High-throughput functional screen of mouse gastrula cDNA libraries reveals new components of endoderm and mesoderm specification

Abstract

This study describes a cross-species functional screen of mouse gastrula cDNA libraries for components of endoderm and mesoderm specification. Pools of 96 cDNAs from arrayed mouse gastrula cDNA libraries were transcribed into mRNA and injected into either the presumptive mesoderm or the ectoderm of one-cell Xenopus laevis embryos. Injected embryos were examined at gastrula stage by in situ hybridization with endoderm or mesoderm markers. Using this approach, we screened over 700 pools or ∼60,000 cDNAs. We identified 17 unique cDNAs that function during mesoderm and/or endoderm specification and 16 that cause general morphology changes. Identified molecules fall into eight general functional groups as follows: cell cycle components (seven), transcription factors (four), extracellular secreted molecules (seven), transmembrane receptors (one), intracellular signaling components (five), microtubule components (two), metabolism molecules (three), and unknown (four). Several of the genes we identified would not have been predicted to be involved in endoderm or mesoderm specification, highlighting the usefulness of nonbiased screening approaches. This includes Otx2, which we show is a downstream target of Xsox17␤. The speed, low cost, and high efficiency of this cross-species screen makes it an ideal method for examining cDNAs from difficult-to-obtain sources. Therefore, this approach complements the current mouse molecular genetics systems and provides a powerful means for the genome-wide examination of mammalian gene function.[The sequence data from this study have been submitted to dbEST/GenBank under accession nos.

Developmental expression patterns of candidate cofactors for vertebrate six family transcription factors

“…Vertebrate Bop-1 was first identified as a mouse gastrula cDNA clone that caused morphological changes in the developing Xenopus embryo in a high-through put screen (Chiao et al, 2005). Mouse Bop-1 is implicated in rRNA processing and ribosome biogenesis and plays a role in cell cycle progression (Strezoska et al, 2000; 2002; Rohrmoser et al, 2007); deregulation of human Bop-1 is associated with colorectal tumors (Killian et al, 2006).…”

HeOtx expression in an indirectly developing polychaete correlates with gastrulation by invagination

“…Farther away within the deuterostome branch, the ascidian Halocynthia roretzi (Wada et al 1996) and the cephalochordate Branchiostoma floridae (Williams and Holland 1998) exhibit robust Otx endomesoderm precursor expression, but Otx ectopic expression does not induce ectopic endoderm differentiation in ascidians, although it can induce ectopic morphogenetic alterations in notochord cells (Wada and Saiga 1999); this was interpreted as indicative of a peripheral or insufficient regulatory role regarding endoderm specification, and a more relevant functional involvement in gastrulation was suggested (Wada and Saiga 1999). As in ascidians, Otx has not been identified as a key endomesoderm regulator in vertebrates (Loose and Patient 2004), although there is circumstantial evidence that suggests a regulator role for Otx in the endomesoderm (Chiao et al 2005). In contrast, Otx is essential for proper mouse gastrulation (Ang et al 1996).…”

Sox17 facilitates the differentiation of mouse embryonic stem cells into primitive and definitive endoderm in vitro

“…2000; Kofron et al . 2004; Chiao et al . 2005), which together with Bix1 and Bix4 in turn regulate the expression of the downstream target Xsox17 (Xanthos et al .…”

“…In Xenopus, a T-box transcription factor, maternal VegT (Chang & Hemmati-Brivanlou 2000;Shivdasani 2002), directly activates Nodal signaling, Bix1 and Bix4 expression (Tada et al 1998;Casey et al 1999). Nodal signaling through Fzr1 contributes to Mixer-like homeodomain proteins and Gata factors (Gata4 and Gata5) expression (Shoichet et al 2000;Kofron et al 2004;Chiao et al 2005), which together with Bix1 and Bix4 in turn regulate the expression of the downstream target Xsox17 (Xanthos et al 2001), then Xsox17 regulates the commitment to endoderm through other cascades of transcription factors (Stainier 2002).…”