Netrin-DCC signaling regulates corpus callosum formation through attraction of pioneering axons and by modulating Slit2-mediated repulsion.

The left and right sides of the nervous system communicate via commissural axons that cross the midline during development using evolutionarily conserved molecules. These guidance cues have been particularly well studied in the mammalian spinal cord, but it remains unclear whether these guidance mechanisms for commissural axons are similar in the developing forebrain, in particular for the corpus callosum, the largest and most important commissure for cortical function. Here, we show that Netrin1 initially attracts callosal pioneering axons derived from the cingulate cortex, but surprisingly is not attractive for the neocortical callosal axons that make up the bulk of the projection. Instead, we show that Netrin-deleted in colorectal cancer signaling acts in a fundamentally different manner, to prevent the Slit2-mediated repulsion of precrossing axons thereby allowing them to approach and cross the midline. These results provide the first evidence for how callosal axons integrate multiple guidance cues to navigate the midline.

Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK.

Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, we investigated potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. We found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-alpha. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis.

A NUDEL-dependent mechanism of neurofilament assembly regulates the integrity of CNS neurons.

The cytoskeleton controls the architecture and survival of central nervous system (CNS) neurons by maintaining the stability of axons and dendrites. Although neurofilaments (NFs) constitute the main cytoskeletal network in these structures, the mechanism that underlies subunit incorporation into filaments remains a mystery. Here we report that NUDEL, a mammalian homologue of the Aspergillus nidulans nuclear distribution molecule NudE, is important for NF assembly, transport and neuronal integrity. NUDEL facilitates the polymerization of NFs through a direct interaction with the NF light subunit (NF-L). Knockdown of NUDEL by RNA interference (RNAi) in a neuroblastoma cell line, primary cortical neurons or post-natal mouse brain destabilizes NF-L and alters the homeostasis of NFs. This results in NF abnormalities and morphological changes reminiscent of neurodegeneration. Furthermore, variations in levels of NUDEL correlate with disease progression and NF defects in a mouse model of neurodegeneration. Thus, NUDEL contributes to the integrity of CNS neurons by regulating NF assembly.

Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression.

The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.

Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.

Neuropilin-1 (Npn-1) is a receptor that binds multiple ligands from structurally distinct families, including secreted semaphorins (Sema) and vascular endothelial growth factors (VEGF). We generated npn-1 knockin mice, which express an altered ligand binding site variant of Npn-1, and npn-1 conditional null mice to establish the cell-type- and ligand specificity of Npn-1 function in the developing cardiovascular and nervous systems. Our results show that VEGF-Npn-1 signaling in endothelial cells is required for angiogenesis. In striking contrast, Sema-Npn-1 signaling is not essential for general vascular development but is required for axonal pathfinding by several populations of neurons in the CNS and PNS. Remarkably, both Sema-Npn-1 signaling and VEGF-Npn-1 signaling are critical for heart development. Therefore, Npn-1 is a multifunctional receptor that mediates the activities of structurally distinct ligands during development of the heart, vasculature, and nervous system.

Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning.

Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), we found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning.

Transient enhancement of proliferation of neural progenitors and impairment of their long-term survival in p25 transgenic mice.

Cyclin-dependent kinase 5 (CDK5) regulates important neuronal functions via p35. p35 undergoes cleavage in response to neuronal activity and neurotoxic conditions to release its subunit p25. Although p25 has been implicated in various neurodegenerative diseases, the mechanisms by which p25 mediates neurodegenerative impairment have not been fully elucidated. We aimed to determine the role of p25-mediated neurodegeneration on neurogenesis in an inducible transgenic mouse line overexpressing p25 (p25 TG) in the forebrain. Adult neuronal progenitor cells (NPCs) were labeled with BrdU in vivo, which were significantly increased in numbers in the subventricular zone, the hippocampus, and the cortex of p25 TG mice. Consistently, more mitotic cells were observed in p25 TG mice than in controls, even in the cortex and the CA1, which are not neurogenic regions. BrdU-positive cells were negative for GFAP or γ-H2AX, suggesting that they are not astrocytes or dying cells. Neurospheres derived from the dentate gyrus and the cortex were significantly increased in p25 TG mice and can be differentiated into astrocytes and neurons. However, p25 TG decreased the long-term survival of proliferating NPCs and severely impaired adult neurogenesis. A Transwell co-culture system was used to assess the influence of p25-expressing primary neurons on adult NPCs. Co-culture with p25-expressing neurons downregulated Ki67 expression and upregulated cleaved caspase-3, indicating that the paracrine signaling in cell-cell communication is essential for NPC survival and proliferation. Moreover, increased CDK5 activity impairs Wnt activation. This study demonstrates that hyperactivation of p25 may temporarily enhance NPC proliferation, but impair their long-term survival.

Slit2 guides both precrossing and postcrossing callosal axons at the midline in vivo.

Commissural axons generally cross the midline only once. In the Drosophila nerve cord and mouse spinal cord, commissural axons are guided by Slit only after they cross the midline, where Slit prevents these axons from recrossing the midline. In the developing corpus callosum, Slit2 expressed by the glial wedge guides callosal axons before they cross the midline, as they approach the corticoseptal boundary. These data highlighted a potential difference between the role of Slit2 in guiding commissural axons in the brain compared with the spinal cord. Here, we investigate whether Slit2 also guides callosal axons after they cross the midline. Because such questions cannot be addressed in conventional gene knock-out animals, we used in utero injections of antisense oligonucleotides to specifically deplete Slit2 on only one side of the brain. We used this technique together with a novel in vitro assay of hemisected brain slices to specifically analyze postcrossing callosal axons. We find that in the brain, unlike the spinal cord, Slit2 mediates both precrossing and postcrossing axonal guidance. Depletion of Slit2 on one side of the brain causes axons to defasciculate and, in some cases, to aberrantly enter the septum. Because these axons do not recross the midline, we conclude that the principle function of Slit2 at the cortical midline may be to channel the axons along the correct path and possibly repel them away from the midline. We find no evidence that Slit2 prevents axons from recrossing the midline in the brain.

Abnormal development of forebrain midline glia and commissural projections in Nfia knock-out mice.

Nuclear factor I (NFI) genes are expressed in multiple organs throughout development (Chaudhry et al., 1997; for review, see Gronostajski, 2000). All four NFI genes are expressed in embryonic mouse brain, with Nfia, Nfib, and Nfix being expressed highly in developing cortex (Chaudhry et al., 1997). Disruption of the Nfia gene causes agenesis of the corpus callosum (ACC), hydrocephalus, and reduced GFAP expression (das Neves et al., 1999). Three midline structures, the glial wedge, glia within the indusium griseum, and the glial sling are involved in development of the corpus callosum (Silver et al., 1982; Silver and Ogawa, 1983; Shu and Richards, 2001). Because Nfia(-)/- mice show glial abnormalities and ACC, we asked whether defects in midline glial structures occur in Nfia(-)/- mice. NFI-A protein is expressed in all three midline populations. In Nfia(-)/-, mice sling cells are generated but migrate abnormally into the septum and do not form a sling. Glia within the indusium griseum and the glial wedge are greatly reduced or absent and consequently Slit2 expression is also reduced. Although callosal axons approach the midline, they fail to cross and extend aberrantly into the septum. The hippocampal commissure is absent or reduced, whereas the ipsilaterally projecting perforating axons (Hankin and Silver, 1988; Shu et al., 2001) appear relatively normal. These results support an essential role for midline glia in callosum development and a role for Nfia in the formation of midline glial structures.

Trekking across the brain: the journey of neuronal migration.

The correct positioning of neurons during development--achieved through directed migration--is the basis for proper brain function. Several decades of research have yielded a comprehensive map illustrating the temporal and spatial events underlying neurogenesis and neuronal migration during development. The discovery of distinct migration modes and pathways has been accompanied by the identification of a large interwoven molecular network that transmits extracellular signals into the cell. Moreover, recent work has shed new light on how the cytoskeleton is regulated and coordinated at the molecular and cellular level to execute neuronal migration.

The glial sling is a migratory population of developing neurons.

For two decades the glial sling has been hypothesized to act as a guidance substratum for developing callosal axons. However, neither the cellular nature of the sling nor its guidance properties have ever been clearly identified. Although originally thought to be glioblasts, we show here that the subventricular zone cells forming the sling are in fact neurons. Sling cells label with a number of neuronal markers and display electrophysiological properties characteristic of neurons and not glia. Furthermore, sling cells are continuously generated until early postnatal stages and do not appear to undergo widespread cell death. These data indicate that the sling may be a source of, or migratory pathway for, developing neurons in the rostral forebrain, suggesting additional functions for the sling independent of callosal axon guidance.

Development of midline glial populations at the corticoseptal boundary.

Three midline glial populations are found at the corticoseptal boundary: the glial wedge (GW), glia within the indusium griseum (IGG), and the midline zipper glia (MG). Two of these glial populations are involved in axonal guidance at the cortical midline, specifically development of the corpus callosum. Here we investigate the phenotypic and molecular characteristics of each population and determine whether they are generated at the same developmental stage. We find that the GW is derived from the radial glial scaffold of the cortex. GW cells initially have long radial processes that extend from the ventricular surface to the pial surface, but by E15 loose their pial attachment and extend only part of the way to the pial surface. Later in development the radial morphology of cells within the GW is replaced by multipolar astrocytes, providing supportive evidence that radial glia can transform into astrocytes. IGG and MG do not have a radial morphology and do not label with the radial glial markers, Nestin and RC2. We conclude that the GW and IGG have different morphological and molecular characteristics and are born at different stages of development. IGG and MG have many phenotypic and molecular characteristics in common, indicating that they may represent a common population of glia that becomes spatially distinct by the formation of the corpus callosum.