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Sonic hedgehog (Shh) is a morphogenic protein that operates through the Gli transcription factor-dependent canonical pathway to orchestrate normal development of many tissues. Because aberrant levels of Gli activity lead to a wide spectrum of diseases ranging from neurodevelopmental defects to cancer, understanding the regulatory mechanisms of Shh canonical pathway is paramount. During early stages of spinal cord development, Shh specifies neural progenitors through the canonical signaling. Despite persistence of Shh as spinal cord development progresses, Gli activity is switched off by unknown mechanisms. In this study we find that Shh inverts its action on Gli during development. Strikingly, Shh decreases Gli signaling in the embryonic spinal cord by an electrical activity- and cAMP-dependent protein kinase-mediated pathway. The inhibition of Gli activity by Shh operates at multiple levels. Shh promotes cytosolic over nuclear localization of Gli2, induces Gli2 and Gli3 processing into repressor forms, and activates cAMP-responsive element binding protein that in turn represses gli1 transcription. The regulatory mechanisms identified in this study likely operate with different spatiotemporal resolution and ensure effective down-regulation of the canonical Shh signaling as spinal cord development progresses. The developmentally regulated intercalation of electrical activity in the Shh pathway may represent a paradigm for switching from canonical to noncanonical roles of developmental cues during neuronal differentiation and maturation.

Calsyntenins form a family of linker proteins between distinct populations of vesicles and kinesin motors for axonal transport. They were implicated in synapse formation and synaptic plasticity by findings in worms, mice and humans. These findings were in accordance with the postsynaptic localization of the Calsyntenins in the adult brain. However, they also affect the formation of neural circuits, as loss of Calsyntenin-1 (Clstn1) was shown to interfere with axonal branching and axon guidance. Despite the fact that Calsyntenins were discovered originally in embryonic chicken motoneurons, their distribution in the developing nervous system has not been analyzed in detail so far. Here, we summarize our analysis of the temporal and spatial expression patterns of the cargo-docking proteins Clstn1, Clstn2 and Clstn3 during neural development by comparing the dynamic distribution of their mRNAs by in situ hybridization in the spinal cord, the cerebellum, the retina and the tectum, as well as in the dorsal root ganglia (DRG).

SUMMARY During vertebrate development, spinal neurons differentiate and connect to generate a system that performs sensorimotor functions critical for survival. Spontaneous Ca2+ activity regulates different aspects of spinal neuron differentiation. It is unclear whether environmental factors can modulate this Ca2+ activity in developing spinal neurons to alter their specialization and ultimately adjust sensorimotor behavior to fit the environment. Here, we show that growing Xenopus laevis embryos at cold temperatures results in an increase in the number of spinal motor neurons in larvae. This change in spinal cord development optimizes the escape response to gentle touch of animals raised in and tested at cold temperatures. The cold-sensitive channel TRPM8 increases Ca2+ spike frequency of developing ventral spinal neurons, which in turn regulates expression of the motor neuron master transcription factor HB9. TRPM8 is necessary for the increase in motor neuron number of animals raised in cold temperatures and for their enhanced sensorimotor behavior when tested at cold temperatures. These findings suggest the environment modulates neuronal differentiation to optimize the behavior of the developing organism., Graphical Abstract, In Brief Spencer et al. discover that Xenopus larvae reared in cold temperature are better equipped to escape upon touch at cold temperature relative to warm-grown siblings. This advantage is dependent on the cold-sensitive channel TRPM8, which is necessary for increased Ca2+ spike frequency in embryonic spinal neurons, their differentiation, and survival.

The mechanisms governing the navigation of commissural axons during embryonic development have been extensively investigated in the past years, often using the drosophila ventral nerve cord and the spinal cord as model systems. Similarities but also specificities in the general strategies, the molecular signals as well as in the regulatory pathways controlling the response of commissural axons to the guidance cues have been found between species. Whether the semaphorin signaling contributes to midline crossing in the fly nervous system remains unknown, while in contrast, it does play a prominent contribution in vertebrates. In this review we discuss the functions of the semaphorins during commissural axon guidance in the developing spinal cord, focusing on the family member semaphorin 3B (Sema3B) in the context of midline crossing in the spinal cord.

The Necl/SynCAM subgroup of immunoglobulin superfamily cell adhesion molecules has been implicated in late stages of neural circuit formation. They were shown to be sufficient for synaptogenesis by their trans-synaptic interactions. Additionally, they are involved in myelination, both in the central and the peripheral nervous system, by mediating adhesion between glia cells and axons. Here, we show that Necls/SynCAMs are also required for early stages of neural circuit formation. We demonstrate a role for Necls/SynCAMs in post-crossing commissural axon guidance in the developing spinal cord in vivo. Necl3/SynCAM2, the family member that has not been characterized functionally so far, plays a crucial role in this process. It is expressed by floorplate cells and interacts with Necls/SynCAMs expressed by commissural axons to mediate a turning response in post-crossing commissural axons.

The eutherian X-chromosome specific family of Armcx genes has been described as originating by retrotransposition from Armc10/SVH, a single Arm-containing somatic gene. Armcx3 and Armc10/SVH are characterized by high expression in the central nervous system and they play an important role in the regulation of mitochondrial distribution and transport in neurons. In addition, Armcx/Arm10 genes have several Armadillo repeats in their sequence. In this study we address the potential role of this gene family in neural development by using the chick neural tube as a model. We show that Armc10/SVH is expressed in the chicken spinal cord, and knocking-down Armc10/SVH by sh-RNAi electroporation in spinal cord reduces proliferation of neural precursor cells (NPCs). Moreover, we analyzed the effects of murine Armcx3 and Armc10 overexpression, showing that both proteins regulate progenitor proliferation, while Armcx3 overexpression also specifically controls neural maturation. We show that the phenotypes found following Armcx3 overexpression require its mitochondrial localization, suggesting a novel link between mitochondrial dynamics and regulation of neural development. Furthermore, we found that both Armcx3 and Armc10 may act as inhibitors of Wnt-β-catenin signaling. Our results highlight both common and differential functions of Armcx/Armc10 genes in neural development in the spinal cord., This work was supported by grants from Spanish MINECO (SAF2013-42445R), CIBERNED (ISCIII) and La Marató de TV3 Foundation to ES; Spanish MINECO (BFU2010-21507) to FU; BFU2013-46477-P to EM

AIM: To demonstrate the effect of glatiramer acetate (GA) in chick embryos on neural tube (NT) development, and to explore its effects of FOXP1, apoptosis, and N-cadherin. MATERIAL and METHODS: One hundred fertile, specific pathogen free eggs were divided into 5 groups for this study. The eggshell was windowed specifically at 24 hours of incubation. The embryos in Group 1 (n=20) were treated with 10 mu l physiological saline; in Group 2 the embryos (n=20) were given 10 mu l GA (equal to daily human therapeutic dose); 20 mu l GA (equal to twice daily human therapeutic dose) was injected to embryos in Group 3 (n=20); in Group 4 and 5, 30 mu l and 40 mu l GA were administered to the embryos (n=20) (equal to x3 and x4 daily human therapeutic dose, respectively). Each egg was re-incubated for 24 hours more. Then, histological and immunohistochemical evaluation of the subjects were done. RESULTS: The embryos with NT defect showed FOXP1 expression without N-cadherin or staining with N-cadherin in another location in our study. We interpreted this result as GA leading to an NT closure defect by increasing FOXP expression. Moreover, we also showed the reverse relation between FOXP1 and N-cadherin at the immunohistochemical level for the first time. CONCLUSION: GA affects the spinal cord development through FOXP in the chick embryo model at high doses.

The basic helix-loop-helix transcription factor Neurogenin2 (NGN2) is expressed in distinct populations of neural progenitor cells within the developing central and peripheral nervous systems. Transgenic mice containing ngn2/lacZ reporter constructs were used to study the regulation of ngn2 in the developing spinal cord. ngn2/lacZ transgenic embryos containing sequence found 5' or 3' to the ngn2 coding region express lacZ in domains that reflect the spatial and temporal expression profile of endogenous ngn2. A 4.4-kb fragment 5' of ngn2 was sufficient to drive lacZ expression in the ventral neural tube, whereas a 1.0-kb fragment located 3' of ngn2 directed expression to both dorsal and ventral domains. Persistent -gal activity revealed that the NGN2 progenitor cells in the dorsal domain give rise to a subset of interneurons that send their axons to the floor plate, and the NGN2 progenitors in the ventral domain give rise to a subset of motor neurons. We identified a discrete element that is required for the activity of the ngn2 enhancer specifically in the ventral neural tube. Thus, separable regulatory elements that direct ngn2 expression to distinct neural progenitor populations have been defined.

Background Axons navigate to their future synaptic targets with the help of choice points, intermediate targets that express axon guidance cues. Once they reach a choice point, axons need to switch their response from attraction to repulsion in order to move on with the next stage of their journey. The mechanisms underlying the change in axonal responsiveness are poorly understood. Commissural axons become sensitive to the repulsive activity of Slits when they cross the ventral midline of the CNS. Responsiveness to Slits depends on surface expression of Robo receptors. In Drosophila, Commissureless (Comm) plays a crucial regulatory role in midline crossing by keeping Robo levels low on precommissural axons. Interestingly, to date no vertebrate homolog of comm has been identified. Robo3/Rig1 has been shown to control Slit sensitivity before the midline, but without affecting Robo1 surface expression. Results We had identified RabGDI, a gene linked to human mental retardation and an essential component of the vesicle fusion machinery, in a screen for differentially expressed floor-plate genes. Downregulation of RabGDI by in ovo RNAi caused commissural axons to stall in the floor plate, phenocopying the effect observed after downregulation of Robo1. Conversely, premature expression of RabGDI prevented commissural axons from entering the floor plate. Furthermore, RabGDI triggered Robo1 surface expression in cultured commissural neurons. Taken together, our results identify RabGDI as a component of the switching mechanism that is required for commissural axons to change their response from attraction to repulsion at the intermediate target. Conclusion RabGDI takes over the functional role of fly Comm by regulating the surface expression of Robo1 on commissural axons in vertebrates. This in turn allows commissural axons to switch from attraction to repulsion at the midline of the spinal cord.

At spinal levels, sensory information pertaining to body positioning (proprioception) is relayed to the cerebellum by the spinocerebellar tracts (SCTs). In the past we revealed the basic helix-loop-helix transcription factor Atoh1 (Math1) to be important for establishing Dorsal Progenitor 1 (DP1) commissural interneurons, which comprise a subset of proprioceptive interneurons. Given there exists multiple subdivisions of the SCT we asked whether Atoh1 may also play a role in specifying other cell types in the spinal cord. Here, we reveal the generation of at least three DP1 derived interneuron populations that reside at spatially restricted positions along the rostral–caudal axis. Each of these cell populations expresses distinct markers and anatomically coincides with the cell bodies of the various subdivisions of the SCT. In addition, we found that as development proceeds (e.g. by E13.5) Atoh1 expression becomes apparent in the dorsal midline in the region of the roof plate (RP). Interestingly, we find that cells derived from Atoh1 expressing RP progenitors express SSEA-1, and in the absence of Atoh1 these progenitors become SOX9 positive. Altogether we reveal the existence of multiple Atoh1 dependent cell types in the spinal cord, and uncover a novel progenitor domain that arises late in development.

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component β-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.

We investigated the expression of ERG channels in immature spinal interneurons, using organotypic embryonic cultures of mouse spinal cord after 1 and 2 weeks of development in vitro. We report here that all the genes of the erg family known so far (erg1a,erg1b, erg2, erg3) are expressed in embryonic spinal cultures. We demonstrate for the first time that three ERG proteins (ERG1A, ERG2 and ERG3) are co-expressed in the same neuronal population, and display a spatio-temporal distribution in the spinal slices. ERG immuno-positive cells, representing mainly GABAergic interneurons, were present in large numbers at early stages of development, while declining later, with a ventral to dorsal gradient. Patch clamp recordings confirmed these data, showing that ventral interneurons expressed functional ERG currents only transiently. Similar expression of the erg genes was observed at comparable ages in vivo. The role of ERG currents in regulating neuronal excitability during the earliest phases of spinal circuitry development will be examined in future studies.

Organotypic cocultures of dorsal root ganglia and spinal cord from embryonic rats provides direct access to spinal interneurons in a culture system in which the cytoarchitectural organization of the spinal cord slice is maintained. This preparation was used to investigate the possible induction of rhythmic behaviour at different times of development in vitro. Spontaneous rhythmic bursts induced by coapplication of strychnine (1 microM) and bicuculline (20 microM) were observed with patch-clamp recordings from ventral interneurons. Ventral horn interneurons consistently developed a very regular pattern of activity which was superimposed on a background of sustained synaptic activity. The pattern of the spontaneous bursting following application of strychnine and bicuculline showed a developmentally regulated difference in frequency between two distinct stages of in vitro development.