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The nervous system requires metabolites and oxygen supplied by the neurovascular network, but this necessitates close apposition of neurons and endothelial cells. We find motor neurons attract vessels with long-range VEGF signaling, but endothelial cells in the axonal pathway are an obstacle for establishing connections with muscles. It is unclear how this paradoxical interference from heterotypic neurovascular contacts is averted. Through a mouse mutagenesis screen, we show that Plexin-D1 receptor is required in endothelial cells for development of neuromuscular connectivity. Motor neurons release Sema3C to elicit short-range repulsion via Plexin-D1, thus displacing endothelial cells that obstruct axon growth. When this signaling pathway is disrupted, epaxial motor neurons are blocked from reaching their muscle targets and concomitantly vascular patterning in the spinal cord is altered. Thus, an integrative system of opposing push-pull cues ensures detrimental axon-endothelial encounters are avoided while enabling vascularization within the nervous system and along peripheral nerves.

The collier/Olf1/EBF (COE) family genes encode helix–loop–helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B-cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.

Pyramidal neurons in layers 2/3 and 5 of primary somatosensory cortex (S1) exhibit somewhat modest synaptic plasticity after whisker input deprivation. Whether neurons involved at earlier steps of sensory processing show more or less plasticity has not yet been examined. Here, we used longitudinalin vivotwo-photon microscopy to investigate dendritic spine dynamics in apical tufts of GFP-expressing layer 4 (L4) pyramidal neurons of the vibrissal (barrel) S1 after unilateral whisker trimming. First, we characterize the molecular, anatomical, and electrophysiological properties of identified L4 neurons in Ebf2-Cre transgenic mice. Next, we show that input deprivation results in a substantial (∼50%) increase in the rate of dendritic spine loss, acutely (4–8 d) after whisker trimming. This robust synaptic plasticity in L4 suggests that primary thalamic recipient pyramidal neurons in S1 may be particularly sensitive to changes in sensory experience. Ebf2-Cre mice thus provide a useful tool for future assessment of initial steps of sensory processing in S1.

Neurogenesis is a tightly regulated process whose success depends on the ability to balance the expansion/maintenance of an undifferentiated neural progenitor pool with the precisely timed birth of sequential generations of neurons. TheZfp423gene encodes a 30-Zn-finger transcription factor (TF) that acts as a scaffold in the assembly of complex transcriptional and cellular machineries regulating neural development. While null mutants forZfp423feature a severe cerebellar hypoplasia, the underlying mechanism is only partially characterized. Mutations of the human orthologZNF423have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), associated with other signs of classical ciliopathy outside the central nervous system (CNS). ZNF423 also plays a role in the DNA damage response (DDR). To further characterize the role of ZFP423 in cerebellar neurogenesis, with a focus on Purkinje cells (PC) development, we analyzed two previously undescribed mutant mouse lines carrying allelic in-frame deletions of the corresponding gene, selectively affecting two functionally characterized protein-protein interaction domains, affecting zinc (Zn) fingers 9-20 or 28-30. Some phenotypic defects are allele specific:Zfp423Δ9-20/Δ9-20mutants exhibit a depletion of the OLIG2+ PC progenitor pool in the cerebellar ventricular zone (VZ). In these mutants, M-phase progenitors display changes in spindle orientation indicative of a precocious switch from symmetric to asymmetric cell division. Conversely, theZfp423Δ28-30/Δ28-30primordium displays a sharp decrease in the expression of PC differentiation markers, including CORL2, despite an abundance of cycling PC progenitors. Moreover, and importantly, in both mutants VZ progenitor cell cycle progression is remarkably affected, and factors involved in the DDR are substantially upregulated in the VZ and in postmitotic precursors alike. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time supports the emerging notion that an impaired DNA damage response may be a key factor in the pathogenesis of JS and other ciliopathies.

Summary The rich functional diversity of the nervous system is founded in the specific connectivity of the underlying neural circuitry. Neurons are often preprogrammed to respond to multiple axon guidance signals because they use sequential guideposts along their pathways, but this necessitates a strict spatiotemporal regulation of intracellular signaling to ensure the cues are detected in the correct order. We performed a mouse mutagenesis screen and identified the Rho GTPase antagonist p190RhoGAP as a critical regulator of motor axon guidance. Rather than acting as a compulsory signal relay, p190RhoGAP uses a non-conventional GAP-independent mode to transiently suppress attraction to Netrin-1 while motor axons exit the spinal cord. Once in the periphery, a subset of axons requires p190RhoGAP-mediated inhibition of Rho signaling to target specific muscles. Thus, the multifunctional activity of p190RhoGAP emerges from its modular design. Our findings reveal a cell-intrinsic gate that filters conflicting signals, establishing temporal windows of signal detection.

The Zfp423/ZNF423 gene encodes a 30-Zn-finger transcription factor involved in key developmental pathways. While null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations have been associated to Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA damage response, raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other one impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DNA-damage response markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DNA damage response may be a key factor in the pathogenesis of JS and other ciliopathies. The Zfp423/ZNF423 gene encodes a 30-Zn-finger transcription factor involved in key developmental pathways. While null Zfp423 mutants develop cerebellar malformations, the underlying mechanism remains unknown. ZNF423 mutations have been associated to Joubert Syndrome, a ciliopathy causing cerebellar vermis hypoplasia and ataxia. ZNF423 participates in the DNA damage response, raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. To characterize in vivo the function of ZFP423 in neurogenesis, we analyzed allelic murine mutants in which distinct functional domains are deleted. One deletion impairs mitotic spindle orientation, leading to premature cell cycle exit and Purkinje cell (PC) progenitor pool deletion. The other one impairs PC differentiation. In both mutants, cell cycle progression is remarkably delayed and DNA-damage response markers are upregulated in cerebellar ventricular zone progenitors. Our in vivo evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DNA damage response may be a key factor in the pathogenesis of JS and other ciliopathies.

By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.

We describe the use of DNA transposons as tools for carrying out functional screenings in murine embryonic stem (ES) cell-derived neural stem (NS) cells. NS cells are a new type of stem cells featuring radial glial properties, that undergoes symmetric cell division for an indefinite number of passages, expanding as a monolayer. In this model, the previously unreported Sleeping Beauty transposase M3A achieves an optimal blend of clone generation efficiency and low redundancy of integrations per clone, compared to the SB100X Sleeping Beauty variant and to the piggyBac transposon. The technology described here makes it possible to randomly trap genes in the NS cell genome and modify their expression or tag them with fluorescent markers and selectable genes, allowing recombinant cells to be isolated and expanded clonally. This approach will facilitate the identification of novel determinants of stem cell biology and neural cell fate specification in NS cells.

Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons of the granular layer. Previous studies have identified three distinct UBC subsets in the mouse cerebellar cortex: one expressing the calcium-binding protein calretinin (CR), a second expressing both the metabotropic glutamate receptor (mGluR)1alpha and phospholipase C(PLC)beta4, and a third expressing PLCbeta4 but not mGluR1alpha. We have investigated UBC topography in two strains of mutant mice: early B-cell factor 2 (Ebf2) null and scrambler. In Ebf2 null mice Purkinje cell topography is disrupted due to Purkinje cell death and ectopic gene expression. The topography of all three classes of UBCs is also abnormal: the CR(+) UBCs, which are normally aligned with zebrin II stripes, become homogeneously distributed; the numerical density of mGluRlalpha(+) UBCs is increased; and many PLCbeta4(+) UBCs are located ectopically. The UBC ectopia is not a cell-intrinsic action of the Ebf2 gene-analysis of the constitutive expression of a beta-galactoside reporter under the control of the Ebf2 promoter reveals no Ebf2 expression in UBCs at any stage of cerebellar development. In scrambler (Dab1(scm)), most Purkinje cells are ectopic but nevertheless have normal adult gene expression patterns. In scrambler, UBCs associate with specific ectopic Purkinje cell clusters. Finally, similar associations with specific Purkinje cell clusters are seen during normal cerebellar development. These data suggest that UBCs become regionally restricted during development through a non-cell-autonomous mechanism involving embryonic interactions with different Purkinje cell subtypes.

Improved and modular tools are needed for the neuroanatomical dissection of CNS axonal tracts, and to study the cell-intrinsic and cell-extrinsic cues that govern their assembly and plasticity. Here we describe a general purpose transgenic tracer that can be used to visualize axonal tracts and synaptic terminals in any region of the embryonic neural tube or postnatal CNS, on any wild type or mutant genetic background. The construct permits CRE-inducible expression of a dicistronic axonal marker encoding two surface reporter proteins: a farnesylated GFP and the human Placental Alkaline Phosphatase (PLAP). Both proteins localize alongside the neuronal surface, permitting the concomitant detection of cell body, neurites, and presynaptic and postsynaptic sites in the same neuron. This provides a CRE-inducible dual system for imaging neural circuits in vivo, and to study their assembly and remodeling in cultured neurons, neural stem cells, and tissue explants derived from the reporter line. Unlike existing lines, this reporter does not encode a ubiquitously expressed, floxable LacZ gene, permitting the simultaneous analysis of beta galactosidase activity in mutant lines.

This article was republished on December 19, 2014 because of incorrect figures. Please download this article again to view the figures.

Despite great progress in the identification of mesenchymal stem cells (MSCs) from bone marrow (BM), our knowledge of their in vivo cellular identity remains limited. We report here that cells expressing the transcription factor Ebf2 in adult BM display characteristics of MSCs. The Ebf2(+) cells are highly clonal and physiologically quiescent. In vivo lineage-tracing experiments, single cell clone transplantations, and in vitro differentiation assays revealed their self-renewal and multilineage differentiation capacity. Gene expression analysis of the freshly sorted Ebf2(+) cells demonstrated the expression of genes previously reported to be associated with MSCs and the coexpression of multiple lineage-associated genes at the single-cell level. Thus, Ebf2 expression is not restricted to committed osteoblast progenitor cells but rather marks a multipotent mesenchymal progenitor cell population in adult mouse BM. These cells do not appear to completely overlap the previously reported MSC populations. These findings provide new insights into the in vivo cellular identity and molecular properties of BM mesenchymal stem and progenitor cells.

Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo.

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., Highlights ► Ebf2 is a novel marker for hem- and septum-derived Cajal–Retzius cells. ► Ebf2 mutant shows a transient migratory defect of hem-derived Cajal–Retzius cells. ► Ebf3 is expressed in similar territories as Ebf2 and rescues Ebf2 mutant phenotype. ► Both Ebf2 and Ebf3 are required for Cajal–Retzius cell migration.

Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.

Background Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs. Results Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor. Conclusions Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances.