Exploring the human “eye-field” and “eye-spot” gene networks using human induced pluripotent stem cells.

Schettler, Raphael (2024) Exploring the human “eye-field” and “eye-spot” gene networks using human induced pluripotent stem cells. Masters thesis, University of East Anglia.

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Abstract

Eye development is dependent on highly organised processes during the development of the human embryo. A domain of neural progenitor cells, termed the eye-field, is formed and splits in half to develop two symmetrical outgrowths and the fully functioning eye. The eye-field is characterised by a class of eight transcription factors. Following the formation of the eye-field, we hypothesise that two pattern forming mechanisms (self-organising Turing networks involving Pax6, TGFβ-2 and Fst and wave-pinning mechanisms) lead to the formation of retinal eye spots. These spots are mediated by the coordinated activity of the eight eye field transcription factors and the Turing network genes.

Mutations or mishaps occurring in any of the involved genes can lead to various ocular phenotypes ranging from mild to severe. Research concerning the eye field has only been conducted on model organisms that aim to recapitulate the human body. However, by using human induced pluripotent stem cells (hiPSCs) as a model, we reduce the need for animal usage while also more accurately representing human development. hiPSCs are also devoid of confounding positional information (which are present in the embryo) which can obscure self-organisational processes. Using hiPSCs allow us to study these processes and can shed light on how the human retina is patterned.

Here, we use qRT-PCR for identifying temporal activation of gene expression and HCR-FISH to investigate the spatial arrangement of gene expression. We show the activation of all EFTF on several days of the retinal differentiation, highlighting these days as “key” days. This correlates with observed spot patterns (on the key days) which are generated by self-organisation. Our data suggests Tbx3 to be irrelevant to the EFTF gene network due to its limited expression and detection. We also identify two distinct spot patterns which we hypothesise to be mediated by the two pattern forming mechanisms and identify that these spots appear within a uniform eye-field.

This research provides the groundwork for EFTF research in human derived stem cells. We aim to create a better understanding of the fundamentals of the EFTF gene network by looking at the very first signs of retinal development. With this, we intend to bridge a gap in fundamental knowledge and contribute to the precise engineering of retinas “in-vitro”.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	20 May 2025 07:57
Last Modified:	20 May 2025 07:57
URI:	https://ueaeprints.uea.ac.uk/id/eprint/99305
DOI:

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