Early function of KCC2 and Wnt genes : cytoskeletal effects in neural stem cells

Abstract

From closure of the neural tube until formation of neuronal networks, cells undergo several crucial changes which depend on the expression of many important molecules. GABA, the principal inhibitory neurotransmitter in the adult nervous system, acts as an excitatory signal during embryonic development. This is a necessary neurotrophic signal resulting from a high level of chloride in neural cells. During neural maturation, the potassium-chloride co-transporter, KCC2, lowers the intracellular chloride level and switches the GABA response to hyperpolarizing. Studies have shown a role for KCC2 in the formation of neuronal dendrites but earlier functions in mammals have not yet been revealed. Among the most well-known factors regulating early nervous system development are the Wnt proteins, which act as extracellular ligands. This thesis describes the action of KCC2 and Wnt genes on the cytoskeleton of neural progenitors and demonstrates how a change in their expression can alter neural cell behaviour.

To correlate the expression pattern of KCC2 with functionality, we recorded the GABA response in fetal rat respiratory neurons. The developmental switch from depolarizing to hyperpolarizing was shown to be around embryonic day (E) 20. The KCC2 expression pattern in the main respiratory-related nuclei preBötzinger complex and parafacial respiratory group changed drastically between E18 and P0. From being essentially cytoplasmic at E18, KCC2 appeared to be translocated to the plasma membrane at E20. At P0, the KCC2 protein was localized predominantly at the plasma membrane and maintained this expression pattern postnatally.

Overexpression of KCC2 in the neural tube of transgenic mouse embryos had a deleterious effect on the nervous system development. Transgenic embryos at E9.5-E13.5 displayed a reduced neuronal differentiation and impaired neural crest migration. Similar results were obtained with a truncated form of KCC2, lacking the sequence for ion transport, implying that the effects were not GABA-dependent. Interestingly, the neural tube of transgenic embryos had an aberrant distribution of the cytoskeletal protein actin, suggesting an interaction between KCC2 and the cytoskeleton.

In a related study, we examined transgenic embryos with neural-specific overexpression of Wnt7a. These embryos have been shown to exhibit cytoskeletal defects discernible as impaired adherens junctions in the rostral neural tube. Analysis of E9.5 and E10.5 transgenic embryos showed a reduced neuronal differentiation, indicating a role for Wnt7a in the control of neuronal progenitor maturation. Moreover, the downstream signalling gene Vangl2 was upregulated in the neural tube. We therefore studied transgenic embryos overexpressing Vangl2 in neural progenitors and loop-tail embryos with a natural mutation in the Vangl2 gene. Both Vangl2 gain-of-function and loss-of-function resulted in cytoskeletal defects in the neural tube, characterized by aberrant distribution of actin and other cytoskeletal components. Moreover, the small GTPase Rac1 was redistributed in the cells of the neural tube, indicating an interaction between Vangl2 and Rac1. Cell studies using HEK293T, MDCK and C17.2 cell lines showed similar effects of Vangl2 on the cytoskeleton as well as altered cell adhesion and motility. Interestingly, these effects could be blocked by a Rac1 knock-down, verifying the interaction between Vangl2 and Rac1 observed in vivo. Taken together, these results demonstrate the significance of a coordinated expression of cytoskeletal-interacting proteins during nervous system development.