Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification.
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.
Brown
Jamie L
JL
Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Snir
Mirit
M
Noushmehr
Houtan
H
Kirby
Martha
M
Hong
Sung-Kook
SK
Elkahloun
Abdel G
AG
Feldman
Benjamin
B
D000818:Animals; D004268:DNA-Binding Proteins / Q000172:deficiency*; D054637:Dual-Specificity Phosphatases / Q000235:genetics* / Q000502:physiology; D004475:Ectoderm / Q000166:cytology / Q000196:embryology; D004625:Embryo, Nonmammalian; D047108:Embryonic Development / Q000235:genetics; D004627:Embryonic Induction / Q000235:genetics; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005855:Germ Layers / Q000166:cytology* / Q000196:embryology; D006609:High Mobility Group Proteins / Q000172:deficiency*; D008648:Mesoderm / Q000166:cytology / Q000196:embryology; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000235:genetics* / Q000502:physiology; D055760:SOXF Transcription Factors; D014157:Transcription Factors / Q000172:deficiency*; D014158:Transcription, Genetic; D029961:Zebrafish Proteins / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D055760:SOXF Transcription Factors; D014157:Transcription Factors; D029961:Zebrafish Proteins; C491603:sox17 protein, zebrafish; D054639:Mitogen-Activated Protein Kinase Phosphatases; C532443:DUSP4 protein, zebrafish; D054637:Dual-Specificity Phosphatases