[Mutant generation of the testis genes and phenotype analyses in Drosophila].

Multiple genes work together to maintain the normal functions of the reproductive system. However, for many of these genes, little is known about their specific functions and mechanisms. In the present study, eight Drosophila genes, including CG4161, CG11475, CG2921, CG10541, CG7276, CG3800, CG8117 and CG16779, were selected for detailed studies based on their testis expression, undefined functions, and having highly homologous and conserved genes in humans (Homo sapiens) and mouse (Mus musculus). We analyzed their expression levels in different tissues, and determined their probably functions in male reproduction. The results showed that the first five genes were mainly expressed in testis, while other three showed ubiquitous expression in all tissues examined. Using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and homology-directed repair (HDR) strategies, we have systematically generated the mutants of these genes and studied their functions in male reproduction. Loss of function of CG7276 and CG3800 resulted in partial sterility and reduction of the offspring number, while other genes showed no significant impact on fertility. CG7276 -/- and CG3800 -/- mutants were partial sterile and showed various types of abnormities, including seminal vesicle atrophy, spermatogonial stem cell reduction and cellular distribution disorders. Results of DNA and F-actin staining also indicated that CG7276 and CG3800 could play important roles in spermiogenesis. The establishments of these mutants have provided means to unravel the functions and mechanisms of these genes in animal models.

Functional conservation study of polarity protein Crumbs intracellular domain.

The transmembrane protein Crumbs (Crb) plays key roles in the establishing and maintaining cell apical-basal polarity in epithelial cells by determining the apical plasma membrane identity. Although its intracellular domain contains only 37 amino acids, it is absolutely essential for its function. In Drosophila, mutations in this intracellular domain result in severe defects in epithelial polarity and abnormal embryonic development. The intracellular domain of Crb shows high homology across species from Drosophila to Mus musculus and Homo sapiens. However, the intracellular domains of the two Crb proteins in C. elegans are rather divergent from those of Drosophila and mammals, raising the question on whether the function of the intracellular domain of the Crb protein is conserved in C. elegans. Using genomic engineering approach, we replaced the intracellular domain of the Drosophila Crb with that of C. elegans Crb2 (CeCrb2), which has extremely low homology with those from the Crb proteins of Drosophila and mammals. Surprisingly, substituting the intracellular domain of Drosophila Crb with that of CeCrb2 did not cause any abnormalities in development of the Drosophila embryo, in terms of expression and localization of Crb and other polarity proteins and apical-basal polarity in embryonic epithelial cells. Our results support the notion that despite their extensive sequence variations, all functionally critical amino acid residues and motifs of the intercellular domain of Crb proteins are fully conserved between Drosophila and C. elegans.