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Optogenetic protein manipulation during asymmetric divisions in vertebrate brain development

Reference	BB/R001103/1
Principal Investigator / Supervisor	Professor Jonathan Clarke
Co-Investigators / Co-Supervisors
Institution	King's College London
Department	Developmental Neurobiology
Funding type	Research
Value (£)	367,790
Status	Completed
Type	Research Grant
Start date	01/01/2018
End date	31/12/2021
Duration	48 months

Abstract

During brain development in vertebrate embryos progenitors divide to generate two daughter cells. In early development the two daughters are essentially both copies of the original progenitor cell, i.e. they are both also progenitors that can themselves go on to divide and make two more cells. These cell divisions are called symmetric because their two daughters have similar identities. Later in development many progenitors will change the way they divide so that their two daughters are generated with different fates. For example one daughter will become another progenitor and be capable of dividing again, while the other daughter becomes a more specialised cell such as a neuron which is incapable of dividing. This type of cell division is called an asymmetric and is of fundamental importance to brain development because it allows the brain to get bigger by repeatedly generating more cells while also making some cells that construct the neural circuits that underlie animal behaviour. This proposal will test a long held hypothesis that asymmetric divisions are determined by the asymmetric inheritance of specific proteins from the progenitor cell to the two daughters, for example one daughter inherits certain proteins that push it towards becoming a neuron, while the other daughter does not inherit the same proteins and therefore has a different identity. We have developed an optogenetic method that allows us to manipulate subcellular protein localisation in individual progenitors during mitosis in the intact zebrafish embryo CNS. This will allow us to manipulate the symmetry of inheritance of specific proteins and then determine the consequences of this manipulation on daughter cell fate. We will concentrate on the role of the apically localised polarity proteins Pard3 and aPKC because previous work in vertebrates suggests they will have key roles and because these proteins are known to regulate asymmetric divisions in simpler animals such as worms and flies.

Summary

During brain development in vertebrate embryos (including man) individual cells known as progenitors divide to generate two daughter cells. In early development the two daughters are essentially both copies of the original progenitor cell, i.e. they are both also progenitors that can themselves go on to divide and make two more cells. These cell divsions are called symmetric cell divisions because their two daughters have similar identities. A little later in development many progenitors will change the way they divide so that their two daughters are generated with different fates. For example one daughter will become another progenitor and be capable of dividing again, while the other daughter becomes a more specialised cell such as a neuron which is incapable of dividing. This type of cell division is called an asymmetric cell division and is of fundamental importance to brain development because it allows the brain to get bigger by repeatedly generating more cells (the daughter that is the progenitor does this) while also making some cells that construct the neural circuits that underlie animal behaviour (the daughter that is the neuron does this by connecting and communicating to other neurons). This proposal aims to understand the biological mechanisms that determine whether a cell division is symmetric or asymmetric. Specifically it will test a long held hypothesis that asymmetric divisions are determined by the asymmetric inheritance of specific proteins from the progenitor cell to the two daughters, for example one daughter inherits certain proteins that push it towards becoming a neuron, while the other daughter does not inherit the same proteins and therefore has a different identity. To achieve asymmetric inheritance of a protein the progenitor cell needs to have a mechanism that moves the protein into only one of its daughters as it splits into two. We have developed a new method that allows us to use light of particular wavelength to manipulate proteinlocalisation in individual progenitor cells in a living embryo. This allows us to experimentally control protein localisation during cell divisions and thus control whether proteins are symmetrically or asymmetrically inherited. This will allow us to determine whether asymmetric protein inheritance can explain the occurrence of asymmetric divisions in a vertebrate embryo.

Impact Summary

Who might benefit from this research? And how might they benefit from this research? 1. The postdoctoral researcher will benefit from training and execution of a novel technique of optogentic manipulation of subcellular protein localisation in a living embryo. This will be excellent experience for their potential future career in in vivo analysis of vertebrate development. 2. Local colleagues will benefit from seeing the successful implementation of a novel optogenetic in vivo technique that may be relevant to their own studies of brain development and cell biology. More distant cell and developmental biology colleagues will also benefit in the same way once we reach the stage of communicating our results through seminars, symposia and publications. 3. Other academic researchers interested in understanding vertebrate brain development and neural stem cell biology will benefit from the knowledge gained in understanding asymmetric cell division which is a very fundamental cell behaviour in embryogenesis and adult stem cell behaviour. 4. Local school children will benefit from the established outreach programmes our research Centre has in place. This programme allows us to enthuse young scientists about the importance of scientific research in general and our area of brain research in particular. Our aim is to encourage young people to become the scientists of tomorrow.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Neuroscience and Behaviour, Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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