Generating physiologically-relevant cells of interest in vitro is a major challenge faced by researchers making cells for therapy. Accordingly, precise knowledge of the fate choices that cells make during embryo development, and the environmental signals governing these choices, is paramount in designing efficient in vitro differentiation protocols.
The ‘textbook’ description of central nervous system development statest that neural tissue is first specified, then subsequently patterned into brain and spinal cord (the ‘activation-transformation’ hypothesis, generated first in amphibians). The activation-transformation hypothesis has formed the rationale for in vitro neural differentiation protocols from pluripotent cells. However we have shown that during embryo development, the spinal cord, backbone, and its associated musculature are built not by committed neural precursors, but by bipotent neural/mesodermal progenitors (NMPs) located at the tail end of the embryo, while the brain is likely to be formed by an independent cell population at the head end. In vitro culture conditions that allow NMP differentiation have now resulted in the production of lower spinal cord neural types, which had not previously been generated using differentiation protocols that follow the ‘activation-transformation idea’ of first specifying indeterminate neural tissue, then ‘transforming’ it to a posterior identity. However the in vivo (and in vitro), sequence of cell fate restrictions leading to brain and spinal cord production is still not clear: do pluripotent cells first commit to a neural identity, then separate into brain and spinal cord- generating NMPs, or are NMPs and brain separately specified from pluripotent cells?
In this project, this question will be investigated both in vitro in mouse pluripotent cells, and in two in vivo systems, mouse and chick embryos.
NMPs are characterised by the coexpression of two transcription factors, T(brachyury) and Sox2. Meanwhile Sox1 expression characterises the site of future brain formation. Since we have fluorescent markers of these populations, this is a tractable in vitro system to follow the sequence of cell fate decisions needed to make brain and spinal cord at the single cell level. Analysis of single cell transcriptomes in parallel with analysis of their commitment will further characterise these cell fate decisions. Furthermore, in vitro culture of manipulated early mouse and embryos where future brain and spinal cord tissue are challenged by transplantation to spinal cord or brain environments respectively will test at what stage these cells are committed to brain or spinal cord fates: before or after their commitment to a neural identity. Our preliminary data suggests a rather similar sequence of events in each organism, but subtle differences in the generation of these anterior and posterior cell types may underpin some of the species differences; this will be analysed by comparison of chick and mouse transcriptomes.
This project therefore challenges a fundamental concept in developmental biology, but also is likely to lead to improvements in the in vitro differentiation of pluripotent cells into cell types of clinical interest for degenerative disease and/or injury.