Appropriate Bmp7 levels are required for the differentiation of midline guidepost cells involved in corpus callosum formation

Guidepost cells are essential structures for the establishment of major axonal tracts. How these structures are specified and acquire their axon guidance properties is still poorly understood. Here, we show that in mouse embryos appropriate levels of Bone Morphogenetic Protein 7 (Bmp7), a member of the TGF-β superfamily of secreted proteins, are required for the correct development of the glial wedge, the indusium griseum, and the subcallosal sling, three groups of cells that act as guidepost cells for growing callosal axons. Bmp7 is expressed in the region occupied by these structures and its genetic inactivation in mouse embryos caused a marked reduction and disorganization of these cell populations. On the contrary, infusion of recombinant Bmp7 in the developing forebrain induced their premature differentiation. In both cases, changes were associated with the disruption of callosal axon growth and, in most animals fibers did not cross the midline forming typical Probst bundles. Addition of Bmp7 to cortical explants did not modify the extent of their outgrowth nor their directionality, when explants were exposed to a focalized source of the protein. Together, these results indicate that Bmp7 is indirectly required for corpus callosum formation by controlling the timely differentiation of its guidepost cells.     

Multiple Slits regulate the development of midline glial populations and the corpus callosum

The Slit molecules are chemorepulsive ligands that regulate axon guidance at the midline of both vertebrates and invertebrates. In mammals, there are three Slit genes, but only Slit2 has been studied in any detail with regard to mammalian brain commissure formation. Here, we sought to understand the relative contributions that Slit proteins make to the formation of the largest brain commissure, the corpus callosum. Slit ligands bind Robo receptors, and previous studies have shown that Robo1(-/-) mice have defects in corpus callosum development. However, whether the Slit genes signal exclusively through Robo1 during callosal formation is unclear. To investigate this, we compared the development of the corpus callosum in both Slit2(-/-) and Robo1(-/-) mice using diffusion magnetic resonance imaging. This analysis demonstrated similarities in the phenotypes of these mice, but crucially also highlighted subtle differences, particularly with regard to the guidance of post-crossing axons. Analysis of single mutations in Slit family members revealed corpus callosum defects (but not complete agenesis) in 100% of Slit2(-/-) mice and 30% of Slit3(-/-) mice, whereas 100% of Slit1(-/-); Slit2(-/-) mice displayed complete agenesis of the corpus callosum. These results revealed a role for Slit1 in corpus callosum development, and demonstrated that Slit2 was necessary but not sufficient for midline crossing in vivo. However, co-culture experiments utilising Robo1(-/-) tissue versus Slit2 expressing cell blocks demonstrated that Slit2 was sufficient for the guidance activity mediated by Robo1 in pre-crossing neocortical axons. This suggested that Slit1 and Slit3 might also be involved in regulating other mechanisms that allow the corpus callosum to form, such as the establishment of midline glial populations. Investigation of this revealed defects in the development and dorso-ventral positioning of the indusium griseum glia in multiple Slit mutants. These findings indicate that Slits regulate callosal development via both classical chemorepulsive mechanisms, and via a novel role in mediating the correct positioning of midline glial populations. Finally, our data also indicate that some of the roles of Slit proteins at the midline may be independent of Robo signalling, suggestive of additional receptors regulating Slit signalling during development.     

The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191(+/-) mice are normal and fertile. Homozygous Zfp191(-/-) embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191(-/-) and Zfp191(+/-) embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191(-/-) cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191(+/-) intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation.     

The degenerative process during development of the corpus callosum

The ontogenesis of the corpus callosum is inextricably linked with the various processes controlling prosencephalic development. Our study is based on series of frontal and sagittal sections through the prosencephalon of 16 and 17 day mouse embryos and on ultrathin sections of the septum, particularly of the zone where the callosal fibres cross. The septum, which contains the first callosal fibres, does not undergo the fusional process described by other authors. The passage of pioneer fibres from one hemisphere to the other is preceded by the degeneration and death of the atrocytes of the cortical plate in the fundus of the interhemispheric issure, and by proliferation of the subependymal cells. The proliferation and migration of the subependymal cells from the medial angles of the lateral ventricles may well assist the passage of pioneering callosal fibres.     

The transcription factor Pax6 is required for development of the diencephalic dorsal midline secretory radial glia that form the subcommissural organ.

During brain development, Pax6 is expressed in specific regions of the diencephalon including secretory cells of the subcommissural organ (SCO), a circumventricular organ at the forebrain-midbrain boundary that originates from the pretectal dorsal midline neuroepithelial cells beneath the posterior commissure (PC). Homozygous small eye (Sey/Sey) mice lack functional Pax6 protein and fail to develop the SCO, a normal PC and the pineal gland. Small eye heterozygotes (Sey/+) show defective development of the SCO's basal processes which normally penetrate the PC, indicating that normal development of the gland requires normal Pax6 gene-dosage. A correlation between the defects of SCO formation and altered R- and OB-cadherin expression patterns in the SCO is observed in mutants suggesting a role for cadherins in SCO development.