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Type: Journal article
Title: A reduction in Npas4 expression results in delayed neural differentiation of mouse embryonic stem cells
Author: Klaric, T.
Thomas, P.
Dottori, M.
Leong, W.
Koblar, S.
Lewis, M.
Citation: Stem Cell Research and Therapy, 2014; 5(3):64-1-64-14
Publisher: BioMed Central
Issue Date: 2014
ISSN: 1757-6512
Statement of
Thomas S Klaric, Paul Q Thomas, Mirella Dottori, Wai Khay Leong, Simon A Koblar, and Martin D Lewis
Abstract: INTRODUCTION 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. 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. METHODS Two 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. RESULTS We 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. CONCLUSIONS Here 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.
Keywords: Neurons
Cell Line
Flow Cytometry
In Situ Hybridization
Polymerase Chain Reaction
Cell Differentiation
Embryonic Development
Basic Helix-Loop-Helix Transcription Factors
Embryonic Stem Cells
Embryo, Mammalian
Neural Stem Cells
Rights: © 2014 Klaric et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.
DOI: 10.1186/scrt453
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