Distinct microglial transcriptomic signatures within the hippocampus.

Microglia, the resident immune cells of the brain, are crucial in the development of the nervous system. Recent evidence demonstrates that microglia modulate adult hippocampal neurogenesis by inhibiting cell proliferation of neural precursors and survival both in vitro and in vivo, thus maintaining a balance between cell division and cell death in the neural stem cell pool. There are increasing reports suggesting these microglia found in neurogenic niches differ from their counterparts in non-neurogenic areas. Here, we present evidence that hippocampal microglia exhibit transcriptomic heterogeneity, with some cells expressing genes associated with neurogenesis. By comprehensively profiling myeloid lineage cells in the hippocampus using single cell RNA-sequencing, we have uncovered a small, yet distinct population of microglia which exhibit depletion in genes associated with homeostatic microglia and enrichment of genes associated with phagocytosis. Intriguingly, this population also expresses a gene signature with substantial overlap with previously characterized phenotypes, including disease associated microglia (DAM), a particularly unique and compelling microglial state.

Novel roles of HP1a and Mcm10 in DNA replication, genome maintenance and photoreceptor cell differentiation.

Both Mcm10 and HP1a are known to be required for DNA replication. However, underlying mechanism is not clarified yet especially for HP1. Knockdown of both HP1a and Mcm10 genes inhibited the progression of S phase in Drosophila eye imaginal discs. Proximity Ligation Assay (PLA) demonstrated that HP1a is in close proximity to DNA replication proteins including Mcm10, RFC140 and DNA polymerase ε 255 kDa subunit in S-phase. This was further confirmed by co-immunoprecipitation assay. The PLA signals between Mcm10 and HP1a are specifically observed in the mitotic cycling cells, but not in the endocycling cells. Interestingly, many cells in the posterior regions of eye imaginal discs carrying a double knockdown of Mcm10 and HP1a induced ectopic DNA synthesis and DNA damage without much of ectopic apoptosis. Therefore, the G1-S checkpoint may be affected by knockdown of both proteins. This event was also the case with other HP family proteins such as HP4 and HP6. In addition, both Mcm10 and HP1a are required for differentiation of photoreceptor cells R1, R6 and R7. Further analyses on several developmental genes involved in the photoreceptor cell differentiation suggest that a role of both proteins is mediated by regulation of the lozenge gene.

The Human Bcl-2 Family Member Bcl-rambo Localizes to Mitochondria and Induces Apoptosis and Morphological Aberrations in Drosophila.

Bcl-2 family proteins play a central role in regulating apoptosis. We previously reported that human Bcl-rambo, also termed BCL2L13, localized to mitochondria and induced apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in Drosophila melanogaster. Bcl-rambo mainly localized to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induced apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induced aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induced the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo was partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, we showed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. The results of the present study demonstrated that human Bcl-rambo localized to mitochondria and at least regulated an apoptosis signaling pathway in Drosophila.

Polycomb-dependent nucleolus localization of Jumonji/Jarid2 during Drosophila spermatogenesis.

Drosophila Jumonji/Jarid2 (dJmj) has been identified as a component of Polycomb repressive complex 2. However, it is suggested that dJmj has both PRC-dependent and -independent roles. Subcellular localization of dJmj during spermatogenesis is unknown. We therefore performed immunocytochemical analyses with specific antibodies to dJmj and tri-methylation at lysine 27 on histone H3 (H3K27me3). Interestingly, dJmj exclusively localizes at nucleolus in the late growth stage. Examination of the dJmj localization in various Polycomb group (PcG) mutant lines at the late growth stage allowed identification of some PcG genes, including Polycomb (Pc), to be responsible for dJmj recruitment to nucleolus. In addition, we found that size of nucleolus was decreased in some of these mutant lines. In a mutant of testis-specific TAF homolog (tTAF) that is responsible for nucleolus localization of Pc, dJmj signals were detected not only at nucleolus but also on the condensed chromatin in the late growth stage. Duolink In situ Proximity ligation assay clarified that Pc interacts with dJmj at nucleolus in the late growth stage. Furthermore, the level of H3K27me3 decreased in nuclei at this stage. Taken together, we conclude that tTAF is responsible for recruitments of dJmj to nucleolus in the late growth stage that appears to be mediated by Pc. Compartmentalization of dJmj in nucleolus together with some of PcG may be necessary to de-repress the expression of genes required to cellular growth and proliferation in the following meiotic divisions.

The Hippo pathway as a target of the Drosophila DRE/DREF transcriptional regulatory pathway.

The DRE/DREF transcriptional regulatory system has been demonstrated to activate a wide variety of genes with various functions. In Drosophila, the Hippo pathway is known to suppress cell proliferation by inducing apoptosis and cell cycle arrest through inactivation of Yorkie, a transcription co-activator. In the present study, we found that half dose reduction of the hippo (hpo) gene induces ectopic DNA synthesis in eye discs that is suppressed by overexpression of DREF. Half reduction of the hpo gene dose reduced apoptosis in DREF-overexpressing flies. Consistent with these observations, overexpression of DREF increased the levels of hpo and phosphorylated Yorkie in eye discs. Interestingly, the diap1-lacZ reporter was seen to be significantly decreased by overexpression of DREF. Luciferase reporter assays in cultured S2 cells revealed that one of two DREs identified in the hpo gene promoter region was responsible for promoter activity in S2 cells. Furthermore, endogenous hpo mRNA was reduced in DREF knockdown S2 cells, and chromatin immnunoprecipitation assays with anti-DREF antibodies proved that DREF binds specifically to the hpo gene promoter region containing DREs in vivo. Together, these results indicate that the DRE/DREF pathway is required for transcriptional activation of the hpo gene to positively control Hippo pathways.

Drosophila Mcm10 is required for DNA replication and differentiation in the compound eye.

Mini chromosome maintenance 10 (Mcm10) is an essential protein, which is conserved from S. cerevisiae to Drosophila and human, and is required for the initiation of DNA replication. Knockdown of Drosophila Mcm10 (dMcm10) by RNA interference in eye imaginal discs induces abnormal eye morphology (rough eye phenotype), and the number of ommatidia is decreased in adult eyes. We also observed a delay in the S phase and M phase in eye discs of dMcm10 knockdown fly lines. These results show important roles for dMcm10 in the progression of S and M phases. Furthermore, genome damage and apoptosis were induced by dMcm10 knockdown in eye imaginal discs. Surprisingly, when we used deadpan-lacZ and klingon-lacZ enhancer trap lines to monitor the photoreceptor cells in eye discs, knockdown of dMcm10 by the GMR-GAL4 driver reduced the signals of R7 photoreceptor cells. These data suggest an involvement of dMcm10 in R7 cell differentiation. This involvement appears to be independent of the apoptosis induced by dMcm10 knockdown. Together, these results suggest that dMcm10 knockdown has an effect on DNA replication and R7 cell differentiation.

Drosophila RecQ4 is directly involved in both DNA replication and the response to UV damage in S2 cells.

The RecQ4 protein shows homology to both the S.cerevisiae DNA replication protein Sld2 and the DNA repair related RecQ helicases. Experimental data also suggest replication and repair functions for RecQ4, but the precise details of its involvement remain to be clarified.Here we show that depletion of DmRecQ4 by dsRNA interference in S2 cells causes defects consistent with a replication function for the protein. The cells show reduced proliferation associated with an S phase block, reduced BrdU incorporation, and an increase in cells with a subG1 DNA content. At the molecular level we observe reduced chromatin association of DNA polymerase-alpha and PCNA. We also observe increased chromatin association of phosphorylated H2AvD--consistent with the presence of DNA damage and increased apoptosis.Analysis of DmRecQ4 repair function suggests a direct role in NER, as the protein shows rapid but transient nuclear localisation after UV treatment. Re-localisation is not observed after etoposide or H2O2 treatment, indicating that the involvement of DmRecQ4 in repair is likely to be pathway specific.Deletion analysis of DmRecQ4 suggests that the SLD2 domain was essential, but not sufficient, for replication function. In addition a DmRecQ4 N-terminal deletion could efficiently re-localise on UV treatment, suggesting that the determinants for this response are contained in the C terminus of the protein. Finally several deletions show differential rescue of dsRNA generated replication and proliferation phenotypes. These will be useful for a molecular analysis of the specific role of DmRecQ4 in different cellular pathways.