| Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules. Cadherins associate laterally within the same plasma membrane to form parallel cis dimers, and cadherins protruding from adjacent plasma membranes associate in an anti-parallel fashion to form transdimers. in particular, primary cadherins (classic cadherins) were identified as synaptic components, and roles for them in neuronal circuitry, synaptic junction formation, and synaptic plasticity have been suggested. Interestingly, many of theprotocadherins in mammals are highly expressed in the central nervous system. Roles in tissue morphogenesis and formation of neuronal circuits during early vertebrate development have been inferred. In the postnatal brain, protocadherins are possibly involved in the modulation of synaptic transmission and the generation of specific synaptic connections.Mouse Pcdhl8(mPcdhl8), which consists of four exons similar to other protocadherin family members, maps to chromosome3. The amino acid sequence of mPcdhl8 contains six extracellular cadherin motifs, a single transmembrane region, and a large intracellular domain. Although Pcdhl8 was described in mice, not much is known about its role in embryonic development. Mouse Pcdhl8 is present throughout the embryo, in particular in the ventricular zone in the forebrain and midbrain, in the olfactory bulb, cerebral cortex, thalamus, cerebellum, and in additional organs of the adult. The expression pattern of pcdhl8 in zebrafish embryos seemed to be quite different from that in mice; pcdhl8 expression is rather systemic in rodents whereas it was limited to the CNS and head structures in zebrafish. Nonetheless, further comparative studies on the functional diversity of pcdhl8 in zebrafish are required.In recent years, zebrafish has emerged as an exciting animal model system for studying vertebrate organ development, in particular, the development of centre neural system, as zebrafish embryos are optically transparent, easily manipulated. Owing to its optical clarity, genetics, and ease of manipulation, zebrafish may be an ideal model system to obtain a comprehensive understanding of pcdhl8b in vivo. In the present study, we turn to the zebrafish embryos to pursue the role of pcdhl8b during embryonic neurogenesis.To study the role of pcdhl8b on embryonic neurogenesis in zebrafish, we injected one type of well designed antisense morpholino oligonucleotide into one or two-cell stage embryos to block the translation of pcdhl8b. After injection, the phenotypes of nervous system were monitored by whole mount in situ hybridization and acridine orange staining. Whole-mount in situ hybridization with neurogl, elav13, gfap and krox20 RNA probes showed that the expression of neural precursor cells, neurons, neuroglia cells and rhombencephalon3,5 was strikingly affected; meanwhile, the expression of MHB (midbrain-hindbrain boundary) markers pax2a and wntl was significantly compromised and showed duplication of neural tube in pcdh18b down regulation group. Acridine orange staining pointed out that down regulation of pcdhl8b increased cell apoptosis in midbrain, hindbrain and MHB region. These results suggest that pcdhl8b plays an important role in the zabrafish neurogenesis.Several platforms for constructing artificial zinc finger arrays using'modular assembly'have been described by J Keith Joung, standardized reagents and protocols that permit rapid, cross-platform'mixing-and-matching'of the various zinc finger modules are available. Meanwhile, we constructed zinc finger nuclease expression vector for knockouting pcdhl8b, and transcribed mRNA in vitro. |
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