Dispersion of the neurons expressing layer specific markers in the reeler brain

Neurons with similar functions including neuronal connectivity and gene expression form discrete condensed structures within the vertebrate brain. This is exemplified within the circuitry formed by the cortical layers and the neuronal nuclei. It is well known that the Reelin protein is required for development of these neuronal structures in rodents and human, but the function of Reelin remains controversial. In this report, we used “layer‐specific markers” of the cerebral cortex to carry out detailed observations of spatial distribution of the neuronal subpopulations in the brain of the Reelin deficient mouse, reeler. We observed a spatially dispersed expression of the markers in the reeler cerebral cortex. These markers are expressed also in other laminated and non‐laminated structures of brain, in which we observed similar abnormal gene expression. Our observations suggest that neurons within the brain structures (such as the layers and the nuclei), which normally exhibit condensed distribution of marker expressions, loosen their segregation or scatter by a lack of Reelin.     

Radial glial cell development and transformation are disturbed in reeler forebrain

Radial glia are among the earliest cell types to differentiate in the developing mammalian forebrain. Glial fibers span the early cortical wall, forming a dense scaffold; this persists throughout corticogenesis, providing a cellular substrate which supports and directs the migration of young neurons. Although the mechanisms regulating radial glial cell development are poorly understood, a secreted cortical radial glial differentiation signal was recently identified in the embryonic mouse forebrain. This signal is abundant at the time radial glia function to support neuronal migration, and down-regulated perinatally, when radial glia are known to undergo transformation into astrocytes. Therefore, it seems that this signal functions as a radial glial maintenance factor, the availability of which regulates the phenotype of cortical astroglia. Here the differentiation signal is further characterized as RF60, a protein with a molecular weight of approximately 60 kD. In addition, the neurologic mutant mouse reeler provides a genetic model for analysis of RF60 function. Radial glia in reeler cortex are shown to be poorly differentiated and the radial scaffold is shown to be maintained for a shorter time than normal. Furthermore, although astroglial cells from normal cortex are induced to elaborate a radial phenotype by RF60, reeler astroglia show an impaired differentiation response to this. These findings suggest that an intrinsic defect in glial differentiation contributes to the phenotype of abnormal cortical lamination seen in reeler mouse, and indicate that RF60 may play a critical role in normal cortical patterning. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 459–472, 1997     

Interdigital cell death in the embryonic limb is associated with depletion of Reelin in the extracellular matrix

Interdigital cell death is a physiological regression process responsible for sculpturing the digits in the embryonic vertebrate limb. Changes in the intensity of this degenerative process account for the different patterns of interdigital webbing among vertebrate species. Here, we show that Reelin is present in the extracellular matrix of the interdigital mesoderm of chick and mouse embryos during the developmental stages of digit formation. Reelin is a large extracellular glycoprotein which has important functions in the developing nervous system, including neuronal survival; however, the significance of Reelin in other systems has received very little attention. We show that reelin expression becomes intensely downregulated in both the chick and mouse interdigits preceding the establishment of the areas of interdigital cell death. Furthermore, fibroblast growth factors, which are cell survival signals for the interdigital mesoderm, intensely upregulated reelin expression, while BMPs, which are proapototic signals, downregulate its expression in the interdigit. Gene silencing experiments of reelin gene or its intracellular effector Dab-1 confirmed the implication of Reelin signaling as a survival factor for the limb undifferentiated mesoderm. We found that Reelin activates canonical survival pathways in the limb mesoderm involving protein kinase B and focal adhesion kinase. Our findings support that Reelin plays a role in interdigital cell death, and suggests that anoikis (apoptosis secondary to loss of cell adhesion) may be involved in this process.     

Reln,an Allele ofReelerIsolated from a Chlorambucil Screen, Is Due to an IAP Insertion with Exon Skipping

Thereeler Albany2mutation (Reln^rl-Alb2) in the mouse is an allele ofreelerisolated during a chlorambucil mutagenesis screen. Homozygous animals had drastically reduced concentrations ofreelinmRNA, in which an 85-nt exon was absent. At the genomic level, the mutation was shown to be due to an intracisternal A-particle insertion leading to exon skipping. This appears to be the first observation of retrotransposon insertion during chlorambucil mutagenesis.     

High-Speed Positional Cloning Based on Restriction Landmark Genome Scanning

Restriction landmark genome scanning (RLGS) was developed as a method of genome analysis that is based on the concept that restriction enzyme sites can be used as landmarks. In this article, we demonstrate how this method can be used for the systematic, successful positional cloning of mouse mutantreelergene. The major advantage of the RLGS method is that it allows the scanning of several thousand spots/loci throughout the genome with one RLGS profile. High-speed positional cloning based on the RLGS method includes (1) high-speed construction of a linkage map (RLGS spot mapping), (2) high-speed detection of RLGS spot markers tightly linked to the mutant phenotype (RLGS spot bombing method), and (3) construction of YAC contigs covering the region where tightly linked spot markers are located (RLGS-based YAC contig mapper). We introduced a series of these procedures by using them to positionally clone thereelergene. High-speed construction of the whole genetic map and spots/loci (less than 1 cM) within the closest flanking markers is demonstrated. The RLGS-based YAC contig mapper also efficiently yielded the YAC physical contig map of the target region. Finally, we cloned thereelergene, which is the causal gene for the perturbation of the three-dimensional brain architecture due to the abnormal migration of neuroblasts inreelermouse. Since the RLGS method itself can be used for any organism, we conclude that the total RLGS-based positional cloning system can be used to identify any mutant gene of any organism.     

Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells

During embryonic development in vertebrates, the neural crest‐derived melanoblasts migrate along the dorsolateral axis and cross the basal membrane separating the dermis from the epidermis to reach their final location in the interfollicular epidermis and epidermal hair follicles. Neoplastic transformation converts melanocytes into highly invasive and metastatic melanoma cells. In vitro, these cells extend various types of protrusions and adopt two interconvertible modes of migration, mesenchymal and amoeboid, driven by different signalling molecules. In this review, we describe the major contributions of natural mouse mutants, mouse models generated by genetic engineering and in vitro culture systems, to identification of the genes, signalling pathways and mechanisms regulating the migration of normal and pathological cells of the melanocyte lineage, at both the cellular and molecular levels.     

Reelin: A novel extracellular matrix protein involved in brain lamination

Normal development of the nervous system is achieved through an elaborate program of guided neuronal migration and axonal growth. In the last few years, a flood of research has dissected the molecular bases of these phenomena, and several cell-surface and extracellular matrix molecules, which are implicated in neuronal and axonal targeting processes, have been recognized. Taking this knowledge a step further, a recent paper by Tom Curran's group⁽¹⁾ reports the molecular cloning of the gene deleted in the autosomal recessive mouse mutation reeler, affecting cortical neuronal migration. This gene encodes reelin, a novel extracellular matrix protein.     

A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development

Although the mechanisms that regulate development of the cerebral cortex have begun to emerge, in large part through the analysis of mutant mice (Boncinelli et al. 2000; Molnar and Hannan 2000; Walsh and Goffinet 2000), many questions remain unanswered. To provide resources for further dissecting cortical development, we have carried out a focused screen for recessive mutations that disrupt cortical development. One aim of the screen was to identify mutants that disrupt the tangential migration of interneurons into the cortex. At the same time, we also screened for mutations that altered the growth or morphology of the cerebral cortex. We report here the identification of thirteen mutants with defects in aspects of cortical development ranging from the establishment of epithelial polarity to the invasion of thalamocortical axons. Among the collection are three novel alleles of genes for which mutant alleles had already been used to explore forebrain development, and four mutants with defects in interneuron migration. The mutants that we describe here will aid in deciphering the molecules and mechanisms that regulate cortical development. Our results also highlight the utility of focused screens in the mouse, in addition to the large-scale and broadly targeted screens that are being carried out at mutagenesis centers.     

Dab2ip Regulates Neuronal Migration and Neurite Outgrowth in the Developing Neocortex

Dab2ip (DOC-2/DAB2 interacting protein) is a member of the Ras GTPase-activating protein (GAP) family that has been previously shown to function as a tumor suppressor in several systems. Dab2ip is also highly expressed in the brain where it interacts with Dab1, a key mediator of the Reelin pathway that controls several aspects of brain development and function. We found that Dab2ip is highly expressed in the developing cerebral cortex, but that mutations in the Reelin signaling pathway do not affect its expression. To determine whether Dab2ip plays a role in brain development, we knocked down or over expressed it in neuronal progenitor cells of the embryonic mouse neocortex using in utero electroporation. Dab2ip down-regulation severely disrupts neuronal migration, affecting preferentially late-born principal cortical neurons. Dab2ip overexpression also leads to migration defects. Structure-function experiments in vivo further show that both PH and GRD domains of Dab2ip are important for neuronal migration. A detailed analysis of transfected neurons reveals that Dab2ip down- or up-regulation disrupts the transition from a multipolar to a bipolar neuronal morphology in the intermediate zone. Knock down of Dab2ip in neurons ex-vivo indicates that this protein is necessary for proper neurite development and for the expression of several major neuronal microtubule associated proteins (MAPs), which are important for neurite growth and stabilization. Thus, our study identifies, for the first time, a critical role for Dab2ip in mammalian cortical development and begins to reveal molecular mechanisms that underlie this function.     

An In Silico Agent-Based Model Demonstrates Reelin Function in Directing Lamination of Neurons during Cortical Development

The characteristic six-layered appearance of the neocortex arises from the correct positioning of pyramidal neurons during development and alterations in this process can cause intellectual disabilities and developmental delay. Malformations in cortical development arise when neurons either fail to migrate properly from the germinal zones or fail to cease migration in the correct laminar position within the cortical plate. The Reelin signalling pathway is vital for correct neuronal positioning as loss of Reelin leads to a partially inverted cortex. The precise biological function of Reelin remains controversial and debate surrounds its role as a chemoattractant or stop signal for migrating neurons. To investigate this further we developed an in silico agent-based model of cortical layer formation. Using this model we tested four biologically plausible hypotheses for neuron motility and four biologically plausible hypotheses for the loss of neuron motility (conversion from migration). A matrix of 16 combinations of motility and conversion rules was applied against the known structure of mouse cortical layers in the wild-type cortex, the Reelin-null mutant, the Dab1-null mutant and a conditional Dab1 mutant. Using this approach, many combinations of motility and conversion mechanisms can be rejected. For example, the model does not support Reelin acting as a repelling or as a stopping signal. In contrast, the study lends very strong support to the notion that the glycoprotein Reelin acts as a chemoattractant for neurons. Furthermore, the most viable proposition for the conversion mechanism is one in which conversion is affected by a motile neuron sensing in the near vicinity neurons that have already converted. Therefore, this model helps elucidate the function of Reelin during neuronal migration and cortical development.     

Lamination of the cerebral cortex is disturbed in Gli3 mutant mice

The layered organization of the cerebral cortex develops in an inside-out pattern, a process which is controlled by the secreted protein reelin. Here we report on cortical lamination in the Gli3 hypomorphic mouse mutant Xt^J/Pdn which lacks the cortical hem, a major source of reelin⁺ Cajal Retzius cells in the cerebral cortex. Unlike other previously described mouse mutants with hem defects, cortical lamination is disturbed in Xt^J/Pdn animals. Surprisingly, these layering defects occur in the presence of reelin⁺ cells which are probably derived from an expanded Dbx1⁺ progenitor pool in the mutant. However, while these reelin⁺ neurons and also Calretinin⁺ cells are initially evenly distributed over the cortical surface they form clusters later during development suggesting a novel role for Gli3 in maintaining the proper arrangement of these cells in the marginal zone. Moreover, the radial glial network is disturbed in the regions of these clusters. In addition, the differentiation of subplate cells is affected which serve as a framework for developing a properly laminated cortex.     

Reelin signaling and Cdk5 in the control of neuronal positioning

Neuronal positioning is important for higher brain function because it is the architectural basis of the formation of precise synaptic circuits. Analysis of neurological mutant mice has led to dramatic progress in the identification and characterization of molecules important for neuronal positioning in the developing mammalian brain. Among these molecules, identification of signal pathways mediated by Reelin and Cdk5 kinase has provided a conceptual framework for exploring the molecular mechanisms underlying proper neuronal positioning in the developing mammalian brain. Recent evidence has implicated synergism between Reelin signaling and Cdk5 in contributing to the proper positioning of selective neuronal populations.     

A landscape of hairy and twisted: hunting for new trichome mutants in the Saskatoon Arabidopsis T‐DNA population

A total of 88 new Arabidopsis lines with trichome variation were recovered by screening 49,200 single‐seed descent T₃ lines from the SK activation‐tagged population and from a new 20,000‐line T‐DNA insertion population (called pAG). Trichome variant lines were classified into 12 distinct phenotype categories. Single or multiple T‐DNA insertion sites were identified for 89% of these mutant lines. Alleles of the well‐known trichome genes TRY, GL2 and TTG1 were recovered with atypical phenotype variation not reported previously. Moreover, atypical gene expression profiles were documented for two additional mutants specifying TRY and GL2 disruptions. In remaining mutants, ten lines were disrupted in genes coding for proteins not implicated in trichome development, five were disrupted in hypothetical proteins and 11 were disrupted in proteins with unknown function. The collection represents new opportunities for the plant biology community to define trichome development more precisely and to refine the function of individual trichome genes.     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.     

Two novel CNRs from the CNR gene cluster have molecular features distinct from those of CNR1 to 8

Cadherin-related neuronal receptor (CNR) family proteins are known as synaptic cadherins and Reelin receptors. Here we have identified two novel mouse CNR genes, CNRc1 and CNRc2, orthologues of human protocadherin (Pcdh) alpha-c1 and Pcdhalpha-c2, respectively. While the variable large exons of CNRc1 and c2 contain six conserved extracellular cadherin repeats (EC1-6) and are linked to the constant exons, both contain several molecular features distinct from CNR1-8. CNRc1 and c2 lack the Arg-Gly-Asp (RGD) sequence that is conserved in the EC1 of CNR1-8, which is necessary for binding to Reelin. The present studies confirm that CNRc1 and c2 failed to immunoprecipitate with Reelin. In addition, the regulation of novel CNR expression patterns during brain development is slightly different from that of CNR1. The identification of these new CNR genes characterized by their distinct extracellular function and expression is indicative of the novel diversity of the processes of brain structuring and synapse regulation.     

Reelin controls progenitor cell migration in the healthy and pathological adult mouse brain

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.     

Identification of alternatively spliced dab1 isoforms in zebrafish

We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

The Reelin signaling pathway in mouse cortical development

Most of the cerebral cortex derives from the cortical plate which, in all mammals, is radially organized and develops from inside to outside. Several genes involved in the organization and inside-outside development of the embryonic cortical plate in the mouse form the so-called Reelin signaling pathway. Biochemical and genetic arguments show that the extracellular matrix protein Reelin binds to two lipoprotein receptors (VLDLR and ApoER2), which relay the Reelin signal inside target neurons by docking the tyrosine kinase adapter disabled-1 (Dab1). In addition, biochemical evidence suggests that the integrins alpha 3/beta 1 and protocadherins of the CNR family may also modulate the Reelin signal. The mechanisms by which the presence of Reelin stops migration and instructs the radial organization of cortical plate cells remains unknown.     

p59fyn and pp60 c-src modulate axonal guidance in the developing mouse olfactory pathway

The Src-family tyrosine kinases p59^fyn and pp60^c-src are localized on axons of the mouse olfactory nerve during the initial stages of axonal growth, but their functional roles remain to be defined. To study the role of these kinases, we analyzed the trajectory of the olfactory nerve in E11.5 homozygous null mutant mice lacking single src or fyn genes and double mutants lacking both genes. Primary olfactory axons of single and double mutants exited the olfactory epithelium and projected toward the telencephalon, but displayed differences in fasciculation. The fyn-minus olfactory nerve had significantly more fascicles than the src-minus nerve. Most strikingly, the primary olfactory nerve of src/fyn double mutants showed the greatest degree of defasciculation. These defects, identified by NCAM labeling, were not due to apparent changes in the size of the olfactory epithelium. With the exception of the src-minus mice, which had fewer fascicles than the wild type, no obvious differences were observed in coalescence of vomeronasal axons from mutant mice. The mesenchyme of the double and single mutants exhibited only subtle changes in laminin and fibronectin staining, indicating that the adhesive environment of the mesenchyme may contribute in part to defects in fasciculation. The results suggest that signaling pathways mediated by p59^fyn and pp60^c-src contribute to the appropriate fasciculation of axons in the nascent olfactory system, and comprise partially compensatory mechanisms for axonal adhesion and guidance. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 53–63, 1998     

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.