insm1a在成年斑马鱼自发性脊髓再生过程中的功能研究

Adult zebrafish was adopted as an excellent model to study recovery from spinal cord injury (SCI) due to their extraordinary ability to regenerate. Axonal regrowth in adult zebrafish achieved restoration of function by approximately 4-6 weeks after complete spinal cord transection. Recent reports have shown that insulinoma-associated protein 1 (insm1) is a zinc finger transcription factor with a limited spatial and temporal embryonic expression pattern in neuronal and neuroendocrine tissues. Insm1 represented an important player in early embryonic neurogenesis, the retina and pancreas development. Interestingly, our preliminary data showed that the expression of insm1a was widespread throughout the developing zebrafish central nervous system including midbrain, hindbrain and spinal cord, whereas insm1a expression was not detectable in spinal cord of adult zebrafish. Furthermore, we observed that knockdown of insm1a delayed the recovery of swimming ability, which suggested that insm1a played important roles in adult zebrafish regeneration of spinal cord. Up to date, there was no report concerning to the functional study of insm1a in the spinal cord regeneration. Thus, we propose to examine the insm1a-positive cell fate after adult zebrafish spinal cord injury by performing cell lineage tracing experiments in Tg((insm1a:Cre-ERT2-2A-ECFP::ubi:loxP-EGFP-stop-loxP-mCherry) transgenic zebrafish, and study the response of insm1a-positive cells to spinal cord injury in adult zebrafish by conditional cell ablation in Tg(insm1a:Cre- ERT2-2A-ECFP::beta-actin:loxP-mCherry-stop-loxP-DAT) transgenic zebrafish. In addition, we plan to identify downstream target genes of insm1a by ChIP-sequencing during adult zebrafish spinal cord regeneration. In current project, for the first time we suppose to investigate the roles of insm1a in regulating adult zebrafish spinal cord regeneration. The outcome of this project will help understanding the biological function of insm1a in the adult zebrafish spinal cord regeneration and allow us to open a new avenue for spinal cord repair strategies.

成年斑马鱼具有很强的脊髓损伤后自主修复的能力，是研究脊髓再生的良好模型。Insm1是锌指结构转录因子，在发育阶段的特异性时空表达与其对发育的调控密切相关，特别是在神经、视网膜以及胰岛发育过程中发挥重要作用，到目前为止Insm1在脊髓再生中的研究还未见报道。我们前期研究结果显示：insm1a/b广泛表达在斑马鱼胚胎发育期的中枢神经系统；而在成年斑马鱼脊髓中未检测到表达。在成年斑马鱼脊髓横断损伤模型中insm1a被重新启动表达。并且insm1a的表达下调导致斑马鱼脊髓损伤后再生过程延迟，这提示insm1a在脊髓再生中发挥重要作用。本课题将进一步分析insm1a在成年斑马鱼脊髓损伤后的功能。拟建立多种转基因品系，通过Cre-loxP重组系统介导的Cell ablation和细胞谱系追踪分析insm1a阳性细胞的命运。利用ChIP-Seq技术鉴定其下游分子，分析其参与调控脊髓再生的分子机制。

近年来，斑马鱼已成为研究各种生物学过程理想的模式动物。胰岛瘤相关1a（insm1a）是锌指结构转录因子，在发育阶段的特异性时空表达与其对发育的调控密切相关。研究发现，insm1a在脊椎动物中枢和外周神经系统和神经内分泌系统的细胞形成和分化中起着一系列的作用。 到目前为止，在运动神经元发育中的作用还未见报道。我们通过原位杂交技术发现insm1a在脊髓中的定位，并对转基因斑马鱼Tg(insm1a:mCherry)ntu805进行成像分析发现insm1a表达于原代运动神经元（PMNs）。通过CRISP/Cas9系统建立insm1a突变体，证实insm1a在斑马鱼胚胎中的缺失将导致PMNs发育缺陷，观察发现CaP和MiP减少、运动神经元轴突分支增加、相邻的CaP之间的距离异常等现象，影响运动神经元分化。利用 Zebralab 斑马鱼行为视频跟踪分析系统自动记录insm1a突变体的运动能力，发现insm1a突变体的运动能力显著降低。此外，我们通过realtime–PCR技术检测发现insm1a功能丧失显着降低了olig2和nkx6.1的转录水平，而显微注射olig2和nkx6.1 mRNA能够挽救insm1a缺失导致的运动神经元发育缺陷。这与 insm1a 在成年斑马鱼脊髓再生过程中的功能相一致，是成年斑马鱼自发性脊髓再生过程中的功能研究的重要补充。本研究结果将进一步帮助理解insm1a在斑马鱼发育和脊髓再生过程中的功能，为临床治疗相关疾病提供新的思路。