A crucial component of the endoderm formation pathway, CASANOVA, is encoded by a novel sox-related gene

Abstract

casanova (cas) mutant zebrafish embryos lack endoderm and develop cardia bifida. In a substractive screen for Nodal-responsive genes, we isolated an HMG box-containing gene, 10J3, which is expressed in the endoderm. The cas phenotype is rescued by overexpression of 10J3 and can be mimicked by 10J3-directed morpholinos. Furthermore, we identified a mutation within 10J3 coding sequence that cosegregates with the cas phenotype, clearly demonstrating that cas is encoded by 10J3. Epistasis experiments are consistent with an instructive role for cas in endoderm formation downstream of Nodal signals and upstream of sox17. In the absence of cas activity, endoderm progenitors differentiate into mesodermal derivatives. Thus, cas is an HMG box-containing gene involved in the fate decision between endoderm and mesoderm that acts downstream of Nodal signals. The endoderm germ layer generates the structures of the digestive and respiratory tracts. In addition, endoderm is crucial in the organization and/or induction of neighboring tissues, such as the head and the heart (Grapin-Botton and Melton 2000). In the zebrafish, endoderm derives from cells positioned at the blastoderm margin of the late blastula (Warga and Nusslein-Volhard 1999). Although endoderm and mesoderm progenitors partially overlap, most mesoderm progenitors come from positions relatively far away from the very margin at this stage.The molecular pathway leading to endoderm formation is only partially understood. Specification of endoderm requires Nodal signaling (Kimelman and Griffin 2000). Zebrafish mutants lacking the Nodal-related factors Squint (Sqt) and Cyclops (Cyc) fail to form endoderm (Feldman et al. 1998;Sampath et al. 1998). Similarly, endoderm does not form in embryos defective in both maternal and zygotic components of one-eyed pinhead (MZoep), which encodes an EGF-CFC protein required for cells to respond to Nodal signals (Schier et al. 1997;Strähle et al. 1997;Zhang et al. 1998;Gritsman et al. 1999). In zebrafish, Nodals induce endoderm presumably via activation of the type I TGF␤ receptor TARAM-A (Tar; Renucci et al. 1996;Peyrieras et al. 1998), the mix-like homeobox transcription factor MIXER (bonnie and clyde, bon; Kikuchi et al. 2000), and the zinc-finger transcription factor GATA5 (faust; Reiter et al. 1999Reiter et al. , 2001). Both transcription factors require a third gene, casanova (cas), to efficiently induce the endoderm-specific sox17 gene (Alexander and Stainier 1999) and to allow marginal cells to achieve the proper endodermal program. At gastrula stages, cas mutant embryos express sox17 neither in endoderm precursors nor in the forerunner cells, a small group of noninvoluting mesendodermal cells at the dorsal margin (Melby et al. 1996). At later stages, cas mutants lack a gut tube and develop a heart condition known as cardia bifida. cas activity is required cell-autonomously for endoderm development and endodermal expression of foxA2 . Thus, cas acts within endoderm precursors, downstream of the Nodal signals Cyc and Sqt and the ...

Expression of Sox transcription factors in the developing mouse pancreas

“…Recently, several Sox genes have been recognized as key players in the regulation of embryonic development and cell fate determination. The analysis of Sox gene mutations in humans, mice, and zebrafish have demonstrated a role for Sox genes in endoderm specification, as well as the development of gonads, lens, heart, lymphocytes, bone, and glial cells (Kent et al, 1996;Schilham et al, 1996;Britsch et al, 2001;Dickmeis et al, 2001;Kamachi et al, 2001;Lefebvre et al, 2001;Stolt et al, 2002). Despite their broad function in embryonic development, the expression and function of Sox genes during development of the pancreas remain to be explored.…”

Identification of nodal signaling targets by array analysis of induced complex probes

“…The identified clones include 15 genes that have previously been identified to be inducible by Nodal/Tar signaling (e.g., lefty2 [Meno et al, 1999], bhikhari [Vogel and Gerster, 1999], foxA2 [axial; Strahle et al, 1993;Peyrieras et al, 1998], casanova [Dickmeis et al, 2001a;Kikuchi et al, 2001]), or to be expressed in the germ ring/organizer (e.g., spadetail [Griffin et al, 1998], otx3 [Mori et al, 1994], forkhead2, [Odenthal and Nusslein-Volhard, 1998], chordin [Schulte-Merker et al, 1997]). Forty-three genes were either related to hypothetical proteins (16), previously identified ESTs (19), or had no obvious sequence homology (8) ( Table 2, and additional data in Table 4 in supplementary material on the Web).…”

“…Their expression restricted to Nodal target tissues together with their responsiveness to Tar* misexpression in the embryo strongly support the notion that Nodals either directly or indirectly control the expression of these genes. Among the genes identified was a novel sox box transcription factor, casanova, mutation of which abolishes endoderm formation in the zebrafish embryo and which was shown to act downstream of the nodal signaling pathway Dickmeis et al, 2001a;Kikuchi et al, 2001).…”

A mutation in the zebrafish Na,K‐ATPase subunit atp1a1a.1 provides genetic evidence that the sodium potassium pump contributes to left‐right asymmetry downstream or in parallel to nodal flow

“…The expression of genes involved in specification of the endoderm, like sox32 (Dickmeis et al, 2001;Kikuchi et al, 2001), sox17 (Alexander and Stainier, 1999), and foxA2 (Strahle et al, 1993;Odenthal and Nusslein-Volhard, 1998) appeared normal in m883 mutant embryos before the 20 somite stage (data not shown). Following formation of an endodermal sheet, and migration to form a rod-shaped intestinal anlage, additional morphogenesis steps follow, such as hollowing of the rod, budding or outgrowth of the endoderm organs, and looping of the gut itself (Horne-Badovinac et al, 2003).…”

“…Probes used were preproinsulin, ipf1 (Milewski et al, 1998), glucagon, preprosomatostatin2 (called somatostatin2 in this study) (Argenton et al, 1999), trypsin (Biemar et al, 2001), islet-1 (Korzh et al, 1993), nkx2.2 (Barth and Wilson, 1995;Kudoh et al, 2001), pax6.2 (Nornes et al, 1998), foxa2 (Strahle et al, 1993;Odenthal and Nusslein-Volhard, 1998), sox32 (Dickmeis et al, 2001;Kikuchi et al, 2001), sox17 (Al-exander and Stainier, 1999), foxA2 (Strahle et al, 1993;Odenthal and Nusslein-Volhard, 1998), foxa1 (Odenthal and Nusslein-Volhard, 1998), prox1 (Glasgow and Tomarev, 1998), cha (Hashimoto et al, 2004), lefty1 (Bisgrove et al, 1999;Thisse and Thisse, 1999), spaw (Long et al, 2003), and pitx2 (Essner et al, 2000).…”