Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and

The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhd mi149 ), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function.␤-pix ͉ CNS ͉ endothelial cell ͉ p21-activated kinase ͉ vasculature

The zebrafish has become a powerful tool for analysis of vertebrate hematopoiesis. Zebrafish, unlike mammals, have a robust primitive myeloid pathway that generates both granulocytes and macrophages. It is not clear how this unique primitive myeloid pathway relates to mammalian definitive hematopoiesis. In this study, we show that the two myeloid subsets can be distinguished using RNA in situ hybridization. Using a morpholino-antisense gene knockdown approach, we have characterized the hematopoietic defects resulting from knockdown of the myeloid transcription factor gene pu.1 and the unique zebrafish gene c/ebp1. Severe reduction of pu.1 resulted in complete loss of primitive macrophage development, with effects on granulocyte development only with maximal knockdown. Reduction of c/ebp1 did not ablate initial macrophage or granulocyte development, but resulted in loss of expression of the secondary granule gene lys C. These data reveal strong functional conservation of pu.1 between zebrafish primitive myelopoiesis and mammalian definitive myelopoiesis. Further, these results are consistent with a conserved role between c/ebp1 and mammalian C/EBPE, whose ortholog in zebrafish has not been identified. These studies validate the examination of zebrafish primitive myeloid development as a model for human myelopoiesis, and form a framework for identification and analysis of myeloid mutants.

Zebrafish are a powerful vertebrate model system for the study of human disease as they share many molecular pathways with mammals. Of note, nearly all of the mammalian coagulation factors are also highly conserved in fish. As part of a whole genome ENU mutagenesis screen, we identified a mutant zebrafish which displayed intraventricular hemorrhage between 2–3 days post fertilization (dpf), which we named redhead. Using an F2 intercross strategy, we mapped this recessive mutant to a 100 kilobase interval on chromosome 2 and identified a splice site mutation in the gene for an ortholog of the p21-activated kinase Pak2. This IVS9 T+2A mutation renders greater than 90% of transcripts nonfunctional, resulting in a hypomorphic allele. Surveying zebrafish expressed sequence tag databases, we identified two Pak2 orthologs in zebrafish, designated Pak2a and Pak2b, with the redhead mutation in pak2a. Central nervous system (CNS) hemorrhage in redhead embryos was rescued by injection of wild type pak2a mRNA. Morpholino knockdown of pak2a in wild type fish phenocopies the redhead mutant, but with an increase in penetrance and severity, including hydrocephalus and pericardial edema secondary to severe hemorrhage. Injection of either pak2a or pak2b mRNA was able to rescue this phenotype. In addition, pak2b knockdown worsened the bleeding phenotype in redhead embryos, with no effect on wild type fish. These results suggest a partial overlap in function between Pak2a and Pak2b. Confocal microscopy was performed at 2.5 dpf in pak2a knockdown embryos transgenic for Gata1-dsRED and Flk1-eGFP, distinctively labeling erythrocytes and endothelial cells, respectively. The sites and magnitude of hemorrhage were variable between individual embryos, but there were no obvious abnormalities in vessel patterning. In summary, we have identified a critical role for Pak2 in maintaining CNS vessel integrity in zebrafish, without apparent effects on other vascular beds. Further analysis of this signaling pathway in the vessel wall may provide novel insight into the mechanisms underlying vascular heterogeneity in mammals, and the tissue specific function of Pak2 in the CNS vasculature.