A reduction in Npas4 expression results in delayed neural differentiation of mouse embryonic stem cells

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.

Activity-dependent expression of neuronal PAS domain-containing protein 4 (npas4a) in the developing zebrafish brain

“…Given the existing similarities between vertebrate Npas4 genes that we have outlined, our results in the zebrafish draw attention to this overlooked area of research by propounding the idea that perhaps the mammalian Npas4 homologs may also have some involvement in CNS development. Interestingly, a recent study demonstrated that the murine Npas4 transcript is also expressed in the developing forebrain (Klaric et al, 2014 ). While these observations support a possible role for Npas4 in mouse forebrain development, the function of Npas4 in this context remains unknown and is an area that requires further investigation.…”

“…For instance, little is known about the expression of the Npas4 genes during embryogenesis and whether or not they play a role in embryonic development. One study has demonstrated that in the mouse embryo Npas4 is expressed in the developing forebrain (Klaric et al, 2014 ), though the functional significance of this expression has not been investigated. The limited amount of data on this subject may be a result of the inherent difficulties of investigating embryonic development in mammals and for this reason certain species may be better suited to addressing these types of questions.…”

The Role of the Neuroprotective Factor Npas4 in Cerebral Ischemia

“…Using immunostaining, Yun et al , reported that, in the subgranular zone (SGZ) of the DG, a small number of Npas4-expressing cells were also found to express markers of neural progenitor cells (NPCs) such as SRY (sex determining region Y)-box 2 (Sox2) and doublecortin (Dcx) [ 14 ]. In line with this, using two independent in vitro models of neurogenesis, our laboratory has found that while Npas4 is not expressed in undifferentiated embryonic stem cells, it becomes transiently upregulated at a stage that is characterized by proliferation of NPCs [ 50 ].…”

Prenatal one-carbon metabolism dysregulation programs schizophrenia-like deficits

“…50 Knockdown of Npas4 expression in mouse embryonic stem cells undergoing neural differentiation inhibits their ability to differentiate appropriately, and results in delayed neural differentiation. 51 Npas4 downregulation in MET mice might be a mechanism for the decreased neurogenesis and delayed differentiation of embryonic stem cells, as well as the cognitive impairment in these animals. The regional differential expression analysis of Npas4 expression in MET mice’ brains shows a decrease in the prefrontal cortex and a similar decrease (albeit not statistically significant) in the hippocampus.…”