Maintenance and elimination of long-term axial progenitors in mouse

Abstract

Elongation of the vertebrate rostrocaudal axis depends on localised populations of axial progenitors. Previous work has demonstrated the presence of Neuromesodermal (NM) progenitors that behave as multipotent stem cells, which contribute to the neurectoderm and mesoderm throughout axis elongation. They have been localised to the Node-­‐Streak Border (NSB) in the primitive streak region, and the Chordoneural Hinge (CNH) in its descendant, the tail bud. At primitive streak stages, the Caudal Lateral Ectoderm (CLE) on either side of the primitive streak itself is also fated for neurectoderm and mesoderm. However, fate mapping studies in mouse and chick have suggested that these progenitors are more transitory than those in the NSB and CNH, leading to the idea that two different types of progenitor cell exist in the primitive streak region; long-­‐term (stem cell-­‐like) and transient progenitors. In this thesis, I have examined the potency of the CLE cells by heterotopically grafting them into the NSB. Anterior CLE cells are exquisitely sensitive to their position and differentiate predominantly as neurectoderm, mesoderm, or both, depending on their exact location in the NSB. Most significantly, their descendants are retained in the CNH, indicating that CLE cells show equal potential to NSB progenitors on transplantation to the border environment. The relationship between fate and potency within the streak stage embryo suggest a mechanism by which stem cells are maintained not only by their intrinsic stem cell program, but are also influenced by their location. Furthermore, I have investigated the expression of candidate markers of NM-­‐progenitors, and have found that the combined expression of Sox2 and T genes in the progenitor area coincides with observed NM-­‐potency, and could serve as a marker for this stem cell population. Over time, axial elongation comes to a halt and NM-­‐progenitors are thereafter not longer active. It is still unclear how exactly this process occurs and specifically whether axial elongation ceases because NM progenitors are eliminated. I have investigated the events occurring immediately before the end of axial elongation. Morphological and gene expression analysis shows that apoptosis reaches a peak only after the complete axis has been laid down, and is not dramatically elevated in the progenitors themselves before that. In order to test signalling pathways that lead to progenitor maintenance, I have developed an in vitro tail growth assay that recapitulates in vivo development, as measured by several morphometric criteria. I show that, even though FGF signalling is critical for most cells in the tail bud including NM-­‐progenitors, it is not sufficient for NM-­‐ progenitor maintenance. In contrast, exposing tail buds to elevated Wnt/β-­‐catenin signalling does prolong the lifetime of NM-­‐progenitors in the ageing tail bud, as judged by the presence of Sox2-­‐T double-­‐positive cells. In this regard we have found that the time of cessation coincides with the disappearance of Sox2-­‐T double-­‐positive cells, the disappearance of Wnt3a and concomitant neuralisation of the progenitor region. This data suggest an important governing role for Wnt signalling in both maintenance and fate decision of NM progenitors. Thus the disappearance of Wnt signalling in the tail bud over time could well be the main reason for triggering the halt of caudal elongation.