NeuroD 於澤蛭神經發生中的功能分析

Abstract

神經源性 basic helix-loop-helix（bHLH）轉錄因子是神經發生中高度保守的調控因子，但不同動物的神經細胞譜系與神經系統形成方式具有顯著差異。NeuroD 通常與神經元分化相關，並在部分研究較完整的系統中位於 Neurogenin 等前神經因子的下游。先前針對冠輪動物 bHLH 基因家族的比較分析在 Helobdella 全基因組資料庫中辨識出 ASC 與 NeuroD 的同源基因，但未找到明確的 Neurogenin 或 Atonal 同源基因。因其高度刻板的胚胎細胞譜系，本研究以澤蛭 Helobdella austinensis 為模式，探討 Hau-NeuroD 在神經發生中的表現及發育功能。本研究首先利用全胚原位雜交比較 Hau-ASCa、Hau-ASCb、Hau-ASC-like 與 Hau-NeuroD 四個候選神經源性 bHLH 基因，其中 Hau-NeuroD 與發育中的中樞神經系統呈現最明顯的空間關聯。Hau-NeuroD 自原腸胚時期開始明顯表現，之後沿發育中的腹神經索由前向後形成節段性訊號。譜系追蹤顯示，軀幹 Hau-NeuroD 表現主要與未來將生成軀幹部中樞神經的 N 譜系衍生區域相關，並出現在前端推定的食道上神經節區域，顯示其與不同胚胎來源的中樞神經區域皆具有關聯。功能分析方面，將以 Hau-NeuroD 為標靶的 CRISPR–Cas9 注射入胚胎後，其目標區域呈現 DNA 片段異質性；多數存活胚胎維持近似正常的 Hau-PCSK2 表現，但其中一個胚胎呈現近乎完全喪失的中樞神經 Hau-PCSK2 訊號。由於注射控制組亦具有較高死亡率，且尚未建立單一胚胎的基因型與表型對應，因此目前僅能視為 Hau-NeuroD 可能參與神經元分化或成熟的初步證據。於 OP 譜系過度表達 Hau-NeuroD 顯著降低表皮細胞報導基因 Hau-cif1 陽性細胞數量，但未顯著增加神經細胞報導基因 Hau-elav4 陽性細胞數量；以 H2B:GFP 追蹤 OP 譜系所有後代亦顯示細胞數量與空間分布可能受到影響。綜合而言，Hau-NeuroD 與 Helobdella 中樞神經發生具有關聯，其功能可能不侷限於神經元分化，而可能更廣泛地涉及神經發生的不同階段，包括細胞命運特化與決定、細胞週期退出、神經元分化及後續功能成熟。然而，目前結果尚不足以證明 NeuroD 的表現能直接將表皮細胞轉換為神經元命運。本研究為進一步探討 NeuroD 如何整合於 Helobdella 高度刻板的神經發生架構提供了基礎。

Neurogenic basic helix-loop-helix (bHLH) transcription factors are highly conserved regulators of neurogenesis, although neural cell lineages and the mechanisms of nervous-system formation differ considerably among animals. NeuroD is generally associated with neuronal differentiation and, in several well-characterized systems, functions downstream of proneural factors such as Neurogenin. Previous comparative analyses of lophotrochozoan bHLH gene families identified ASC and NeuroD homologs in Helobdella, whereas recognizable Neurogenin or Atonal homologs were absent. Because of its highly stereotyped embryonic cell lineage, this study used the leech Helobdella austinensis as a model to investigate the expression and potential developmental functions of Hau-NeuroD during neurogenesis. Whole-mount in situ hybridization was first used to compare four neurogenic bHLH candidates, Hau-ASCa, Hau-ASCb, Hau-ASC-like, and Hau-NeuroD, among which Hau-NeuroD showed the strongest spatial association with the developing central nervous system. Hau-NeuroD expression became clearly detectable during gastrulation and subsequently extended posteriorly in a segmental pattern along the developing ventral nerve cord. Lineage tracing showed that trunk Hau-NeuroD expression was predominantly associated with the N-lineage-derived territories, corresponding to the trunk ventral nerve cord, and was also detected in the anterior region corresponding to the putative supraesophageal ganglion, indicating its association with CNS territories of distinct embryonic origins. In functional analyses, injection of CRISPR–Cas9 reagents targeting Hau-NeuroD into embryos produced DNA-fragment heterogeneity at the target region; most surviving embryos retained near-normal Hau-PCSK2 expression, whereas one embryo showed near-complete loss of CNS-associated Hau-PCSK2 expression. Because relatively high mortality also occurred in injected control groups and genotype–phenotype relationships were not established for individual embryos, these results provide only preliminary evidence that Hau-NeuroD may participate in neuronal differentiation or maturation. In gain-of-function experiments, Hau-NeuroD overexpression in the OP lineage significantly reduced the number of cells positive for the epithelial reporter Hau-cif1 but did not significantly increase the number of cells positive for the neural reporter Hau-elav4; preliminary lineage-tracing experiments using single-cell-resolution tracer H2B:GFP also suggested possible alterations in cell number and spatial distribution. Together, these findings suggest a role for Hau-NeuroD in CNS neurogenesis. Furthermore, its functions may not be restricted to neuronal differentiation but may extend more broadly across different stages of neurogenesis, including cell fate specification and determination, cell-cycle exit, neuronal differentiation, and subsequent functional maturation. Although the present results are insufficient to demonstrate that expression of NeuroD can result in an epidermal-to-neuronal fate conversion, this study provides a basis for further investigating how NeuroD is integrated into the highly stereotyped neurogenic program of Helobdella.