Lsd1 Restricts the Number of Germline Stem Cells by Regulating Multiple Targets in Escort Cells

Abstract

Specialized microenvironments called niches regulate tissue homeostasis by controlling the balance between stem cell self-renewal and the differentiation of stem cell daughters. However the mechanisms that govern the formation, size and signaling of in vivo niches remain poorly understood. Loss of the highly conserved histone demethylase Lsd1 in Drosophila escort cells results in increased BMP signaling outside the cap cell niche and an expanded germline stem cell (GSC) phenotype. Here we present evidence that loss of Lsd1 also results in gradual changes in escort cell morphology and their eventual death. To better characterize the function of Lsd1 in different cell populations within the ovary, we performed Chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq). This analysis shows that Lsd1 associates with a surprisingly limited number of sites in escort cells and fewer, and often, different sites in cap cells. These findings indicate that Lsd1 exhibits highly selective binding that depends greatly on specific cellular contexts. Lsd1 does not directly target the dpp locus in escort cells. Instead, Lsd1 regulates engrailed expression and disruption of engrailed and its putative downstream target hedgehog suppress the Lsd1 mutant phenotype. Interestingly, over-expression of engrailed, but not hedgehog, results in an expansion of GSC cells, marked by the expansion of BMP signaling. Knockdown of other potential direct Lsd1 target genes, not obviously linked to BMP signaling, also partially suppresses the Lsd1 mutant phenotype. These results suggest that Lsd1 restricts the number of GSC-like cells by regulating a diverse group of genes and provide further evidence that escort cell function must be carefully controlled during development and adulthood to ensure proper germline differentiation.

Yorkie and Hedgehog independently restrict BMP production in escort cells to permit germline differentiation in the Drosophila ovary

“…5D,H) despite producing a very strong germline differentiation defect. It is possible that our lsd1 RNAi experiment, which allowed some egg chamber budding, produced lower Lsd1 inhibition than in previous analyses using the same RNAi approach and lsd1 null mutations (Eliazer et al, 2014). The set of phenotypes that we observed allow us to deduce that reduction of Lsd1 can produce germline differentiation defects in the absence of an apparent transformation of ECs towards cap cell identity, as observed for loss of Smo and Yki function.…”

“…The Lsd1 phenotype was previously shown to be partially suppressed by reducing the activity of En or Hh, which is normally induced by En in cap cells, and was partially phenocopied by ectopic En expression but not by ectopic Hh (Eliazer et al, 2014). That study suggested that En induction might be one of several crucial effectors in ECs with reduced Lsd1.…”

“…The lsd1 mutant phenotype was reported to include ectopic induction of two characteristic markers of cap cells, Engrailed (En) and Lamin C (LamC), in ECs, suggesting a transformation of EC identity towards that of cap cells, which normally produce Dpp and Gbb ligands (Eliazer et al, 2014). We observed no ectopic expression of En in ECs expressing yki RNAi or smo RNAi (Fig.…”

“…Conversely, Yki can associate with Trr complexes that include Nuclear receptor coactivator 6 (Ncoa6) to increase H3K4 methylation (Qing et al, 2014). Loss of Lsd1 in ECs was reported to induce ectopic Hh expression (Eliazer et al, 2014), while Hh signaling regulates Yki activity in FSCs (Huang and Kalderon, 2014). We therefore investigated whether the related phenotypes induced by inhibition of Lsd1, Smo and Yki in ECs reflect direct causal connections.…”

Novel Tools for Genetic Manipulation of Follicle Stem Cells in the Drosophila Ovary Reveal an Integrin-Dependent Transition from Quiescence to Proliferation

“…Clear conclusions can be drawn for the effects of these changes on IGS cells located anterior to the region 2A-2B border. However, both Gal4 drivers are also expressed in FSCs and their prefollicle cell daughters in addition to IGS cells (Song et al 2004;Decotto and Spradling 2005;Eliazer et al 2014), making it challenging to definitively distinguish labeled posterior IGS cells from FSCs at the region 2A-2B border in order to analyze IGS cell dynamics and identify the mechanisms that control their behavior. Fifteen Gal4 lines that drove expression of UAS-GFP in IGS cells were identified in our screen.…”