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.

Developmental guidance of the retroflex tract at its bending point involves Robo1-Slit2-mediated floor plate repulsion.

The retroflex tract contains medial habenula efferents that target the hindbrain interpeduncular complex and surrounding areas. This tract displays a singular course. Initially, habenular axons extend ventralwards in front of the pretectum until they reach the basal plate. Next, they avoid crossing the local floor plate, sharply changing course caudalwards (the retroflexion alluded by the tract name) and navigate strictly antero-posteriorly across basal pretectum, midbrain and isthmus. Once they reach rhombomere 1, the habenular axons criss-cross the floor plate several times within the interpeduncular nuclear complex as they innervate it. Here we described the timing and details of growth phenomena as these axons navigate to their target. The first dorsoventral course apparently obeys Ntn1 attraction. We checked the role of local floor plate signaling in the decision to avoid the thalamic floor plate and bend caudalwards. Analyzing the altered floor and basal plates of Gli2 knockout mice, we found a contralateral projection of most habenular axons, plus ulterior bizarre navigation rostralwards. This crossing phenotype was due to a reduced expression of Slit repulsive cues, suggesting involvement of the floor-derived Robo-Slit system in the normal guidance of this tract. Using Slit and Robo mutant mice, open neural tube and co-culture assays, we determined that Robo1-Slit2 interaction is specifically required for impeding that medial habenular axons cross the thalamic floor plate. This pathfinding mechanism is essential to establish the functionally important habenulo-interpeduncular connection.

Down-regulation of Slit-Robo pathway mediating neuronal cytoskeletal remodeling processes facilitates the antidepressive-like activity of Gastrodia elata Blume.

Nowadays, depression is a serious psychological disorder that causes extreme economic loss and social problems. Previously, we discovered that the water extract of Gastrodia elata Blume (WGE) improved depressive-like behavior by influencing neurotransmitters in rats subjected to the forced swimming test. To elucidate possible mechanisms, in the present study, we performed a proteomics and bioinformatics analysis to identify the related pathways. Western blot-validated results indicated that the core protein network modulated by WGE administration was closely associated with down-regulation of the Slit-Robo pathway, which modulates neuronal cytoskeletal remodeling processes. Although Slit-Robo signaling has been well investigated in neuronal development, its relationship with depression is not fully understood. We provide a potential hint on the mechanism responsible for the antidepressive-like activity of WGE. In conclusion, we suggest that the Slit-Robo pathway and neuronal cytoskeleton remodeling are possibly one of the pathways associated with the antidepressive-like effects of WGE.

FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons.

BACKGROUND: Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown. RESULTS: Here, we show that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence of Slit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance in vivo. CONCLUSIONS: These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain.

[Effect of electroacupuncture of "Neiguan" (PC 6) and "Zusanli" (ST 36) on expression of cerebral cortical slit 2/Robo 1 in the focal cerebral infarction rats ].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on expression of Slit 2 and its transmembrane receptor Robo 1 in the cortex tissue of cerebral infarction rats so as to study its mechanism underlying improvement of cerebral ischemia. METHODS: Ninety male Sprague Dawley rats were randomly divided into control group (n = 10), model group (n = 40) and EA group (n = 40), and the latter two groups were further randomized into four subgroups: 1 d, 3 d, 7 d, and 14 d (n = 10 in each subgroup) according to the cerebral ischemia duration. Cerebral infarction model was established by occlusion of the middle cerebral artery (MCAO). EA (80-100 Hz, 1-3 mA) was applied to bilateral "Neiguan" (PC 6) and "Zusanli" (ST 36) for 30 min, once daily for 1 d, 3 d, 7 d, and 14 d, respectively. The animals' neurological defect was assessed using Zea-Longa scoring. The expression of Slit 2 and Robo 1 proteins in the cerebral cortex on the ischemic side was assayed using immunohistochemistry and Western blotting, respectively. RESULTS: In comparison with the control group, the neurological score was significantly higher in the model group (P < 0.05), and reduced considerably on day 7 and 14 after MCAO in the EA group compared with the model group (P < 0.05). Immunohistochemical results showed that in comparison with the control group, the immunoactivity levels of cerebral Slit 2 and Robo 1 were remarkably upregulated on day 1, 3 and 7 after MCAO in the model group (P < 0.05, P < 0.01), and backed to the control levels on day 14 (P > 0.05). While compared with the model group, the immunoactivity levels of cerebral Slit 2 and Robo 1 were further obviously upregulated on day 1, 3, 7 and 14 after cerebral ischemia in the EA group (P < 0.05, P < 0.01). The results of Western blotting about the expression levels of cerebral Slit 2 and Robo 1 proteins were nearly the same to those of immunohistochemical outcomes in the 4 subgroups apart from that the expression levels of both Slit 2 and Robo 1 proteins were still obviously higher on day 14 after MCAO in the model group (P < 0.01). CONCLUSION: EA intervention can significantly improve cerebral ischemia rats' neurological function and obviously upregulate the expression of cerebral Slit 2 and Robo 1 proteins, which may be one of the mechanisms of EA therapy for relieving cerebral infarction in clinic.

Cooperative slit and netrin signaling in contralateralization of the mouse trigeminothalamic pathway.

Ascending somatosensory pathways are crossed pathways representing each side of the body in the contralateral neocortex. The principal sensory nucleus of the trigeminal nerve (PrV) relays the facial sensations to the contralateral somatosensory cortex via the ventrobasal thalamus. In the companion article (Kivrak and Erzurumlu [2012] J. Comp. Neurol. 12-0013) we described the normal development of the trigeminal lemniscal pathway in the mouse. In this study we investigated the role of midline axon navigation signals, the netrin and slit proteins. In situ hybridization assays revealed that both netrin and slit mRNAs are expressed along the midline facing the PrV axons and their receptors are expressed in developing PrV neurons. In wild-type mouse embryos, PrV axons cross the midline and take a sharp rostral turn heading toward the contralateral thalamus. Examination of trigeminal lemniscal axons in dcc knockout mice revealed absence of midline crossing between E11 and E15. However, a few axons crossed the midline at E17 and reached the contralateral thalamus, resulting in a bilateral PrV lemniscal pathway at P0. We also found that slit1, -2 or -3 single or double knockout mice have impaired development of the trigeminal-lemniscal pathway. These include axon stalling along the midline, running within the midline, and recrossing of axons back to the site of origin. Collectively, our studies indicate a cooperative role for netrin and slit proteins in midline attraction and crossing behavior of the ascending facial somatosensory projections during development.

Gbx2 regulates thalamocortical axon guidance by modifying the LIM and Robo codes.

Combinatorial expression of transcription factors forms transcriptional codes to confer neuronal identities and connectivity. However, how these intrinsic factors orchestrate the spatiotemporal expression of guidance molecules to dictate the responsiveness of axons to guidance cues is less understood. Thalamocortical axons (TCAs) represent the major input to the neocortex and modulate cognitive functions, consciousness and alertness. TCAs travel a long distance and make multiple target choices en route to the cortex. The homeodomain transcription factor Gbx2 is essential for TCA development, as loss of Gbx2 abolishes TCAs in mice. Using a novel TCA-specific reporter, we have discovered that thalamic axons are mostly misrouted to the ventral midbrain and dorsal midline of the diencephalon in Gbx2-deficient mice. Furthermore, conditionally deleting Gbx2 at different embryonic stages has revealed a sustained role of Gbx2 in regulating TCA navigation and targeting. Using explant culture and mosaic analyses, we demonstrate that Gbx2 controls the intrinsic responsiveness of TCAs to guidance cues. The guidance defects of Gbx2-deficient TCAs are associated with abnormal expression of guidance receptors Robo1 and Robo2. Finally, we demonstrate that Gbx2 controls Robo expression by regulating LIM-domain transcription factors through three different mechanisms: Gbx2 and Lhx2 compete for binding to the Lmo3 promoter and exert opposing effects on its transcription; repressing Lmo3 by Gbx2 is essential for Lhx2 activity to induce Robo2; and Gbx2 represses Lhx9 transcription, which in turn induces Robo1. Our findings illustrate the transcriptional control of differential expression of Robo1 and Robo2, which may play an important role in establishing the topography of TCAs.

Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth.

Developing axons must control their growth rate to follow the appropriate pathways and establish specific connections. However, the regulatory mechanisms involved remain elusive. By combining live imaging with transplantation studies in mice, we found that spontaneous calcium activity in the thalamocortical system and the growth rate of thalamocortical axons were developmentally and intrinsically regulated. Indeed, the spontaneous activity of thalamic neurons governed axon growth and extension through the cortex in vivo. This activity-dependent modulation of growth was mediated by transcriptional regulation of Robo1 through an NF-κB binding site. Disruption of either the Robo1 or Slit1 genes accelerated the progression of thalamocortical axons in vivo, and interfering with Robo1 signaling restored normal axon growth in electrically silent neurons. Thus, modifications to spontaneous calcium activity encode a switch in the axon outgrowth program that allows the establishment of specific neuronal connections through the transcriptional regulation of Slit1 and Robo1 signaling.

The lhx2 transcription factor controls thalamocortical axonal guidance by specific regulation of robo1 and robo2 receptors.

The assembly of neural circuits is dependent upon the generation of specific neuronal subtypes, each subtype displaying unique properties that direct the formation of selective connections with appropriate target cells. Actions of transcription factors in neural progenitors and postmitotic cells are key regulators in this process. LIM-homeodomain transcription factors control crucial aspects of neuronal differentiation, including subtype identity and axon guidance. Nonetheless, their regulation during development is poorly understood and the identity of the downstream molecular effectors of their activity remains largely unknown. Here, we demonstrate that the Lhx2 transcription factor is dynamically regulated in distinct pools of thalamic neurons during the development of thalamocortical connectivity in mice. Indeed, overexpression of Lhx2 provokes defective thalamocortical axon guidance in vivo, while specific conditional deletion of Lhx2 in the thalamus produces topographic defects that alter projections from the medial geniculate nucleus and from the caudal ventrobasal nucleus in particular. Moreover, we demonstrate that Lhx2 influences axon guidance and the topographical sorting of axons by regulating the expression of Robo1 and Robo2 guidance receptors, which are essential for these axons to establish correct connections in the cerebral cortex. Finally, augmenting Robo1 function restores normal axon guidance in Lhx2-overexpressing neurons. By regulating axon guidance receptors, such as Robo1 and Robo2, Lhx2 differentially regulates the axon guidance program of distinct populations of thalamic neurons, thus enabling the establishment of specific neural connections.

Human ROBO1 regulates interaural interaction in auditory pathways.

In rodents, the Robo1 gene regulates midline crossing of major nerve tracts, a fundamental property of the mammalian CNS. However, the neurodevelopmental function of the human ROBO1 gene remains unknown, apart from a suggested role in dyslexia. We therefore studied axonal crossing with a functional approach, based on magnetoencephalography, in 10 dyslexic individuals who all share the same rare, weakly expressing haplotype of the ROBO1 gene. Auditory-cortex responses were recorded separately to left- and right-ear sounds that were amplitude modulated at different frequencies. We found impaired interaural interaction that depended on the ROBO1 in a dose-dependent manner. Our results indicate that normal crossing of the auditory pathways requires an adequate ROBO1 expression level.

Emergent growth cone responses to combinations of Slit1 and Netrin 1 in thalamocortical axon topography.

How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.

Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections.

Early events in the axonal tract formation from mammillary bodies remain poorly understood. In the present study, we reported an aberrant pattern of axonal projections from mammillary bodies to the dorsal thalamus in mice lacking the transcription factor Pax6. We found that Netrin-1 was ectopically up-regulated and that both Slit1 and Slit2 were down-regulated in the presumptive dorsal thalamus of Pax6 mutant mice. We then examined the effects of Netrin-1 and Slit2 on the mammillary axons by in utero electroporation techniques. Netrin-1 had an attractive action toward the mammillary axons. Moreover, mammillary trajectories were disorganized in Netrin-1-deficient mice. On the other hand, Slit2 had a repulsive effect on the mammillary axons. These findings suggest that the combination of Netrin and Slit may be involved in proper axonal projection from the mammillary bodies and that their misexpression in the diencephalon may cause the misrouting of these axons in Pax6 mutant mice.

Substantial migration of SVZ cells to the cortex results in the generation of new neurons in the excitotoxically damaged immature rat brain.

Mammalian SVZ progenitors continuously generate new neurons in the olfactory bulb. After injury, changes in SVZ cell number suggest injury-induced migration. Studies that trace the migration of SVZ precursors into neurodegenerating areas are lacking. Previously, we showed a decrease in BrdU+SVZ cells following excitotoxic damage to the immature rat cortex. Here, we demonstrate that NMDA-induced injury forces endogenous Cell Tracker Green (CTG) labeled VZ/SVZ precursors out of the SVZ into the neurodegenerating cortex. CTG+/Nestin+/Filamin A+ precursors are closely associated with vimentin+/GFAP+/GLAST+ filaments and express both chemokine receptor CXCR4 and Robo1. In the cortex, SVZ-derived progenitors show a progressive expression of developing, migrating and mature neurons and glial markers. CTG+/GFAP+ astrocytes greatly outnumber CTG+/MAP2+/NeuN+ neurons. SVZ-derived progenitors differentiate into both tbr1+ cortical glutamatergic neurons and calretinin+ interneurons. But, there is little integration of these neurons into the existing circuitry, as seen by Fluorogold retrograde tracing from the internal capsule.

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.

Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain.

The function of the nervous system depends on the precision of axon wiring during development. Previous studies have demonstrated that Slits, a family of secreted chemorepellent proteins, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 or, even more so, in both Slit1 and Slit2 have defects in multiple axonal pathways, including corticofugal, thalamocortical, and callosal connections. In the spinal cord, members of the Robo family of proteins help mediate the function of Slits, but the relative contribution of these receptors to the guidance of forebrain projections remains to be determined. In the present study, we addressed the function of Robo1 and Robo2 in the guidance of forebrain projections by analyzing Robo1-, Robo2-, and Robo1;Robo2-deficient mice. Mice deficient in Robo2 and, more dramatically, in both Robo1 and Robo2, display prominent axon guidance errors in the development of corticofugal, thalamocortical, and corticocortical callosal connections. Our results demonstrate that Robo1 and Robo2 mostly cooperate to mediate the function of Slit proteins in guiding the major forebrain projections.

Frequent but borderline methylation of p16 (INK4a) and TIMP3 in medulloblastoma and sPNET revealed by quantitative analyses.

Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 (INK4A), p14 (ARF), TIMP3, CDH1, p15 (INK4B )and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 (INK4A) (17/43), p14 (ARF) (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 (INK4A )and TIMP3 were methylated consistently in medulloblastomas (p16 (INK4A ) 14%, TIMP3 11%) and p16 (INK4A) also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1-5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.

Sim1 and Sim2 are required for the correct targeting of mammillary body axons.

The mammillary body (MB), and its axonal projections to the thalamus (mammillothalamic tract, MTT) and the tegmentum (mammillotegmental tract, MTEG), are components of a circuit involved in spatial learning. The bHLH-PAS transcription factors SIM1 and SIM2 are co-expressed in the developing MB. We have found that MB neurons are generated and that they survive at least until E18.5 in embryos lacking both Sim1 and Sim2 (Sim1(-/-);Sim2(-/-)). However, the MTT and MTEG are histologically absent in Sim1(-/-);Sim2(-/-) embryos, and are reduced in embryos lacking Sim1 but bearing one or two copies of Sim2, indicating a contribution of the latter to the development of MB axons. We have generated, by homologous recombination, a null allele of Sim1 (Sim1(tlz)) in which the tau-lacZ fusion gene was introduced, allowing the staining of MB axons. Consistent with the histological studies, lacZ staining showed that the MTT/MTEG is barely detectable in Sim1(tlz/tlz);Sim2(+/-) and Sim1(tlz/tlz);Sim2(-/-) brains. Instead, MB axons are splayed and grow towards the midline. Slit1 and Slit2, which code for secreted molecules that induce the repulsion of ROBO1-producing axons, are expressed in the midline at the level of the MB, whereas Robo1 is expressed in the developing MB. The expression of Rig-1/Robo3, a negative regulator of Slit signalling, is upregulated in the prospective MB of Sim1/Sim2 double mutants, raising the possibility that the growth of mutant MB axons towards the midline is caused by a decreased sensitivity to SLIT. Finally, we found that Sim1 and Sim2 act along compensatory, but not hierarchical, pathways, suggesting that they play similar roles in vivo.

Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor.

Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. We show that Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. We further show that the ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. Our results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels.

Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain.

We report that Slit proteins, a family of secreted chemorepellents, are crucial for the proper development of several major forebrain tracts. Mice deficient in Slit2 and, even more so, mice deficient in both Slit1 and Slit2 show significant axon guidance errors in a variety of pathways, including corticofugal, callosal, and thalamocortical tracts. Analysis of multiple pathways suggests several generalizations regarding the functions of Slit proteins in the brain, which appear to contribute to (1) the maintenance of dorsal position by prevention of axonal growth into ventral regions, (2) the prevention of axonal extension toward and across the midline, and (3) the channeling of axons toward particular regions.