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A cascade of RNA binding proteins controls the switch from proliferation to differentiation in an adult stem cell lineage [array]

ABSTRACT: ABSTRACT: The mechanisms that regulate the switch from precursor cell proliferation to onset of differentiation in the adult stem cell lineages that maintain or repair many tissues in the body must be carefully regulated to assure production of sufficient progeny to maintain tissues yet prevent overproliferation that may lead to oncogenic progression. We are investigating the mechanisms regulating the switch from transit amplifying divisions to onset of differentiation in the Drosophila male germ line adult stem cell lineage, where spermatogonia undergo a limited number of mitotic divisions before switching to the spermatocyte program that sets up differentiation and meiosis. The number of transit amplifying spermatogonia divisions is set by accumulation of the Bag of marbles (Bam) protein to a critical threshold. Here we identify repression of Held Out Wings (how), homolog of mammalian Quaking, as the key target of Bam enabling the mitosis to differentiation switch. Knock down of how in germ cells was sufficient to allow spermatogonia mutant for bam or its partner bgcn to differentiate, while forced expression of nuclear-targeted How protein in otherwise wild-type spermatogonia resulted in continued proliferation of spermatogonia at the expense of differentiation. Our findings suggest that Bam targets how RNA for degradation by acting as an adapter to recruit the CCR4-NOT deadenylation complex via binding its subunit, Caf40. Our data support the model that the switch from proliferation to differentiation in this model adult stem cell lineage is regulated by a cascade of RNA-binding proteins. We used microarray to profile a time-course gene expression change from spermatogonia to spermatocytes for 48 hours in 8-hour intervals. We used the Bam-induced synchronous differentiation strategy we developed (PMID: 28522526). bam mutant testes are filled with developmentally arrested, overproliferating spermatogonia. By a pulse of Bam expression using 30 minutes of heat shock for the bam mutant flies bearing heat shock promoter driven Bam transgene, we induced synchronous differentiation of spermatogonia to spermatocytes. These cells stopped dividing and initiated the expression of early spermatocyte genes by 24 hours post-heat shock (PHS). By 48 PHS, they became young spermatocytes expressing hundreds of testis-specific genes. As a control, we used bam mutant flies without hs-Bam transgene but received the same 30 minutes of heat shock treatment.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE274384 | GEO | 2025/06/04

REPOSITORIES: GEO

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Project description:The piRNA pathway is studied in great detail in Drosophila female germline. In this study we show that unlike the female germline where all Piwi proteins are expressed throughout oogenesis, Ago3 - a Piwi family protein shows a spatial expression male germline. To understand dynamics of piRNA pathway during spermatogonia and primary spermatocyte stages of male germline development, we used arrest mutants. The bag of marbles (bam) and benign gonial cell neoplasm (bgcn) mutants have only early mitotic dividing germline cells in the testes due to failure to progress to primary spermatocyte stage, the cannonball (can) and spermatocyte arrest (sa) mutant germline cells cannot progress beyond primary spermatocyte stage. To investigate the dynamics of the piRNA pathway during spermatogenesis in spermatogonia and primary spermatocyte stages, we used testicular tissues from these stage-specific arrested mutants. While we used entire bam and bgcn mutant testes for spermatogonia purification, we while we manually removed the apical regions of can and sa mutant testes to exclude mitotically dividing undifferentiated germline cells for primary spermatocytes purification. Our results show that piRNAs mapping to transposons are more abundant in spermatogonia, whereas those mapping to Suppressor of Stellate [Su(Ste)] and AT-chX are mostly expressed in primary spermatocytes. Furthermore we observed that transposon-mapping piRNAs with ping-pong signature are more abundant in spermatogonia albeit still detectable in primary spermatocytes where Ago3 is not expressed. These results suggest that robust piRNA production via ping-pong cycle takes place in spermatogonia, and to a lesser extent in primary spermatocytes even in the absence of Ago3. Consistently, piRNAs from ago3 mutant testes also exhibit the ping-pong signature, confirming that a non-canonical ping-pong cycle is acting during spermatogenesis. Our study provides a developmental dimension to the piRNA pathway and uncovers a new mechanism used in the male germline to silence transposons. The difference in piRNA from spermatogonia and primary spermatocyte stages was studied by comparing small RNAs from bam and bgcn mutant testis, which represent spermatogonia stages with the small RNAs from apex removed can and sa testis, representing primary spermatocyte stages. In the study we also studied effect of loss of Piwi family proteins Aub and Ago3, which have different spatial expression during male germline development.

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A cascade of RNA binding proteins controls the switch from proliferation to differentiation in an adult stem cell lineage [array]

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