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Weber H
,
Symes CE
,
Walmsley ME
,
Rodaway AR
,
Patient RK
.
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The endoderm gives rise to the gut and tissues that develop as outgrowths of the gut tube, including the lungs, liver and pancreas. Here we show that GATA5, a zinc-finger transcription factor, is expressed in the yolk-rich vegetal cells of Xenopus embryos from the early gastrula stage onwards, when these cells become committed to form endoderm. At mid-gastrula stages, GATA5 is restricted to the sub-blastoporal endoderm and is the first molecular marker for this subset of endodermal cells so far identified. We show that GATA4 and GATA5 are potent inducers of endodermal marker genes in animal cap assays, while other GATA factors induce these genes only weakly, if at all. When injected into the dorsal marginal zone, GATA5 respecifies prospective mesoderm towards an endodermal fate, thereby disrupting the convergence and extension movements normally undergone by the dorsal mesoderm. The resulting phenotype is very similar to those seen after injection of dominant negative versions of the FGF-receptor or the T-box transcription factor, Xbra and can be rescued by eFGF. The ability of GATA5 to respecify ectodermal and mesodermal cells towards endoderm suggests an important role for GATA5 in the formation of this germlayer. In animal cap assays, GATA5 is induced by concentrations of activin above those known to induce dorsal mesoderm and heart, in an FGF-independent manner. These data indicate that the emerging view for endodermal induction in general, namely that it is specified by high levels of TGF-beta in the absence of FGF signalling, is specifically true for sub-blastoporal endoderm.
Fig. 1. GATA5 is expressed in prospective endoderm during Xenopus gastrulation. Whole-mount in situ hybridisations of embryos viewed from the vegetal pole (A,C,E) and in situ hybridisations on sections of corresponding stages (B,D,F), using a GATA5-specific probe. No GATA5-specific staining is obvious in blastula stage (st. 9) embryos (A,B). At the early gastrula stage (st. 10.25), staining is seen in bottle cells and in the sub-blastoporal endoderm (C,D). In the advanced gastrula (st. 11.5), GATA5 accumulates in the sub-blastoporal endoderm and appears to be excluded from the involuting mesendoderm at the dorsal and ventral side of the embryo (E,F). Arrows in C,D,F mark the dorsal blastoporal lip.
Fig. 2. GATA5 is restricted to the sub-blastoporal endoderm at mid- gastrula stages. In situ hybridisation of serial sections of stage 11.5 embryos using GATA5 (A) and cerberus (B) or stage 11 embryos using GATA5- (C), goosecoid- (D) and Sox17a- (E) specific probes. GATA5 overlaps with cerberus in the dorsoanterior yolk-rich cells (arrowheads), but not in more posterior located smaller cells, which appear to represent the leading edge of the involuting mesendoderm (arrows in A,B). GATA5 is excluded from prechordal plate mesendoderm stained by goosecoid and the supra-blastoporal endoderm, which is strongly stained by Sox17a (arrows in C,E).
Fig. 4. GATA5 respecifies prospective ectoderm towards endoderm. Whole-mount in situ hybridisation to endodermin of GATA5- (A), GATA2a- (B) or Myf5- (C) injected animal caps at stage 31 equivalent. Injection of GATA5 mRNA induces patches of ectopic endodermin. (D,E) GATA2a (top row) or GATA5 (bottom rows) injected embryos at stage 22, showing GATA5 induced ectopic pigment (D, arrow) or stage 36 embryos showing GATA5 induced loss of eyes (E). Fertilised eggs were injected in the animal pole with 100 pg Myf5 or GATA2a RNAs or 50 pg GATA5 RNA. beta-Galactosidase-stained larvae at stage 34 (F) injected in the animal pole with RNA coding for nuclear localised β-galactosidase on its own (top row) or coinjected with 50 pg GATA5 RNA (bottom rows). Arrowheads mark the areas with strongest β- galactosidase staining in GATA5-injected embryos. Vibratome cross sections of β-galactosidase-stained control (G) or GATA5-injected embryos (H) through the midgut region (arrowheads in F). β-Galactosidase staining is restricted to the ectoderm in control embryos and covers several cell layers in GATA5- injected embryos (arrowheads in G and H). Ectopic pigment is marked with an arrow in H. (I,J) Control (top row) or GATA5-injected embryos (bottom rows) at stage 34 after β-galactosidase staining (I,J) and in situ hybridisation with an endodermin specific probe (J). In control embryos, lacZ-positive (turquoise) and endodermin-expressing cells (purple) do not overlap. In GATA5-injected embryos, many lacZ-positive cells in the ventral region (arrowheads) and in isolated patches (arrows) express endodermin.
Fig. 5. GATA4 and GATA5 induce endodermal marker genes in animal cap explants. (A) RT-PCR of whole embryos (we), uninjected or with 50 pg Myf5 or GATA5 injected animal caps at stage 33/34, using primers for the indicated genes. GATA5 induces Xlhbox8 and IFABP. To assess for genomic contamination an RT reaction without reverse transcriptase was performed on whole embryo RNA (we-RT) and general contamination was assessed by taking a mock tube through RNA preparation, RT-reaction and PCR. ODC served as a loading control and sample titrations were performed to ensure that signals were in the linear range (oading. (B) In vitro translation of GATA1 6 mRNAs in reticulolysate lysate, demonstrating that all GATA RNAs translate with similar efficiencies. (C) RT-PCR of whole embryos (we), uninjected (lane 4) or with 50 pg of GATA1, 2a, 3, 4, 5 or 6 RNAs injected (lane 5-10) animal caps at stage 11 using primers for the indicated genes. GATA4 and 5 clearly induce Sox17α and HNF1β and downregulate cytokeratin. Controls were as for part A.
Fig. 6. GATA5 respecifies prospective mesoderm towards endoderm. Whole-mount in situ hybridisations of control embryos (A-C top row, D-G,L) or GATA5 injected embryos (A-C bottom row, H-K, M) at stage 11 (A-C) or the stages indicated (D-M) using the indicated probes. Embryos were injected with either 50 pg GATA2a or 50 pg GATA5 RNA per blastomere as indicated in the figure and described in the text. Expression of Xbra (A) and chordin (B) is reduced in GATA5 injected embryos (arrows), while Sox17α is ectopically induced
(C, arrows). Embryos injected at the four-cell stage into two dorsal blastomeres with RNA coding for nuclear β-galactosidase on its own (D-G) or coinjected with 50 pg GATA5 RNA (H-K) were stained for β- galactosidase at the indicated stages. Dorsal injection of GATA5 disrupts convergence and extension movements. The dorsal blastopore is marked by arrowheads, axial mesoderm by arrows and anteriormesendoderm by asterisks. (L,M) Whole mount in situ hybridisations of β-galactosidase stained embryos in E and I, respectively, with a Sox17α specific probe. (N,O) Vibratome cross sections of stage 12 embryos shown in L and M, respectively. In control embryos the majority of lacZ-positive cells (turquoise) do not express Sox17α (purple), while in GATA5-injected embryos lacZ-positive cells also express Sox17α (arrows in O; cytoplasm as well as nuclei now blue). Arrowheads mark the dorsal blastopore lip and the arrows in L,N indicate the Sox17α positive dorsoanterior endoderm. Embryos in A-E,H-J are viewed from the vegetal pole. Anterior is towards the left in the other whole mount panels.
Fig. 7. The GATA5 induced open blastopore phenotype can be rescued by eFGF or BMP4. Embryos were injected into the marginal zone of two dorsal blastomeres at the 4-cell stage with 25 pg GATA5 RNA per blastomere (bottom row in A; panel B),
25 pg GATA5 and 1 fg eFGF (C), or 25 pg GATA5 and 250 pg BMP4 (D) and cultured until stage 30. Embryos in the bottom row of A and in B, except for the top leftembryo in B, are characterised by an open blastopore. 1 fg eFGF rescues to the wild type phenoptype (C) and embryos rescued with 250 pg BMP4 are either wild type or ventralised (D), as assessed by the loss of head structures (arrows). ((A) In situ hybridisation with a cardiac actin specific probe at stage 30 demonstrates the reduced somitic tissue in GATA5-injected embryos compared to control embryos).
Fig. 8. High concentrations of activin induce GATA5 in an FGF independent manner. (A) RT-PCR of animal caps at stage 11.5 equivalent incubated with the indicated concentrations of activin. (B) Stage 11.5 animal caps injected with RNAs for a control receptor (d50) or a dominant negative FGF-receptor (XFD) before treatment with the indicated concentrations of activin. 500 pg d50 or XFD RNAs were injected into the animal pole of each blastomere of two- cell embryos. Controls were as described in the legend to Fig.5A.
Fig. 9. Injection of a dominant negative activin receptor (dnXAR) inhibits GATA5 expression. Double in situ hybridisation for Xbra (arrow in A) and GATA5 (arrowhead in A) on stage 11.5 embryos. These were injected with either 1 ng β-galactosidase (A) or 1 ng dnXAR (B) RNA vegetal of the marginal zone into one blastomere of two-cell embryos.
