Student Theses and Dissertations
Date of Award
2015
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Ali H. Brivanlou
Keywords
corticogenesis, human pluripotent stem cells (hPSCs), TGFβ signaling, neural induction, WNT signaling, projection neurons
Abstract
The formation of the mammalian cerebral cortex is a complex multi‐tiered process that involves three major milestones: 1) neural induction and folding of the neuroepithelium, 2) areal patterning and generation of various progenitor types, and 3) corticogenesis. Our current understanding of the molecular and cellular basis of cortical development comes largely from mouse studies due to the genetic tractability of this model system. However, as primate studies have shown, the primate brain is unique in terms of its progenitor and neuronal composition, cortical areas, scale, and gene expression. Limitations in the availability of non‐human primate and human fetal material, the longer timescale of developmental processes, as well as the ethical considerations involved preclude direct experimental observations in both these organisms. However, human pluripotent stem cells (hPSCs) allow a window into early human fetal development and permit experimental manipulation of developmental events, thereby enabling molecular and cellular dissection of corticogenesis. The default model of neural induction, described originally in amphibians, posits that induction of telencephalic (forebrain) fate in an embryo requires elimination of TGFβ signaling. Using transgenic hPSCs, we show that inducible expression of a cell‐intrinsic inhibitor of canonical TGFβ signaling – SMAD7 – is sufficient to directly convert hPSCs to forebrain fate as seen by gene expression and immunocytochemical analysis of several transcription factors. Moreover, this conversion is direct and does not involve induction of non‐neural fates. Our findings suggest a conservation of the default model in humans. Additionally, we also show that FGF‐MEK signaling has no direct role in hPSC neural induction, thereby resolving an existing debate in the field. We were able to derive neuroepithelium from PSCs of multiple species (mouse, primates, and human) with small molecule inhibitors of TGFβ signaling. In order to demonstrate that in vitro generated neuroepithelium is comparable to the in vivo germinal zone of an embryo, we utilized transgenic lines expressing FUCCI (fluorescently ubiquitination cell cycle indicators). Together with single‐cell analysis, we were able to demonstrate self‐organization in radial progenitors, interkinetic nuclear migration, as well as requirement for Notch signaling for progenitor maintenance, all of which are hallmarks of human neural progenitors in vivo. Region‐specific marker analysis, we conclude that in vitro hPSC‐ derived neural progenitors are similar to their in vivo counterparts based on several measurements. Within telencephalic territory generated by TGFβ inhibition, we demonstrate that the in vitro derived neuroepithelium can be patterned on the rostrocaudal axis by manipulating WNT signaling. Small molecule inhibitors of WNT signaling promoted expression of frontal markers; conversely, small molecule activators of WNT promoted expression of occipitotemporal markers. Importantly, WNT activation had to be moderate; higher levels of WNT activation resulted in switch of neuroepithelial identity from forebrain to midbrain. This finding provides insights into arealization in the mammalian cortex and suggests that exposure to different levels of WNT signaling early on may act as a selector between generation of a six‐layered neocortex or a three‐layered archicortex. Lastly, we utilize our reductionist in vitro corticogenesis model to establish that neural progenitors can be differentiated into various classes of projection neurons. By utilizing genome modification strategies in hPSCs, we observe that single neural progenitors possess the capacity to give rise to callosal projection neurons (CPNs) and subcortical projection neurons (SCPNs), thereby lending support to the "Progressive Restriction" model of corticogenesis. Additionally, based on analysis of human fetal tissue at various stages, and further confirmation with BrdU labeling studies during in vitro differentiation, we propose that progressive progenitor restriction manifests during corticogenesis as a progressive limitation in the ability of their daughter neurons to express various projection neuron class‐determinants over time. Co‐expression of CPN and SCPN fate determinants is rarely observed during mouse development, even though recent mouse studies clearly show the requirement of CPN genes in SCPN formation. This suggests that neuronal restriction may be evolutionary conserved and this may have implications for diseases such as autism where laminar differentiation is affected. Neuronal restriction provides a conceptual link between how defects in progenitors could affect laminar development and neuronal hodology, which in turn underlies neuronal circuit formation. Finally, by lineage‐tracing CUX2 positive progenitors in transgenic hPSC‐derived neuroepithelium, we show that a small fraction of CPNs in our in vitro corticogenesis paradigm may be derived from lineage‐restricted progenitors. Given the large numbers of progenitors that display multi‐laminar differentiation ability, we conclude that lineage‐ restricted progenitors are not a major source of CPNs in our system, but may represent a transient population derived from multipotential progenitors. Thus, by combining human genome modification technologies with the default model of neural induction, we are now able to probe fundamental questions of human corticogenesis. Using genetic tools, we have established that hPSC‐derived neural progenitors retain most characteristics of their in vivo mammalian counterparts and have begun to uncover mechanistic principles for generation of the cortical projection neuron classes. Together, our findings open new avenues for study of human brain development, disease modelling, and drug screening using hPSCs. Moreover, the genetic and analytical tools we describe here can be used for in vitro studies of cell cycle dynamics, self‐organization, lineage tracing, and live imaging of multiple PSC‐derived tissue types.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Ozair, Mohammad Zeeshan, "A Reductionist Approach to Modeling Human Corticogenesis" (2015). Student Theses and Dissertations. 493.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/493
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy