Axonal Wiring Research Articles

Autism-Linked Mutations in α2δ-1 and α2δ-3 Reduce Protein Membrane Expression but Affect Neither Calcium Channels nor Trans-Synaptic Signaling

Background: α2δ proteins regulate membrane trafficking and biophysical properties of voltage-gated calcium channels. Moreover, they modulate axonal wiring, synapse formation, and trans-synaptic signaling. Several rare missense variants in CACNA2D1 (coding for α2δ-1) and CACNA2D3 (coding for α2δ-3) genes were identified in patients with autism spectrum disorder (ASD). However, the pathogenicity of these variants is not known, and the molecular mechanism by which α2δ proteins may contribute to the pathophysiology of autism is, as of today, not understood. Therefore, in this study we functionally characterized two heterozygous missense variants in α2δ-1 (p.R351T) and α2δ-3 (p.A275T), previously identified in patients with ASD. Methods: Electrophysiological recordings in transfected tsA201 cells were used to study specific channel-dependent functions of mutated α2δ proteins. Membrane expression, presynaptic targeting, and trans-synaptic signaling of mutated α2δ proteins were studied upon expression in murine cultured hippocampal neurons. Results: Homologous expression of both mutated α2δ proteins revealed a strongly reduced membrane expression and synaptic localization compared to the corresponding wild type α2δ proteins. Moreover, the A275T mutation in α2δ-3 resulted in an altered glycosylation pattern upon heterologous expression. However, neither of the mutations compromised the biophysical properties of postsynaptic L-type (CaV1.2 and CaV1.3) and presynaptic P/Q-type (CaV2.1) channels when co-expressed in tsA201 cells. Furthermore, presynaptic expression of p.R351T in the α2δ-1 splice variant lacking exon 23 did not affect trans-synaptic signaling to postsynaptic GABAA receptors. Conclusions: Our data provide evidence that the pathophysiological mechanisms of ASD-causing mutations of α2δ proteins may not involve their classical channel-dependent and trans-synaptic functions. Alternatively, these mutations may induce subtle changes in synapse formation or neuronal network function, highlighting the need for future α2δ protein-linked disease models.

Developmental refinement of the active zone nanotopography and axon wiring at the somatosensory thalamus

Functional refinement of neural circuits is a crucial developmental process in the brain. However, how synaptic maturation and axon wiring proceed cooperatively to establish reliable signal transmission is unclear. Here, we combined nanotopography of release machinery at the active zone (AZ), nanobiophysics of neurotransmitter release, and single-neuron reconstruction of axon arbors of lemniscal fibers (LFs) in the developing mouse somatosensory thalamus. With development, the cluster of Cav2.1 enlarges and translocates closer to vesicle release sites inside the bouton, and LFs drastically shrink their arbors and form larger boutons on the perisomatic region of target neurons. Experimentally constrained simulations show that the nanotopography of mature synapses enables not only rapid vesicular release but also reliable transmission following repetitive firing. Sensory deprivation impairs the developmental shift of molecular nanotopography and axon wiring. Thus, we uncovered the cooperative nanotopographical and morphological mechanisms underlying the developmental establishment of reliable synaptic transmission.

Nr4a1 regulates cell-specific transcriptional programs in inhibitory GABAergic interneurons

The patterns of synaptic connectivity and physiological properties of diverse neuron types are shaped by distinct gene sets. Our study demonstrates that, in the mouse forebrain, the transcriptional profiles of inhibitory GABAergic interneurons are regulated by Nr4a1, an orphan nuclear receptor whose expression is transiently induced by sensory experiences and is required for normal learning. Nr4a1 exerts contrasting effects on the local axonal wiring of parvalbumin- and somatostatin-positive interneurons, which innervate different subcellular domains of their postsynaptic partners. The loss of Nr4a1 activity in these interneurons results in bidirectional, cell-type-specific transcriptional switches across multiple gene families, including those involved in surface adhesion and repulsion. Our findings reveal that combinatorial synaptic organizing codes are surprisingly flexible and highlight a mechanism by which inducible transcription factors can influence neural circuit structure and function.

Impaired Direction Selectivity in the Nucleus of the Optic Tract of Albino Mice.

Human albinos have a low visual acuity. This is partially due to the presence of spontaneous erroneous eye movements called pendular nystagmus. This nystagmus is present in other albino vertebrates and has been hypothesized to be caused by aberrant wiring of retinal ganglion axons to the nucleus of the optic tract (NOT), a part of the accessory optic system involved in the optokinetic response to visual motion. The NOT in pigmented rodents is preferentially responsive to ipsiversive motion (i.e., motion in the contralateral visual field in the temporonasal direction). We compared the response to visual motion in the NOT of albino and pigmented mice to understand if motion coding and preference are impaired in the NOT of albino mice. We recorded neuronal spiking activity with Neuropixels probes in the visual cortex and NOT in C57BL/6JRj mice (pigmented) and DBA/1JRj mice with oculocutaneous albinism (albino). We found that in pigmented mice, NOT is retinotopically organized, and neurons are direction tuned, whereas in albino mice, neuronal tuning is severely impaired. Neurons in the NOT of albino mice do not have a preference for ipsiversive movement. In contrast, neuronal tuning in visual cortex was preserved in albino mice and did not differ significantly from the tuning in pigmented mice. We propose that excessive interhemispheric crossing of retinal projections in albinos may cause the disrupted left/right direction encoding we found in NOT. This, in turn, impairs the normal horizontal optokinetic reflex and leads to pendular albino nystagmus.

SEMA6C: a novel adhesion-independent FAK and YAP activator, required for cancer cell viability and growth

Transmembrane semaphorins are signaling molecules, controlling axonal wiring and embryo development, which are increasingly implicated in human diseases. Semaphorin 6C (Sema6C) is a poorly understood family member and its functional role is still unclear. Upon targeting Sema6C expression in a range of cancer cells, we observed dramatic growth suppression, decreased ERK phosphorylation, upregulation of cell cycle inhibitor proteins p21, p27 and p53, and the onset of cell senescence, associated with activation of autophagy. These data are consistent with a fundamental requirement for Sema6C to support viability and growth in cancer cells. Mechanistically, we unveiled a novel signaling pathway elicited by Sema6C, and dependent on its intracellular domain, mediated by tyrosine kinases c-Abl and Focal Adhesion Kinase (FAK). Sema6C was found in complex with c-Abl, and induced its phosphorylation, which in turn led to FAK activation, independent of cell–matrix adhesion. Sema6C-induced FAK activity was furthermore responsible for increased nuclear localization of YAP transcriptional regulator. Moreover, Sema6C conferred YAP signaling-dependent long-term cancer cell survival upon nutrient deprivation. In conclusion, our findings demonstrate that Sema6C elicits a cancer promoting-signaling pathway sustaining cell viability and self-renewal, independent of growth factors and nutrients availability.

Regional redistribution of CB1 cannabinoid receptors in human foetal brains with Down's syndrome and their functional modifications in Ts65Dn+/+ mice.

The endocannabinoid system with its type 1 cannabinoid receptor (CB1 R) expressed in postmitotic neuroblasts is a critical chemotropic guidance module with its actions cascading across neurogenic commitment, neuronal polarisation and synaptogenesis in vertebrates. Here, we present the systematic analysis of regional CB1 R expression in the developing human brain from gestational week 14 until birth. In parallel, we diagrammed differences in CB1 R development in Down syndrome foetuses and identified altered CB1 R signalling. Foetal brains with normal development or with Down's syndrome were analysed using standard immunohistochemistry, digitalised light microscopy and image analysis (NanoZoomer). CB1 R function was investigated by in vitro neuropharmacology from neonatal Ts65Dn transgenic mice brains carrying an additional copy of ~90 conserved protein-coding gene orthologues of the human chromosome 21. We detected a meshwork of fine-calibre, often varicose processes between the subventricular and intermediate zones of the cortical plate in the late first trimester, when telencephalic fibre tracts develop. The density of CB1 Rs gradually decreased during the second and third trimesters in the neocortex. In contrast, CB1 R density was maintained, or even increased, in the hippocampus. We found the onset of CB1 R expression being delayed by ≥1month in age-matched foetal brains with Down's syndrome. In vitro, CB1 R excitation induced excess microtubule stabilisation and, consequently, reduced neurite outgrowth. We suggest that neuroarchitectural impairments in Down's syndrome brains involve the delayed development and errant functions of the endocannabinoid system, with a particular impact on endocannabinoids modulating axonal wiring.

Protein tyrosine phosphatase 69D is a substrate of protein O-mannosyltransferases 1-2 that is required for the wiring of sensory axons in Drosophila

Mutations in protein O-mannosyltransferases (POMTs) result in severe brain defects and congenital muscular dystrophies characterized by abnormal glycosylation of α-dystroglycan (α-Dg). However, neurological phenotypes of POMT mutants are not well understood, and the functional substrates of POMTs other than α-Dg remain unknown. Using a Drosophila model, here we reveal that Dg alone cannot account for the phenotypes of POMT mutants, and identify Protein tyrosine phosphatase 69D (PTP69D) as a gene interacting with POMTs in producing the abdomen rotation phenotype. Using RNAi-mediated knockdown, mutant alleles, and a dominant-negative form of PTP69D, we reveal that PTP69D is required for the wiring of larval sensory axons. We also found that PTP69D and POMT genes interact in this process, and that their interactions lead to complex synergistic or antagonistic effects on axon wiring phenotypes, depending on the mode of genetic manipulation. Using glycoproteomic approaches, we further characterized the glycosylation of the PTP69D transgenic construct expressed in genetic strains with different levels of POMT activity. We found that the PTP69D construct carries many O-linked mannose modifications when expressed in Drosophila with wild-type or ectopically upregulated expression of POMTs. These modifications were absent in POMT mutants, suggesting that PTP69D is a substrate of POMT-mediated O-mannosylation. Taken together, our results indicate that PTP69D is a novel functional substrate of POMTs that is required for axon connectivity. This mechanism of POMT-mediated regulation of receptor-type protein tyrosine phosphatase functions could potentially be conserved in mammals and may shed new light on the etiology of neurological defects in muscular dystrophies.

ER stress transforms random olfactory receptor choice into axon targeting precision.

Olfactory sensory neurons (OSNs) convert the stochastic choice of one of >1,000 olfactory receptor (OR) genes into precise and stereotyped axon targeting of OR-specific glomeruli in the olfactory bulb. Here, we show that the PERK arm of the unfolded protein response (UPR) regulates both the glomerular coalescence of like axons and the specificity of their projections. Subtle differences in OR protein sequences lead to distinct patterns of endoplasmic reticulum (ER) stress during OSN development, converting OR identity into distinct gene expression signatures. We identify the transcription factor Ddit3 as a key effector of PERK signaling that maps OR-dependent ER stress patterns to the transcriptional regulation of axon guidance and cell-adhesion genes, instructing targeting precision. Our results extend the known functions of the UPR from a quality-control pathway that protects cells from misfolded proteins to a sensor of cellular identity that interprets physiological states to direct axon wiring.

Self-avoidance alone does not explain the function of Dscam1 in mushroom body axonal wiring

Alternative splicing of Drosophila Dscam1 into 38,016 isoforms provides neurons with a unique molecular code for self-recognition and self-avoidance. A canonical model suggests that the homophilic binding of identical Dscam1 isoforms on the sister branches of mushroom body (MB) axons supports segregation with high fidelity, even when only a single isoform is expressed. Here, we generated a series of mutant flies with a single exon 4, 6, or 9 variant, encoding 1,584, 396, or 576 potential isoforms, respectively. Surprisingly, most of the mutants in the latter two groups exhibited obvious defects in the growth, branching, and segregation of MB axonal sister branches. This demonstrates that the repertoires of 396 and 576 Dscam1 isoforms were not sufficient for the normal patterning of axonal branches. Moreover, reducing Dscam1 levels largely reversed the defects caused by reduced isoform diversity, suggesting a functional link between Dscam1 expression levels and isoform diversity. Taken together, these results indicate that canonical self-avoidance alone does not explain the function of Dscam1 in MB axonal wiring.

A neural progenitor mitotic wave is required for asynchronous axon outgrowth and morphology.

Spatiotemporal mechanisms generating neural diversity are fundamental for understanding neural processes. Here, we investigated how neural diversity arises from neurons coming from identical progenitors. In the dorsal thorax of Drosophila, rows of mechanosensory organs originate from the division of sensory organ progenitor (SOPs). We show that in each row of the notum, an anteromedial located central SOP divides first, then neighbouring SOPs divide, and so on. This centrifugal wave of mitoses depends on cell-cell inhibitory interactions mediated by SOP cytoplasmic protrusions and Scabrous, a secreted protein interacting with the Delta/Notch complex. Furthermore, when this mitotic wave was reduced, axonal growth was more synchronous, axonal terminals had a complex branching pattern and fly behaviour was impaired. We show that the temporal order of progenitor divisions influences the birth order of sensory neurons, axon branching and impact on grooming behaviour. These data support the idea that developmental timing controls axon wiring neural diversity.

Gli2-Mediated Shh Signaling Is Required for Thalamocortical Projection Guidance.

The thalamocortical projections are part of the most important higher level processing connections in the vertebrates and follow a highly ordered pathway from their origin in the thalamus to the cerebral cortex. Their functional complexities are not only due to an extremely elaborate axon guidance process but also due to activity-dependent mechanisms. Gli2 is an intermediary transcription factor in the Sonic hedgehog (Shh) pathway. During neural early development, Shh has an important role in dorsoventral patterning, diencephalic anteroposterior patterning, and many later developmental processes, such as axon guidance and cell migration. Using a Gli2 knockout mouse line, we have studied the role of Shh signaling mediated by Gli2 in the development of the thalamocortical projections during embryonic development. In wild-type brains, we have described the normal trajectory of the thalamocortical axons into the context of the prosomeric model. Then, we have compared it with the altered thalamocortical axons course in Gli2 homozygous embryos. The thalamocortical axons followed different trajectories and were misdirected to other territories probably due to alterations in the Robo/Slit signaling mechanism. In conclusion, the alteration of Gli2-mediated Shh signaling produces an erroneous specification of several territories related with the thalamocortical axons. This is translated into a huge modification in the pathfinding signaling mechanisms needed for the correct wiring of the thalamocortical axons.

Cortical development coupling between surface area and sulcal depth on macaque brains.

Postnatal development of cerebral cortex is associated with a variety of neuronal processes and is thus critical to development of brain function and cognition. Longitudinal changes of cortical morphology and topology, such as postnatal cortical thinning and flattening have been widely studied. However, thorough and systematic investigation of such cortical change, including how to quantify it from multiple spatial directions and how to relate it to surface topology, is rarely found. In this work, based on a longitudinal macaque neuroimaging dataset, we quantified local changes in gyral white matter's surface area and sulcal depth during early development. We also investigated how these two metrics are coupled and how this coupling is linked to cortical surface topology, underlying white matter, and positions of functional areas. Semi-parametric generalized additive models were adopted to quantify the longitudinal changes of surface area (A) and sulcal depth (D), and the coupling patterns between them. This resulted in four classes of regions, according to how they change compared with global change throughout early development: slower surface area change and slower sulcal depth change (slowA_slowD), slower surface area change and faster sulcal depth change (slowA_fastD), faster surface area change and slower sulcal depth change (fastA_slowD), and faster surface area change and faster sulcal depth change (fastA_fastD). We found that cortex-related metrics, including folding pattern and cortical thickness, vary along slowA_fastD-fastA_slowD axis, and structural connection-related metrics vary along fastA_fastD-slowA_slowD axis, with which brain functional sites align better. It is also found that cortical landmarks, including sulcal pits and gyral hinges, spatially reside on the borders of the four patterns. These findings shed new lights on the relationship between cortex development, surface topology, axonal wiring pattern and brain functions.

Gyral Hinges Account for the Highest Cost and the Highest Communication Capacity in a Corticocortical Network.

Prior studies reported the global structure of brain networks exhibits the "small-world" and "rich-world" attributes. However, the underlying structural and functional architecture highlighted by these graph theory findings hasn't been explicitly related to the morphology of the cortex. This could be attributed to the lower resolution of used folding patterns, such as gyro-sulcal patterns. By defining a novel gyral folding pattern, termed gyral hinge (GH), which is the conjunction of ordinary gyri from multiple directions, we found GHs possess the highest length and cost in the white matter fiber connective network, and the shortest paths in the network tend to travel through GHs in their middle part. Based on these findings, we would hypothesize GHs could reside in the centers of a network core, thereby accounting for the highest cost and the highest communication capacity in a corticocortical network. The following results further support our hypothesis: 1) GHs possess stronger functional network integration capacity. 2) Higher cost is found on the connection with GHs to hinges and GHs to GHs. 3) Moving GHs introduces higher extra network cost. Our findings and hypotheses could reveal a profound relationship among the cortical folding patterns, axonal wiring architectures, and brain functions.

New Functions of Vav Family Proteins in Cardiovascular Biology, Skeletal Muscle, and the Nervous System.

Simple SummaryIn this review, we provide information on the role of Vav proteins, a group of signaling molecules that act as both Rho GTPase activators and adaptor molecules, in the cardiovascular system, skeletal muscle, and the nervous system. We also describe how these functions impact in other physiological and pathological processes such as sympathoregulation, blood pressure regulation, systemic metabolism, and metabolic syndrome.Vav proteins act as tyrosine phosphorylation-regulated guanosine nucleotide exchange factors for Rho GTPases and as molecular scaffolds. In mammals, this family of signaling proteins is composed of three members (Vav1, Vav2, Vav3) that work downstream of protein tyrosine kinases in a wide variety of cellular processes. Recent work with genetically modified mouse models has revealed that these proteins play key signaling roles in vascular smooth and skeletal muscle cells, specific neuronal subtypes, and glia cells. These functions, in turn, ensure the proper regulation of blood pressure levels, skeletal muscle mass, axonal wiring, and fiber myelination events as well as systemic metabolic balance. The study of these mice has also led to the discovery of new physiological interconnection among tissues that contribute to the ontogeny and progression of different pathologies such as, for example, hypertension, cardiovascular disease, and metabolic syndrome. Here, we provide an integrated view of all these new Vav family-dependent signaling and physiological functions.

Intron-targeted mutagenesis reveals roles for Dscam1 RNA pairing architecture-driven splicing bias in neuronal wiring.

Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. Here, we provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. We generate mutant flies with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D-/- and DscamΔ9D-/-), respectively. In contrast to DscamΔ4D-/-, DscamΔ9D-/- exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. We conclude that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling.

The Rac-GAP alpha2-Chimaerin Signals via CRMP2 and Stathmins in the Development of the Ocular Motor System.

A precise sequence of axon guidance events is required for the development of the ocular motor system. Three cranial nerves grow toward, and connect with, six extraocular muscles in a stereotyped pattern, to control eye movements. The signaling protein alpha2-chimaerin (α2-CHN) plays a pivotal role in the formation of the ocular motor system; mutations in CHN1, encoding α2-CHN, cause the human eye movement disorder Duane Retraction Syndrome (DRS). Our research has demonstrated that the manipulation of α2-chn signaling in the zebrafish embryo leads to ocular motor axon wiring defects, although the signaling cascades regulated by α2-chn remain poorly understood. Here, we demonstrate that several cytoskeletal regulatory proteins-collapsin response mediator protein 2 (CRMP2; encoded by the gene dpysl2), stathmin1, and stathmin 2-bind to α2-CHN. dpysl2, stathmin1, and especially stathmin2 are expressed by ocular motor neurons. We find that the manipulation of dpysl2 and of stathmins in zebrafish larvae leads to defects in both the axon wiring of the ocular motor system and the optokinetic reflex, impairing horizontal eye movements. Knockdowns of these molecules in zebrafish larvae of either sex caused axon guidance phenotypes that included defasciculation and ectopic branching; in some cases, these phenotypes were reminiscent of DRS. chn1 knock-down phenotypes were rescued by the overexpression of CRMP2 and STMN1, suggesting that these proteins act in the same signaling pathway. These findings suggest that CRMP2 and stathmins signal downstream of α2-CHN to orchestrate ocular motor axon guidance and to control eye movements.SIGNIFICANCE STATEMENT The precise control of eye movements is crucial for the life of vertebrate animals, including humans. In humans, this control depends on the arrangement of nerve wiring of the ocular motor system, composed of three nerves and six muscles, a system that is conserved across vertebrate phyla. Mutations in the protein alpha2-chimaerin have previously been shown to cause eye movement disorders (squint) and axon wiring defects in humans. Our recent work has unraveled how alpha2-chimaerin coordinates axon guidance of the ocular motor system in animal models. In this article, we demonstrate key roles for the proteins CRMP2 and stathmin 1/2 in the signaling pathway orchestrated by alpha2-chimaerin, potentially giving insight into the etiology of eye movement disorders in humans.

Temporal Order of Neural Progenitor Division Controls Neural Connectivity

Spatiotemporal mechanisms generating neural diversity are fundamental for understanding neural processes. Here, we investigated how neural connection diversity arises from neurons coming from identical progenitors. In the dorsal thorax of Drosophila, rows of mechanosensory organs originate from the division of sensory organ progenitor (SOPs). We show that in each row of the notum, a central SOP divides first, then neighboring SOPs divide, and so on. This centrifugal wave of mitoses depends on cell-cell inhibitory interactions mediated by SOP cytoplasmic protrusions and Scabrous, a secreted protein interacting with the Delta/Notch complex. When scabrous was downregulated, the mitotic wave was abolished, axonal growth was more synchronous, axonal terminals had a complex branching pattern and fly behavior was impaired. We propose that the temporal order of progenitor divisions influences the birth order of sensory neurons which is critical for correct axon wiring and appropriate grooming behavior, supporting the idea that developmental timing controls neural connectivity.

βIII spectrin controls the planarity of Purkinje cell dendrites by modulating perpendicular axon-dendrite interactions.

The mechanism underlying the geometrical patterning of axon and dendrite wiring remains elusive, despite its crucial importance in the formation of functional neural circuits. The cerebellar Purkinje cell (PC) arborizes a typical planar dendrite, which forms an orthogonal network with granule cell (GC) axons. By using electrospun nanofiber substrates, we reproduce the perpendicular contacts between PC dendrites and GC axons in culture. In the model system, PC dendrites show a preference to grow perpendicularly to aligned GC axons, which presumably contribute to the planar dendrite arborization in vivo We show that βIII spectrin, a causal protein for spinocerebellar ataxia type5, is required for the biased growth of dendrites. βIII spectrin deficiency causes actin mislocalization and excessive microtubule invasion in dendritic protrusions, resulting in abnormally oriented branch formation. Furthermore, disease-associated mutations affect the ability of βIII spectrin to control dendrite orientation. These data indicate that βIII spectrin organizes the mouse dendritic cytoskeleton and thereby regulates the oriented growth of dendrites with respect to the afferent axons.

Human neocortical development as a basis to understand mechanisms underlying neurodevelopmental disabilities in extremely preterm infants.

Recent advances in perinatal and neonatal medicine have resulted in marked improvements in the survival rates of extremely preterm infants (born before 28 gestational weeks) around the world, and Japan is among the countries with the highest reported survival rates of extremely preterm infants. However, it remains a major concern that many survivors develop neurodevelopmental disabilities, including cognitive dysfunctions and neurodevelopmental disorders later in life. In order to understand the pathophysiological mechanisms underlying the neurodevelopmental disabilities observed in the survivors of extremely preterm births, we reviewed recently reported findings about the development of the human neocortex. First, we have summarized the current knowledge about the development of the neocortex, including recently reported human- and/or primate-specific developmental events. Next, we discussed the possible causal mechanisms underlying the development of neurodevelopmental disabilities in extremely preterm infants. Around the birth of extremely preterm infants, neurogenesis and succeeding neuronal migrations are ongoing in the neocortex of human brain. Expansion and maturation of the subplate, which is thought to reflect the axonal wiring in the neocortex, is also prominent at this time. Brain injuries that occur around the birth of extremely preterm infants are presumed to affect the dynamic developmental events in the neocortex, such as neurogenesis, neuronal migrations and maturation of the subplate, which could underlie the neurodevelopmental disabilities that often develop subsequently in extremely preterm infants. These possibilities should be borne in mind while considering maternal and neonatal care to further improve the long-term outcomes of extremely preterm infants.

Modular transcriptional programs separately define axon and dendrite connectivity.

Patterns of synaptic connectivity are remarkably precise and complex. Single-cell RNA sequencing has revealed a vast transcriptional diversity of neurons. Nevertheless, a clear logic underlying the transcriptional control of neuronal connectivity has yet to emerge. Here, we focused on Drosophila T4/T5 neurons, a class of closely related neuronal subtypes with different wiring patterns. Eight subtypes of T4/T5 neurons are defined by combinations of two patterns of dendritic inputs and four patterns of axonal outputs. Single-cell profiling during development revealed distinct transcriptional programs defining each dendrite and axon wiring pattern. These programs were defined by the expression of a few transcription factors and different combinations of cell surface proteins. Gain and loss of function studies provide evidence for independent control of different wiring features. We propose that modular transcriptional programs for distinct wiring features are assembled in different combinations to generate diverse patterns of neuronal connectivity.