Forward Genetic Screens in Zebrafish Identify Pre-mRNA-Processing Pathways Regulating Early T Cell Development

Abstract

SummaryLymphocytes represent basic components of vertebrate adaptive immune systems, suggesting the utility of non-mammalian models to define the molecular basis of their development and differentiation. Our forward genetic screens in zebrafish for recessive mutations affecting early T cell development revealed several major genetic pathways. The identification of lineage-specific transcription factors and specific components of cytokine signaling and DNA replication and/or repair pathways known from studies of immunocompromised mammals provided an evolutionary cross-validation of the screen design. Unexpectedly, however, genes encoding proteins required for pre-mRNA processing were enriched in the collection of mutants identified here. In both zebrafish and mice, deficiency of the splice regulator TNPO3 impairs intrathymic T cell differentiation, illustrating the evolutionarily conserved and cell-type-specific functions of certain pre-mRNA-processing factors for T cell development.

Uncovering the Role of RNA-Binding Proteins in Gene Expression in the Immune System

“…In pro-B cells, PI3K signaling supresses the function of FOXO-1 that is required for the correct splicing of Ikzf1 , a transcription factors which enables VDJ recombination in cooperation with PAX5 ( 126 ). Forward genetic screens in zebrafish have extended the list of RBPs involved in pre-mRNA-processing in T cells in thymus and they support the idea that global splicing pathways control lymphoid development ( 127 ). Expression of the splicing factors FUS, SC35, hnRPNPL, and hnRPNPLL is important for B- and T-cell development and for T-cell activation ( 128 , 129 ).…”

Transgenerational inheritance of impaired larval T cell development in zebrafish

“…In a forward genetic screen for aberrant T cell development, we identified a large number of recessive mutations, all characterized by reduced numbers of rag1-expressing immature thymocytes in larvae 5 days after fertilization (dpf) 23,24 . The mutation in the IY071 line 23 (Fig. 1a) affected the gene encoding the maintenance DNA methyltransferase DNMT1.…”

“…The recessive viable dnmt1 allele (dnmt1 t25501 ) exhibits a missense mutation (N1391K) in the target recognition domain (TRD) of the catalytic domain of the enzyme (Fig. 1b) 23 ; since a previously identified dnmt1 mutant allele is embryonic lethal 20 , we consider the dnmt1 t25501 allele to be a hypomorph. The mutation in dnmt1 t25501 occurs in an evolutionarily conserved region of the protein (equivalent to N1510K in the mouse protein) (Fig.…”

“…Here, using a forward genetics approach 23 , we describe the identification and characterization of viable recessive allele of zebrafish dnmt1. Our results provide evidence for the transgenerational inheritance of aberrant DNA methylation patterns associated with impaired larval T cell development.…”

Making Thymus Visible: Understanding T-Cell Development from a New Perspective

“…The teleost fishes, zebrafish ( Danio rerio ) and medaka ( Oryzias latipes ), offer a number of attractive features for studying T-cell development ( 9 , 35 – 38 ). Forward and reverse genetic approaches have been applied to characterize mechanisms underlying T-cell development in these model organisms ( 38 – 40 ). Apart from their strength as genetic model systems, the most important advantage of these fish is the optical access they provide.…”