Supervisors: Clare Buckley and Kristian Franze
Project 1: Mechanical properties of neural tube polarisation
This project aims to investigate the role of mechanical force in initiating and organising apico-basal polarity within the zebrafish neural tube.
The vertebrate brain arises from a tube-like structure made from polarised cells called neuroepithelial cells. These have a strict apico-basal orientation. The zebrafish neural tube polarises ‘de novo’, establishing 2 adjacent polarised epithelia within the centre of an initially unpolarised tissue. This provides a tractable model to investigate the fundamental cell biology underlying how neuroepithelial (NE) cells polarise and how they then coordinate this polarity with their neighbours. Work from the Buckley lab suggests that cadherin-based adhesions play a role in the initiation of de novo polarisation (Buckley et al 2013 and unpublished). However, it is not clear what role the mechanical forces transduced through these adhesions play in polarity initiation. The Franze lab has demonstrated that tissue mechanics is instructive in a wide range of neural development. For example, the stiffness of Xenopus brain tissue was found to change rapidly during development, instructing the turning of retinal ganglion cells (Thompson et al 2019).
The student will use iAFM techniques in the Franze lab to first measure stiffness gradients as apico-basal polarity arises and then to exert a compressive force to test whether mechanical force influences the location and timing of apical polarity initiation during neuroepithelial polarisation. They will do this both at the tissue level in vivo, along the developing zebrafish neural rod and at the cell level within an in vitro ‘neural tube’, of zebrafish neuroepithelial progenitor cells seeded within hydrogels of different matrix stiffnesses. Time permitting, we will use optogenetic methods (Buckley 2016) to spatiotemporally alter actomyosin contractility and cadherin cell-cell adhesion localisation within the in vitro model to assess the interplay between internal and external mechanical forces and cell polarity establishment.
Relevant references:
1. Thompson et al. 2019 Elife. Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain.
https://www.ncbi.nlm.nih.gov/pubmed/30642430
2. Buckley et al. 2016. Dev Cell. Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.
https://www.ncbi.nlm.nih.gov/pubmed/26766447
3. Buckley et al. 2013. EMBO J. Mirror-symmetric microtubule assembly and cell interactions drive lumen formation in the zebrafish neural rod.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545300/.
Project 2: Optogenetically untangling cell polarity and cell division during neural tube morphogenesis
This project aims to use optogenetics to determine the interrelationship between cell polarity and cell division during zebrafish neural tube epithelialisation.
Understanding the relationship between cell polarity and division during epithelial organ establishment is fundamental to understand body patterning and organogenesis. Rather than occurring via the folding of an already established epithelium, teleost neurulation occurs via de novo polarisation and cavitation within a solid tissue. It therefore provides an ideal model to investigate the cell biology underlying progressive polarisation and epithelialisation of epithelial cells during organ development. Progenitor cell division is thought to be a major driving force behind the specific localisation of the apical membrane initiation site (AMIS) in epithelial tubes. However, our previous work in the developing zebrafish neural tube uncovered a surprising mechanism of division-independent de novo cell polarisation; individual neuroepithelial cells are able to localise apical polarity proteins such as Pard3 to the midline of the neural keel prior to and in absence of cell divisions, even if this is part-way along the cell length (Buckley et. al., 2013). This raises the questions of how cell polarity and cell division are coordinated in the development of a whole organ.
To test the interrelationship between cell polarity and cell division in directing midline development, the student will use optogenetics within the developing zebrafish neural rod (Buckley 2016, 2019) to manipulate the subcellular position of key polarity and division regulators during epithelial establishment. The effect of these manipulations on cell behaviour and morphogenesis of the zebrafish neural tube in vivo will be analysed using confocal 4D imaging. These experiments will be complemented by functional knock down experiments using CRISPR to confirm the role of these proteins in epithelial establishment. Together, these experiments will test both the necessity and sufficiency of polarity proteins in directing neural tube morphogenesis.
Relevant references:
1. Buckley C.E. (2019) Optogenetic Control of Subcellular Protein Location and Signaling in Vertebrate Embryos. In: Pelegri F. (eds) Vertebrate Embryogenesis. Methods in Molecular Biology, vol 1920. Humana Press, New York, NY
2. Buckley et al. 2016. Dev Cell. Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo.
https://www.ncbi.nlm.nih.gov/pubmed/26766447
3. Buckley et al. 2013. EMBO J. Mirror-symmetric microtubule assembly and cell interactions drive lumen formation in the zebrafish neural rod.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545300/.