Rho及其相關分子在斑馬魚胚分裂及腔腸化細胞遷徙過程中所扮演角色之探討

The roles of Rho and its associated proteins in cytokinesis and gastrulation cell migration in zebrafish, Danio rerio.

賴時磊(Shih-Lei Lai)

指導教授 ： 李士傑

共同指導教授 ： 羅秀婉(Show-Wan Lou)

國立臺灣大學/生命科學院/動物學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.02780

摘要

細胞分裂 (cleavage)與腔腸化 (gastrulation)發生於動物胚早胚發育過程中，此等過程由非常動態的細胞骨架尤其是細肌絲細胞骨架 (actin cytoskeleton)重組及大量細胞遷移來完成。 根據前人的研究，控制細肌絲細胞骨架重組的主要分子是Rho與其下游調控蛋白，例如Rho-kinase (ROCK)和Diaphanous-related formin。 這些蛋白在細胞中能夠調控細肌絲細胞骨架，產生支撐細胞突起的構造或是具有收縮力的收縮絲 (stress fiber)，而這些構造對細胞質分裂(cytokinesis)、細胞突起 (protrusion)與細胞遷移 (migration)都扮演著相當重要的角色，在此我們以斑馬魚為模式試著探討Rho與其下游調控蛋白在早胚發育過程中如何調控細胞分裂與腔腸化。
我們首先利用Rho的抑制劑Clostridium botulinum C3-exoenzyme (C3)注射到一細胞期的斑馬魚胚中，發現到C3會阻礙魚胚發育早期的細胞質分裂過程，而C3的抑制性可為同時注射自體活化RhoA所減弱，證明了此一結果的專一性。 在這些細胞質分裂受阻的魚胚中，我們發現原本應該分佈在分裂溝 (cleavage furrow)的細肌絲構造，因為Rho的抑制而消失。 當我們利用肌動蛋白 (myosin)的抑制劑blebbistatin來進行一樣的實驗時，我們也得到類似的結果。 這顯示Rho藉由調控肌凝蛋白 (actin)與肌動蛋白 (myosin)在分裂溝的分佈來控制細胞質分裂。 除此之外，我們也發現一部分C3注射過的卵出現外包作用 (epiboly)受阻的現象，在這些卵上利用原位雜合技術 (in situ hybridization)標定最前緣的囊胚細胞 (marginal blastoderm cells)與背唇細胞 (dorsal lip of blastopore)也顯示外包過程中這些細胞的遷移受到影響。我們同時發現ROCK抑制劑Y27632的注射對魚胚發育會造成類似上述的影響。這些結果顯示Rho-ROCK調控路徑至少控制了正常的細胞質分裂與外包作用的細胞遷移過程。 針對Rho的另一個下游調控蛋白Diaphanous，我們利用morpholino oligonucleotides (MO)抑制其蛋白質的正確表現，並發現這影響到魚胚的外包作用與胚體趨中及擴展作用 (convergent extension)。 經由對邊緣囊胚層(marginal blastoderm)、前脊索版細胞 (prechordal plate cells)和側邊下胚層 (lateral hypoblasts)等不同細胞群進行詳細的動態錄影分析，我們發現到這些細胞的細胞突起能力受到diaphanous MO影響而降低。 此外，一個在邊緣囊胚層前緣對外包作用很重要的環狀細肌絲構造也因為diaphanous MO的抑制而消失。 而且針對抑制肌凝蛋白調控因子profilin I設計的MO不但同樣會對魚胚發育產生類似的影響，而且與diaphanous MO一起注射時會產生協同作用 (synergistic effect)。 這些結果顯示Rho下游的Diaphanous和Profilin I對斑馬魚腔腸化過程中的細胞遷移是必需的。
經由這一連串的實驗，我們的結果勾勒出Rho在斑馬魚早期胚發育所扮演的角色。 Rho經由ROCK調控肌動與肌凝蛋白在分裂溝的分佈與產生的收縮力控制細胞質分裂。 同樣地，Rho經由Diaphanous與Profilin影響邊緣囊胚層前緣環狀細肌絲構造之組成，與腔腸化過程中的細胞突起及遷移，進而調控斑馬魚胚腔腸化過程中之外包及其他細胞運動。

關鍵字

斑馬魚 ； 外包作用 ； 胚體趨中及擴展作用 ； Rho ； ROCK ； Diaphanous ； Profilin

並列摘要

Cytokinesis and gastrulation cell movements occur during early embryonic development which relies heavily on dynamic actin assembly. Rho and its associated proteins, including the Rho-associated kinase and Rho-activated formin, diaphanous, have been demonstrated to be key regulators of actomyosin contractility in cytokinesis and actin assembly in cellular protrusion and migration. However, roles of Rho and its associated proteins play in early development remains unclear.
To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated β-catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in marginal blastoderm cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. These results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via at lease a ROK/ROCK-dependent pathway. We also found that Knockdown of another Rho associated protein Diaphanous-related formin, Dia2, by antisense morpholino oligonucleotides (MOs) blocked epiboly formation and convergent extension in a dose-dependent manner. Time-lapse recording showed that cellular protrusions of marginal blastoderm cells, prechordal plate cells and lateral hypoblasts were inhibited in the zdia2 morphants. Furthermore, a ring-like actin structure normally forms during late epiboly was abolished in zdia2 morphants. Lastly, co-injection of antisense MOs of zdia2 and profilin I, but not profilin II, resulted in a synergistic inhibition of gastrulation. These results suggest that zDia2 and profilin I are required for cell movements during gastrulation in zebrafish.
In conclusion, our findings draw the outline of Rho function in early developments. Rho regulates cytokinesis, probably through the assembly of furrow proteins and controlling actomyosin contractility via ROCK. Nevertheless, Rho also controls gastrulation cell migrations, including epiboly, convergence and extension, through regulating cell protrusions and actin condensation at front edge of migrating cells via zDia2 and profilin I.

並列關鍵字

zebrafish ； epiboly ； convergent extension ； Rho ； ROCK ； Diaphanous ； Profilin

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