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Development of Epithelium Apical Polarity: Does the mechanical cell-cell adhesions play a role?

Periodic Reporting for period 1 - DEAP (Development of Epithelium Apical Polarity: Does the mechanical cell-cell adhesions play a role?)

Reporting period: 2019-05-01 to 2021-04-30

During development, most organs in the body arise from epithelial tubes. Whilst some tubes arise via the folding of an already polarised epithelial tissue, others arise via de novo polarisation in the centre of a previously unpolarised, solid tissue. De novo polarisation happens, for example, during mammalian epiblast formation when the inner-cell-mass (ICM) cells within embryos establish apical-basal polarity and form the amniotic cavity; as well as during teleost neural tube (NT) development when the neuroepithelium progenitor cells epithelialise and open a central lumen. How polarity is initiated in the precisely correct location within these epithelial tubes is therefore a fundamental question in organ development and has great implications in understanding diseases and tissue engineering. This project was proposed to understand how de novo polarisation is initiated during epithelial tube formation, by using mouse embryonic stem cell (mESC) culture, zebrafish models, optogenetics and live cell imaging. Specifically, the project was designed to address the following scientific questions: 1). Whether de novo polarization of epithelial organs is dependent on cell-cell adhesions. 2). Whether polarity establishment depends on cell-cell adhesion-based force. Due to career development, I am ending this fellowship 12 months earlier than the initial proposal. Most of the work proposed till this 12-month time point in the initial proposal have been achieved. No publications have been achieved till the end of period, but I am continuing work in the host laboratory and institution on the project and expecting to publish the results from the project in the next 12-18 months.
My research at Dr. Clare Buckley lab in Department of Physiology, Development and Neuroscience, University of Cambridge, focused on the role of cell-cell adhesion mediated force during de novo polarisation of epithelial tubes. I used two models for my study to explore the topic. First, I used a mESC culture model that phenocopies the morphogenesis of the very early mammalian embryo during implantation, when the epiblast forms a centrally located, polarised lumen. This minimal model allowed me to control cell number and cell-cell interaction so that I can analyse the initiation of absolutely naïve junctional adhesions in relation to polarisation. My work in this system with immunofluorescence, stable cell lines, as well as live cell imaging, labelled the location of key apical proteins (e.g. Pard3 and Pard6) during de novo polarisation in real time in the system. Second, I used the zebrafish neural tube as an in vivo model of epithelial tube formation. I designed to use CRISPR-CAS9 to manipulate cell adhesion molecules and a cutting-edge optogenetic approach to both image and manipulate the process of apicobasal polarisation and junctional formation, and were at the initial stage of trialing these techniques with some key preliminary data collected. My work have found that 1) mESC cultured into clumps can polarise de novo at the cell-cell interface and this can happen dispensable of cell divisions; 2) the position of de novo polarisation in mESC clumps can be guided by E-cadherin-based cell-cell adhesions; 3) During zebrafish neural tube opening, the mechanical force generator - myosin may play a important role in morphogenesis. The implement of the project transferred my knowledge in general cell culture and epithelium cell biology to the host, while the host helped me to develop my knowledge in mouse and zebrafish embryonic study and developmental biology. I have presented my work as posters in conference and workshops, as well as had the opportunity to share my knowledge in public engagements (e.g. university open days and Cambridge Science Festival). With the knowledge I acquired from the implement of the project, I have supervised and will teach and supervise research degree students, as well as teaching zebrafish developmental biology and optogenetics in workshops and courses.
This study of using two different systems were expected to uncover conserved principles of driving de novo polarisation during vertebrate morphogenesis. The results were able to extend our understanding of the biological importance of cell adhesions and advance our knowledge of the fundamental principles of tissue patterning during development. Beyond this, it might also help us to understand diseases such as the progress of cancer during which dysregulation of cell polarity happens.
Apical protein (Pard3) accumulation at cell-cell adhesions in cell division-disabled cell aggregates
Optogenetics to localise protein LARG to manipulate myosin activity in zebrafish neural tube.