by Remarkably, can functionally substitute for in ES cell-derived EBs and transgenic embryos. integrity of the epicardial sheet and disturbed organization of the Incyclinide 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. Incyclinide and expression patterns in the developing mouse embryo (Haines et al., 2006; Maretto et al., 2008). is weakly co-expressed in the AVE, but with a delayed onset beginning at E7.5 (Maretto et al., 2008). One day later, shows very strong expression in the trunk mesenchyme beneath the forming heart (Maretto et al., 2008). By E10.5, and expression domains broadly overlap within the pharyngeal mesenchyme, developing heart, somites and central nervous system (CNS) (Haines et al., 2006; Maretto et al., 2008). By contrast, expression is restricted to the developing brain. In and expression domains in the developing heart. Interestingly, and are both strongly expressed in the PEO. However, is selectively downregulated following attachment and outgrowth of the epicardial cells, and beginning at around E10 only expression is maintained in the fully delaminated epicardium. By contrast, and co-expression is maintained throughout the myocardium RGS3 until around E12.5. To investigate functional activities in vivo, we generated a null allele via gene targeting. Nearly all homozygous mutant embryos arrest at mid-gestation (E12.5) owing to cardiac insufficiency. Consistent with its unique expression domain, mutant AVE, we also observe here disruptions in the epicardial cell layer, suggesting that Incyclinide Flrt2 and Flrt3 might contribute similar functional activities in the epicardium and AVE, respectively. To test this possibility, we exploited in vitro and in vivo reconstitution assays. Interestingly, expression efficiently corrects the abnormal morphology and disorganized BM phenotype of mutant embryoid bodies (EBs), and similarly ectopic expression in the epicardium of expression is independent of Fgf signalling. MATERIALS AND METHODS Mouse strains and genotyping The resistance cassette. The flanking genomic regions comprised of the 3 kb 5 (probe. From 960 drug-resistant colonies, we recovered two correctly targeted clones that were injected into C57BL/6J blastocysts to generate germ line chimeras. The mutation was maintained on a 129/C57Bl/6J genetic background. Genotyping by PCR The following three primers were used: F2-4FW, 5-CTGCCACATCCCCAACAACATGAGAT; F2-5RV, 5-TCAACAGCGAAGTGGTTAATCTGCATC; and HYG-FW, 5-TTTGAATGGAAGGATTGGAGCTACGGGG. After 36 cycles of 94C for 45 seconds, 64C for 1 minute, a 72C extension for 1 minute and a final extension at 72C for 10 minutes, 419 bp (wild type) and 500 bp (mutant) products were resolved on a 2% agarose gel. To generate transgenic constructs full-length or cDNAs (Maretto et al., 2008) were subcloned into the pCAGGs expression vector (Niwa et al., 1991; Dunn et al., 2005). The expression cassettes comprising Incyclinide a CAG promoter with either and expression was subsequently tested by RT-PCR and western blot analysis on adult tissues taken from representative transgenic offspring. and transgenic mice were then crossed to (Maretto et al., 2008) or heterozygous animals carrying the null alleles. Transgene positive heterozygous offspring were subsequently backcrossed to or heterozygotes to evaluate possible rescue of the embryonic lethalities. Cell lines Cos7 cells were transfected using Lipofectamine2000 (Invitrogen, Incyclinide Carlsbad, CA, USA) according to the manufacturer’s guidelines. homozygous mutant ES cell lines were isolated from blastocysts from heterozygous intercrosses as described (Robertson, 1987), and genotyped by Southern blot analysis (Maretto et al., 2008). mutant ES cell lines were transfected with linearized pCAGG and expression vectors, and drug resistant colonies screened by Southern blot analysis were subsequently tested for protein expression by western analysis. Embryoid bodies were generated using standard protocols (Robertson, 1987). The LyEnd.1 endothelial cell line (a kind gift from Dr Friedemann Kiefer, Mnster, Germany) was grown as described (Ong et al., 2001). Production of Flrt2 antibodies and western blots The region encoding the Flrt2 extracellular domain corresponding to.
