XB-ART-36327
Mech Dev
August 1, 2007;
124
(7-8):
518-31.
Retinoic acid-mediated patterning of the pre-pancreatic endoderm in Xenopus operates via direct and indirect mechanisms.
Abstract
Early patterning of the
endoderm as a prerequisite for
pancreas specification involves retinoic acid (RA) as a critical signalling molecule in
gastrula stage Xenopus embryos. In extension of our previous studies, we made systematic use of early embryonic endodermal and mesodermal explants. We find RA to be sufficient to induce
pancreas-specific gene expression in dorsal but not
ventral endoderm. The differential expression of retinoic acid receptors (RARs) in
gastrula stage endoderm is important for the distinct responsiveness of dorsal versus
ventral explants. Furthermore, BMP signalling, that is repressed dorsally, prevents the formation of pancreatic precursor cells in the
ventral endoderm of
gastrula stage Xenopus embryos. An additional requirement for
mesoderm suggests the production of one or more further
pancreas inducing signals by this
tissue. Finally, recombination of manipulated early embryonic explants, and also inhibition of RA activity in whole embryos, reveal that RA signalling, as it is relevant for
pancreas development, operates simultaneously on both mesodermal and endodermal germ layers.
PubMed ID:
17643968
Article link:
Mech Dev
Species referenced:
Xenopus laevis
Genes referenced:
gal.2
hhex
ins
mixer
pdia2
pdx1
ptf1a
tbxt
Article Images:
[+] show captions
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Fig. 5. Blocking RA signalling specifically in either the endoderm or the mesoderm of whole embryos interferes with pancreas specification. To block RA signalling specifically in the endoderm, DN-RARα2 was injected into the vegetal pole of 8-cell stage embryos together with the lineage tracer β-gal. In order to block RA signalling in the mesoderm, DN-RARα2 and β-gal were co-injected into the marginal zone of the two dorsal blastomeres in 4-cell stage embryos. The injected embryos were cultivated until control siblings had reached stage 36 or stage 39, and subjected to β-gal staining before whole mount in situ hybridization was carried out. (panels 1–3) Lateral view of stage 39 embryos stained for XlHbox8 expression (anterior towards the left), (panels 4–6) Ptf1a/p48 expression, (panels 7–9) insulin expression, (panels 10–12) XPDIp expression, (panels 13–15) Xhex expression. Red arrowheads demarcate the position of the ventral pancreas, white arrowheads the position of the dorsal pancreas. The statistics obtained are indicated at the right bottom corner of each panel.
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pdia2 (protein disulfide isomerase family A, member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior left, dorsal up.
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Fig. 2. Differential expression of RARs contributes to the competence to activate pancreas gene expression in response to RA. (a) Stage 11 embryos were dissected to generate dorsal endoderm (DE), dorsal mesoderm (DM), ventral endoderm (VE) and ventral mesoderm (VM). The isolated explants were collected immediately after isolation and subjected to semi-quantitative RT-PCR analysis for expression of various xRARs and xRXRs, as indicated. xRARα2.1 and xRARγ2.1 were found to be predominantly expressed in the dorsal portion of the endoderm and mesoderm, respectively. Xbra and Mixer are markers for controlling the purity of the isolated mesodermal and endodermal explants. (b) xRARγ2.1, either alone or in combination with xRARα2.1, is able to induce a pancreatic fate in the VEM explants in the presence of its co-receptor xRXRβ. xRARγ2.1 (500 pg/embryo), either alone or in combination with xRARα2.1 (500 pg/embryo) or/and xRXRβ (500 pg/embryo) was injected into four vegetal cells of 8-cell stage embryos. Ventral endoderm/mesoderm (VEM) explants were isolated from the injected embryos at stage 11. The isolated VEM was treated with or without 5 μM RA for 1 h right after isolation and cultured until control embryos had reached stage 39. These explants were subjected to semi-quantitative and quantitative RT-PCR analysis for pancreas marker expression. An average of 10 explants per assay was used for each RNA preparation.
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