PRUEBA TESTIMONIAL: PRIMER DÍA:

1. The Spleen is the Major Secondary Lymphoid Organ in Teleosts Primary lymphoid organs are the sites where T and B cell production occurs. Clusters of lymphoid cells can first be observed in lampreys (Zapata et al., 1981; Hagen et al., 1983) and in the Atlantic hagfish in the ‘‘central mass’’ of the pronephros (Zapata et al., 1984; Zapata and Amemiya, 2000), which is the site of bone marrow hematopoiesis in many lower vertebrates. However, there are no distinct secondary lymphoid organs in these primitive vertebrates (Zapata et al., 1996; Zapata and Amemiya, 2000). It is only with the advent of modern fishes that primary and secondary lymphoid organs are truly discernible. In chondrichthyes and osteichthyes B cells are produced in different organs that represent sites of hematopoiesis. These can range from the meninges to the gonads, but in most species involve the kidney (reviewed in Zapata and Amemiya, 2000), while T cells are always produced in the thymus (see above).

Secondary lymphoid organs in vertebrates serve as trapping and processing devices for antigen. It is here that antigen-presenting cells communicate with T cells, and where T cell–B cell interactions take place. The major secondary lymphoid organ consistently found in fishes is the spleen. The mammalian

spleen is compartmentalized into areas of erythroid predominance (red pulp) and lymphoid follicles (white pulp). At the border between red and white pulp is the marginal zone, where antigen trapping occurs and cells transit in and out of the white pulp. Upon exposure to antigen, B–T cell cooperation leads to activation of both cell types. Activated B cells along with the antigen-specific T helper cells migrate to the primary follicles of the spleen, where the B cells form distinct structures, the germinal centers. It is here that B cell repertoire selection occurs through isotype class switching and somatic hypermutation- mediated affinity maturation of the B cell receptor for antigen (BCR). In birds and rabbits gene conversion in secondary lymphoid organs is the major event that confers diversification to the Ig repertoire. There appears to be no direct correlation between germinal center formation and efficient affinity maturation. For example in birds, which form germinal centers during secondary immune response, affinity maturation is rather poor (Du Pasquier et al., 1998).

In contrast to higher vertebrates, the marginal zone separating the splenic white and red pulp is not fully developed in fishes. White pulp, consisting of lymphocytes, APCs and plasma cells are intermingled with red pulp (Zapata and Amemiya, 2000). Following antigen stimulation white pulp in teleosts increases in size. However, germinal centers are conspicuously absent in ectotherms, including fishes. Class switching is a process that has its earliest evolutionary roots in amphibians and does not occur in fishes. On the other hand, somatic hypermutation has been clearly demonstrated in lower vertebrates, including fishes. However, affinity maturation of immunoglobulins is inferior in fishes compared to higher vertebrates and the anatomic site where this process occurs in ectotherms is unknown.

The three processes affecting diversification of the immunoglobulin repertoire, somatic hypermutation, class switching and gene conversion (found in birds and rabbits) depends on the activity of a recently discovered enzyme, activation-induced cytidine deaminase (AID). The presence of this enzyme has been documented and its activity studied in mammals. Given the evidence for hypermutation of the shark heavy and in particular light chains (Lee et al., 2002b), AID has been presumed to be present in lower jawed vertebrates (see above). It is conceivable that the mechanism of hypermuta- tion evolutionarily precedes adaptive immunity (Flajnik, 2002). Verification of this hypothesis awaits cloning of the AID gene and study of its function in agnathan vertebrates.

The gut is the other anatomic site where lymphocytes are consistently found in fishes. Lymphoid aggregates are found in the lamina propria of the teleostian gut, but they are not encapsulated and hence do not represent true, isolated lymphoid organs. These aggregates represent mostly Ig positive

plasma cells (Rombout et al., 1993). DLT15 þ T cells were also found in the intestine of the sea bass (Dicentrarchus labrax) (Picchietti et al., 1997; Romano et al., 1997) and probably represent a majority of the Ig negative intraepithelial lymphocytes (Rombout et al., 1993).

2. Secondary Lymphoid Tissues in Zebrafish

As mentioned above, teleosts do not have lymph nodes. This makes the spleen the major secondary lymphoid organ that can be traced throughout vertebrate phylogeny (reviewed in Zapata and Amemiya, 2000). In mammals, spleen development has been shown to be critically dependent on the homeobox gene Hox-11 (Roberts et al., 1994). During zebrafish development, Tom Look’s group first identified the putative splenic primordium by WISH using the Hox-11/Tlx-1 probe (Langenau et al., 2002). Figure 5 shows developmental expression of Hox-11/Tlx-1 in wild-type zebrafish larvae. We first detected evidence of Hox-11 as an asymmetric focus of expression on the left anterior gut, in the region of the putative spleen primordium on day 4 pf. Over the next several days, signal intensity increased in this area. Cross- sections confirm the identity of this focus of expression with an organ on the left side of the larva in proximity to the gut (Fig. 5, T. Palomero, D. Langenau, A. Ferrando, J. Kanki and Tom Look, personal communication).

In adult zebrafish the spleen is a highly cellular organ. Danilova and Steiner found predominantly erythrocytes in the adult spleen and no significant expression of either Rag-1 or Ig (Danilova and Steiner, 2002). Figure 7 shows a FACS profile of cells populating the spleen and confirms the erythroid predominance of the teleostian spleen. However, there is a subpopulation of lymphocytes, which can be distinguished by light scatter characteristics (see Section IV.A). These cells presumably represent mature lymphocytes, which do not rearrange their receptors for antigen (TCR and BCR, respectively), explaining the absence of Rag-expression, which we also confirmed in Rag-2- GFP transgenics (Fig. 7C). Germinal centers are not found in teleost spleen and class switching does not occur due to the absence of isotypes other than IgM and an IgD equivalent (see Section III.C.1). However, evidence for somatic hypermutation is found in teleost B cells (reviewed in Flajnik, 2002). We have therefore conducted a search for a zebrafish homolog of AID. A stretch of sequence homology to human AID was found in the zebrafish database Sanger Center. Further examination of the genetic region surroun- ding the sequence homology revealed the entire ZF AID gene encompassing 5700 bp. Several lines of evidence support the identity of zebrafish AID as the true ortholog of human AID. There is a high degree of sequence homology between zebrafish AID with human (60% identity at the protein level) and mouse AID (64% identity at the protein level). Compared with human AID,

zebrafish AID has conserved intron-exon boundaries for exons 1–5. Expression studies are under way. We were able to amplify a correctly spliced AID transcript from day 2 embryos. This implies that zebrafish AID has a role beyond B cell antigen receptor maturation, as B cells are first detected in the zebrafish pancreas at 4 dpf (Danilova and Steiner, 2002) and

FIG. 5. Expression of Hox-11 in Zebrafish Larvae. A–D. Hox-11 expression in wild-type zebrafish. WISH analysis reveals Hox-11 in pharyngeal endoderm (asterisks) in side view on d4 (A) and 6 (C), brain in dorsal view on d6 (D), and in the left anterior gut (arrows). E. Cross-section of d4 wild-type zebrafish larva after WISH. Black arrowhead indicates Hox-11 expression on left side in proximity of gut (G). YC ¼ yolk cell, NC ¼ notochord, NT ¼ neural tube. (Cross-section courtesy of T. Palomero, D. Langenau, A. Ferrando, J. Kanki, T. Look.)

predicts that AID may also be found to be expressed in more primitive vertebrates, where it could fulfill a role in DNA metabolism.

The gut is a site of lymphoid activity in zebrafish, although no distinct secondary lymphoid structures, such as Peyer’s patches, can be identified histologically. Thus, Ig and Rag-1 positive cells were detected in the lamina propria of the straight part of the intestine (Danilova and Steiner, 2002) reminiscent of Rag-1 expression detected in the activated B cells that populate murine Peyer’s patch germinal centers (Han et al., 1996). Interestingly, Danilova and Steiner report that patches co-expressing Ig and Rag-1 in the zebrafish gut resembled Peyer’s patches, previously not reported in lower vertebrates (Danilova and Steiner, 2002).

IV. Phenotypic Characterization of Zebrafish Hematolymphoid Cells

Mutagenesis screens in zebrafish have led to the discovery of a wide array of mutants that fail to correctly develop embryonic blood cells. Due to the early time points analyzed, the vast majority of these mutants show defects in the maturation of primitive erythrocytes. As discussed above, definitive, multilineage hematopoiesis does not occur until several days post fertilization in larval development, making simple visualization of non- erythroid mutants difficult. Current screens aimed to uncover deficiencies in definitive hematopoiesis have thus relied upon in situ-based approaches for both the lymphoid (Trede and Zon, 1998; Schorpp et al., 2000; Trede et al., 2001) and myeloid lineages (Bennett et al., 2001; Lyons et al., 2001a,b; Lieschke et al., 2001, 2002). These screens have yielded mutants that fail to specify early larval lymphoid and myeloid populations. It remains to be determined whether these mutants also show defects in adult blood cell production in the kidney, the teleost equivalent of mammalian bone marrow. Additionally, since primitive erythrocytes are thought to derive from different populations of hematopoietic stem cells (HSCs) than their adult, definitive counterparts, it remains to be determined whether the embryonic blood mutants also show defects in producing adult red blood cells. To this end, we have undertaken a thorough characterization of the adult zebrafish hematopoietic system.