RESUMEN
The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium.
Asunto(s)
Corazón/embriología , Glicoproteínas de Membrana/metabolismo , Organogénesis/genética , Pericardio/metabolismo , Animales , Western Blotting , Línea Celular , Movimiento Celular/genética , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFbeta target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1-3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior-posterior (A-P) patterning, primitive streak formation and left-right (L-R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.
Asunto(s)
Movimiento Celular/fisiología , Embrión de Mamíferos/fisiología , Endodermo/fisiología , Glicoproteínas de Membrana/metabolismo , Morfogénesis , Animales , Adhesión Celular/fisiología , Embrión de Mamíferos/anatomía & histología , Endodermo/citología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Genes Reporteros , Genotipo , Humanos , Hibridación in Situ , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Transducción de Señal/fisiologíaRESUMEN
Genomic imprinting is a developmentally important mechanism that involves both differential DNA methylation and allelic histone modifications. Through detailed comparative characterization, a large imprinted domain mapping to chromosome 7q21 in humans and proximal chromosome 6 in mice was redefined. This domain is organized around a maternally methylated CpG island comprising the promoters of the adjacent PEG10 and SGCE imprinted genes. Examination of Dnmt3l(-/+) conceptuses shows that imprinted expression for all genes of the cluster depends upon the germline methylation at this putative "imprinting control region" (ICR). Similarly as for other ICRs, we find its DNA-methylated allele to be associated with trimethylation of lysine 9 on histone H3 (H3K9me3) and trimethylation of lysine 20 on histone H4 (H4K20me3), whereas the transcriptionally active paternal allele is enriched in H3K4me2 and H3K9 acetylation. Our study reveals a novel placenta-specific transcript, TFPI2, which is expressed from the maternal allele in both humans and mice. Deficiency for the histone methyltransferase EHMT2 (also known as G9A) or for the Polycomb group protein EED, involved in repressive H3K9me2 and H3K27me3 respectively, leads to biallelic expression of Tfpi2 in the extra-embryonic lineages, whereas the other genes in the cluster maintain correct imprinting. Apart from the putative ICR, however, no other promoter regions within the domain exhibited allele-specific repressive histone modifications. This unexpected general lack of repressive histone modifications suggests that this domain may utilize a different silencing mechanism as compared to other imprinted domains.