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Dev Growth Differ
1998 Feb 01;401:75-84. doi: 10.1046/j.1440-169x.1998.t01-5-00009.x.
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Regulation of ectodermal differentiation in Xenopus laevis animal caps treated with TPA and ammonium chloride.
Sotgia C
,
Fascio U
,
Pennati R
,
De Bernardi F
.
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Animal caps isolated from Xenopus laevis embryos at the blastula stage were treated sequentially with NH4Cl, a known cement gland inducer, and with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a known neural inducer. The two artificial inducers were also used in reverse order to see if they can mimic the natural inducers acting during the progressive determination of the ectodermal organ. Immunofluorescence and whole-mount in situ hybridization were used to study the expression of tubulin, taken to indicate an early step on the pathway of cell elongation, and neural cell adhesion molecule (N-CAM) taken to indicate an early step in the determination of the nervous system. The expression of XCG-1, a marker of early specification of the cement gland, was also studied. The results showed that the two artificial inducers can mimic the effects of the natural inducers in animal cap explants. The TPA behaves like a neural inducer, reducing the number and the extension of the cement gland when added to the medium in addition to NH4Cl, before or after NH4Cl treatment. In the process of cement gland/neural induction, it is possible to redirect the ectoderm already specified as cement gland to neural tissue, but it does not seem possible to respecify the neural tissue as cement gland. Moreover, the animal caps were also cut into dorsal and ventral parts and the two halves were treated separately. The results were similar to those obtained with treatment of the entire animal cap, suggesting that a dorsal-ventral pattern is not yet established before the gastrula stage, and that in normal embryos there are boundaries between the effects of different inducers.
Fig. 1. Responses of animal cap to a single inducer. (A) Hematoxylin-eosin stained section of animal cap treated with NH4CI showing
the large induced cement gland (arrows) with irregularly arranged cells. (B) Confocal microscope image of an induced cement
gland stained with FITC-conjugated anti-tubulin antibody. The cells are normally elongated with bundles of microtubules (arrow). (C)
TPA-treated animal cap showing induced neural tissue inside the explant (arrow). (D) Confocal microscope images of bundles of
microtubules inside elongated cells of a TPA-induced neural tube. Bars, 20 um.
Fig. 2. Confocal microscope images of cement glands stained with FITC-conjugated anti-tubulin antibody. (A) Cement gland of a
control embryo at stage 37. (B) Cement gland induced in animal cap by treatment with NH4CI, showing bright fluorescence at the apex
of the elongated cells. (C) Small and poorly differentiated cement gland in a TPA-treated animal cap. Epidermally differentiated cells
with cilia (arrow) are present with cement gland cells. Bars, 20 um.
Fig. 3. Sequential treatment of animal caps. (A) NH4CI and TPA-treated animal cap showing a small, well differentiated cement gland
(CG) at the surface and an induced neural tube (NT) inside. Section stained with hematoxylin-eosin. (B) Confocal microscope image
of bundles of microtubules in the elongated cells of the induced cement gland stained by FITC-conjugated anti-tubulin antibody. (C)
TPA- and NH4CI treated animal cap showing an induced neural tube inside the explant. Groups of not well elongated cells are present
on the surface. (D) Confocal microscope image of the poorly elongated cells. Bars, 20 um.
Fig. 4. Whole-mount in situ hybridization with XCG-1 antisense RNA (A) Control explant and whole albino embryo at stage 26. (B)
Animal caps NH4CI-treated: large cement glands are blue-stained. (C) TPA-treated animal caps: small cement glands are recognizable
(arrows) (D) NH4CI- and TPA-treated animal caps: cement glands are limited in extension and in percentage. (E) TPA- and NH4CItreated
animal caps: small cement glands similar to those obtained in explants treated with TPA alone. Bar, 200 um.
Fig. 5. Whole-mount in situ hybridization with N-CAM antisense
RNA (A) Control explants. (B) Animal caps TPA-treated: neural
masses are blue-stained (arrows). (C) TPA-treated dorsal half of
animal cap. (D) TPA-treated ventral half of animal cap. Arrows
mark the blue-stained neural masses. Bar, 200 umol.
Fig. 6. Neural induction in TPA-treated dorsal (A. C. E) and ventral (B.D.F) explants of animal caps. (A) Confocal image of anti-tubulin
immunofluorescence. (B) Confocal image of anti-N-CAM immunofluorescence. The same explants in (A) and (B) were embedded. sectioned
and stained with methylene blue. (C) Neural cells are arranged around a lumen. (E) Enlargement of (C). Arrows indicate the
boundary between neural tissue and the surrounding undifferentiated ectoderm. (D.F) The ventral explant in (B) embedded and sectioned.
A mass of neural cells is recognizable inside the explant. Bars. 20 um.
