NeuroD 2/6 regulate expression balance of transcription factors controlling neurocortical cytoarchitecture

Abstract

BACKGROUND: Genes of NeuroD family that includes NeuroD1, NeuroD2, NeuroD6 control neuronal survival, differentiation, maturation and neurite patterning in nervous system. In the mouse brain, the deletion of NeuroD1 results in complete loss of the dentate gyrus as a consequence of neuronal apoptosis. NeuroD2 is required for neuron survival in the cerebellum, and it is essential for the integration of thalamo-cortical connections into the neocortex as well as the formation of somatosensory whisker barrel cortex. We have recently reported that, in NeuroD2/6 double deficient (DKO) mice, callosal axon projections are defective due to abnormal EfnA4 signaling. In order to to investigate NeuroD2/6 controlled molecular cascade further, we investigated the expression of key transcription factors that control various aspects of cortical development in the brains of NeuroD2 and NeuroD6 deficient mutants. AIM: To investigate possible changes of differentiation programs downstream of NeuroD2/6 transcription factors. METHODS: Embryos with NeuroD2/6 double deficient were used in the experiments. Pregnant mice carrying E13.5 embryos were operated for in utero electroporation. To study the expression pattern of target genes, in situ hybridization with preliminary synthesis of probes at different stages of embryonic development was performed. To analyze the activity of a gene promoter, genomic DNA fragments containing Neurod2/6 motifs were cloned into the pMCS-GL vector to measure luciferase activity. The charts were made with GraphPad Prism software and presented as mean standard error. RESULTS: We have shown that NeuroD1 expression is ectopically upregulated in postmitotic neurons of NeuroD2/6 DKO neocortex and hippocampus. We detected changes in expression of key transcription factors, Cux1, Tbr1, Lhx2, Id2. Moreover, Cux1 is a direct target of NeuroD2/6. In addition, Olig2+ progenitors are increased in NeuroD2/6 DKO neocortex and expression of NeuroD2/6 and Olig2 is mutually exclusive. Thus, NeuroD2/6 regulate the expression of transcription factors in the developing brain. CONCLUSION: We discovered that cumulative action of NeuroD2 and NeuroD6 genes is required to initiate and maintain the expression of Cux1, Tbr1, Lhx2, Id2 transcription factors. On the other hand, both genes are required to prevent premature differentiation of Olig2 positive glial precursors.