Catherine Lambert de Rouvroit

ABERRANT SPLICING OF A MOUSE DISABLED HOMOLOG, MDAB1, IN THE SCRAMBLER MOUSE

Although accurate long-distance neuronal migration is a cardinal feature of cerebral cortical development, little is known about control of this migration. The scrambler (scm) mouse shows abnormal cortical lamination that is indistinguishable from reeler. Genetic and physical mapping of scm identified yeast artificial chromosomes containing an exon of mdab1, a homolog of Drosophila disabled, which encodes a phosphoprotein that binds nonreceptor tyrosine kinases. mdab1 transcripts showed abnormal splicing in scm homozygotes, with 1.5 kb of intracisternal A particle retrotransposon sequence inserted into the mdab1 coding region in antisense orientation, producing a mutated and truncated predicted protein. Therefore, mdab1 is most likely the scm gene, thus implicating nonreceptor tyrosine kinases in neuronal migration and lamination in developing cerebral cortex.

The central fragment of Reelin, generated by proteolytic processing in vivo, is critical to its function during cortical plate development.

Reelin is a large extracellular protein that controls cortical development. It binds to lipoprotein receptors very-low-density lipoprotein receptor and apolipoprotein-E receptor type 2, thereby inducing phosphorylation of the adapter Dab1. In vivo, Reelin is cleaved into three fragments, but their respective function is unknown. Here we show the following: (1) the central fragment is necessary and sufficient for receptor binding in vitro and for Dab1 phosphorylation in neuronal cultures; (2) Reelin does not bind the protocadherin cadherin-related neuronal receptor (CNR1) as reported previously; (3) Reelin and its central fragment are equally able to rescue the reeler phenotype in a slice culture assay; and (4) anti-receptor antibodies can induce Dab1 phosphorylation but do not correct the reeler phenotype in slices. These observations show that the function of Reelin is critically dependent on the central fragment generated by processing but primarily independent of interactions with CNR1 and on the N-terminal region. They also indicate that events acting in parallel to Dab1 phosphorylation might be required for full activity.

Brain Development in Normal and reeler Mice: the Phenotype

In spite of close scrutiny during the last decades, most of the mechanisms that govern neural development remain unknown. Yet, in addition to an obvious theoretical interest, understanding of these principles is requisite to the effective management of human brain malformations and mental retardation. Present views on the development of the central nervous system can be schematized as follows. The CNS derives from the embryonic epiblast. An initial neural plate becomes hollow (neural groove) and closes into the neural tube. As it does so, it sends into the mesoderm a contingent of cells known as the neural crest. The cavity of the neural tube gives rise to the ventricular system, whereas the neural parenchyma derives from its walls. It is worth pointing out that the development of the CNS is largely intra-epithelial and thus different from the development of most other organs. Of course, this does not imply that interactions between the neural epithelium and the mesoderm are negligible. For example, the observation that Cajal-Retzius cells keep contact with the basal lamina by radial processes may be relevant to their function (Derer 1979).

Early forebrain wiring: genetic dissection using conditional Celsr3 mutant mice.

Development of axonal tracts requires interactions between growth cones and the environment. Tracts such as the anterior commissure and internal capsule are defective in mice with null mutation of Celsr3. We generated a conditional Celsr3 allele, allowing regional inactivation. Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex, subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These observations provide in vivo evidence that Celsr3-mediated interactions between axons and guidepost cells govern axonal tract formation in mammals.

The evolution of cortical development. An hypothesis based on the role of the Reelin signaling pathway

Expression of the genes encoding Reelin and Dab1 during cortical development in turtle, lizard, chick and mammals correlates with architectonic patterns. In all species, Reelin is secreted by marginal zone cells, whereas Dab1, which mediates the response to Reelin, is synthesized by cortical plate neurons. This pattern was presumably present in stem amniotes. In mammals, the cortical plate is radially organized and develops from inside to outside, these features depend on amplification of reelin synthesis in the marginal zone. In lizards, the cortical plate develops from outside to inside, similar to other non-mammals, but is radially organized, with an additional layer of Reelin added in the subcortex. Thus, the Reelin pathway played a key role in cortical architectonic evolution in mammalian and squamate lineages.

A new view of early cortical development

Recently, several genes that regulate the development of the cerebral cortex and are potential pharmacological targets have been cloned. Reelin, an extracellular matrix glycoprotein secreted by Cajal-Retzius cells in the marginal zone, instructs the radial organization of the cortical plate. The response of cortical plate cells to reelin requires the tyrosine kinase adaptor disabled-1 (Dab1). Cyclin-dependent kinase 5 and its activator p35 are necessary for the development of the cortical plate, probably at a later stage than reelin/Dab1. The transcription factor Tbr-1 is essential for differentiation of preplate and Cajal-Retzius cells and for formation of thalamocortical connections, while D1x-1/2 are required for tangential migration. Some neurotrophin systems such as neurotrophin 4, brain-derived neurotrophic factor, and neuregulin and its receptor ErbB are also thought to assist in the regulation of cortical development. In addition, a few genes implicated in human cortical dysplasias have been characterized. LIS1 encodes a protein related to platelet-activating factor acetyl hydrolase that is defective in lissencephaly-1 of the Miller-Dieker type, while the double cortex malformation is related to mutations of a new gene dubbed doublecortn.

Epidermal morphogenesis during progressive in vitro 3D reconstruction at the air-liquid interface.

Keratinocyte monolayers, cultured in immersed conditions, constitute a frequently used in vitro model system to study keratinocytes behaviour in response to environmental assaults. However, monolayers lack the keratinocyte terminal differentiation and the organization of the epidermal tissue, which are observed in vivo. Advancements of in vitro techniques were used to reconstruct three‐dimensional equivalents that mimic human epidermis in terms of layering, differentiation and barrier function. Here, we update a published method and illustrate the progressive morphogenesis responsible for in vitro reconstruction. The analysis of cell proliferation, expression of differentiation markers and barrier efficacy demonstrate the excellent similarity of the reconstructed tissue with normal human epidermis. Availability of epidermal tissue during its reconstruction phase in culture appears crucial for studies intending to challenge the barrier function.

Disabled‐1 mRNA and protein expression in developing human cortex

Disabled‐1 (Dab1) forms part of the Reelin–Dab1 signalling pathway that controls neuronal positioning during brain development; Dab1 deficiency gives rise to a reeler‐like inversion of cortical layers. To establish a timetable of Dab1 expression in developing human brain, Dab1 mRNA and protein expression were studied in prenatal human cortex. The earliest Dab1 signal was detected at 7 gestational weeks (GW), the stage of transition from preplate to cortical plate, suggesting a role of the Reelin–Dab1 signalling pathway in preplate partition. From 12 to 20 GW, the period of maximum cortical migration, Dab1 expression was prominent in the upper tiers of the cortical plate, to decline after midgestation. Radially orientated apical dendrites of Dab1‐expressing neurons indicated a predominant pyramidal phenotype. Pyramidal cells in hippocampus and entorhinal cortex displayed a more protracted time of Dab1 expression compared to neocortex. In addition, at later stages (18–25 GW), Dab1 was also expressed in large neurons scattered throughout intermediate zone and subplate. From 14 to 22 GW, particularly high levels of Dab1 mRNA and protein were observed in cells of the ventricular/subventricular zone displaying the morphology of radial glia. The partial colocalization of vimentin and Dab1 in cells of the ventricular zone supported a radial glia phenotype. The concentration of Dab1 protein in ventricular endfeet and initial portions of radial processes of ventricular‐zone cells points to a possible involvement of Dab1 in neurogenesis. Furthermore, a subset of Cajal–Retzius cells in the marginal zone colocalized Dab1 and Reelin, and may thus represent a novel target of the Reelin–Dab1 signalling pathway.

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.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.