Functions of the Krüppel-like Factor 7 protein in cellular differentiation and neuronal morphogenesis

Abstract

KLF7 is a transcription factor that is expressed during embryogenesis mostly in the nervous system and its inactivation in mice leads to perinatal lethality associated with multiple neuronal defects. To identify KLF7 functions I have used an in vitro approach performed on PC12, primary and embryonic stem (ES) cells in order to elucidate KLF7 role in differentiation processes. Subsequently I have investigated KLF7 role during mouse embryogenesis using Klf7 null mice. The major finding in all the in vitro experiments was that KLF7 depletion both by RNA interference (RNAi) and gene deletion alters the appropriate differentiation mechanisms in neurogenic, cardiogenic, adipogenic and osteogenic processes. Moreover I have analysed the KLF7 potential role in ES cells self-renewal pathways and I have found by RNAi that Oct4 or Nanog increase Klf7 gene expression. In the course of the in vivo experiments I have analysed Klf7 expression during wt brain ontogenesis and found a sharp peak concomitant with development of dopaminergic system in the midbrain. Thus the transcriptional characterization of the Klf7-KO mouse brain suggested a dopaminergic deficit in Klf7-KO mice. Indeed the newborn mutant analysed showed a significant decrease in Tyrosine Hydroxylase (Th) and Dopamine Transporter (Dat) mRNA both in the ventral midbrain and olfactory bulbs (OB). TH and DAT are specific markers of the dopaminergic function. In accordance with the biochemical data, immunohistochemical analysis showed reduction of TH positive fibres in the striatum and TH positive neurons in the midbrain and OB. To further evaluate the Klf7 function in the central nervous system development, I have compared self-renewal and neuronal differentiation ability of wt and Klf7-KO derived neurospheres. To this aim I have used counting of cells positive for BLBP and western blot analysis. Both revealed a deficit in this neural stem cells marker in the Klf7-KO neurospheres. Altogether the results of this work support the hypothesis that KLF7 plays a wide role in cellular differentiation and in a number of developmental pathways. Among these a potential role for KLF7 in the maintenance of the dopaminergic phenotype emerges.