epidermis showing several cornified (8Ct and SC~) 13
and granular (SG1 to SGs) layers. A dense filamentous layer (mc) or "mucous coat" is Ja
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seen along the outer skin surface. The cytoplasm of the cornified cells appears dense and ua
without clearly recognizable cytoplasmic organelles. The granular cells contain numerous ry 2
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clusters of ribosomes (r), a few mitochondria (m), and occasional cisternae of the endo- 23
plasmic reticulum (er) and of the Golgi complex (g), concentrated mostly near the nucleus.
In addition, they contain numerous dense granules of two distinct types: one type (lg)
is relatively large (300 to 900 m#), and is found in greatest numbers in the perinuclear
cytoplasm of the outermost granular cell (SG1). The other type of granule (sg) is smaller
(100 to 051 m/~); is absent from the outer granular cell; and is concentrated near the
distal cell membranes of the second and third granular layers (SG2 and 8Ga).
Numerous desmosomes )d( can be made out between cells of the lower layers. The
intercellular spaces (Is) contain a material of low density (in all probability, just em-
bedding plastic), except between the outer granular and inner cornified cells where they
are occupied by a material (din) of high density.
Specimen and section preparation as for Fig. .~ X 17,000.
268 THE ~*OURNAL OF CELL BIOLOGY • VOLUME ~6, 1965
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M. G. RAIfUQRAF DNA G. E. PAI,ADE Cell Junctions in Amphibian Skin 269
fully keratinized cells. When cells move from the Cell Membranes
s. granulosum to the s. corneum, they acquire a shell
The cell membranes of all except the cornified
of dense cytoplasmic material along the cell mem-
elements are of the thin, asymmetrical type,
brane (vide infra) and the latter becomes thicker,
with a total thickness of about 70 to 80 A, and an
as already noted by Rhodin and Reith (14) in
outer leaflet thinner and less dense than the inner
mammalian keratinizing epithelia.
one. As pointed out elsewhere (1), the outer
Special Cell Types within the Epidermis leaflets of membranes of this type are difficult to
see in OsO4-fixed specimens, but can be regu-
M ( itoehondria- Rich Cells)
larly demonstrated after fixation or staining in
As is well known, various specialized cell Wpes KMnO4 (compare Figs. 31 and 18, which were
occur scattered throughout the epidermis. In the not stained in KMnO4, with Figs. 14, 15, and 71
frog, occasional lymphocytcs, macrophages, and which were). The outer leaflets are especially
pigment cells can be recognized, particularly in well demonstrated after staining in block with
the deep strata. In addition, we have regularly uranyl acetate (Figs. 16, 19, and 20). In tissues
encountered another type of cell, located pre- fixed in glutaraldehyde followed by OsO4, the
dominantly in the s. spinosum and s. granulosum. usual asymmetry is reversed by this procedure
D
Such cells are typically shaped like an elongated (see Figs. 61 and 19). ow
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flask with a perikaryon in the s. spinosum and a The entire cell membrane of the cornified cell loa
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thick main process extending up to the s. corneum is of the thick (100 A), nearly symmetrical type ed
where it sometimes branches into a number of (cf. 1). The dense outer leaflet is clearly visible in from
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secondary processes. Cytologically, the most dis- OsO4- or glutaraldehyde-fixed tissue stained only ttp
tinctive features of these cells are a relatively light with lead (Figs. 7 to 9 and 11). The light layer ://ru
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matrix and the presence of large numbers of stands out sharply because of the generally high ss.o
mitochondria, especially in the main process. density of the cornified cell cytoplasm (Figs. 8 rg
We refer to these elements as "mitochondria-rich and 9). This thick membrane of the cornified /jcb/a
cells," as done by Choi (15) for similar cells found cells is backed all around the cell surface by a rticle
in the toad bladder (15, 16). They contain the continuous (200 to 300 A thick) shell of dense -pd
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usual set of cell organelles, but usually no secretion material located in the cytoplasm immediately 6/1
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granules, and are attached to neighboring epider- adjacent to the inner leaflet (Figs. 7, l0 to 13). 6
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mal cells by desmosomes which are similar to, but The surface membrane of the cells of the outer 38
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noticeably less frequent than, those between usual cornified layer is covered by a dense fibrillar 80
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epidermal cells at this level. coating (Figs. 5, 7 to 9, and 1 l) which varies in /26
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ERUGIF 4 Cells from the s. granulosum of the frog epidermis, to illustrate the two dif- Ja
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fine rent size, granule populamteimonbsr ane-bonnded, variable in found in the cells shape, of thaen d middle occurs stratpar.i marily One in type the (lg) perinuclear is large ary 2
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cytoplasm. The other type (sg), also membrane-limlted, is much smaller in size, ovoid or 23
tear-shaped, and accumulates along the cell membrane at the distal cell front, facing the
expanded intercellular spaces (Is).
Numerous free ribosomes, bundles of cytoplasmic filaments, and mitochondria (m)
occupy the remaining cytoplasm. Some of the filaments (f) converge on desmosomes )d(
at the cell junctions. Part of a Golgi complex is seen at g on the lower left. Near it there
is an irregularly shaped body (fg) with a moderately dense content which is believed to
represent a "condensing vacuole," i.e., a granule in formation within the Golgi complex.
Part of another Golgi complex ,)~g( consisting of a pile of 5 to 6 flattened cisternae, is
seen in an adjoining cell on the upper right. A small granule appears to be forming by
"budding" from the tip of the innermost Golgi cisterna (arrow).
Specimen and section preparation as for Fig. .~ <) ~8,000.
270 THE LANRUOJ FO CELL YOOLOIB • EMULOV ,6~ 1965
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M. G. RAHUQRAF: DNA G. E. PALADE Cell Junctions in Amphibian Skin 271
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Fmmm 5 Outer front of the frog epidermis showing portions of a cornified (8C) and of a granular cell /1
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(SG). The intercellular spaces between the two layers are occupied by a material (din) of high dcasity 63
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aud rclativcly finc tcx-turc whi¢~ completely fills the~ spaces and outlines their connections. The cort- 3
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tours of thc spathes arc more elaborateo n thc s. musodznarg sidc owing to the presence of numerous pseudo- 28
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podia (ps) projecting from the cell surface. of Remmqnt_s cytoplasmic organelles (arrow) arc barely visiblc /2
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in the cytopla.qm of the cornified cells. A fl~lffy mucous coat (me) can be made out along the outer mem- .p
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brane of the same t~lls. Several bacteria ruc~co in close aaso~iation with the outer skin surface. f b
Specimca fixcd in 2 pcrccnt OsO4 in acetate-Veronal buffer (pH 7.6) with sucrose, dehydrated in y gu
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alcohol, stained with PTA, and embedded in Araldite. Section doubly stained with uranyl and lead. st o
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thickness and appearance according to the method CeU Junctions ry 2
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of fixation and staining: it appears particularly 23
NI EHT MUTARTS :M-DENROC On the outer front
thick and well preserved after fixation in phos-
of the epidemis, near the point at which the mem-
phate-buffered OsO, or in glutaraldehyde fol- brmlcs of two adjacent ceils turn from thc dista]
lowed by ()sOu, but is less abundant after fixation to the lateral cell surface, the outer leaflets of the
in acetate-Veronal-buffered OsO(. Its fibrillar converging membranes come together and. fuse
texture is particularly evident after PTA staining. into a single dense band ,'~30 to 40 A thick (Fig s.
This layer presumably corresponds to the "mucous 7 to 9). The two fused membranes measure 170
coats" previously described in the epithelium of to 180 A across. The extraneous fibrillar coating
the toad bladder (15, 16), gastric (17) and intes- of each membrane stops at, or shortly before, the
tinal (18) mucosa. line of mergence..Usually, the band of fusion runs
272 Tim ~l.~_~mrtol, FO C~.r.r, YOOLOIB • ~athVLOV 26, 1965