Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 in zebrafish

Ikzf1 is a Krüppel-like zinc-finger transcription factor that plays indispensable roles in T and B cell development. Although the function of Ikzf1 has been studied extensively, the molecular mechanism underlying T lymphopoiesis remains incompletely defined during the embryonic stage. Here we report that the genetic ablation of ikzf1 in mutant zebrafish resulted in abrogated embryonic T lymphopoiesis. This was ascribed to impaired thymic migration, proliferation, and differentiation of hematopoietic stem/progenitor cells (HSPCs). Ccr9a and Irf4a, two indispensable factors in T lymphopoiesis, were the direct targets of Ikzf1 and were absent in the ikzf1 mutants. Genetic deletion of either ccr9a or irf4a in the corresponding mutant embryos led to obvious T cell development deficiency, which was mainly caused by disrupted thymic migration of HSPCs. Restoration of ccr9a in ikzf1 mutants obviously promoted HSPC thymus homing. However, the HSPCs then failed to differentiate into T cells. Additional replenishment of irf4a efficiently induced HSPC proliferation and T cell differentiation. Our findings further demonstrate that Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 and provide new insight into the genetic network of T lymphocyte development.

As a key cellular component in adaptive immunity, T lymphocytes are considered to originate from hematopoietic stem cells (HSCs) 2 through a tightly controlled hierarchy (1,2). Although common lymphoid progenitors are regarded as the major sources of T cells (3), the identification of lymphoid-primed multipotent progenitors, which are endowed with both myeloid and lymphoid potential, has suggested that myeloid and lymphoid lineages share a common precursor (4). The seeding of the thymus by these hematopoietic progenitor cells is required for the commitment, proliferation, differentiation, and maturation of T lymphocytes (5). Although the processes of T cell differentiation and maturation have been investigated extensively, the molecular mechanisms governing the migration of hematopoietic progenitor cells to the thymus and their differentiation during embryonic stages are poorly understood.
Zebrafish (Danio rerio) are widely used as a model system for studying hematopoiesis because they present several unique characteristics, including extrauterine development and optical transparency. Zebrafish T lymphoid cells result from the definitive hematopoiesis that occurs in the ventral wall of the dorsal aorta (VDA), where hematopoietic stem/progenitor cells (HSPCs) emerge from the aortic endothelium through endothelial hematopoietic transition (6 -8). These newly formed HSPCs subsequently migrate to the caudal hematopoietic tissue (CHT), thymus, and kidney, where they proliferate and differentiate to produce distinct blood lineages (9 -12). At ϳ60 h post-fertilization (hpf), VDA-born hematopoietic precursors appear to start colonizing the developing thymus (13), where they undergo rapid proliferation and differentiation and give rise to mature T cells (13).
The IKAROS family zinc finger 1 (Ikzf1) transcription factor, also known as Ikaros, was initially detected in mouse liver rudiment (14). The Ikzf1 gene generated various spliced isoforms that translated into a complex group of proteins characterized by a DNA-binding domain and proteinprotein interaction domain in the N and C terminus, respectively (15,16). Ikzf1 proteins are detected in various subsets of hematopoietic cells and are especially enriched in lymphocytes (15,17,18). Mice carrying distinct Ikzf1 mutant alleles display similar lymphopoiesis defects and other hematopoietic phenotypes (19 -26). These studies indicate that Ikzf1 plays critical roles in multiple steps of hematopoietic cell growth, especially in T cell development. Ikzf1 targets the nucleosome-remodeling-deacetylase complex (NuRD) to promote lymphoid priming of HSPCs (27). Ikzf1 cooperates with other members of the IKAROS family (28,29) to regulate several key molecules in T cell differentiation.
Similar to the observations in mice, the zebrafish ikzf1 mutant alleles present a significant early T cell development deficiency (30,31), suggesting an evolutionarily conserved role in T cell development. Although the function of Ikzf1 has been investigated extensively for decades, the corresponding molecular mechanisms in embryonic hematopoiesis, especially T lymphopoiesis, remain largely undefined.
In this study, two ikzf1 mutant alleles that affect the functional domains were generated. We show that loss of Ikzf1 function in zebrafish impairs thymus migration, proliferation, and differentiation of HSPCs, which results in failure of embryonic T lymphopoiesis. We further reveal that ccr9a and irf4a are directly regulated by Ikzf1 and are functionally indispensable in the T lymphopoiesis process.

Embryonic T lymphopoiesis is abolished in ikzf1 mutants
To explore the function of ikzf1 in early hematopoiesis, including T lymphopoiesis, ikzf1 expression patterns and levels in different embryonic blood cells were examined in zebrafish. Ikzf1 clearly appeared in runx1-GFP ϩ (32) and cmyb-GFP ϩ (33) cells (Fig. S1A) in the VDA regions at 36 hpf. Ikzf1 was then detected in the larval hematopoietic organs (CHT, thymus, and kidney; Fig. S1B) (11). Ikzf1 transcript levels were highest in rag2-DsRed ϩ lymphoid cells (34), followed by CD41-GFP lo HSPCs (10), mpx-GFP ϩ myeloid cells (35), and gata1-DsRed ϩ erythroid cells (36). In CD41-GFP hi thrombocytes (37), a limited amount of ikzf1 transcript was detected (Fig. S1C). The hematopoietic expression of ikzf1 suggested its critical role during hematopoiesis, especially in T cell development. Therefore, the ikzf1 gene was edited (Fig. 1A). Two types of ikzf1 mutants were identified. One mutant allele was 1 bp short in exon 3 (deletion of 4 bp but addition of another 3 bp), and the other showed a 1-bp insertion in exon 9 (Fig. S1, D-F). Both mutations led to a premature stop codon in their coding sequence ( Fig. 1B and Fig. S1F), which did not cause an obvious reduction in expression but resulted in synthesis of truncated Ikzf1 proteins in both mutant alleles ( Fig. 1B and Fig. S1, G and H). This suggested loss of function in both alleles. Similar hematopoietic phenotypes were seen in both mutants (Fig. S2, A and B). Therefore, the ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutant was selected for intensive investigation.
The enrichment of the ikzf1 transcript in rag2-DsRed ϩ cells drew our attention to the T cell phenotypes. As expected, T cell formation, as indicated by the expression of ccr9a, ccr9b, lck, and rag1, was severely compromised in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants (Fig. 1C). We confirmed that this T cell deficiency phenotype in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants was caused by the ikzf1 mutation; forced expression of ikzf1 in hematopoietic cells, driven by the hematopoiesis-specific coro1a promoter and overexpressed in hematopoietic progenitors (Fig. S1I) and leukocytes (38,39), was sufficient for restoring T cell development in mutants (Fig. 1C). The expression of ccl25a, a chemokine gene expressed in thymic epithelial cells (13), was not detectably altered (Fig. S1J). Collectively, these data demonstrate that Ikzf1 is required for T cell development, in accordance with previous findings (30). A, the target sites for ikzf1 gene editing. The AlwI restriction enzyme was used to identify one mutant allele (purple). The AGG and GGG sequences (red) are the protospacer adjacent motifs. Red triangles indicate the mutation sites in two mutant alleles. B, the ikzf1 ⌬4 ϩ 3 and ikzf1 ϩ1 mutations lead to generation of truncated Ikzf1 proteins lacking zinc finger domains. The ikzf1 ⌬4 ϩ 3 mutation (in amino acid (aa) 28) and ikzf1 ϩ1 mutation (in aa 398) caused formation of 57-and 403-aa truncated proteins, respectively. The purple squares indicate the mismatched amino acid caused by the frameshift in the mutant compared with the WT. C, whole-mount in situ hybridization (WISH) of ccr9a, ccr9b, lck, and rag1 in the thymus of ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 and their siblings (Sib). The values in the bottom right corner of each panel indicate counts with a typical appearance as presented (first number) in the total number of examined samples (last number). The red circles indicate the thymi.

Compromised HSPC expansion in ikzf1 mutants
The reduction of coro1a-Kaede ϩ cells in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutant kidneys (Fig. 2G) suggested HSPC pool shrinkage at later stages. Indeed, the cmyb ϩ population in ikzf1 mutants was significantly decreased from 4 dpf compared with those of siblings ( Fig. 3, A and B, and Fig. S2B). Consistently, the confocal microscopy images and statistical analysis indicated that the CD41-GFP lo (10) and runx1-GFP ϩ (32) cells were significantly reduced in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants compared with WT siblings (Fig. S3, A-C). The ratio of EdU ϩ cells and expression levels of several cell cycle-related genes in the CD41-GFP lo and runx1-GFP ϩ population were clearly decreased in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants compared with WT siblings (Fig. 3, C-F, and Fig. S3, D and E). However, TUNEL ϩ cells remained unchanged (Fig. S3, F and G). These results indicated that the reduction of HSPCs in ikzf1 ⌬ 4 ϩ 3/⌬4 ϩ 3 mutants was caused by impaired cellular proliferation rather than increased cell apoptosis. Collectively, ikzf1 was essential for thymic migration and proliferation of HSPCs at embryonic stages, which play indispensable roles in T cell development.

Ccr9a was a key factor mediating thymus homing in HSPCs
The downstream factors accounting for HSPC thymus homing were investigated by focusing on the chemokine (C-C motif) receptor Ccr9, a critical factor involved in HSPC recruit-ment to the thymus (43)(44)(45). Two orthologues, ccr9a and ccr9b, exist in zebrafish. Both molecules were expressed in the thymic T lymphoid cells from 4 dpf onward in WT siblings, but neither was detected in the ikzf1 mutants ( Fig. 1C and Fig. S2A). However, the ccr9a transcript was seen in the WT CHT much earlier than ccr9b (2.5-3 dpf) and was barely observed in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants (Fig. 4A). Genomic sequence analysis of the ccr9a promoter indicated that there were elements directly targeted by Ikzf1 (Fig. 4B). Notably, the results of ChIP-qPCR (46) demonstrated that the DNA fragment containing the binding site was enriched in the anti-HA immunoprecipitation group by using Tg(coro1a:HA-ikzf1) ( Fig. 4C and Fig. S4, A-D). Furthermore, Dual-Luciferase reporter analysis (47) revealed that deletion of the Ikzf1 binding sites markedly reduced the activity of the ccr9a promoter (Fig. 4D), suggesting that ccr9a might be a critical mediator of ikzf1 in the early migration of HSPCs. Therefore, a ccr9a mutant allele with a 10-bp genomic fragment deletion in exon 3 was generated, which resulted in synthesis of a truncated protein (Fig. S5, A-D). The populations of cmyb ϩ , rag1 ϩ , tcrb ϩ , and lck ϩ cells in the thymus were notably reduced but not totally lost in the ccr9a ⌬10/⌬10 mutants compared with WT siblings (Fig. 4, E and F, and Fig. S5E). This phenomenon was reversed by restoration of ccr9a in coro1a ϩ hematopoietic cells (Fig. 4E). In contrast, cmyb ϩ and irf4a ϩ cells were unaltered in the CHT and kidney of ccr9a ⌬10/⌬10 mutants compared with siblings ( Fig. 4F and Fig. S5, F and G). Thus, ccr9a function deficiency might impair the thymic trafficking of HSPCs. To prove this, similar time-lapse imaging and transient lineagetracing assays utilized to study ikzf1 mutants were performed.

Ikzf1 regulates T lymphopoiesis via Ccr9 and Irf4
Time-lapse imaging showed that the frequency of Kaede ϩ cells entering the thymus was strikingly decreased in ccr9a ⌬10/⌬10 mutants (Fig. 4, G and H, and Movies S3 and S4), and the numbers of red-Kaede ϩ and red-Dendra2 ϩ cells were obviously decreased in the ccr9a ⌬10/⌬10 mutant thymus compared with siblings (Fig. 4, I and J, and Fig. S5, H and I). These data suggest defective homing of hematopoietic progenitors in ccr9a ⌬10/⌬10 mutants.

Restoration of Ccr9a rescued the thymus-settlement of HSPCs in ikzf1 ⌬4 ؉ 3/⌬4 ؉ 3 mutants
The importance of ccr9a in HSPC thymus-homing and their disappearance in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants prompted supply of this factor and an exploration of T cell phenotypes. To this end, the coro1a:ccr9a transgenic line was generated (Fig. S6, A and  B). Overexpression of ccr9a had no detectable effects on hematopoietic development, as shown by the comparable expression

Homing of hematopoietic progenitors was disrupted in irf4a ⌬18/⌬18 mutants
Irf4, a member of the interferon regulatory factor family that is essential in zebrafish T lymphoid determination (40), was then examined. Similar to ccr9a, the expression of irf4a was barely detected in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants (Fig. 6A). The data from ChIP and Dual-Luciferase reporter experiments showed that irf4a was directly regulated by Ikzf1 (Fig. 6, B-E), suggesting that irf4a may be the candidate factor. To this end, an irf4a ⌬18/⌬18 mutant line (harboring an 18-bp deletion) was created (Fig. S7, A and B). This mutation resulted in removal of six amino acids from the conserved DNA-binding domain of the protein (48) and, consequently, in a drastic reduction in Irf4a protein level (Fig. S7, C and D). Neither cmyb ϩ , ikzf1 ϩ , or ccr9a ϩ cells in the CHT (Fig. S7, E and F) nor thymic structure/ chemokines were affected by the irf4a mutation (Fig. S7G). However, most of the irf4a ⌬18/⌬18 larvae exhibited complete loss of ccr9a ϩ and ikzf1 ϩ cells in the thymus at 3 dpf, and the remaining larvae only showed a trace amount of these cells, which suggests that early T cell development was interrupted (Fig. 6, F and G, and Fig. S7H). Intriguingly, the T cell number partially recovered in irf4a ⌬18/⌬18 mutants as the embryos grew. At 6 dpf, only a small group of irf4a ⌬18/⌬18 larvae showed complete lack of T cells, whereas a large proportion of the mutants displayed relatively smaller but detectable T cell foci in the thymus (Fig. 6, F and G, and Fig. S7H). The normal population of hematopoietic progenitors and the gradual recovery of T cells in the irf4a ⌬18/⌬18 mutants suggest that the T cell deficiency was probably caused by inefficient homing but was not due to the cell fate alteration (40) (Fig. S7I), as that found in the ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 and ccr9a mutants. Indeed, time-lapse imaging revealed the presence of only a limited number of spherical coro1a-DsRed ϩ lyz-GFP Ϫ cells in the irf4a ⌬18/⌬18 thymus, in contrast with that of the WT control (Fig. 6, H and I, and Movies S1 and S8). Furthermore, transient tracing of the coro1a-Kaede ϩ cells in the CHT (somites [15][16][17] showed that the marked cells presented notably compromised movement to the thymus of irf4a ⌬18/⌬18 mutants relative to that in the siblings (Fig. 6, J and K), recapitulating what occurred in the ikzf1 and ccr9a mutants. Collectively, these data suggested that the progenitors in the irf4a ⌬18/⌬18 mutants, as with those in the ikzf1 and ccr9a mutants, were compromised by a thymus homing blockade.

Ikzf1 regulates T lymphopoiesis via Ccr9 and Irf4
migration and the crucial function of irf4a in HSPC proliferation and T cell differentiation. Supporting this assumption, supplementation with both irf4a and ccr9a in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants effectively recovered great numbers of rag1 ϩ and lck ϩ cell foci (ϳ65%) in the thymus compared with irf4a alone (ϳ36%) (Fig. 7, C  and D). As a previous study suggested that ccr9a was directly regulated by Irf4a (40), Ikzf1 and Irf4a probably functioned together to regulate ccr9a activity. This prediction was validated by the results from the coimmunoprecipitation and Dual-Luciferase reporter experiments, which indicated that GFP-Ikzf1 or Ikzf1-GFP interacted with HA-Irf4a (Fig. 7E) and that cotransfection of both factors elevated the transcriptional activity of ccr9a more obviously than transfection with either one (although Ikzf1 played a major role) (Fig. 7F). Taken together, Ikzf1 regulated the thymic migration, proliferation, and differentiation of HSPCs via ccr9a and irf4a. These factors worked closely together to accomplish T lymphopoiesis in embryonic zebrafish (Fig. 7G).

Discussion
In addition to the compromised embryonic T lymphopoiesis that recovered at later stages and persisted for over 17 months in a reported zebrafish mutant (30), the newly created ikzf1 mutant alleles in this study presented a drastic reduction of T cells in the adult stage as well as diminished hematopoietic progenitors with impaired proliferation. The difference was ascribed to the more significant impairment of Ikzf1 function in mutants that lacked functional domains compared with previous mutants (30,31) that carried a point mutation in the sequence encoding the C-terminal protein interaction domain. The mutants were not considered a dominant-negative mutation because no overt hematopoietic phenotype was detected in heterozygous mutant embryos.
T cells were completely absent in ikzf1 mutant zebrafish at the embryonic stage as a result of impaired HSPC thymus homing, proliferation, and differentiation. However, it is challenging to define whether the compromised homing of HSPCs was the primary cause of T cell loss or whether this simply reflected a defect in the specification or commitment of lymphoid cells in ikzf1 mutants. Although ikzf1 has been recognized previously  n ϭ 3). G, a model of our study. Ikzf1 promotes T lymphopoiesis via thymus migration, proliferation, and differentiation of HSPCs, which are mediated by the downstream targets irf4a and ccr9a. Loss of ikzf1 functions in mutant fish leads to compromised migration and arrested proliferation of HSPCs. Compromised production of T cells was then observed in ikzf1 mutant embryos. The red circles indicate the thymi (A-D). The values in the bottom right corner in A-D indicate counts with a typical appearance as presented (first number) in the total number of examined samples (last number). ns, not significant; *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001.

Ikzf1 regulates T lymphopoiesis via Ccr9 and Irf4
as a lymphoid progenitor marker (13), our study indicated that ikzf1 was also expressed in hematopoietic progenitors for multiple lineages and plays a critical role in their proliferation. Consistently, Ikzf1 was seen to be highly expressed in mouse KTLS ϩ long-term HSCs, and Ikzf1 Plastc/Plastc mutant mice initially generated long-term HSCs but quickly failed to maintain and expand this pool at the embryonic stage (49). Similar HSPC phenotypes were observed in our ikzf1 mutant zebrafish, suggesting a functionally conserved role of Ikzf1 as a self-renewing regulator in HSC maintenance. However, it is difficult to exactly identify which HSPC populations were affected in ikzf1 mutant zebrafish because of the shortage of specific markers for different subtypes in zebrafish HSPCs.
Ccr9 was essential in the thymic seeding of hematopoietic progenitors in mice (43)(44)(45). One orthologue of ccr9, ccr9a, was absent in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutant larvae. The obvious impairment of hematopoietic progenitor migration was seen in ccr9a mutants. Restoration of ccr9a in ikzf1 mutants efficiently promoted thymic migration of HSPCs but resulted in failed differentiation, implying that thymic migration of HSPCs was necessary but not sufficient for T cell development. Irf4a was another critical factor downstream of Ikzf1 in T lymphopoiesis. Irf4a has been reported to regulate lymphoid versus myeloid determination in zebrafish (40). In this study, irf4a expression was lost, and continual replenishment of irf4a only partially rescued the T cell deficiency in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants by promoting both CHT expansion and ccr9a-mediated thymic seeding of HSPCs, implying a requirement for additional factors and more complicated regulatory networks in this process.
Recently, IRF4 was reported to cooperate with Ikzf1 and serve as a transcriptional complex to target the zinc finger-IRF composite elements (ZICEs) that appeared in the promoters of genes that functioned essentially during plasma cell differentiation in mice (50). This report suggested the possibility that irf4a, when turned on by Ikzf1 in HSPCs with lymphoid potential, functioned together with Ikzf1 to further guide lymphoid differentiation in zebrafish. However, unlike the suppressive roles of the Ikzf1-IRF4 complex on the ZICE motif in mice (50), zebrafish Ikzf1 interacted with Irf4a and more effectively promoted ccr9a expression working together than singly. This difference was probably caused by the fact that there was no ZICE element in the ccr9a promoter. A previous study revealed the Ets-IRF and AP-1-IRF composite elements in the ccr9a promoter (40), which were located adjacent to the Ikzf1 binding sites. The highest transcriptional activity of Ikzf1/Irf4a in the ccr9a promoter, like the Ikzf1-IRF4 complex, acted as a transcriptional activator in the presence of E26 transformation-specific family members such as PU.1 (50), suggesting the involvement of other factors. This process should be a focus of future investigation.
Supplementation with both ccr9 and irf4 in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants restored thymic T cell phenotypes more efficiently than either one alone. Therefore, we propose that, during zebrafish embryonic T lymphopoiesis, ikzf1 activates irf4a expression to protect the progenitor pool and assure their lymphoid potential. Then ikzf1 works together with irf4a to produce sufficient amounts of ccr9a that efficiently accomplish thymic seeding of hematopoietic progenitors, where T cell dif-ferentiation is finally achieved. However, there was still approximately 30% T cell failure in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants supplied with both ccr9a and irf4a. One possibility for this phenomenon was that the ccr9a and irf4a levels, even in transgenic zebrafish lines, were insufficient for final thymic T cell formation. Concordant with this hypothesis, neither a transient supply of irf4a and ccr9a mRNA nor the weak transgenic Tg(coro1a:irf4a) allele rescued thymic T cells in ikzf1 ⌬4 ϩ 3/⌬4 ϩ 3 mutants (data not shown). In addition, it remains possible that other factors were involved. Transcription factors such as Bcl11b, E2A, and Hes1 are crucial for T cell commitment (2). The P-selectin/ PSGL-1 axis plays a role in recruiting progenitors to the thymus (2,51,52), and CCR9, CCR7, and CXCR4 chemokine receptors function together to support cells found in the thymus (43)(44)(45). Therefore, achieving thymic T cell development under physiological conditions depends on the coordinated actions of an ensemble of transcriptional factors, cytokines, adhesion molecules, and chemoattractants. Overall, our findings extend the mechanistic understanding of embryonic T lymphopoiesis in zebrafish and are valuable for further elucidation of Ikzf1 function in hematopoiesis.

Fish lines
The

Generation of mutants and transgenic lines
The ikzf1 and ccr9a mutants were created by the CRISPR/ Cas9 system (54). The in vitro synthesized guide RNA and hCas9 mRNA were injected into one-cell WT embryos. The irf4a mutants were obtained using transcription activator-like effector nucleases (55). Constructs targeting 5Ј-CCTGCTC-TTGACGACTgcaggcctgggctttatttaAGGGCAAATACAGGG-3Ј were created. The mutants were identified by sequencing or restriction enzymes. To generate Tg(coro1a:HA-ikzf1), Tg(coro1a: ccr9a), and Tg(coro1a:irf4a), their complementary DNA was amplified with specific primers (Table S1) and constructed into the pTol2 vector with the coro1a promoter (39). To facilitate screening, a Cryaa-Cerulean-BGHpA element (56) was reversely inserted. The constructs were injected into one-cell WT embryos. The transgenic lines were identified based on both the fluorescent signals in the eyes and a similar ikzf1/ccr9a/irf4a expression pattern as that of coro1a.

Ikzf1 regulates T lymphopoiesis via Ccr9 and Irf4
gen REPLI-g WTA Single Cell Kit (150063) according to the manufacturer's protocols. Total RNA of the whole embryos was extracted using TRIzol reagent. The amplified mRNA was used for qPCR. Each sample was tested in triplicate. sep15 (selenoprotein F precursor 15) (57), ␤-actin, and ef1a (58) expression was measured and used to normalize signals for each queried transcript by using the ⌬⌬Ct method. The primers of cell cycle-related genes (58) and other genes are listed in Table S2.

ChIP
For the ChIP assay, the tails of 300 Tg(coro1a:HA-ikzf1) embryos were collected at 3 dpf. After treatment with lysis buffer, the suspension was fragmentized by 0.5 units of MNase (37°C for 45 s) to generate 300ϳ1000 fragments. Cross-linked chromatin was immunoprecipitated with anti-HA antibody (Abcam, ab9110) or anti-IgG antibody (Sigma, A6154, negative control), according to the procedure in Ref. 46. The resultant immunoprecipitation samples were subjected to semi-quantitative PCR or qPCR using primers (Table S1).

Reporter assay
The luciferase assays were performed with the Dual-Luciferase Reporter Assay System. The luciferase activity was measured with a GloMax 20/20 luminometer (Promega) according to the manufacturer's instructions. The full length of ikzf1 CDS amplified with primers 5Ј-CGGAATTCAATGGAGAC-TGAGGAGGCACA-3Ј/5Ј-GCTCTAGAGTGGTTTACGTA-CACCATTG-3Ј and the full length of irf4a CDS amplified with primers 5Ј-CCATCGATATGAACTTAGATGGGGACTG-3Ј/5Ј-GCTCTAGATCACTCTGTCAGGTGTTGTA-3Ј were cloned into the pCS2 vector. The Ϫ2.8-kb ccr9a promoter was amplified with the primers (Table S1) and cloned into the pGL3-basic vector (Promega). The Ikzf1-binding sites were mutated by site-directed mutagenesis using the designed sitespecific oligonucleotides primers 5Ј-GAAGGATCGGCAAA-AGACCTCGATGAACTCGGGTCGCAGCAAG-3Ј/5Ј-CTT-GCTGCGACCCGAGTTCATCGAGGTCTTTTGCCGATC-CTTC-3Ј for MBS1 (mutant Ikzf1 binding site 1) and 5Ј-CTT-AAACAAACAAACCCAACTCAACTCAACCTTACTTAGT-3Ј/5Ј-ACTAAGTAAGGTTGAGTTGAGTTGGGTTTGTT-TGTTTAAG-3Ј for MBS2 (mutant Ikzf1 binding site 2) according to the manufacturer's instructions. To study the ccr9a promoter regulated by Ikzf1, Irf4a or both, the plasmids (200 ng of pGL3-ccr9a promoter, 200 ng of pGL3-ccr9a promoter-MBS1, 200 ng of pGL3-ccr9a promoter-MBS2, 200 ng of pGL3-ccr9a promoter-2MBS, 200 ng of pCS2-ikzf1 CDS, 200 ng of pCS2-irf4a CDS, 200 of ng pCS2, and 10 ng of pRL-CMV) were transfected into HEK293T cells by using Lipo8000 TM (Beyotime, C0533) with different combinations. pRL-CMV plasmids were added to each combinational group as an internal control. pCS2 empty vector was used as a negative control. HEK293T cells were cultured in DMEM with 10% fetal bovine serum in 24-well plates. After 36 h, cells were harvested for the luciferase assays according to the manufacturer's protocols. Analysis of the irf4a promoter was performed using zebrafish embryos. The irf4a promoter regions with the putative Ikzf1 binding site were amplified with the primers 5Ј-TCCCCCGG-GCAGAAGAGCATCTCTGAACA-3Ј/5Ј-CCGCTCGAGCA-GCTGAACCGCTGATACAA-3Ј and cloned into the pGL3-Enhancer vector (Promega). The binding site was mutated by site-directed mutagenesis using the designed site-specific oligonucleotides primers 5Ј-GCCGCTCCTGTCAGCTAAG-ATGAGGCTACAATTCACACAG-3Ј/5Ј-CTGTGTGAATT-GTAGCCTCATCTTAGCTGACAGGAGCGGC-3Ј according to the manufacturer's instructions. The designed plasmids (30 pg of pGL3-irf4a promoter or pGL3-irf4a promoter-MBS1 together with 1 pg of pRL-CMV) and 150 pg of ikzf1 mRNA were injected into the embryos at the one-cell stage. Doubledistilled H 2 O was used to replace ikzf1 mRNA in the control groups at the same time. After 28 hpf, the embryos were collected for the luciferase assays according to the manufacturer's protocols and as described previously (47).

Histological analysis, WISH, immunostaining, double staining, TUNEL staining, and EdU incorporation
H&E staining was performed on 10-m paraffin sections as described previously (60). After H&E staining, images were taken under an Axio Imager.Z2 Vario (Carl Zeiss). Antisense RNA probes and WISH were performed using a standard protocol (61). Single-color FISH and double staining were performed as described previously (38). TUNEL staining was performed according to the manufacturer's instruction or as reported previously (62). For the EdU incorporation assay, the Click-iT EdU Imaging Kit (Invitrogen, C10340) was used. Zebrafish larvae at different developmental stages were injected intravenously with 10 mM EdU and incubated for 2 h. The subsequent experiments followed the standard protocol. Goat anti-GFP antibody (Abcam, ab6658, 1:250, 4°C, overnight) and

Ikzf1 regulates T lymphopoiesis via Ccr9 and Irf4
Alexa Fluor 488 donkey anti-goat secondary antibody (Invitrogen, 1:400, 4°C, overnight) were used successively to visualize the signals. The WISH signals were observed under a SteREO Discovery.V20 microscope, and the fluorescence signals were imaged under an LSM700 confocal microscope (Carl Zeiss).

Time-lapse live imaging and transient lineage tracing
The fish embryos were anesthetized, mounted in 1% agarose, and subsequently imaged under an LSM700 confocal microscope (Carl Zeiss) with a ϫ20 objective. For live imaging, the targeted cells (DsRed ϩ , GFP ϩ , or Kaede ϩ cells) located around the thymi were imaged and calculated. To achieve transient tracing, cells in the targeted regions of Tg(coro1a:Kaede) and Tg(kdrl:Dendra2) embryos (somites 8 -10 in the VDA and 15-17 in the CHT) were selected and stimulated using the ROI mode via a 405-nm laser for around 15-20 s. The labeled cells were revealed and calculated 1-2 days later in the CHT, kidney, and thymus regions.

Quantification, calculation, and statistical methods
The positive signals in larval CHT were manually scored and double-confirmed blindly. All quantified data (mean Ϯ S.D.) were analyzed by GraphPad Prism 6. Student's t test (onetailed) was used.