Transcription factor codes patterning neuronal groundplans of the cerebrum

Article Transcription factor codes patterning neuronal groundplans of the cerebrum https://doi.org/10.1038/s41586-026-10526-3 Received: 18 June 2025 Najia A. Elkahlah1, Yunzhi Lin2, Yijie Pan2, Joseph A. Carter2, Troy R. Shirangi3 & E. Josephine Clowney2,4 ✉ Accepted: 10 April 2026 Published online: xx xx xxxx Open access Check for updates Brain regions that regulate motivated behaviours, including the vertebrate hypothalamus and arthropod cerebrum, house bespoke neural circuits dedicated to perceptual and internal regulation of many behavioural states1,2. These circuits are built to purpose from complex sets of cell types whose patterning has been challenging to elucidate. Here we developed methods in Drosophila melanogaster to embed well-studied neurons that regulate mating in the transcriptional contexts of the neuronal lineages that generate them3–5. By comparing transcription within and between lineages, we identified a large set of transcription factors expressed in complex combinations that delineate cerebral hemilineages—classes of postmitotic neurons born from the same stem cell and sharing Notch status6,7. Hemilineages comprise the major anatomic classes in the cerebrum8–10 and these transcription factors are required to generate their gross features. We show that subtypes of the same hemilineage can provide a common computational module to circuits regulating different drives, and identify an orthogonal set of transcription factors that stratify hemilineage subtypes of differing birth order. Our findings suggest that distinct sets of transcription factors operate in a hierarchical system to build, diversify and sexually differentiate lineally related neurons that compose motivated behaviour circuits. By linking developmental patterning to separable transcriptional axes that produce gross versus fine aspects of information flow, we provide a logical framework for cerebral control of diverse drives. Motivated behaviours, such as mating, feeding or aggression, are regulated by neurons that each perform singular information-processing functions1,2. These neurons are organized into unique feed-forward circuits in vertebrate subcortical nuclei and the arthropod cerebrum. The genome contains the information necessary to assign meaning to sensory inputs and to organize relationships among different drives into motivated behaviour circuits. Yet the immense diversity of contributing neurons has made their patterning too difficult to address. To explain the anatomic and functional principles that structure these circuits and states, we need to describe the logical principles and molecular mechanisms through which their constituent neurons are patterned during development. Four logical axes diversify neuronal cell types. They are used distinctly across brain regions in organisms, and in different kinds of neurogenesis programs across clades11–13: neural stem cells are (1) spatially differentiated and (2) progress through temporal expression windows as they divide asymmetrically; differentiating daughter cells can be further separated by (3) a Notch switch; and (4) sex further differentiates a subset of cell types. In the arthropod cerebrum, approximately 200 anatomic units called hemilineages form an information highway system traversing brain regions, as their constituent neurons share neurite outgrowth tracts, gross morphology and, often, neurotransmitter systems8–10,14–17. In adult Drosophila melanogaster (D. melanogaster), cerebral hemilineages—like those of the ventral nerve cord (VNC)—are composed of the NotchON or NotchOFF cousins from the same neuroblast7–10,18,19. Within hemilineage ground plans, individual cells have identifiable differences in fine axonal and dendritic anatomy and connectivity, linked to their birth order15,18,20,21. Although lineages of nerve cords, visual systems or cortices repeat across segments or columns, the unique functions of the cerebrum are produced by mostly singular lineages. Thus cerebral cell types, defined at the level of anatomy, connectivity or circuit function, are often specific birth-order cohorts from within one hemilineage. This singularity is what makes the complex circuit computations of the cerebrum possible; at the same time, the rarity of these neuroblasts and their daughter cells has impeded efforts to describe the transcriptional mechanisms that link neurogenic patterning to the formation of circuit architecture. Here we break this impasse by starting with the unusually wellunderstood circuit that regulates male mating in D. melanogaster. This circuit is built from over 60 populations of neurons that arise as subtypes of defined lineages and that are sexually differentiated by the Fruitless and/or Doublesex transcription factors (TFs)3,4,10,18,22,23. We transcriptionally characterized neuronal subtypes in the mating circuit in the context of their lineages and identified groups of TFs that play separable logical roles in diversifying hemilineage ground plans, 1 Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, MI, USA. 2Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA. 3Department of Biology, Villanova University, Villanova, PA, USA. 4Michigan Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA. ✉e-mail: Nature | www.nature.com | 1 Article Hemilineages provide anatomic groundplans Neuroblast identity and Notch combine to create distinct hemilineages Hemilineage subtypes are generated across time NB e.g. CREa1A CREa1B SMPad1A NotchOFF (B) neurons Pdfr R19C05 CREa1 d c CREa1 ∩Dpn p35 R19C05Pdfr-lineages, ♂ adult d′ ♀ adult mAL P1 vAB3 ♂ fru-expressing (mAL) ♀ R19C05Pdfr-lineages fru mRNA ♂ R19C05Pdfr-lineages fru mRNA LexA fluor ♂ 15 h APF Cre ♀ ♂ -GAL4 UAS-KD ♀ (Undergo apoptosis) CREa1B GMC b Specific subtypes are sexually differentiated ♀ NotchON (A) neurons R19C05Pdfr-lineages 15 h APF, +p35 (apoptotic block) a e ♂ fru mRNA grim mRNA ♀ fru mRNA grim mRNA e′ 12 and 48 h APF (without p35) CREa1A SMPad1A CREa1B UMAP2 UMAP2 CREa1A UMAP1 12 and 48 h APF (with p35) UMAP1 SMPad1A CREa1B Fig. 1 | Transcriptional analysis of developing cerebral lineages. a, Type I neuroblasts generate hemilineages, which form anatomic highways structuring arthropod cerebral circuits. The D. melanogaster CREa1B hemilineage produces fruitless mAL neurons (black) and other morphologies across time. Furthest right: mAL neurons receive gustatory pheromone input from vAB3 and inhibit P1 neurons to gate courtship15,18,26,27. b, Intersection of R19C05 Pdfr enhancer with deadpan allows lineage tracing of CREa1 and other neuroblasts (magenta)5,22, including their fruitless progeny (RNA FISH, green). Dashed lines outline the central brain. Further information is provided in Extended Data Fig. 1 and the Methods. Scale bars, 50 μm. c, Clonal induction of p35 in the CREa1 (...truncated)