Calcium waves, induced by extracellular agonists and sustained by intraorganellar calcium stores, have been suggested to influence the proliferation of neural stem and progenitor cells (NSPCs) of the developing cerebral cortex. It remains unclear, however, how NSPC calcium stores are regulated and how calcium dynamics are transduced into NSPC behaviors. This thesis work begins to tackle this central question by investigating how store operated calcium entry (SOCE), a calcium influx pathway tied to emptying of endoplasmic reticulum (ER) calcium stores, regulates calcium signaling in cortical NSPCs to influence their behavior and output. To this end, we used calcium imaging to successfully record SOCE in mouse and human cortical NSPCs. By isolating enriched populations of embryonic cortical cells using an in vivo labeling technique called “FlashTag,” we also found that Orai1, Orai2 and Stim2 encode the primary mediators of SOCE during development. Moreover, their expression is dynamically regulated during NSPC lineage progression. In line with this, conditional deletion of Orai1 and Orai2 in the excitatory lineage eliminates SOCE in embryonic NSPCs. Orthogonal pharmacological experiments revealed that SOCE is required in both mouse and human primary cortical NSPCs to maintain proliferation, and robust SOCE responses in proliferative NSPCs are significantly diminished upon cell cycle exit. Supporting this idea, in utero electroporation of STIM2 isoforms with opposing effects on SOCE bidirectionally regulates cell cycle exit of cortical NSPCs.
In an effort to define upstream regulators of SOCE in the developing cortex, we found that activation of P2Y1 receptor-mediated purinergic signaling induces SOCE. Importantly, SOCE is abrogated upon purinergic receptor activation in NSPCs lacking Orai1 and Orai2, suggesting that SOCE may transduce purinergic signals in the developing cortex to modulate proliferation. Interestingly, our preliminary analyses suggest that dual conditional deletion of Orai1/2 does not grossly alter NSPC proliferation across the entire NSPC compartment in the embryonic cortex. This suggests the possibility that proliferation may be regulated at a cell type-specific level in the context of conditional Orai1/2 inactivation, an idea that has been suggested in the context of other modulators of NSPC proliferation. Collectively, our data suggest that dynamic regulation of SOCE mediators and downstream calcium signaling plays indispensable roles in the control of stem/progenitor cell biology in the developing cortex. As aberrant calcium signaling has been implicated in neurodevelopmental disorders, our studies defining the molecules and mechanisms involved in transducing calcium signals during cortical development will provide essential insights into normal and dysfunctional corticogenesis.
Chapter 1 and 2 of this thesis provide an introduction to calcium signaling in the developing cortex, with an emphasis on roles of aberrant calcium regulation and electrical activity in the etiology of neurodevelopmental disease. Through a detailed discussion of the known mechanisms by which calcium directs cellular behaviors in the developing cortex, we posit that understanding the normal events that build the nervous system relies on gaining insight into cell type-specific calcium signaling mechanisms and that these mechanisms may be disrupted or reactivated in the context of neurological disease. Chapter 3 focuses on the regulation of SOCE during embryonic cortical development. Finally, Chapter 4 concludes this thesis with a discussion of the intersection of different modes of calcium entry, the reactivation of developmental calcium signaling mechanisms in disease states, and a brief overview of ongoing work that we have been pursuing aimed at defining cell type specific regulators of calcium signaling using long isoform sequencing.
