Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification

Abstract

A major goal for developmental biologists is to define the behaviors and molecular contents of differentiating cells. We have devised a strategy for isolating cells from diverse embryonic regions and stages in the zebrafish, using computer-guided laser photoconversion of injected Kaede protein and flow cytometry. This strategy enabled us to perform a genome-wide transcriptome comparison of germ layer precursor cells. Mesendoderm and ectoderm precursors cells isolated by this method differentiated appropriately in transplantation assays. Microarray analysis of these cells reidentified known genes at least as efficiently as previously reported strategies that relied on artificial mesendoderm activation or inhibition. We also identified a large set of uncharacterized mesendoderm-enriched genes as well as ectoderm-enriched genes. Loss-of-function studies revealed that one of these genes, the MAP kinase inhibitor dusp4, is essential for early development. Embryos injected with antisense morpholino oligonucleotides that targeted Dusp4 displayed necrosis of head tissues. Marker analysis during late gastrulation revealed a specific loss of sox17, but not of other endoderm markers, and analysis at later stages revealed a loss of foregut and pancreatic endoderm. This specific loss of sox17 establishes a new class of endoderm specification defect. microarray ͉ zgc:55423 ͉ gastrulation ͉ mkp2 ͉ sox17

In Vivo Regulation of the Zebrafish Endoderm Progenitor Niche by T-Box Transcription Factors

“…Intriguingly, our analyses also indicate that a subset of CRMs are bound by combinations of Eomesa, Smad2, Mixl1, Nanog, Mxtx2, and Pou5f3, including those proximal to other genes implicated in endoderm formation, such as dusp4 (Brown et al., 2008), cxcr4a (Stückemann et al., 2012), and spns2 (Osborne et al., 2008). It will be interesting to learn more about how these TFs collectively contribute to the function of the identified CRMs.…”

Pre-gastrula expression of zebrafish extraembryonic genes

“…We assessed the Nodal regulation of several YSL, EVL and margin genes identified in this and a previous screen [24]. As expected from previous reports, expression of the two margin genes we tested was downregulated in the margin of MZoep mutant embryos, which carry null mutations in an EGF-CFC-type receptor that is essential for Nodal signaling (Figure 5I-L) [25,26].…”

Incomplete splicing, cell division defects, and hematopoietic blockage in dhx8 mutant zebrafish

“…RNA amplification, labeling and hybridization to a 33k In-house oligo microarray chips were carried out as previously described (Pei et al, 2007; Brown et al, 2008). The hybridizations were repeated once to make sure the results were reproducible.…”