Integrin restriction by miR-34 protects germline progenitors from cell death during aging.

During aging, regenerative tissues must dynamically balance the two opposing processes of proliferation and cell death. While many microRNAs are differentially expressed during aging, their roles as dynamic regulators of tissue regeneration have yet to be described. We show that in the highly regenerative Drosophila testis, miR-34 levels are significantly elevated during aging. miR-34 modulates germ cell death and protects the progenitor germ cells from accelerated aging. However, miR-34 is not expressed in the progenitors themselves but rather in neighboring cyst cells that kill the progenitors. Transcriptomics followed by functional analysis revealed that during aging, miR-34 modifies integrin signaling by limiting the levels of the heterodimeric integrin receptor αPS2 and ßPS subunits. In addition, we found that in cyst cells, this heterodimer is essential for inducing phagoptosis and degradation of the progenitor germ cells. Together, these data suggest that the miR-34-integrin signaling axis acts as a sensor of progenitor germ cell death to extend progenitor functionality during aging.

Sedentary Behavior Impacts on the Epigenome and Transcriptome: Lessons from Muscle Inactivation in Drosophila Larvae.

The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. Our findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating our findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.

Live Imaging of Phagoptosis in ex vivo Drosophila Testis.

Phagoptosis is a prevalent type of programmed cell death (PCD) in adult tissues in which phagocytes non-autonomously eliminate viable cells. Therefore, phagoptosis can only be studied in the context of the entire tissue that includes both the phagocyte executors and the targeted cells doomed to die. Here, we describe an ex vivo live imaging protocol of Drosophila testis to study the dynamics of phagoptosis of germ cell progenitors that are spontaneously removed by neighboring cyst cells. Using this approach, we followed the pattern of exogenous fluorophores with endogenously expressed fluorescent proteins and revealed the sequence of events in germ cell phagoptosis. Although optimized for Drosophila testis, this easy-to-use protocol can be adapted to a wide variety of organisms, tissues, and probes, thus providing a reliable and simple means to study phagoptosis.

The phagocytic cyst cells in Drosophila testis eliminate germ cell progenitors via phagoptosis.

Phagoptosis is a frequently occurring nonautonomous cell death pathway in which phagocytes eliminate viable cells. While it is thought that phosphatidylserine (PS) "eat-me" signals on target cells initiate the process, the precise sequence of events is largely unknown. Here, we show that in Drosophila testes, progenitor germ cells are spontaneously removed by neighboring cyst cells through phagoptosis. Using live imaging with multiple markers, we demonstrate that cyst cell-derived early/late endosomes and lysosomes fused around live progenitors to acidify them, before DNA fragmentation and substantial PS exposure on the germ cell surface. Furthermore, the phagocytic receptor Draper is expressed on cyst cell membranes and is necessary for phagoptosis. Significantly, germ cell death is blocked by knockdown of either the endosomal component Rab5 or the lysosomal associated protein Lamp1, within the cyst cells. These data ascribe an active role for phagocytic cyst cells in removal of live germ cell progenitors.

microRNAs selectively protect hub cells of the germline stem cell niche from apoptosis.

Genotoxic stress such as irradiation causes a temporary halt in tissue regeneration. The ability to regain regeneration depends on the type of cells that survived the assault. Previous studies showed that this propensity is usually held by the tissue-specific stem cells. However, stem cells cannot maintain their unique properties without the support of their surrounding niche cells. In this study, we show that exposure of Drosophila melanogaster to extremely high levels of irradiation temporarily arrests spermatogenesis and kills half of the stem cells. In marked contrast, the hub cells that constitute a major component of the niche remain completely intact. We further show that this atypical resistance to cell death relies on the expression of certain antiapoptotic microRNAs (miRNAs) that are selectively expressed in the hub and keep the cells inert to apoptotic stress signals. We propose that at the tissue level, protection of a specific group of niche cells from apoptosis underlies ongoing stem cell turnover and tissue regeneration.

miR-9a modulates maintenance and ageing of Drosophila germline stem cells by limiting N-cadherin expression.

Ageing is characterized by a decline in stem cell functionality leading to dampened tissue regeneration. While the expression of microRNAs across multiple species is markedly altered with age, the mechanism by which they govern stem cell-sustained tissue regeneration is unknown. We report that in the Drosophila testis, the conserved miR-9a is expressed in germline stem cells and its levels are significantly elevated during ageing. Transcriptome and functional analyses show that miR-9a directly regulates the expression of the adhesion molecule N-cadherin (N-cad). miR-9a null mutants maintain a higher number of stem cells even in the aged tissue. Remarkably, this rise fails to improve tissue regeneration and results in reduced male fertility. Similarly, overexpression of N-cad also results in elevated stem cell number and decreased regeneration. We propose that miR-9a downregulates N-cad to enable adequate detachment of stem cells toward differentiation, thus providing the necessary directionality toward terminal differentiation and spermatogenesis.In the Drosophila testis, ageing leads to loss of germline stem cells. Here, the authors show that, during ageing in Drosophila, miR-9a is upregulated in male germline stem cells and regulates their proliferation by targeting N-cadherin.