ABSTRACT
Rho GTPases play an important and versatile role in several biological processes. In this study, we identified the zebrafish ortholog of the mammalian Rho A guanine exchange factor, synectin-binding guanine exchange factor (Syx), and determined its in vivo function in the zebrafish and the mouse. We found that Syx is expressed specifically in the vasculature of these organisms. Loss-of-function studies in the zebrafish and mouse point to a specific role for Syx in angiogenic sprouting in the developing vascular bed. Importantly, vasculogenesis and angioblast differentiation steps were unaffected in syx knockdown zebrafish embryos, and the vascular sprouting defects were partially rescued by the mouse ortholog. Syx knockdown in vitro impairs vascular endothelial growth factor-A-induced endothelial cell migration and angiogenesis. We have also uncovered a potential mechanism of endothelial sprout guidance in which angiomotin, a component of endothelial cell junctions, plays an additive role with Syx in directing endothelial sprouts. These results identify Syx as an essential contributor to angiogenesis in vivo.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Neovascularization, Physiologic , Zebrafish Proteins/metabolism , Zebrafish/embryology , rhoA GTP-Binding Protein/metabolism , Angiomotins , Animals , Cell Movement/physiology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Guanine Nucleotide Exchange Factors/genetics , Intercellular Junctions/genetics , Intercellular Junctions/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Zebrafish/genetics , Zebrafish Proteins/genetics , rhoA GTP-Binding Protein/geneticsABSTRACT
Heteroduplex screening identified 74 small mutations in the factor VIII genes of 72 families with hemophilia A. In addition, patients from 3 families with high titer inhibitors had partial gene deletions and 5 unrelated families that were negative for heteroduplex formation had a mutation on direct sequencing. The latter had mild hemophilia A with an inhibitor, and sequencing their exon 23 fragments found a transition predicting a recurrent Arg2150 to His. Of 69 distinct mutations (including the 3 partial gene deletions), 47 are novel. Of small mutations, 51 were missense (one possibly a normal variant and two that could also alter splicing) at 39 sites, 13 were small deletions or insertions (3 inframe and one a normal variant in an intron), 13 were nonsense at 12 sites and 2 altered intron splice junctions. In 24 families, at least one affected member had evidence for an alloimmune response to factor VIII: of these, 11 were associated with missense mutations. In 14 families, de novo origin was demonstrated.
