以果蝇、小鼠为模式解析LAMMER蛋白激酶在生殖细胞分化过程中的功能

Germ cells' transition from the mitotic to the meiotic state is tightly regulated.and crucial for the sustainable reproduction. Drosophila spermatogenesis is.an ideal system to dissect the regulatory program of the mitosis/meiosis.decision. The timely accumulation of the pro-differentiation factor Bam has.been shown to be central in this process. In a Drosophila genetic screen, we discovered that the mutations in Doa, a known gene encoding a member of the highly conserved LAMMER protein kinase family, cell-autonomously prevented the amplifying germ cells from exiting the mitotic cycle. Remarkably, the human or murine homolog of Doa could restore the mitosis/meiosis transition and even the fertility of Doa mutant flies. Such spermatogenic function of Doa or its human homolog requires the conserved residue in their respective kinase catalytic domain, previously shown to be essential for the LAMMER kinase activity. Furthermore, additional Bam expression in Doa mutant germline promoted the differentiation from the mitotic to the meiotic state. Our findings suggest that LAMMER protein kinase, represented by Doa and its mammalian homologs, is a critical and conserved component in the regulatory program of the mitosis-to-meiosis switch. We will explore the function of the LAMMER kinases in mouse spermatogenesis by generating CLK2 knockout mice. Additionally, we will further dissect the regulatory mechanism of Doa/CLK2 in the germline differentiation.

生殖细胞从有丝向减数分裂转变是生殖过程的关键并受严格调控。果蝇精子发生是研究这一过程的理想系统。前人发现分化因子Bam量决定生殖细胞何时终止有丝分裂（精原细胞）进入减数分裂（精母细胞）。我们发现LAMMER激酶家族成员Doa突变阻断精原细胞向精母细胞分化，同时精原细胞过度增殖。人类和小鼠的LAMMER激酶CLK2可以替代Doa在果蝇精子发生过程中的功能，甚至可以恢复Doa突变体的育性。另外，去除LAMMER激酶域、或改变其中1个保守氨基酸，都能使Doa或humanCLK2丧失功能。Doa作用在Bam的上游促进精原细胞向精母细胞分化。基于上述结果，我们推测：LAMMER激酶在调控生殖细胞从有丝分裂向减数分裂转变的过程中具有跨物种的保守性。我们将以小鼠的精子发生为模式研究哺乳动物LAMMER激酶的功能；将在果蝇、小鼠2个模式中以Doa/CLK2为切入点进一步解析生殖细胞分化的调控机制。

摘要. 生殖细胞从有丝向减数分裂转变是生殖过程的关键并受严格调控。果蝇精子发生是研究这一过程的理想系统。前人发现分化因子Bam 的量决定生殖细胞何时终止有丝分裂（精原细胞）进入减数分裂（精母细胞）。我们通过前期EMS突变筛选找到一个调控有丝分裂向减数分裂转变的基因Doa,该基因属于LAMMER激酶家族，在果蝇与哺乳动物中非常保守。我们推测：LAMMER激酶在调控生殖细胞从有丝分裂向减数分裂转变的过程中具有跨物种的保守性。在本面上项目资助的四年研究过程中，我们主要以果蝇和基因敲除小鼠为实验体系，研究了LAMMER蛋白激酶在生殖细胞分化过程中的功能。我们发现,LAMMER激酶家族成员Doa突变阻断精原细胞向精母细胞分化，同时精原细胞过度增殖。人类和小鼠的LAMMER激酶CLK2可以替代Doa在果蝇精子发生过程中的功能，甚至可以恢复Doa 突变体的育性。Doa作用在Bam的上游促进精原细胞向精母细胞分化。但是,我们目前尚未发现Doa激酶的底物。.我们通过Cre/LoxP系统和CRISPR/Cas9技术分别构建了两种CLK2基因敲除小鼠。在CLK2基因敲除小鼠中，雄性小鼠[睾丸重量/体重]比明显下降, 并出现个别不育或逐渐低育的雄性小鼠。我们在果蝇、小鼠2个模式动物中以Doa/CLK2 为切入点，进一步解析生殖细胞分化的调控机制。.项目执行期间，发表论文2篇，培养博士2名。