Analysis of Ceylon and TC4, two zebrafish mutants with immunodeficiency

Understanding the developmental underpinnings of the immune system is essential to comprehend how defects in this process lead to disease. T cells are a crucial part of the adaptive immune response and perturbation of T cell development leads to illnesses such as leukemia and immunodeficiency. To understand how defects in T cell development result in disease, we use zebrafish because of their many advantageous features. We focus our analysis on two zebrafish mutants lacking T cells that were identified in a screen for mutants with defects in T cells. By identifying the mutant genes and characterizing the mutant phenotypes, we will gain a better understanding of the molecular mechanisms underlying immunodeficiency. Ceylon, a zebrafish mutant that has severely reduced T cells was our first mutant of interest. Our goal is to use mRNA in situ hybridization to observe which hematopoietic cells are affected in the ceylon mutant. In addition to a severe decrease of T cells hematopoietic stem/progenitor cells (HSC/HSPCs) at 6 days post fertilization (dpf) are severely reduced despite the fact that HSPCs at 2 dpf are normal. Based on our observation of normal definitive HSPCs, our hypothesis is that there is a defect specifically in the niche required for HSPC proliferation and/or survival at later ages, causing loss of HSPCs. To identify defects in other tissues that could contribute to defects in the HSPC niche or HSPC development in general, we have also analyzed ceylon for defects in other non-hematopoietic organs and are studying cell proliferation and cell death within the HSC niche to identify the cause of HSC and T cell loss. The second mutant lacking T cells we are studying is known as TC4, complements ceylon and also has a loss of HSPCs. To identify the specific blood cells affected, we are also using double fluorescent in situs on both ceylon and TC4 mutants. In addition, we are employing a positional cloning approach to identify the genetic lesion that causes these defects. The data from our study will lead to important insights into the cause and mechanism of immunodeficiency and other related diseases.