Reevaluation of the Role of the med-1 and med-2 Genes in Specifying the Caenorhabditis elegans Endoderm

Abstract

The med-1 and med-2 genes encode a pair of essentially identical GATA factor-related transcription factors that have been proposed to be necessary for specification of the C. elegans endoderm (intestine or E lineage) as well as part of the C. elegans mesoderm. med-1 and med-2 are proposed to be the direct downstream targets and the principal effectors of the maternally provided SKN-1 transcription factor; med-1 and med-2 would thus occupy the pivotal interface between maternal and zygotic control of gene expression. The conclusion that med-1 and med-2 are necessary for C. elegans endoderm specification was based on a partially penetrant (50%) loss of endoderm markers produced by RNA-mediated interference (RNAi). To determine whether this partial penetrance reflects: (i) inefficient RNAi against early zygotic transcripts, (ii) experimental uncertainty in the expected level of endoderm loss in skn-1 nulls, or (iii) additional redundancy in the pathway of endoderm specification, we constructed worm strains that segregate embryos lacking both the med-1 gene (because of a gene-specific deletion) and the med-2 gene (using either of two chromosomal deficiencies). Contrary to expectations, we observe that only 3-20% of med-2(ÿ); med-1(ÿ) embryos do not express markers of endoderm differentiation. Furthermore, we found no evidence for a maternal contribution of the med genes to endoderm specification. We conclude that the major pathway(s) for endoderm specification in C. elegans must be independent of the med-1 and med-2 genes. T HE Caenorhabditis elegans endoderm (intestine or E lineage) is clonally derived from a single cell, the E cell, in the eight-cell embryo (Sulston et al. 1983). The early endoderm is one of the few C. elegans lineages for which a plausible specification pathway has been proposed in molecular detail, beginning with maternally provided transcription factors, progressing through several waves of zygotically produced transcription factors, and ending with gene products that function in the terminally differentiated intestine (see review by Maduro and Rothman 2002; see also Baugh et al. 2003Baugh et al. , 2005Robertson et al. 2004). Figure 1 summarizes the regulatory cascade proposed for specification of the C. elegans endoderm. The maternally provided b-ZIP-like transcription factor SKN-1 is essential for correct specification of the fate of the EMS blastomere of the four-cell embryo (Bowerman et al. 1992(Bowerman et al. , 1993. Within the EMS cell, SKN-1 is proposed to directly activate the zygotic expression of two genes called med-1 and med-2, which encode zinc-finger proteins related to GATA-type transcription factors but with atypical binding sites (Maduro et al. 2001;Maduro and Rothman 2002;Broitman-Maduro et al. 2005).These two small intronless genes are 98% identical and, for convenience, are often referred to simply as the med genes (mesendoderm-determining); they are the principal subjects of this article. The med genes are proposed to specify both the C. elegans endoderm and that p...

Specification of theC. elegansMS blastomere by the T-box factor TBX-35

“…We have previously reported that 50% of med-1,2(RNAi) embryos lack endoderm (Maduro et al, 2001). However, a recent study has shown that zygotic loss of med-1 and med-2 results in a weak or undetectable endoderm defect (Goszczynski and McGhee, 2005). We have since found that a significant maternal contribution of the MED genes exists, explaining this discrepancy (M.F.M., G.B.-M., I.…”

“…In the E cell, the MEDs contribute to the activation of the Especifying genes end-1,3, but are dispensable for E specification much of the time Goszczynski and McGhee, 2005;Maduro et al, 2001). We have previously reported that 50% of med-1,2(RNAi) embryos lack endoderm (Maduro et al, 2001).…”

The NK-2 class homeodomain factor CEH-51 and the T-box factor TBX-35 have overlapping function inC. elegansmesoderm development

“…In EMS (the mother of MS and E), SKN-1 activates the zygotic med-1 med-2 (med-1,2) divergent GATA factor gene pair (Coroian et al, 2005;Maduro et al, 2001). Loss of med-1,2 has a similar effect on MS specification as loss of skn-1, but a much weaker effect on E specification owing to parallel contributions to endoderm from SKN-1 and other factors (Goszczynski and McGhee, 2005;Maduro et al, 2005a;Maduro et al, 2001). In MS, MED-1,2 activate the T-box factor gene tbx-35 (Broitman-Maduro et al, 2006).…”

Neither Maternal nor Zygotic med-1/med-2 Genes Play a Major Role in Specifying the Caenorhabditis elegans Endoderm

“…In the first protocol, embryos were collected 7-9 hr following injection of concentrated doublestranded RNA (dsRNA) into the maternal gonad; the rationale for such an early and limited observation period was to more effectively target the transient expression of the med-1/2 genes in the early embryo (Maduro et al 2001;Coroian et al 2006). In the more standard protocol, the RNAi effect is found to be maximally effective from 1 to 3 days following injection (Fire et al 1998;Zipperlen et al 2001;Goszczynski and McGhee 2005;Ahringer 2006); however, these more usual methods of administering RNAi show no effect with the med-1/2 genes (Kamath et al 2003;Goszczynski and McGhee 2005;Sonnichsen et al 2005). A second method of administering med-1/2 RNAi (Maduro et al 2001) was by means of a transgenic heat-inducible promoter driving expression of double-stranded med RNA (Tavernarakis et al 2000).…”

“…Thus, the interpretation of Maduro et al (2007) requires that the putative maternal med transcripts are somehow susceptible to cosuppression and susceptible to RNAi for only a narrow window of several hours following injection (Maduro et al 2001), but are not susceptible to conventional (and usually much more powerful) RNAi effects that routinely persist for days. a med-1 dsRNA was injected at a concentration of $1 mg/ml by the protocol described previously , which is highly effective against either maternal or zygotic transcripts; i.e., it produces 100% arrested embryos/larvae when skn-1/elt-2 dsRNA is injected (Fukushige et al 2005;Goszczynski and McGhee 2005). Because of their high sequence similarity (Maduro et al 2001), injection of med-1 dsRNA should also target med-2 transcripts.…”