Biology subjects

Manning, L.

Publications and source records attributed to Manning, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Rapid GeneXpert surveillance of influenza A virus in seabirds and the environment provides early warning for wildlife health in Aotearoa New Zealand

The global expansion of highly pathogenic avian influenza (HPAI) virus A(H5N1) underscores the need for rapid surveillance at high-risk wildlife interfaces. Taiaroa Head (45.7828{degrees} S, 170.7333{degrees} E) in the South Island of Aotearoa New Zealand hosts a plethora of aquatic wildlife including a large red-billed gull (Chroicocephalus novaehollandiae scopulinus) colony as well as the only mainland breeding colony of northern royal albatross (Diomedea sanfordi). The Royal Albatross Centre is also a major nature tourism destination, attracting tens of thousands of visitors annually, thereby creating a dense ecological and human-wildlife interface vulnerable to viral incursion. We evaluated the GeneXpert II platform using the Xpert(R) Xpress Flu/RSV cartridge as a field-deployable tool for avian influenza virus detection in environmental and wildlife-associated samples. The assay detected synthetic influenza A viral RNA and multiple endemic low pathogenic avian influenza virus subtypes (A(H3N8), A(H1N9), A(H5N2) and A(H7N7)) circulating in New Zealand birds. Influenza A virus was reliably identified in spiked environmental water samples with no consistent PCR inhibition as well as naturally occurring avian influenza virus in duck pond water. Field deployment demonstrated that the system could be operated by non-laboratory personnel with minimal training in a non-clinical setting. This study establishes the feasibility of near-real-time environmental monitoring. Repurposing clinical cartridge-based point-of-care diagnostics offers a practical early warning approach for avian influenza virus surveillance at ecologically and economically significant locations.

microbiology↗

bioRxiv2025

Sparse mesenchymal cell networks as a fluid under tension

Sparse mesenchymal cellular networks are ubiquitous across animals, shaping both embryonic and adult structures through dynamic interactions with epithelia. Yet, the physical principles underlying their collective behaviors remain elusive, as their stellate cells and large extracellular spaces--filled with matrix or interstitial fluid--pose significant experimental and computational challenges. Here, we demonstrate that the avian presomitic mesoderm (PSM), a canonical embryonic mesenchymal tissue, behaves as a fluid under tension, exhibiting structural organization that cannot be explained by simple Brownian-like cell motion. Through quantitative modeling, we identify contact inhibition of locomotion (CIL)--where cells actively retract and move away upon contact--as a key mechanism that enables sparse mesenchymal networks to sustain macroscopic tension while flowing like a fluid. Simple continuum equations relate observable cell-scale parameters to the emergent remodeling dynamics observed in both experiments and simulations. Together, these findings put forward an unrecognized mechanical role for CIL, extending its influence beyond collective migration, and establish the fluid-under-tension state as a distinct class of tissue behavior that describes key developing embryonic tissues and may illuminate how matrix-rich adult tissues become fluidized during tumorigenesis.

biophysics↗

bioRxiv2025

Tight translation regulation of the canonical C9ORF72 through a multi-component system

C9ORF72 is involved in multiple neuronal functions and a major factor in Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disease. Pathogenicity is implemented through an intronic repeat expansion in the genes mRNA leader that produces long, repetitive RNA and dipeptides when translated through a non-canonical mechanism. In contrast, despite the presence of ribosome-occupied, regulatory elements in the mRNA leader, nothing is known about C9ORF72 translation regulation under normal conditions. Surprisingly, when analyzing a series of mutants of the C9ORF72 mRNA leader, we found that translation of C9ORF72 is tightly regulated through a multi-layer system. First, non-canonical, multi-initiation upstream open reading frames (nc-uORFs) in all three frames, a start-stop element in frame 2, and a canonical uORF in frame 1work together to keep baseline C9ORF72 translation very low. Second, a strong secondary structure enhances these repressive elements, mainly the two nc-uORFs in frames 0 and 2. Finally, as initiation at the nc-uORFs reduces initiation at the downstream start-stop, the nc-uORFs effectively dampen the start-stops repressive function, forming a feedforward loop. We hypothesize that this buffered repressor system has likely evolved to ensure reliable, noise-insensitive expression of this critical regulator of neuronal function.

molecular biology↗

bioRxiv2025

Morphogenesis-on-chip: A minimal in vitro assay for cell intercalation highlights the importance of interfacial tension and migratory forces

Cell intercalation, the dynamic exchange of cellular neighbors, is fundamental to embryonic morphogenesis, tissue homeostasis, and wound healing. Despite extensive study in complex tissues, the minimal mechanical requirements driving intercalation remain poorly understood due to confounding tissue level interactions. Here, we present a novel morphogenesis-on-chip assay utilizing micropatterned cell quadruplets. This system isolates the elementary unit of intercalation while enabling quantitative force and shape measurements. Cross-shaped micropatterns generate stable four cell configurations in MDCK epithelial cells. Surprisingly, these cells spontaneously undergo T1 transitions autonomously. We combined live imaging with force inference and traction force microscopy, which revealed that intercalation emerges from two distinct mechanisms: interfacial tension dynamics and differential cell migration. Specifically, we show a correlation between central junction shrinkage and increased relative tension. Similarly, we show a correlation between central junction shrinkage and migratory forces. We successfully adapted the assay to Xenopus mesoderm cells, revealing conserved mechanical principles across cell types. Furthermore, experimentally derived effective energy landscapes closely match theoretical vertex model predictions, and suggest a dominant role for migratory forces in driving intercalation. This confirms that our minimal system recapitulates the fundamental physics of intercalation. This approach provides the first quantitative framework for studying intercalation mechanics in isolation and establishes a versatile platform for investigating morphogenetic processes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/662274v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@41db6aorg.highwire.dtl.DTLVardef@1ac1cb1org.highwire.dtl.DTLVardef@8fcb2corg.highwire.dtl.DTLVardef@63e320_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Seven-up acts in neuroblasts to specify adult central complex neuron identity and initiate neuroblast decommissioning

An open question in neurobiology is how diverse neuron cell types are generated from a small number of neural stem cells. In the Drosophila larval central brain, there are eight bilateral Type 2 neuroblast (T2NB) lineages that express a suite of early temporal factors followed by a different set of late temporal factors and generate the majority of the central complex (CX) neurons. The early-to-late switch is triggered by the orphan nuclear hormone receptor Seven-up (Svp), yet little is known about this Svp-dependent switch in specifying CX neuron identities. Here, we (i) birthdate the CX neurons P-EN and P-FN (early and late, respectively); (ii) show that Svp is transiently expressed in all early T2NBs; and (iii) show that loss of Svp expands the population of early born P-EN neurons at the expense of late born P-FN neurons. Furthermore, in the absence of Svp, T2NBs fail decommissioning and abnormally extend their lineage into week-old adults. We conclude that Svp is required to specify CX neuron identity, as well as to initiate T2NB decommissioning. SummarySeven-up acts in Type 2 neuroblasts to specify adult central complex columnar neuron identity and to initiate neuroblast decommissioning.

developmental biology↗

bioRxiv2023

Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport

Mitochondria transport is crucial for mitochondria distribution in axons and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 recruitment to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1 and metaxin2. We conclude that polarized transport complexes containing kinesin-1 and RIC-7 form at the leading edge of mitochondria, and that these complexes are required for anterograde axonal transport. Summary statementAnterograde transport of axonal mitochondria is critical for maintenance of the mitochondria pool and neuronal health. Wu et al. show that the endogenous kinesin-1 and RIC-7 localize at the leading end of mitochondria to drive axonal anterograde transport in vivo.

cell biology↗

bioRxiv2022

Mechanical forces across compartments coordinate cell shape and fate transitions to generate tissue architecture

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. An excellent paradigm to understand the coupling of these processes is mammalian hair follicle development, initiated by the formation of an epithelial invagination - termed placode - that coincides with the emergence of a designated hair follicle stem cell population. The mechanisms directing the deformation of the epithelium, cell state transitions, and physical compartmentalization of the placode are unknown. Here, we identify a key role for coordinated mechanical forces stemming from contractile, proliferative, and proteolytic activities across the epithelial and mesenchymal compartments in generating the placode structure. A ring of fibroblast cells gradually wraps around the placode cells to generate centripetal contractile forces, which in collaboration with polarized epithelial myosin activity promote elongation and local tissue thickening. These mechanical stresses further enhance and compartmentalize Sox9 expression to promote stem cell positioning. Subsequently, proteolytic remodeling locally softens the basement membrane to facilitate release of pressure on the placode, enabling localized cell divisions, tissue fluidification, and epithelial invagination into the underlying mesenchyme. Together, our experiments and modeling identify dynamic cell shape transformations and tissue-scale mechanical co-operation as key factors for orchestrating organ formation.

developmental biology↗

bioRxiv2022

Universal features of rigidity transitions in vertex models for biological tissues

Simple vertex models, where the cell shape is defined as a network of edges and vertices, have made useful predictions about the collective behavior of confluent biological tissues, including rigidity transitions. Quite a few different versions of vertex models have appeared in the literature, and they propose substantial differences in how the mechanical energy depends on vertex positions, yet all of them seem to make correct predictions. To understand how this is possible, we search for universality in the emergent mechanical behavior - including the shear modulus defined in the limit of zero strain rate and the viscoelastic response at finite strain rates - of six different vertex models. We identify a class of models with a well-defined shear modulus, and demonstrate that these models all exhibit a cross-over from a soft or floppy regime to a stiff regime. While the parameter that controls the crossover is different in each model, we find that the observed cell shape index (the ratio of the cell perimeter to the square root of the cell area) is a good observable order parameter for the crossover. We also find that the finite strain-rate viscoelastic response of all models exhibits a universal scaling with frequency, following the Zener model in the rigid phase and Burgers model in the fluid phase. This suggests there is a broad class of vertex models with universal mechanical features, and helps to explain why many different vertex models are able to robustly predict these features in experiments.

biophysics↗

bioRxiv2021

Collective chemotaxis in a Voronoi model for confluent clusters

Collective chemotaxis, where single cells cannot climb a biochemical signaling gradient but clusters of cells can, has been observed in different biological contexts, including confluent tissues where there are no gaps or overlaps between cells. Although particle-based models have been developed that predict important features of collective chemotaxis, the mechanisms in those models depend on particle overlaps, and so it remains unclear if they can explain behavior in confluent systems. Here, we develop an open-source code that couples a 2D Voronoi simulation for confluent cell mechanics to a dynamic chemical signal that can diffuse, advect, and/or degrade, and use the code to study potential mechanisms for collective chemotaxis in cellular monolayers. We first study the impact of advection on collective chemotaxis, and delineate a regime where advective terms are important. Next, we investigate two possible chemotactic mechanisms, contact inhibition of locomotion and heterotypic interfacial tension, and demonstrate that both can drive collective chemotaxis in certain parameter regimes. We further demonstrate that the scaling behavior of cluster motion is well-captured by simple analytic theories. 1 Statement of SignificanceThe ability of cell collectives to respond to biochemical signals, called collective chemotaxis, is crucial for many important processes including embryonic development and wound healing. We developed an open-source computational model that couples biochemical signaling gradients to confluent cell layers, where there are no gaps between cells. Our model demonstrates that two experimentally observed local cell behaviors - neighbor-induced changes to interfacial tension or a tendency of cells to repel one another after they come into contact - can drive collective chemotaxis. We also highlight a regime in which the motion of migrating cells can alter the gradient.

biophysics↗

bioRxiv2021

The active zone protein Clarinet regulates ATG-9 trafficking at synapses and presynaptic autophagy

In neurons, autophagy is temporally and spatially regulated to occur near presynaptic sites. How trafficking of autophagy proteins is regulated to support synaptic autophagy is not well understood. From forward genetic screens, we identify a role for the long isoform of the active zone protein Clarinet (CLA-1L) in regulating trafficking of autophagy protein ATG-9 at synapses, and presynaptic autophagy. ATG-9 is a transmembrane protein that undergoes activity-dependent exo-endocytosis at synapses, and mutations in CLA-1L result in abnormal accumulation of ATG-9 into clathrin-rich endocytic intermediates. CLA-1L extends from the active zone to the periactive zone, and genetically interacts with periactive zone proteins required for clathrin-dependent endocytosis. We find that CLA-1L is specifically required for sorting of ATG-9 at synapses, likely via endosome-mediated endocytosis, and for activity-dependent presynaptic autophagy. Our findings provide mechanistic insights into how active zone proteins regulate key steps of ATG-9 exo-endocytosis, a process that could couple the activity state of the neuron and autophagy. HighlightsO_LI* The long isoform of the active zone protein Clarinet (CLA-1L) regulates ATG-9 trafficking at synapses C_LIO_LI* CLA-1L extends from the active zone to the periactive zone and cooperates with the periactive zone endocytic proteins EHS-1/EPS15 and ITSN-1/ intersectin 1 in ATG-9 trafficking during exo-endocytosis C_LIO_LI* Mutations in CLA-1L, or in clathrin-associated adaptor molecules, result in abnormal accumulation of ATG-9 into clathrin-rich endocytic intermediates C_LIO_LI* CLA-1L mutants which affect ATG-9 trafficking are also defective in activity-dependent presynaptic autophagy C_LI

neuroscience↗

bioRxiv2021

Phylodynamic modelling of bacterial outbreaks using nanopore sequencing

Nanopore sequencing and phylodynamic modelling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Here, we implement Random Forest models for single nucleotide polymorphism (SNP) polishing to estimate divergence and effective reproduction numbers (Re) of two community-associated, methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in remote Far North Queensland and Papua New Guinea (n = 159). Successive bar-coded panels of S. aureus isolates (2 x 12 per MinION) sequenced at low-coverage (> 5x - 10x) provided sufficient data to accurately infer assembly genotypes with high recall when compared with Illumina references. De novo SNP calling with Clair was followed by SNP polishing using intra- and inter-species models trained on Snippy reference calls. Models achieved sufficient resolution on ST93 outbreak sequence types (> 70 - 90% accuracy and precision) for phylodynamic modelling from lineage-wide hybrid alignments and birth-death skyline models in BEAST2. Our method reproduced phylogenetic topology, geographical source of the outbreaks, and indications of sustained transmission (Re > 1). We provide Nextflow pipelines that implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks using nanopore sequencing.

bioinformatics↗

bioRxiv2021

Phylodynamic signatures in the emergence of community-associated MRSA

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 years. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole genome sequencing data for the Australian sequence type (ST) 93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australia, dissemination of the ST93-MRSA-IV geno-type into population centers on the Australian East Coast, and sub-sequent importation into the highlands of Papua New Guinea and Far North Queensland. Analysis of a ST772-MRSA-V cluster in Pakistan suggests that sustained transmission in the community following importation of resistant genotypes may be more common than previously thought. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia and Europe. Surges in Re were observed at the divergence of antibiotic resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed amongst drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA and MSSA lineages in the late 20th century was driven by a combination of antibiotic resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

evolutionary biology↗

bioRxiv2021

3D viscoelastic drag forces drive changes to cell shapes during organogenesis in the zebrafish embryo

The left-right organizer in zebrafish embryos, Kupffers Vesicle (KV), is a simple organ that undergoes programmed asymmetric cell shape changes that are necessary to establish the left-right axis of the embryo. We use simulations and experiments to investigate whether 3D mechanical drag forces generated by the posteriorly-directed motion of the KV through the tailbud tissue are sufficient to drive such shape changes. We develop a fully 3D vertex-like (Voronoi) model for the tissue architecture, and demonstrate that the tissue can generate drag forces and drive cell shape changes. Furthermore, we find that tailbud tissue presents a shear-thinning, viscoelastic behavior consistent with those observed in published experiments. We then perform live imaging experiments and particle image velocimetry analysis to quantify the precise tissue velocity gradients around KV as a function of developmental time. We observe robust velocity gradients around the KV, indicating that mechanical drag forces must be exerted on the KV by the tailbud tissue. We demonstrate that experimentally observed velocity fields are consistent with the viscoelastic response seen in simulations. This work also suggests that 3D viscoelastic drag forces could be a generic mechanism for cell shape change in other biological processes. HighlightsO_LInew physics-based simulation method allows study of dynamic tissue structures in 3D C_LIO_LImovement of an organ through tissue generates viscoelastic drag forces on the organ C_LIO_LIthese drag forces can generate precisely the cell shape changes seen in experiment C_LIO_LIPIV analysis of experimental data matches simulations and probes tissue mechanics C_LI Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

developmental biology↗

bioRxiv2021

Hunting Eagles with glass mice: revisiting the inoculum effect for Streptococcus pyogenes with a hollow fibre infection model

BackgroundSevere, invasive Streptococcus pyogenes (Strep A) infections result in greater than 500,000 deaths annually. First line treatment for such infections is combination benzylpenicillin and clindamycin, but treatment failure can occur with this regimen. This failure has been partially attributed to the inoculum effect, which presents as reduced antibiotic susceptibility during high bacterial density and plateau-phase growth. Hollow fibre infection models (HFIM) have been proposed as an alternative to in vivo research to study these effects. ObjectivesTo re-evaluate the inoculum effect for benzylpenicillin, clindamycin, linezolid and trimethoprim-sulfamethoxazole using a Strep A HFIM. MethodsDifferential antibiotic susceptibility of Strep A was measured in a HFIM starting from low- and high-density inocula. Dynamic antibiotic concentrations were delivered over 48 hours to simulate human pharmacokinetics. Differences in antibiotic susceptibility were determined at 24 and 48 hours by plate count of remaining viable colony-forming units. ResultsInoculum effects were seen in benzylpenicillin and linezolid at 24 hours, and benzylpenicillin, linezolid and clindamycin at 48 hours. The effect size was greatest for continuously infused benzylpenicillin. No inoculum effect was seen in trimethoprim-sulfamethoxazole. ConclusionsInoculum effects were seen in the HFIM model using benzylpenicillin, linezolid and clindamycin, which may predict reduced clinical efficacy following treatment delay. The model has proven robust and largely in agreeance with published data, recommending it for further Strep A study.

microbiology↗

bioRxiv2020

Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9

Autophagy is a cellular degradation pathway essential for neuronal health and function. Autophagosome biogenesis occurs at synapses, is locally regulated and increases in response to neuronal activity. The mechanisms that couple autophagosome biogenesis to synaptic activity remain unknown. In this study we determine that trafficking of ATG-9, the only transmembrane protein in the core autophagy pathway, links the synaptic vesicle cycle with autophagy. ATG-9 positive vesicles in C. elegans are generated from the trans-Golgi network via AP3-dependent budding, and delivered to presynaptic sites. At presynaptic sites, ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt endocytosis, including one associated with Parkinsons disease, result in abnormal ATG-9 accumulation at clathrin-rich synaptic foci and defects in activity-dependent presynaptic autophagy. Our findings uncover regulated key steps of ATG-9 trafficking at presynaptic sites, and provide evidence that ATG-9 exo-endocytosis couples autophagosome biogenesis at presynaptic sites with the activity-dependent synaptic vesicle cycle. HighlightsO_LIIn C. elegans, ATG-9 is delivered to presynaptic sites in vesicles generated from the trans-Golgi network via AP-3-dependent budding C_LIO_LIATG-9 vesicles undergo activity-dependent exo-endocytosis at presynaptic sites C_LIO_LIMutations in endocytic proteins, including a mutation associated with Parkinsons disease, result in abnormal ATG-9 accumulation at clathrin-rich foci C_LIO_LIAbnormal accumulation of ATG-9 at clathrin-rich foci is associated with defects in activity-dependent presynaptic autophagy C_LI

neuroscience↗

bioRxiv2020

Transcriptional control of parallel-acting pathways that remove discrete presynaptic proteins in remodeling neurons

Synapses are actively dismantled to mediate circuit refinement, but the developmental pathways that regulate synaptic disassembly are largely unknown. We have previously shown that the epithelial sodium channel UNC-8 triggers an activity-dependent mechanism that drives the removal of presynaptic proteins liprin-/SYD-2, Synaptobrevin/SNB-1, RAB-3 and Endophilin/UNC-57 in remodeling GABAergic neurons in C. elegans (Miller-Fleming et al., 2016). Here, we report that the transcription factor Iroquois/IRX-1 regulates UNC-8 expression as well as an additional pathway, independent of UNC-8, that functions in parallel to dismantle functional presynaptic terminals. We show that the additional IRX-1-regulated pathway is selectively required for the removal of the presynaptic proteins, Munc13/UNC-13 and ELKS, which normally mediate synaptic vesicle fusion and neurotransmitter release. Our findings are notable because they highlight the key role of transcriptional regulation in synapse elimination and reveal parallel-acting pathways that orchestrate synaptic disassembly by removing specific active zone proteins.

neuroscience↗

bioRxiv2019

ADAMTS-family protease MIG-17 regulates synaptic allometry by modifying the extracellular matrix and modulating glia morphology during growth

Synapses are largely established during embryogenesis and maintained during growth. The mechanisms that regulate synaptic allometry--the maintenance of synaptic positions during growth--are largely unknown. We performed forward genetic screens in C. elegans for synaptic allometry mutants and identified mig-17, a secreted metalloprotease of the conserved ADAMTS family. Through proteomic mass spectrometry analyses, cell biological and genetic studies we determined that MIG-17 is expressed by muscle cells to modulate glia location and morphology. Glia are proximal to synapses, and the glial location and morphology determine synaptic position during growth. Mig-17 regulates synapse allometry by influencing epidermal-glia crosstalk through the regulation of basement membrane proteins, including collagen type IV, SPARC and fibulin. Our findings underscore the importance of glia location in the maintenance of synaptic allometry, and uncover a muscle-epidermal-glia signaling axis, mediated through the extracellular matrix, in the regulation of glia morphology and synaptic positions during growth.

neuroscience↗

bioRxiv2019

The role of lineage, hemilineage and temporal identity in establishing neuronal targeting and connectivity in the Drosophila embryo

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, we mapped the developmental origin of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and identify them in a synapse-scale TEM reconstruction of the Drosophila larval CNS. We find that lineages concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, we show that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, we demonstrate that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.

neuroscience↗