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Proc Natl Acad Sci U S A
2000 Apr 11;978:4076-81. doi: 10.1073/pnas.97.8.4076.
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Action of the Caenorhabditis elegans GATA factor END-1 in Xenopus suggests that similar mechanisms initiate endoderm development in ecdysozoa and vertebrates.
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In ecdysozoan protostomes, including arthropods and nematodes, transcription factors of the GATA family specify the endoderm: Drosophila dGATAb (ABF/Serpent) and Caenorhabditis elegans END-1 play important roles in generating this primary germ layer. end-1 is the earliest expressed endoderm-specific gene known in C. elegans and appears to initiate the program of gene expression required for endoderm differentiation, including a cascade of GATA factors required for development and maintenance of the intestine. Among vertebrate GATA proteins, the GATA-4/5/6 subfamily regulates aspects of late endoderm development, but a role for GATA factors in establishing the endoderm is unknown. We show here that END-1 binds to the canonical target DNA sequence WGATAR with specificity similar to that of vertebrate GATA-1 and GATA-4, and that it functions as a transcriptional activator. We exploited this activity of END-1 to demonstrate that establishment of the vertebrate endoderm, like that of invertebrate species, also appears to involve GATA transcriptional activity. Like the known vertebrate endoderm regulators Mixer and Sox17, END-1 is a potent activator of endoderm differentiation in isolated Xenopus ectoderm. Moreover, a dominant inhibitory GATA-binding fusion protein abrogates endoderm differentiation in intact embryos. By examining these effects in conjunction with those of Mixer- and Sox17beta-activating and dominant inhibitory constructs, we further establish the likely relationships between GATA activity and these regulators in early development of the vertebrate endoderm. These results suggest that GATA factors may function sequentially to regulate endoderm differentiation in both protostomes and deuterostomes.
Figure 1
END-1 is a sequence-specific DNA-binding protein and transcriptional activator. (A) Electrophoretic mobility shift assay with a radiolabeled GATA probe and nuclear extracts from COS cells transfected with XGATA-4 or END-1 cDNA. Cold competitor oligonucleotides (100× excess) either had the same sequence as the probe (GATA) or replacement of this sequence by GATC or CTTA. In the last two lanes, reactions included preimmune or End-1-specific rabbit antiserum. (B) Fold activation of the GATA-driven luciferase reporter construct αD3 (black bars) or the mutant reporter construct αD4 (in which the GATA site in the promoter is replaced by CTGA; gray bars) after transfection of plasmids encoding END-1, END-1Δ3′, XGATA-4, or XGATA-5.
Figure 2
Induction of endoderm in Xenopus ectodermal explants by GATA proteins. Embryos were injected with 250–500 pg of mRNA into the animal pole, and animal cap explants were cultured until sister tadpoles reached Stage 40. RNA was analyzed by RT-PCR (A, C, and D) or whole-mount in situ hybridization (B and E) for endodermin (Edd), IFABP, LFABP, Sox17α, XlHbox8, brachyury (Xbra), and Xtwist. Equal input of RNA samples was established by RT-PCR for elongation factor (EF)-1α or ODC. A low background of endodermin and Xsox17α is occasionally observed in controls.
Figure 3
A dominant inhibitory GATA-binding protein represses endoderm development in Xenopus embryos. (A) Design of the dominant-inhibitory construct End-1∷EnR fusing the putative DNA-binding domain of END-1 (aa 106–221) to the transcriptional repression domain (aa 2–298) of Drosophila Engrailed. (B) Phenotype of Xenopus embryos at stage 42/43 after microinjection of 100 pg of End-1∷EnR or control (End-1δ3′) mRNA into the vegetal hemisphere at the one-cell stage. (C) Rescue of lethal developmental effects of vegetal expression of End-1∷EnR by coinjection of wild-type End-1. Two-cell embryos were injected in the vegetal hemisphere of each blastomere with 400 pg of Sox17β∷EnR or End-1∷EnR mRNA, and 400 pg of either End-1 (panel 3) or End-1δ3′ (panels 1 and 2) mRNA. (D) Down-regulation of mature markers of Xenopus endoderm in vegetal explants treated with End-1∷EnR. Two-cell embryos were injected as above with 250 pg of Sox17β∷EnR or End-1∷EnR mRNA and 250 pg of End-1 (lane 6) or End-1δ3′ (lanes 3–5) mRNA. Vegetal explants were cultured until the equivalent of Stage 35. RT-PCR for ODC serves as a control for equal RNA input.
Figure 4
GATA activity functions early in vertebrate endoderm development. One-cell Xenopus embryos were injected with 250 pg of mRNA, animal cap explants were cultured until sister tadpoles reached Stage 11.5, and RNA was analyzed by RT-PCR for the early endoderm markers Mixer and XSox17α and for EF-1α.
Figure 5
Placing GATA activity within the context of Mixer/Mix.3 and Sox17 functions in Xenopus endoderm development. One-cell embryos were injected with 200 pg of endoderm-inducing mRNAs, and 400 pg of either mRNA-encoding dominant-inhibitory EnR fusion proteins or EF-1α “filler†mRNA. Animal cap explants were cultured and RNA analyzed by RT-PCR for Sox17α when sister tadpoles reached Nieuwkoop-Faber Stage 15 (A), for endodermin (Edd) at the equivalent of Stage 40 (B), and for ODC as a loading control.
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