Bioenergetics of nervous system development

Background:
The brain and spinal cord develop from an embryonic structure called the neural tube, which develops early in development by the folding and fusion of a sheet of neuroepithelial cells. Determining 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 (e.g. spina bifida), caused by failure of this process.

As neural tube closure propagates down the body the zippering process involves coordinated cell movement and rearrangements to establish and reinforce new cell-cell contacts. Closure occurs during a period of development when embryonic metabolism transitions from glycolysis to oxidative phosphorylation. This project will investigate the hypothesis that progression of neural tube closure is an energy-intensive process that is dependent on localised ATP production and regulation of glycolysis along the body axis.

Aims/Objectives:
Using wild-type and genetic mutant mouse strains, the aims are to:
1. Test whether axial variation in glycolytic activity modulates rates of neural tube zippering.
2. Investigate the local regulation of energy metabolism by analysing ATP:ADP ratio at cellular resolution using fluorescent biosensors in live embryos.
3. Determine the contribution of impaired energy metabolism to development of neural tube defects (NTDs) in mouse genetic mutants and test possible protective treatments.

The overall objective is to ask how energy metabolism contributes to neural tube closure and/or can be modulated to provide novel means to prevent neural NTDs.

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 whole embryo culture, immunostaining advanced imaging using light, fluorescent and confocal microscopy 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: Learning mouse embryo collection, microscopy 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).