• hipsc-derived
• human neurogenesis
• in vitro dentate gyrus
• ZBTB20

During vertebrate development, a wide variety of cell types and tissues emerge from a single fertilized oocyte. One of these tissues, the central nervous system, contains many types of neurons and glial cells that were born during the period of embryonic and post‐natal neuro‐ and gliogenesis(Paridaen & Huttner, 2014). As to neurogenesis, neural progenitors initially divide symmetrically to expand their pool and switch to asymmetric neurogenic divisions at the onset of neurogenesis. While neurogenesis is ubiquitous throughout embryonic stages, this process is also known to persist in the subventricular and subgranular zones postnatally(Altman & Das, 1965). Herein, these new neurons are generated at the subgranular zone (SGZ) of the DG and integrate into the existing hippocampal circuitry, where they play a fundamental role in learning and spatial memory formation by performing pattern separation on inputs from the entorhinal cortex, an area of the brain located in the medial temporal lobe (Shi et al., 2012).
This process involves various mechanisms involving intrinsic as well as extrinsic factors.
Among the many factors contributing to hippocampal neurogenesis, a recently characterized zinc finger protein and transcription factor has accrued evidence suggesting its involvement in newborn hippocampal neurons.
Zinc finger and BTB-binding domain protein 20 (ZBTB20) was first demonstrated as a potential marker for the developing hippocampus (Doeppner et al., 2019; Nagao et al., 2016) and later across all hippocampal neurons(Koul, 2014) .Though its role not fully investigated in either study, Nielsen et al 2014 did attribute ZBTB20’s role in the mouse hippocampus a transcriptional repressor of Satb2 and Ctip2 (Bcll11b) in the developing mouse hippocampus. Zbtb20 was laterally demonstrated as imperative to hippocampal formation development and that its embryogenic knock-out resulted in complete ablation of CA1, 2, and 3 and the DG.
Despite its evident role in hippocampal neurogenesis, Zbtb20’s function is still not well understood in neurogenic facilitation and its sustained physiological implications.
Here, I discuss ZBTB20 role in regulation of neurogenesis in the developing human dentate gyrus. To this aim, I generated, through a novel differentiation protocol, DG-like neural progenitors from hiPSCs as an in vitro model that recapitulates many features of the process of hippocampal neurogenesis.
The ability to mimic the developmental process of disease-relevant cell types in an in vitro setting is important for providing insights into the generation and the pathogenesis of CNS disorders with a strong neurodevelopmental component(Diana Xuan Yu, di Giorgio, et al., 2014).
In this thesis, I first demonstrate that CHIR99021, a known WNT actuator/GSK-3β inhibitor, induces a hippocampal identity in hiPSC cultures and that ZBTB20 is downstream of co-inhibition of NOTCH and GSK-3β pathways, both in young (DIV35) and older (DIV170) NPCs populations.
Then, to dissect ZBTB20’s role in the anticipated cultures, I performed a ZBTB20 loss of function experiments either via CRISPR system and by the overexpression of a dominant negative ZBTB20 plasmid.
However, both CRISPR either interfering mutant derivative of ZBTB20 system reported a similar effect upon cell cycle regulators expression.
From these results I concluded that ZBTB20 plays an important role in the modulation of human embryonic neurogenesis as it is mainly involved in the differentiation of DG-like progenitors in the hippocampus.
Furthermore, these experiments elucidate that while not all differentiation physiologies and trends are conserved from younger counterparts, they share some major functions in terms of NOTCH inhibition, ZBTB20 upregulation, and suggest that ZBTB20’s function is not conserved over time as it shows a heterochronic expression.
Nevertheless, ZBTB20 molecular mechanisms need further investigations since still little is known about ZBTB proteins interactome and how these evolutionarily conserved proteins contact basal transcriptional machinery in mammals.
Altogether these findings are relevant to studies of human in vitro hippocampal neurogenesis and represent a promising tool for the screening of drugs that could have clinical application in a disease setting, as they can provide advancements in understanding human neural evolution, function, and dysfunction.