Expression profiling analysis of mouse P4 cerebellum in CitK mutant mice proficient or knockout for P53

Purpose: Citron kinase (CitK) knockout mice show a severe form of primary microcephaly, associated with ataxia and lethal epilepsy. This phenotype is caused by massive apoptosis occuring during embryonic and post-natal brain development, associated with cytokinesis failure. Cerebellum is the tissue showing highest sensitivity to CitK loss. The clinical phenotype of CitK knockout mice is significantly resued by P53 inactivation. In addition, CitK/P53 double knockout brains have almost normal levels of apoptosis, but display high percentage of binucleated and multinucleated cells. The aim of this study was to analyze the gene expression changes produced in developing neural tissue by CitK loss and to determine which alterations are P53-dependent. expression changes Methods: We analyzed by RNA sequencing total RNA extracted from P4 cerebellum of mice characterized by the following genotypes: 1. CitK +/-, P53 +/- (CTRL); 2. CitK -/-, P53 +/- (CitK-KO); 3. CitK +/-, P53 -/- (P53-KO); 4. CitK -/-, P53 -/- (D-KO). Biological triplicates were analyzed per every genotype. Conclusions: The loss of CitK leads to a strong reduction of the expression of pro-neural genes and induces a P53-related pro-apoptotic gene sets. The analysis of D-KO mice reveals that most of these changes are P53-dependent, but many genes implicated in growth arrest are induced through P53-independent mechanisms. Cerebellar mRNA profiles of 4-day old mice of CTRL, CitK-KO, P53-KO and D-KO mice were generated by deep sequencing, in triplicate, using Illumina HiScan SQ研究目的 香橼激酶（Citron kinase, CitK）敲除小鼠会表现出严重的原发性小头畸形，伴随共济失调与致死性癫痫。该表型由胚胎及出生后脑发育过程中发生的大量细胞凋亡引发，且与胞质分裂失败相关。小脑是对CitK缺失敏感性最高的组织。P53失活可显著挽救CitK敲除小鼠的临床表型。此外，CitK/P53双敲除小鼠的脑组织凋亡水平几乎恢复正常，但会出现高比例的双核及多核细胞。本研究旨在分析CitK缺失对发育中神经组织的基因表达影响，并确定哪些改变为P53依赖性的。 研究方法 我们通过RNA测序（RNA-seq）分析了四种基因型小鼠出生后第4天（P4）小脑组织提取的总RNA：1. CitK+/-、P53+/-（对照组，CTRL）；2. CitK-/-、P53+/-（CitK敲除组，CitK-KO）；3. CitK+/-、P53-/-（P53敲除组，P53-KO）；4. CitK-/-、P53-/-（双敲除组，D-KO）。每种基因型均设置3次生物学重复。 研究结论 CitK缺失会显著降低促神经基因的表达水平，并诱导与P53相关的促凋亡基因集。对双敲除小鼠的分析显示，此类改变大多为P53依赖性，但许多涉及生长阻滞的基因是通过P53非依赖性机制被诱导的。本研究通过Illumina HiScan SQ平台对4日龄对照组、CitK-KO、P53-KO及D-KO小鼠的小脑mRNA表达谱进行了三次重复深度测序。