Thymic organogenesis and T-cell commitment, hallmarks of adaptive immunity, are initiated at the end of embryogenesis and are unique to vertebrates. In this study, we describe the first early pressure forward genetic screen for lymphoid defects in zebrafish. This type of screening provides a new tool to the field of immunology. Our results characterize mutations that disrupt normal T-cell and/or thymic epithelial cell development.
Advantages and Challenges Using Early Pressure Genetic Screening in Zebrafish
In conventional saturating forward genetic screens, 5,000 F2 families of heterozygous mutants (five times coverage of the genome) are raised and mutant phenotypes are detected by F2 incrossing in the F3 generation (Mullins et al.,1994; Solnica-Krezel et al.,1994). Our strategy involved the use of gynogenetic diploid embryos using EP (Streisinger et al.,1981; Beattie et al.,1999). This procedure has the advantage of allowing immediate visualization of mutant phenotypes in the F2 generation. As this approach obviates the need to raise large numbers of families, it is particularly well suited for addressing specific aspects of a developmental process, where the number of genes involved is restricted. This type of screen can also be carried out in laboratories where the logistics do not allow for large-scale screens. The disadvantages of this procedure reside in the suboptimal recovery of viable embryos and the nonmendelian distribution of mutant phenotypes after the EP procedure (Beattie et al.,1999). As shown in Table 1, only 50% of the clutches had more than 50 viable embryos, the number required for detecting at least two mutants in a clutch with 95% confidence (Beattie et al.,1999). Small clutch size may be caused by early lethal mutations, reduced egg viability in F1 females, and epigenetic effects from the EP procedure (Streisinger et al.,1981). It is possible that mutations were missed in clutches with low number of individuals, because the frequency of mutation detection in telomeric regions is only approximately 5% (Streisinger et al.,1986). This suggests that we have confidently reached one-thirtieth saturation (165 F1 females with >50 offspring analyzed) with our current screen. Given that we have identified 6 complementation groups of mutants, a total of 150–180 genes are expected to affect T-cell and/or thymic organogenesis to a degree that their inactivation leads to absent rag-1 expression. This number may be an underestimate of all genes that lead to an immune defect because we may have missed mutations that lead to reduced, but not absent rag-1 expression. Such phenotypes are observed in the mouse by disrupting several genes, including Pax-1 (Wallin et al.,1996), IL-7 (von Freeden-Jeffry et al.,1995), IL-7 receptor (Peschon et al.,1994), p56lck (Molina et al.,1992), and pTα (Fehling et al.,1995). Furthermore, we may have missed incompletely penetrant or hypomorphic mutations of genes that lead to a rag-1 negative phenotype if completely disrupted. In a recent screen in medaka, an F2 screen for nonexpression of rag-1, 22 mutations were identified in 538 F2-families screened (Iwanami et al.,2004). As this screen reached approximately 1/9 saturation, the estimate for the total number of genes affecting T-cell and/or thymic development is 200. In addition, our estimate closely matches the observed frequency of rag-1 deficiency in a saturation screen carried out by the group of T. Boehm in association with the 2000 European Consortium, where 141 rag-1 deficient mutants were identified (Schorpp et al.,2006). Recovery of mutants in our screen was approximately 35% (8 of 23 potential mutants confirmed in the F2 generation). Failure to outcross F1 females and false positive results after the EP procedure are the most plausible explanation for this finding. Despite nonsaturating conditions in the present screen, the egy mutation was detected in offspring of three independent F1 females. This suggests that this mutation was likely present in the germline of the males before mutagenesis, and was confirmed by positional cloning of the affected p110/sart3 gene (Trede et al.,2007).
Characterization of Mutants
Mutants with absent rag-1 expression identified in this screen fall into six complementation groups that can be further subdivided into two broad categories: Mutants with normal pharyngeal arches and mutants with abnormal pharyngeal arches (Table 2). These mutants likely affect the early steps in the homing to and/or expansion of pro-T cells in the thymus (class I) and early steps in the formation of the thymic anlage (class II). Mutants with normal pharyngeal arches could have a leaky phenotype, where small numbers of pro-T cells are attracted to the thymic anlage, but cannot expand. These mutants could have been missed in our screen because a positive rag-1 signal, albeit weak, is expected to be present.
In class I mutants pharyngeal arch architecture is grossly normal, so that neural crest contribution to thymic organogenesis is expected to be normal. Normal expression of nkx2.3 also suggests an intact endodermal compartment. While thymic stroma in these mutants is mildly (asm) to markedly decreased (jas), lymphoblasts are undetectable by electronmicroscopy in jas. The defect in this group of mutants could, therefore, primarily affect thymic stromal development. In this scenario thymic epithelial cells are unable to differentiate to a point where they are capable of attracting pro-T cells efficiently, or to allow for expansion of attracted pro-T cells. Alternatively, defects in the T-cell compartment can also severely affect the competence of pro-T cells to home to the thymus or their capacity to expand once having reached the thymic rudiment. For example, mutations in the GATA-3 gene (Ting et al.,1996), the Ikaros gene (Georgopoulos et al.,1994), or the c-kit−/−γc−/− mice (Rodewald et al.,1997) lead to complete absence or severe reduction of pro-T cells in the thymic rudiment. In all these cases, the thymic anlage remains in a rudimentary state, characterized by cyst formation and lack of corticomedullary differentiation (Rodewald and Fehling,1998; van Ewijk et al.,2000). If class I mutants have a T-cell autonomous defects, the affected gene is expected to act at an early stage in thymocyte maturation. The asm phenotype most closely resembles that of the FOXN1 deficient nude mouse. In the latter, Ikaros-positive cells are observed in the mesenchyme surrounding the thymic anlage, but they do not enter into the anlage that fails to proliferate and grow (Itoi et al.,2001). However, the murine FOXN1 mutation does not cause an eye phenotype or gestational lethality. The disappearance of ikaros positive cells in asm mutants at 8 dpf also points to a difference to the nude mouse phenotype. In support of this conclusion, the thymic rudiment forms normally in asm, as evidenced by normal initial foxn1 expression. In conclusion, the rudimentary state of the thymic anlage in class I mutants may be primary or secondary to absent T-cell/thymic epithelial cell interaction.
Embryologically class II defects could arise from a defect in neurectoderm or a lack of neural crest/endoderm interaction, as described for example in the Hoxa-3 −/− mice (Manley and Capecchi,1995; Su and Manley,2000) or in sucker zebrafish. In the latter example postmigratory neural crest response to endodermal signals is missing secondary to a mutation in the endodermal endothelin-1 gene (Miller et al.,2000). In each of these models, the integrity of the pharyngeal arch architecture is compromised to varying degrees. This mirrors the phenotypes of the class II mutants described in our screen. The fact that the caudal pharyngeal arches, which critically depend on neural crest cell (NCC) contribution, are abnormal in class II mutants, suggests that the primary defect in this group may reside in abnormal migration or function of NCCs. This conclusion is further supported by the observed abnormalities in craniofacial skeleton of class II mutants. Despite these abnormalities, the thymic rudiment forms in all of the mutants analyzed, as evidenced by expression of the TEC marker foxn1.
Recent work has demonstrated that in zebrafish normal eye development is a prerequisite for normal anterior NCC migration (Langenberg et al.,2008). Hence, a primary eye defect could explain craniofacial abnormalities observed in our microphthalmia mutants, even if NCCs were not primarily affected. The association of abnormalities in thymic development with microphthalmia is an unexpected finding in our screen, as no such correlation has been described in mammals. Interestingly, in a recent screen for T-cell and/or thymic abnormalities in medaka, a similar correlation was described, where 21 out of 22 mutants with absence or reduction of rag-1 had microphthalmia (Iwanami et al.,2004). As both, retina and thymic epithelium are highly proliferative tissues, an underlying mutation could be a rate-limiting factor in rapidly cycling cells. Given that production of erythrocytes, which relies on high cell-turnover, is normal in all mutants, such a mutation is not expected to be a general cell cycle factor. This conclusion is bolstered by the recent cloning of the egy mutation (Trede et al.,2007), where disruption of the p110/sart3, a U4/U6 recycling factor essential in rapidly proliferating cells (Medenbach et al.,2004), leads to tissue-specific defects, including eye and thymus epithelium. Additionally, genes that influence patterning of both thymus and eye in fish could be affected.
Isolation of the Mutant Genes
All six complementation groups of mutants have already been mapped to a chromosomal region on the zebrafish genome (Table 2). Closely linked markers (between 0.5 and 4 cM) have been identified for these mutations. No obvious candidate genes have so far been identified. As the cloning of the egy mutation demonstrates, the isolation of the mutant genes may reveal novel genes or novel functions of known genes that disrupt normal thymic and/or T-cell development. Taken together, the above data establish zebrafish screens for lymphoid mutants as complementary to murine genetic approaches, and as a valuable additional tool in the analysis of critical developmental steps in the vertebrate adaptive immune system.