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We have analyzed two gene products expressed in the early endoderm of Xenopus laevis: Xlhbox-8, a pancreas-specific transcription factor and intestinal fatty acid binding protein (IFABP), a marker of small intestinal epithelium. Expression of the pancreas marker relies on cell signaling mediated by both the TGF-beta and FGF classes of secreted peptide growth factors, whereas, expression of the more posterior small intestinal marker does not. Endodermal explants devoid of mesoderm express both markers in a regionalized manner. Cortical rotation is required for the expression of the more anterior marker, Xlhbox-8, but not for the small intestinal marker, IFABP. These findings suggest that endodermal patterning is dependent, in part, on the same events and signals known to play important roles in mesodermal development. Furthermore, inhibition of TGF-beta signaling in the endoderm leads to ectopic expression of both mesodermal and ectodermal markers, suggesting the TGF-beta signaling may play a general role in the segregation of the three embryonic germ layers.
Fig. 1. Autonomous expression of endodermal markers. (A) Animal caps or vegetal poles were dissected from blastula embryos (stage 8-9) as shown. Vegetal pole explants were also dissected into dorsal and ventral halves. Explants were cultured in isolation until midtailbud stages (stages 33-36). RNA levels were assayed by RT-PCR. (B) Vegetal pole explants express Xlhbox-8, IFABP and insulin, but not the mesodermal markers cardiac actin, Xtwist or globin. Animal caps do not autonomously express either mesodermal or endodermal markers. (C) The capacity for Xlhbox-8 expression is localized to the dorsal side of the vegetal pole whereas IFABP expression is seen in both the dorsal and ventral vegetal pole explants. WE lanes represent RT-PCR products where mRNA from an unmanipulated embryo was used for cDNA synthesis. The WE-RT lanes represent assays in which reverse transcriptase was left out of the cDNA synthesis step with whole embryo RNA as a template. This serves as a control for DNA contamination. Elongation factor 1 alpha (EF-1a) is included as a control to insure that similar amounts of RNA are used in each lane.
Fig. 2. Expression patterns of Xlhbox-8 and IFABP
both in whole embryos and vegetal pole explants.
(A) Sagittal section through a stage 38 embryo that
was subsequently immunostained with an anti-
Xlhbox-8 antibody (Gamer and Wright, 1995). A
thin band of endodermal nuclei stretching from the
dorsal pancreas, through the duodenum and down to
the ventralpancreas is indicated by the bracketed
area. (B) Sagittal section through a stage 38 embryo
that was in situ hybridized to an anti-sense probe
that detects IFABP mRNA. (C) Section of a stage
38 vegetal pole explant that was probed with an
anti-sense IFABP probe. (D) The adjacent section to
that in C except that a control sense IFABP probe
was used in the in situ. (E) Section of a stage 38
explant that was subsequently immunostained with
an anti-Xlhbox-8 antibody. Positively stained nuclei
are contained within the bracketed area which
represents the dorsally derived side of the explant.
Abbreviations: n, notochord; p, pancreas; ph,
pharnyx; si, small intestine.
Fig. 3. Xlhbox-8 expression relies on TGF-b and FGF signals. (A) At the 1-cell
stage, synthetic mRNA encoding inhibitory proteins were injected into the vegetal
pole. At stage 9, vegetal pole explants were dissected and cultured to the mid-tailbud
period (stage 33-36). RNA levels were assayed by RT-PCR. (B) Both a truncated
activin (tAR) and truncated FGF receptor (XFD) block expression of Xlhbox-8 but a
truncated BMP receptor has no effect. IFABP expression is unaffected by all three
truncated receptors. 4-5 nanograms of mRNA for each receptor was used in this
experiment and the result does not differ from experiments using 400-500 picograms
of mRNA except that the truncated BMP receptor does not efficiently induce cardiac
actin expression at 400 picograms (data not shown). (C) Induction of the ectodermal
markers NCAM and epidermal cytokeratin in vegetal poles expressing a truncated
activin receptor. The activin-specific antagonist follistatin (XFS) neuralizes vegetal
poles but does not induce expression of epidermal cytokeratin. 2 nanograms and 500
picograms of mRNA were used in the truncated activin receptor and follistatin
injections, respectively. (D) A dominant negative form of RAS (2 nanograms) blocks
expression of Xlhbox-8 but has no effect on IFABP expression. The activin-specific
antagonist follistatin has no effect on the expression of either IFABP or Xlhbox-8.
Neither follistatin nor the dominant negative RAS induce expression of the
mesodermal marker cardiac actin. (E) The follistatin mRNA used in parts C and D of
this figure blocks induction in animal caps by injected activin mRNA as judged by
expression of the pan-mesodermal marker Brachyury (Xbra). 1 nanogram of mRNA
encoding follistatin was co-injected with 2 picograms of mRNA encoding activin
bB. WE lanes represent RT-PCR products where mRNA from an unmanipulated
embryo was used for cDNA synthesis. The WE-RT lanes represent assays in which
reverse transcriptase was left out of the cDNA synthesis step with whole embryo
RNA as a template. This serves as a control for DNA contamination. Elongation
factor 1 alpha (EF-1a) is included as a control to insure that similar amounts of RNA
are used in each lane.
Fig. 4. Induction of endodermal markers by activin and Vg1 protein. (A) At the 1-cell
stage synthetic mRNA encoding a TGF-b ligand was injected into the animal pole.
Animal caps were dissected from blastula stage embryos (stage 9) and cultured in
isolation until the mid-tailbud period (Stage 33-36). Gene expression was monitored by
RT-PCR. (B) To express the mature forms of Vg1 and BMP-2, mRNA encoding chimeric
forms of each gene were used in our injections. In the case of Vg1, a chimeric mRNA was
used where the pro region of BMP-2 was fused to the mature region of Vg1 (B-Vg1)
(Thomsen and Melton 1993). For BMP-2, the pro region of activin bB was fused to the
mature region of BMP-2 (A-BMP-2). The B-Vg1 chimera induces expression of cardiac
actin, IFABP and Xlhbox-8 in animal caps consistent with previously published results
that demonstrated the ability of B-Vg1 to induce axial mesoderm (Thomsen and Melton
1993). The A-BMP-2 chimera induces expression of globin, a ventral mesodermal
marker, in animal caps. Neither IFABP nor Xlhbox-8 expression is efficiently induced in
animal caps by BMP-2. The amount of RNA injected is indicated in picograms.
(C) Activin can induce cardiac actin, IFABP (data not shown) and Xlhbox-8 in animal
caps. The amount of injected RNA is indicated in femtograms. WE lanes represent RTPCR
products where mRNA from an unmanipulated embryo was used for cDNA
synthesis. The WE-RT lanes represent assays in which reverse transcriptase was left out
of the cDNA synthesis step with whole embryo RNA as a template. This serves as a
control for DNA contamination. Elongation factor 1 alpha (EF-1a) is included as a
control to insure that similar amounts of RNA are used in each lane.
Fig. 5. (A) Explants dissected from UV-ventralized
blastula embryos fail to express Xlhbox-8 but express
normal levels of IFABP. The dorsoanterior index (D.A.I.)
was determined at middle tailbud stages and in the
experiment shown the UV irradiation was sufficient to
give a D.A.I. of zero (explants dissected from embryos
with a D.A.I. of 2, 3 and 4 all express Xlhbox-8).
Explants dissected from embryos that were UV irradiated
during the first cell cycle, followed by injection of 50
picograms of B-Vg1 mRNA into a single vegetal
blastomere at the 8-cell stage express both Xlhbox-8 and
IFABP. In UV-ventralized whole embryos both Xlhbox-8
and IFABP are not expressed. Injection of B-Vg1
recovers IFABP and Xlhbox-8 expression in whole UV
embryos. (B) Endodermal explants dissected from
embryos injected at the 1-cell stage, into the vegetal pole,
with 80 picograms of B-Vg1 mRNA. A slight increase in
Xlhbox-8 expression results with no effect on IFABP
expression. As a control, in the same experiment animal caps were dissected from embryos that were injected into the animal pole with 80
picograms of B-Vg1 mRNA at the one cell stage. WE lanes represent RT-PCR products where mRNA from an unmanipulated embryo was
used for cDNA synthesis. The WE-RT lanes represent assays in which reverse transcriptase was left out of the cDNA synthesis step with whole
embryo RNA as a template. This serves as a control for DNA contamination. Elongation factor 1 alpha (EF-1a) is included as a control to
insure that similar amounts of RNA are used in each lane.
