HOXA13 directly regulates EphA6 and EphA7 expression in the genital tubercle vascular endothelia.

Hypospadias, a common defect affecting the growth and closure of the external genitalia, is often accompanied by gross enlargements of the genital tubercle (GT) vasculature. Because Hoxa13 homozygous mutant mice also exhibit hypospadias and GT vessel expansion, we examined whether genes playing a role in angiogenesis exhibit reduced expression in the GT. From this analysis, reductions in EphA6 and EphA7 were detected. Characterization of EphA6 and EphA7 expression in the GT confirmed colocalization with HOXA13 in the GT vascular endothelia. Analysis of the EphA6 and EphA7 promoter regions revealed a series of highly conserved cis-regulatory elements bound by HOXA13 with high affinity. GT chromatin immunoprecipitation confirmed that HOXA13 binds these gene-regulatory elements in vivo. In vitro, HOXA13 activates gene expression through the EphA6 and EphA7 gene-regulatory elements. Together these findings indicate that HOXA13 directly regulates EphA6 and EphA7 in the developing GT and identifies the GT vascular endothelia as a novel site for HOXA13-dependent expression of EphA6 and EphA7.

Elucidation, quantitative refinement, and in vivo utilization of the HOXA13 DNA binding site.

Mutations in Hoxa13 cause malformations of the appendicular skeleton and genitourinary tract, including digit loss, syndactyly, and hypospadias. To determine the molecular basis for these defects, the DNA sequences bound by HOXA13 were empirically determined, revealing a novel high affinity binding site. Correlating the utilization of this high affinity binding site with genes exhibiting perturbed expression in Hoxa13 mutant limbs, we identified that HOXA13 suppresses the expression of the BMP antagonist, Sostdc1. In the absence of HOXA13 function, Sostdc1 is ectopically expressed in the distal limb, causing reduced expression of BMP-activated genes and decreased SMAD phosphorylation. Limb chromatin immunoprecipitation revealed HOXA13 binding at its high affinity site in two conserved Sostdc1 regulatory sites in vivo. In vitro, HOXA13 represses gene expression through the Sostdc1 high affinity binding sites in a dosage-dependent manner. Together, these findings confirm that the high affinity HOXA13 binding site deduced by quantitative analyses is used in vivo to facilitate HOXA13 target gene regulation, providing a critical advance toward understanding the molecular basis for defects associated with the loss of HOXA13 function.

Crucial role of Bysl in mammalian preimplantation development as an integral factor for 40S ribosome biogenesis.

Blastocyst formation during mammalian preimplantation development is a unique developmental process that involves lineage segregation between the inner cell mass and the trophectoderm. To elucidate the molecular mechanisms underlying blastocyst formation, we have functionally screened a subset of preimplantation embryo-associated transcripts by using small interfering RNA (siRNA) and identified Bysl (bystin-like) as an essential gene for this process. The development of embryos injected with Bysl siRNA was arrested just prior to blastocyst formation, resulting in a defect in trophectoderm differentiation. Silencing of Bysl by using an episomal short hairpin RNA expression vector inhibited proliferation of embryonic stem cells. Exogenously expressed Bysl tagged with a fluorescent protein was concentrated in the nucleolus with a diffuse nucleoplasmic distribution. Furthermore, the loss of Bysl function by using RNA interference or dominant negative mutants caused defects in 40S ribosomal subunit biogenesis. These findings provide evidence for a crucial role of Bysl as an integral factor for ribosome biogenesis and suggest a critical dependence of blastocyst formation on active translation machinery.