Dysregulation of Neuropilin-2 Expression in Inhibitory Neurons Impairs Hippocampal Circuit Development Leading to Autism-Epilepsy Phenotype.

Dysregulation of development, migration, and function of interneurons, collectively termed interneuronopathies, have been proposed as a shared mechanism for autism spectrum disorders (ASDs) and childhood epilepsy. Neuropilin-2 (Nrp2), a candidate ASD gene, is a critical regulator of interneuron migration from the median ganglionic eminence (MGE) to the pallium, including the hippocampus. While clinical studies have identified Nrp2 polymorphisms in patients with ASD, whether dysregulation of Nrp2-dependent interneuron migration contributes to pathogenesis of ASD and epilepsy has not been tested. We tested the hypothesis that the lack of Nrp2 in MGE-derived interneuron precursors disrupts the excitation/inhibition balance in hippocampal circuits, thus predisposing the network to seizures and behavioral patterns associated with ASD. Embryonic deletion of Nrp2 during the developmental period for migration of MGE derived interneuron precursors (iCKO) significantly reduced parvalbumin, neuropeptide Y, and somatostatin positive neurons in the hippocampal CA1. Consequently, when compared to controls, the frequency of inhibitory synaptic currents in CA1 pyramidal cells was reduced while frequency of excitatory synaptic currents was increased in iCKO mice. Although passive and active membrane properties of CA1 pyramidal cells were unchanged, iCKO mice showed enhanced susceptibility to chemically evoked seizures. Moreover, iCKO mice exhibited selective behavioral deficits in both preference for social novelty and goal-directed learning, which are consistent with ASD-like phenotype. Together, our findings show that disruption of developmental Nrp2 regulation of interneuron circuit establishment, produces ASD-like behaviors and enhanced risk for epilepsy. These results support the developmental interneuronopathy hypothesis of ASD epilepsy comorbidity.

Dysregulation of Neuropilin-2 Expression in Inhibitory Neurons Impairs Hippocampal Circuit Development Leading to Autism-Epilepsy Phenotype.

Dysregulation of development, migration, and function of interneurons, collectively termed interneuronopathies, have been proposed as a shared mechanism for autism spectrum disorders (ASDs) and childhood epilepsy. Neuropilin-2 (Nrp2), a candidate ASD gene, is a critical regulator of interneuron migration from the median ganglionic eminence (MGE) to the pallium, including the hippocampus. While clinical studies have identified Nrp2 polymorphisms in patients with ASD, whether dysregulation of Nrp2-dependent interneuron migration contributes to pathogenesis of ASD and epilepsy has not been tested. We tested the hypothesis that the lack of Nrp2 in MGE-derived interneuron precursors disrupts the excitation/inhibition balance in hippocampal circuits, thus predisposing the network to seizures and behavioral patterns associated with ASD. Embryonic deletion of Nrp2 during the developmental period for migration of MGE derived interneuron precursors (iCKO) significantly reduced parvalbumin, neuropeptide Y, and somatostatin positive neurons in the hippocampal CA1. Consequently, when compared to controls, the frequency of inhibitory synaptic currents in CA1 pyramidal cells was reduced while frequency of excitatory synaptic currents was increased in iCKO mice. Although passive and active membrane properties of CA1 pyramidal cells were unchanged, iCKO mice showed enhanced susceptibility to chemically evoked seizures. Moreover, iCKO mice exhibited selective behavioral deficits in both preference for social novelty and goal-directed learning, which are consistent with ASD-like phenotype. Together, our findings show that disruption of developmental Nrp2 regulation of interneuron circuit establishment, produces ASD-like behaviors and enhanced risk for epilepsy. These results support the developmental interneuronopathy hypothesis of ASD epilepsy comorbidity.

Balancing act of small GTPases downstream of plexin-A4 signaling motifs promotes dendrite elaboration in mammalian cortical neurons.

The precise development of neuronal morphologies is crucial to the establishment of synaptic circuits and, ultimately, proper brain function. Signaling by the axon guidance cue semaphorin 3A (Sema3A) and its receptor complex of neuropilin-1 and plexin-A4 has multifunctional outcomes in neuronal morphogenesis. Downstream activation of the RhoGEF FARP2 through interaction with the lysine-arginine-lysine motif of plexin-A4 and consequent activation of the small GTPase Rac1 promotes dendrite arborization, but this pathway is dispensable for axon repulsion. Here, we investigated the interplay of small GTPase signaling mechanisms underlying Sema3A-mediated dendritic elaboration in mouse layer V cortical neurons in vitro and in vivo. Sema3A promoted the binding of the small GTPase Rnd1 to the amino acid motif lysine-valine-serine (LVS) in the cytoplasmic domain of plexin-A4. Rnd1 inhibited the activity of the small GTPase RhoA and the kinase ROCK, thus supporting the activity of the GTPase Rac1, which permitted the growth and branching of dendrites. Overexpression of a dominant-negative RhoA, a constitutively active Rac1, or the pharmacological inhibition of ROCK activity rescued defects in dendritic elaboration in neurons expressing a plexin-A4 mutant lacking the LVS motif. Our findings provide insights into the previously unappreciated balancing act between Rho and Rac signaling downstream of specific motifs in plexin-A4 to mediate Sema3A-dependent dendritic elaboration in mammalian cortical neuron development.

Using Simulated Patient Avatar and Monitor (SPA©M) to Advance Learner's Knowledge of Pathophysiology and Pharmacology.

A novel method was implemented in the Interprofessional education (IPE) program, which incorporated a Simulated-Avatar© case presentation preceding the virtual breakout segments. Simulated real-time clinical interactions replaced the in-person encounters, leading to the translation of the participants' basic science knowledge of pathophysiology and pharmacology (P&P) into effective treatment of the patient-avatar's condition.

Semaphorin signaling restricts neuronal regeneration in C. elegans.

Extracellular signaling proteins serve as neuronal growth cone guidance molecules during development and are well positioned to be involved in neuronal regeneration and recovery from injury. Semaphorins and their receptors, the plexins, are a family of conserved proteins involved in development that, in the nervous system, are axonal guidance cues mediating axon pathfinding and synapse formation. The Caenorhabditis elegans genome encodes for three semaphorins and two plexin receptors: the transmembrane semaphorins, SMP-1 and SMP-2, signal through their receptor, PLX-1, while the secreted semaphorin, MAB-20, signals through PLX-2. Here, we evaluate the locomotion behavior of knockout animals missing each of the semaphorins and plexins and the neuronal morphology of plexin knockout animals; we described the cellular expression pattern of the promoters of all plexins in the nervous system of C. elegans; and we evaluated their effect on the regrowth and reconnection of motoneuron neurites and the recovery of locomotion behavior following precise laser microsurgery. Regrowth and reconnection were more prevalent in the absence of each plexin, while recovery of locomotion surpassed regeneration in all genotypes.

An appropriate verbal probability lexicon for communicating surgical risks is unlikely to exist.

Effective risk communication about medical procedures is critical to ethical shared decision-making. Here, we explore the potential for development of an evidence-based lexicon for verbal communication of surgical risk. We found that Ear, Nose and Throat (ENT) surgeons expressed a preference for communicating such risks using verbal probability expressions (VPEs; e.g., "high risk"). However, there was considerable heterogeneity in the expressions they reported using (Study 1). Study 2 compared ENT surgeons' and laypeople's (i.e., potential patients) interpretations of the ten most frequent VPEs listed in Study 1. While both groups displayed considerable variability in interpretations, lay participants demonstrated more, as well as providing systematically higher interpretations than those of surgeons. Study 3 found that lay participants were typically unable to provide unique VPEs to differentiate between the ranges of (low) probabilities required. Taken together, these results add to arguments that reliance on VPEs for surgical risk communication is ill-advised. Not only are there systematic interpretational differences between surgeons and potential patients, but the coarse granularity of VPEs raises severe challenges for developing an appropriate evidence-based lexicon for surgical risk communication. We caution against the use of VPEs in any risk context characterized by low, but very different, probabilities. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery.

Human pluripotent stem-cell-derived organoids are models for human development and disease. We report a modified human kidney organoid system that generates thousands of similar organoids, each consisting of 1-2 nephron-like structures. Single-cell transcriptomic profiling and immunofluorescence validation highlighted patterned nephron-like structures utilizing similar pathways, with distinct morphogenesis, to human nephrogenesis. To examine this platform for therapeutic screening, the polycystic kidney disease genes PKD1 and PKD2 were inactivated by gene editing. PKD1 and PKD2 mutant models exhibited efficient and reproducible cyst formation. Cystic outgrowths could be propagated for months to centimeter-sized cysts. To shed new light on cystogenesis, 247 protein kinase inhibitors (PKIs) were screened in a live imaging assay identifying compounds blocking cyst formation but not overall organoid growth. Scaling and further development of the organoid platform will enable a broader capability for kidney disease modeling and high-throughput drug screens.

Neuropilin 2/Plexin-A3 Receptors Regulate the Functional Connectivity and the Excitability in the Layers 4 and 5 of the Cerebral Cortex.

The functions of cortical networks are progressively established during development by series of events shaping the neuronal connectivity. Synaptic elimination, which consists of removing the supernumerary connections generated during the earlier stages of cortical development, is one of the latest stages in neuronal network maturation. The semaphorin 3F coreceptors neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) may play an important role in the functional maturation of the cerebral cortex by regulating the excess dendritic spines on cortical excitatory neurons. Yet, the identity of the connections eliminated under the control of Nrp2/PlxnA3 signaling is debated, and the importance of this synaptic refinement for cortical functions remains poorly understood. Here, we show that Nrp2/PlxnA3 controls the spine densities in layer 4 (L4) and on the apical dendrite of L5 neurons of the sensory and motor cortices. Using a combination of neuroanatomical, ex vivo electrophysiology, and in vivo functional imaging techniques in Nrp2 and PlxnA3 KO mice of both sexes, we disprove the hypothesis that Nrp2/PlxnA3 signaling is required to maintain the ectopic thalamocortical connections observed during embryonic development. We also show that the absence of Nrp2/PlxnA3 signaling leads to the hyperexcitability and excessive synchronization of the neuronal activity in L5 and L4 neuronal networks, suggesting that this system could participate in the refinement of the recurrent corticocortical connectivity in those layers. Altogether, our results argue for a role of semaphorin-Nrp2/PlxnA3 signaling in the proper maturation and functional connectivity of the cerebral cortex, likely by controlling the refinement of recurrent corticocortical connections.SIGNIFICANCE STATEMENT The function of a neuronal circuit is mainly determined by the connections that neurons establish with one another during development. Understanding the mechanisms underlying the establishment of the functional connectivity is fundamental to comprehend how network functions are implemented, and to design treatments aiming at restoring damaged neuronal circuits. Here, we show that the cell surface receptors for the family of semaphorin guidance cues neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) play an important role in shaping the functional connectivity of the cerebral cortex likely by trimming the recurrent connections in layers 4 and 5. By removing the supernumerary inputs generated during early development, Nrp2/PlxnA3 signaling reduces the neuronal excitability and participates in the maturation of the cortical network functions.

Neuronal Microsurgery with an Yb-Doped Fiber Femtosecond Laser.

Laser microsurgery allows the user to ablate cell bodies or disconnect nerve fibers by using a laser microbeam focused through a microscope. This technique was pioneered in C. elegans where it led to exciting discoveries in the fields of development and neurobiology. All neurons studied so far in C. elegans can regenerate and regrow axons and dendrites after injury, allowing studies of the molecular and cellular basis of neuroregeneration. In this chapter, we describe how to assemble and operate a platform for Yb-doped fiber laser microsurgery. The novel laser setup described here is a more robust, lower cost, and user-friendly alternative to other femtosecond-pulsed laser systems.

Semaphorin3A/PlexinA3 association with the Scribble scaffold for cGMP increase is required for apical dendrite development.

The development of the apical dendrite from the leading process of the bipolar pyramidal neuron might be directed by spatially organized extrinsic cues acting on localized intrinsic determinants. The extracellular cues regulating apical dendrite polarization remain elusive. We show that leading process and apical dendrite development are directed by class III Semaphorins and mediated by a localized cGMP-synthesizing complex. The scaffolding protein Scribble that associates with the cGMP-synthesizing enzyme soluble guanylate cyclase (sGC) also associates with the Semaphorin3A (Sema3A) co-receptor PlexinA3. Deletion or knockdown of PlexinA3 and Sema3A or disruption of PlexinA3-Scribble association prevents Sema3A-mediated cGMP increase and causes defects in apical dendrite development. These manipulations also impair bipolar polarity and leading process establishment. Local cGMP elevation or sGC expression rescues the effects of PlexinA3 knockdown or PlexinA3-Scribble complex disruption. During neuronal polarization, leading process and apical dendrite development are directed by a scaffold that links Semaphorin cue to cGMP increase.

Loss of miR-34 in Drosophila dysregulates protein translation and protein turnover in the aging brain.

Aging is a risk factor for neurodegenerative disease, but precise mechanisms that influence this relationship are still under investigation. Work in Drosophila melanogaster identified the microRNA miR-34 as a modifier of aging and neurodegeneration in the brain. MiR-34 mutants present aspects of early aging, including reduced lifespan, neurodegeneration, and a buildup of the repressive histone mark H3K27me3. To better understand how miR-34 regulated pathways contribute to age-associated phenotypes in the brain, here we transcriptionally profiled the miR-34 mutant brain. This identified that genes associated with translation are dysregulated in the miR-34 mutant. The brains of these animals show increased translation activity, accumulation of protein aggregation markers, and altered autophagy activity. To determine if altered H3K27me3 was responsible for this proteostasis dysregulation, we studied the effects of increased H3K27me3 by mutating the histone demethylase Utx. Reduced Utx activity enhanced neurodegeneration and mimicked the protein accumulation seen in miR-34 mutant brains. However, unlike the miR-34 mutant, Utx mutant brains did not show similar altered autophagy or translation activity, suggesting that additional miR-34-targeted pathways are involved. Transcriptional analysis of predicted miR-34 targets identified Lst8, a subunit of Tor Complex 1 (TORC1), as a potential target. We confirmed that miR-34 regulates the 3' UTR of Lst8 and identified several additional predicted miR-34 targets that may be critical for maintaining proteostasis and brain health. Together, these results present novel understanding of the brain and the role of the conserved miRNA miR-34 in impacting proteostasis in the brain with age.

Reduced hippocampal inhibition and enhanced autism-epilepsy comorbidity in mice lacking neuropilin 2.

The neuropilin receptors and their secreted semaphorin ligands play key roles in brain circuit development by regulating numerous crucial neuronal processes, including the maturation of synapses and migration of GABAergic interneurons. Consistent with its developmental roles, the neuropilin 2 (Nrp2) locus contains polymorphisms in patients with autism spectrum disorder (ASD). Nrp2-deficient mice show autism-like behavioral deficits and propensity to develop seizures. In order to determine the pathophysiology in Nrp2 deficiency, we examined the hippocampal numbers of interneuron subtypes and inhibitory regulation of hippocampal CA1 pyramidal neurons in mice lacking one or both copies of Nrp2. Immunostaining for interneuron subtypes revealed that Nrp2-/- mice have a reduced number of parvalbumin, somatostatin, and neuropeptide Y cells, mainly in CA1. Whole-cell recordings identified reduced firing and hyperpolarized shift in resting membrane potential in CA1 pyramidal neurons from Nrp2+/- and Nrp2-/- mice compared to age-matched wild-type controls indicating decrease in intrinsic excitability. Simultaneously, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) are reduced in Nrp2-deficient mice. A convulsive dose of kainic acid evoked electrographic and behavioral seizures with significantly shorter latency, longer duration, and higher severity in Nrp2-/- compared to Nrp2+/+ animals. Finally, Nrp2+/- and Nrp2-/- but not Nrp2+/+, mice have impaired cognitive flexibility demonstrated by reward-based reversal learning, a task associated with hippocampal circuit function. Together these data demonstrate a broad reduction in interneuron subtypes and compromised inhibition in CA1 of Nrp2-/- mice, which could contribute to the heightened seizure susceptibility and behavioral deficits consistent with an ASD/epilepsy phenotype.

Spatial transcriptional mapping of the human nephrogenic program.

Congenital abnormalities of the kidney and urinary tract are among the most common birth defects, affecting 3% of newborns. The human kidney forms around a million nephrons from a pool of nephron progenitors over a 30-week period of development. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlage, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single-cell RNA sequencing identified progenitor states, which were spatially mapped to the nephron anatomy, enabling the generation of functional gene networks predicting interactions within and between nephron cell types. Network mining identified known developmental disease genes and predicted targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease and enhance efforts to generate new kidney structures.

Alcohol-induced aggression in Drosophila.

Alcohol-induced aggression is a destructive and widespread phenomenon associated with violence and sexual assault. However, little is understood concerning its mechanistic origin. We have developed a Drosophila melanogaster model to genetically dissect and understand the phenomenon of sexually dimorphic alcohol-induced aggression. Males with blood alcohol levels of 0.04-mg/ml BAC were less aggressive than alcohol-naive males, but when the BAC had dropped to ~0.015 mg/ml, the alcohol-treated males showed an increase in aggression toward other males. This aggression-promoting treatment is referred to as the post-ethanol aggression (PEA) treatment. Females do not show increased aggression after the same treatment. PEA-treated males also spend less time courting and attempt to copulate earlier than alcohol-naive flies. PEA treatment induces expression of the FruM transcription factor (encoded by a male-specific transcript from the fruitless gene), whereas sedating doses of alcohol reduce FruM expression and reduce male aggression. Transgenic suppression of FruM induction also prevents alcohol-induced aggression. In male flies, alcohol-induced aggression is dependent on the male isoform of the fruitless transcription factor (FruM). Low-dose alcohol induces FruM expression and promotes aggression, whereas higher doses of alcohol suppress FruM and suppress aggression.

Alcohol potentiates a pheromone signal in flies.

For decades, numerous researchers have documented the presence of the fruit fly or Drosophila melanogaster on alcohol-containing food sources. Although fruit flies are a common laboratory model organism of choice, there is relatively little understood about the ethological relationship between flies and ethanol. In this study, we find that when male flies inhabit ethanol-containing food substrates they become more aggressive. We identify a possible mechanism for this behavior. The odor of ethanol potentiates the activity of sensory neurons in response to an aggression-promoting pheromone. Finally, we observed that the odor of ethanol also promotes attraction to a food-related citrus odor. Understanding how flies interact with the complex natural environment they inhabit can provide valuable insight into how different natural stimuli are integrated to promote fundamental behaviors.

Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Controls Dendrite Morphogenesis.

Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.SIGNIFICANCE STATEMENT The proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/Plexin-A4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that three specific amino acids, the KRK motif within the Plexin-A4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guidance. Our results unravel a novel Semaphorin3A-Plexin-A4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo.

Cellular Recruitment by Podocyte-Derived Pro-migratory Factors in Assembly of the Human Renal Filter.

Analysis of kidney disease-causing genes and pathology resulting from systemic diseases highlight the importance of the kidney's filtering system, the renal corpuscles. To elucidate the developmental processes that establish the renal corpuscle, we performed single-nucleus droplet-based sequencing of the human fetal kidney. This enabled the identification of nephron, interstitial, and vascular cell types that together generate the renal corpuscles. Trajectory analysis identified transient developmental gene expression, predicting precursors or mature podocytes express FBLN2, BMP4, or NTN4, in conjunction with recruitment, differentiation, and modeling of vascular and mesangial cell types into a functional filter. In vitro studies provide evidence that these factors exhibit angiogenic or mesangial recruiting and inductive properties consistent with a key organizing role for podocyte precursors in kidney development. Together these studies define a spatiotemporal developmental program for the primary filtration unit of the human kidney and provide novel insights into cell interactions regulating co-assembly of constituent cell types.

Neuropilin 2 Signaling Mediates Corticostriatal Transmission, Spine Maintenance, and Goal-Directed Learning in Mice.

The striatum represents the main input structure of the basal ganglia, receiving massive excitatory input from the cortex and the thalamus. The development and maintenance of cortical input to the striatum is crucial for all striatal function including many forms of sensorimotor integration, learning, and action control. The molecular mechanisms regulating the development and maintenance of corticostriatal synaptic transmission are unclear. Here we show that the guidance cue, Semaphorin 3F and its receptor Neuropilin 2 (Nrp2), influence dendritic spine maintenance, corticostriatal short-term plasticity, and learning in adult male and female mice. We found that Nrp2 is enriched in adult layer V pyramidal neurons, corticostriatal terminals, and in developing and adult striatal spiny projection neurons (SPNs). Loss of Nrp2 increases SPN excitability and spine number, reduces short-term facilitation at corticostriatal synapses, and impairs goal-directed learning in an instrumental task. Acute deletion of Nrp2 selectively in adult layer V cortical neurons produces a similar increase in the number of dendritic spines and presynaptic modifications at the corticostriatal synapse in the Nrp2-/- mouse, but does not affect the intrinsic excitability of SPNs. Furthermore, conditional loss of Nrp2 impairs sensorimotor learning on the accelerating rotarod without affecting goal-directed instrumental learning. Collectively, our results identify Nrp2 signaling as essential for the development and maintenance of the corticostriatal pathway and may shed novel insights on neurodevelopmental disorders linked to the corticostriatal pathway and Semaphorin signaling.SIGNIFICANCE STATEMENT The corticostriatal pathway controls sensorimotor, learning, and action control behaviors and its dysregulation is linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here we demonstrate that Neuropilin 2 (Nrp2), a receptor for the axon guidance cue semaphorin 3F, has important and previously unappreciated functions in the development and adult maintenance of dendritic spines on striatal spiny projection neurons (SPNs), corticostriatal short-term plasticity, intrinsic physiological properties of SPNs, and learning in mice. Our findings, coupled with the association of Nrp2 with ASD in human populations, suggest that Nrp2 may play an important role in ASD pathophysiology. Overall, our work demonstrates Nrp2 to be a key regulator of corticostriatal development, maintenance, and function, and may lead to better understanding of neurodevelopmental disease mechanisms.

In Vivo Developmental Trajectories of Human Podocyte Inform In Vitro Differentiation of Pluripotent Stem Cell-Derived Podocytes.

The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.