NudC associates with Lis1 and the dynein motor at the leading pole of neurons.

NUDC is a highly conserved protein important for nuclear migration and viability in Aspergillus nidulans. Mammalian NudC interacts with Lis1, a neuronal migration protein important during neocorticogenesis, suggesting a conserved mechanism of nuclear movement in A. nidulans and neuronal migration in the developing mammalian brain (S. M. Morris et al., 1998). To further investigate this possibility, we show for the first time that NudC, Lis1, and cytoplasmic dynein intermediate chain (CDIC) colocalize at the microtubule organizing center (MTOC) around the nucleus in a polarized manner facing the leading pole of cerebellar granule cells with a migratory morphology. In neurons with stationary morphology, NudC is distributed throughout the soma and colocalizes with CDIC and tubulin in neurites as well as at the MTOC. At the subcellular level, NudC, CDIC, and p150 dynactin colocalize to the interphase microtubule array and the MTOC in fibroblasts. The observed colocalization is confirmed biochemically by coimmunoprecipitation of NudC with CDIC and cytoplasmic dynein heavy chain (CDHC) from mouse brain extracts. Consistent with its expression in individual neurons, a high level of NudC is detected in regions of the embryonic neocortex undergoing extensive neurogenesis as well as neuronal migration. These data suggest a biochemical and functional interaction of NudC with Lis1 and the dynein motor complex during neuronal migration in vivo.

Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system.

Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking.

Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization.

Lissencephaly is a severe brain malformation in humans. To study the function of the gene mutated in lissencephaly (LIS1), we deleted the first coding exon from the mouse Lis1 gene. The deletion resulted in a shorter protein (sLIS1) that initiates from the second methionine, a unique situation because most LIS1 mutations result in a null allele. This mutation mimics a mutation described in one lissencephaly patient with a milder phenotype. Homozygotes are early lethal, although heterozygotes are viable and fertile. Most strikingly, the morphology of cortical neurons and radial glia is aberrant in the developing cortex, and the neurons migrate more slowly. This is the first demonstration, to our knowledge, of a cellular abnormality in the migrating neurons after Lis1 mutation. Moreover, cortical plate splitting and thalomocortical innervation are also abnormal. Biochemically, the mutant protein is not capable of dimerization, and enzymatic activity is elevated in the embryos, thus a demonstration of the in vivo role of LIS1 as a subunit of PAF-AH. This mutation allows us to determine a hierarchy of functions that are sensitive to LIS1 dosage, thus promoting our understanding of the role of LIS1 in the developing cortex.

Temporally and spatially regulated expression of a candidate G-protein-coupled receptor during cerebral cortical development.

Genes expressed in layer-specific patterns in the mammalian cerebral cortex may play a role in specifying the identity of different cortical layers. Using PCR-differential display, we identified a cDNA that encodes rCNL3, a gene cloned previously by sequence homology to G-protein-coupled receptors. rCNL3 is expressed predominantly in layers 2-4 of the young rat cortex and in the developing and adult striatum. Cortical expression of rCNL3 begins postnatally at P3 and continues at high levels until around P15, while striatal expression begins at E20 and continues through adulthood. rCNL3 expression is not detectable in the ventricular zone precursors that generate the neurons of layers 2-4. The expression pattern of rCNL3 in the developing cortex suggests that rCNL3 is not involved in the initial specification of laminar fate, but rather may be involved with later differentiation events within the superficial cortical layers.

Developmental expression pattern of the cdo gene.

CDO is a cell-surface protein of the immunoglobulin/fibronectin type III repeat family that positively regulates myogenic differentiation in vitro. To gain a better understanding of the role of cdo during vertebrate development, we carried out an extensive in situ hybridization study to characterize its expression pattern from postimplantation to late stages of mouse embryogenesis and in rat brain from E13 to adult. Our results show a broad pattern of cdo expression that is spatially and temporally restricted during embryogenesis. In the central nervous system (CNS), cdo expression is detected as early as E7.5 and maintained in the dorsal ventricular zones of the brain and spinal cord, becoming increasingly restricted in the adult. High levels of cdo are detected in developing sensory organs, such as the eye and ear. Outside the CNS, cdo is expressed mainly in neural crest and mesodermal derivatives, including skeletal muscle precursors. Overall, the highest levels of cdo expression are seen from E9.0 to E15.5. The temporal onset and restricted expression of cdo suggest that cdo plays a role in the determination and/or differentiation of a number of cell types during embryogenesis.

Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed.

Partial genetic linkage maps, based on microsatellite markers, were constructed for two tilapia species, Oreochromis aureus and Oreochromis niloticus using an interspecific backcross population. The linkage map for O. aureus comprised 28 markers on 10 linkage groups and covered 212.8 CM. Nine markers were mapped to four linkage groups on an O. niloticus female linkage map covering 40.6 CM. Results revealed a high degree of conservation of synteny between the linkage groups defined in O. aureus and the previously published genetic linkage map of O. niloticus.

Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures.

The use of genetics to study the development of the telencephalon and derivatives such as the cerebral cortex has been limited. The telencephalon begins to form midway through gestation, and targeted mutations in genes suspected of playing roles in its development often lead to early phenotypes that preclude analysis of their role at later stages. This problem can be circumvented using a Cre/loxP recombination system. A mouse line was produced in which cre was targeted to the Foxg1 (BF-1) locus, a gene expressed specifically in the telencephalon and discrete head structures. Crosses between Foxg1-Cre mice and three separate loxP reporter mice generated embryos with recombination patterns matching that expected from the normal pattern of Foxg1 expression. Recombination occurs invariably in the telencephalon, anterior optic vesicle, otic vesicle, facial and head ectoderm, olfactory epithelium, mid-hindbrain junction, and pharyngeal pouches. Recombination in some animals also occurs less efficiently in tissues not known to express Foxg1. We show that the genetic background of the parental mice and the loxP target allele can each contribute to differences in the exact pattern of recombination. Collectively, these data show that Foxg1-Cre mice should be useful in the deletion or ectopic expression of any floxed target gene in a Foxg1-like pattern.

Progressive restriction in fate potential by neural progenitors during cerebral cortical development.

During early stages of cerebral cortical development, progenitor cells in the ventricular zone are multipotent, producing neurons of many layers over successive cell divisions. The laminar fate of their progeny depends on environmental cues to which the cells respond prior to mitosis. By the end of neurogenesis, however, progenitors are lineally committed to producing upper-layer neurons. Here we assess the laminar fate potential of progenitors at a middle stage of cortical development. The progenitors of layer 4 neurons were first transplanted into older brains in which layer 2/3 was being generated. The transplanted neurons adopted a laminar fate appropriate for the new environment (layer 2/3), revealing that layer 4 progenitors are multipotent. Mid-stage progenitors were then transplanted into a younger environment, in which layer 6 neurons were being generated. The transplanted neurons bypassed layer 6, revealing that layer 4 progenitors have a restricted fate potential and are incompetent to respond to environmental cues that trigger layer 6 production. Instead, the transplanted cells migrated to layer 4, the position typical of their origin, and also to layer 5, a position appropriate for neither the host nor the donor environment. Because layer 5 neurogenesis is complete by the stage that progenitors were removed for transplantation, restrictions in laminar fate potential must lag behind the final production of a cortical layer. These results suggest that a combination of intrinsic and environmental cues controls the competence of cortical progenitor cells to produce neurons of different layers.

Cortical degeneration in the absence of neurotrophin signaling: dendritic retraction and neuronal loss after removal of the receptor TrkB.

To examine functions of TrkB in the adult CNS, TrkB has been removed from neurons expressing CaMKII, primarily pyramidal neurons, using Cre-mediated recombination. A floxed trkB allele was designed so that neurons lacking TrkB express tau-beta-galactosidase. Following trkB deletion in pyramidal cells, their dendritic arbors are altered, and cortical layers II/III and V are compressed, after which there is an apparent loss of mutant neurons expressing the transcription factor SCIP but not of those expressing Otx-1. Loss of neurons expressing SCIP requires deletion of trkB within affected neurons; reduction of neuronal ER81 expression does not, suggesting both direct and indirect effects of TrkB loss. Thus, TrkB is required for the maintenance of specific populations of cells in the adult neocortex.

Regional differences in the developing cerebral cortex revealed by ephrin-A5 expression.

The development of axonal connections between thalamic nuclei and their cortical target areas occurs in a highly specific manner. To explore the mechanisms of thalamocortical axon pathfinding, we investigated the expression of several members of the ephrin and Eph gene families in the forebrain. The Eph ligand ephrin-A5 was expressed in three distinct gradients during the development of the telencephalon. The first gradient occurred in the cortical ventricular zone and established ephrin-A5 as one of the earliest markers distinguishing cells residing in the anterior versus posterior cortical neuroepithelium. The second gradient was apparent in the subplate and occurred in spatial opposition to a distinct gradient for the low-affinity NGF receptor p75. This finding reveals that different regions of the early subplate are molecularly heterogeneous. Third, we confirmed that ephrin-A5 is expressed in a bi-directional gradient in the cortical plate, with highest levels in the somatomotor cortex. Three putative receptors for ephrin-A5 -- EphA3, EphA4 and EphA5 -- showed distinct expression patterns in the developing thalamus. The graded distributions of ephrin-A5 in the developing subplate and cortex and the expression of its receptors in the thalamus are consistent with the notion that the Eph ligands and their receptors may function in the topographic mapping of thalamic axons to specific cortical areas.

The isolation and mapping of 19 tetranucleotide microsatellite markers in the chicken.

Microsatellites consisting of tetranucleotide repeats are more easily, and consequently efficiently, scored than loci consisting of dinucleotides. However, they are much less frequent in the genome. A hybridisation selection protocol was therefore employed to generate a chicken genomic library enriched for inserts containing the tetranucleotide repeat motif (TTTC)n. Forty-five new microsatellite sequences were isolated that mainly consisted of perfect repeats of the tetranucleotide (TTTC) motif. Nineteen markers were mapped in one or both of the East Lansing and Compton international chicken reference populations.

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.

To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.

Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets.

Information processing in the nervous system depends on the creation of specific synaptic connections between neurons and targets during development. The homeodomain transcription factor Otx1 is expressed in early-generated neurons of the developing cerebral cortex. Within layer 5, Otx1 is expressed by neurons with subcortical axonal projections to the midbrain and spinal cord. Otx1 is also expressed in the precursors of these neurons, but is localized to the cytoplasm. Nuclear translocation of Otx1 occurs when layer 5 neurons enter a period of axonal refinement and eliminate a subset of their long-distance projections. Otx1 mutant mice are defective in the refinement of these exuberant projections, suggesting that Otx1 is required for the development of normal axonal connectivity and the generation of coordinated motor behavior.

Intrinsic polarity of mammalian neuroepithelial cells.

Progenitor cells in the mammalian forebrain can undergo either symmetric or asymmetric cell divisions by varying their cleavage orientation. In asymmetric divisions, cells distribute apically and basally localized proteins differentially to their daughters. Here we explore the intrinsic polarity of neuroepithelial cells in the developing telencephalon. Actin microfilaments are concentrated apically, forming beltlike structures that encircle spots of gamma-tubulin immunoreactivity. Staining for N-cadherin, beta-catenin, and the tyrosine kinase substrates pp120 and paxillin is also enriched at the lumenal surface, presumably due to the localization of these proteins at adherens junctions. Phosphotyrosine immunoreactivity is concentrated apically in rings, suggesting that adherens junctions are enriched for signaling molecules. In mitotic cells it appears that adherens junction proteins and phosphotyrosine immunoreactivity may be inherited either symmetrically or asymmetrically, depending on the cell's cleavage orientation during mitosis. The differential inheritance of junctional proteins may determine whether a daughter cell can respond to extrinsic signals after mitosis.

Determination of the migratory capacity of embryonic cortical cells lacking the transcription factor Pax-6.

The cerebral cortex forms by the orderly migration and subsequent differentiation of neuronal precursors generated in the proliferative ventricular zone. We studied the role of the transcription factor Pax-6, which is expressed in the ventricular zone, in cortical development. Embryos homozygous for a mutation of Pax-6 (Small eye; Sey) had abnormalities suggesting defective migration of late-born cortical precursors. When late-born Sey/Sey precursors were transplanted into wild-type embryonic rat cortex, they showed similar integrative, migrational and differentiative abilities to those of transplanted wild-type mouse precursors. These results suggest that postmitotic cortical cells do not need Pax-6 to acquire the capacity to migrate and differentiate, but that Pax-6 generates a cortical environment that permits later-born precursors to express their full developmental potential.

Postmitotic neurons migrate tangentially in the cortical ventricular zone.

Patterns of cell movement play a key role in the establishment of the brain's functional architecture during development. The migration of neuronal progenitor cells has been hypothesized to disperse clonally related cells among different areas of the developing cerebral cortex. To test this model, we explored the migratory patterns of cells in the proliferative zone of the intact cortex of the ferret. After focal injections of DiI, labeled cells migrated in all directions and over long distances within the ventricular and subventricular zones. These cells expressed the neuron-specific marker TuJ1 and did not incorporate BrdU after cumulative labeling. Our results reveal an extensive tangential dispersion of cortical cells mediated predominantly or exclusively by the non-radial migration of postmitotic neurons.

Induction of deep layer cortical neurons in vitro.

Transplantation studies suggest that the laminar fates of cerebral cortical neurons are determined by environmental signals encountered just before mitosis. In ferret, E29 progenitor cells normally produce neurons of layers 5 and 6. When transplanted during S-phase into an older ventricular zone, E29 progenitors produce neurons that change their fates and migrate to layer 2/3; however, cells transplanted later in the cell cycle migrate to their normal deep-layer positions even in an older environment (McConnell and Kaznowski, 1991). Here we utilize three culture systems to investigate the nature of the environmental signals involved in laminar specification. E29 cells were first cultured at low density to ascertain whether cell contact and/or short-range cues are required for deep layer specification. Neurons transplanted after a short time in low-density culture failed to adopt their normal fates and migrated instead to the upper layers. When crude cell contacts were restored by pelleting E29 cells together, most transplanted neurons cells became specified to their normal deep layer fates. Finally, E29 cells were transplanted after being cultured in explants that maintained the architecture of the cerebral wall. Explants allowed normal deep layer specification to occur, as transplanted cells migrated to layers 5 and 6. These results suggest that short-range cues induce multipotent progenitors to produce deep layer neurons.

Restriction of late cerebral cortical progenitors to an upper-layer fate.

Early in development, neural progenitors in cerebral cortex normally produce neurons of several layers during successive cell divisions. The laminar fate of their daughters depends on environmental cues encountered just before mitosis. At the close of neurogenesis, however, cortical progenitors normally produce neurons destined only for the upper layers. To assess the developmental potential of these cells, upper-layer progenitors were transplanted into the cerebral cortex of younger hosts, in which deep-layer neurons were being generated. These studies reveal that late cortical progenitors are not competent to generate deep-layer neurons and are instead restricted to producing the upper layers.

Strategies for the generation of neuronal diversity in the developing central nervous system.

During development, the neural tube produces a large diversity of neuronal phenotypes from a morphologically homogeneous pool of precursor cells. In recent years, the cellular and molecular mechanisms by which specific types of neurons are generated have been explored, in the hope of discovering features common to development throughout the nervous system. This article focuses on three strategies employed by the CNS to generate distinct classes of neuronal phenotypes during development: dorsal-ventral polarization in the spinal cord, segmentation in the hindbrain, and a lamination in the cerebral cortex. The mechanisms for neurogenesis exemplified by these three strategies range from a relatively rigid, cell lineage-dependent specification with a high degree of subservance to early patterns of gene expression, to inductions and cell-cell interactions that determine cell fates more flexibly.