Roles of the transcriptional regulator Id1 in pluripotency and differentiation

The transition from pluripotency to differentiation is a key event in the life of all complex multicellular organisms. In the development of the mouse, the pluripotent epiblast undergoes gastrulation and gives rise to three multipotent germ layers, which will in turn form the tissues of the adult body. The events leading up to gastrulation have been extensively studied in vivo in developing embryos, and modelled in vitro making use of embryonic stem (ES) cells. Bone morphogenic protein (BMP) signalling plays a key role in these processes. BMP can in fact maintain ES cells in a self-renewing state by inhibiting their differentiation into neural ectoderm, whilst at the same time being required for the specification of mesoderm in the developing embryo (Winnier et al. 1995, Ying et al. 2003a). A key intracellular target of BMP is the transcriptional regulator Id1, which can recapitulate the effects of BMP in the preservation of ES cell pluripotency and in the inhibition of neural specification from pluripotent cells (Ying et al. 2003a). This thesis will focus on understanding the roles of this molecule in the early decisions affecting the transition from pluripotency to differentiation. In particular, I aim to study the expression pattern of Id1 in cultures of pluripotent cells, and to clarify which extracellular and intracellular molecules regulate the expression of the factor; I aim to understand how forced Id1 expression inhibits the differentiation of pluripotent cells, and whether Id1 may play a similar role in the regulation of the asynchronous exit from pluripotency observed in differentiating wild-type cells; finally, I aim to characterise the expression pattern of Id1 in the early stages of post-implantation development at the single-cell resolution, and to understand how the expression of the molecule correlates with the previously characterised expression patterns of key signalling molecules and transcription factors. The generation of a reporter ES cell line expressing the yellow fluorescent protein Venus fused to the C-terminus of Id1 allowed me to assess the expression of the factor in culture on a single-cell basis, making use of immunofluorescence and flow cytometry. I observed that expression of Id1 is reliant on active BMP signalling and low Activin/Nodal signalling, and I characterised the combinatory effects of the two pathways on Id1 expression. Furthermore, I demonstrated that high Nanog expression is incompatible with high Id1 expression in ES cell cultured in the presence of LIF and serum, which raises the possibility that Nanog may be affecting the expression of Id1 in vivo, both in pre-implantation and in post-implantation embryos. I generated ES cell lines overexpressing Id1 and observed that the factor inhibits differentiation of pluripotent cells into neural ectoderm by delaying their exit from a post-implantation epiblast-like pluripotent state, and ultimately favouring mesodermal specification. This suggests that Id1 is acting at a specific stage of differentiation and that the differentiation process itself is following a similar developmental pathway to what is observed in the peri-gastrulation stage embryo. I performed single-cell transcriptional analysis on differentiating wild-type ES cells and observed that Id1 is not expressed at an appropriate point in time to affect the asynchronous the exit from pluripotency observed in neural adherent monolayer differentiation, which suggests that other factors must be responsible for this phenomenon. Finally, I addressed the expression pattern of Id1 protein in the embryonic tissue of gastrulating mouse embryos by imaging chimaeric embryos generated using the Id1- Venus reporter ES cells. I observed that Id1 is expressed in the proximal regions of streak stage embryos; in the epiblast and migrating mesendoderm of bud stage embryos; in cardiac, lateral and allantoic mesoderm and in foregut endoderm in headfold stage embryos. These expression patterns fit with the reported expression of BMP molecules at these stages of development, and suggest that Id1 expression is primarily dependent on BMP expression in early post-implantation embryos. However, I also observed Id1 expression in a ring of cells surrounding the node in headfold stage embryos, a previously uncharacterised expression pattern not directly attributable to BMP expression. This raises the intriguing question of what is regulating Id1 expression and what roles Id1 may be playing in this key embryonic structure

Enabling neighbour-labelling: using synthetic biology to explore how cells influence their neighbours

Cell-cell interactions are central to development, but exploring how a change in any given cell relates to changes in the neighbour of that cell can be technically challenging. Here, we review recent developments in synthetic biology and image analysis that are helping overcome this problem. We highlight the opportunities presented by these advances and discuss opportunities and limitations in applying them to developmental model systems

SyNPL:Synthetic notch pluripotent cell lines to monitor and manipulate cell interactions in vitro and in vivo

Cell-cell interactions govern differentiation and cell competition in pluripotent cells during early development, but the investigation of such processes is hindered by a lack of efficient analysis tools. Here, we introduce SyNPL: clonal pluripotent stem cell lines that employ optimised Synthetic Notch (SynNotch) technology to report cell-cell interactions between engineered ‘sender’ and ‘receiver’ cells in cultured pluripotent cells and chimaeric mouse embryos. A modular design makes it straightforward to adapt the system for programming differentiation decisions non-cell-autonomously in receiver cells in response to direct contact with sender cells. We demonstrate the utility of this system by enforcing neuronal differentiation at the boundary between two cell populations. In summary, we provide a new adaptation of SynNotch technology that could be used to identify cell interactions and to profile changes in gene or protein expression that result from direct cell-cell contact with defined cell populations in culture and in early embryos, and that can be customised to generate synthetic patterning of cell fate decisions

Cadherins in early neural development

During early neural development, changes in signalling inform the expression of transcription factors that in turn instruct changes in cell identity. At the same time, switches in adhesion molecule expression result in cellular rearrangements that define the morphology of the emerging neural tube. It is becoming increasingly clear that these two processes influence each other; adhesion molecules do not simply operate downstream of or in parallel with changes in cell identity but rather actively feed into cell fate decisions. Why are differentiation and adhesion so tightly linked? It is now over 60 years since Conrad Waddington noted the remarkable "Constancy of the Wild Type" (Waddington in Nature 183: 1654-1655, 1959) yet we still do not fully understand the mechanisms that make development so reproducible. Conversely, we do not understand why directed differentiation of cells in a dish is sometimes unpredictable and difficult to control. It has long been suggested that cells make decisions as 'local cooperatives' rather than as individuals (Gurdon in Nature 336: 772-774, 1988; Lander in Cell 144: 955-969, 2011). Given that the cadherin family of adhesion molecules can simultaneously influence morphogenesis and signalling, it is tempting to speculate that they may help coordinate cell fate decisions between neighbouring cells in the embryo to ensure fidelity of patterning, and that the uncoupling of these processes in a culture dish might underlie some of the problems with controlling cell fate decisions ex-vivo. Here we review the expression and function of cadherins during early neural development and discuss how and why they might modulate signalling and differentiation as neural tissues are formed.Peer reviewe

Gene expression analysis of subpopulations of mouse embryonic stem cells sorted based on Id1 and Nanog expression

Overall design: Id1-Venus Nanog-tagRFP (IVNR) reporter mouse ES cells cultured in LIF+FCS were sorted into Nanog-tagRFP-high, Id1-Venus-high, or double low subpopulations. Unsorted IVNR cells cultured in LIF+FCS, as well as Id1-Venus (Id1V) reporter ES cells cultured in 2i and in 2i+LIF were included as controls. All samples were collected in duplicate. Total 12 samples. Experiment type: Expression profiling by array.Analysis of gene expression in sorted subpopulations of mouse embryonic stem cells. We set out to investigate whether expression of Id1 in Nanog-low cells affected the expression of pluripotency factors and signalling molecules.Malaguti M, Lowell S, 2018, Gene expression analysis of subpopulations of mouse embryonic stem cells sorted based on Id1 and Nanog expression, Gene Expression Omnibus, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE10822

Image6_Repurposing the lineage-determining transcription factor Atoh1 without redistributing its genomic binding sites.TIF

Although the lineage-determining ability of transcription factors is often modulated according to cellular context, the mechanisms by which such switching occurs are not well known. Using a transcriptional programming model, we found that Atoh1 is repurposed from a neuronal to an inner ear hair cell (HC) determinant by the combined activities of Gfi1 and Pou4f3. In this process, Atoh1 maintains its regulation of neuronal genes but gains ability to regulate HC genes. Pou4f3 enables Atoh1 access to genomic locations controlling the expression of sensory (including HC) genes, but Atoh1 + Pou4f3 are not sufficient for HC differentiation. Gfi1 is key to the Atoh1-induced lineage switch, but surprisingly does not alter Atoh1’s binding profile. Gfi1 acts in two divergent ways. It represses the induction by Atoh1 of genes that antagonise HC differentiation, a function in keeping with its well-known repressor role in haematopoiesis. Remarkably, we find that Gfi1 also acts as a co-activator: it binds directly to Atoh1 at existing target genes to enhance its activity. These findings highlight the diversity of mechanisms by which one TF can redirect the activity of another to enable combinatorial control of cell identity.</p