Abstract
IntroductionNpas4 is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. It is known that in the adult brain Npas4 plays an important role in several key aspects of neurobiology including inhibitory synapse formation, neuroprotection and memory, yet very little is known about the role of Npas4 during neurodevelopment. The aim of this study was to examine the expression and function of Npas4 during nervous system development by using a combination of in vivo experiments in the developing mouse embryo and neural differentiation of embryonic stem cells (ESCs) as an in vitro model of the early stages of embryogenesis.MethodsTwo different neural differentiation paradigms were used to investigate Npas4 expression during neurodevelopment in vitro; adherent monolayer differentiation of mouse ESCs in N2B27 medium and Noggin-induced differentiation of human ESCs. This work was complemented by direct analysis of Npas4 expression in the mouse embryo. The function of Npas4 in the context of neurodevelopment was investigated using loss-of-function experiments in vitro. We created several mouse ESC lines in which Npas4 expression was reduced during neural differentiation through RNA interference and we then analyzed the ability of these Npas4 knockdown mouse ESCs lines to undergo neural differentiation.ResultsWe found that while Npas4 is not expressed in undifferentiated ESCs, it becomes transiently up-regulated during neural differentiation of both mouse and human ESCs at a stage of differentiation that is characterized by proliferation of neural progenitor cells. This was corroborated by analysis of Npas4 expression in the mouse embryo where the Npas4 transcript was detected specifically in the developing forebrain beginning at embryonic day 9.5. Finally, knockdown of Npas4 expression in mouse ESCs undergoing neural differentiation affected their ability to differentiate appropriately, resulting in delayed neural differentiation.ConclusionsHere we provide the first evidence that Npas4 is expressed during embryonic development and that it may have a developmental role that is unrelated to its function in the adult brain.
Highlights
Neuronal basic helix-loop-helix Per-Arnt-Sim (PAS) domain protein 4 (Npas4) is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity
When the change in Npas4 expression was quantified using Quantitative RT PCR (qRT-PCR), we found that the highest expression of Npas4 occurred at Day 4 when a 10-fold induction was observed relative to undifferentiated mouse embryonic stem cells (ESCs) (mESC) (Figure 1B)
While these findings suggest that Npas4 may be involved in aspects of neural progenitor cells (NPCs) biology, such as NPC maintenance and/or differentiation, further research is needed to confirm the developmental role of Npas4 in vivo
Summary
Npas is a calcium-dependent transcription factor expressed within neurons of the brain where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. Neuronal PAS domain protein 4 (Npas4) is a transcription factor belonging to the basic helix-loop-helix Per-ArntSim (bHLH PAS) family of regulatory proteins [1,2,3]. Members of this family are involved in a wide range of signaling [8] making it an immediate-early gene that acts as a direct link between changes in intracellular Ca2+ levels and rapid changes in gene expression. It has been shown that Npas is induced by learning and has a functional role in memory formation [8,11]
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