Syk and Zap-70 function redundantly to promote angioblast migration.

Spleen tyrosine kinase (Syk) plays critical roles in B-cell and T-cell development, the maintenance of vascular integrity, and proper partitioning of the blood vascular and lymphatic vascular system. Here, we utilize the zebrafish as an in vivo system to demonstrate novel roles for Syk and the related kinase Zeta associated protein (Zap-70) in promoting angioblast migration. Partial knockdown of either gene results in early angiogenic delay of the intersegmental vessels, dorsal intersegmental vessel patterning defects, and partial loss of the thoracic duct. Higher dose knockdown of both genes results in little to no angiogenic sprouting of the intersegmental vessels, a phenotype which resembles knockdown of vegfa. Di-phosphorylated ERK, an effector of the vegfa pathway, is also downregulated in the aorta of syk:zap double morphants. Over-expression of syk under the control of a blood-specific or vascular-specific promoter rescues sprouting defects after loss of vegfa. Together these results suggest that syk and zap-70 function redundantly in an early progenitor to promote the migration of intersegmental vessel angioblasts and lymphangioblasts that contribute to the thoracic duct, either downstream of, or in parallel to vegfa.

Zebrafish collapsin response mediator protein (CRMP)-2 is expressed in developing neurons.

The collapsin response mediator proteins (CRMPs) are highly expressed in the vertebrate nervous system. CRMP2 has been shown to function in Semaphorin and lysophosphatidic acid induced growth cone collapse. Correspondingly, the highest levels of CRMP2 protein are found in the distal portion of growing axons. To understand the role of CRMP2 during embryonic development we have documented its expression pattern in zebrafish embryos at multiple stages. We find that CRMP2 is expressed in the major neural clusters of the embryonic brain during the primary stages of neurogenesis. From 20 somites through 30 hpf CRMP2 is expressed in the dorsal rostral cluster of the telencephalon, the ventral rostral cluster of the diencephalon, the ventral caudal cluster of the mesencephalon, and the hindbrain clusters. CRMP2 is also expressed in the trigeminal sensory ganglia and the Rohon Beard cells of the neural tube from 15 somites. By 48 hpf, we find expression of CRMP2 throughout the developing brain, trigeminal sensory ganglia, and Rohon Beard cells. CRMP2 is also detected in the retinal ganglion cell layer of the eye, and in the otic vesicle. Finally, we have compared the expression of CRMP2 to PlexinA4, a Semaphorin receptor expressed in sensory neurons, and find that their expression partially overlaps.

Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin.

In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat and the control of vasomotor responses in the vascular system. Connexins, the protein subunits of gap junctions, are coded by a multigene family. In this study, a connexin gene (zfCx45.6), which exhibits 53% amino acid identity to chick Cx42, was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 h postfertilization (hpf) and then again beginning at about 12 hpf, after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver, and ovary, but not the lens. Whole mount in situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo and in both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4, zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system.

Expression of connexin48.5, connexin44.1, and connexin43 during zebrafish (Danio rerio) lens development.

Connexins (Cx), the protein units of gap junctions, play important roles in lens development and homeostasis. Here, we report the mRNA expression patterns of zebrafish Cx48.5, Cx44.1, Cx43 during lens development. The expression of all three connexins in the adult lens was first confirmed by reverse transcriptase-polymerase chain reaction. By whole-mount in situ hybridization, we detected Cx48.5 expression throughout the lens, except the lateral lens epithelium, at 36 hours postfertilization (hpf). The pattern remained the same at 2 days postfertilization (dpf). By 3 and 4 dpf, Cx48.5 expression was restricted to the differentiating lens fibers in the equatorial and medial regions. Cx44.1 was expressed in a similar manner as Cx48.5 from 36 hpf to 4 dpf. However, Cx44.1 expression was also detected in the lens at 24 hpf. Cx43 expression was detected throughout the lens at 24 and 36 hpf but became restricted to the lateral epithelium at later stages.