Molecular design of hypothalamus development.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.

Vascular-Derived Vegfa Promotes Cortical Interneuron Migration and Proximity to the Vasculature in the Developing Forebrain.

Vascular endothelial growth factor (Vegfa) is essential for promoting the vascularization of the embryonic murine forebrain. In addition, it directly influences neural development, although its role in the forming forebrain is less well elucidated. It was recently suggested that Vegfa may influence the development of GABAergic interneurons, inhibitory cells with crucial signaling roles in cortical neuronal circuits. However, the mechanism by which it affects interneuron development remains unknown. Here we investigated the developmental processes by which Vegfa may influence cortical interneuron development by analyzing transgenic mice that ubiquitously express the Vegfa120 isoform to perturb its signaling gradient. We found that interneurons reach the dorsal cortex at mid phases of corticogenesis despite an aberrant vascular network. Instead, endothelial ablation of Vegfa alters cortical interneuron numbers, their intracortical distribution and spatial proximity to blood vessels. We show for the first time that vascular-secreted guidance factors promote early-migrating interneurons in the intact forebrain in vivo and identify a novel role for vascular-Vegfa in this process.

Cdk5 phosphorylation of ErbB4 is required for tangential migration of cortical interneurons.

Interneuron dysfunction in humans is often associated with neurological and psychiatric disorders, such as epilepsy, schizophrenia, and autism. Some of these disorders are believed to emerge during brain formation, at the time of interneuron specification, migration, and synapse formation. Here, using a mouse model and a host of histological and molecular biological techniques, we report that the signaling molecule cyclin-dependent kinase 5 (Cdk5), and its activator p35, control the tangential migration of interneurons toward and within the cerebral cortex by modulating the critical neurodevelopmental signaling pathway, ErbB4/phosphatidylinositol 3-kinase, that has been repeatedly linked to schizophrenia. This finding identifies Cdk5 as a crucial signaling factor in cortical interneuron development in mammals.

Robo1 modulates proliferation and neurogenesis in the developing neocortex.

The elaborate cytoarchitecture of the mammalian neocortex requires the timely production of its constituent pyramidal neurons and interneurons and their disposition in appropriate layers. Numerous chemotropic factors present in the forebrain throughout cortical development play important roles in the orchestration of these events. The Roundabout (Robo) family of receptors and their ligands, the Slit proteins, are expressed in the developing forebrain, and are known to play important roles in the generation and migration of cortical interneurons. However, few studies have investigated their function(s) in the development of pyramidal cells. Here, we observed expression of Robo1 and Slit genes (Slit1, Slit2) in cells lining the telencephalic ventricles, and found significant increases in progenitor cells (basal and apical) at embryonic day (E)12.5 and E14.5 in the developing cortex of Robo1(-/-), Slit1(-/-), and Slit1(-/-)/Slit2(-/-), but not in mice lacking the other Robo or Slit genes. Using layer-specific markers, we found that both early- and late-born pyramidal neuron populations were significantly increased in the cortices of Robo1(-/-) mice at the end of corticogenesis (E18.5). The excess number of cortical pyramidal neurons generated prenatally appears to die in early postnatal life. The observed increase in pyramidal neurons was due to prolonged proliferative activity of their progenitors and not due to changes in cell cycle events. This finding, confirmed by in utero electroporation with Robo1 short hairpin RNA (shRNA) or control constructs into progenitors along the ventricular zone as well as in dissociated cortical cell cultures, points to a novel role for Robo1 in regulating the proliferation and generation of pyramidal neurons.

Slit2 and Robo3 modulate the migration of GnRH-secreting neurons.

Gonadotropin-releasing hormone (GnRH) neurons are born in the nasal placode and migrate along olfactory and vomeronasal axons to reach the forebrain and settle in the hypothalamus, where they control reproduction. The molecular cues that guide their migration have not been fully identified, but are thought to control either cell movement directly or the patterning of their axonal substrates. Using genetically altered mouse models we show that the migration of GnRH neurons is directly modulated by Slit2 and Robo3, members of the axon guidance Slit ligand and Robo receptor families. Mice lacking Slit2 or Robo3 have a reduced number of GnRH neurons in the forebrain, but a normal complement of their supporting axons, pointing to a direct role for these molecules in GnRH neuron migration.

Slit-roundabout signaling regulates the development of the cardiac systemic venous return and pericardium.

RATIONALE: The Slit-Roundabout (Robo) signaling pathway has pleiotropic functions during Drosophila heart development. However, its role in mammalian heart development is largely unknown. OBJECTIVE: To analyze the role of Slit-Robo signaling in the formation of the pericardium and the systemic venous return in the murine heart. METHODS AND RESULTS: Expression of genes encoding Robo1 and Robo2 receptors and their ligands Slit2 and Slit3 was found in or around the systemic venous return and pericardium during development. Analysis of embryos lacking Robo1 revealed partial absence of the pericardium, whereas Robo1/2 double mutants additionally showed severely reduced sinus horn myocardium, hypoplastic caval veins, and a persistent left inferior caval vein. Mice lacking Slit3 recapitulated the defects in the myocardialization, alignment, and morphology of the caval veins. Ligand binding assays confirmed Slit3 as the preferred ligand for the Robo1 receptor, whereas Slit2 showed preference for Robo2. Sinus node development was mostly unaffected in all mutants. In addition, we show absence of cross-regulation with previously identified regulators Tbx18 and Wt1. We provide evidence that pericardial defects are created by abnormal localization of the caval veins combined with ectopic pericardial cavity formation. Local increase in neural crest cell death and impaired neural crest adhesive and migratory properties underlie the ectopic pericardium formation. CONCLUSIONS: A novel Slit-Robo signaling pathway is involved in the development of the pericardium, the sinus horn myocardium, and the alignment of the caval veins. Reduced Slit3 binding in the absence of Robo1, causing impaired cardiac neural crest survival, adhesion, and migration, underlies the pericardial defects.

CXC chemokine receptor 7 (CXCR7) affects the migration of GnRH neurons by regulating CXCL12 availability.

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells, located in the hypothalamus, that play an essential role in mammalian reproduction. These neurons originate in the nasal placode and migrate during embryonic development, in association with olfactory/vomeronasal nerves, first in the nose, then through the cribriform plate to enter the forebrain, before settling in the hypothalamus. One of the molecules required for their early migration in the nose is the chemokine CXCL12, which is expressed in the embryonic nasal mesenchyme in an increasing ventral to dorsal gradient, presumably guiding GnRH neurons toward the forebrain. Mice lacking CXCR4, the receptor for CXCL12, exhibit defective GnRH cell movement and a significant reduction in their number, suggesting that CXCL12/CXCR4 signaling is important in the migration and survival of these neurons. Here, we investigated the role of the more recently identified second CXCL12 receptor, CXCR7, in GnRH neuron development. We demonstrate that CXCR7 is expressed along the migratory path of GnRH neurons in the nasal cavity and, although not expressed by GnRH neurons, it affects their migration as indicated by the ectopic accumulation of these cells in the nasal compartment in CXCR7(-/-) mice. Absence of CXCR7 caused abnormal accumulation of CXCL12-RFP at CXCR4-positive sites in the nasal area of CXCL12-RFP-transgenic mice and excessive CXCL12-dependent intracellular clustering of CXCR4 in GnRH neurons, suggesting internalization. These findings imply that CXCR7 regulates CXCL12 availability by acting as a scavenger along the migratory path of GnRH neurons and, thus, influences the migration of these cells in a noncell-autonomous manner.

VEGF signalling controls GnRH neuron survival via NRP1 independently of KDR and blood vessels.

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. We previously reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, we show here that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. We also demonstrate that survival signalling relies on neuronal, but not endothelial, NRP1 expression and that it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, we show that the VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system.

Robo1 regulates the migration and laminar distribution of upper-layer pyramidal neurons of the cerebral cortex.

Laminar organization is a key feature of the mammalian cerebral cortex, but the mechanisms by which final positioning and "inside-out" distribution of neurons are determined remain largely unknown. Here, we demonstrate that Robo1, a member of the family of Roundabout receptors, regulates the correct positioning of layers II/III pyramidal neurons in the neocortex. Specifically, we used RNA interference in mice to suppress the expression of Robo1 in a subset of layers II/III neurons, and observed the positions of these cells at distinct developmental stages. In contrast to control neurons that migrated toward the pial surface by P1, Robo1-suppressed neurons exhibited a delay in entering the cortical plate at respective stages. Unexpectedly, after the first postnatal week, these neurons were predominantly located in the upper part of layers II/III, in contrast to control cells that were distributed throughout these layers. Sequential electroporation studies revealed that Robo1-suppressed cells failed to establish the characteristic inside-out neuronal distribution and, instead, they accumulated beneath the marginal zone regardless of their birthdate. These results demonstrate that Robo receptors play a crucial role in neocortical lamination and particularly in the positioning of layers II/III pyramidal neurons.

Early B-cell factors 2 and 3 (EBF2/3) regulate early migration of Cajal-Retzius cells from the cortical hem.

Cajal-Retzius (CR) cells play a crucial role in the formation of the cerebral cortex, yet the molecules that control their development are largely unknown. Here, we show that Ebf transcription factors are expressed in forebrain signalling centres-the septum, cortical hem and the pallial-subpallial boundary-known to generate CR cells. We identified Ebf2, through fate mapping studies, as a novel marker for cortical hem- and septum-derived CR cells. Loss of Ebf2 in vivo causes a transient decrease in CR cell numbers on the cortical surface due to a migratory defect in the cortical hem, and is accompanied by upregulation of Ebf3 in this and other forebrain territories that produce CR cells, without affecting proper cortical lamination. Accordingly, using in vitro preparations, we demonstrated that both Ebf2 and Ebf3, singly or together, control the migration of CR cells arising in the cortical hem. These findings provide evidence that Ebfs directly regulate CR cell development.

Defective gonadotropin-releasing hormone neuron migration in mice lacking SEMA3A signalling through NRP1 and NRP2: implications for the aetiology of hypogonadotropic hypogonadism.

Kallmann syndrome (KS) is a genetic disease characterized by hypogonadotropic hypogonadism and impaired sense of smell. The genetic causes underlying this syndrome are still largely unknown, but are thought to be due to a developmental defect in the migration of gonadotropin-releasing hormone (GnRH) neurons. Understanding the causes of the disease is hampered by lack of appropriate mouse models. GnRH neurons are hypothalamic cells that centrally control reproduction in mammals by secreting the GnRH decapeptide into the portal blood vessels of the pituitary to stimulate the production of gonadotropins. During development, these cells are born in the nasal placode outside the brain and migrate in association with olfactory/vomeronasal axons to reach the forebrain and position themselves in the hypothalamus. By combining the analysis of genetically altered mice with in vitro models, we demonstrate here that a secreted guidance cue of the class 3 semaphorin family, SEMA3A, is essential for the development of the GnRH neuron system: loss of SEMA3A signalling alters the targeting of vomeronasal nerves and the migration of GnRH neurons into the brain, resulting in reduced gonadal size. We found that SEMA3A signals redundantly through both its classical receptors neuropilin (NRP) 1 and, unconventionally, NRP2, while the usual NRP2 ligand SEMA3F is dispensable for this process. Strikingly, mice lacking SEMA3A or semaphorin signalling through both NRP1 and NRP2 recapitulate the anatomical features of a single case of KS analysed so far, and may therefore be used as genetic models to elucidate the pathogenesis of KS.

Robo1 regulates semaphorin signaling to guide the migration of cortical interneurons through the ventral forebrain.

Cortical interneurons, generated predominantly in the medial ganglionic eminence, migrate around and avoid the developing striatum in the subpallium en route to the cortex. This is attributable to the chemorepulsive cues of class 3 semaphorins expressed in the striatal mantle and acting through neuropilin (Nrp1 and Nrp2) receptors expressed in these cells. Cortical interneurons also express Robo receptors, and we show here that in mice lacking Robo1, but not Robo2, these cells migrate aberrantly through the striatum. In vitro experiments demonstrated that interneurons lacking Robo1 function are significantly less responsive to the effects of semaphorins. Failure to respond to semaphorin appears to be attributable to a reduction in Nrp1 and PlexinA1 receptors within these cells. Biochemical studies further demonstrated that Robo1 binds directly to Nrp1, but not to semaphorins, and this interaction is mediated by a region contained within its first two Ig domains. Thus, we show for the first time that Robo1 interacts with Nrp1 to modulate semaphorin signaling in the developing forebrain and direct the migration of interneurons through the subpallium and into the cortex.

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3.

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34⁺ HSPCs. Human CD34⁺ HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34⁺ HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34⁺ HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34⁺ HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34⁺ HSPCs.

Connexin 43 mediates the tangential to radial migratory switch in ventrally derived cortical interneurons.

The adult cerebral cortex is composed of excitatory and inhibitory neurons that arise from progenitor cells in disparate proliferative regions in the developing brain and follow different migratory paths. Excitatory pyramidal neurons originate near the ventricle and migrate radially to their position in the cortical plate along radial glial fibers. On the other hand, inhibitory interneurons arise in the ventral telencephalon and migrate tangentially to enter the developing cortex before migrating radially to reach their correct laminar position. Gap junction adhesion has been shown to play an important mechanistic role in the radial migration of excitatory neurons. We asked whether a similar mechanism governs the tangential or radial migration of inhibitory interneurons. Using short hairpin RNA knockdown of Connexin 43 (Cx43) and Cx26 together with rescue experiments, we found that gap junctions are dispensable for the tangential migration of interneurons, but that Cx43 plays a role in the switch from tangential to radial migration that allows interneurons to enter the cortical plate and find their correct laminar position. Moreover this action is dependent on the adhesive properties and the C terminus of Cx43 but not the Cx43 channel. Thus, the radial phase of interneuron migration resembles that of excitatory neuron migration in terms of dependence on Cx43 adhesion. Furthermore, gap junctions between migrating interneurons and radial processes were observed by electron microscopy. These findings provide mechanistic and structural support for a gap junction-mediated interaction between migrating interneurons and radial glia during the switch from tangential to radial migration.

Differential gene expression in migratory streams of cortical interneurons.

Cortical interneurons originate in the ganglionic eminences of the subpallium and migrate into the cortex in well-defined tangential streams. At the start of corticogenesis, two streams of migrating neurons are evident: a superficial one at the level of the preplate (PPL), and a deeper one at the level of the intermediate zone (IZ). Currently, little is known about the signalling mechanisms that regulate interneuron migration, and almost nothing is known about the molecules that may be involved in their choice of migratory stream. Here, we performed a microarray analysis, comparing the changes in gene expression between cells migrating in the PPL and those migrating in the IZ at embryonic day 13.5. This analysis identified genes, many of them novel, that were upregulated in one of the two streams. Moreover, polymerase chain reaction, in situ hybridization experiments and immunohistochemistry showed the expression of these genes in interneurons migrating within the PPL or IZ, suggesting that they play a role in their migration and choice of stream.

Robo2 is required for Slit-mediated intraretinal axon guidance.

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.

Cortical interneurons require p35/Cdk5 for their migration and laminar organization.

Projection neurons and interneurons populate the cerebral cortex in a layer-specific manner. Here, we studied the role of Cyclin-dependent kinase 5 (Cdk5) and its activator p35 in cortical interneuron migration and disposition in the cortex. We found that mice lacking p35 (p35(-/-)) show accumulation of interneurons in the upper part of the cortex. We also observed an inverted distribution of both early- and late-born interneurons, with the former showing a preference for the upper and the latter for the lower aspects of the cortex. We investigated the causes of the altered laminar organization of interneurons in p35(-/-) mice and found a cell-autonomous delay in their tangential migration that may prevent them from reaching correct positions. Incomplete splitting of the preplate in p35(-/-) mice, which causes accumulation of cells in the superficial layer and defects in the "inward" and "outward" components of their radial movement, may also account for the altered final arrangement of interneurons. We, therefore, propose that p35/Cdk5 plays a key role in guiding cortical interneurons to their final positions in the cortex.

The role of Robo3 in the development of cortical interneurons.

A number of studies in recent years have shown that members of the Roundabout (Robo) receptor family, Robo1 and Robo2, play significant roles in the formation of axonal tracks in the developing forebrain and in the migration and morphological differentiation of cortical interneurons. Here, we investigated the expression and function of Robo3 in the developing cortex. We found that this receptor is strongly expressed in the preplate layer and cortical hem of the early cortex where it colocalizes with markers of Cajal-Retzius cells and interneurons. Analysis of Robo3 mutant mice at early (embryonic day [E] 13.5) and late (E18.5) stages of corticogenesis revealed no significant change in the number of interneurons, but a change in their morphology at E13.5. However, preliminary analysis on a small number of mice that lacked all 3 Robo receptors indicated a marked reduction in the number of cortical interneurons, but only a limited effect on their morphology. These observations and the results of other recent studies suggest a complex interplay between the 3 Robo receptors in regulating the number, migration and morphological differentiation of cortical interneurons.

Cell-autonomous roles of ARX in cell proliferation and neuronal migration during corticogenesis.

The aristaless-related homeobox (ARX) gene has been implicated in a wide spectrum of disorders ranging from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of X-linked mental retardation without apparent brain abnormalities. To better understand its role in corticogenesis, we used in utero electroporation to knock down or overexpress ARX. We show here that targeted inhibition of ARX causes cortical progenitor cells to exit the cell cycle prematurely and impairs their migration toward the cortical plate. In contrast, ARX overexpression increases the length of the cell cycle. In addition, we report that RNA interference-mediated inactivation of ARX prevents cells from acquiring multipolar morphology in the subventricular and intermediate zones, resulting in decreased neuronal motility. In contrast, ARX overexpression appears to promote the development of tangentially oriented processes of cells in the subventricular and intermediate zones and affects radial migration of pyramidal neurons. We also demonstrate that the level of ARX expression is important for tangential migration of GABA-containing interneurons, because both inactivation and overexpression of the gene impair their migration from the ganglionic eminence. However, our data suggest that ARX is not directly involved in GABAergic cell fate specification. Overall, these results identify multiple and distinct cell-autonomous roles for ARX in corticogenesis.

The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons.

Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1(-/-), but not Robo2(-/-), animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1(-/-) mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1(-/-)/Slit2(-/-) mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1(-/-), Robo2(-/-) and Slit1(-/-)/Slit2(-/-), but not in Slit1(-/-) mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons.