The secreted ligand Reelin is thought to regulate the translocation and positioning of prospective layer 6 (L6) neurons into the preplate, a plexus of neurons overlying the ventricular zone. We examined wild type and Reelin-deficient cortices and found that L6 neurons were equivalently positioned beneath the pia during the period of preplate splitting and initial cortical plate (CP) formation. The absence of detectable L6 ectopia in “reeler” cortices at this developmental time point indicates that Reelin-signaling might not regulate L6 neuron migration or gross positioning during preplate splitting. To explore the acute response of L6 neurons to Reelin, subpial injections of Reelin were made into Reelin-deficient explants. Reelin injection caused L6 neurons to orient their nuclei and polarize their Golgi toward the pia while initiating exuberant dendritic (MAP2+) outgrowth within 4 h. This rapid Reelin-dependent neuronal orientation and alignment created CP-like histology without any significant change in the mean position of the population of L6 neurons. Conversely, subplate cells and chondroitin sulfate proteoglycan immunoreactivity were found at significantly deeper positions from the pial surface after injection, suggesting that Reelin partially rescues preplate splitting within 4 h. Thus, Reelin has a direct role in promoting rapid morphological differentation and orientation of L6 neurons during preplate splitting.

The mechanisms controlling cortical dendrite initiation and targeting are poorly understood. Multiphoton imaging of developing mouse cortex reveals that apical dendrites emerge by direct transformation of the neuron9s leading process during the terminal phase of neuronal migration. During this ∼110 min period, the dendritic arbor increases ∼2.5-fold in size and migration arrest occurs below the first stable branch point in the developing arbor. This dendritic outgrowth is triggered at the time of leading process contact with the marginal zone (MZ) and occurs primarily by neurite extension into the extracellular matrix of the MZ. In reeler cortices that lack the secreted glycoprotein Reelin, a subset of neurons completed migration but then retracted and reorganized their arbor in a tangential direction away from the MZ soon after migration arrest. For these reeler neurons, the tangential oriented primary neurites were longer lived than the radially oriented primary neurites, whereas the opposite was true of wild-type (WT) neurons. Application of Reelin protein to reeler cortices destabilized tangential neurites while stabilizing radial neurites and stimulating dendritic growth in the MZ. Therefore, Reelin functions as part of a polarity signaling system that links dendritogenesis in the MZ with cellular positioning and cortical lamination. SIGNIFICANCE STATEMENT Whether the apical dendrite emerges by transformation of the leading process of the migrating neuron or emerges de novo after migration is completed is unclear. Similarly, it is not clear whether the secreted glycoprotein Reelin controls migration and dendritic growth as related or separate processes. Here, multiphoton microscopy reveals the direct transformation of the leading process into the apical dendrite. This transformation is coupled to the successful completion of migration and neuronal soma arrest occurs below the first stable branch point of the nascent dendrite. Deficiency in Reelin causes the forming dendrite to avoid its normal target area and branch aberrantly, leading to improper cellular positioning. Therefore, this study links Reelin-dependent dendritogenesis with migration arrest and cortical lamination.

Background: The secreted ligand Reelin is believed to regulate the translocation of prospective layer 6 (L6) neocortical neurons into the preplate, a loose layer of pioneer neurons that overlies the ventricular zone. Recent studies have also suggested that Reelin controls neuronal orientation and polarized dendritic growth during this period of early cortical development. To explicitly characterize and quantify how Reelin controls this critical aspect of neurite initiation and growth we used a new ex utero explant model of early cortical development to selectively label a subset of L6 cortical neurons for complete 3-D reconstruction. Results: The total neurite arbor sizes of neurons in Reelin-deficient (reeler mutant) and Dab1-deficient (Reelin-non-responsive scrambler mutant) cortices were quantified and unexpectedly were not different than control arbor lengths (p = 0.51). For each mutant, however, arbor organization was markedly different: mutant neurons manifested more primary processes (neurites emitted directly from the soma) than wild type, and these neurites were longer and displayed less branching. Reeler and scrambler mutant neurites extended tangentially rather than radially, and the Golgi apparatus that normally invests the apical neurite was compact in both reeler and scrambler mutants. Mutant cortices also exhibited a neurite “exclusion zone” which was relatively devoid of L6 neuron neurites and extended at least 15 μm beneath the pial surface, an area corresponding to the marginal zone (MZ) in the wild type explants. The presence of an exclusion zone was also indicated in the orientation of mutant primary neurite and neuronal somata, which failed to adopt angles within ~20˚ of the radial line to the pial surface. Injection of recombinant Reelin to reeler, but not scrambler, mutant cortices fully rescued soma orientation, Golgi organization, and dendritic projection defects within four hrs. Conclusions: These findings indicate Reelin promotes directional dendritic growth into the MZ, an otherwise exclusionary zone for L6 neurites.

Despite the recent identification of over 40 missense heterozygous Reelin gene (RELN) mutations in autism spectrum disorder (ASD), none of these has been functionally characterized. Reelin is an integral signaling ligand for proper brain development and post‐natal synapse function – properties likely disrupted in ASD patients. We find that the R2290C mutation, which arose de novo in an affected ASD proband, and other analogous mutations in arginine‐amino acid‐arginine domains reduce protein secretion. Closer analysis of RELN R2290C heterozygous neurospheres reveals up‐regulation of Protein Disulfide Isomerase A1, best known as an endoplasmic reticulum‐chaperone protein, which has been linked to neuronal pathology. This effect is recapitulated in a heterozygous RELN mouse mutant that is characterized by defective Reelin secretion. These findings suggest that both a deficiency in Reelin signaling and pathologic impairment of Reelin secretion may contribute to ASD risk. image

Paxillin and Hic-5 are homologous focal adhesion adaptor proteins that coordinate cytoskeletal rearrangements in response to integrin signaling, but their role(s) in cortical development are unknown. Here, we find that Hic-5-deficient mice are postnatal viable with normal cortical layering. Mice with a neural-specific deletion of paxillin are also postnatal viable, but show evidence of a cortical neuron migration delay that is evident pre- and perinatally, but is not detected at postnatal day 35 (P35). This phenotype is not modified by Hic-5 deficiency (double knockout). Specific deletion of paxillin in postmitotic neurons using Nex-Cre-mediated recombination as well as in utero electroporation of a Cre-expression construct identified a cell-autonomous requirement for paxillin in migrating neurons. Paxillin-deficient neurons have shorter leading processes that exhibited multiple swellings in comparison with control. Multiphoton imaging revealed that paxillin-deficient neurons migrate ∼30% slower than control neurons. This phenotype is similar to that produced by deletion of focal adhesion kinase (FAK), a signaling partner of paxillin, and suggests that paxillin and FAK function cell-autonomously to control migrating neuron morphology and speed during cortical development.

Background: Cortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomes + (Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled. Results: We report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97%) were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP) across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate. Conclusions: This analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways), and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease.