Sanger Institute Fellowship Project Outline Project 5: Charting the Zebrafish Immune System One Cell at a Time Supervisors: Sarah Teichmann and Ana Cvejic Email: Sarah Teichmann - [email protected] Ana Cvejic - [email protected] Webpage: Sarah Teichmann - http://www.sanger.ac.uk/research/faculty/steichmann/ Anan Cvejic - http://www.sanger.ac.uk/research/faculty/acvejic/ The immune system is one of the fastest-evolving biological processes, and it has become clear that all kingdoms of life, bacteria, plants and animals, have developed their own forms of adaptive immune system. Within the animal kingdom, genome analysis suggests that all vertebrates have rearranging molecules in the form of T- and B-cell receptors. However, the precise nature of the T and B cells in which these receptors are expressed is known only in human and mouse. Using the power of high-throughput single cell transcriptomics, we can now discover the cellular landscape of the adaptive immune system in model organisms ab initio. Here, we propose to profile T cells in a lower vertebrate, in order to gain insight into the origins of mammalian adaptive immunity. We will begin by profiling T cells in the zebrafish, a genetically tractable vertebrate model organism that is increasingly emerging as a model for immunity and disease (Tobin et al., PLOS Pathog., 2012). Initially, we will focus on CD4+ T helper cells, which are white blood cells that initiate the body's response to infections, and are an essential part of the human immune system. T helper cell subtypes (e.g. Th1, Th2, Th17) have been well characterised in higher vertebrates and can be distinguished by the specific interleukins that serve as activators, repressors or products of these cell types. However, the presence and identity of T helper cell subtypes in bony fish is still an open question. Through computational analysis, we have identified several of the interleukins and their receptors in zebrafish, suggesting that most genes characteristic of T helper cell differentiation are present. In particular, we have compiled evidence of IL1, IL4, IL5, IL6, IL10, IL13, IL17, IL21, IL23 and IL27 based on the literature (Secombes et al., Dev. Comp. Imm., 2011; Laing and Hansen, Dev. Comp. Imm., 2011) and gene prediction. Moreover, synteny and the presence of their receptors indicate that IL2 and IL33 could also be present in zebrafish. We will answer this intriguing question through massively parallel single cell RNA-sequencing of cells from existing Tg(lck:EGFP) and custom Tg(cd4:EGFP) zebrafish reporter lines, as well as FACS-sorted CD4+ cells with a new commercially available antibody. These experiments will provide a rich data set to mine bioinformatically, and gain unparalleled insights into the evolution of immunity. The project will benefit from combined expertise of the Teichmann group in single cell RNA-sequencing (Brennecke et al., Nat. Methods, 2013; Mahata et al., Cell Reports, 2014) and the Cvejic group in zebrafish biology (Cvejic et al., Nat. Genet, 2013; Bielczyk-Maczyńska, PLOS Genet., 2014, in print). It will involve a combination of in vivo zebrafish experiments, genomics and bioinformatics methods. Page 1 of 1