Investigating the bioenergetics of early nervous system development

Background:

Development of the brain and spinal cord depends on the formation of the neural tube, a structure which is formed by the folding and fusion of a sheet of neuroepithelial cells on the back of the embryo. Understanding the mechanisms that underlie neural tube closure is important for understanding both nervous system development and the basis of common birth defects, neural tube defects (NTDs; eg. spina bifida), caused by failure of this process. Neural tube closure propagates down the body by a zippering process which involves coordinated movement and rearrangement of cells which depends on cytoskeletal function and formation of new cell-cell contacts. These processes are energy-intensive and it is notable that the closure process occurs during when central carbon metabolism transitions from glycolysis to oxidative phosphorylation.

Aims/Objectives:

This project will test the hypothesis that progression of neural tube closure is an energy-intensive process that is dependent on localised ATP production and that impaired regulation of energy metabolism causes NTDs. The aims are to:
1. Test whether neural tube zippering depends on glycolysis and whether this varies at differing axial levels, for example, in the future brain and spinal cord.
2. Investigate the local regulation of energy metabolism by analysing ATP:ADP ratio at cellular resolution using fluorescent biosensors in embryos using live imaging. 
3. Determine the contribution of impaired energy metabolism to development of NTDs in mouse genetic mutants, including with impaired folate metabolism. The possible prevention of NTDs will be tested using treatments which modulate energy metabolism.
Findings may be directly relate to understanding the cause and possible prevention of the equivalent conditions in humans.

Methods:

The project will focus on mouse neurulation, as a model system which is directly relevant to human development. The student will gain experience in a range of techniques in developmental biology including advanced imaging using light, fluorescent and confocal microscopy, whole embryo culture, immunostaining, and use of conditional knockout and transgenic mice. In parallel, metabolic assays will be carried out using a Seahorse analyser, with opportunity to gain experience in mass spectrometry and molecular biology techniques depending on the interests of the student.
Collaboration with University of Toronto: The PhD student will have the opportunity to carry out a 6 month research placement in Dr Sevan Hopyan’s laboratory in the Developmental & Stem Cell Biology Program at The Hospital for Sick Children, Toronto. The Hopyan group aims to understand morphogenesis and pattern formation by application of advanced imaging, mouse genetics and mathematical modelling. During the placement the student will gain experience in fluorescent biosensor imaging and analysis.

Timeline:

Month 1-12: Training in embryo collection and culture, fluorescence microscopy and introduction to and Seahorse assays. Month 13-18: Research visit to Hopyan Lab (Toronto) – flurorescent biosensor, embryo live imaging and analysis. Month19-36: Application of metabolic assays, biosensors and live imaging in mouse NTD models.  Month 37-42: Finalise experiments & write-up.

References:

1.    Nikolopoulou et al. Neural tube closure: cellular, molecular and biomechanical mechanisms. Development 144, 552-566 (2017).
2.    Mole et al. Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure. Dev Cell 52, 321-334 e326 (2020)
3.    Leung et al. Partitioning of One-Carbon Units in Folate and Methionine Metabolism Is Essential for Neural Tube Closure. Cell Reports 21, 1795-1808 (2017).
4.    Bulusu et al. Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development. Dev. Cell 40, 331-341 (2017).
5.    Tantama et al. Imaging energy status in live cells with a fluorescent biosensor of the intracellular ATP-to-ADP ratio. Nat Commun 4, 2550 (2013).