PTEN在斑馬魚胚發育過程中之基因表現及功能研究

Abstract

蛋白質去磷酸酶，Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) 為已知腫瘤抑制因子之一，其異常是多種人類癌症發生的主因。PTEN的主要功能是去除調控細胞方向性遷移的訊息傳遞分子PIP3 在D3位置上的磷酸根。雖然許多細胞株已經被用來研究PTEN對細胞遷移的影響，但其在生物體內功能角色的研究仍十分有限。在斑馬魚有兩種不同PTEN同功異構物，分別為PTENA與PTENB。在先前之研究證明PTENA與PTENB在斑馬魚仔稚魚發育可能有不同功能，但其對於PTEN在早期胚發育尤其是對細胞遷移的影響並無探討。因此在本研究中，我乃針對PTENB在斑馬魚原腸期細胞遷移所扮演之角色進行探討。藉由morpholino oligos (MO) 抑制ptenb的表現，我們發現降低ptenb會阻礙原腸期胚細胞遷移方向性。同時，抑制PI3-kinase活性可防止ptenb所造成之缺陷，進一步證明了由PI3-kinase所調控產生之PIP3極可能因ptenb活性之抑制而累積而造成所觀察到之缺陷。更進一步檢查PIP3下游的目標分子中，我們觀察到肌動蛋白 (actin) 聚合化增加之現象。因此，我們推測在斑馬魚原腸期，ptenb及PI3-kinase會經由調節PIP3之濃度掌控肌動蛋白聚合化以調控原腸期胚細胞方向性遷移。

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a well-known tumor suppressor whose mutation induces a variety of cancers in human. By dephosphorylating the D3 position of PIP3 (phosphatidylinositol-3,4,5- triphosphate), PTEN functions as a negative regulator to reduce PIP3, a signaling molecule for polarized cell migration in cellular level studies. However, the role of PTEN in cellular migration in organism has been difficult to study. In this regards, the study of PTEN in embryogenesis is a step further toward the understanding of PTEN in organismal level. Here, I used zebrafish as a model to investigate the role of PTEN in embryogenesis, in particular during gastrulation. I demonstrated that PTENB, one of the PTEN isoforms, regulates convergence and extension during zebrafish gastrulation. Knocking down ptenb by antisense morpholino oligonucleotide interfered with normal dorsoventral morphogenesis without perturbing dorsoventral patterning. The defects could be rescued by inhibiting PI3-kinase which demonstrated that ptenb may affect the process through controlling the PIP2/PIP3 balance. At the cellular level, the ptenb knockdown affects polarity and persistency of lamellapodia of lateral convergent cells. Furthermore, actin polymerization was increased in the ptenb-knockdown embryos. These results correlate to the PTEN study in culture cells to show that PTEN acts via the PIP3-, Rac 1- and Cdc42-dependent signaling pathway. Therefore, in this work I not only first confirm this pathway in an animal study but reveal the convergence and extension in zebrafish require ptenb to control the balance of PIP3 and PIP2 for normal cell migrating behaviors.