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Probing the assembly and segregation of phase-separating RNA granules in live zebrafish embryos using optogenetic tools

Primary Supervisor: Professor Karuna Sampath, WMS

Secondary supervisors: Daniel Hebenstreit, SLS & Masanori Mishima, WMS

PhD project title: Probing the assembly and segregation of phase-separating RNA granules in live zebrafish embryos using optogenetic tools

University of Registration: University of Warwick

Project outline:

Background: Phase separation is a process that occurs in a variety of cell types and organisms, through which cellular content is organised in membrane-less compartments within cells. These dynamic structures, also known as ‘phase separating granules’ or ‘liquid condensates’, have functions in sequestering biomolecules, facilitating reactions and channelling intracellular signalling within cells. In animal cells, phase-separating condensates have been found to organise cell fate determinants. In the germline, the tissue that gives rise to the next generation, progenitor cells (stem cells) have phase-separating condensates called “germ plasm” comprising of RNAs and RNA-binding proteins (RNPs). Germ granules are essential for germ cell fate and the next generation. How germ plasm forms and how it is distributed is not fully understood.

Zebrafish is a simple vertebrate that is highly amenable to in vivo cell biology owing to the optical clarity of embryos and has been used to understand the cellular basis of many fundamental processes. Zebrafish shares 70% of the genes in humans and counterparts for >83% of human disease genes. How germ plasm forms in zebrafish germline progenitors and how it is distributed is poorly understood. For instance, it is not known if germ granule size regulates germ plasm distribution in progenitor cells.

Objectives: To develop optogenetic tools for probing the assembly and distribution of phase separating RNP granules in zebrafish embryos.

Methods: The student will combine experimental work (molecular, cell and developmental biology) with computational analysis of imaging. The student will use zebrafish transgenic reporter lines labelling the germ granules (Tg Buc-gfp) to visualise germ plasm in vivo. An ‘optogenetic’ system (i.e. light-controlled switch) to manipulate germ granules in zebrafish will be developed, based on systems used in mammalian cells (Shin et al., 2017). Transgenic lines for manipulating germ granules will be generated. In vivo time-lapse imaging will be performed to determine if the new optogenetic system can be used to generate phase separating germ granules of different sizes in a controllable manner in developing zebrafish. Imaging will be done using lattice light sheet microscopy (LLSM), spinning disc confocal microscopy (SDM) and multiphoton FLIM. Computational analysis of the imaging data will be performed to measure the dynamics of phase separating germ granules in vivo in embryos. The findings can provide a conceptual framework to build mathematical models of the process.

For the mini-project, the optogenetic system will be generated by molecular biology methods (PCR, Gibson cloning, plasmid preps, RNA synthesis, etc) and preliminary tests of the system will be performed using transient micro-injection assays in zebrafish embryos.

Doctoral project: Upon successful generation and tests of the optogenetic module, stable transgenic lines will be generated in the Sampath lab and used in the doctoral project for investigating germ granule assembly and segregation in vivo in zebrafish.

Supervisory arrangement: The experimental work and in particular, zebrafish work will be supervised by Sampath; Imaging and computational analysis will be overseen by Mishima and Hebenstreit. The student will meet with the main supervisor at least 1 x per week, and all supervisors 1x per month (or more frequently, when required) to discuss the project.

References:

  1. Shin et al., Cell (2017). http://dx.doi.org/10.1016/j.cell.2016.11.054
  2. Vong YH, Sivashanmugam L, Zaucker A, Jones A and Sampath K. The RNA-binding protein Igf2bp3 is critical for embryonic and germline development in zebrafish. bioRxiv (2020). doi:https://doi.org/10.1101/2020.06.23.167163).
  3. Krishnakumar et al. PLoS Genetics (2018). https://doi.org/10.1371/journal.pgen.1007696

BBSRC Strategic Research Priority: Understanding the Rules of Life: Stem Cells & Systems Biology. Integrated Understanding of Health: Ageing

Techniques that will be undertaken during the project:

  • Molecular biology: Standard molecular biology including DNA and RNA extraction, RNA synthesis, PCR and qRT-PCR, plasmid cloning, ligation and bacterial transformation.
  • Zebrafish embryonic development and genetics, cell biology techniques including microinjections, genetic crosses, transgenic reporter generation and analysis
  • Imaging: standard light and fluorescence microscopy, lattice light sheet microscopy, spinning disc confocal microscopy and multiphoton FLIM
  • Image analysis: 3D particle tracking and segmentation, Scripting with Fiji/ImageJ
  • Data analysis: Statistical analysis using R

Contact: Professor Karuna Sampath, University of Warwick