The Founction Of Dhhc2 During Zebrafish Cental Nervous System Development And Underlying Mechanism

Dhhc2 belongs to the dhhc molecule family which has the characteristic domain Asp-His-His-Cys (dhhc). These molecules are widely present in various species, and have the palmitoyl transferase (PAT) activity due to the dhhc domain effect.In order to get the biological activity, most protein moleculors need to go through a series of complex post-translational modifications, including phosphorylation, glycosylation, ubiquitination, and lipid modification. Lipid modification include various modification including palmitoylation、myristoylation、prenylation and so on. For all these lipid modification, palmitoylation modification is the most common one, also it is the only lipid modification way that is reversible. As a post-translational modification, palmitoylation could increase the lipid affinity of its modificated protein, then promote the transport, membrane localization of these proteins. At last, the midificated protein get its biological activity and play its role in the right location.Palmitoylation modification occurs more often in brain compared with the other tissues and organs, partially because of the fact that the brain is a lipid-rich organ. Since most of the dhhc molecules have PAT activity, many dhhc proteins have been proved to be closely related to brain development, maturation, function execution and diseases rising, for example, dhhc 17, also known as hip 14. In our pre experiments, WISH results of zebrafish embryos showed that most of the dhhc proteins, including dhhc2, expressed in the nervous system during the early developmental period from 1 cell stage to 72hpf. So we get the hypothesis that these molecules could have their roles during the central nervous system development in zebrafish embryos.Dhhc2, as a member of the dhhc family, also has the PAT activity, which has been proved in the nervous system, dhhc2 palmitoylates psd95, gap43 and regulate synaptic activity, what’s more, this molecule has also been proved as an anti-tumor molecular in various kinds of tumors with ckap4 as its substrate. However, there is no relevant studies and reports about the molecular mechanisms and the specific role during development of the nervous system.On the basis of the zebrafish animal model, we make specific inhibition of dhhc2 expression by injecting dhhc2 morpholino (mo) in zebrafish embryos of 1-4 cell stage, so that we can study the phenotype of zebrafish embryos and investigate the relevant mechanisms In order to study the role of dhhc2 molecules during zebrafish embryonic development, we use the whole-mount in situ hybridization(WISH) and RT-PCR method to detect dhhc2 expression in zebrafish embryos. WISH and RT-PCR results showed that, in addition to four-cell stage significant maternal expression, dhhc2 rarely expressed in zebrafish embryos the from 1000-cell stage to bud stage, and express again since 18hpf significantly increased. In situ hybridization results suggest that the main expression in zebrafish dhhc2 position in the area of the brain. These results suggest that:dhhc2 brain during development may play an important role in zebrafishTo study the role of dhhc2 molecules during embryonic development in zebrafish, we injected dhhc2 mo to inhibite dhhc2 expression and observed the embryonic development. The results showed that the mo injected embryos developed normally before 18hpf and showed obvious difference in the hindbrain since 24hpf compared to the con mo injected embryos. Then we detected the expression of the forebrain marker gene foxg1, midbrain marker gene pax2.1, hindbrain marker gene krox2.0, and rhombomere boundary marker gene rfng. WISH results showed that the expression of krox2.0 and rfng were obviously abnormal in dhhc2 mo group:krox2.0 showed little expression and rfng expressed ectopically. This suggests that the inhibition of expression dhhc2 zebrafish embryos in hindbrain development.To further clarify the reasons for the zebrafish hindbrain abnormalities, we examined the expression of neuron precursor marker neurod4 and glial markers gfap. In situ hybridization showed that after dhhc2 mo injection, gfap expression decreased in the center of rhombomeres. So we came up with the hypothesis that, during the development of zebrafish hindbrain, dhhc2 contributes to the formation of gliagenesis zone in the center of rhombomeres, so that the hindbrain can develop well with normal structure and normal function.In order to investigate the underlying mechanism, we tested two major genes contribute to the gliagenesis in the center of rhombomeres----cyp26b1 and sox9b. RT-PCR and WISH results showed that the expression of cyp26b1 rarely varied with the injection of dhhc2 mo, while the expression of sox9b was obviously decreased after we knock-down the expression of dhhc2. When we injected the sox9b mRNA together with dhhc2 mo in embryos, we can see that the phenotype of these zebrafish embryos was normal compared with the embyos injected with only dhhc2 mo, also both krox2.0 and rfng expressed normally in these embryos. All these results proved that sox9b was the major reason that leads to the abnormal hindbrain development in dhhc2 knock-down embryos.Previous studies have proved that both cyp26bl and sox9b are regulated by fgfr2 signaling pathway in zebrafish hindbrain. There three classic fgf signaling pathways----fgf-erk pathway, fgf-jnk pathway and fgf-p38 pathway. It had been proved that cyp26bl is regulated by fgf-erk pathway. Since cyp26b1 showed little difference after dhhc2 knock-down, we just tested the fgf-erk pathway and fgf-p38 pathway in our research. Western blot results showed that the phosphorylation of p38 decreased obviously in dhhc2 mo group. Anisomycin is a widely used p38 activatior, it can specifically improve phosphorylation of p38. In our research, when we use anisomysin at proper concentration, the phenotype of embryos injected with dhhc2 mo became much more normal, also anisomysin increased the sox9b expression in hindbrain.So we can conclude that dhhc2 regulates the expression of sox9b by participating in fgf-p38 signaling pathway, so that contribute to the formation of gliagenesis zone during the development of zebrafish hindbrain.