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”).
Project description:Cell type identity in the nervous system is encoded within cis-regulatory landscapes that integrate transcription factor activity with chromatin accessibility. However, how these regulatory programs are organized and remodeled during post-embryonic neural development remains poorly understood. We generate a temporally resolved single-cell chromatin accessibility atlas of ~95,000 zebrafish brain and retina nuclei spanning larval, juvenile, and adult stages. We define 212 discrete chromatin states and uncover widespread, cell type-specific chromatin reorganization across development. By integrating with transcriptomic data, we link motif accessibility to transcription factor expression and identify regulatory programs that are either maintained or reconfigured during post-embryonic development of each cell type. Leveraging this atlas, we systematically identify and functionally validate candidate enhancers in vivo. Focusing on the slc1a3b locus in radial glia, we define evolutionarily conserved, compact enhancer modules that act combinatorially to drive gene expression. Together, these findings provide a systems-level framework for decoding neural regulatory logic and enable functional dissection of conserved cis-regulatory programs in the vertebrate nervous system.
Project description:The cerebral cortex comprises diverse excitatory and inhibitory neuron subtypes, each with distinct laminar positions and connectivity patterns. Yet, the molecular logic underlying their precise wiring remains poorly understood. To identify ligand–receptor (LR) interactions involved in cortical circuit assembly, we tracked gene expression dynamics across major neuronal populations at 17 developmental stages using single-cell transcriptomics. This generated a comprehensive atlas of LR-mediated communication between excitatory and inhibitory neuron subtypes, capturing known and novel interactions. Notably, we identify neogenin-1 as the principal receptor for Cbln4 during the perinatal period, mediating synapse formation between somatostatin-expressing interneurons and glutamatergic neurons. We also identify cadherin superfamily members as candidate regulators of perisomatic inhibition onto deep and superficial excitatory neurons by parvalbumin-expressing basket cells, with opposing effects on synapse formation. These findings suggest a context-dependent role for cadherins in synaptic specificity and underscore the power of single-cell transcriptomics for decoding molecular mechanisms of cortical wiring.
Project description:In order to generate data suitable to decipher cis-regulatory logic, we generated ~100 million synthetic promoters (in yeast) comprised of random DNA and measured their expression by FACS (sorting into 18 bins).
Project description:The cerebral cortex comprises diverse excitatory and inhibitory neuron subtypes, each with distinct laminar positions and connectivity patterns. Yet, the molecular logic underlying their precise wiring remains poorly understood. To identify ligand–receptor (LR) interactions involved in cortical circuit assembly, we tracked gene expression dynamics across major neuronal populations at 17 developmental stages using single-cell transcriptomics. This generated a comprehensive atlas of LR-mediated communication between excitatory and inhibitory neuron subtypes, capturing known and novel interactions. Notably, we identify neogenin-1 as the principal receptor for Cbln4 during the perinatal period, mediating synapse formation between somatostatin-expressing interneurons and glutamatergic neurons. We also identify cadherin superfamily members as candidate regulators of perisomatic inhibition onto deep and superficial excitatory neurons by parvalbumin-expressing basket cells, with opposing effects on synapse formation. These findings suggest a context-dependent role for cadherins in synaptic specificity and underscore the power of single-cell transcriptomics for decoding molecular mechanisms of cortical wiring.