Analysis of cell fate choice regulation in organiser/primitive streak stem cells and neuroectoderm progenitors.

The body axis, backbone and spinal cord are formed very early in development; during the first month of pregnancy in humans, between days 8 and 12 of development in the mouse and between days 1 and 4 in the chick. These structures form from a group of cells called axial progenitors that are similar to stem cells. Stem cells have two essential properties: they can divide to give more copies of themselves and they can produce a variety of specialised cell types in a process called differentiation. Disruption to the genes that regulate the functions of these particular progenitor cells, in animal models or human pathologies, causes a loss of axial tissue development, loss of patterning of the spinal cord, and can lead to severe holoprosencephaly or lethality of embryos. These tissues and processes are thus of fundamental importance to the developing embryo and a detailed understanding of how they develop and the molecules mediating their respective functions is crucial both to the understanding of embryo development and the generation of means of treating embryonic defects.
It is vitally important for the developing embryo that the right number of these axial progenitor cells chooses to differentiate into each of the specialised cell types so that the embryo develops into the right size and shape. We aim to understand what molecules make these cells choose which cell type to differentiate into. To date, very little was known about what regulates the cell fate choice of these particular stem cell-like progenitors. We have recently identified a key molecular pathway regulating this event and we plan to investigate the biochemical basis of this regulation.
In mice, unlike Embryonic Stem cells, these cells do not form malignant tumours when transplanted into adults and may therefore become a useful therapeutic cell type for degenerative diseases and injuries involving the spinal cord, and the back bone.

A fundamental challenge in stem cell biology is to understand how cells acquire distinct fates. During early vertebrate embryogenesis a stem cell-like progenitor population located in a structure called the primitive streak gives rise to structures that run the length of the body, notably, the neural tube (future spinal cord) and somites (future muscle and skeleton). The organizer is a specialised structure at the anterior tip of the primitive streak. Stem cell-like progenitors located in the organiser give rise to two specific tissues namely the floor plate, which lies in the ventral floor of the neural tube and the notochord, a rod of mesoderm lying directly below the floor plate. The floor plate and notochord provide a critical signal, sonic hedgehog, which patterns the neural tube regulating where and when distinct neuronal subtypes differentiate. The organiser/primitive streak are thus of fundamental importance to the developing embryo and a detailed understanding of the molecular mechanism that regulates allocation of appropriate numbers of these stem-like progenitor cells to these various embryonic tissues is crucial both to the understanding of embryogenesis and the generation of means of treating embryonic defects. Very little is known about which signalling pathways regulate cell fate choice among these progenitors in amniote embryos. We have recently shown Notch regulates cell fate choice in the organiser. We identified a second novel role for Notch which is to influence cell fate decisions of neural progenitors (by modifying their competence to respond to SHH), and thereby affect dorsoventral patterning of the neural tube.
We aim to 1) Identify which pathways Notch interacts with in the organiser to ensure appropriate numbers of cells adopt each fate. 2) Establish if Notch also regulates cell fate choice in the adjacent primitive streak stem-like progenitors. 3) Investigate how Notch regulates the response of neuroectodermal cells to the morphogen Shh. 4) We will determine if these are conserved roles in higher vertebrates. The analyses will involve gain and loss of function assays treating progenitors to pharmacological reagents or electroporation of relevant constructs followed by grafting progenitors to a host embryo. We will then lineage trace the derivatives of the treated progenitors. We have also devised a screen using Next Generation Sequencing technology to identify pathways regulating these cell fate choices. For the Notch-Shh interplay we will analyse the effect of Notch misexpression on components of the Shh pathway known to affect efficacy of signal transduction.