| Summary: | 碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 93 === Netrin and Hh protein families have critical functions in regulating early developmental processes of embryo. Both families have been known to participate in establishing central and peripheral nervous system. In the past years, many scientists reported that both family members have also much influence on peripheral tissues or cells other than nervous system.
We choose zebrafish as a model animal in our study. The aim of this study was to analyze the biological function of Netrin-1a and Twhh, which are expressed in different successive timing in RPE during development of eye. According to published data, endogenous netrin-1a is expressed transiently from 16 hpf to 20 hpf in the region of RPE primordia, while twhh is expressed in RPE cells beginning at 40 hpf. During this period, eye develops quickly and displays a distinctive phenotype that the surface of RPE has been covered by lots of melanophores and iridophores. In the preliminary data, under IRES system, using zebrafish lens-specific βB1-Crystallin 1.3 kb promoter fragment (Cr1.3) to drive ectopic over-expressed shh gene in lens can induce ectopic aggregates of melanocyte-like cells in the pupil region (Wang, 2005). Preliminarily, we supposed that melanocyte-like cells could be melanophores. However, endogenous Shh expression in RPE is beginning at 40 hpf and according to Lister’s report (2002), melanophore precursor cells have migrated to RPE marginal zone before 24 hpf. Moreover, according to the precise timing of Netrin-1a expression in RPE primordia, we addressed our hypothesis that in the early stage Netrin-1a may function as long range chemoattractive signal to guide melanophore precursor cells migrating to RPE primordia, while Twhh and Shh function as short range signals to maintain the attachment, survival and proliferation of melanophores and iridophores at later stage.
We use gain of function strategy to ectopically over-express Netrin-1a or Twhh in the lens under Cr1.3 promoter. The results showed that, when ectopically over-expressing Netrin-1a, we could not observe any aggregates of cells in transient experiments or transgenic stable lines. Several reasons may account for these results, including dosage and timing of Netrin-1a protein, potential barrier role of RPE and other endogenous factors like Shh that could disturb function of Netrin-1a. In the future, we may use transplatation experiment or Morpholino gene knockdown approach to resolve the problems on this topic.
On the other side, when ectopically over-expressing Twhh in the lens, we observed aggregates of melanocyte-like cells and iridophores in the pupil region. When treating twhh transgenic stable lines with cyclopamine, the frequency of aggregates of cells declined largely. In addition, we also noticed that the larvae of twhh transgenic stable lines possessed smaller pupil. It is shown significant difference in statistic analysis. For the present, we have not yet got direct evidence to support the notion that melanocyte-like cells should be melanophores. However, I have cloned pMITFa-2.5 kb-DsRed1 construct which can specifically label melanophore precursor cells by DsRed1. In the future, we can cross MITFa transgenic stable line with twhh transgenic stable line. And then, some critical issues could be answered.
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