Abstract-Vascular endothelial growth factor (Vegf) plays central roles in the establishment of stereotypic vascular patterning in vertebrates. However, it is not fully understood how the network of blood vessels is established and maintained during vascular development. A zebrafish ko095 mutant presented the disorganized vessels with abnormal branching of the established intersegmental vessels (ISVs) after 60 hours postfertilization. The gene responsible for ko095 encodes seryl-tRNA synthetase (Sars) with a nonsense mutation. The abnormal branching of ISVs in ko095 mutant was suppressed by the introduction of either wild-type Sars or a mutant Sars (T429A) lacking the enzymatic activity that catalyzes aminoacylation of transfer RNA for serine (canonical activity), suggesting that the abnormal branching is attributable to the loss of function of Sars besides its canonical activity. We further found the increased expression of vegfa in ko095 mutant at 72 hours postfertilization, which was also reversed by the introduction of Sars (T429A). Furthermore, the abnormal branching of ISVs in the mutant was suppressed by knockdown of vegfa or vegfr2 (kdra and kdrb). Knockdown of vegfc or vegfr3 rescued the abnormal ISV branching in ko095 mutant. These results suggest that the abnormal ISV branching in ko095 mutant is caused by the activated Vegfa-Vegfr2 signal and requires the Vegfc-Vegfr3 signal, because the latter is needed for general angiogenesis. Hence, we conclude that noncanonical activity of Sars is involved in vascular development presumably by modulating the expression of vegfa. 4,5 The network of trunk vessels along somite boundaries is well conserved among vertebrates. Primary angiogenic sprouts emanate bilaterally from the dorsal aorta and forms the intersegmental vessels (ISVs) along the somites from ventral to dorsal and the dorsal longitudinal anastomotic vessels along the head to tail axis at the most dorsal side. The secondary sprouts from the posterior cardinal vein connecting to the primary ISVs becomes the intersegmental veins. The remaining secondary sprouts branch to form the parachordal vessels near the horizontal myoseptum that develops toward the head or tail. 5,6 Angiogenic sprouting is regulated by tip cells and the cells following the tip cells, the stalk cells. 2,3,7 In these 2 types of cells, the importance of vascular endothelial growth factor (Vegf)-mediated regulation has been unraveled. Vegfa induces the migration of the tip cells and the expression of a Notch ligand Dll4 that is exclusively expressed in the endothelial cells, via Vegfr2 in the tip cells. Subsequently, Dll4 represses the expression of Vegfr2 and Vegfr3 via Notch expressed in the stalk cells to inhibit the responses to Vegfs. 2,3,8 Primary ISV sprouts express both Vegfr2 and Vegfr3, suggesting that differential expression of Vegfrs affects the vascular development via modulating the responsiveness of endothelial cells to Vegfs. 7,9 Consistently, deletion of a single Dll4 allele in mice and knockdown of dll4 in zebrafish r...

The glial cells missing (gcm) gene in Drosophila encodes a GCM-motif transcription factor that functions as a binary switch to select between glial and neuronal cell fates. To understand the function of gcm in vertebrates, we isolated the zebrafish gcmb and analyzed the function of this gene using antisense morpholino oligonucleotides against gcmb mRNA (gcmb-MO) and transgenic overexpression. Zebrafish gcmb is expressed in the pharyngeal arch epithelium and in cells of the macrophage lineage. gcmb-MO-injected larvae show significantly reduced branchial arch cartilages. fgf3-MO-injected larvae display a similar phenotype to that of gcmb-MO-injected larvae with respect to the lack of pharyngeal cartilage formation. In addition, gcmb expression in the pharyngeal arches is down-regulated in fgf3-MO-injected larvae. The gcmb transgenic larvae show a protrusion of the lower jaw and abnormal spatial arrangement of the pharyngeal cartilage elements. These results suggest that gcmb is required for normal pharyngeal cartilage formation in zebrafish and that its expression is dependent on fgf3 activity.

Somites in vertebrates are periodic segmented structures that give rise to the vertebrae and muscles of body. Somites are generated from presomitic mesoderm (PSM), but it is not fully understood how cellular differentiation and segment formation are achieved in the anterior PSM. We report here that zebrafish gadd45␤1 and gadd45␤2 genes are periodically expressed as paired stripes adjacent to the neural tube in the anterior PSM region where presomitic cells mature. In mammals, it is known that GADD45 (growth arrest and DNA damage) family proteins play a role in cell-cycle control. We found that both knockdown and overexpression of gadd45␤ genes caused somite defects with different consequences for marker gene expression. Knockdown of gadd45␤ genes with antisense morpholino oligonucleotides caused a broad expansion of mesp-a in the PSM, and both cyclic expression of her1 and segmented expression of MyoD were disorganized. On the other hand, injection of gadd45␤1 or gadd45␤2 suppressed expression of mesp-a and her1 in anterior PSM and MyoD in paraxial mesoderm. These results indicate that regulated expression of gadd45␤ genes in the anterior PSM is required for somite segmentation.presomitic mesoderm ͉ periodicity ͉ patterning ͉ knockdown

Hemoglobin consists of heme and globin proteins and is essential for oxygen transport in all vertebrates. Although biochemical features of heme synthesis enzymes have been well characterized, the function of these enzymes in early embryogenesis is not fully understood. We found that the sixth heme synthesis enzyme, coproporphyrinogen oxidase (CPO), is predominantly expressed in the intermediate cell mass (ICM) that is a major site of zebrafish primitive hematopoiesis. Knockdown of zebrafish CPO using anti-sense morpholinos (CPO-MO) leads to a significant suppression of hemoglobin production without apparent reduction of blood cells. Injection of human CPO RNA, but not a mutant CPO RNA that is similar to a mutant responsible for a hereditary coproporphyria (HCP), restores hemoglobin production in the CPO-MO-injected embryos. Furthermore, expression of CPO in the ICM is severely suppressed in both vlad tepes / gata1 mutants and in biklf -MO-injected embryos. In contrast, over-expression of biklf and gata1 significantly induces ectopic CPO expression. The function of CPO in heme biosynthesis is apparently conserved between zebrafish and human, suggesting that CPO-MO-injected zebrafish embryos might be a useful in vivo assay system to measure the biological activity of human CPO mutations.

Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.