RESUMO
Several signalling cascades are implicated in the formation and patterning of the three principal germ layers, but their precise temporal-spatial mode of action in progenitor populations remains undefined. We have used conditional gene deletion of mouse ß-catenin in Sox17-positive embryonic and extra-embryonic endoderm as well as vascular endothelial progenitors to address the function of canonical Wnt signalling in cell lineage formation and patterning. Conditional mutants fail to form anterior brain structures and exhibit posterior body axis truncations, whereas initial blood vessel formation appears normal. Tetraploid rescue experiments reveal that lack of ß-catenin in the anterior visceral endoderm results in defects in head organizer formation. Sox17 lineage tracing in the definitive endoderm (DE) shows a cell-autonomous requirement for ß-catenin in midgut and hindgut formation. Surprisingly, wild-type posterior visceral endoderm (PVE) in midgut- and hindgut-deficient tetraploid chimera rescues the posterior body axis truncation, indicating that the PVE is important for tail organizer formation. Upon loss of ß-catenin in the visceral endoderm and DE lineages, but not in the vascular endothelial lineage, Sox17 expression is not maintained, suggesting downstream regulation by canonical Wnt signalling. Strikingly, Tcf4/ß-catenin transactivation complexes accumulated on Sox17 cis-regulatory elements specifically upon endoderm induction in an embryonic stem cell differentiation system. Together, these results indicate that the Wnt/ß-catenin signalling pathway regulates Sox17 expression for visceral endoderm pattering and DE formation and provide the first functional evidence that the PVE is necessary for gastrula organizer gene induction and posterior axis development.
Assuntos
Endoderma/embriologia , Endoderma/metabolismo , Proteínas HMGB/metabolismo , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , Fatores de Transcrição SOXF/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/deficiência , Proteínas HMGB/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Organizadores Embrionários/citologia , Gravidez , Fatores de Transcrição SOXF/deficiência , Fatores de Transcrição SOXF/genética , Tetraploidia , Fator de Transcrição 4 , beta Catenina/deficiência , beta Catenina/genéticaRESUMO
Sox17 encodes an SRY-related high-mobility group (HMG) box transcription factor that is essential for endoderm formation and fetal hematopoietic stem cell maintenance. In the mouse, expression of Sox17 is first observed in the extraembryonic endoderm and is subsequently seen in the definitive endoderm as well as in blood and the endothelial cells of the developing vasculature. To conditionally inactivate genes in these domains, we have targeted the Sox17 locus to generate a bicistronic mRNA linking Sox17 to a codon improved Cre recombinase (iCre) via a viral 2A sequence. Here we report a new Cre knock-in mouse line, Sox17-2A-iCre, with activity in the developing endoderm, the vascular endothelial cells of the cardiovascular system and the hematopoietic system. Our results indicate that the Sox17-2A-iCre is active in an early endoderm progenitor and recombination of the Rosa26 reporter was observed in all previously reported expression domains of Sox17. The Sox17-2A-iCre line will be an excellent tool to conditionally inactivate genes in the definitive endoderm as well as in the vasculature and hematopoietic system.
Assuntos
Endoderma/metabolismo , Células Endoteliais/metabolismo , Integrases/metabolismo , Modelos Animais , Fatores de Transcrição SOXF/metabolismo , Animais , Southern Blotting , Primers do DNA/genética , Células-Tronco Embrionárias/metabolismo , Vetores Genéticos/genética , Genótipo , Camundongos , Camundongos Transgênicos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The HMG-box transcription factor Sox17 has been shown to play important roles in both endoderm formation and cardiovascular development. To conditionally inactivate genes in these domains, we have targeted a codon improved Cre Recombinase (iCre) into exon 1 of the Sox17 gene. Surprisingly, Cre-mediated recombination in the Rosa26 reporter mouse line revealed largely specific activity within the vasculature rather than in endoderm-derived tissues. Here we report a new Cre knock-in mouse line, Sox17(iCre) with activity in the vascular endothelial cells of arteries in the cardiovascular system but not in veins. Cre-mediated recombination was also strongly detected in the liver and spleen, the two organs associated with hematopoiesis. Thus, these results indicate that the Sox17(iCre) would be an appropriate tool for conditional mutagenesis of genes in the vasculature and could be used in studies of blood vessel development and angiogenesis. Additionally, we provide evidence that two different promoters drive Sox17 expression in the endodermal and vascular system.
