Nodal-Dependent Mesendoderm Specification Requires the Combinatorial Activities of FoxH1 and Eomesodermin

Abstract

Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA–binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals.

Transcriptional Control by the SMADs

“…Importantly, in zebrafish, loss of both Foxh1 and Mixer resulted in a phenotype consistent with a substantial reduction of Nodal activity (Kunwar et al 2003). Complete loss of Nodal signaling in zebrafish, however, results in a more severe phenotype, suggesting that additional Smad2-recruiting transcription factors exist (Kunwar et al 2003;Slagle et al 2011).…”

Genome-wide view of TGFβ/Foxh1 regulation of the early mesendoderm program

“…S11). Eomes, a T-box protein, binds Smad2 and has been implicated in Nodal-mediated mesendoderm induction in Xenopus, zebrafish and mammalian epiblast stem cells (Arnold et al, 2008;Picozzi et al, 2009;Slagle et al, 2011;Teo et al, 2011). Recently, ChIP-seq analysis of Eomes has been reported in Xenopus (Gentsch et al, 2013), allowing for a direct test of whether both Smad2/3 and Eomes are bound to the aforementioned 15 genes.…”

“…In mouse, Foxh1 −/− embryos display a spectrum of phenotypes from severe gastrulation defects to milder anterior and midline deficiencies (Hoodless et al, 2001;Yamamoto et al, 2001). In zebrafish, schmalspur and midway mutants, both defective in the foxh1 gene, are deficient in prechordal plate, notochord and some axial mesoderm development (Pogoda et al, 2000;Sirotkin et al, 2000;Slagle et al, 2011). Loss of Foxh1 in Xenopus laevis results in reduced expression of mesendodermal markers, including gsc, nodal1 and mix1, but axial mesoderm was still present, albeit obviously abnormal (Howell et al, 2002;Kofron et al, 2004b;Watanabe and Whitman, 1999).…”

“…The stronger phenotypes from loss of Nodal signaling, compared with foxh1 loss of function (LOF) in frog, fish and mouse, suggests that Nodal signals through both Foxh1-dependent and -independent pathways. Mixer, Foxh1.2, Gtf2ird1, Gtf2i, Tp53, Eomes and Tcf3 (also known as E2a) have been implicated in activation of Nodal targets in Xenopus (Cordenonsi et al, 2003;Germain et al, 2000;Howell et al, 2002;Ku et al, 2005;Ring et al, 2002;Slagle et al, 2011;Teo et al, 2011;Yoon et al, 2011), although the extent that any of these TFs play in the broader regulation of Nodal signaling gene batteries in developing embryos remains ill defined. Thus, despite advances made in dissecting the Nodal molecular cascade, significant gaps remain in our knowledge.…”

Biphasic wnt8a expression is achieved through interactions of multiple regulatory inputs

“…Additionally, while the presence of consensus FoxH1-binding sites may generally be suggestive of Nodal-dependent regulation, FoxH1 is not the sole effector for Nodal-dependent functions in zebrafish. A recent report identified the Tbox protein Eomesodermin (Ryan et al, 1996) as an additional mediator of Nodal-dependent functions in the zebrafish (Slagle et al, 2011). In this study, inhibition of both FoxH1 and Eomesodermin function was found to recapitulate the phenotype observed upon complete inhibition of Nodal signaling.…”