Project description:Stem cells reside in a specialized microenvironment, called niche, which provides essential signals controlling stem cell behavior. Proper niche architecture is a key for normal stem cell function, yet only few upstream regulators are known. Here we report that the Hox transcription factor Abd-B, active in pre-meiotic spermatocytes, affects niche positioning in the Drosophila testis by regulating integrin localization in differentiated somatic cyst cells. Loss of Abd-B results in cell non-autonomous effects within the niche including centrosome misorientation in germline stem cells (GSCs) and reduced GSC divisions in larval testis, leading to a dramatic reduction of pre-meiotic stages in adult testes. By identifying Abd-B binding regions throughout the genome, we find that Abd-B mediates its effects on niche function by directly controlling at multiple levels the localization and thus signaling activity of the Sevenless (Sev) ligand, Bride of Sevenless (Boss), via its direct targets src42A and sec63. In sum, our data show for the first time that Abd-B through local signaling provides positional cues for integrin localization, which is critical for niche localization and architecture, and ensures proper niche function and GSC activity. DamID (DNA adenine methyltransferase identification) method was used to identify direct Abd-B target genes in the Drosophila 3rd instar larval testis Dam was fused to the N terminus of Abd-B and transgenic flies were generated. For identifying Abd-B targets in the Drosophila testis the fusion protein was expressed from the uninduced minimal Hsp70 promoter of the UAS vector pUAST (Brand and Perrimon, 1993). As a control for nonspecific Dam activity, transgenic flies expressing the Dam alone were used (Choksi et al., 2006). Subsequently, genomic DNA was extracted from 3rd instar larval testes, expressing either the Dam-Abd-B fusion protein or the Dam protein alone using a specific protocol (Tolhuis et al., 2011); and van Steensel personal communication). Two individual replicates, for Dam-AbdB and Dam alone, have been generated. Following a methylation-sensitive DNA digestion and PCR amplification, DNA fragments from Dam-Abd-B and control DNA were labeled and hybridized to genomic Affymetrix arrays in duplicates (Protocol available at M-bM-^@M-^\www.flychip.org.ukM-bM-^@M-^]).

Project description:The importance of the niche to provide regulatory inputs to balance stem cell self-renewal and differentiation has become clear. However, the regulatory interplay between stem cells and their niche at the whole genome level is still poorly understood. To elucidate the mechanisms controlling stem cells and their progenies as they progress through development at the transcriptional level, we recorded the regulatory program of two independent cell lineages in the Drosophila testis. We identified genes active in the soma or germline as well as genome-wide binding profiles of two transcription factors, Zfh-1 and Abd-A, expressed in somatic support cells and crucial for fate acquisition of both cell lineages. In order to uncover gene activities in the testis soma, we first determined the transcriptome of the somatic and germline lineages by RNA polymerase II Targeted DamID (TaDa) (Southall et al., 2013), followed by the identification of genes bound by two regulators active in somatic sub-populations and controlling their development using regular DNA adenine methyltransferase identification (DamID) (Van Steensel et al., 2001). For identifying abd-A and Zfh1 binding regions in the Drosophila testis the fusion protein was expressed from the uninduced minimal Hsp70 promoter of the UAS vector pUAST. As a control for nonspecific Dam activity, transgenic flies expressing the Dam alone were used (Choksi et al., 2006). To express the AbdA-Dam fusion protein, we first generated a pNDam-Myc-abdA construct by cloning abdA in the pNDam-Myc vector (van Steensel et al., 2001) and then subcloned the NDam-Myc-abdA fragment into the pUAST-attB and to express the Zfh1-Dam fusion protein, we first generated a pNDam-Myc-zfh1 construct by cloning zfh1 in the pNDam-Myc vector (van Steensel et al., 2001) and then subcloned the NDam-Myc-zfh1 fragment into the pUAST-attB. For targeted DamID (TaDa) in Drosophila 3rd instar testes cyst and germline cells. Cell-type specific DamID was performed in cyst cells (CySCs and SCCs) and early germline of 3rd instar larval testes for profiling RNA Pol II occupancy in these cells by crossing UAS-LT3-Dam-Pol II and UAS-LT3-Dam control flies to c587-GAL4 (somatic lineage) or Nanos-GAL4 (early germline) drivers. For Dam-ID two individual replicates of Dam-abd-A, Dam-Zfh1 and Dam alone have been generated whereas for TaDa, two individual replicates of c587>UAS-LT3-Dam-PolII, Nanos>UAS-LT3-Dam-Pol II and c587>UAS-LT3-Dam (control), Nanos>UAS-LT3-Dam (control) have been used. Following a methylation-sensitive DNA digestion and PCR amplification, DNA fragments from the above samples were labeled and hybridized to genomic Affymetrix arrays in duplicates (Protocol available at “www.flychip.org.uk”).

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Drosophila melanogaster

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