Ptf1a 调控热带爪蛙早期神经及胰腺发育的分子机制探究

MOLECULAR MECHANISM OF PTF1A REGULATING EARLY NEURAL AND PANCREATIC DEVELOPMENT IN XENOPUS TROPICALIS

肖园慧

11849370

硕士

生物学

陈永龙

2020-06-05

2020-07-07

哈尔滨工业大学

深圳

胰腺转录因子 1a（pancreatic transcription factor 1a， Ptf1a） 编码一种碱性螺旋-环-螺旋（basic helix-loop-helix， bHLH）蛋白，在脊椎动物的中枢神经系统和胰腺中特异性表达，在神经元亚型的分化，胰腺多能祖细胞的命运决定以及成体外分泌胰腺的维持中都起着重要作用，但目前关于其调控神经及胰腺早期发育的下游靶点还知之甚少。本研究对 CRISPR/Cas9 技术构建的热带爪蛙 ptf1a-KI-3×HA-Td-tomato 品系进行了传代和鉴定，在 F2 代获得了健康的纯合敲进个体。 通过观察报告基因Td-tomato 的红色荧光发现， Ptf1a 在热带爪蛙视网膜、后脑及胰腺的表达均晚于已报道的 mRNA 信号，结合 qRT-PCR 和 Western blot 分析发现 Ptf1a 在早期胚胎发育期间的表达水平呈上升趋势。基于热带爪蛙 34 期 ptf1a 敲除整体胚胎的转录组测序分析及已发表文献，本研究在差异表达基因中筛选到 7 个与神经发育相关的基因： purb，dvl2，nect1，psen2， purne2， slc4a3， slc6a1。 对 34 期和 43 期 ptf1a 敲除整体胚胎的转录组测序分析提示，该时期 Ptf1a 可能通过 MAPK， ERbB， GNRH 和 PPAR 等信号通路调控多基因的表达。本文以 ptf1a-KI-3×HA-Td-tomato 43 期蝌蚪的胰腺组织分离后，利用 HA 标签的抗体进行了染色质免疫共沉淀和高通量测序。基于对ChIP-seq 富集基因的 GO 分析和 KEGG 分析结果，本文筛选到了 7 个与胰腺发育相关的基因： selenot， hk2， prx， zfp36l2， mapk10， slc30a8， hes1。总之，本研究在蛋白水平揭示了 Ptf1a 的表达变化，已获得的 ptf1a-KI-3×HATd-tomato 热带爪蛙品系能够用于后续对 Ptf1a 的功能分析，筛选到的差异表达基因和 ChIP-seq 富集基因也为进一步研究 Ptf1a 调控中枢神经系统及胰腺的发育机制奠定了基础。

Pancreatic transcription factor 1a (Ptf1a) encodes a basic helix-loop-helix (bHLH) protein, which is specifically expressed in the vertebrate nervous system and pancreas. Ptf1a plays important roles in neuron subtype specification, pancreatic progenitor cell fate specification, and the maintenance of adult exocrine pancreas. But little is known about its downstream targets in regulating early neural and pancreatic development.In this study, healthy F2 homozygotes were obtained from a Xenopus tropicalis ptf1a-KI-3×HA-Td-tomato knock-in strain established by CRISPR/Cas9 technology. The red fluorescent signal of Td-tomato reflecting Ptf1a expression in retina, hindbrain, and pancreas appeared later than the reported mRNA signal. qRT-PCR and Western blot analyses indicated that the expression level of Ptf1a increased during early embryonic development. Based on the RNA-seq data of stage 34 ptf1a knockout (KO) whole embryos and published literatures, seven genes related to neurodevelopment were identified from the differentially expressed genes, namely purb, dvl2, nect1, psen2, purne2, slc4a3 and slc6a1. The RNA-seq analyses of the stage 34 and stage 43 ptf1a-KO whole embryos suggest that Ptf1a might regulate the expression of multiple genes through MAPK, ERbB, GNRH and PPAR signaling pathways. ChIP-seq (chromatin immunoprecipitation-sequencing) analysis was carried out with dissected pancreata from stage 43 ptf1a-KI-3×HA-Td-tomato tadpoles using the HA-tag antibody. Based on the GO analysis and KEGG analysis of ChIP-seq data, seven genes related to pancreatic development were identified，namely selenot，hk2，prx，zfp36l2，mapk10，slc30a8 and hes1.In conclusion, this study revealed the Ptf1a protein expression profile during Xenopus tropicalis early embryogenesis. The established ptf1a-KI-3×HA-Td-tomato strain is useful for studying Ptf1a function. The preliminary RNA-seq and ChIP-seq data are helpful for further research on the mechanism by which Ptf1a regulates the development of central nervous system and pancreas.

Ptf1a 中枢神经系统 胰腺 RNA-seq ChIP-seq

Ptf1a central nervous system pancreas RNA-seq ChIP-seq

中文

联合培养

南方科技大学

肖园慧. Ptf1a 调控热带爪蛙早期神经及胰腺发育的分子机制探究[D]. 深圳. 哈尔滨工业大学,2020.