RESUMO
Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by small brain size with mental retardation. CPAP (also known as CENPJ), a known microcephaly-associated gene, plays a key role in centriole biogenesis. Here, we generated a previously unreported conditional knockout allele in the mouse Cpap gene. Our results showed that conditional Cpap deletion in the central nervous system preferentially induces formation of monopolar spindles in radial glia progenitors (RGPs) at around embryonic day 14.5 and causes robust apoptosis that severely disrupts embryonic brains. Interestingly, microcephalic brains with reduced apoptosis are detected in conditional Cpap gene-deleted mice that lose only one allele of p53 (also known as Trp53), while simultaneous removal of p53 and Cpap rescues RGP death. Furthermore, Cpap deletion leads to cilia loss, RGP mislocalization, junctional integrity disruption, massive heterotopia and severe cerebellar hypoplasia. Together, these findings indicate that complete CPAP loss leads to severe and complex phenotypes in developing mouse brain, and provide new insights into the causes of MCPH.
Assuntos
Microcefalia , Animais , Encéfalo/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Humanos , Camundongos , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
We previously isolated Aurora-C (Aurkc/Aie1) in a screen for kinases expressed in mouse sperm and eggs. Aurora-C kinase was reported to be a chromosomal passenger protein that plays critical roles in chromosome alignment, segregation, kinetochore-microtubule attachment, and cytokinesis in female mouse meiosis. This chapter describes experimental approaches for examining the subcellular localization and function of Aurora-C kinase during female mouse meiosis, presenting detailed methods for introducing exogenous Aurora-C wild-type and kinase-dead mutant mRNAs into mouse oocytes by cytosolic microinjection, and preparing whole-mount meiotic oocytes and chromosome spreads for confocal immunofluorescence microscopy.
Assuntos
Ensaios Enzimáticos/métodos , Meiose , Oócitos/citologia , Oócitos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase C , Aurora Quinases , Separação Celular , Cromossomos de Mamíferos/metabolismo , Citosol/enzimologia , Feminino , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Oócitos/metabolismo , Ovário/citologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Transcrição GênicaRESUMO
We previously isolated Aurora-C/Aie1 in a screen for kinases expressed in mouse sperm and eggs. Here, we show the localization of endogenous Aurora-C and examine its roles during female mouse meiosis. Aurora-C was detected at the centromeres and along the chromosome arms in prometaphase I-metaphase I and was concentrated at centromeres at metaphase II, in which Aurora-C also was phosphorylated at Thr171. During the anaphase I-telophase I transition, Aurora-C was dephosphorylated and relocalized to the midzone and midbody. Microinjection of the kinase-deficient Aurora-C (AurC-KD) mRNA into mouse oocytes significantly inhibited Aurora-C activity and caused multiple defects, including chromosome misalignment, abnormal kinetochore-microtubule attachment, premature chromosome segregation, and cytokinesis failure in meiosis I. Furthermore, AurC-KD reduced Aurora-C and histone H3 phosphorylation and inhibited kinetochore localization of Bub1 and BubR1. Similar effects also were observed in the oocytes injected with INCNEP-delIN mRNAs, in which the Aurora-C binding motif was removed. The most dramatic effect observed in AurC-KD-injected oocytes is cytokinesis failure in meiosis I, resulting in producing large polyploid oocytes, a pattern similar to Aurora-C deficiency human spermatozoa. Surprisingly, we detected no Aurora-B protein in mouse oocytes. We propose that Aurora-C, but not Aurora-B, plays essential roles in female mouse meiosis.