all vertebrates share the same basic principle of lymphocyte differentiation along distinct t - cell - like and b - cell - like lineages ( boehm , 2011 , hirano et  al . , 2013 ) , suggesting that the genetic program regulating the developmental pathways of lymphocytes must have already existed in a common ancestor for all vertebrates . taking advantage of the apparent evolutionary conservation of lymphocyte - based immunity , we conducted genetic screens in zebrafish aimed at identifying previously unknown regulators of t lymphocyte development . zebrafish is particularly suited for such an analysis , as t  cell development already begins in the embryo during the third day after fertilization ( langenau and zon , 2005 ) . during these early stages of embryonic development , maternal factors are expected to buffer , at least partially , the phenotypic consequences of zygotic defects in mutant fish unless particular cell types ( t cells in the present case ) exhibit a specific requirement for the unimpaired activity of a certain gene(s ) . we therefore expected that this unique biological feature of t  cell development in zebrafish would allow us to identify cell - type - specific functions of genes that are also required for stages of development prior to the onset of lymphopoiesis . a similar approach would not be feasible in mammals , because their lymphocytes develop at a considerably later point in embryogenesis ; as a consequence , in mice , for instance , embryonic lethality often masks subsequent lineage - specific functions of pleiotropically acting genes , for example gata3 ( pandolfi et  al . , 1995 ) . in the mouse , specific networks of transcription factors have been shown to regulate the three major phases of t  cell development . in the initial phase , t  cell progenitors are generated and recruited to the thymus ; subsequently , they are induced to adopt  a t  cell fate ; finally , they become responsive to signals emanating from the t  cell receptor ( yui and rothenberg , 2014 ) . hence , assuming that these regulatory circuits emerged at an early stage in vertebrate evolution , a comprehensive genetic screen of t  cell development in zebrafish would be predicted to identify at least some of the factors governing these three phases . in keeping with this expectation , we identified mutations in genes encoding lymphoid lineage - specific transcription factors , and components of cytokine signaling and dna replication / repair pathways . quite unexpectedly , however , pre - mrna - processing factors were also found to play a specific role in t  cell development . using genetic interaction analysis and transcriptome profiling , we established a functional network of certain components of the pre - mrna splicing machinery and demonstrated that the role of this network for t  cell development is evolutionarily conserved . two genetic screens were conducted in zebrafish to identify recessive mutations affecting t lymphocyte development ( boehm et  al . , 2003 , 2006 ) . to this end , the expression of rag1 was determined at 5  days post - fertilization ( dpf ) by rna in  situ hybridization . the product of the rag1 gene is essential for t  cell receptor assembly in developing t  cells in the thymus , the first site of lymphopoiesis in zebrafish embryos . only mutant fish with no overt developmental abnormalities apart from impaired intrathymic t  cell development were considered for further characterization . together with the tbingen 2000 screen consortium , we screened f3 clutches of 4,584 f2 families , representing 4,253 mutagenized haploid genomes ; so far , 42 lines carrying recessive mutations affecting rag1 expression levels could be established . the freiburg gynogenetic screen of 281 genomes led to the establishment of three lines , all of which were found to harbor recessive mutations . owing to the considerable efforts associated with isolating mutated genes by positional cloning , we conducted an interim analysis after the identification of more than one - third of affected genes . the pertinent features of the first 15 complementation groups , for which the affected genes were identified by linkage analysis and positional cloning ( in two cases , aided by whole - genome sequencing ) , are summarized in table 1 . the fact that , among the first 17 of the 45 mutant lines analyzed here , two genes ( ikzf1 and top3a ) were represented by two distinct alleles each suggests that our screen was near saturation . according to their known functions , it was possible to group the affected genes into three categories : regulators of hematopoiesis and lymphopoiesis ; regulators of dna replication , repair , and cell cycle ; and regulators of pre - mrna processing . the products of the five genes in the third functional group ( snapc3 [ small nuclear rna - activating protein complex protein  3 ] , lsm8 [ like - sm protein 8 ] , gemin5 [ gem nuclear organelle associated protein 5 ] , tnpo3 [ transportin 3 ] , and cstf3 [ cleavage stimulation factor subunit 3 ] ) are implicated in pre - mrna processing . the mutations are predicted to affect different aspects of this multi - layered process , such as transcription of small nuclear rnas ( snrnas ) ( snapc3 ) ( hernandez , 2001 ) , formation of small nuclear rna - containing ribonucleoprotein ( snrnp ) splicing complexes ( gemin5 ; lsm8 ) ( friesen and dreyfuss , 2000 ) , nuclear import of splice regulators ( tnpo3 ) ( kataoka et  al . , 1999 ) , and polyadenylation ( cstf3 ) ( xiang et  al . , 2014 ) . as our screen was focused on t  cell development , the prevalence of this group of ubiquitously expressed genes was unexpected , particularly because , to the best of our knowledge , there is no precedence for their hematopoietic / lymphopoietic roles from studies in mammals . previous biochemical studies indicated that the number of spliceosome - associated factors is in the order of 170 ( wahl et  al . , 2009 ) , whereas the gene ontology term  rna processing  is associated with about 500 genes . assuming a coding capacity of the zebrafish genome of about 26,000 genes ( kettleborough et  al . , 2013 ) , and using a conservative estimate that a total of 2,000 genes encode the various components of pre - mrna - processing pathways , the degree of enrichment of genes in this functional category in our screen ( 5/15 ) is significant ( hypergeometric test , p  = 0.0036 ) . the unexpected prevalence of mutations in genes encoding components of the pre - mrna - processing machinery in our screen prompted us to examine whether the cell - type - specific functions of the gene products of this group are mirrored in functional similarities , i.e. , whether they belong to the same genetic network and whether their functions are evolutionarily conserved . despite the fact that the identified mutant alleles of lsm8 , gemin5 , and snapc3 most likely encode non - functional variants , larval development and hematopoietic development initially proceed normally apart from a pronounced defect in t  cell development ( figures s1s3 ) . we monitored the presence of developing t  cells in the thymus of 5 dpf larvae by evaluating the signal emanating from rag1-expressing thymocytes in both thymic lobes ; on a per - cell basis , rag1 expression levels are the same in all genotypes analyzed , allowing us to use the rag1 hybridization signal as a proxy for the number of rag1-expressing thymocytes . the signal emanating from growth hormone ( gh ) gene - expressing cells in the hypophysis was found to be unchanged in mutant fish and thus subsequently used as an internal standard ( figure  1a ) . the extent of t  cell development was  then expressed as the rag1/gh ratio and referred to as the  thymopoietic index . the double - probe rna in  situ analyses indicate severe reductions of rag1 signals in the thymic lobes of homozygous lsm8 , gemin5 , and snapc3 mutants ; heterozygotes exhibit no detectable decrease in the thymopoietic indices ( figures 1b1d ) . for example , in lsm8 and gemin5 mutants , the development of lymphoid precursors  visualized in the ikaros : egfp transgenic background ( hess and boehm , 2012)is initially not affected ( figures 1e and 2a ) . however , by 5 dpf , fewer lymphoid precursors are present in the thymus  as identified by ikaros expression ( figures 1e and 2b ) , in line with the  reduced rag1 signal ( figures 1b , 1c , and 2c ) . by 8 dpf , t  cell development in gemin5 fish this defect is lymphocyte intrinsic , because the stromal microenvironment of the thymus appears to be normal . the mutant thymic anlage expresses foxn1 ( figure  2e ) , the gene encoding the master regulator of thymic epithelial cell differentiation ( nehls et  al . , 1996 ) , and is colonized normally by transplanted wild - type lymphoid precursors ( figure  2f ) . in contrast to impaired t  cell development , several other features indicate that gemin5 embryos and larvae initially develop normally . for instance , development of different types of neuronal tissues  appears undisturbed , including the hypophysis ( exhibiting normal numbers of growth - hormone expressing cells [ figure  2c ] ) , the hindbrain ( visualized by ikaros - expressing neurons [ figure  2 g ] ) , and the retina ( exhibiting the characteristic multi - layered organization [ figure  2h ] ) . moreover , the swim bladder develops normally and the body size is indistinguishable from that of wild - type siblings [ figure  2i ] ) . collectively , these observations suggest a surprisingly tissue - restricted consequence of gemin5 mutation during early stages of zebrafish development , well beyond the stages during which maternally supplied protein and/or mrna could be expected to compensate for the loss of  zygotic gemin5 function . likewise , fish mutant for lsm8 ( figures 1 and s1 ) and snapc3 ( figure  s3 ) also exhibit tissue - restricted phenotypes . importantly , the phenotypes that are observed in pre - mrna - processing mutants can not be explained simply by impairment of the rapid cell proliferation occurring in developing zebrafish embryos and larvae , as this would also cause , for instance , severe perturbations in the development of cranio - facial and neuronal structures in addition to affecting t  cell development . rather , our findings point to a particular sensitivity of developing thymocytes to aberrations in certain components of snrnps . in order to gain insight into the functional interrelationships between the three regulators of pre - mrna processing identified here , epistasis analysis was performed . the smn ( survival of motor neurons ) complex ( part of which is gemin5 ) contributes to the formation of u1 , u2 , u4 , and u5 snrnps , and to the formation of the spliceosomal u6 snrnp ( containing the lsm2 - 8 protein ring ) ( friesen and dreyfuss , 2000 ) . the phenotype of impaired intrathymic t  cell development in gemin5 fish was considerably more severe when the fish originated from gemin5;lms8 double - heterozygous parents rather than from single heterozygous gemin5 ( that is , gemin5;lms8 ) parents ; by contrast , the phenotype of lsm8 fish was not affected by parental heterozygosity of gemin5 ( figure  3a ) . the more severe phenotype of gemin5 mutants arising from lsm8 heterozygosity has a maternal origin ( figure  3b ) , suggesting that reduced levels of lsm8 mrna and/or protein in the oocyte affect the function of gemin5 in the early stages of embryogenesis . by contrast , maternal contribution of gemin5 mrna and/or protein appears to be less important than that of lsm8 , at least with respect to t  cell development , because the extent of t  cell development in lsm8 fish was unaffected by parental gemin5 heterozygosity ( figure  3a ) . impaired translation of maternal and zygotic lsm8 mrna ( using antisense oligonucleotides directed against the translational start site ) phenocopies the genetic lsm8 defect and affects t  cell development at later stages of development , whereas interference with splicing of zygotic lsm8 pre - mrnas alone ( using antisense oligonucleotides directed against the splice donor site of exon 3 ) , has no effect on the thymopoietic index ( figures s4a and s4b ) . collectively , these observations indicate that reduced levels of lsm8 in the early embryo have a long - lasting effect that becomes apparent at much later stages of development and specifically affects t  cell differentiation . of note , the effect of maternal heterozygosity of lsm8 was not observed in combination with a null mutation of the il7r gene , encoding a component of the il7 cytokine receptor ( figures s4c and s4d ) , which by itself also affects t  cell development ( table 1 ) ( iwanami et  al . , 2011 ) . collectively , our observations support the notion that gemin5 and lsm8 function predominantly in separate pathways with redundant or complementary roles ( mani et  al . , 2008 ) . epistasis analysis of snapc3 mutants was complicated by the fact that their thymopoietic index is very low ; hence , it proved to be difficult to establish by analysis of compound mutants the presence and the direction ( synthetic or alleviating ) of genetic interactions between snapc3 and lsm8 mutations and snapc3 and gemin5 ( data not shown ) . next , we determined the effect of snapc3 and lsm8 mutations on the global splicing patterns in 4 dpf whole larvae using rna sequencing ( rna - seq ) , as it is technically not feasible to isolate the few developing thymocytes for transcriptome analysis . the alterations of splicing patterns observed in both mutants are dominated by the occurrence of exon - skipping events ( figures 4a and 4b ; table s1 ) . however , in lsm8 mutants , a significant number of genes ( including tnpo3 ) were additionally affected by incomplete splicing events , resulting in the frequent occurrence of retained introns ( figure  4b ; table s2 ) . other types of splicing events , such as the aberrant usage of mutually exclusive exons and the use of alternative donor and acceptor sites , did not appreciably contribute to the global changes in either genotype ( figures 4a and 4b ) . intriguingly , we observed a considerable degree of overlap between the genes that are affected by  splicing aberrations in the two mutants ( figures 4c and 4d ;  tables s1 and s2 ) . interestingly , in snapc3 mutant fish ,  skipping of the first coding exon of the gemin8 gene ( ensdarg00000053496 ; ensdart00000142315 ; table s1 ; inclusion levels , 0.86  0.05 [ mean  sem ] for wild type ; 0.46  0.02 [ mean  sem ] for mutant ; p  = 0.002 ; t test , two - tailed ) was detected , predicting the formation of an n - terminally truncated protein . because gemin8 is , like gemin5 , part of the smn complex ( battle et  al . , 2006 , wahl et  al . , 2009 ) , this observation supports the notion that snapc3 and gemin5 act in the same molecular pathway . to further address the functional consequences of the identified mutations , we also analyzed the transcriptomes of snapc3 and lsm8 mutants and their wild - type counterparts for changes in gene expression levels ( 2-fold ; false discovery rate [ fdr ]  < 5% ) ( table s3 ) . in snapc3 mutants , 140 genes were significantly upregulated ( figure  4e ) , including 17 protein - coding genes with known functions in pre - mrna splicing ( table s4 ) . most notable was the increase in expression levels for genes encoding components of the sm ( snrpa1 , snrpd2 , snrpd3 , snrpe , snrpf ) and the lsm ( lsm7 , lsm8 ) heptameric complexes ( table s4 ) . in lsm8 mutants , the expression levels of 427 genes were increased ( figure  4e ) , including 35 protein - coding genes , the functions of which have been linked to pre - mrna splicing ( table s4 ) . as is the case in snapc3 mutants , genes encoding components of the sm ( snrpb , snrpd1 , snrpd2 , snrpd3 , snrpe , snrpf ) and lsm ( lsm6 , lsm7 , lsm8 ) ring structures are upregulated ( table s4 ) . of the 58 genes upregulated in both snapc3 and lsm8 mutants , 16 ( 28% ) encode splicing - related proteins ( figure  4f ) . this includes snapc4 , encoding one component of the transcriptional activation complex of snrna genes , and of genes ( snrpd2 , snrpd3 , snrpe , snrpf , lsm7 , lsm8 ) encoding 6 of the 14 components of the sm and lsm heptameric ring structures containing snrnas ( table s4 ) . in both lsm8 and snapc3 mutants , expression of the foxg1d gene is reduced ( table s3 ) ; the mouse homolog of this gene ( foxg1 ) is implicated in the regulation of thymic epithelial cell differentiation ( wei and condie , 2011 ) , possibly contributing to impaired t  cell development . we then tested the generality of the presumed transcriptional feedback regulation among components of snrnps by examining transcriptional responses in gemin5 mutants , focusing on the expression levels of sm and lsm genes . in support of our hypothesis , the qpcr results indicate that snapc3 , lsm8 , and gemin5 mutations all result in strong upregulation of lsm7 and lsm8 mrnas ( figure  4 g ) , defining a core group of genes co - regulated by perturbation of snapc3 , lsm8 , and gemin5 functions . by contrast , expression levels of mrnas of genes encoding the eight known gemin protein family members are largely unaffected in the three mutants ( table s5 ) , supporting the notion of the specific nature of the transcriptional feedback regulation . collectively , the present genetic interaction studies and transcriptome analyses suggest the presence of a network connecting snapc3 , lsm8 , and gemin5 genes encoding key components of snrnps that appears to converge on the transcriptional regulation of lsm7 and lsm8 genes . tnpo3 has been shown to participate in the import of splice regulators to the nucleoplasm ( maertens et  al . , 2014 ) . hence , in contrast to the gene products of snapc3 , lsm8 , and gemin5 , tnpo3 is predicted to indirectly affect pre - mrna processing . in zebrafish tnpo3 mutant larvae , the number of lymphoid precursors in hematopoietic tissues is not affected at 1 dpf when definitive hematopoiesis has begun in zebrafish ; at later stages of development , lymphoid progenitors are found in the thymus ( albeit in somewhat reduced numbers ) , indicating that the homing process is largely unaffected by tnpo3 deficiency ( figures 5a and s5 ) . however , only few of thymocytes in tnpo3 mutants express rag1 ( figures 5b and 5c ) , despite normal expression levels of a thymocyte marker , ccr9b ( figure  5b ) , suggesting that intrathymic defects contribute to impaired t  cell development in tnpo3-deficient fish . at the time point of our analyses ( 5 dpf ) , only t  cells are present in zebrafish larvae , because b cells develop considerably later in development ( danilova and steiner , 2002 ) . hence , we reasoned that the splicing pattern of ptprc ( encoding the zebrafish ortholog of the mammalian thymocyte maturation marker cd45 ) might provide further evidence for  impaired intrathymic t  cell differentiation in tnpo3-deficient larvae . indeed , the mutant splicing pattern is dominated by lower  molecular weight isoforms ( figure  5d ) that are characteristically found in immature mouse thymocytes ( lefrancois and goodman , 1987 ) . at present , it is not possible to determine the cause - and - effect relationship of differential ptprc splicing in the absence of tnpo3 function . however , tnpo3-deficient zebrafish larvae exhibit aberrant splicing of the nck2b gene ( table s1 ) , encoding the zebrafish homolog of the essential adaptor protein nck required for proper tcr signaling in the mouse ( alarcn et  al . , 2003 ) , suggesting that differential splicing of ptprc is an indirect effect of tnpo3 deficiency . tnpo3 appears to be functionally connected to the snpac3/lsm8/gemin5 network of splice regulators , as indicated by alleviating genetic interaction between tnpo3 and gemin5 mutations . in double - deficient fish , the thymopoietic index is lower than in each of the single mutants , yet 3-fold higher than expected if the two genes acted independently ( mani et  al . , 2008 ) ( figure  5e ) , suggesting that tnpo3 and gemin5 are active in the same pathway . as expected , splicing defects were abundant in 4 dpf tnpo3 mutants possibly owing to faulty transport of splice regulators to the nucleoplasm ( maertens et  al . , 2014 ) . we recorded a considerably greater number of exon - skipping events in mutant fish compared to their wild - type siblings , alongside other splicing changes , such as the presence of retained introns and the use of mutually exclusive exons or alternative splice donor and splice acceptor sites ( figures 5f and 5 g ; tables s1 and s2 ) . interestingly , we observed that both lsm8 and tnpo3 deficiency are associated with aberrant splicing of gemin5 and tnpo3 pre - mrna , and snapc3- and tnpo3-deficient larvae both exhibit aberrant splicing patterns of gemin8 ( table s1 ) , further emphasizing the functional interrelationships in the snpac3/lsm8/gemin5/tnpo3 network . comparatively few alterations were observed at the level of gene expression ( 2-fold ; fdr  < 5% [ table s3 ] ) in tnpo3 mutants . thirty - eight genes are upregulated in tnpo3 mutants , however , with little overlap to genes upregulated in snapc3 and lsm8 mutants , indicating the presence of mechanistic differences with respect to the regulation of snrnp assembly among the three mutants ( figure  5h ) . the presence of pervasive autoregulation affecting the processing of pre - mrnas of splice regulator genes as a consequence of mutations in snpac3 , lsm8 , and tnpo3 is suggested by our finding that among the 32 genes commonly affected by exon - skipping events ( figure  5 g ) , two genes srsf3a and ptbp3encode known regulators of pre - mrna processing ; and of the four genes commonly affected by intron retention , two encode splice regulators ( srsf1b , prpf39 ) ( table s2 ) . when we examined transcriptomes for changes common to all  three mutants and potentially involved in t  cell / thymus development , we noted that splicing of the dnmt4 ( also known as dnmt3bb.1 ) gene , which is expressed in the hematopoietic lineage ( takayama et  al . , 2014 ) , is similarly affected in all three mutants ; the splice pattern suggests the presence of different compositions of the n - terminal non - catalytic domains of the predicted protein ( figure  5 g ; table s6 ) . many of the 34 downregulated genes ( figure  5i ) are signature genes of the exocrine pancreas , identifying a second cell type exhibiting particular sensitivity to the malfunction of  the pre - mrna splicing regulators identified here . the lower  expression levels of elastase , trypsin , amylase , and carboxypeptidase , etc . ( ela3l , try , amy2a , cpa1 ) genes suggest that the downstream effects of snapc3 , lsm8 , and tnpo3 mutations converge on malfunction of the exocrine pancreas ( table s7 ) . this conclusion is supported by the fact that in all three mutants , aberrant splicing of the nr5a2 transcription factor also occurs ( table s6 ) , which is required for liver and exocrine pancreas development ( nissim et  al . , 2016 ) . we also note that the gene encoding the brush - border membrane glycoprotein trehalase ( treh ) is affected by intron retention in all three mutants , adding to impaired absorption of carbohydrates . although more work is needed to assess the functional significance of these findings , we hypothesize that malfunction of the exocrine pancreas and the intestine at least partly underlies the phenotype of premature death in the mutant fish . these results also underscore the notion of cell - type - restricted functions of snapc3 , lsm8 , and tnpo3 genes . having established the role of the snpac3/lsm8/gemin5/tnpo3 network ( figure  5j ) in  zebrafish t  cell development , we next examined a possible evolutionary conservation of functions of these genes in mammalian t  cell development ; we chose the mouse tnpo3 gene for our studies . to this end , we generated conditional knockout mice using a floxed allele of tnpo3 ( exon 7 is flanked by loxp sites ) and lck : cre ( orban et  al . . under these conditions , complete inactivation of the tnpo3 gene was achieved ( figure  s6 ) . the predicted tnpo3 mutant protein consisting of the first 291 amino acids closely resembles the mutant form identified in zebrafish ( 202 amino acids ) ( figure  s6 ) ; notably , they both lack the region required for interaction with rs cargo proteins ( maertens et  al . , 2014 ) . in the thymus , tnpo3 deficiency was accompanied by a moderate , but non - significant reduction of cd4/cd8-double - positive ( dp ) t  cell progenitors ; by contrast , single - positive ( sp ) thymocytes were significantly reduced , cd4 cells by about 40% and cd8 cells by about 60% ( figures 6a and 6b ) . as expected , the number of  t  cells remained unchanged , because the lck : cre transgene is not expressed in this lineage ( figure  s6 ) . reduced levels of cd5 staining of tnpo3-deficient  thymocytes ( particularly of those with cd8 surface phenotype ) indicate that tcr signaling is impaired in mutant cells ( figure  6c ) ; this feature is also evident in peripheral t  cells ( figure  s6 ) . these findings suggest that loss of tnpo3 in thymocytes is associated with an incomplete maturation block affecting the dp - to - sp transition . because expression levels of rag genes are unchanged in mutant dp cells ( data not shown ) this finding is reminiscent of the features of impaired tcr signaling identified in the tnpo3-deficient zebrafish larvae . the loss of single - positive cells in mutant thymi does not appear to be due to increased apoptosis , because the fractions of tunel - positive and annexin v - positive thymocytes remained unchanged ( data not shown ) ; these findings call for further mechanistic studies addressing migration , survival , etc . , of mutant t  cells in the periphery . as expected for the lymphopenic condition in tnpo3-deficient mice  ( figure  6d ) , increased proliferation levels of peripheral t  cells are observed ( figure  6e ) , compatible with the notion that tnpo3 deficiency per se does not impair cell proliferation . collectively , the present data suggest an evolutionarily conserved role of tnpo3 in t  cell differentiation . our unbiased genetic screens identified an extended genetic  network linking genes of several distinct functional categories  ( hematopoietic differentiation ; dna replication / repair ; pre - mrna processing ) required for early stages of intrathymic t  cell development in zebrafish . the present study has established additional animal models for mammalian ( particularly human ) immunodeficiency syndromes and presents previously unknown candidate genes whose mutations might underlie failing immune function . indeed , we note that some of the genes that we have identified are implicated in human immunodeficiency disorders , such as il7r ( puel et  al . , 1998 ) , jak3 ( russell et  al . , 1995 ) , and pole1 ( pachlopnik schmid et  al . , 2012 ) ( table  1 ) . the value of forward genetic screens to identify candidate genes is underscored by our finding of an enrichment of genes encoding factors involved in pre - mrna processing . this outcome was unexpected , because mutations in this group of genes have so far been associated mostly with neuronal disease , such as spinal muscular atrophy or retinitis pigmentosa ( singh and cooper , 2012 ) . mutations in spliceosomal factors manifest themselves as hypomorphic alleles ( singh and cooper , 2012 ) , indicating that the resulting phenotypes most likely arise from subtle perturbations in the multi - component protein complexes regulating pre - mrna splicing . for example , insufficient levels of the smn complex are associated with impaired maturation of snrnps and aberrant splicing reactions in neuronal tissues ( zhang et  al . , 2008 , zhang et  al . , 2013 ) , whereas complete loss of smn function is lethal ( hsieh - li et  al . , 2000 ) . however , because defects in early development are buffered by maternal factors , our results also illustrate how the unique biological features of the zebrafish model allow the identification of tissue - restricted functions of  pleiotropic genes also in the case of null alleles , which in the  mammalian system can only be studied by conditional mutagenesis . although the results of the present epistasis analysis are consistent with those in previously published molecular studies on snrnp assembly and function ( wahl et  al . , 2009 ) , an unexpected finding in the in  vivo studies described here is the presence of a feedback loop connecting impaired snapc3 , lsm8 , and gemin5 function with the transcriptional regulation of the lsm7 and lsm8 genes . in contrast to many other genes in zebrafish , lsm genes do not possess paralogs . hence , the transcriptional response in our mutants is unlikely to be part of a direct compensatory mechanism , but rather appears to be a general consequence of snrnp biogenesis perturbation , because expression levels of lsm7 and lsm8 are also upregulated in zebrafish larvae mutant for the p110 splicing regulator ( trede et  al . , 2007 ) , which is required for recycling of the u4/u6 snrnp from singular u4 and u6 snrnps ( bell et  al . , 2002 ) another notable feature of transcriptional changes emerging from our studies is the presence of pervasive autoregulatory loops affecting the splicing of pre - mrnas - encoding splice regulators , as illustrated here by , for instance , the srsf3a gene , a homolog of the mammalian srsf3 gene previously shown to be regulated by variable inclusion of so - called poison - cassette exons ( lareau et  al . , 2007 ) . on a more general level , our findings provide evidence for the emerging notion of tissue - restricted roles played by ubiquitous components of basic cellular pathways . future work may thus reveal an  rna splicing code  of context- and cell - type - dependent functions of some general - purpose factors involved in pre - mrna processing ; our mutants provide an entry point into the identification of the precise mechanism(s ) by which pre - mrna splicing affects t  cell development . with respect to the mechanism(s ) underlying failing t  cell development in zebrafish larvae , our work indicates that , despite mechanistic differences , certain commonalities exist among the aberrations caused by mutations in the snapc3/lsm8/gemin5/tnpo3 network . one particularly notable observation is the apparent association with dna methylation , because the splicing of several genes encoding putative de novo dna methylases is affected by perturbations in this network ; most importantly , aberrant processing of the dnmt3bb.1 gene is a common feature of snapc3 , lsm8 , and tnpo3 mutants . we consider this mechanistic link to be particularly intriguing , because our screen has independently uncovered a mis - sense  mutation in the gene encoding the maintenance dna methylase dnmt1 ( table 1 ) . given that the status of dna methylation affects the differentiation of hematopoietic stem cells ( challen et  al . , 2014 ) and modulates immune functions including t  cell differentiation ( jin et  al . , 2008 ) , it will be interesting to examine the possible presence of common changes in gene - specific dna methylation patterns to identify those genes whose methylation signatures are crucial to normal t  cell development . finally , an important aspect of our present work addresses the  evolutionarily conserved function of the identified network of splice regulators with respect to t  cell differentiation . we chose tnpo3 to verify that the observations made in the zebrafish model also hold for the mammalian system . our conditional mouse tnpo3 knockout model exhibits a phenotype of impaired t  cell differentiation , resembling the phenotype observed in tnpo3-deficient zebrafish larvae . the comparative analysis of mutant zebrafish tnpo3 and mouse tnpo3 genes highlights the advantages of the zebrafish model to identify cell - type - restricted functions of pleiotropic regulators , because tnpo3-deficient mice exhibit early embryonic lethality . future work will have to be aimed at addressing the degree of functional conservation for the other members of the identified genetic networks and the precise molecular mechanism(s ) underlying the exquisite sensitivity of t  cells to perturbed pre - mrna splicing . the zebrafish ( danio rerio ) strains ekkwill ( ekk ) , tpfel long fin ( tl ) , wild - type - in - kalkutta ( wik ) , ab , assam ( ass ) , and tbingen ( t ) are maintained in the animal facility of the max planck institute of immunobiology and epigenetics . the ikaros : egfp transgenic line was described previously ( hess and boehm , 2012 ) . all animal experiments were approved by the institute s review committee and conducted under licenses from the local government ( regierungsprsidium freiburg ) . a detailed description of the screen design , coverage , complementation analysis , and mutant identification by positional cloning and whole genome sequencing can be found in supplemental experimental procedures . the esc line epd0318_3_g02 ( genetic background : c57bl/6n agouti(a / a ) ; allele name : tnpo3 ) was obtained from the komp repository and used to derive chimeric mice using standard procedures . for rescue experiments , mrnas were injected into fertilized eggs and analysis carried out by gh and rag1 rna in  situ hybridization at various time points after injection . egfp cells were sorted from single - cell suspensions of kidney marrow cells isolated from adult ikaros : egfp transgenic zebrafish and injected into the sinus venosus of embryos from heterozygous intercrosses at 2 dpf . gfp cells in the thymic region were counted using imager.z1 ( zeiss ) at 3  days post - injection . the procedures for live imaging using the ikaros : egfp transgenic background were described previously ( hess and boehm , 2012 ) . for analysis of tnpo3 mutant mice , analytical flow cytometry was carried out as described in caldern and boehm ( 2012 ) . procedures for rna in  situ hybridization and probes were described previously ( schorpp et  al . , 2006 ) . total rna was extracted using tri reagent ( sigma ) following the manufacturer s instructions . after treatment with cloned dnasei ( takara ) , rna extraction using tri reagent was repeated . superscript ii reverse transcriptase ( invitrogen ) and random hexamer primers were used for cdna synthesis from total rna . qpcr was carried out as described ( rode and boehm , 2012 ) ; primers are listed in table s8 . total rna was extracted from whole zebrafish larvae at 4 dpf and subjected to  transcriptome analysis . the libraries were sequenced in paired - end 75-bp  mode on 0.8 lanes per sample on an illumina hiseq 2500 instrument . the high - throughput rna sequencing analysis pipeline , version 0.4.2 , written by fabian kilpert ( https://github.com/kilpert/rna-seq-qc ) was applied as described in supplemental experimental procedures . rna - seq data are deposited in ncbi s geo ( edgar et  al . , 2002 ) and are accessible through geo : gse77480 . for single comparisons of independent groups , student s t test or the mann - whitney test the statistical models applied to rna - seq data are described in supplemental experimental procedures . , f.m . , and m.s . identified and characterized zebrafish mutants by positional cloning .
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 .