The Multiple Tasks Endured by PI3K during neural tube development

Abstract

Development of the spinal cord involves coordination between exposure to localized extracellular signals and controlled activation of intracellular signaling pathways. This way, neuroepithelial cells firstly proliferate apically to increase the progenitor pool and, later on, initiate neurogenic divisions giving rise to a variety of neuronal cell types. Class IA PI3Ks are heterodimeric enzymes (catalytic+regulatory subunits) activated by receptors tyrosine kinase (RTKs) or G protein coupled receptors (GPCRs) that, upon extracellular stimuli, modulate diverse target proteins through local production of PtdIns(3,4,5)P3 lipids. Vertebrates express three Class IA catalytic subunits (p110-alpha, p110-beta, and p110-delta), all important for the development of the central nervous system. However, it is unclear to what extent these p110-alpha isoforms have overlapping or distinct biological roles, and what exact functions they hold in neural development.

Analysis of PI3Kalpha (p110-alpha-alpha+regulatory subunit) expression in the embryonic spinal cord revealed abundant mRNA and protein levels in cycling progenitors followed by restriction of PI3K-alpha exclusively to differentiating neurons. To examine the role of PI3K-alpha in progenitors and neurons, we interfered with normal PI3K-alpha regulation by expressing active mutants or knocking down of p110-alpha-alpha in the chicken neural tube. Loss of p110-alpha resulted in high apoptotic rates in both progenitors and neurons, sustaining a role for PI3K-alpha in neural survival as seen in other studies. Instead, uncontrolled upregulation of PI3K-alpha activity resulted in severely disrupted neural tubes, with abnormal cell masses in the luminal face of the neuroepithelium and ectopic mitosis. Additionally, we observed alterations in the neural lamination characterized by basement membrane breaches followed by enhanced neural migration and misoriented axonal growth. A thorough analysis of the tissue unveiled loss of polarity as the main cellular mechanism driving the luminal structural aberrations, suggesting a major role of PI3K-alpha in neuroepithelial apico basal polarity. Moreover, the rescue of the depolarization phenotype with a dominant negative form of RhoA proposes local regulation of the Rho family of small GTPases as the molecular mechanism responsible for the PIP3 dependent regulation of adherens junction dynamics. Alternatively, we found the neural overmigration caused by excess of PI3K-alpha activity explained by increased basal accumulation of PIP3, leading to actin based membrane protrusions and basement membrane breaches. Coherently, when we assessed the neural positioning after p110-alpha knock down, we detected neurons inserted in the proliferative layer and reduction of the neuronal cytoskeletal component beta III tubulin, suggesting that PI3K-alpha also modulates morphological maturation and apico basal positioning of differentiating neurons. Interestingly, PIP3 induced overmigration seemed to be carried out through local activation of other two members of the Rho GTPases, Cdc42 and Rac1. These results shed some light upon the PI3K-alpha/PIP3 specific roles during early neural tube development, stressing out its function in cell polarity. Furthermore, we propose a mechanism that may partially explain how the PI3K-alpha /PIP3 signaling is able to control different types of polarity corresponding to different developmental moments. This could help to understand the initial events leading to some neurodevelopmental disorders caused by hyperactivation of PI3K signaling.

El desarrollo de la médula espinal requiere una fina coordinación entre señales extracelulares y la activación de vías intracelulares específicas. De este modo se da una primera fase de proliferación de las células neuroepiteliales en la zona apical para aumentar el número de progenitores y una segunda fase de neurogénesis, a partir de la cual se originan diferentes tipos de neuronas. La clase IA de las PI3Ks se encuentra implicada en la transducción de señales a través de receptores tirosina quinasa (RTKs) o receptores acoplados a proteínas G (GPCRs). En respuesta a estímulos extracelulares, controlan la actividad de distintas proteínas diana a través de la producción local de lípidos PtdIns(3,4,5)P3. La clase IA de las PI3Ks, formada por enzimas heterodiméricas, consta de tres tipos de subunidades catalíticas (p110-alfa, p110-beta y p110-delta). Todas ellas son importantes para el desarrollo del sistema nervioso, sin embargo no están claras las funciones específicas de cada isoforma.

El análisis de la expresión de PI3K a nivel de RNAm y proteína en la médula espinal embrionaria reveló una expresión diferencial según el estadío, siendo alta en progenitores antes de la neurogénesis y restringida a neuronas en estadíos más tardíos. Para estudiar su función en progenitores y neuronas, transfectamos formas activas de PI3K-alfa o suprimimos transitoriamente la p110-alfa en el tubo neural de embriones de pollo. La pérdida de p110-alfa provocó una alta tasa de apoptosis en ambas poblaciones, revelando su importancia en supervivencia. La sobreexpresión de la PI3K-alfa activa, en cambio, generó disrupciones muy severas del tejido neural caracterizadas por la presencia de masas celulares en la pared ventricular y mitosis ectópicas. En el lado basal, se observaron alteraciones en la laminación neuronal con células atravesando la lámina basal y crecimiento axonal aberrante. Nuestros resultados apuntan hacia la pérdida de polaridad como la principal causa de las aberraciones estructurales apicales, indicando que la PI3K-alfa tiene una función en la regulación de la polaridad apico basal. Asimismo, la PI3K-alfa parece implicada en la maduración del citoesqueleto neural y en el posicionamiento de las neuronas en el eje apico basal, funciones parcialmente mediadas por miembros de las Rho GTPasas.