Zebrafish B Cell Development Without a Pre–B Cell Stage, Revealed By

Zebrafish B Cell Development without a Pre− B Cell Stage, Revealed by CD79 Fluorescence Reporter Transgenes

This information is current as Xingjun Liu, Yue-Sheng Li, Susan A. Shinton, Jennifer of September 28, 2021. Rhodes, Lingjuan Tang, Hui Feng, Cicely A. Jette, A. Thomas Look, Kyoko Hayakawa and Richard R. Hardy J Immunol published online 24 July 2017 http://www.jimmunol.org/content/early/2017/07/22/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 24, 2017, doi:10.4049/jimmunol.1700552 The Journal of Immunology

Zebraﬁsh B Cell Development without a Pre–B Cell Stage, Revealed by CD79 Fluorescence Reporter Transgenes

Xingjun Liu,* Yue-Sheng Li,*,† Susan A. Shinton,* Jennifer Rhodes,* Lingjuan Tang,* Hui Feng,‡ Cicely A. Jette,x A. Thomas Look,{ Kyoko Hayakawa,* and Richard R. Hardy*

CD79a and CD79b proteins associate with Ig receptors as integral signaling components of the B cell Ag receptor complex. To study B cell development in zebraﬁsh, we isolated orthologs of these genes and performed in situ hybridization, ﬁnding that their expression colocalized with IgH-m in the kidney, which is the site of B cell development. CD79 transgenic lines were made by linking the promoter and upstream regulatory segments of CD79a and CD79b to enhanced GFP to identify B cells, as demonstrated by PCR analysis of IgH-m expression in sorted cells. We crossed these CD79-GFP lines to a recombination activating gene (Rag)2:mCherry transgenic line to identify B cell development stages in kidney marrow. Initiation of CD79:GFP expression in Rag2:mCherry+ cells

and the timing of Ig H and L chain expression revealed simultaneous expression of both IgH-m–andIgL-k–chains, without Downloaded from progressing through the stage of IgH-m–chain alone. Rag2:mCherry+ cells without CD79:GFP showed the highest Rag1 and Rag2 mRNAs compared with CD79a and CD79b:GFP+ B cells, which showed strongly reduced Rag mRNAs. Thus, B cell development in zebraﬁsh does not go through a Raghi CD79+IgH-m+ pre–B cell stage, different from mammals. After the generation of CD79:GFP+ B cells, decreased CD79 expression occurred upon differentiation to Ig secretion, as detected by alteration from membrane to secreted IgH-m exon usage, similar to in mammals. This conﬁrmed a conserved role for CD79 in B cell development

and differentiation, without the requirement of a pre–B cell stage in zebraﬁsh. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/

awed vertebrates, including the fish, express a number of cell–specific protein tyrosine kinase (Lck)–GFP reporter transgenic innate and adaptive immune system receptors, such as TLR line (5). It has been established that the thymus is a common pri- J and NOD-like receptor for innate immunity, and recombinase mary site for T cell development, as confirmed by examination of activating gene (RAG) and TCR and BCR genes for adaptive Rag1 and Rag2 and TCR gene expression (6, 7). The Rag genes immunity, initially found in mice and humans (1). The zebrafish is encode proteins necessary for rearrangement of both T and B cell a bony fish, a teleost, with ancestry that was generated .300 mil- Ag receptor chains (8, 9), and a Rag2:GFP reporter identified the lion years ago as one of the early jawed vertebrates. The zebrafish presence of Rag2:GFP+ cells in thymus (10, 11). has both an innate as well as an adaptive immune system, and it is B cells are the other major adaptive immune cell type. However, the by guest on September 28, 2021 thereby regarded as a good model organism for the study of im- details of the B cell development in zebrafish are still not well un- mune responses (2–4). Presence of one of the major cell types in derstood. In mice, B cells are generated from hematopoietic stem cells adaptive immunity, the T cells, has been identified in zebrafish and that reside in the liver before birth and in the bone marrow of adults (12, studied by detection of relevant mRNAs and use of a lymphocyte 13). Mouse B cell development is a highly orchestrated process, wherein precursors initiate Ig H chain rearrangement at the pro–B stage (14), then assemble the H chain with a surrogate L chain to form *Fox Chase Cancer Center, Philadelphia, PA 19111; †DNA Sequencing and Genomic a pre-BCR that signals clonal expansion of pre–B stage cells, pro- Core, National Institutes of Health, Bethesda, MD 20892; ‡The Center for Cancer gression to later stages of development, and initiation of Ig L chain Research, Boston University School of Medicine, Boston, MA 02118; xHuntsman { rearrangement (15). Upon successful completion of L chain rear- Cancer Institute, University of Utah, Salt Lake City, UT 84103; and Department of Pediatric Oncology, Dana–Farber Cancer Institute, Boston, MA 02215 rangement, the BCR is expressed on the surface of newly formed ORCIDs: 0000-0002-7012-7053 (X.L.); 0000-0002-1393-3913 (Y.-S.L.); 0000-0003- B cells that then undergo further maturation to become fully func- 2649-1904 (S.A.S.); 0000-0001-7298-1213 (J.R.); 0000-0003-1318-821X (H.F.); tional B cells. A similar process has been identified in the generation 0000-0003-3157-5897 (K.H.). of B cells in humans (16) and in rabbits (17). However, not all ver- Received for publication April 18, 2017. Accepted for publication June 27, 2017. tebrate species construct B cells in this fashion. For example, chicken This work was supported by National Institutes of Health Grant P30 CA006927 (to B cells are produced by simultaneous rearrangement of Ig H and L the Fox Chase Cancer Center), an appropriation from the Commonwealth of Penn- sylvania, and by the Blood Cell Development and Cancer Keystone Program at the chains in the bursa of fabricius, with no distinct pre–B stage (18). Fox Chase Cancer Center. This work was also supported by the Zebrafish Core VpreB together with l5 form a surrogate L chain (19), also known as Facility in the Fox Chase Cancer Center. pseudo–L chain, to generate progression through the pre-BCR to pre– Address correspondence and reprint requests to Dr. Kyoko Hayakawa, Fox Chase B cell stage in mammals. In general, existence of the pseudo–L chain Cancer Center, Reimann Building, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail address: [email protected] has not been clearly established in nonmammals (20). In zebrafish, neither a pseudo–L chain nor pre-Ta that creates a pre-TCR has been The online version of this article contains supplemental material. detected (20, 21). Thus, it has not been clear whether the zebrafish Abbreviations used in this article: BAC, bacterial artificial chromosome; dpf, day postferilization; EGFP, enhanced GFP; Fr., fraction; Lck, lymphocyte cell–specific generates pre–B and/or pre–T cell stage in development. protein tyrosine kinase; PI, propidium iodide; qRT-PCR, quantitative RT-PCR; Rag, We have now investigated B cell development in the zebrafish recombinase activating gene. model organism, seeking to determine similarities and differences This article is distributed under The American Association of Immunologists, Inc., from mammalian B cells. The zebrafish is a small fish where Reuse Terms and Conditions for Author Choice articles. embryos develop most organs by 5 d after fertilization, allowing Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 visual tracking of maturation (22). Although Abs to detect and

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700552 2 ZEBRAFISH B CELL DEVELOPMENT distinguish adaptive immune cell types in zebrafish have not yet PCR products were amplified with PFU DNA polymerase, cloned into the + been developed, a powerful approach in this model organism has pCR2.1-TOPO vector (Invitrogen), and subcloned into the PCS2 vector. been the generation of fluorescent reporter transgenic fish, an Clones were then characterized and validated by sequencing. CD79a mRNA was identical to NM_001326470, and CD79b was close to XM_009306430.2 approach that has been used to identify and characterize erythroid, (as a predicted sequence) in GenBank (see Supplemental Fig. 1). myeloid, T cell, and early lymphoid cells (5, 23, 24). Fluorescent reporter lines have revealed early T cell development in zebrafish Analysis of mRNA expression thymus (7, 25), and such reporters have been used to extensively Tissue RNA was prepared using TRIzol (Invitrogen) and phenol extraction, study T cells (5). Thus, we sought to assess B cell development by then precipitated with isopropanol and washed with 70% ethanol; pelleted generating CD79 fluorescent reporter transgenic zebrafish. RNA was resuspended in DEPC. In flow cytometry experiments, cells were sorted directly into RNA lysis buffer and RNA was prepared as previously B cell Ag receptors use heterodimers of the adapter proteins, described (47). RNA was reverse transcribed with random hexamers us- CD79a (Iga) and CD79b (Igb), to couple membrane-bound Ig, the ing the SuperScript II kit (Invitrogen). PCR reactions (RT-PCR and quan- external Ig Ag-sensing molecule, with cytoplasmic signaling titative RT-PCR [qRT-PCR]) were performed with the primers listed in cascades (26–28). These CD79 proteins are essential for Ig cell Table I. surface expression and B cell signal transduction, including the Preparation of RNA probes for in situ hybridization pre-BCR and pre–B cell stage in mammals. Both CD79 peptides contain an ITAM, and this motif is also found in other trans- The CD79a antisense RNA probe was prepared by 59 linearization with NotI and synthesis with SP6 polymerase; the sense control was prepared membrane receptor signaling adapter proteins such as CD3 com- by 39 linearization with HindIII and synthesis with T7 polymerase. The ponents, TCRz and DAP12 (29). These CD79 molecules serve as CD79b antisense RNA probe was prepared by 59 linearization with BamHI robust B cell markers. Mammalian CD79 expression is first de- and synthesis with T7 polymerase; the sense control was prepared by 39 Downloaded from tected at the stage of B lineage commitment, including the pre– linearization with XhoI and synthesis with SP6 polymerase. The IgH-m clone (AY643753; GenBank) was obtained from Addgene (plasmid 11301); B cell stage (30), and continues until the plasma cell stage, when the antisense RNA probe was prepared by 59 linearization with XbaI and they are downregulated (31). Both CD79a and CD79b have been synthesis with T7 polymerase; the sense probe was prepared by 39 line- described in chickens (32, 33), and orthologous genes have been arization with BamHI and synthesis with SP6 polymerase. Polymerases are identified in some bony fish species, including fugu (34) and from Roche Biotech, probes are labeled with DIG, and posttranscription cleanup was done with SigmaSpin columns (Sigma-Aldrich). In situ hy- channel catfish (29), as well as in a boneless jawed vertebrate, the http://www.jimmunol.org/ bridization of zebrafish thin sections was carried out as described by Smith spiny dogfish (shark) (35). The presence of CD79a in zebrafish et al. (48). Staining in sections hybridized with antisense and sense (control) B cells has also been demonstrated by single-cell quantitative PCR probes was developed with NBT/5-bromo-4-chloro-3-indolyphosphate, and (36). However, these CD79 genes have not been characterized in then images were acquired using a spectral imaging camera and processed the context of zebrafish B cell development. Thus, to unambigu- with Nuance software for visualization of the staining. ously identify and characterize B cells and B cell development in Generation of CD79a-GFP and CD79b-GFP transgenic lines zebrafish, we cloned orthologs of mammalian CD79a and CD79b, To generate CD79a -and CD79b-GFP transgenes, bacterial artificial then generated GFP reporter lines with transgenic constructs chromosome (BAC) clones CH211-64K10 and CH211-93E11, containing containing regulatory sequences for these genes driving fluores- CD79a and CD79b genes and an upstream genomic sequence, respectively, cence protein expression. We then used these lines to cross with a were obtained from the Children’s Hospital Oakland Research Institute by guest on September 28, 2021 transgenic Rag2-mCherry line to assess B cell development and and reengineered by galk recombineering (49), replacing the first exon differentiation. In zebrafish B cells, three Ab classes have been from the ATG start codon of CD79a and CD79b by an enhanced GFP (EGFP)–polyA segment. CD79a and CD79b recombineered BAC plasmids identified, IgM, IgD, and IgZ (also named as IgT) (37–40). IgM is (containing EGFP) were used as a PCR template for transgene generation the main Ab class in all jawed vertebrates, and IgZ/T is specific in with the following primers: CD79a (14k) (forward), 59-GGCAGAAATTCC- teleost fish (38). IgZ/T is specialized in mucosal immunity and CAAAGACA-39; CD79a (reverse), 59-GGCGTCATACATGGACAGTG- present in gut mucus, closely resembling IgA in mammalian 39; CD79b (5k) (forward), 59-TAGGTTTTGCTCGCCTCTGT-39; CD79b (reverse), 59-GCCAGGTACACAACGTTCCT-39. The restriction site RsrII mucosal immunity (41). Three Ig L chain isotypes, named L1, L2, was incorporated into both forward and reverse PCR primers for easy and L3, have been detected in zebrafish. L1 and L3 are Ig-k release of transgenes. Transgenes were amplified using the proofreading orthologous (IgL-k), and L2 is orthologous to Xenopus s (38, 42). LA Taq (TaKaRa) enzyme and cloned into a TOPO TA vector (Life Thus, we focused on Rag, IgH-m, IgL-k, and CD79 expression in Technologies). Then the plasmids were digested with RsrII to release the kidney marrow where Rag-expressing B cell development occurs transgene from the vector backbone; following agarose electrophoresis, transgenes were gel purified using a QIAquick gel extraction kit (Qiagen) as the major site of hematopoiesis in adult zebrafish (43, 44). and the DNA was resuspended in double-distilled H2O at 200–500 ng/ml, then injected (30–50 pg per oocyte) into zebrafish eggs at the one- to two- Materials and Methods cell stage. Larvae were monitored for transient GFP expression, discarding those that lacked expression. Founders were identified by PCR. This screen Zebrafish maintenance eventually yielded two CD79a (14k)–GFP lines and two CD79b (5k)–GFP Zebrafish adults were bred and embryos were staged using standard lines. practices (45). Rag2-mCherry and Lck-GFP transgenic lines were previously described (5, 46). All procedures using experimental animals were Flow cytometry carried out under a protocol approved by the Fox Chase Cancer Center Tissues were dissected from adult fish and dissociated by grinding between Institutional Animal Care and Use Committee. two frosted glass slides. Material was rinsed into a petri dish with staining Cloning of zebrafish CD79a and CD79b cDNA medium (deficient RPMI 1640 [lacking phenol red, riboflavin and biotin], 3% newborn calf serum, 0.1% sodium azide) and this suspension was passed Predicted zebrafish CD79a (XM_001339187) and CD79b (XM_002663902) through a 70-mm Nitex cloth screen into a 15-ml conical centrifuge tube. mRNA sequences were found in the National Center for Biotechnology Cells were pelleted by centrifugation (1200 rpm; 273 3 g; 7 min; 4˚C) and Information database. 59-RACE was performed to detect and amplify the washed twice more with staining medium. Cell pellets were resuspended in 59 untranslated region of CD79a and CD79b mRNA. Primers included: 1) staining medium that included propidium iodide (PI; 1 mg/ml) to facilitate cd79a reverse 1, 59-CTACGGCTTCTCCAGCTGAAT-39 for the first round; exclusion of dead cells (also evaluation of DNA content in cell cycle) and cd79a reverse 2, 59-CAGCTGAATGTCTTCCTCACA-39 for second round; transferred to sample tubes. Clutches of larval fish (day 7 to day 28) were and 2) cd79b reverse 1, 59-TCACTCTTGGCATGGAGACT-39 for first round; dissociated by grinding between frosted glass slides as above, but after cd79b reverse 2, 59-CTCTTGGCATGGAGACTCAA-39 for second round. initial pelleting, the sample was resuspended in 1 ml of staining medium RT-PCR and PCR reactions were performed according to the manufac- and layered over 1 ml of Lympholyte M (Accurate Chemical & Scientific) turer’s recommendations (59RACE kit; Invitrogen). CD79a and CD79b and centrifuged (2800 rpm; 1762 3 g; 20 min; room temperature). Cells The Journal of Immunology 3 were recovered from the interface and washed twice with staining medium. Results Cell pellets were resuspended as above. Flow cytometry was performed Identification and characterization of zebrafish CD79a and with a FACSAria II (BD Biosciences). CD79b Microscope imaging Zebrafish CD79a and CD79b cDNA sequences were amplified by Images of whole fish or larvae were acquired with a Nikon SMZ1500 59RACE using primers based on partial clones that showed ho- stereomicroscope using a Nikon DS-Fi1 camera and NIS-Elements soft- mology to channel catfish CD79a and CD79b. The full-length ware. Fish frozen sections were mounted with Vectashield mounting media CD79a and CD79b cDNA sequences and their deduced amino (Vector Laboratories), overlaid with coverslips, and images were acquired with a Nuance camera (CRi, Hopkinton, MA) on a Nikon Eclipse 80i acid sequences are shown in Supplemental Fig. 1. Both CD79a microscope. WISH white-light images were also acquired with the same and CD79b zebrafish sequences contain the ITAM domain, and system. Nuance spectral images were unmixed using CRi software. Con- strong homology around this domain was found between zebra- focal images were acquired with a Nikon C1 spectral imaging confocal fish, catfish, mouse, and human amino acid sequences (Fig. 1, bold microscope, using 488 nm excitation for GFP and detecting fluorescence line), indicating that zebrafish CD79a and CD79b mediate the between 415 and 570 nm. mCherry fluorescence was excited at 561 nm and detected between 570 and 655 nm. same function as CD79a and CD79b in other vertebrates. These ITAM domains are known to be important in BCR signaling, as Infection with bacteria they provide two tyrosine phosphorylation sites (Fig. 1, asterisk The bacterial strain used for infection is Pseudomonas aeruginosa PA14 marked) for docking Syk kinase and initiating downstream sig- (p66TDC1) strain with Tomato Red expression (50). A stab of this strain naling pathways (51). (obtained from Dr. J. Singer, Maine School of Marine Sciences, University Fish lack bone marrow and lymph nodes, and B cell lympho- Downloaded from of Maine) was plated out onto L agar with 750 mg of ampicillin per poiesis in zebrafish occurs mainly in the kidney, as a kidney marrow, milliliter. Bacteria were grown in Luria–Bertani medium and concentration was determined by comparing OD600 of a dilution series, simultaneously after the larval stage. The pronephros is the first kidney to be formed plating dilutions in plates, and then counting colonies. For infection, adult during embryogenesis, then the mesonephros forms as an extended CD79a-GFP and CD79b-GFP transgenic zebrafish were anesthetized with kidney, and pronephros head kidney is the major site of B cell 4 5 160 mg/ml tricaine and injected i.p. with 10 or 10 CFU of P. aeruginosa lymphopoiesis in the adult (52). In the adult zebrafish, expression of (RFP labeled). A third (control) group was injected with PBS. Bacterial CD79a and CD79b was strongly detected in kidney marrow sec- exposures were performed in triplicate per dose. After recovery from an- http://www.jimmunol.org/ esthesia, each group was moved to a small offline tank in a quarantine tions by in situ hybridization using antisense RNA probes, localized room and maintained for 28 d. Fish were observed daily for signs of in areas similar to IgH-m (Fig. 2A). To further assess tissue distri- disease and morbidity. Fish were anesthetized with 160 mg/ml tricaine and bution of zebrafish CD79a and CD79b, RT-PCR with primers of observed two times per week by microscopy (fluorescence detection of CD79a and CD79b was performed (Fig. 2B, Table I) (53). This GFP and RFP) to track changes in B cell numbers and localization and bacterial growth. Three fish were selected from 7, 14, and 28 d postin- showed predominant expression in head kidney marrow where fection for each group; dissected tissues, including liver, intestine, spleen, B cells develop, and also in spleen, a site of mature B cells by and kidney, were used for FACS analysis. reentry from circulation after B cell development in kidney. by guest on September 28, 2021

FIGURE 1. Zebrafish CD79a and CD79b protein alignment. (A and B) Alignment of zebrafish CD79a and CD79b amino acid sequences with channel catfish, mouse, and human orthologs. ITAM domains are indicated by bold lines below the sequences; the two key tyrosines are labeled by asterisks. 4 ZEBRAFISH B CELL DEVELOPMENT

FIGURE 2. Predominant expression of CD79a and CD79b in kidney by in situ hybridization. (A) In situ hybridization of IgH-m, CD79a, and CD79b antisense RNA probes to thin sections of zebraﬁsh kidney. Images were processed with Nuance software to highlight hybridization signal stained by NBT/5-bromo-4-chloro-3-indolyphosphate. Right panels show background with sense probes. Scale bars, 400 mm. (B) RT-PCR analysis of CD79a and CD79binzebraﬁshtissues.Imagecolor is inverted for clarity.

Although at lower transcript levels, CD79 and IgH-m were also mRNA. Thus, CD79:GFP expression clearly distinguishes be- Downloaded from detected in intestine. tween B and T lymphocytes in zebrafish, not expressed by the T lineage. We conclude that GFP+ cells in the CD79a-GFP and Generation of CD79a-GFP and CD79b-GFP CD79b-GFP transgenic lines label the B-lineage cells in kidney transgenic zebrafish marrow. Next, two lines each of CD79a-GFP and CD79b-GFP transgenic Analysis of CD79a-GFP and CD79b-GFP transgenic zebrafish reporter lines (Fig. 3A) were established. These four lines were http://www.jimmunol.org/ crossed to Rag2-mCherry bred to the F3 generation and showed similar stable GFP expression patterns. In all lines, GFP expression was clearly detected in In mice, CD79a and CD79b are expressed early in B-lineage cell the primary immune site, head kidney marrow (Fig. 3B). These development with the Rag1 and Rag2 genes before generation of GFP-labeled cells in the kidney were distributed around the pro- BCR-expressed immature B cells (30). In zebrafish, Rag1 and nephric tubules, in a pattern similar to the antisense RNA probe Rag2 are also both present. Thus, to determine timing of CD79a staining originally detected in Fig. 2A. GFP-labeled cells showed and CD79b expression in zebrafish B-lineage cells, we crossed a small round morphology, resembling lymphocytes (Fig. 3B, one of each CD79a-GFP and CD79b-GFP transgenic fish lines right panels), supporting their identification as B cells. Scattered with the Rag2-mCherry transgenic zebrafish line, which bears a

GFP+ cells were also visualized microscopically in other tissues, Rag2 regulatory segment driving expression of mCherry. by guest on September 28, 2021 including in intestine of both CD79a-GFP and CD79b-GFP cell Fig. 4A shows the screening of early developing zebrafish from lines (Fig. 3C), as originally found by RT-PCR in Fig. 2B. the first 7 d postfertilization (7 dpf) to the 28 dpf (4 wk) stage by To determine whether GFP in these transgenic lines labels the flow cytometry analyses of cell suspensions prepared from B lineage, we used flow cytometry to sort GFP+ and GFP2 cells clutches of dual transgenic line, in comparison with nontransgenic from kidney marrow (Fig. 3D) and isolated RNA from the sorted zebrafish as a control (wild-type AB). Before this dual transgenic samples. We also sorted GFP+ and GFP2 cells from Lck-GFP line analysis, we noticed that offspring from female CD79:GFP transgenic zebrafish from kidney to identify T cells for compari- transgenic carriers exhibited diffuse maternal GFP mRNA. Thus, son. We then used RT-PCR to examine expression of CD79a, experiments with larval clutches were established by crossing CD79b, IgH-m, and Lck (Fig. 3E). The GFP+ fraction from both transgenic male parents from both CD79a-GFP and CD79b-GFP lines showed expression of CD79a, CD79b, and IgH-m, but not lines. Even under these conditions, we detected GFP in the mid- Lck. In contrast, GFP+ cells from the Lck reporter line lack ex- brain, ventral branch arches, and otic vesicle during the first 6 d pression of the B cell genes, but they are strongly positive for Lck postfertilization in CD79a-GFP zebrafish by sequential screening.

Table I. Primers used for RT-PCR and qRT-PCR analysis of gene expression in zebraﬁsh

Target Gene Forward Primer Reverse Primer CD79a 59-TCAAGAATACTCCCGCCATC-39 59-GGCTTCTCCAGCTGAATGTC-39 CD79b 59-GCTCACTTACGAATGACCAGAGAATAAC-39 59-GTCCTCATACACATCTCCACCAACC-39 VH-Cm 59-CCTCCTCAGACTCTGTGGTGA-39 59-TTGCTGATCCACCTTCTAATTC-39 VH-Cz 59-CCTCCTCAGACTCTGTGGTGA-39 59-GATTGTTTGCTGTAGAATGCTG-39 Total m 59-GGAGCCCCAAAAACAGCTTC-39 59-TCAGCATGTCTTCAGGGGTG-39 IgH-m (secreted) 59-CACCCCTGAAGACATGCTGA-39 59-ACAAACACCTCCTTGGGCAA-39 IgH-m (membrane) 59-CACCCCTGAAGACATGCTGA-39 59-GCAATGCCACTGTCATCTGC-39 IgL-k (L1) 59-GGCACCAAACTGGATGTTG-39 59-AGCAGGAGTGTGTGTGTGCT-39 RAG-1 59-AAGAACCAGGTGAAGACATTTGCC-39 59-TGCCACATCATAACGGAATCGTC-39 RAG-2 59-TTCCAGCACCAAACAGCTCTCAG-39 59-GAGGTAACATTCAGGTTTGCCGTC-39 Lck 59-AGATGAATGGTGTGACCAGTGTA-39 59-GATCCTGTAGTGCTTGATGATGT-39 b-Actin 59-GCCAACAGGGAAAAGATGACACAG-39 59-GAGTATTTACGCTCAGGTGGGGC-39

VH used for Cm and Cz forward primer is VH1 (AF273897; GenBank), detected from early age (day 4) of zebraﬁsh (53). IgL-k is zebraﬁsh L1 (AF246185; GenBank). qRT-PCR primers for Rag1, Rag2, and Lck were from Life Technologies. The Journal of Immunology 5

FACS analysis of macerated clutches of the CD79a lines at 1 wk line in Fig. 4D. Fluorescence microscopy of kidney and thymus to ,3 wk postfertilization consistently showed 5- to 10-fold sections of these adults revealed strikingly different patterns of higher frequencies of GFP+ cells compared with the CD79b lines, expression and localization of these genes (Fig. 4C). In kidney and most of these CD79a:GFP+ cells found in the larval stage were marrow, we observed a broad dispersal of red-only (Rag2+ negative for Rag2:mCherry (Fig. 4A). Thus, the expression of CD79b2), yellow (Rag2+CD79b+), and green (Rag22CD79b+) GFP in the B lineage was unclear in younger larvae. Then, in cells throughout the kidney tissue. In contrast, green fluorescent subsequent analyses of the 3-wk-old (21 dpf) larval–juvenile cells were only localized along the border of the thymus, and a transition stage, fish showed low numbers of double-positive cells large number of red cells were observed within the thymus organ. detected with either the CD79a-GFP or CD79b-GFP reporter, and Fig. 4C (bottom) shows high-power images obtained with a con- double-positive cells became abundant by 4 wk postfertilization focal microscope that reinforced this difference. T-lineage cells (28 dpf) as juveniles. rearrange TCR genes during their development in the thymus. As After 4 wk postfertilization (1 mo), CD79a- and CD79b-GFP/ expected from the Fig. 4C microscope images, the flow cytometry Rag2-mCherry analysis was performed with kidney marrow. As pattern shown in Fig. 4D demonstrated fewer CD79b:GFP+ cells shown in Fig. 4B, CD79a:GFP+ cells in the CD79a-GFP/Rag2- in thymus than in kidney, and such GFP+ cells mostly expressed mCherry transgenic line increased by 16 wk (4 mo) in adult decreased levels of Rag2:mCherry. kidney, and part of them showed the clearly decreased Rag2: mCherry expression, which was more prominent at 32 wk (8 mo). Zebrafish B cell development without the pre–B cell stage The CD79b-GFP/Rag2-mCherry transgenic line showed a similar Our major question was whether the CD79+m+ pre–B cell stage increase in CD79a:GFP+ cells in kidney during 1–8 mo aging, as exists in zebrafish. In the CD79b-GFP/Rag2-mCherry line, RT- Downloaded from shown in a 5-mo adult CD79b-GFP/Rag2-mCherry transgenic PCR of the few GFP+ cells isolated by sorting from 3 wk (21 http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. GFP expression in CD79a and CD79b transgenic zebrafish identify B cells. (A) Diagram of production of transgenic constructs, made by long PCR from BACs containing CD79a and CD79b, where the first coding exon of each was replaced by an EGFP-PolyA segment. (B) Low power (left panels) and higher power (right panels) images of GFP expression in thin sections of kidney region of adult (4–6 mo) CD79a-GFP and CD79b-GFP zebrafish. Images were processed with Nuance software to reduce background autofluorescence. Scale bars, 100 mm (left), 200 mm (right). (C)PresenceofCD79a:GFP+ and CD79b:GFP+ cells outside head kidney in adult fish. CD79a-GFP, 34 cryosection. Scale bar, 100 mm (left), and original magnification 310 image of cryosection. Scale bar, 200 mm (right). CD79a-GFP transgenic zebrafish also showing intestinal region. Scale bar, 200 mm. (D) Flow cytometry histogram plots of kidney marrow tissue from CD79a-GFP and CD79b-GFP lines. (E)GFP2 and GFP+ cell fractions were purified by electronic cell sorting from Lck-GFP, CD79a-GFP, and CD79b-GFP kidney tissue. Figure shows ethidium bromide staining of DNA amplified by RT-PCR from these samples. The lines indicate where parts of the image were joined. 6 ZEBRAFISH B CELL DEVELOPMENT

FIGURE 4. Identification of developing and mature B cells in CD79a- GFP and CD79b-GFP 3 Rag2- mCherry double-transgenic zebrafish. (A) B cells in young CD79a- GFP and CD79b-GFP zebrafish larvae identified by coexpression with Rag2:mCherry, analyzed by flow cytometry. Wild-type zebrafish AB line is shown as a control. Repre-

sentative data of four to seven sample Downloaded from analyses from each day postferilization zebrafish are shown. (B) Kidney marrow of 1–8 mo CD79a-GFP 3 Rag2-mCherry. Dotted region is GFP2mCherry2.(C) Five month CD79b-GFP 3 Rag2-mCherry zebrafish. Low-power (top) and high- http://www.jimmunol.org/ power (bottom) images of thin sections of kidney and thymus are shown. Scale bars, 200 mm (top) and 100 mm (bottom). (D) Flow cytometry analysis of adult CD79b-GFP 3 Rag2-mCherry zebrafish kidney and thymus. Dotted region is GFP2 mCherry2. Frequency of CD79b: GFP+ cells in kidney is 26.4%, in by guest on September 28, 2021 thymus is 5%. Data are representative of five separate CD79b/Rag2 transgenic fish kidney analyses.

dpf) larval clutches showed weak coexpression of IgH-m and IgL-k, shown in Fig. 5C, Fr. 4–6 cells with higher levels of CD79b:GFP but not IgH-m alone (Fig. 5A). In adult kidney, GFP+Rag2- together with middle to diminishing levels of Rag2:mCherry are mCherry+ cells showed further increased IgH-m and IgL-k levels small cells, and they have exited the cell cycle, because PI staining (Fig. 5A). We divided these adult kidney marrow cells into several showed a ,0.5% increased cell fraction, in contrast to 12–22% for fractions, with different CD79:GFP levels in Rag2:mCherry+ cells, Fr. 1–3 (Fig. 5D). Fr. 3 cells coexpressing IgH-m and IgL-k including the Rag2:mCherry dimmest cell fraction, and charac- contained cycling cells with slightly increased cell size and DNA terized Ig gene expression (by RT-PCR, Fig. 5B), cell size (by content. This suggested that Fr. 3 cells become a mature quiescent forward light scatter, Fig. 5C), and cell cycle (by PI staining of stage as Fr. 4. cell DNA, Fig. 5D). This analysis revealed that Rag2:mCherry+ Although Rag2:mCherry was consistently detected from a high cells with dim to intermediate CD79b:GFP+ fractions (Fr. 2–3) to diminishing level in all CD79b:GFP+ cells, we anticipated the expressed IgH-z and IgL-k, the alternate fish IgH known as IgZ/T. possibility that this long-term detection was due to the long half- This IgZ/T was predominant in cells with the lowest GFP levels as life of fluorescent proteins after Rag2 expression is extinguished, Fr. 2. Fr. 3 showed a mixture of IgH-z and IgH-m, whereas IgH-m as elongated stability. Previous work in mice found that the GFP+ was predominant in cells in Fr. 4 and later. It was striking that IgL-k level in Rag2-GFP immature B cells was higher than comparative was continuously found through all CD79b:GFP+ cell fractions mRNA levels (54). Similarly, analysis of the thymus in Rag2-GFP coexpressing with H chain. This pattern was very different from zebrafish also suggested longer persistence of GFP protein than what is seen in the mouse, where H chain precedes L chain ex- Rag2 protein, because GFP was detected in both cortical and pression at a distinct cytoplasmic IgH m+ pre–B cell stage. As medullary regions of the thymus (5). In the zebrafish CD79-GFP/ The Journal of Immunology 7

Rag2-mCherry lines shown in this study, although Rag2-mCherry Rag2:mCherry and expression of both IgH and IgL in Fr. 2–5, was clearly detectable, mCherry levels were lower by Fr. 2 than Fr. distinct from Fr.1, was also found in the CD79a-GFP/Rag2- 1 and further declined in later fractions (Fig. 5E). This decline of mCherry transgenic line (Fig. 5F). When qRT-PCR was performed Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Characterization of B cell development in kidney marrow of CD79-GFP 3 Rag2:mCherry zebrafish. (A) RT-PCR detection of both IgH-m and IgL-k genes at 3 wk (21 dpf) age for CD79b:GFP+ cells sorted from macerated zebrafish clutches. In adults, CD79b:GFP+ cells were sorted from kidney. (B) Left: 4 mo. CD79b/Rag2 transgenic fish kidney. Two-color flow cytometry plot shows regions of cells sorted for analysis. Right: RT-PCR analysis of kidney fractions for Ig expression. (C) Differences in cell size detected by light scatter. (D) Differences in cell cycle profiles detected by PI staining. (E) Reduction of Rag2-mCherry level from Fr. 1 stage. Gray is mCherry2GFP2 fraction. (F) Three month CD79a/Rag2 transgenic fish kidney. Fractions and RT-PCR analysis. (G) qRT-PCR of fractions from 3 to 4 mo. CD79a and CD79b/Rag2 transgenic lines (n = 4–5 for each fraction, mean 6 SE). CD79a/Rag2 and CD79b/Rag2 data were similar, and total data are shown. 8 ZEBRAFISH B CELL DEVELOPMENT on these fractions prepared from both CD79a- and CD79b-GFP/ Rag2-mCherry lines, we consistently found strong downregulation of both Rag1 and Rag2 mRNAs after the Fr. 1 stage (Rag1 was reduced by .30-fold from Fr. 1 to Fr. 2) and undetectable by Fr. 5 (Fig. 5G, left panel). In opposition to Rag mRNAs, both CD79a and CD79b mRNAs were upregulated after the Fr. 1 stage, and increased by Fr. 3/4, together with IgM mRNA (Fig. 5G, right). This confirmed the absence of a RaghiCD79+IgH-m+ pre–B cell stage, in contrast to the presence of RaghiCD792IgH-m2 cells and RagloCD79+IgH-m+IgL-k+ B cells. Interestingly, Fr. 5 lacked Rag mRNAs and consistently showed decreased CD79a and CD79b mRNA levels as compared with Fr. 3/4, and often showed further increased IgH-m mRNA. This suggested that after generation of mature B cells, reduced expression of CD79 may be occurring during differentiation. Reduction of CD79 by B cell responses to bacterial infection To confirm whether alterations in the level of CD79 occur during zebrafish B cell differentiation, we examined B cell responses to Downloaded from pathogen by exposing CD79a-GFP and CD79a-GFP lines to bacteria. As shown with the CD79a-GFP line, injection of adult fish i.p. with P. aeruginosa, which also contained a plasmid coding for the RFP Tomato Red (50), resulted in changes in GFP+ cell numbers in secondary tissues, including a prominent increase in intestine ++

(Fig. 6A). We noted an increased frequency of both CD79a and http://www.jimmunol.org/ CD79a+:GFP cells in intestine, but also in spleen and liver, in contrast to kidney (Fig. 6B). Fig. 6C shows an increased incidence of CD79a+:GFP cells as compared with CD79a++:GFP cells in intestine at 1 wk postinfection. We also found an increase in B cell size, particularly in the intestine early postinfection (1 wk), that resolved by 4 wk postinfection (Fig. 6D). At this 4 wk stage postinfection (and control), we then purified B cell fractions in kidney and assessed expression of membrane and secreted IgH-m by RT-PCR. Clearly, infection-induced B cells with low to inter- by guest on September 28, 2021 mediate CD79a:GFP levels were present in kidney, showing decreased levels of membrane IgH-m and greatly increased levels of secreted IgH-m, indicating the presence of plasma cells in kidney that had been generated in response to infection (Fig. 6E). This confirmed that downregulation of CD79 occurs in zebrafish, in parallel with the differentiation of mature B cells to Ig-secreting plasma cells, as found in mammals.

Discussion By generation of CD79-GFP lines, we investigated zebrafish B cell development and differentiation, seeking to determine similarities and differences from mammalian B cells. In adult mouse B cell development, a surrogate L chain is involved in progression to the pre–B cell stage. Although it has already FIGURE 6. Zebrafish B cell response to bacterial infection with CD79 been known that the zebrafish lacks a surrogate L chain (20), it was reduction. (A) Low-power images of intestinal region of PBS control and unclear whether an alternative pre–B cell stage exists, because early bacteria injected CD79a-GFP fish at 1 wk postinfection. Scale bars, 1 mm. ++ + fetal/neonatal B-1 B cell development in mice can occur in the (B) Percentage of CD79a and CD79a cells in indicated tissues at 1, 2, 6 C absence of a surrogate L chain (12, 55), and this surrogate L chain– and 4 wk postinfection; n = 3 each, mean SE. ( ) Flow cytometry histograms of CD79a++ and CD79a+ cells in kidney and intestine tissue 1 independent mouse B-1 development still progresses through a + hi + + wk postinfection, showing the clear increase in low CD79 cells in in- Rag CD79 IgH-m pre–B cell stage. In this study, we demonstrate testine. (D) Increase in size of CD79a+ cells from intestine of infected fish, that the zebrafish does not require the pre–B cell developmental detected by flow cytometry using forward light scatter (FSC). (E) CD79a+ stage. Both H and L chains rearrange simultaneously and B cells cells are enriched for secreted IgM compared with CD79a++ cells. RT-PCR expressing complete BCRs initially proliferate with slightly in- analysis of b-actin, membrane IgH-m (m), and secreted IgH-m (s). creased cell size, before entering a mature quiescent stage, more similar to chickens (18) than mammals. the B cells that exhibit decreased levels of Rag2 with further in- Previously, it was reported that the pre–B cell stage exists in creased IgM+. This result led to the conclusion that a Rag2+IgH-m+ zebrafish by using a newly generated IgM1:eGFP line crossed with pre–B cell stage exists. However, our new generation of CD79a-GFP a Rag2:DsRed line (56). In the context of this dual IgM/Rag2 and CD79b-GFP lines then crossed with the Rag2-mCherry line transgenic reporter line, it was concluded that IgM2,IgMlo,andIgM+ allowed us to reveal further details of the B cell development pro- cells all express a similar level of Rag2+ in kidney, as compared with cess in zebrafish. Previous IgM/Rag2 data showed the presence of The Journal of Immunology 9

IgZ in the Rag2+IgM2 cell fraction. Now, our CD79b/Rag2 trans- Lin28 expression was also found during embryonic development genic system revealed that the Rag2+IgM2 fraction consists of the in both chicken and zebrafish (59, 60). In zebrafish, lin28a and mixture of two distinct cell fractions, Rag2hiCD792IgM2IgZ2 pre- lin28b were found to be ubiquitously expressed early after em- cursor cells (Fr. 1) and Rag2loCD79lo/+IgM2/+IgZ+ cells (Fr. 2) bryonic fertilization, then expression decreased, and Let7a/let7b (Fig. 5B). Thus, IgZ+ cells are already Raglo, as further confirmed by microRNA expression increased at 3 dpf (59). Ikaros is a marker thepresenceofRagloIgZ+ cells in the CD79a/Rag2 line. Importantly, for lymphoid progenitors, and higher expression is found in fetal our data demonstrate that a strong reduction of Rag mRNAs occurs hematopoietic stem cells than adult hematopoietic stem cells in when B cells are initially generated, and a further reduction of Rag mice (61). In zebrafish, elimination of the Ikaros gene resulted in mRNAs results at differentiated B cell stages often in conjunction absolute loss of IgH-z–bearing B cells (IgZ) but sparing of some with increased IgM. Thus, our discovery of Rag2hiCD792 cells in our IgH-m B cells (IgM) (62). In mice, fetal/neonatal generated B1 CD79/Rag2 transgenic system allowed us to clarify the Rag2 levels. B cells can enter the intestine and generate IgA, independent of Analysis of our CD79/Rag2 transgenic lines also allowed us to T cell help (63, 64). Perhaps this issue of distinctive develop- find nonlymphoid cells with CD79:GFP expression without Rag2- mental origin of B cells in zebrafish can be further addressed by mCherry at the larval stage (Fig. 4A). In the CD79a-GFP line, all sorting early progenitors in culture. There is a report of a culture CD79a:GFP+ cells showed more expression in non–B-lineage system using a zebrafish stromal cell line, ZKS (65), that might cells than in the CD79b-GFP line at the larval stage. Under mi- facilitate such work together with cells from our CD79-GFP lines. croscopy, we also detected some tissue-restricted GFP at 24–48 h Developing a culture system would also facilitate direct testing of postfertilization in both CD79a-GFP and CD79b-GFP lines, with the proposed progression of B cell stages. neural expression by CD79a-GFP lines and muscle expression by In addition to enabling tracking of B cell immune responses and Downloaded from CD79b-GFP lines. An explanation for this nonlymphoid cell ex- a more precise examination of their development, the identification pression of GFP remains unclear. However, a previous report of B cell–specific regulatory elements may also provide an ap- described expression of CD79a with the ITAM domain by im- proach for the development of B cell cancer models. In contrast to mature myeloid cells in mouse bone marrow, and expression of mice, the development of B cell leukemia models in zebrafish has CD79a has been found in some cases of acute myeloid leukemia proven difficult to date, with one report of B cell acute lympho-

in human (57). Thus, there is a possibility of CD79 expression in blastic leukemia driven by a TEL-AML1 translocation fusion with http://www.jimmunol.org/ nonlymphoid cells at the larval stage, particularly CD79a. This an actin promoter, but not Rag (66). Both the CD79a-GFP and requires further larval analysis to confirm the presence of CD79- CD79b-GFP transgenic reporter lines drive high GFP expression, expressing nonlymphoid cells in early zebrafish development. so these are candidates for expressing oncogenes selectively in A major remaining question in teleost fish is whether the distinct B cells. In the future we expect that these lines and the regulatory early generated B cell development occurs before adult B cell de- elements defined in the course of this work will lead to the development. Although green+red+ (CD79+Rag2+) double-positive velopment of leukemic B cell models that may yield insights into cells were difficult to detect at the larval stage, our finding of their growth, with potential therapeutic implications. CD79lo IgZ B cells in adults supports the possibility that distinct B cell development may exist in early life. IgZ, also known as IgT in Acknowledgments by guest on September 28, 2021 teleost fish, has been known to be expressed in early B cells in We thank the staff from the Zebrafish Core Facility, as well as Dietmar Kappes mucosal immunity (37, 39, 41). Importantly, IgH sequencing of and Kerry Campbell for discussion and comments on the manuscript. zebrafish discovered that the expression of zebrafish (DJC)z precedes to (DJC)m, leading to early IgH-z transcript before IgH-m, Disclosures thereby generating IgZ B cells (37). This resembles TCRd expres- The authors have no financial conflicts of interest. sion preceding TCRa in T cell development. 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