Precision Medicine DTP - Dissecting the biophysics of ocular transcription factors in vivo

Background

Of the ~1,600 human TF genes, up to ~25% (~400 genes) are expected to be haploinsufficient with a plethora of them leading to developmental disorders1, underlining that transcription factor (TF) abundance (dosage) plays a major role during cell and tissue specification. Next to the questions of “where” and “when” TFs are expressed, it is also important to investigate “how much” TF there is at specific developmental time points and “how uniformly” intranuclear TF concentrations are maintained across cells during development. However, how TF concentration and chromatin-binding dynamics quantitatively regulate developmental processes, and how cell-to-cell heterogeneities in TF concentration influence these processes, all remain poorly understood.

We are interested in understanding how the abundance and dynamic intranuclear behaviour of conserved eye-specific TFs regulate the early induction of the optic field in vivo. Optic cup induction and development is a complex process dependent upon the correct regulation of key TFs such as Pax6, Sox2 and Otx22-4. Mutations in one of two alleles of eye specification TFs, as well as variations in copy number are responsible for the majority of ocular malformations (i.e micropthalmia, aniridia or anopthalmia) in humans2-5. Although the specification of the eye field by transcription factors has been thoroughly investigated and the genetic relationships of key TFs has been described4, we lack quantitative information on the contribution of TF numbers, their cell-to-cell variability, their relative stoichiometries and their dynamic binding to chromatin during eye specification induction and maintenance.

Aims

1) Investigate the relative endogenous protein concentrations of Pax6, Sox2 and Otx2 and how these impact upon their molecular interactions among themselves and with chromatin; and how these interactions contribute to ocular development and maintenance. For this, we will use CRISPR/Cas9 editing to endogenously tag the mouse ocular TFs in ES cells and differentiate those into optic cup organoids.

2) In collaboration with the Karolinska Institute in Stockholm, Sweden, we aim to investigate the variability in TF numbers among different cells during optic cup induction and differentiation. We will use single- point Fluorescence Correlation Spectroscopy (FCS) in our lab and a uniquely developed system (Karolinska Institute), which can perform multi-point FCS measurements. FCS is a well-established biophysical methodological approach that allows the study of the spatiotemporal properties of fluorescently tagged molecules in live cells, by performing temporal autocorrelation analysis of fluorescence intensity fluctuations and subsequently quantitatively studying TF-chromatin and TF-TF interactions, as well as their absolute concentrations in live cells and tissues6. The multi-point FCS system uses a matrix of detectors to simultaneously record 1024 FCS measurements (32x32 array of Avalanche PhotoDiodes) to provide ‘heat maps’ of molecular diffusion, chromatin-binding dynamics and cell-to-cell variability of the concentrations of fluorescent molecules7,8.

3) We will investigate how TF concentration perturbations (increase or decrease of concentration and cell-to-cell variabilities) influence optic vesicle induction and specification. We will use partial degradation technologies for Halo and Auxin Inducible Degron tags to mildly increase or mildly increase (by means of third copy inducible alleles) the amount of ocular TFs and study the developmental outcomes of these manipulations.

Training outcomes

The student will benefit from state-of-the-art genome editing methodologies, the understanding of mechanisms of gene expression and developmental genetics, as well as biophysical methodologies (single- point and multi-point FCS) to derive in vivo large scale datasets of ocular TF mobilities (diffusion), kinetic interactions with chromatin during optic cup induction and differentiation, as well the tolerable degree of variability in TF numbers, all required for correct development of eye structures. The synergy and interactions with physical chemists involved in the development of customized FCS systems will equip the student with robust understanding of biophysical properties, microscopy optics and large-scale data analysis and automation.

This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.

All applications should be made via the University of Edinburgh, irrespective of project location. For those applying to a University of Glasgow project, your application along with any supporting documents will be shared with University of Glasgow.

http://www.ed.ac.uk/studying/postgraduate/degrees/index.php?r=site/view&id=919

Please note, you must apply to one of the projects and you must contact the primary supervisor prior to making your application. Additional information on the application process is available from the link above.

For more information about Precision Medicine visit:
http://www.ed.ac.uk/usher/precision-medicine