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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biology

Collaborative Specialization

Developmental Biology

Supervisor

Kelly, Gregory M.

Abstract

The Hedgehog (Hh) and Wnt protein signaling pathways are essential in the differentiation of neurons and astrocytes. As there are many known and new players involved in regulating these pathways, the role of the regulators Suppressor of Fused (SUFU) and Never in Mitosis Kinase 2 (Nek2) have either not been previously reported or have not been thoroughly explored. To address this shortfall CRISPR gene editing was used to target SUFU and Nek2 in the mouse P19 embryonal carcinoma cell model of neural differentiation. Hh and Wnt signaling were explored in normal P19 neural differentiation, which occurs in the presence of retinoic acid. Both pathways are required for the establishment of neural cell fates, however, neither is sufficient to induce both neurons and astrocyte lineages alone. SUFU was found to be required in the differentiation of astrocytes, but not neurons, and its loss resulted in the loss of the Gli3 transcription factor. The loss of Gli3 in SUFU-deficient cells resulted in constitutive expression of Hh target genes. Nek2 was required for the differentiation of both neurons and astrocytes, however, loss of function experiments did not find a clear link between Nek2, and the Hh or Wnt pathways. Instead, a mechanism was discovered where Nek2 is required to destabilize hypoxia inducible factor 1a, allowing for a required metabolic shift from glycolysis to oxidative phosphorylation during the differentiation of neural cell types. Together this work shows novel mechanisms of regulating neural cell fate through the presence or absence of intracellular regulatory proteins.

Summary for Lay Audience

During development, how specific cell types are determined requires the activation of specific information pathways. Becoming a cell that belongs to the nervous system requires, in part, activation of the Hedgehog (Hh) and Wnt signaling pathways. The cell types being explored here were neurons, cells that send and receive electrical impulses, and astrocytes, cells that support neurons. To answer questions regarding the development of these cell types a method where cells were grown in a dish that does not require live animals was used. These stem-like cells comprise the mouse P19 embryonal carcinoma model and were isolated from a mouse tumor. These cells can become any cell type in the body, and when given the vitamin A derivative, retinoic acid (RA), can become neurons and astrocytes. Through chemical studies Hh and Wnt signaling were determined to be essential during neural cell development. Two proteins involved in regulating these signaling pathways, Suppressor of Fused (SUFU) and Never in Mitosis Kinase 2 (Nek2), were investigated through loss of function experiments where a targeted gene editing approach, CRISPR, was used. Loss of SUFU showed it is vital in the development of astrocytes but not neurons; Nek2 is vital in the development of both cell types. When SUFU was lost there was over-activation of Hh signaling, resulting in the loss of Gli3, a transcription factor normally required to inhibit the Hh pathway. The connection between Nek2, Hh and Wnt signaling pathways were explored, however, there was no clear evidence showing a link. Instead, Nek2 was found to be involved in regulating how cells use sugar molecules as without it, the cells metabolize sugar via a pathway that does not require oxygen. In normal cells, however, the development of neurons and astrocytes requires a shift from an oxygen-deficient metabolic pathway, to relying exclusively on one that requires oxygen. Thus, this work sheds new light on how proteins regulate the development of specific cell types in the central nervous system and makes connections between signaling pathways, development, and metabolism.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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