Effect of the Oesophageal Mesenchyme on the Differentiation of the Digestive-tract Endoderm of the Chick Embryo

9 Pages·1993·3.3 MB·English

by  Susumu Matsushita

ZOOLOGICAL SCIENCE 10: 439-447 (1993) 1993ZoologicalSocietyofJapan
Effect of the Oesophageal Mesenchyme on the Differentiation
of the Digestive-tract Endoderm of the Chick Embryo
Susumu Matsushita
Department ofBiology, Tokyo Women's Medical College,
Tokyo 162, Japan
—
ABSTRACT The endoderm of various regions of the digestive tract of 6-day chick embryos was
culturedin vitro in recombinationwith theoesophagealorotherdigestive-tract mesenchymesof6-day
chick embryos, and the differentiation of the epithelium was examined with attention given to the
appearanceofthestratifiedsquamousepitheliumcharacteristicofthematureoesophagealepithelium.
The oesophageal endoderm developed stratified squamous epithelium in high frequency in the
presence of the oesophageal mesenchyme, but in low frequency when recombined with the other
mesenchymes, which suggested the supporting action ofthe oesophageal mesenchyme on the proper
differentiationoftheoesophagealendoderm. Amongtheotherendodermsofdigestivetract, onlythe
proventricular endoderm developed stratified squamous epithelium, when cultured in recombination
with the oesophageal mesenchyme. The squamous cells of this epithelium contained numerous
tonofilaments as those ofthe stratified squamous epithelium ofthe intact oesophagus. The ability to
elicit stratified squamous epithelium in the proventricular endoderm was shown to be confined to the
oesophageal mesenchyme. Thus, the oesophageal mesenchyme was likely to induce oesophagus-type
differentiation at least in the proventricular endoderm.
endoderm to become an epithelium forming tubu-
INTRODUCTION
lar glands with prominent mucus production,
It is well known that the epithelial-mesenchymal whichresembledtheintactgizzardepithelium [12],
interaction is prerequisite for the development of though it is not yet proved whether the chemical
the digestive tractofthe Aves [1, 14, 24]. Numer- nature ofthe mucus produced in the recombinates
ous studies have shown that the mesenchyme of wasthesameasthatofthegizzardmucus. Thus,it
the digestive tract can induce some endoderms to may be possible that the mesenchymes of most
differentiate in a mesenchyme-dependent fashion. regions of the digestive tract of the young chick
The duodenal mesenchyme of the young chick embryo possess the ability to induce the region-
embryo was reported to elicit, in the gizzard specificdifferentiationatleastinsomeendoderms.
endodermassociatedtoit,theintestine-likesimple The effect of the oesophageal mesenchyme on
columnar epithelium forming villus- or previllous the differentiation of the endodermal epithelium
ridge-like structures, which developed brush- was also studied so far. The undifferentiated
border structure and its enzymes [2, 5-7]. The allantoic endoderm was reported to become pluri-
proventricularmesenchymeinducedtheassociated stratifiedepithelium showingsome resemblance to
gizzard or oesophageal endoderm to form com- the embryonic oesophageal epithelium when re-
pound gland and to produce embryonic pepsi- combined with the oesophageal mesenchyme of
nogenorpepsinogenmRNA,whichwasspecificto theyoung avian embryo and cultured in vitro orin
the differentiated proventricular epithelium [3, vivo on the chorioallantoicmembrane ofthe chick
18-20]. Recently, it was demonstrated that the embryo [21,22], butitbecameintesinalepithelium
gizzard mesenchyme could induce the duodenal with sucrase as well as cloacal epithelium when
culturedlongenoughtoachievefulldifferentiation
Accepted March 30, 1993 in the coelomic cavity of the chick embryo [11].
Received December28, 1992 The oesophageal mesenchyme was also reported
440 S. Matsushita
to elicit the yolk sac endoderm to become pluri- digestive tract of
6-day chick embryo
stratified epithelium [9], but the intestinal epithe- collagenase
lium with brush border and its enzyme activities treatment
also developed in the same type ofrecombination
[9, 10]. The recombination study usingthe epithe-
lia and mesenchymes of digestive tract demon-
strated the heterotypic differentiation of some
endoderms cultured in recombination with the
intact recombinate
oesophageal mesenchyme [23]. Thus, it is still fragment
obscurewhetherthe oesophageal mesenchymehas
the inductive ability of mesenchyme-dependent
differentiation. The present study intended to
examine whether the oesophageal mesenchyme
has the region-specific inductive influence or not, explant
by the recombination experiment carried out
underin vitroculturecondition. Asforthemarker
of oesophageal differentiation, appearance of the agar medium ,- id medium
stratified squamous epithelium was examined, 1/2day
since this epithelium was the characteristics ofthe Fig. 1. Diagram showing the mode of combination of
fully differentiated oesophageal endoderm and epithelia and mesenchymes of the digestive tract.
was far from being mistaken for other types of OE, oesophagus; PV, proventriculus; GZ, gizzard;
epithelium. Though histological is the marker of SI, small intestine; epi., epithelium; mes., mesen-
chyme.
oesophageal-type differentiation adopted in this
study, the stratified squamous epithelium could be
regarded as closely related to the cytodifferentia- only the posterior part was used for the proven-
tion of the oesophageal epithelial cells, since it tricularendodermortheintactproventricularfrag-
appears only in the terminally-differentiated ment, for the purpose of eliminating the possible
oesophagusofthechicknearandafterhatching [4, contamination of the apparent oesophageal tissue
8, 15, 16]. (Fig. 1). For the oesophageal mesenchyme of
olderstages, the fragment just anteriortothe crop
MATERIALS AND METHODS wastakenfrom8-day, 10-dayand 12-dayembryos.
The isolated endoderm and mesenchyme were
recombined after washing in serum-supplemented
Animal
(50%) Tyrode's solution and then in Tyrode's
Embryos ofthe White Leghorn chick were used solution.
throughout the experiments.
In Vitro culture technique
Preparation oftissuefragments After cultivation on an agar medium for about
Themiddleportionoftheoesophagusandofthe half a day at 38°C to ascertain the coherence of
small intestinal fragment between the bile duct endoderm andmesenchyme [23], the recombinates
entrance and the yolk stalk, and the apex of were transfered onto a millipore filter (Nihon
gizzard body were removed from 6-day-old chick Millipore Kogyo K. K., pore size 0.8/urn). The
embryos (Fig. 1), and the endodermal epithelium filter with the recombinate was placed on a stain-
and mesenchyme were separated with the aid of less-steel grid in a small culture dish containing a
collagenase (Worthington, Code CLS, 0.03% in liquid medium up to the level of the membrane
Tyrode's solution at 38°C for 1 h). Whole portion filter, and the recombinate was cultured at 38°C in
of the proventriculus was taken from 6-day chick 95% air and 5% CO;>. The intact fragments of
embryosforthe proventricularmesenchyme, while digestive tract were also cultured in the same way.
Inductive Effect of Oesophageal Mesoderm 441
Theculture mediumconsistedof75% Medium 199 turedfor 12or 18days(Table 1, Fig. 2). Columnar
with Earle's salt (Nissui Seiyaku), 20% 12-day epithelium producing AB-stained mucus that
chickdigestive organ- andeye-free embryo extract might probably correspond to the oesophageal
(50% inTyrode'ssolution), 5% fetalbovineserum mucous gland epithelium was also found in the
(GIBCO Lab.), and antibiotics (penicillin 100 oesophageal explants (Fig. 2). In the proventricu-
units/ml, streptomycin 100//g/ml). The medium lar explants, columnar epithelium with apical AB-
was changed every third day. Since the stratified staining differentiated, part of which occasionally
squamous epithelium was shown in a preliminary developed glandular invagination (Fig. 3). Giz-
experiment to appear in the intact oesophageal zard explants often formed lots of tubulargland-
explant after 12 days' cultivation in a liquid like structures or intra-epithelial invaginations,
medium, explants were cultivated for 12 days or which were lined with AB-positive mucus-
longer up to 18 days. secretory epithelium. In the small-intestinal ex-
plants, simple columnar epithelium with goblet
Histology
cells appeared. Thus, the appearance ofthe stra-
After cultivation, the explants were fixed with tified squamous epithelium was confirmed to be
ice-cold 95% ethanol for 4hr. Six/um paraffin specific to the oesophageal-type differentiation.
sections were stained with alcian blue (AB)- When cultured in the presence of the oeso-
hematoxylin. Some explants were fixed in 2.5% phageal mesenchyme, the proventricular en-
M
glutaraldehyde in 0.1 sodium cacodylate buffer doderm as well as the oesophageal endoderm
at 4°C for 2 h and then in 1% Os04 in the same developedthe stratifiedsquamousepitheliumafter
buffer at 4°C for 1 h. They were dehydrated with 12 days' cultivation (Table 1). It appeared in the
ethanols and embedded in Embedding Resin recombinates with the oesophageal endoderm in
(TAAB). Ultra-thin sections were stained with wide area, while in those with the proventricular
methanolicsolutionofuranylacetateandwithlead endoderm it appeared in various degree, which
citrate. varied from only a small number of a tiny focus
consisting of several squamous cells to a rather
RESULTS wide area (Figs. 4, 5). Transmission electron mi-
croscopy showed that numerous tonofilaments
were contained in the squamous cells of this stra-
Appearanceofstratifiedsquamousepitheliuminthe tified squamous epithelium, and ciliated cells were
digestive-tractendoderm in the intactdigestive-tract often found (Figs. 6a, b). These were characteris-
fragments and in the recombinates with the tics found in the intact oesophagus during normal
oesophageal mesenchyme
development [4, 15] and in the explants of intact
In the explants of intact digestive-tract frag- oesophagus in thepresent study (Figs. 7a, b). The
ment, thestratifiedsquamousepitheliumappeared possibility that the stratified squamous epithelium
only in the explants of oesophageal fragment cul- found in these recombinates originated from the
Table 1. Appearance of stratified squamous epithelium in the digestive-tract
endodermintheintactdigestive-tractfragmentsandintherecombinateswith
the oesophageal mesenchyme cultured in vitro
Grafts developing stratified squamous epithelium
Origin of endoderm
Intact fragments Recombinates
Oesophagus 16/17 (94%) II 9 (78%)
Proventriculus 0/17 ( 0%) 20/21 (95%)
Gizzard 0/11 ( 0%) 0/11 ( 0%)
Small intestine 0/15 ( 0%) 0/12 ( 0%)
442 S. Matsushita
ojS
-•
Inductive Effect of Oesophageal Mesoderm 443
oesophageal epithelial cells contaminated in the As shown in Table2, the proventricular en-
oesophageal mesenchyme was denied by the fol- doderm developed stratified squamous epithelium
lowing findings. Serial sections of the isolated only in the recombinates with the oesophageal
oesophageal mesenchyme revealed the absence of mesenchyme, in which AB-positive mucous
oesophageal epithelial cells, and the cultivation of epithelium also appeared. TheAB-stained epithe-
the mesenchyme alone for 12 days or longer never lium with occasional glandular invagination differ-
developed stratifiedsquamous epithelium. In rare entiated in the presence of the proventricular
cases (1 out of 12), the isolated mesenchyme mesenchyme, and the mucus-secretory epithelium
contained a small epithelial vesicle of unknown forming intra-epithelial invaginations or tubular
origin in the presumptive adventitial tissue outside gland-like structures developed in the presence of
of the muscular layer, and this epithelial cells did thegizzardmesenchyme. Intherecombinateswith
not become stratified squamous epithelium after the small-intestinal mesenchyme, AB-positive
cultivation. In the other region of the recombi- epithelium was less frequently found.
nateswith the proventricularendoderm and in the The oesophageal endoderm developed stratified
recombinates with the gizzard endoderm, mucus- squamous epithelium in high frequency in the
secretingepitheliumwith abundant undulations or presence ofthe oesophageal mesenchyme. In the
shortinvaginationsappeared (Figs. 4, 5, 8). These presence of other digestive-tract mesenchymes,
epithelial cells may be regarded as the differenti- stratified squamous epithelium was found in low
ated proventricular or gizzard mucous cells, but frequency (Table2). In the recombinates with the
discrimination from the oesophageal mucous cells proventricular mesenchyme, AB-positive epithe-
was impossible in the present study. The small- lium with occasional glandular invagination differ-
intestinal endoderm became simple columnar entiated but even a small focus of stratified
epithelium with goblet cells. In a few explants, squamous epithelium rarely developed (Table2,
non-goblet mucous cells were also found. Fig. 9). In the recombinates with the gizzard
mesenchyme, AB-positive stratified or simple
Effect of various digestive-tract mesenchymes on epithelium and stratified squamous epithelium
the differentiation of the proventricular and appeared (Fig. 10). In the recombinates with the
oesophageal endoderm small-intestinal mesenchyme, AB-positive colum-
The proventricular and oesophageal endoderms nar epithelium appeared. Stratified squamous
were found to develop stratified squamous epithe- epithelium was found only in small areas of a few
lium in the presence of the oesophageal mesen- recombinates.
chyme. Then, the effectofothermesenchymeson
these endoderms was analyzed.
Fig. 2. An explant of intact oesophageal fragment cultured for 18 days. Stratified squamous epithelium (s) and
mucous epithelium (arrows) differentiated. X230.
Fig. 3. An explant ofintact proventricularfragment cultured for 18 days. A glandular invagination (g) is formed,
and the epithelium is often stained with alcian blue in the apical portion (arrowheads). X230.
Fig.4. A recombinate of proventricular endoderm and oesophageal mesenchyme cultured for 18 days. Typical
stratified squamous epithelium (s) and mucous epithelium (arrow) are seen. X230.
Fig. 5. Small foci (arrows) of stratified squamous epithelium that appeared in the mucous epithelium found in a
recombinate ofproventricular endoderm and oesophageal mesenchyme cultured for 18 days. X230.
Fig. 6. Transmission electron microscopy of an epithelium of a recombinate of proventricular endoderm and
oesophagealmesenchymeculturedfor18days. a. Cellsinthemiddlelayerofthestratifiedsquamousepithelium,
which are interconnected with each other by desmosomes (arrowheads) and contained abundant tonofilaments
(t). n, nucleus. X24000. b. A ciliated cell. X12000.
7. Transmissionelectronmicroscopyofanepitheliumofanexplantofintactoesophagealfragmentculturedfor
18 days. a. Cells in the middle layer ofthe stratified squampous epithelium. Desmosomes (arrowheads) and
numerous tonofilaments (t) are seen. X24000. b. A ciliated cell. X12000.
444 S. Matsushita
Effect of the oesophageal mesenchyme of aged
embryos
As shown in Table3, the stratified squamous
epithelium appeared only in the presence of the
oesophageal mesenchyme of 6-day embryos and
not in the presence of the mesenchyme of older
embryos, suggesting that the inductive ability of
the oesophageal mesenchyme declined rather
'^s>""* v _ "^^""^i^'-'ai. quickly after6 days ofincubation. The epithelium
in the recombinateswith the mesenchyme ofolder
embryos became mucus-secreting epithelium with
abundant undulations or short invaginations.
Table3. Appearance of stratified squamous
epithelium in the 6-day proventricular endoderm
cultured in the presence of oesophageal mesen-
chyme of various developmental stages
Stage Grafts developing stratified
squamous epithelium
6-day 20/21 (95%)
8-day 0/10 0%)
(
10-day 0/ 8 ( 0%)
12-day 0/ 6 ( 0%)
Fig. 8. A recombinate of gizzard endoderm and
oesophageal mesenchyme cultured for 18 days.
Mucus-secreting epithelium with abundant undula-
tions or short invaginations appeared. X230.
Fig. 9. A recombinate of oesophageal endoderm and
proventricular mesenchyme cultured for 18 days.
Columnar epithelium with apical alcian blue-
staining forming glandular invaginations appeared.
X230.
Fig. 10. A recombinate ofoesophageal endoderm and
gizzard mesenchyme cultured for 18 days. A small
areaofstratifiedsquamousepithelium(s)isseen. X
230.
Table2. Appearanceofstratifiedsquamousepithelium inthe proventricularand
oesophageal endoderms cultured in the presence of various digestive-tract
mesenchymes
Grafts developing stratified squamous epithelium
Origin of mesenchyme
Proventricular endoderm Oesophageal endoderm
Oesophagus 20/21 (95%) 23/26 (88%)a
Proventriculus 0/21 ( 0%)a 1/18 ( 6%)
Gizzard 0/11 ( 0%) 6/11 (55%)
Small intestine 0/12 ( 0%) 3/11 (27%)
a, Explants of the intact fragments as well as the recombinates were included.
Inductive Effect of Oesophageal Mesoderm 445
thedigestive-tractendoderms, andwasfoundtobe
DISCUSSION
confined to the endoderm of the proventriculus,
The present study clearly showed that the stra- which situated next to the oesophagus, at least in
tified squamous epithelium appeared in high fre- the 6-day-old chick embryo. That the competence
quencyin the proventricularendodermcultured in for the inductive action of the mesenchyme was
recombinationwith the oesophageal mesenchyme, high in the endoderm of its neighboring region(s)
demonstrating the ability of the oesophageal was reported also in the case of intestinal dif-
mesenchyme to induce the differentiation of stra- ferentiation induced by the duodenal mesenchyme
tified squamous epithelium. The previous study [5]oroftheinductionofpepsinogenorpepsinogen
[23] also reported an occasional appearance ofthe mRNA by the proventricular mesenchyme [3, 20].
stratified squamous epithelium in the same type of As for the high responsiveness ofthe proventricu-
recombination, but in the present study the stra- larendoderm, thefollowingthreepossibilitiesmay
tified squamous epithelium appeared in much be considered; 1) some of the proventricular en-
higher frequency, which might be due to a better dodermal cells might be induced to change their
culture condition or a longer cultivation period. fate and to become oesophageal-type epithelium;
The ultrastructural features of the stratified 2) undifferentiated cells capable to adopt oeso-
squamous epithelium found in the recombinates phageal or proventricular fate according to the
were shown to resemble those of the stratified influence of the mesenchyme might be contained
squamous epithelium of intact oesophageal ex- in the proventricularendoderm; 3) asmallnumber
plants, suggesting that both epithelia might repre- of oesophageal cells which are to be lost through
sent the same cytodifferentiated state. As shown some selective mechanism(s) during normal de-
in the present study, the appearance of stratified velopment in the proventriculus might be distri-
squamous epithelium in the proventricular en- butedevenintheposteriorpartoftheproventricu-
dodermwas confined to the recombinateswiththe lus. It needs further extensive analysis to know
oesophagealmesenchyme. Thus, itwas likelythat which is the case.
the oesophageal mesenchyme had the region- The present study also showed that the dif-
specificmesenchyme-dependentinducingabilityto ferentiation of the oesophageal endoderm was
elicitthe oesophagus-like differentiation atleast in influencedbythemesenchyme. Theappearanceof
the proventricular endoderm. The small focus of stratified squamous epithelium was low in the
stratified suamous epithelium would have de- oesophageal endoderm cultured in recombination
veloped from a single induced cell or a group ofa with the mesenchyme other than that of the
fewinducedcellsin the recombinedproventricular oesophagus. Thus, the other mesenchyme might
endoderm. The inductive ability of the oeso- provide only an inappropriate condition for the
phageal mesenchyme to elicit stratified squamous differentiation of the stratified squamous epithe-
epithelium in the proventricular endoderm was liumor exert an inhibitoryeffect on itsdifferentia-
shown to decline rather quickly after 6 days of tion. The proventricular mesenchyme was known
incubation, while the ability to elicit pluristratified to possess an ability to induce proventricular dif-
epithelium inthe allantoicendodermwasreported ferentiation [3, 20], and this effect may have
to be retained until the 11th days of incubation contributed to rare appearance of the stratified
[21]. This discrepancy might be due to the differ- squamous epithelium in the recombinateswith the
ence in the responding tissue and/or to the differ- proventricular mesenchyme. On the contrary, the
ence in the examined histological marker in the oesophageal mesenchyme provided asuitable con-
two experiments. dition for the normal differentiation of the
Regional difference in the responsiveness ofthe oesophagealendoderm. Whetherthiseffectofthe
digestive-tract epithelium to the inductive stimuli oesophageal mesenchyme on the oesophageal en-
ofthe mesenchyme was repetitivelyreported [3, 5, doderm may be a nutritional one or be related to
20, 23]. The responsiveness to the oesophageal theinductiveinfluence asexertedoverthe proven-
mesenchyme also showed regional difference in tricular endoderm is not known. The appearance
446 S. Matsushita
of the stratified squamous epithelium even in the induced by the duodenal mesenchyme. J Embryol
recombinateswith the mesenchyme otherthan the Exp Morphol 82: 163-176
oesophageal one, though in a rather low frequen- 6 Ishizuya-Oka A, Mizuno T (1985) Chronological
cy, suggests that the oesophageal endoderm ofthe analysis of the intestinalization of chick stomach
endoderm induced in vitro by duodenal mesen-
6-day chick embryo has already been determined
chyme. Roux's Arch Dev Biol 194: 301-305
to some extent, as suggested by the presence of 7 Ishizuya-Oka A, Mizuno T (1992) Demonstration
self-differentiation potency in the oesophageal en- of sucrase immunoreactivity of the brush border
doderm of this stage [17]. induced by duodenal mesenchyme in chickstomach
At least at 6 days ofincubation and probably at endoderm. Roux's Arch Dev Biol 201: 389-392
later stages during normal development, the 8 IveyWD, EdgarSA (1952) Thehistogenesisofthe
esophagus and crop of the chicken, turkey, guinea
oesophageal mesenchyme would interact mainly
fowl and pigeon, with special reference to ciliated
with the oesophageal endoderm likely to be en- epithelium. Anat Rec 114: 189-211
dowed with the oesophageal fate. Since the 9 Masui T (1981) Differentiation of the yolk-sac
oesophageal endoderm of 6-day embryos has also endoderm under the influence ofthe digestive-tract
a potency of heterotypic differentiation into mesenchyme. J Embryol Exp Morphol 62: 277-289
proventricular-type [3, 20], the inductive effect of 10 MasuiT(1982) Intestinalizationofthearea-vitellina
the oesophageal mesenchyme might play, in con- endoderm cultured in association with digestive-
tract mesenchymes. J Embryol Exp Morphol 72:
cert with its probable supporting action that pro- 117-124
vides the appropriate condition(s), an important 11 Matsushita S (1984) Appearance of brush-border
role in assurring the correct differentiation of the antigens and sucrase in the allantoic endoderm
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mesenchymes. Roux's Arch Dev Biol 193: 211-218
(1975) [13].
12 Matsushita S (1988) Effect of the mesenchyme on
the differentiation of the duodenal and gizzard
ACKNOWLEDGMENTS epithelia ofthe chick embryo. Zool Sci 5: 1264
13 MizunoT(1975) Unehypothesesurl'organogenese
The author wishes to express his deep gratitude to du tractus digestif. C R Soc Biol 169: 1096-1098
Professor Kazuo Utsugi of Tokyo Women's Medical 14 MizunoT, Yasugi S (1990) Susceptibility ofepithe-
College. lia to directive influences of mesenchymes during
organogenesis: Uncoupling of morphogenesis and
cytodifferentiation. CellDifferDevelop31: 151-159
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