Specification of the endoderm precursor, the E cell, in Caenorhabditis elegans requires a genomic region called the Endoderm Determining Region (EDR). We showed previously that end-1, a gene within the EDR encoding a GATA-type transcription factor, restores endoderm specification to embryos deleted for the EDR and obtained evidence for genetic redundancy in this process. Here, we report molecular identification of end-3, a nearby paralog of end-1 in the EDR, and show that end-1 and end-3 together define the endoderm-specifying properties of the EDR. Both genes are expressed in the early E lineage and each is individually sufficient to specify endodermal fate in the E cell and in non-endodermal precursors when ectopically expressed. The loss of function of both end genes, but not either one alone, eliminates endoderm in nearly all embryos and results in conversion of E into a C-like mesectodermal precursor, similar to deletions of the EDR. While two putative end-1 null mutants display no overt phenotype, a missense mutation that alters a residue in the zinc finger domain of END-3 results in misspecification of E in approximately 9% of mutant embryos. We report that the EDR in C. briggsae, which is estimated to have diverged from C. elegans approximately 50--120 myr ago, contains three end-like genes, resulting from both the ancient duplication that produced end-1 and end-3 in C. elegans, and a more recent duplication of end-3 in the lineage specific to C. briggsae. Transgenes containing the C. briggsae end homologs show E lineage-specific expression and function in C. elegans, demonstrating their functional conservation. Moreover, RNAi experiments indicate that the C. briggsae end genes also function redundantly to specify endoderm. We propose that duplicated end genes have been maintained over long periods of evolution, owing in part to their synergistic function.

The endoderm in the nematode Caenorhabditis elegans is clonally derived from the E founder cell. We identified a single genomic region (the endoderm-determining region, or EDR) that is required for the production of the entire C. elegans endoderm. In embryos lacking the EDR, the E cell gives rise to ectoderm and mesoderm instead of endoderm and appears to adopt the fate of its cousin, the C founder cell. end-1, a gene from the EDR, restores endoderm production in EDR deficiency homozygotes. end-1 transcripts are first detectable specifically in the E cell, consistent with a direct role for end-1 in endoderm development. The END-1 protein is an apparent zinc finger-containing GATA transcription factor. As GATA factors have been implicated in endoderm development in other animals, our findings suggest that endoderm may be specified by molecularly conserved mechanisms in triploblastic animals. We propose that end-1, the first zygotic gene known to be involved in the specification of germ layer and founder cell identity in C. elegans, may link maternal genes that regulate the establishment of the endoderm to downstream genes responsible for endoderm differentiation.

POP-1, a Tcf/Lef-1-like target of the convergent Wnt and MAP kinase (MAPK) signaling pathways, functions throughout Caenorhabditis elegans development to generate unequal daughters during asymmetric cell divisions. A particularly prominent such asymmetric division occurs when the EMS blastomere divides to produce MS, a mesoderm precursor, and E, the sole endoderm progenitor. POP-1 allows mesoderm development in the MS lineage by repressing the endoderm-promoting end-1 and end-3 genes. This repression is relieved in the E lineage by Wnt/MAPK signaling, which results in phosphorylation and export of POP-1 from the E nucleus. Here, we report that, in addition to repressing E development in MS, POP-1 also functions positively in endoderm development, in conjunction with the well-characterized endoderm-promoting SKN-1-->MED regulatory cascade. While removal of POP-1 alone results in derepression of endoderm development in the MS lineage, mutations in several genes that result in impenetrant loss of endoderm are strongly enhanced by loss of pop-1 function. A Lef-1-like binding site is essential for activation of an end-1 promoter fusion, suggesting that POP-1 may act directly on end-1. Thus, POP-1 may generate developmental asymmetry during many cell divisions in C. elegans by reiteratively switching from repressive and activating states. Furthermore, we report that the Caudal-like homeodomain protein PAL-1, whose role in early embryogenesis was thought to be exclusive specification of mesectodermal development in the lineage of the C blastomere, can act with POP-1 to activate endoderm specification in the absence of the SKN-1-->MED transcriptional input, accounting for the impenetrance of mutants lacking SKN-1 or MED-1,2 activity. We conclude that the combined action of several separate transcriptional regulatory inputs, including SKN-1, the MEDs, PAL-1, and the Wnt/MAPK-activated form of POP-1, are responsible for activating end gene transcription and endoderm development.

The END-1 GATA factor has been implicated in specifying endoderm in Caenorhabditis elegans and is the earliest known zygotic protein expressed in the lineage of E, the clonal endoderm progenitor. We report that ubiquitous end-1 expression during a critical period in embryogenesis causes all non-endodermal lineages to produce endoderm instead of ectoderm and/or mesoderm. END-1 expression bypasses the requirement for maternal SKN-1 and the maternal Wnt signaling pathway in endoderm formation. This suggests that a primary function of these maternal factors is to regulate zygotic end-1 expression, which is then sufficient to initiate the entire program for endoderm development. Received September 2, 1998; revised version accepted October 19, 1998. How and when during development is a cell committed to a particular fate? Studies on embryos of the nematode Caenorhabditis elegans reveal that blastomeres appear to become committed to distinct patterns of differentiation during the first few embryonic cleavages by the combined action of cell-cell interactions and cell-autonomous maternal factors (for review, see Schnabel and Priess 1997;Bowerman 1998). These events control the identity of the embryonic founder cells, each of which gives rise to a unique set of differentiated cell types. The ability to contribute to each germ layer is differentially allocated to the founder cells. For example, production of endoderm, consisting of only the intestine, is confined to a single founder cell, the E blastomere, in the seven-cell embryo (Sulston et al. 1983). The E cell identity is determined by maternally contributed factors, including the SKN-1 transcription factor (Bowerman et al. 1992(Bowerman et al. , 1993Blackwell et al. 1994) and the Wnt signaling pathway, which causes the E cell parent, EMS, to undergo an asymmetric division that segregates endoderm and mesoderm lineages (Rocheleau et al. 1997;Thorpe et al. 1997). When either maternal SKN-1 activity or the maternal Wnt signal transduction pathway is eliminated, the E cell adopts the fate of another early blastomere and endoderm development does not occur.Specification of endoderm in C. elegans also requires zygotic expression of a genomic region identified by chromosomal deletions (Zhu et al. 1997). Although there appear to be multiple genes in this region that regulate endoderm development (Zhu et al. 1997; E. NewmanSmith, M. Maduro, and J. Rothman, unpubl.), the end-1 gene can restore endoderm production to embryos carrying deletions of the region. end-1 is normally expressed specifically in the E cell and early E lineage. end-1 encodes an apparent zinc finger transcription factor of the GATA factor family (Zhu et al. 1997). GATA factors have also been implicated in regulating endoderm development in Drosophila (Reuter 1994;Rehorn et al. 1996), and in the developing endoderm of vertebrates (Laverriere et al. 1994;Soudais et al. 1995). Moreover, at least two other GATA factors, in addition to END-1, function in endoderm differentiation in C. elegans (Fukushige et al...