A quantitative gene network underlying robust epidermal stem cell fate patterning

Understanding the robustness underlying stem cell maintenance and cell fate patterning is a fundamental problem in developmental biology. It has applications in many disciplines from regenerative medicine to cancer treatment. Robustness is defined as resistance to phenotypic change in the face of internal and external perturbations. One of the most developmentally robust metazoan animals is C. elegans. Its whole developmental process is highly stereotyped including the developmental path of individual cells, to the extent that we know the exact number and type of divisions each cell has undergone in an adult nematode over the course of its development. C. elegans have exactly 959 cells upon reaching adulthood, of these 32 correspond to the lateral seam cells. Seam cells have the essential properties of stem cells. They are able to both self-renew and differentiate into neuronal and epidermal cell fates. The choices that these cells make are regulated by a network of transcription factors, the exact architecture of which is not well resolved.



We will start from a network focused on the three transcription factors involved in seam cell maintenance. These genes are ceh-16, elt-1 and egl-18. ELT-1 is a GATA-binding transcription factor and is required for the specification of the entire epidermis, including the seam cells and its expression is maintained in seam cells throughout development. EGL-18 is also a GATA factor and has been hypothesised to be regulated by ELT-1. CEH-16 is an ortholog of human Engrailed Homeobox 1/2 genes. It is thought to have a role in preventing seam cell fusion and has been thought to influence both elt-1 and egl-18 expression. All three of these are highly important genes in development. Full knockouts of any of the three results in severe malformations of the nematode, which in the case of elt-1 and ceh-16 are embryonic lethal. Due to this we will be working mostly with tissue-specific modifications and partial loss of function mutants.



There are two main approaches that we will take. The first is to evaluate individual connections established by an initial Boolean network in more depth. This approach would require the confirmation of certain behaviours predicted by the Boolean network. Therefore, double and triple mutants of these core genes will be evaluated in terms of their molecular and phenotypic consequences in seam cells. This will allow us to test the regulatory connections between the genes proposed by the model. On top of this, we aim to evaluate the behaviour of our three genes over time through larval development. We aim on using live imaging of transcriptional reporters to collect detailed information on the behaviour of the three genes. Additionally, the use of transcriptional reporters could allow us to identify transcription and degradation rates, which would be essential for more complex modelling of the system's behaviour.



Additionally, the second approach is to expand the network through the addition of more genes and connections. For this purpose, we will investigate the genes thought to be connected to our selected three genes in the literature. Additionally, we will integrate additional connections through targeted DamID (TaDa) experiments in our lab revealing downstream targets as well as confirming interactions between core components.



Overall, we aim to elucidate the connections in the gene network underlying the mechanisms required for seam cell maintenance and division and understand the network's dynamics. We aim to provide a quantitative model of the interactions between their transcription factors in order to have a more in depth understanding of the causes underlying seam cells robustness.