Role of Slit and Robo in the development of midline glia and corpus callosum

Slits, a family of secreted proteins, are crucial for axon guidance during the development of the central nervous system. In rats, Slit1, Slit2 and Slit3 mRNA are expressed in midline glial populations surrounding the developing corpus callosum (Marillat et al., 2002). A previous study has shown that mice lacking the Slit2 gene have an acallosal phenotype (Bagri et al., 2002). However, the role of Slit1 and Slit3 in the development of the corpus callosum is unknown. In the present study expression analysis of Slit1, Slit2 and Slit3 mRNA performed on mice, showed that these molecules were expressed around the developing corpus callosum. To understand the role each Slit might play in the development of the corpus callosum, 4 Slit mutant mouse strains, Slit1-/-, Slit2-/-, Slit1-/-;Slit2-/- and Slit3-/- mice were analysed. This study confirmed that Slit2-/- mice displayed dysgenesis of the corpus callosum across its rostro-caudal extent. Mice lacking the Slit1 gene did not exhibit an acallosal phenotype, however, Slit1-/-;Slit2-/- mice displayed a more severe phenotype compared to mice deficient in Slit2 alone, indicating a role for Slit1 in corpus callosum development. A subset of the Slit3-/- mice displayed an axon guidance defect in the corpus callosum, where the callosal axons in the dorsal region of the tract failed to cross the midline. This defect was observed in embryonic as well as postnatal ages. The callosal axon guidance defects in Slit2-/- and Slit1-/-;Slit2-/- mice were also observed at earlier stages during the development of the cingulate pioneering axons of the corpus callosum. Apart from axon guidance Slit1, Slit2 and Slit3 were involved in the development of the glial populations at the midline of the cerebral cortex. Slit2-/- and Slit1-/-;Slit2-/- mice exhibited a positional defect where the indusium griseum glia was ventrally displaced. The glial-positioning defect in Slit1-/-;Slit2-/- mice was more severe compared to Slit2-/- mice. No positional defect was observed in Slit3-/- mice, however the development of the indusium griseum glia was affected. To compare the axon guidance phenotype of the Slit2-/- mice, and the knockout of its known receptor Robo1-/- mice, diffusion tensor magnetic resonance imaging and tractography was performed on Slit2-/-, and Robo1-/- mice. These experiments showed that the axon guidance defect in Slit2-/- mice is similar, but more severe compared to Robo1-/- mice. The axons of the CC are displaced rostrocaudally in both Slit2-/- and Robo1-/- mice. In Robo1-/- mice only the pre-crossing axons are affected while in Slit2-/- mice both the pre-crossing as well as post-crossing axons are affected. These results suggest a possible role for Slit in the development of the telencephalic midline, where it is first involved in the proper positioning and development of the midline glial populations and then acts as an axon guidance molecule for the crossing of callosal axons to their homotypic targets in the contralateral hemisphere

Analysis of the growth cone turning assay for studying axon guidance

The "pipette" or "growth cone turning" assay is widely used for studying how axons respond to diffusible guidance cues in their environment. However, little quantitative analysis has been presented of the gradient shapes produced by this assay, or how they depend on parameters of the assay. Here we used confocal microscopy of fluorescent gradients to characterize these shapes in 3 dimensions. We found that the shape, and more specifically the concentration at the position usually occupied by the growth cone in this assay, varied in sometimes unexpected ways with the molecular weight of the diffusible factor, charge, pulse duration and pulse frequency. These results suggest that direct observation of the gradient of the particular guidance factor under consideration may be necessary to quantitatively determine the signal to which the growth cone is responding

Solution processable deep-red phosphorescent Pt(II) complex: direct conversion from its Pt(IV) species via a base promoted reduction

Color purity is a critical prerequisite for full color displays. Creation of deep-red phosphorescent materials with high PLQYs is particularly challenging because of “energy gap law”. Simultaneously achieving high yielding solution-processable Pt(II) complexes further complicates this challenge. In this report, we developed a high-yielding synthetic route to a solution processable/deep-red Pt(II) complex with a rigid tetradentate structure, in which we identified octahedral Pt(IV) as a major side product formed under the standard complexation conditions. We managed to effectively transform the octahedral Pt(IV) into a luminescent deep-red square planar Pt(II) complex through a base promoted reduction. We found the Pt(II) complex has high solution and blend film PLQYs. X-ray crystal structure and DFT calculations of the Pt(II) complex showed that perpendicular orientation of molecular dipoles enhanced the luminescence properties. In neat films, there was no luminescence enhancement due to interdigitation of the attached hexyloxy tails, preventing strong Pt∙∙∙Pt interactions in the solid state. Solution processed OLEDs based on the Pt(II) complex showed a low turn-on voltage of 3.3 V (at 1 cd/m2) with a maximum brightness of 2,000 cd/m2 and a maximum EQE of ≈6% (4% at 100 cd/m2). A narrow electroluminescence with a full-width-at-half-maximum of ≈50 nm was observed with a peak at 623 nm, and provided a deep-red emission with 1931 CIE co-ordinates of (0.65, 0.35). Transient electroluminescence measurements were used to investigate the EQE roll-off of the OLEDs

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. (C) 2012 Elsevier Inc. All rights reserved

International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases

© 2017 The Author(s) Provision of a molecularly confirmed diagnosis in a timely manner for children and adults with rare genetic diseases shortens their “diagnostic odyssey,” improves disease management, and fosters genetic counseling with respect to recurrence risks while assuring reproductive choices. In a general clinical genetics setting, the current diagnostic rate is approximately 50%, but for those who do not receive a molecular diagnosis after the initial genetics evaluation, that rate is much lower. Diagnostic success for these more challenging affected individuals depends to a large extent on progress in the discovery of genes associated with, and mechanisms underlying, rare diseases. Thus, continued research is required for moving toward a more complete catalog of disease-related genes and variants. The International Rare Diseases Research Consortium (IRDiRC) was established in 2011 to bring together researchers and organizations invested in rare disease research to develop a means of achieving molecular diagnosis for all rare diseases. Here, we review the current and future bottlenecks to gene discovery and suggest strategies for enabling progress in this regard. Each successful discovery will define potential diagnostic, preventive, and therapeutic opportunities for the corresponding rare disease, enabling precision medicine for this patient population