title: Multiple Slits regulate the development of midline glial populations and the corpus callosum.
abstract: 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.
authors.affiliation: The Queensland Brain Institute, The University of Queensland, Brisbane 4072, Australia. divya.unni@gmail.com
authors.forename: Divya K
authors.initials: DK
authors.lastname: Unni
authors.forename: Michael
authors.initials: M
authors.lastname: Piper
authors.forename: Randal X
authors.initials: RX
authors.lastname: Moldrich
authors.forename: Ilan
authors.initials: I
authors.lastname: Gobius
authors.forename: Sha
authors.initials: S
authors.lastname: Liu
authors.forename: Thomas
authors.initials: T
authors.lastname: Fothergill
authors.forename: Amber-Lee S
authors.initials: AL
authors.lastname: Donahoo
authors.forename: John M
authors.initials: JM
authors.lastname: Baisden
authors.forename: Helen M
authors.initials: HM
authors.lastname: Cooper
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Richards
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D018920:Coculture Techniques; D003337:Corpus Callosum / Q000166:cytology / Q000196:embryology* / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D008279:Magnetic Resonance Imaging; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D009457:Neuroglia / Q000166:cytology / Q000502:physiology; D011971:Receptors, Immunologic / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein; C120010:Slit1 protein, mouse; C432251:Slit3 protein, mouse