title: miR-744 and miR-224 Downregulate Npas4 and Affect Lineage Differentiation Potential and Neurite Development During Neural Differentiation of Mouse Embryonic Stem Cells.
abstract: Neuronal PAS domain protein 4 (Npas4) is a brain-specific transcription factor whose expression is enriched in neurogenic regions of the brain. In addition, it was demonstrated that Npas4 expression is dynamic and highly regulated during neural differentiation of embryonic stem cells (ESCs). While these findings implicate a role for Npas4 in neurogenesis, the underlying mechanisms of regulation remain unknown. Given that growing evidence suggests that microRNAs (miRNAs) play important roles in both embryonic and adult neurogenesis, we reasoned that miRNAs are good candidates for regulating Npas4 expression during neural differentiation of ESCs. In this study, we utilized the small RNA sequencing method to profile miRNA expression during neural differentiation of mouse ESCs. Two differentially expressed miRNAs were identified to be able to significantly reduce reporter gene activity by targeting the Npas4 3'UTR, namely miR-744 and miR-224. More importantly, ectopic expression of these miRNAs during neural differentiation resulted in downregulation of endogenous Npas4 expression. Subsequent functional analysis revealed that overexpression of either miR-744 or miR-224 delayed early neural differentiation, reduced GABAergic neuron production and inhibited neurite outgrowth. Collectively, our findings indicate that Npas4 not only functions at the early stages of neural differentiation but may also, in part, contribute to neuronal subtype specification and neurite development.
authors.affiliation: School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia.
authors.forename: Fong Chan
authors.initials: FC
authors.lastname: Choy
authors.affiliation: School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Klarić
authors.affiliation: School of Medicine, The University of Adelaide, Adelaide, SA, Australia.
authors.forename: Simon A
authors.initials: SA
authors.lastname: Koblar
authors.affiliation: School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia. martin.lewis@sahmri.com.|South Australian Health & Medical Research Institute, North Terrace, Adelaide, SA, Australia. martin.lewis@sahmri.com.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Lewis
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D020413:3' Untranslated Regions; D051792:Basic Helix-Loop-Helix Proteins; D035683:MicroRNAs; C584584:MIRN224 microRNA, mouse; C571521:MIRN744 microRNA, mouse; C533005:Npas4 protein, mouse
keywords: Deep sequencing; GABAergic neurons; MicroRNAs; Neurite outgrowth; Neurogenesis; Npas4