Dlx homeobox genes and their role in interneuronal differentiation and migration in the developing forebrain.
Le, Trung Ngoc
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Understanding the specificity of homeobox genes has been hampered by the lack of verified direct transcriptional targets. The Dlx family of homeobox genes is expressed in the ganglionic eminences of the developing forebrain. Dlx1/Dlx2 double knockout (DKO) mice die at birth. Phenotypic analyses demonstrate abnormal development of the basal telencephalon, including defects in neuronal differentiation in the basal ganglia, reduced expression of GABA in the basal telencephalon, and loss of migration of GABAergic inhibitory interneurons to the neocortex. The mechanisms underlying DLX protein regulation of differentiation and migration of GABAergic interneurons are poorly defined. We have successfully applied chromatin immunoprecipitation to identify potential direct transcriptional targets of DLX homeoproteins from embryonic tissues in vivo. Reporter gene assays demonstrated the transcriptional significance of the binding of DLX proteins to different downstream regulatory elements, which were confirmed in vitro by electrophoretic mobility shift assay and site-directed mutagenesis. The functional significance of DLX mediated transcriptional regulation of these targets was further elaborated through several series of loss-of-function assays including gene expression in Dlx1/2 knockout embryonic forebrain tissues, as well as siRNA or Lentiviral mediated shRNA knockdown experiments with primary forebrain cultures. Quantitative analysis of the regulatory effect of Dlx genes on various forebrain markers of differentiation and migration was performed using in situ hybridization, high-performance liquid chromatography coupled with cell counting. Neuronal migration was assessed by forebrain explants and diI labelling of migratory cells from ganglionic eminence to neocortex. We have demonstrated that DLX1 and DLX2 can transcriptionally activate (Gad1, Gad2) or repress (Nrp2) different downstream targets. In the Dlx1/2 DKO, reduction of GABA expression and failure of GABAergic interneurons to migrate to the neocortex is partly due to loss or aberrant expression of these DLX downstream targets. In the triple Dlx1/2; Nrp2KO, partial restoration of tangential migration of GABAergic interneurons from basal ganglia to the neocortex was successfully established signifying the importance of DLX regulation of Semaphorin-Neuropilin signalling during forebrain development.