The mouse brain after foot shock in four dimensions: Temporal dynamics at a single-cell resolution.

Acute stress leads to sequential activation of functional brain networks. A biologically relevant question is exactly which (single) cells belonging to brain networks are changed in activity over time after acute stress across the entire brain. We developed a preprocessing and analytical pipeline to chart whole-brain immediate early genes' expression-as proxy for cellular activity-after a single stressful foot shock in four dimensions: that is, from functional networks up to three-dimensional (3D) single-cell resolution and over time. The pipeline is available as an R package. Most brain areas (96%) showed increased numbers of c-fos+ cells after foot shock, yet hypothalamic areas stood out as being most active and prompt in their activation, followed by amygdalar, prefrontal, hippocampal, and finally, thalamic areas. At the cellular level, c-fos+ density clearly shifted over time across subareas, as illustrated for the basolateral amygdala. Moreover, some brain areas showed increased numbers of c-fos+ cells, while others-like the dentate gyrus-dramatically increased c-fos intensity in just a subset of cells, reminiscent of engrams; importantly, this "strategy" changed after foot shock in half of the brain areas. One of the strengths of our approach is that single-cell data were simultaneously examined across all of the 90 brain areas and can be visualized in 3D in our interactive web portal.

Distinct spatial arrangements of ACE2 and TMPRSS2 expression in Syrian hamster lung lobes dictates SARS-CoV-2 infection patterns.

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli.

Prebiotic diet normalizes aberrant immune and behavioral phenotypes in a mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a cluster of neurodevelopmental disorders characterized by deficits in communication and behavior. Increasing evidence suggests that the microbiota-gut-brain axis and the likely related immune imbalance may play a role in the development of this disorder. Gastrointestinal deficits and gut microbiota dysfunction have been linked to the development or severity of autistic behavior. Therefore, treatments that focus on specific diets may improve gastrointestinal function and aberrant behavior in individuals with ASD. In this study, we investigated whether a diet containing specific prebiotic fibers, namely, 3% galacto-oligosaccharide/fructo-oligosaccharide (GOS/FOS; 9:1), can mitigate the adverse effects of in utero exposure to valproic acid (VPA) in mice. Pregnant BALB/cByJ dams were injected with VPA (600 mg/kg, sc.) or phosphate-buffered saline (PBS) on gestational day 11 (G11). Male offspring were divided into four groups: (1) in utero PBS-exposed with a control diet, (2) in utero PBS-exposed with GOS/FOS diet, (3) in utero VPA-exposed with a control diet, and (4) in utero VPA-exposed with GOS/FOS diet. Dietary intervention started from birth and continued throughout the duration of the experiment. We showed that the prebiotic diet normalized VPA-induced alterations in male offspring, including restoration of key microbial taxa, intestinal permeability, peripheral immune homeostasis, reduction of neuroinflammation in the cerebellum, and impairments in social behavior and cognition in mice. Overall, our research provides valuable insights into the gut-brain axis involvement in ASD development. In addition, dietary interventions might correct the disbalance in gut microbiota and immune responses and, ultimately, might improve detrimental behavioral outcomes in ASD.

Dorsal Root Ganglia Macrophages Maintain Osteoarthritis Pain.

Pain is the major debilitating symptom of osteoarthritis (OA), which is difficult to treat. In OA patients joint tissue damage only poorly associates with pain, indicating other mechanisms contribute to OA pain. Immune cells regulate the sensory system, but little is known about the involvement of immune cells in OA pain. Here, we report that macrophages accumulate in the dorsal root ganglia (DRG) distant from the site of injury in two rodent models of OA. DRG macrophages acquired an M1-like phenotype, and depletion of DRG macrophages resolved OA pain in male and female mice. Sensory neurons innervating the damaged knee joint shape DRG macrophages into an M1-like phenotype. Persisting OA pain, accumulation of DRG macrophages, and programming of DRG macrophages into an M1-like phenotype were independent of Nav1.8 nociceptors. Inhibition of M1-like macrophages in the DRG by intrathecal injection of an IL4-IL10 fusion protein or M2-like macrophages resolved persistent OA pain. In conclusion, these findings reveal a crucial role for macrophages in maintaining OA pain independent of the joint damage and suggest a new direction to treat OA pain.SIGNIFICANCE STATEMENT In OA patients pain poorly correlates with joint tissue changes indicating mechanisms other than only tissue damage that cause pain in OA. We identified that DRG containing the somata of sensory neurons innervating the damaged knee are infiltrated with macrophages that are shaped into an M1-like phenotype by sensory neurons. We show that these DRG macrophages actively maintain OA pain remotely and independent of joint damage. The phenotype of these macrophages is crucial for a pain-promoting role. Targeting the phenotype of DRG macrophages with either M2-like macrophages or a cytokine fusion protein that skews macrophages into an M2-like phenotype resolves OA pain. Our work reveals a mechanism that contributes to the maintenance of OA pain distant from the affected knee joint and suggests that dorsal root ganglia macrophages are a target to treat osteoarthritis chronic pain.

Sensory Axon Growth Requires Spatiotemporal Integration of CaSR and TrkB Signaling.

Neural circuit development involves the coordinated growth and guidance of axons. During this process, axons encounter many different cues, but how these cues are integrated and translated into growth is poorly understood. In this study, we report that receptor signaling does not follow a linear path but changes dependent on developmental stage and coreceptors involved. Using developing chicken embryos of both sexes, our data show that calcium-sensing receptor (CaSR), a G-protein-coupled receptor important for regulating calcium homeostasis, regulates neurite growth in two distinct ways. First, when signaling in isolation, CaSR promotes growth through the PI3-kinase-Akt pathway. At later developmental stages, CaSR enhances tropomyosin receptor kinase B (TrkB)/BDNF-mediated neurite growth. This enhancement is facilitated through a switch in the signaling cascade downstream of CaSR (i.e., from the PI3-kinase-Akt pathway to activation of GSK3α Tyr279). TrkB and CaSR colocalize within late endosomes, cotraffic and coactivate GSK3, which serves as a shared signaling node for both receptors. Our study provides evidence that two unrelated receptors can integrate their individual signaling cascades toward a nonadditive effect and thus control neurite growth during development.SIGNIFICANCE STATEMENT This work highlights the effect of receptor coactivation and signal integration in a developmental setting. During embryonic development, neurites grow toward their targets guided by cues in the extracellular environment. These cues are sensed by receptors at the surface that trigger intracellular signaling events modulating the cytoskeleton. Emerging evidence suggests that the effects of guidance cues are diversified, therefore expanding the number of responses. Here, we show that two unrelated receptors can change the downstream signaling cascade and regulate neuronal growth through a shared signaling node. In addition to unraveling a novel signaling pathway in neurite growth, this research stresses the importance of receptor coactivation and signal integration during development of the nervous system.

An Image-Based miRNA Screen Identifies miRNA-135s As Regulators of CNS Axon Growth and Regeneration by Targeting Krüppel-like Factor 4.

During embryonic development, axons extend over long distances to establish functional connections. In contrast, axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing CNS regeneration. Here, we performed one of the first miRNome-wide functional miRNA screens to identify miRNAs with robust effects on axon growth. High-content screening identified miR-135a and miR-135b as potent stimulators of axon growth and cortical neuron migration in vitro and in vivo in male and female mice. Intriguingly, both of these developmental effects of miR-135s relied in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon growth and regeneration. These results prompted us to test the effect of miR-135s on axon regeneration after injury. Our results show that intravitreal application of miR-135s facilitates retinal ganglion cell (RGC) axon regeneration after optic nerve injury in adult mice in part by repressing KLF4. In contrast, depletion of miR-135s further reduced RGC axon regeneration. Together, these data identify a novel neuronal role for miR-135s and the miR-135-KLF4 pathway and highlight the potential of miRNAs as tools for enhancing CNS axon regeneration.SIGNIFICANCE STATEMENT Axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing regeneration. By performing an miRNome-wide functional screen, our studies identify miR-135s as stimulators of axon growth and neuron migration and show that intravitreal application of these miRNAs facilitates CNS axon regeneration after nerve injury in adult mice. Intriguingly, these developmental and regeneration-promoting effects rely in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon regeneration. Our data identify a novel neuronal role for the miR-135-KLF4 pathway and support the idea that miRNAs can be used for enhancing CNS axon regeneration.

Frizzled3 Controls Axonal Polarity and Intermediate Target Entry during Striatal Pathway Development.

The striatum is a large brain nucleus with an important role in the control of movement and emotions. Medium spiny neurons (MSNs) are striatal output neurons forming prominent descending axon tracts that target different brain nuclei. However, how MSN axon tracts in the forebrain develop remains poorly understood. Here, we implicate the Wnt binding receptor Frizzled3 in several uncharacterized aspects of MSN pathway formation [i.e., anterior-posterior guidance of MSN axons in the striatum and their subsequent growth into the globus pallidus (GP), an important (intermediate) target]. In Frizzled3 knock-out mice, MSN axons fail to extend along the anterior-posterior axis of the striatum, and many do not reach the GP. Wnt5a acts as an attractant for MSN axons in vitro, is expressed in a posterior high, anterior low gradient in the striatum, and Wnt5a knock-out mice phenocopy striatal anterior-posterior defects observed in Frizzled3 mutants. This suggests that Wnt5a controls anterior-posterior guidance of MSN axons through Frizzled3. Axons that reach the GP in Frizzled3 knock-out mice fail to enter this structure. Surprisingly, entry of MSN axons into the GP non-cell-autonomously requires Frizzled3, and our data suggest that GP entry may be contingent on the correct positioning of "corridor" guidepost cells for thalamocortical axons by Frizzled3. Together, these data dissect MSN pathway development and reveal (non)cell-autonomous roles for Frizzled3 in MSN axon guidance. Further, they are the first to identify a gene that provides anterior-posterior axon guidance in a large brain nucleus and link Frizzled3 to corridor cell development. SIGNIFICANCE STATEMENT: Striatal axon pathways mediate complex physiological functions and are an important therapeutic target, underscoring the need to define how these connections are established. Remarkably, the molecular programs regulating striatal pathway development remain poorly characterized. Here, we determine the embryonic ontogeny of the two main striatal pathways (striatonigral and striatopallidal) and identify novel (non)cell-autonomous roles for the axon guidance receptor Frizzled3 in uncharacterized aspects of striatal pathway formation (i.e., anterior-posterior axon guidance in the striatum and axon entry into the globus pallidus). Further, our results link Frizzled3 to corridor guidepost cell development and suggest that an abnormal distribution of these cells has unexpected, widespread effects on the development of different axon tracts (i.e., striatal and thalamocortical axons).

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids.

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes.

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.

Dysregulation of Semaphorin7A/ß1-integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility.

Reproduction in mammals is dependent on the function of specific neurons that secrete gonadotropin-releasing hormone-1 (GnRH-1). These neurons originate prenatally in the nasal placode and migrate into the forebrain along the olfactory-vomeronasal nerves. Alterations in this migratory process lead to defective GnRH-1 secretion, resulting in heterogeneous genetic disorders such as idiopathic hypogonadotropic hypogonadism (IHH), and other reproductive diseases characterized by the reduction or failure of sexual competence. Combining mouse genetics with in vitro models, we demonstrate that Semaphorin 7A (Sema7A) is essential for the development of the GnRH-1 neuronal system. Loss of Sema7A signaling alters the migration of GnRH-1 neurons, resulting in significantly reduced numbers of these neurons in the adult brain as well as in reduced gonadal size and subfertility. We also show that GnRH-1 cells differentially express the Sema7 receptors ß1-integrin and Plexin C1 as a function of their migratory stage, whereas the ligand is robustly expressed along developing olfactory/vomeronasal fibers. Disruption of Sema7A function in vitro inhibits ß1-integrin-mediated migration. Analysis of Plexin C1(-/-) mice did not reveal any difference in the migratory process of GnRH-1 neurons, indicating that Sema7A mainly signals through ß1-integrin to regulate GnRH-1 cell motility. In conclusion, we have identified Sema7A as a gene implicated in the normal development of the GnRH-1 system in mice and as a genetic marker for the elucidation of some forms of GnRH-1 deficiency in humans.

Purkinje cell microzones mediate distinct kinematics of a single movement.

The classification of neuronal subpopulations has significantly advanced, yet its relevance for behavior remains unclear. The highly organized flocculus of the cerebellum, known to fine-tune multi-axial eye movements, is an ideal substrate for the study of potential functions of neuronal subpopulations. Here, we demonstrate that its recently identified subpopulations of 9+ and 9- Purkinje cells exhibit an intermediate Aldolase C expression and electrophysiological profile, providing evidence for a graded continuum of intrinsic properties among PC subpopulations. By identifying and utilizing two Cre-lines that genetically target these floccular domains, we show with high spatial specificity that these subpopulations of Purkinje cells participate in separate micromodules with topographically organized connections. Finally, optogenetic excitation of the respective subpopulations results in movements around the same axis in space, yet with distinct kinematic profiles. These results indicate that Purkinje cell subpopulations integrate in discrete circuits and mediate particular parameters of single movements.

MICALs in control of the cytoskeleton, exocytosis, and cell death.

MICALs form an evolutionary conserved family of multidomain signal transduction proteins characterized by a flavoprotein monooxygenase domain. MICALs are being implicated in the regulation of an increasing number of molecular and cellular processes including cytoskeletal dynamics and intracellular trafficking. Intriguingly, some of these effects are dependent on the MICAL monooxygenase enzyme and redox signaling, while other functions rely on other parts of the MICAL protein. Recent breakthroughs in our understanding of MICAL signaling identify the ability of MICALs to bind and directly modify the actin cytoskeleton, link MICALs to the docking and fusion of exocytotic vesicles, and uncover MICALs as anti-apoptotic proteins. These discoveries could lead to therapeutic advances in neural regeneration, cancer, and other diseases.

Anatomical Development of the Cerebellothalamic Tract in Embryonic Mice.

The main connection from cerebellum to cerebrum is formed by cerebellar nuclei axons that synapse in the thalamus. Apart from its role in coordinating sensorimotor integration in the adult brain, the cerebello-thalamic tract (CbT) has also been implicated in developmental disorders, such as autism spectrum disorders. Although the development of the cerebellum, thalamus and cerebral cortex have been studied, there is no detailed description of the ontogeny of the mammalian CbT. Here we investigated the development of the CbT at embryonic stages using transgenic Ntsr1-Cre/Ai14 mice and in utero electroporation of wild type mice. Wide-field, confocal and 3D light-sheet microscopy of immunohistochemical stainings showed that CbT fibers arrive in the prethalamus between E14.5 and E15.5, but only invade the thalamus after E16.5. We quantified the spread of CbT fibers throughout the various thalamic nuclei and found that at E17.5 and E18.5 the ventrolateral, ventromedial and parafascicular nuclei, but also the mediodorsal and posterior complex, become increasingly innervated. Several CbT fiber varicosities express vesicular glutamate transporter type 2 at E18.5, indicating cerebello-thalamic synapses. Our results provide the first quantitative data on the developing murine CbT, which provides guidance for future investigations of the impact that cerebellum has on thalamo-cortical networks during development.

Molecular signatures and cellular diversity during mouse habenula development.

The habenula plays a key role in various motivated and pathological behaviors and is composed of molecularly distinct neuron subtypes. Despite progress in identifying mature habenula neuron subtypes, how these subtypes develop and organize into functional brain circuits remains largely unknown. Here, we performed single-cell transcriptional profiling of mouse habenular neurons at critical developmental stages, instructed by detailed three-dimensional anatomical data. Our data reveal cellular and molecular trajectories during embryonic and postnatal development, leading to different habenular subtypes. Further, based on this analysis, our work establishes the distinctive functional properties and projection target of a subtype of Cartpt+ habenula neurons. Finally, we show how comparison of single-cell transcriptional profiles and GWAS data links specific developing habenular subtypes to psychiatric disease. Together, our study begins to dissect the mechanisms underlying habenula neuron subtype-specific development and creates a framework for further interrogation of habenular development in normal and disease states.

Wnt/planar cell polarity signaling controls the anterior-posterior organization of monoaminergic axons in the brainstem.

Monoaminergic neurons [serotonergic (5-HT) and dopaminergic (mdDA)] in the brainstem project axons along the anterior-posterior axis. Despite their important physiological functions and implication in disease, the molecular mechanisms that dictate the formation of these projections along the anterior-posterior axis remain unknown. Here we reveal a novel requirement for Wnt/planar cell polarity signaling in the anterior-posterior organization of the monoaminergic system. We find that 5-HT and mdDA axons express the core planar cell polarity components Frizzled3, Celsr3, and Vangl2. In addition, monoaminergic projections show anterior-posterior guidance defects in Frizzled3, Celsr3, and Vangl2 mutant mice. The only known ligands for planar cell polarity signaling are Wnt proteins. In culture, Wnt5a attracts 5-HT but repels mdDA axons, and Wnt7b attracts mdDA axons. However, mdDA axons from Frizzled3 mutant mice are unresponsive to Wnt5a and Wnt7b. Both Wnts are expressed in gradients along the anterior-posterior axis, consistent with their role as directional cues. Finally, Wnt5a mutants show transient anterior-posterior guidance defects in mdDA projections. Furthermore, we observe during development that the cell bodies of migrating descending 5-HT neurons eventually reorient along the direction of their axons. In Frizzled3 mutants, many 5-HT and mdDA neuron cell bodies are oriented abnormally along the direction of their aberrant axon projections. Overall, our data suggest that Wnt/planar cell polarity signaling may be a global anterior-posterior guidance mechanism that controls axonal and cellular organization beyond the spinal cord.

Protocol for tissue clearing and 3D analysis of dopamine neurons in the developing mouse midbrain.

Advances in tissue clearing enable analysis of complex migratory patterns of developing neurons in whole intact tissue. Here, we implemented a modified version of 3DISCO to study migration of midbrain dopamine (DA) neurons. We provide a detailed protocol starting from whole-brain immunostaining, tissue clearing, and ultramicroscopic imaging to post-acquisition quantification and analysis. This protocol enables precise quantification of DA neuron migration but can also be applied more generally for analyzing neuron migration throughout the nervous system. For complete details on the use and execution of this protocol, please refer to Brignani et al. (2020).

Pharmacological validation of TDO as a target for Parkinson's disease.

Parkinson's disease patients suffer from both motor and nonmotor impairments. There is currently no cure for Parkinson's disease, and the most commonly used treatment, levodopa, only functions as a temporary relief of motor symptoms. Inhibition of the expression of the L-tryptophan-catabolizing enzyme tryptophan 2,3-dioxygenase (TDO) has been shown to inhibit aging-related α-synuclein toxicity in Caenorhabditis elegans. To evaluate TDO inhibition as a potential therapeutic strategy for Parkinson's disease, a brain-penetrable, small molecule TDO inhibitor was developed, referred to as NTRC 3531-0. This compound potently inhibits human and mouse TDO in biochemical and cell-based assays and is selective over IDO1, an evolutionary unrelated enzyme that catalyzes the same reaction. In mice, NTRC 3531-0 increased plasma and brain L-tryptophan levels after oral administration, demonstrating inhibition of TDO activity in vivo. The effect on Parkinson's disease symptoms was evaluated in a rotenone-induced Parkinson's disease mouse model. A structurally dissimilar TDO inhibitor, LM10, was evaluated in parallel. Both inhibitors had beneficial effects on rotenone-induced motor and cognitive dysfunction as well as rotenone-induced dopaminergic cell loss and neuroinflammation in the substantia nigra. Moreover, both inhibitors improved intestinal transit and enhanced colon length, which indicates a reduction of the rotenone-induced intestinal dysfunction. Consistent with this, mice treated with TDO inhibitor showed decreased expression of rotenone-induced glial fibrillary acidic protein, which is a marker of enteric glial cells, and decreased α-synuclein accumulation in the enteric plexus. Our data support TDO inhibition as a potential therapeutic strategy to decrease motor, cognitive, and gastrointestinal symptoms in Parkinson's disease.

Semaphorin 3F is a bifunctional guidance cue for dopaminergic axons and controls their fasciculation, channeling, rostral growth, and intracortical targeting.

Dopaminergic neurons in the mesodiencephalon (mdDA neurons) make precise synaptic connections with targets in the forebrain via the mesostriatal, mesolimbic, and mesoprefrontal pathways. Because of the functional importance of these remarkably complex ascending axon pathways and their implication in human disease, the mechanisms underlying the development of these connections are of considerable interest. Despite extensive in vitro studies, the molecular determinants that ensure the perfect formation of these pathways in vivo remain mostly unknown. Here, we determine the embryonic origin and ontogeny of the mouse mesoprefrontal pathway and use these data to reveal an unexpected requirement for semaphorin 3F (Sema3F) and its receptor neuropilin-2 (Npn-2) during mdDA pathway development using tissue culture approaches and analysis of sema3F(-/-), npn-2(-/-), and npn-2(-/-);TH-Cre mice. We show that Sema3F is a bifunctional guidance cue for mdDA axons, some of which have the remarkable ability to regulate their responsiveness to Sema3F as they develop. During early developmental stages, Sema3F chemorepulsion controls previously uncharacterized aspects of mdDA pathway development through both Npn-2-dependent (axon fasciculation and channeling) and Npn-2-independent (rostral growth) mechanisms. Later on, chemoattraction mediated by Sema3F and Npn-2 is required to orient mdDA axon projections in the cortical plate of the medial prefrontal cortex. This latter finding demonstrates that regulation of axon orientation in the target field occurs by chemoattractive mechanisms, and this is likely to also apply to other neural systems. In all, this study provides a framework for additional dissection of the molecular basis of mdDA pathway development and disease.

Nolz1 expression is required in dopaminergic axon guidance and striatal innervation.

Midbrain dopaminergic (DA) axons make long longitudinal projections towards the striatum. Despite the importance of DA striatal innervation, processes involved in establishment of DA axonal connectivity remain largely unknown. Here we demonstrate a striatal-specific requirement of transcriptional regulator Nolz1 in establishing DA circuitry formation. DA projections are misguided and fail to innervate the striatum in both constitutive and striatal-specific Nolz1 mutant embryos. The lack of striatal Nolz1 expression results in nigral to pallidal lineage conversion of striatal projection neuron subtypes. This lineage switch alters the composition of secreted factors influencing DA axonal tract formation and renders the striatum non-permissive for dopaminergic and other forebrain tracts. Furthermore, transcriptomic analysis of wild-type and Nolz1-/- mutant striatal tissue led to the identification of several secreted factors that underlie the observed guidance defects and proteins that promote DA axonal outgrowth. Together, our data demonstrate the involvement of the striatum in orchestrating dopaminergic circuitry formation.

Remotely Produced and Axon-Derived Netrin-1 Instructs GABAergic Neuron Migration and Dopaminergic Substantia Nigra Development.

The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviors and show select disease vulnerability, including in Parkinson's disease. Despite progress in identifying mDA neuron subtypes, how these neuronal subsets develop and organize into functional brain structures remains poorly understood. Here we generate and use an intersectional genetic platform, Pitx3-ITC, to dissect the mechanisms of substantia nigra (SN) development and implicate the guidance molecule Netrin-1 in the migration and positioning of mDA neuron subtypes in the SN. Unexpectedly, we show that Netrin-1, produced in the forebrain and provided to the midbrain through axon projections, instructs the migration of GABAergic neurons into the ventral SN. This migration is required to confine mDA neurons to the dorsal SN. These data demonstrate that neuron migration can be controlled by remotely produced and axon-derived secreted guidance cues, a principle that is likely to apply more generally.