Molecular mechanisms involved in the cerebral cortex development and their implications in neurodevelopmental disorders

Abstract

During the process of corticogenesis, cortical radial glial cells (RGs) generate sequentially most of the distinct types of neurons and glial cells that will populate the neocortex. The generation of these cells at the proper time and adequate numbers is key for the establishment of fully functional cerebral circuits. During the phase of neuronal production, RGs experience important changes in morphology, division mode and fate of their neuronal progeny. To provide evidence of the genetic networks underlying these changes, in the first part of this work we have compared the expression of protein-coding genes at the onset of neurogenesis (embryonic day (E)11.5) and during middle-late neurogenesis (E15.5). We found that the expression levels of 25% of these genes are regulated during neurogenesis, and a quarter of them are targets of developmentally regulated miRNAs. Down-regulated genes at E15.5 are mainly associated to cell cycle regulation, while most of the up-regulated genes encode for ion-transport proteins that may be involved in modulating the membrane potential of RGs. Moreover, we found significant up-regulation of synapse-related genes, supporting published reports that claim for the existence of a transcriptional “pre-patterning” in RGs. Among the regulated genes, we found significant enrichment in TEAD2 targets. YAP-TEAD transcriptional activity is regulated by mechanical cues and we found that a great number of differentially expressed genes in RGs encode for structural extracellular matrix (ECM) proteins, suggesting a dynamic remodelling of the ECM along neurogenesis. This remodelling likely varies the physical properties of the ECM, and thereby YAP-TEAD transcriptional activity in RGs. The integrated analysis of the developmental regulated mRNAs and miRNAs suggests that let-7 miRNAs are involved in the regulation of collagen and other structural ECM genes. Lastly, our RNA-seq data shows that alternative splicing (AS) is also developmentally regulated in RGs and affects the expression of splicing events in 19% of the expressed protein-coding genes. Functional enrichment analysis of these genes indicates that AS in RGs may be involved in silencing synapse-related genes and the regulation of critical cellular functions such as the biogenesis of the cilium. In the second part of this work, we addressed the effects of DYRK1A haploinsufficiency in the generation and differentiation of cortical glial cells. DYRK1A is a kinase that has a conserved role in brain growth through evolution and regulates the generation, survival and differentiation of different neuron types. Mutations in DYRK1A cause a syndrome, named DYRK1A-haploinsufficiency syndrome, that is characterized by the presence of microcephaly and other clinical features including intellectual disability, autistic traits and epilepsy. Gliosis and hypomyelination have also been reported in affected children. Previous work of the laboratory has shown that the Dyrk1a+/- mouse mimics several of the phenotypic traits of the syndrome, including an increased number of astrocytes and hypomyelination of the corpus callosum (CC). Here, we show that the augmented number of astrocytes in Dyrk1a+/- neocortices results from an enhanced developmental astrogliogenesis. Moreover, we have observed a decreased generation of oligodendrocyte precursor cells (OPCs) in Dyrk1a+/- embryos that correlates with a delay in the appearance of oligodendrocytes in the CC. Focal demyelination experiments in the adult CC indicate that Dyrk1a+/- OPCs differentiate properly, suggesting that the oligodendroglial phenotype in Dyrk1a+/- mutants results from alterations in the generation of OPCs during development. In addition to hypomyelination, the CC of adult Dyrk1a+/- mice shows altered proportion of the myelin proteins PLP and MBP, shorter nodes of Ranvier and a deficit in the expression of the nodal sodium channel NAV1.6. Collectively, these results show that deficits in the generation and differentiation of glial cells may contribute to the neurological defects reported in individuals with DYRK1A-haploinsufficiency syndrome.

La mayoría de las neuronas y células gliales de la corteza cerebral se generan secuencialmente durante el desarrollo a partir de progenitores pluripotentes denominados glía radial (GR). La correcta generación y diferenciación de estas células es crítica para el establecimiento de circuitos funcionales. En la primera parte de esta Tesis, hemos estudiado los cambios de expresión génica que tienen lugar en la GR durante la neurogénesis. Los resultados obtenidos muestran cambios de expresión en el 25% de los genes codificantes e indican que la expresión de una cuarta parte de éstos está regulada por micro-ARNs. Entre los genes expresados diferencialmente encontramos un enriquecimiento en targets del complejo YAP-TEAD, cuya actividad transcripcional responde a estímulos mecánicos. Además, encontramos enriquecimiento en genes que codifican componentes de la matriz extracelular (MEC), sugiriendo que cambios en las propiedades físicas de ésta podrían modular la actividad YAP-TEAD en la GR. Por último, nuestros resultados indican que la regulación por splicing alternativo durante la neurogénesis es muy extensa (afectando al 19% de los genes codificantes) y podría tener un papel en silenciar genes neuronales en la GR y en otras funciones como la biogénesis del cilio. En la segunda parte, hemos estudiado los efectos de la haploinsuficiencia de DYRK1A en la generación y diferenciación de células gliales. DYRK1A codifica una quinasa con funciones clave en el desarrollo cerebral. Mutaciones de novo en DYRK1A causan un síndrome de discapacidad intelectual y autismo. Los cerebros de niños afectados presentan gliosis e hipomielinización. El ratón Dyrk1a+/- recapitula varios de los fenotipos del síndrome, incluyendo un incremento de astrocitos cerebrales e hipomielinización del cuerpo calloso (CC). Nuestros estudios en este modelo indican que la generación de células gliales está alterada durante el desarrollo y correlaciona con un retraso en la aparición de oligodendrocitos en el CC. La corteza adulta del ratón Dyrk1a+/- muestra alteraciones en los nodos de Ranvier y en la composición de proteínas de la mielina. Este fenotipo glial podría contribuir a los problemas neurológicos observados en el modelo Dyrk1a+/- y abrir nuevas vías de terapia para los individuos con mutaciones en el gen DYRK1A.