The Ciliogenic Transcription Factor RFX3 Regulates Early Midline Distribution of Guidepost Neurons Required for Corpus Callosum Development

The corpus callosum (CC) is the major commissure that bridges the cerebral hemispheres. Agenesis of the CC is associated with human ciliopathies, but the origin of this default is unclear. Regulatory Factor X3 (RFX3) is a transcription factor involved in the control of ciliogenesis, and Rfx3–deficient mice show several hallmarks of ciliopathies including left–right asymmetry defects and hydrocephalus. Here we show that Rfx3–deficient mice suffer from CC agenesis associated with a marked disorganisation of guidepost neurons required for axon pathfinding across the midline. Using transplantation assays, we demonstrate that abnormalities of the mutant midline region are primarily responsible for the CC malformation. Conditional genetic inactivation shows that RFX3 is not required in guidepost cells for proper CC formation, but is required before E12.5 for proper patterning of the cortical septal boundary and hence accurate distribution of guidepost neurons at later stages. We observe focused but consistent ectopic expression of Fibroblast growth factor 8 (Fgf8) at the rostro commissural plate associated with a reduced ratio of GLIoma-associated oncogene family zinc finger 3 (GLI3) repressor to activator forms. We demonstrate on brain explant cultures that ectopic FGF8 reproduces the guidepost neuronal defects observed in Rfx3 mutants. This study unravels a crucial role of RFX3 during early brain development by indirectly regulating GLI3 activity, which leads to FGF8 upregulation and ultimately to disturbed distribution of guidepost neurons required for CC morphogenesis. Hence, the RFX3 mutant mouse model brings novel understandings of the mechanisms that underlie CC agenesis in ciliopathies

The dynamic cilium in human diseases

Cilia are specialized organelles protruding from the cell surface of almost all mammalian cells. They consist of a basal body, composed of two centrioles, and a protruding body, named the axoneme. Although the basic structure of all cilia is the same, numerous differences emerge in different cell types, suggesting diverse functions. In recent years many studies have elucidated the function of 9+0 primary cilia. The primary cilium acts as an antenna for the cell, and several important pathways such as Hedgehog, Wnt and planar cell polarity (PCP) are transduced through it. Many studies on animal models have revealed that during embryogenesis the primary cilium has an essential role in defining the correct patterning of the body. Cilia are composed of hundreds of proteins and the impairment or dysfunction of one protein alone can cause complete loss of cilia or the formation of abnormal cilia. Mutations in ciliary proteins cause ciliopathies which can affect many organs at different levels of severity and are characterized by a wide spectrum of phenotypes. Ciliary proteins can be mutated in more than one ciliopathy, suggesting an interaction between proteins. To date, little is known about the role of primary cilia in adult life and it is tempting to speculate about their role in the maintenance of adult organs. The state of the art in primary cilia studies reveals a very intricate role. Analysis of cilia-related pathways and of the different clinical phenotypes of ciliopathies helps to shed light on the function of these sophisticated organelles. The aim of this review is to evaluate the recent advances in cilia function and the molecular mechanisms at the basis of their activity

Pax6 controls centriole maturation in cortical progenitors through Odf2

Cortical glutamatergic neurons are generated by radial glial cells (RGCs), specified by the expression of transcription factor (TF) Pax6, in the germinative zones of the dorsal telencephalon. Here, we demonstrate that Pax6 regulates the structural assembly of the interphase centrosomes. In the cortex of the Pax6-deficient Small eye (Sey/Sey) mutant, we find a defect of the appendages of the mother centrioles, indicating incomplete centrosome maturation. Consequently, RGCs fail to generate primary cilia, and instead of staying in the germinative zone for renewal, RGCs detach from the ventricular surface thus affecting the interkinetic nuclear migration and they exit prematurely from mitosis. Mechanistically, we show that TF Pax6 directly regulates the activity of the Odf2 gene encoding for the appendage-specific protein Odf2 with a role for the assembly of mother centriole. Our findings demonstrate a molecular mechanism that explains important characteristics of the centrosome disassembly and malfunctioning in developing cortex lacking Pax6

Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation

The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Upon birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia thus provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. Here we show that Arl13b, a cilia-specific small GTPase mutated in Joubert syndrome patients, is critical for the initial formation of the polarized radial progenitor scaffold. Through developmental stage-specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b leads to a reversal in the apical-basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulates ciliary signaling necessary for radial glial polarity. Our findings demonstrate that Arl13b signaling in primary cilia is important for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome-related ciliopathies

Developmental disruptions underlying brain abnormalities in ciliopathies

Primary cilia are essential conveyors of signals underlying major cell functions. Cerebral cortical progenitors and neurons have a primary cilium. The significance of cilia function for brain development and function is evident in the plethora of developmental brain disorders associated with human ciliopathies. Nevertheless, the role of primary cilia function in corticogenesis remains largely unknown. Here we delineate the functions of primary cilia in the construction of cerebral cortex and their relevance to ciliopathies, using an shRNA library targeting ciliopathy genes known to cause brain disorders, but whose roles in brain development are unclear. We used the library to query how ciliopathy genes affect distinct stages of mouse cortical development, in particular neural progenitor development, neuronal migration, neuronal differentiation and early neuronal connectivity. Our results define the developmental functions of ciliopathy genes and delineate disrupted developmental events that are integrally related to the emergence of brain abnormalities in ciliopathies