The adaptive immune system, present in all vertebrates from teleost fish to humans, mediates specific delayed and long-term responses to pathogen invasion. This is executed by T and B lymphocytes, two types of white cells that arise from a common lymphoid progenitor (CLP) along sequential biological events that take place in different primary lymphoid organs, through a process globally termed lymphopoiesis. The correct maturation and formation of the different lymphocytes depends on the fine control of complex molecular mechanisms that regulate gene expression both in the lymphocytes themselves and in other cells that interact with them around the primary lymphoid niches. Thus, the action of individual genes can only be properly understood when viewed in the context of the environment in which they are expressed and function. Teleost fish, the most abundant group of vertebrates, represent an excellent tool for establishing possible correlations between the histological organization of their lymphoid organs and their immunological capabilities. In teleosts, the posterior kidney represents a true equivalent of the lymphohematopoietic bone marrow of higher vertebrates, while the thymus participates in T-cell maturation. Apart from the B and T lymphocytes that mature in these organs through a series of immature stages, both the posterior kidney and thymus contain a whole repertoire of both immune and non-immune cells that interact with the lymphocytes during the maturational processes. Currently, it is almost completely unknown how these cells are positioned in the different lymphoid niches of these organs, which in turn prevents a thorough understanding of the functions that each cell type can perform. The MaLymChes project aims to implement state-of-the-art technologies of spatial transcriptomics and single cell epigenetics to give an important boost to the knowledge of lymphopoiesis in teleost fish and more specifically in rainbow trout, a species of high economic interest. The execution of the present proposal will allow 1) to explore cellular heterogeneity in primary lymphoid niches of rainbow trout by capturing mRNA at 3'. Both immune and non-immune cells will be captured, and their transcriptome will be fully characterized. 2) Resolve the positional context of all cells along the posterior kidney and thymus, which will allow us to determine the microenvironment of B and T cells at different maturation stages. 3) Analyze the genome-wide chromatin accessibility of thousands of cells in parallel, which will allow us to examine gene regulation in immune and non-immune cell populations, and 4) identify potential DNA regulatory elements that might be associated with transcription factors expressed in the same cell types. The in-depth understanding of the processes involved in lymphocyte maturation will represent an important advance in knowledge with the potential to be applied in breeding programs and aquaculture.

The adaptive immune system mediates specific long-term responses to face pathogen invasion. This defense system, is formed by T and B lymphocytes, which arise from a common lymphoid progenitor (CLPs) throughout sequential biological events that take place in different primary lymphoid organs, through a process globally designated as lymphopoiesis. During lymphocyte maturation, the formation of T cell receptors (TCRs) and B cell receptors (BCRs), located in the surface of lymphocytes T and B respectively, which are able to recognize a wide range of antigenic epitopes is a key process. The production of these receptors requires a complex genetic recombination mediated by RAG1, RAG2 and TdT, a set of proteins highly conserved along vertebrate’s evolution. By this biological process a set of genes formed by V gene (variable), D gene (diversity) J gene (joining) and C gene (constant) are selected from a large repertoire available in germline. The combination V(D)J represents the variable region of BCRs and TCRs, where these molecules bind to their specific antigen. On the other hand the C gene define the type of receptor. In mammals, the successive steps of lymphocyte maturation have been deeply characterized resulting in the design of a large repertoire of markers applicable in several techniques that allow the isolation and dissection of specific lymphocyte populations or differential maturation states. In fish, this work has not been yet undertaken. Attending to the expression of fish orthologues for RAG1, RAG2 and TdT, the thymus and the head kidney (considered the equivalent of mammals bone marrow) have been identified as the main primary lymphoid organs in fish involved in T and B cell development respectively. To date, according to the expression of these genes, it has been commonly assumed that receptors rearrangement in fish follow the ordered model described in mammals. However, recent evidences seems to indicate strong differences attending to the absence or the unexpected expression of key components involved in lymphocyte maturation. Among the evidence; 1) No orthologues of key genes, like those encoding surrogate light chains VpreB/λ5 or the pre-Tα receptor, have been found in fish during maturation. 2) Several light chain subisotypes are transcriptionally active at the same time in single blood B cells which seems to contrast the allelic exclusion described during mammalian B cell maturation. 3) The presence of pre-B cells in different tissues like spleen, thymus, or peripheral blood leukocytes is in contrast with the maturation model exclusive to bone marrow in mammals. Making use of the single cell sequencing technologies (scRNA-seq), an innovative tool that have revolutionized the state-of-art of cell biology and immunology research TeLymSeq project aims to explore the different maturation states of cells from adaptive immune system of teleost, allowing us to build a specific immune cell atlas of cells in different stages of lymphopoiesis as well as to generate the needed tools to study these different subpopulations, under different circumstances.

Since small non-coding RNAs (ncRNAs) were discovered researchers have been rethinking the “central dogma of molecular biology”. Two types of these small molecules, microRNA (miRNA) and small interfering RNA (siRNA), have been highlighted as critical mediators of biological processes along the different species carrying out their functions though a complex post-transcriptional regulation. In plants, small ncRNAs regulate the expression of genes playing diverse roles, among others those genes involved in abiotic stress responses. Some results have shown that the differential regulation of miRNAs in different tissues is important for adaptation to stress and tree-specific processes. In spite of the importance in regulation of molecular response to stress, the information on gymnosperms miRNAs is scarce.

The aim of the present proposal is the identification of novel and conserved small ncRNAs in Pinus pinaster which are involved in drought stress response as well as predicting the specific target genes controlled by these molecules. The study of post-trascriptional regulation carried out by small RNAs represents a novel and exciting field, really unexplored in conifers to date.