Major vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. We show that proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and we identify mutations in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis.

Exon enhancers are accessory pre-mRNA splicing signals that stimulate exon splicing. One class of proteins, the serine-arginine-rich (SR) proteins, have been demonstrated to bind enhancers and activate splicing. Here we report that A/C-rich exon enhancers (ACE elements) are recognized by the human YB-1 protein, a non-SR protein. Sequence-specific binding of YB-1 was observed both to an ACE derived from an in vivo iterative selection protocol and to ACE elements in an alternative exon (v4) from the human CD44 gene. The ACE element that was the predominant YB-1 binding site in CD44 exon v4 was required for maximal in vivo splicing and in vitro spliceosome assembly. Expression of wild-type YB-1 increased inclusion of exon v4, whereas a truncated form of YB-1 did not. Stimulation of exon v4 inclusion by wild-type YB-1 required the ACE necessary for YB-1 binding in vitro, suggesting that YB-1 stimulated exon inclusion in vivo by binding to an exonic ACE element. These observations identify a protein in addition to SR proteins that participates in the recognition of exon enhancers.

The vasculature tailors to the needs of different tissues and organs. Molecular, structural, and functional specializations are observed in different vascular beds, but few genetic models give insight into how these differences arise. We identify a unique cerebrovascular mutation in the zebrafish affecting the integrity of blood vessels supplying the brain. The zebrafish bubblehead (bbh) mutant exhibits hydrocephalus and severe cranial hemorrhage during early embryogenesis, whereas blood vessels in other regions of the embryo appear intact. Here we show that hemorrhages are associated with poor cerebral endothelial-mesenchymal contacts and an immature vascular pattern in the head. Positional cloning of bbh reveals a hypomorphic mutation in ␤Pix, a binding partner for the p21-activated kinase (Pak) and a guanine nucleotide exchange factor for Rac and Cdc42. ␤Pix is broadly expressed during embryonic development and is enriched in the brain and in large blood vessels. By knockdown of specific ␤Pix splice variants, we show that they play unique roles in embryonic vascular stabilization or hydrocephalus. Finally, we show that Pak2a signaling is downstream of ␤Pix. These data identify an essential in vivo role for ␤Pix and Pak2a during embryonic development and illuminate a previously unrecognized pathway specifically involved in cerebrovascular stabilization.angiogenesis ͉ hemorrhage ͉ hydrocephalus

The regulation of air quality is important for ensuring the health of a population. Current air quality decision support systems are very useful if the user possesses sufficient data to operate them and the necessary expertise to interpret their results. In general, these systems suffer as a result of their excessive complexity. The present study describes the development of a scalable air quality decision support system using the CALPUFF air dispersion model and a Geospatial Information System (GIS). This system uses receptor level exposure modeling and outputs from CALPUFF to estimate the relative impacts on human populations from multiple air pollution sources by calculating intake, defined as the amount of pollution that is inhaled by a population and intake fraction, defined as the fraction of pollutant emitted by a pollution source that is inhaled by a population. Unlike ground level pollution concentration, intake and intake fraction consider receptors and offer a more valuable estimate of pollution exposure, especially when faced with limited input data. The system also leverages the inherent strength of GIS to improve accessibility of geospatial data by generating maps of ground level pollutant concentration, intake, and intake fraction using graduated color schemes. This enables any user to identify potentially hazardous pollution sources and prioritize decisions such as development, maintenance, and decommission.

Mutations in RNA binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb, and neurological symptoms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription, and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. We show a developmental role for Hnrnpul1 in zebrafish, resulting in reduced body and fin growth and missing bones. Defects in craniofacial tendon growth and adult-onset caudal scoliosis are also seen. We demonstrate a role for Hnrnpul1 in alternative splicing and transcriptional regulation using RNA sequencing, particularly of genes involved in translation, ubiquitination, and DNA damage. Given its cross-species conservation and role in splicing, it would not be surprising if it had a role in human development. Whole exome sequencing detected a homozygous frameshift variant in HNRNPUL1 in two siblings with congenital limb malformations, which is a candidate gene for their limb malformations. Zebrafish Hnrnpul1 mutants suggest an important developmental role of hnRNPUL1 and provide motivation for exploring potential conservation of ancient regulatory circuits involving hnRNPUL1 in human development.

180 words) 3 2 Mutations in RNA binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, 3 3 limb and neurological symptoms. Heterogeneous Nuclear Ribonucleoproteins (hnRNPs) are involved 3 4in nucleic acid binding, transcription and splicing through direct binding to DNA and RNA, or through 3 5interaction with other proteins in the spliceosome. Here, we show a developmental role for hnrnpul1 in 3 6zebrafish fin and craniofacial development, and in adult onset scoliosis. Furthermore, we demonstrate 3 7 a role of hnrnpul1 in alternative splicing regulation. In two siblings with congenital limb malformations, 3 8whole exome sequencing detected a frameshift variant in HNRNPUL1; the developmental role of this 3 9gene in humans has not been explored. Our data suggest an important developmental role of 4 0 hnRNPUL1 in both zebrafish and humans. Although there are differences in phenotypes between 4 1 species, our data suggests potential conservation of ancient regulatory circuits involving hnRNPUL1 in 4 2 these phylogenetically distant species. 4 3

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We have investigated the effects of 3' noncoding elements in enhancing translation of messengers having translation-inhibiting 5' untranslated regions (UTRs). The translation of transcripts bearing the 5' UTRs of either human c-myc or a synthetic hairpin structure upstream of a chloramphenicol acetyltransferase (CAT) reporter sequence is greatly attenuated in early embryos of Xenopus laevis. Translation of transcripts bearing the human c-myc-5' UTR was markedly stimulated by the presence of 3' poly(A). Transcripts bearing the 5' hairpin element were insensitive to the presence of poly(A), but they were extremely sensitive to the composition of the 3' UTR. A GC-rich distal sequence repressed translation, whereas a proximal GGAAU sequence promoted translation of these transcripts. Our results support the concept that long-range interactions between the 5' and 3' ends of transcripts are important in regulating translation in Xenopus embryos.