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Neurons must be made in the correct proportions to communicate with the appropriate synaptic partners and form functional circuits. In the Drosophila visual system, multiple subtypes of distal medulla (Dm) inhibitory interneurons are made in distinct, reproducible numbers-from 5 to 800 per optic lobe. These neurons are born from a crescent-shaped neuroepithelium called the outer proliferation center (OPC), which can be subdivided into specific domains based on transcription factor and growth factor expression. We fate mapped Dm neurons and found that more abundant neural types are born from larger neuroepithelial subdomains, while less abundant subtypes are born from smaller ones. Additionally, morphogenetic Dpp/BMP signaling provides a second layer of patterning that subdivides the neuroepithelium into smaller domains to provide more granular control of cell proportions. Apoptosis appears to play a minor role in regulating Dm neuron abundance. This work describes an underappreciated mechanism for the regulation of neuronal stoichiometry., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless ( tim ), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region., Competing Interests: Competing interests statement:The authors declare no competing interest.