Functional Analysis of the Dynein Light Chain Genes, Dnali1 and Tcte3

In the present study, the function of two murine genes, Dnali1 and Tcte3 were investigated. Murine Dnali1 gene is a homologue of Chlamydomonas p28 and belongs to the family of inner arm dynein light chains. The immunolocalization studies indicate that Dnali1 is localized along the entire length of sperm flagella and tracheal cilia. Moreover, Dnali1 is also detected in cilia of brain lateral ventricle lining, suggesting that Dnali1 is an integral component of axoneme. However, the expression of Dnali1 and its human orthologue DNALI1 in tissues, which lack the axonemal structures, supports a putative role of this dynein light chain in the cytoplasmic dynein complex. The results of the co-localisation studies of Dnali1 and Dnchc1 in the mouse Neuroblastoma and ES cells sustain this assumption. Moreover, yeast-two-hybrid assay and co-immunoprecipitation studies demonstrated that Dnali1 might be interacting with the carboxy-terminal of cytoplasmic dynein heavy chain. Targeted disruption of Dnali1 gene was carried out by homologous recombination. On subsequent breedings, the heterozygous animals were found to be fertile and had no apparent abnormalities. However, no Dnali1-/- offspring were obtained by heterozygous breedings, indicating that Dnali1 gene deficiency leads to prenatal embryonic lethality. The Dnali1 deficient embryos die shortly after implantation. To further characterize the reason for embryonic lethality, in vitro culture experiments were performed. It was revealed that putative Dnali1 null mutants exhibited lack of a distinguishable ICM-like structure, although the trophectoderm layer differentiated into giant cells. Subsequently, it was observed that Dnali1 was associated with the Golgi apparatus even in the ES cells treated with depolymerising drug, indicating that the Dnali1 interacts with the Golgi apparatus in a microtubule-independent manner. Thus, it can be speculated that Dnali1 function is necessary for the organization of the Golgi apparatus and is required for the proper function of this organelle presumably as a putative component of the cytoplasmic dynein complex machinery.The Tcte3 gene encodes a member of outer arm dynein light chain family and is part of both axonemal and cytoplasmic dynein complexes. To elucidate the function of Tcte3 gene and its role in spermatogenesis, an attempt was made to inactivate the entire gene. No Tcte3 deficient mice were identified among offspring derived from the F1 intercrosses by conventional genotyping approach. On genomic and cDNA analyses, it was revealed that more than one copy of Tcte3 gene is present in the mouse genome. However, the other copies seem to be either not expressing at a substantial level or are pseudogenes. Therefore, quantitative real-time PCR analysis was performed, which demonstrated the presence of Tcte3-/- mice in normal Mendelian ratios among the F2 progeny. Following Northern and Western blot analyses confirmed the real-time PCR result. Mice lacking Tcte3 displayed male infertility due to multiple defects in spermatogenesis and due to a high incidence of cell death at the spermatocyte and spermatid stages.