Barhl2 limits growth of the diencephalic primordium through Caspase3 inhibition of beta-catenin activation.

Little is known about the respective contributions of cell proliferation and cell death to the control of vertebrate forebrain growth. The homeodomain protein barhl2 is expressed in the diencephalons of Xenopus, zebrafish, and mouse embryos, and we previously showed that Barhl2 overexpression in Xenopus neuroepithelial cells induces Caspase3-dependent apoptosis. Here, barhl2 is shown to act as a brake on diencephalic proliferation through an unconventional function of Caspase3. Depletion of Barhl2 or Caspase3 causes an increase in diencephalic cell number, a disruption of the neuroepithelium architecture, and an increase in Wnt activity. Surprisingly, these changes are not caused by decreased apoptosis but instead, are because of an increase in the amount and activation of ß-catenin, which stimulates excessive neuroepithelial cell proliferation and induces defects in ß-catenin intracellular localization and an up-regulation of axin2 and cyclinD1, two downstream targets of ß-catenin/T-cell factor/lymphoïd enhancer factor signaling. Using two different sets of complementation experiments, we showed that, in the developing diencephalon, Caspase3 acts downstream of Barhl2 in limiting neuroepithelial cell proliferation by inhibiting ß-catenin activation. Our data argue that Bar homeodomain proteins share a conserved function as cell type-specific regulators of Caspase3 activities.

Integration of H-2Z1, a somatosensory cortex-expressed transgene, interferes with the expression of the Satb1 and Tbc1d5 flanking genes and affects the differentiation of a subset of cortical interneurons.

H-2Z1 is an enhancer trap transgenic mouse line in which the lacZ reporter delineates the somatosensory area of the cerebral cortex where it is expressed in a subset of layer IV neurons. In the search of somatosensory specific genes or regulatory sequences, we mapped the H-2Z1 transgene insertion site to chromosome 17, 100 and 460 kb away from Tbc1d5 and Satb1 flanking genes. We show here that insertion of the H-2Z1 transgene results in three distinct outcomes. First, a genetic background-sensitive expression of lacZ in several brain and body structures. While four genes in a 1 Mb region around the insertion are expressed in the barrel cortex, H-2Z1 expression resembles more that of its two direct neighbors. Moreover, H-2Z1 closely reports most of the body and brain expression sites of the Satb1 chromatin remodeling gene including tooth buds, thymic epithelium, pontine nuclei, fastigial cerebellar nuclei, and cerebral cortex. Second, the H-2Z1 transgene causes insertional mutagenesis of Tbc1d5 and Satb1, leading to a strong decrease in their expressions. Finally, insertion of H-2Z1 affects the differentiation of a subset of cortical GABAergic interneurons, a possible consequence of downregulation of Satb1 expression. Thus, the H-2Z1 "somatosensory" transgene is inserted in the regulatory landscape of two genes highly expressed in the developing somatosensory cortex and reports for a subdomain of their expression profiles. Together, our data suggest that regulation of H-2Z1 expression results from local and remote genetic interactions.

Origins and control of the differentiation of inhibitory interneurons and glia in the cerebellum.

Cerebellar GABAergic interneurons and glia originate from progenitors that delaminate from the ventricular neuroepithelium and proliferate in the prospective white matter. Even though this population of progenitor cells is multipotent as a whole, clonal analysis indicates that different lineages are already separated during postnatal development and little is known about the mechanisms that regulate the specification and differentiation of these cerebellar types at earlier stages. Here, we investigate the role of Ascl1 in the development of inhibitory interneurons and glial cells in the cerebellum. This gene is expressed by maturing oligodendrocytes and GABAergic interneurons and is required for the production of appropriate quantities of these cells, which are severely reduced in Ascl1(-/-) mouse cerebella. Nevertheless, the two lineages are not related and the majority of oligodendrocytes populating the developing cerebellum actually derive from extracerebellar sources. Targeted electroporation of Ascl1-expression vectors to ventricular neuroepithelium progenitors enhances the production of interneurons and completely suppresses astrocytic differentiation, whereas loss of Ascl1 function has opposite effects on both cell types. Our results indicate that Ascl1 directs ventricular neuroepithelium progenitors towards inhibitory interneuron fate and restricts their ability to differentiate along the astroglial lineage.

Cerebral cortex development: From progenitors patterning to neocortical size during evolution.

The central nervous system is composed of thousands of distinct neurons that are assembled in a highly organized structure. In order to form functional neuronal networks, distinct classes of cells have to be generated in a precise number, in a spatial and temporal hierarchy and to be positioned at specific coordinates. An exquisite coordination of appropriate growth of competent territories and their patterning is required for regionalization and neurogenesis along both the anterior-posterior and dorso-ventral axis of the developing nervous system. The neocortex represents the brain territory that has undergone a major increase in its relative size during the course of mammalian evolution. In this review we will discuss how the fine tuning of growth and cell fate patterning plays a crucial role in the achievement of the final size of central nervous system structures and how divergence might have contributed to the surface increase of the cerebral cortex in mammals. In particular, we will describe how lack of precision might have been instrumental to neocortical evolution.

Multiple origins of Cajal-Retzius cells at the borders of the developing pallium.

Cajal-Retzius cells are critical in cortical lamination, but very little is known about their origin and development. The homeodomain transcription factor Dbx1 is expressed in restricted progenitor domains of the developing pallium: the ventral pallium (VP) and the septum. Using genetic tracing and ablation experiments in mice, we show that two subpopulations of Reelin(+) Cajal-Retzius cells are generated from Dbx1-expressing progenitors. VP- and septum-derived Reelin(+) neurons differ in their onset of appearance, migration routes, destination and expression of molecular markers. Together with reported data supporting the generation of Reelin(+) cells in the cortical hem, our results show that Cajal-Retzius cells are generated at least at three focal sites at the borders of the developing pallium and are redistributed by tangential migration. Our data also strongly suggest that distinct Cajal-Retzius subtypes exist and that their presence in different territories of the developing cortex might contribute to region-specific properties.

The particles of the embryonic cerebrospinal fluid: how could they influence brain development?

During brain development, the embryonic cerebrospinal fluid (E-CSF) allows brain expansion and promotes neuroepithelial cell survival, proliferation or differentiation. Previous analyses of E-CSF content have revealed a high protein concentration and the presence of membranous particles. The role of these particles in the E-CSF remains poorly investigated. In this study we showed that the E-CSF contains at least two pools of particles: lipoproteins and exosome-like particles. We showed that these two populations of particles strongly interact with neuropithelial cells via an endocytic process, which display regional specificity along the developing neural tube. Finally, we explore and discuss the possibility that these interactions may influence brain development through the regulation of morphogen and growth factor signaling transduction.

Novel sites of expression of the bHLH gene NSCL1 in the developing nervous system.

We report on novel sites of expression of the bHLH transcription factor NSCL1 in the developing forebrain, hindbrain and spinal cord in chick and mouse. In the hindbrain in particular, NSCL1 is the first bHLH transcription factor detected so far in rhombomere boundaries and its expression is coincident with boundary formation and maintenance. Novel sites of expression of this gene include the hippocampus, septum, tectum and hypothalamic nuclei. NSCL1 is thus expressed in various neuronal populations that are either not actively proliferating or postmitotic.

Regionalization of the isthmic and cerebellar primordia.

The complex migrations of neurons born in the dorsal neural tube of the isthmic and rhombomere l (rl) domains complicate the delineation of the cerebellar primordium. We show that Purkinje cells (P) are likely generated over a wide territory before gathering in the future cerebellar primordium under the developing external granular layer. Later expansion of the cerebellum over a restricted ependymal domain could rely on mutual interations between P cells and granule cell progenitors (GCP). P are attracted by GCP and in turn stimulate their proliferation, increasing the surface of the developing cortex. At later stages, regionalization of the developing and adult cerebellar cortex can be detected through regional variations in the distribution of several P cell markers. Whether and how the developmental and adult P subtypes are related is still unknown and it is unclear if they delineate the same sets of cerebellar subdivisions. We provide evidence that the early P regionalization is involved in intrinsic patterning of the cerebellar primordium, in particular it relate to the organization of the corticonuclear connection. We propose that the early P regionalization provides a scaffold to the mature P regionalization but that the development of functional afferent connections induces a period of P plasticity during which the early regional identity of P could be remodeled.

Novel sites of expression of the bHLH gene NSCL1 in the developing nervous system.

We report on novel sites of expression of the bHLH transcription factor NSCL1 in the developing forebrain, hindbrain and spinal cord in chick and mouse. In the hindbrain in particular, NSCL1 is the first bHLH transcription factor detected so far in rhombomere boundaries and its expression is coincident with boundary formation and maintenance. Novel sites of expression of this gene include the hippocampus, septum, tectum and hypothalamic nuclei. NSCL1 is thus expressed in various neuronal populations that are either not actively proliferating or postmitotic.

Positive and negative regulations by FGF8 contribute to midbrain roof plate developmental plasticity.

The roof plate (RP) of the midbrain shows an unusual plasticity, as it is duplicated or interrupted by experimental manipulations involving the mid/hindbrain organizer or FGF8. In previous experiments, we have found that FGF8 induces a local patterning center, the isthmic node, that is essential for the local development of a RP. Here, we show that the plasticity of the midbrain RP derives from two apparently antagonistic influences of FGF8. On the one hand, FGF8 widens beyond the neural folds the competence of the neuroepithelium to develop a RP by inducing the expression of LMX1B and WNT1. Ectopic overexpression of these two factors is sufficient to induce widely the expression of markers of the mature RP in the midbrain. On the other hand, FGF8 exerts a major destabilizing influence on RP maturation by controlling signaling by members of the TGFbeta superfamily belonging to the BMP, GDF and activin subgroups. We show in particular that FGF8 tightly modulates follistatin expression, thus progressively restraining the inhibitory influence of activin B on RP differentiation. These regulations, together with FGF8 triggered apoptosis, allow the formation of a RP progress zone at some distance from the FGF8 source. Posterior elongation of the RP is permitted when the source of FGF8 withdraws. Growth of the posterior midbrain neuroepithelium and convergent extension movements induced by FGF8 both contribute to increase the distance between the source of FGF8 and the maturing RP. Normally, the antagonistic regulatory interactions spread smoothly across the midbrain. Plasticity of midbrain RP differentiation probably results from an experimentally induced imbalance between regulatory pathways.

Does the isthmic organizer influence D/V patterning of the midbrain?

Early brain and spinal cord regionalization along the dorsoventral axis are thought to be governed by similar mechanisms. Subsequently, the size of the alar plate of the neural tube increases dramatically in the midbrain and anterior forebrain, compared to the spinal cord. This suggests that additional mechanisms may be required to refine A/P and D/V patterning in these structures. The isthmic organizer is a signaling center that controls both growth and patterning in the midbrain and anterior hindbrain through the production of several secreted molecules, in particular FGF8. Several studies have indicated that the isthmic organizer is involved in the positioning and development of the midbrain roof and floor plates, the two structures that respectively mark the dorsal and ventral axis of the neural tube. It remains unclear whether its influence on axis formation in the midbrain is a consequence of a more general function of the isthmic organizer/FGF8 as a modulator of DV patterning or if selection of an axis is a necessary and general by-product of its organizing function not directly related to D/V patterning. In this paper, we review the current data supporting each possibility.

The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures.

During vertebrate development, an organizing signaling center, the isthmic organizer, forms at the boundary between the midbrain and hindbrain. This organizer locally controls growth and patterning along the anteroposterior axis of the neural tube. On the basis of transplantation and ablation experiments in avian embryos, we show here that, in the caudal midbrain, a restricted dorsal domain of the isthmic organizer, that we call the isthmic node, is both necessary and sufficient for the formation and positioning of the roof plate, a signaling structure that marks the dorsal midline of the neural tube and that is involved in its dorsoventral patterning. This is unexpected because in other regions of the neural tube, the roof plate has been shown to form at the site of neural fold fusion, which is under the influence of epidermal ectoderm derived signals. In addition, the isthmic node contributes cells to both the midbrain and hindbrain roof plates, which are separated by a boundary that limits cell movements. We also provide evidence that mid/hindbrain roof plate formation involves homeogenetic mechanisms. Our observations indicate that the isthmic organizer orchestrates patterning along the anteroposterior and the dorsoventral axis.

Expression of CRABP I mRNA in fastigial cells of the developing cerebellum.

The expression of the cellular retinoic acid binding protein type I (CRABP I) was examined in the early phase of cerebellar development in the mouse. The CRABP I was expressed from embryonic day (E) 10.5 to E15.5 in the cerebellar plate. The expression was diffused at E10.5-E11.5 and thereafter localized in a small rostrodorsal area of the cerebellar territory of both sides. By using in situ hybridization and both immunohistochemistry and carbocyanine tracing procedures, we identified the fastigial cells as the population that expresses CRABP I in the cerebellum. The results suggest that these cells play a critical role in the early development of the cerebellum.

Early differences in axonal outgrowth, cell migration and GABAergic differentiation properties between the dorsal and lateral cortex.

The regionalization of the cerebral cortex proceeds gradually from early embryonic stages under the control of transcription factors that are expressed in gradients. Two phases can be distinguished at the beginning of cortical development: the genesis of a precocious and transient structure, the preplate, which is followed by development of the cortical plate within the preplate. Cellular indices of early regionalization have not yet been described either in the preplate or in the early cortical plate. In the present study, we identify two regions, lateral and dorsal, in the mouse cortex embryo, which differ strongly in the functional properties of their early neurons. By using culture experiments and grafts on organotypic slices, we show that the earliest neurons in the dorsal cortex extend axons before and more rapidly than the earliest neurons in the lateral cortex. In contrast to the lateral cortex, the dorsal cortex differentiates neurons migrating along axons in vitro. These cells express markers of the GABAergic lineage. Early differences between the two regions suggest that the dorsal part of the cortex generates early neurons with particular intrinsic properties that may in turn specifically influence the later development of the cortical plate in this domain.

A combination of chain and neurophilic migration involving the adhesion molecule TAG-1 in the caudal medulla.

Neuronal populations destined to form several precerebellar nuclei are generated by the rhombic lip in the caudal hindbrain. These immature neurons gather into the olivary and the superficial migratory streams and migrate tangentially around the hindbrain to reach their final position. We focus on the cells of the superficial stream that migrate ventrally, cross the midline and form the lateral reticular (LRN) and external cuneate (ECN) nuclei. The cells of the superficial steam are preceded by long leading processes; in the dorsal neural tube, they migrate in close apposition to each other and form distinct chains, whereas they disperse and follow Tuj-1 immunoreactive axons on reaching the ventral hindbrain. This suggests that, in the superficial stream, neuronal migration combines both homotypic and heterotypic mechanisms. We also show that the adhesion molecule TAG-1 is expressed by the migrating cells. Blocking TAG-1 function results in alterations in the superficial migration, indicating that TAG-1 is involved in the superficial migration. Other members of the immunoglobulin superfamily and known ligands of TAG-1 are also expressed in the region of the migration but are not involved in the migration. These findings provide evidence that the TAG-1 protein is involved as a contact-dependent signal guiding not only axonal outgrowth but also cell migration.

The isthmic neuroepithelium is essential for cerebellar midline fusion.

The cerebellum comprises a medial domain, called the vermis, flanked by two lateral subdivisions, the cerebellar hemispheres. Normal development of the vermis involves fusion of two lateral primordia on the dorsal midline. We investigated how the cerebellum fuses on the midline by combining a study of mid/hindbrain cell movements in avian embryos with the analysis of cerebellar fusion in normal and mutant mouse embryos. We found that, in avian embryos, divergent cell movements originating from a restricted medial domain located at the mid/hindbrain boundary produce the roof plate of the mid/hindbrain domain. Cells migrating anteriorly from this region populate the caudal midbrain roof plate whereas cells migrating posteriorly populate the cerebellar roof plate. In addition, the adjacent paramedial isthmic neuroepithelium also migrates caudalward and participates in the formation of the cerebellar midline region. We also found that the paramedial isthmic territory produces two distinct structures. First, the late developing velum medullaris that intervenes between the vermis and the midbrain, and second, a midline domain upon which the cerebellum fuses. Elimination or overgrowth of this isthmic domain in Wnt1(sw/sw) and En1(+/Otx2lacZ) mutant mice, respectively, impair cerebellar midline fusion. Because the isthmus-derived midline cerebellar domain displays a distinct expression pattern of genes involved in BMP signaling, we propose that the isthmus-derived cells provide both a substratum and signals that are essential for cerebellar fusion.