澤蛭 N teloblast 的轉錄體分析

Abstract

細胞內物質的不對稱分離對澤蛭的早期胚胎發育至關重要。在卵裂過程中，一部分被稱為 teloplasm 的細胞質隨著細胞分裂分配至五對左右對稱的eloblasts，決定其細胞類型。Teloblasts 進行多次幹細胞式不對稱分裂，產生一連串的初級胚母細胞 (primary blast cells)，進而發育為體節構造。胚胎學實驗顯示，N teloblasts 及初級胚母細胞的細胞命運並不需要細胞之間的交互作用，而是由細胞不對稱分裂自主調控產生，其分子機制仍不清楚。本研究首先對於Hau-EGL13a進行基因表現位置及功能性研究。Hau-EGL13a是透過EST-based 原位雜交染色檢測所發現、對teloblast 具有專一性的基因，透過原位雜交染色，我發現該基因除了在N teloblast以外，還會表現在M teloblast。以 Cas13d對N teloblast進行Hau-EGL13a knockdown後並未造成可觀測的發育異常，表示該基因可能僅參與較為晚期的胚胎發育而非決定N細胞命運的決定因子。接著，我透過轉錄體分析找出在N teloblasts中有較高表現量的基因，並鑑定尋找可能影響 N teloblasts 命運的發育基因。而原位雜交染色顯示，在 N teloblasts 中表現較高的基因大多並非專一地表現在 N teloblasts，而是廣泛分布於各 teloblasts。Gene Ontology 分析顯示，在 N teloblasts 中高表現的基因主要與細胞生理活動相關，而非參與在胚胎發育或是細胞分化的過程。綜合而言，由母源mRNA不對稱分布所形成的細胞質決定物 (cytoplasmic determinant) 模型應該不是決定N teloblasts 命運的機制。除了以RNA作為決定因子影響細胞命運外，透過蛋白質的不對稱分布引起區域性的轉譯調控同樣能在細胞分裂時使兩顆細胞產生不同的命運。參考上述模型，未來的研究方向應著重於蛋白質層面的分析，包含轉譯以及後轉錄修飾的調控。

Asymmetric segregation of cytoplasmic determinants plays a critical role in the early development of the leech. During cleavage, a pool of cytoplasm called teloplasm segregates into five bilateral pairs of teloblasts to specify teloblast identities. A teloblast then undergoes iterated stem-cell-like asymmetric divisions to produce a bandlet of primary blast cells, or segment founder cells, each gives rise to a set of serially homologous progeny. Among the five teloblast pairs, the N eloblast mainly contributes to the central nervous system. Embryological experiments revealed that, fate specification of N teloblasts and primary blast cells is cell autonomous. However, molecular identities for the determinants of teloblasts and primary blast cells remain elusive. In this study, I first investigated the expression pattern and function of Hau-EGL13a, a teloblast-specific gene identified through an EST-based in situ hybridization screen. Although initially detected in the N teloblast, Hau-EGL13a was also expressed in the M teloblast. Cas13d-mediated knockdown of Hau-EGL13a in the N teloblast did not result in observable phenotypic changes, suggesting that it functions as a regulator of later developmental processes in teloblasts rather than serving as a determinant of initial cell fate. Subsequently, I performed transcriptome analysis to identify potential RNA determinants of N fate by searching for transcripts enriched in N teloblasts. Unexpectedly, most statistically significant candidates are only slightly enriched in N teloblast. In situ hybridization analysis showed that they are broadly distributed and not specifically localized to the N teloblast. GO analysis indicated that these N-enriched genes are most related to metabolic process, and are not associated with embryonic development or cell differentiation. Together, our results suggest that the molecular mechanism for the specification of N teloblast does not follow the standard model of cytoplasmic determinant in which transcripts encoding for developmental regulatory genes is specifically segregated into a blastomere to specify its developmental fate. In addition to the RNA segregation model, another prevalent mechanism for specifying cell identity during embryonic development is protein localization, which is commonly observed across diverse species. Accordingly, future research should focus on protein-level processes, including translational control and transcriptional regulation.