Netrin-1a及Twhh在色素視網膜發育 過程中潛在扮演的生化誘引之功能

Functional Analyses of Chemoattraction of Netrin-1a and Twhh during RPE Development by Transgenic Ectopic Over-expression Assay

林文郎(Wen-Lang Lin)

指導教授 ： 張百恩

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.00560

摘要

Netrin與Hh (hedgehog)蛋白家族對胚胎早期的正常發育相當重要。已知兩者參與中樞和周邊神經系統的建立，近年來陸續發現兩家族成員對神經系統以外的周邊組織細胞有著廣泛性的影響。
本研究以斑馬魚為材料，目的在探討眼睛發育過程中，Netrin-1a與Twhh蛋白兩者在色素視網膜RPE (retinal pigmented epithelium)組織中的先後表現所扮演的角色與功能。根據文獻資料，已知內生性的netrin-1a在16 hpf至20 hpf (hours post-fertilization)時段內，短暫地表現在RPE primordia的位置；而twhh從40 hpf開始表現在RPE的位置，此過程中，眼睛發育快速且外觀上出現一明顯的變化：RPE的表面被大量黑色素細胞與銀細胞覆蓋。在先前研究中，利用水晶體專一表現的斑馬魚βB1-Crystallin 1.3 kb啟動子(Cr1.3)，在IRES系統之下，異位過量表現Shh蛋白，可發現幼魚瞳孔的位置有黑色細胞點塊的聚集(王偉庭，2005)。初步研究中，我們認為黑色細胞點塊為黑色素細胞的可能性較大。然而，內生性的Shh在RPE位置表現的時間較晚(40 hpf)，且由Lister (2002年)發表的資料顯示：黑色素前驅細胞在24 hpf之前已出現在RPE的邊緣，因此推測在眼睛發育早期有不同的因子負責指引黑色素前驅細胞向眼睛的位置移動，而根據斑馬魚Netrin-1a表現的時空分佈，我們提出實驗的假說：Netrin-1a早期提供長距離的吸引訊號(long range signal)，吸引黑色素前驅細胞向RPE位置移動，而Twhh或Shh蛋白則以短距離的訊號(short range signal)，維持RPE表面黑色素細胞與銀細胞的附著(attachment)、存活(survival)與增生(proliferation)。
本實驗利用gain of function的方法，透過斑馬魚βB1-Crystallin 1.3 kb啟動子的驅動，在水晶體異位過量表現Netrin-1a或Twhh蛋白，結果顯示：當異位過量表現Netrin-1a蛋白，在過渡表現或轉殖恆定品系當中，外觀上，皆沒有任何細胞團塊的聚集，推究其原因可能與Netrin-1a異位表現的劑量、時機、RPE屏障以及其他內生性因子(如Shh)的干擾有關。此部分的研究，在未來可以移植實驗或Morpholino gene knockdown的方法以釐清相關問題。
另外在異位表現Twhh的部分，在過渡表現或轉殖恆定品系的實驗中，皆可觀察到瞳孔的位置出現黑色細胞團塊與銀細胞，當以cyclopamine浸泡處理後，這些特殊外表型出現的頻率明顯降低。另外，異位表現Twhh蛋白亦造成瞳孔縮小的現象，此現象在統計分析上有顯著的差異。關於異位出現的黑色細胞團塊是否就是黑色素細胞，目前仍缺乏直接證據加以證實，而我們已構築可專一標示黑色素細胞的pMITFa-2.5 kb-DsRed1結構體，未來只要將此轉殖恆定品系與twhh品系交配，即可釐清相關問題。

關鍵字

色素視網膜 ； 生化誘引 ； 黑色素細胞

並列摘要

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.

並列關鍵字

Netrin-1a ； Twhh ； RPE ； Chemoattraction

參考文獻

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