Expression and function of Npas4 during early development.

Abstract

Npas4 is an activity-dependent bHLH PAS transcription factor expressed within neurons of the mammalian central nervous system where it regulates the expression of several genes that are important for neuronal survival and synaptic plasticity. In the adult brain, Npas4 plays an important role in several key aspects of neurobiology including inhibitory synapse formation, neuroprotection and memory formation. Consequently, abnormal Npas4 expression has been implicated in a number of neurological disorders such as autism, Down’s syndrome, epilepsy, cerebral ischaemia and Alzheimer’s disease. While the expression and function of Npas4 are beginning to be elucidated in the adult brain, to date little is known regarding the role of Npas4 during neuro-development. The aim of this study was to investigate the expression and function of Npas4 during early development. The expression of Npas4 during early neuro-development was investigated using several different developmental model systems. Npas4 was found to be transiently up-regulated during neural differentiation of both mouse and human embryonic stem (ES) cells at a stage of differentiation that is marked by an increase in neural progenitor cell (NPC) proliferation. This was corroborated by analysis of Npas4 expression in the developing mouse embryo where Npas4 mRNA was found to be expressed between embryonic day 7.5 and 9.5. The function of Npas4 in the context of development was investigated by using RNA interference to decrease endogenous Npas4 expression in mouse ES cells undergoing neural differentiation. Npas4-specific small hairpin RNA expression constructs were delivered to mouse ES cells using lentiviral transduction to create two independent Npas4 knock-down mouse ES cell lines. An Empty vector control line was also generated by transducing mouse ES cells with a construct that does not produce small hairpin RNAs. When the cell lines were assessed for their ability to undergo neural differentiation, it was found that aspects of NPC identity and neuronal maturation were affected by a reduction in Npas4 expression. The percentage of cells expressing the neuroectoderm marker Sox1 was significantly diminished in Npas4 knock-down cultures while expression of Nestin was not affected. In addition, neurite sprouting defects were also observed at a later stage of differentiation in cultures having a more severe reduction in Npas4 expression. These data suggest that Npas4 acts upstream of Sox1 during neural differentiation and is involved in aspects of NPC maintenance and neuritogenesis. When taken together, the data presented in this thesis provide the first evidence that Npas4 is expressed developmentally by a population of early NPCs and that it may have a developmental role that is unrelated to its function in the adult brain.