A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods

Developmental and Comparative Immunology 39 (2013) 103–109

Contents lists available at SciVerse ScienceDirect

Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci

Review

A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods Yi Sun a,⇑, Zhiguo Wei b, Ning Li a, Yaofeng Zhao a,c,⇑ a

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, National Engineering Laboratory for Animal Breeding, China Agricultural University, Beijing 100193, PR China b College of Animal Science and Technology, Henan University of Science and Technology, Henan 471003, PR China c Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, PR China

a r t i c l e

i n f o

Article history: Available online 23 February 2012 Keywords: Immunoglobulin gene Diversity Tetrapod

a b s t r a c t In the past several decades, immunoglobulin (Ig) genes have been extensively characterized in many tetrapod species. This review focuses on the expressed Ig isotypes and the diversity of Ig genes in mammals, birds, reptiles, and amphibians. With regard to heavy chains, five Ig isotypes – IgM, IgD, IgG, IgA, and IgE – have been reported in mammals. Among these isotypes, IgM, IgD, and IgA (or its analog, IgX) are also found in non-mammalian tetrapods. Birds, reptiles, and amphibians express IgY, which is considered the precursor of IgG and IgE. Some species have developed unique isotypes of Ig, such as IgO in the platypus, IgF in Xenopus, and IgY (DFc) in ducks and turtles. The j and k light chains are both utilized in tetrapods, but the usage frequencies of j and k chains differ greatly among species. The diversity of Ig genes depends on several factors, including the germline repertoire and recombinatorial and post-recombinatorial diversity, and different species have evolved distinct mechanisms to generate antibody diversity. Ó 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

4.

5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Placentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. The heavy- and light-chain isotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. The germline, recombinatorial, and post-recombinatorial diversity of the heavy and light chains . . . . . . . . . . . . . . . . . . . . . . . 2.2. Monotremes and marsupials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. The heavy- and light-chain isotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. The germline and recombinatorial diversity of the heavy and light chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The heavy- and light-chain isotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The germline, recombinatorial, and post-recombinatorial diversity of the heavy and light chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reptiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. The heavy- and light-chain isotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The germline and recombinatorial diversity of the heavy and light chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amphibians. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The heavy- and light-chain isotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. The germline and recombinatorial diversity of the heavy and light chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding authors. Tel.: +86 10 62731142x2012; fax: +86 10 62733904 (Y. Sun), tel.: +86 10 62734945; fax: +86 10 62733904 (Y. Zhao). E-mail addresses: [email protected] (Y. Sun), [email protected] (Y. Zhao). 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2012.02.008

104 104 104 104 104 105 105 105 105 105 105 106 106 106 106 106 107 107 107 107

104

Y. Sun et al. / Developmental and Comparative Immunology 39 (2013) 103–109

1. Introduction The genomic organization of the tetrapod IgH and IgL loci is in a ‘‘translocon’’ configuration, in which many tandemly duplicated variable (V) genes are localized in a chromatin domain followed by many similarly arranged diversity (D) genes (only for heavy chains) and joining (J) genes. Compared with the cluster configuration of the Ig gene locus in cartilaginous fish, this type of organization both allows for somatic rearrangement among different V, D, and J segments (known as VDJ recombination) to greatly increase combinatorial diversity and further expands this diversity after rearrangement by somatic gene conversion (GC), a mechanism that utilizes upstream V genes to unidirectionally modify a pre-rearranged V gene. In the tetrapod IgH loci, constant (C)-region genes, which encode various Ig isotypes with different effector functions, are also tandemly arranged downstream of the V, D, and J segments. This arrangement of the C-region genes facilitates the production of different IgH isotypes through a class switch recombination (CSR) process while maintaining the same antigen-binding specificity. Therefore, the tetrapod Ig genes have evolved to produce antibodies with highly diversified multiple effector functions. This review will analyze the features of Ig genes in four lineages of tetrapods: mammals, birds, reptiles, and amphibians, focusing on the multiple IgH and IgL isotypes and the mechanisms responsible for generating Ig diversity. 2. Mammals Mammals (Mammalia) are taxonomically divided into two subclasses: the Prototheria and the Theria. Prototheria contains only one living order, Monotremata (the monotremes), and the Theria consists of the infraclasses Metatheria (including marsupials) and Eutheria (the placentals). 2.1. Placentals 2.1.1. The heavy- and light-chain isotypes Most of our knowledge of the mammalian Ig genes is based on studies of placental mammals. The structure and/or diversity of IgH and/or IgL genes are described in many species belonging to at least nine orders. With the exception of rabbits, whose d gene is lost in the genomic IgH loci, most placental mammals express five classes of Igs: IgM, IgD, IgG, IgA, and IgE (Lanning et al., 2003; Ros et al., 2004). A remarkable feature of the placental IgH locus is the large copy number of Cc and Ca genes, which encode multiple IgG or IgA subclasses. For example, the number of Cc genes in the haploid genome of the elephant, horse, cattle, mouse, and human are eight, seven, three, four, and four, respectively (Guo et al., 2011; Schroeder, 2006; Wagner et al., 2004; Zhao et al., 2003), and the pig genome also possesses a large number of Cc genes (Butler et al., 2006). Moreover, the rabbit germline IgH locus contains 13 non-allelic Ca genes (Burnett et al., 1989). Different IgG subclasses have evolved to exert diverse effector functions by interacting with FccRs and activating the classical complement pathway, and this is well illustrated in the human and in the mouse (Burton and Woof, 1992; Nimmerjahn and Ravetch, 2008; Ravetch and Bolland, 2001). In addition, cattle seem to have a second IgH locus, including a l-like C-region sequence, on another chromosome (Hayes and Petit, 1993; Tobin-Janzen and Womack, 1992). To date, two light-chain types, k and j, have been found in placental mammals. Usually, the k locus consists of a few Jk–Ck clusters, and the j locus contains several Jj segments followed by one Cj gene. The rabbit genome possesses a duplicated Cj gene, Cj2, which is normally expressed at a low level (Heidmann and Rougeon, 1983; Hole et al., 1991).

2.1.2. The germline, recombinatorial, and post-recombinatorial diversity of the heavy and light chains A large number of germline and rearranged VH and VL genes, as well as cDNAs, have been identified in various placental mammals. According to these sequences, VH elements of placental mammals are classified into three clusters: mammalian clans I, II, and III (Ota and Nei, 1994; Sitnikova and Su, 1998). The germline and rearranged VH repertoires of the mouse and human are diverse. The mouse germline VH repertoire contains nearly 200 VH segments (113 potentially functional, 79 pseudogenes, and six ORF VH segments for C57BL/6), which are grouped into 16 subgroups. Among the VH segments, at least 39 VH segments from nine subgroups participate in rearrangement (Lefranc, 2003). In the human IgH locus, 38–44 potentially functional VH segments, most of which can be rearranged and expressed, belong to seven subgroups (Lefranc, 2003). In addition, the VH repertoires expressed in the mouse and human are distributed across all three mammalian VH clans (Lefranc, 2003; Schroeder et al., 1990). However, a preference for a single VH subgroup in the expressed VH repertoire is demonstrated in the rabbit (only VH1 is preferentially utilized), dog, horse, and several artiodactyl lineages, although a few recent studies indicate that the germline VH repertoires of the sheep and horse are more complex and diverse than previously thought. All of these preferred VH segments belong to either clan II (including the horse, cattle, and sheep) or clan III (including the rabbit, dog, and pig) (Almagro et al., 2006; Bao et al., 2010; Becker and Knight, 1990; Berens et al., 1997; Charlton et al., 2000; Dufour et al., 1996; Sun et al., 1994, 2010). Mammalian VL genes are grouped into six clans – five containing Vk sequences and one exclusively containing Vj genes – which are further divided into 10 subgroups (Sitnikova and Nei, 1998; Sitnikova and Su, 1998). Light chain usage in placental mammals has two characteristics. First, in most species, the overall complexity of the germline Vk and Vj repertoire seems to correlate with the preferential use of one light-chain isotype over another (Almagro et al., 1998). Obvious examples include the human and the mouse. The former has numerous and diverse Vk and Vj segments and uses both extensively (j:k, 60%:40%), and the latter has many Vj but only three functional Vk segments; as a result, it primarily uses Vj (j:k, 95%:5%) (Almagro et al., 1998). This rule may also partially explain the predominance of the k isotype in the horse and the j isotype in the rabbit (Sun et al., 2010 and our unpublished data). Second, the placental mammals that express relatively restricted VH repertoires also seem to express limited VL repertoires. Horse and cattle, two predominant lambda chain usage species, mainly utilize a single Vk subgroup in their combinatorial repertoires (Saini et al., 2003; Sun et al., 2010). Pigs show nearly equal usage of j and k light chains, and their pre-immune VL repertoire prefers only one Vj and two Vk subgroups (Butler et al., 2006, 2004). The rabbit may be one exception. Rabbits predominantly use the j light chain type, and most of their Vj genes belong to one subgroup with at least 80% identity (Ros et al., 2005; Sehgal et al., 1999). However, the Vj–Jj junctions seem to be diverse mainly because of the heterogeneity of the CDR3 length (Sehgal et al., 1999). In some cases, the diversity of the IgL chain is not necessary for the overall diversity of the antibody in camelids (camels and llamas), which express unique classes of IgG (IgG2 and IgG3) that lack light chains entirely (De Genst et al., 2006). With regard to the mammals in which V(D)J recombinatorial diversity is limited, two post-rearrangement somatic strategies, somatic hypermutation (SHM) and/or GC, contribute to the generation of a diverse primary antibody repertoire. Unlike GC, SHM can introduce template-independent point mutations into the V regions of Ig genes. In the rabbit, sheep, and cattle and likely in the horse and pig, the gut-associated lymphoid tissue (GALT), especially the appendix in the rabbit and the ileal Peyer’s patch

Y. Sun et al. / Developmental and Comparative Immunology 39 (2013) 103–109

in artiodactyls, provides critical sites for the somatic antibody diversification process in a primary immune response (Lucier et al., 1998; Reynaud et al., 1991b; Vajdy et al., 1998; Weinstein et al., 1994). Additionally, SHM and GC occur during specific antigen-dependent immune responses within rabbit germinal centers (Schiaffella et al., 1999; Winstead et al., 1999). 2.2. Monotremes and marsupials 2.2.1. The heavy- and light-chain isotypes Monotremes branched from the mammalian lineage at least 166 million years ago (Bininda-Emonds et al., 2007). Monotremes are considered primitive mammals because, like reptiles and birds, they lay eggs and have a single cloaca. Like other mammals, however, monotremes produce milk through mammary glands to nourish their young. Modern monotremes include only five living species: the platypus and four echidna species. Although studies from 2002 to 2004 confirm that the monotremes express typical mammalian IgM, IgG, IgA, and IgE (Belov and Hellman, 2003; Belov et al., 2002a–c; Vernersson et al., 2004, 2002), Zhao et al. (2009) have identified two additional isotypes, IgO and IgD, in the duckbilled platypus (Ornithorhynchus anatinus). The o gene (encoding IgO) contains four Ig CH domains and a hinge, and it appears to be a structural intermediate between the t and c genes. The d gene encodes 10 Ig CH domains and no hinge. This structure is similar to the d gene in reptiles, amphibians and fish but is strikingly different from that in eutherian mammals. The Ig heavy-chain constant gene locus of the platypus contains eight Ig-encoding genes, which are arranged in a ldoc2c1a1ea2 order and encode six distinct isotypes (Zhao et al., 2009). Marsupials and placental mammals split from the monotremes approximately 147 million years ago (Bininda-Emonds et al., 2007). Marsupials are characterized by giving birth to relatively undeveloped young, and 258 surviving species are classified into seven orders (Bininda-Emonds et al., 2007). The gray, short-tailed opossum (Monodelphis domestica) is a model species for marsupials. From analyses of the whole genomic sequence of the opossum, single and functional l, c, a, and e genes were identified, but the d gene is absent (Aveskogh and Hellman, 1998; Aveskogh et al., 1999; Wang et al., 2009). The j and k light-chain loci of the known monotremes and marsupials are highly conserved with those of placentals (Johansson et al., 2005; Nowak et al., 2004; Wang et al., 2009). 2.2.2. The germline and recombinatorial diversity of the heavy and light chains All platypus VH sequences analyzed so far likely belong to several closely related V-gene subgroups, which form a separate branch within clan III of mammalian V gene sequences in a distance tree. However, the long and highly diversified CDR3 region increases the V(D)J recombination diversity (Johansson et al., 2002). In contrast to the platypus, the short-beaked echidna (Tachyglossus aculeatus) actively uses at least four germline VH subgroups belonging to all three mammalian VH clans (Belov and Hellman, 2003). In both the platypus and the short-beaked echidna, the k light chain accounts for more than 90% of the light-chain diversity, which is observed in both the sequence and in the length of all three CDRs (Johansson et al., 2005). However, like the VH sequences of platypus, all expressed monotreme Vk sequences appear to belong to only two Vk gene subgroups, which form a separate branch on a distance tree (Johansson et al., 2005). Compared to Vk, the expressed Vj repertoires appear to include at least four and nine Vj subgroups in the platypus and echidna, respectively, although the j light chain is less frequently used in both animals (Nowak et al., 2004). This phenomenon indicates that in monotremes, a large diversity in the CDRs contributes more to the whole repertoire size than the total number of V-gene subgroups (Johansson et al., 2005).

105

Like the platypus, the expressed VH repertoire of the opossum is derived from only two VH subgroups, both of which are related to clan III (Miller et al., 1998). In contrast to the VH families, the opossum-expressed Vk and Vj segments are more diverse, and both types are phylogenetically dispersed into distinct VL subgroups in placental mammals (Lucero et al., 1998; Miller et al., 1999; Wang et al., 2009). Another distantly related marsupial, the Australian brush-tail possum (Trichosurus vulpecula), also has a limited VH repertoire and a diverse VL repertoire (Baker et al., 2005). The similar pattern of Ig V complexity in these two marsupials suggests that the generation of an effective Ig repertoire does not always depend primarily on the H-chain diversity (Baker et al., 2005). 3. Birds 3.1. The heavy- and light-chain isotypes Birds are second only to mammals as the most highly evolved vertebrates, and their Ig genes exhibit several unusual characteristics in their genomic organization and forms of expression. Three Ig isotypes – IgM, IgY and IgA – have been identified in the chicken, turkey, pheasant, quail (Galliformes), and duck (Anseriformes) (Choi et al., 2010; Dahan et al., 1983; Magor et al., 1994b, 1998; Mansikka, 1992; Parvari et al., 1988), and the arrangement of the three C genes in both the chicken and duck IgH loci is in the order lat, where the a gene is inserted in the middle of the locus with an inverted transcriptional orientation to the l and t genes. There is no evidence indicating the presence of a d gene in either the chicken or duck genome (Lundqvist et al., 2001; Zhao et al., 2000). IgY is the major low-molecular-weight antibody identified in non-mammalian tetrapods and is considered to be the progenitor of IgG and IgE both in structure and function (Warr et al., 1995). The chicken, ostrich (Struthio camelus, Struthioniformes) and pigeon (Columbiformes) express only the full-length form of IgY, with four CH domains (Parvari et al., 1988 and our unpublished data), and the duck expresses an additional truncated form of IgY, IgY(DFc), which lacks the CH3 and CH4 domains, causing it to lose its secondary effector functions, such as antigen internalization, opsonization and complement fixation (Magor et al., 1994b). The expression ratio of IgY(DFc) to IgY increases in ducks when they are repeatedly immunized with one antigen (Grey, 1967; Humphrey et al., 2004). Humphrey et al. (2004) found that the phagocytosis of the immune complexes (IC) by duck peripheral blood monocytes dramatically declined once a low percentage of IgY(DFc) appeared in the IC, which was accompanied by an increased IgY(DFc) concentration and a decreased mRNA expression of splenic IL-1b (a pro-inflammatory factor), but hepatic IL-1b showed a biphasic response of decreasing first and then increasing (Humphrey et al., 2004). This suggests that the duck IgY(DFc) might play a role as an immunomodulator during the progression of an infection from acute to chronic phase (Humphrey et al., 2004). IgY(DFc) might perform a similar function to protect the antigen-presenting cells from influenza virus infection, although there is no clear evidence that IgY(DFc) participates in influenza defense (Magor, 2011). Another unusual characteristic of bird Ig genes is that only a single k light-chain locus has been found in some birds, including the chicken, duck, zebra finch (Passeriformes) and ostrich (Das et al., 2010; Magor et al., 1994a; Ratcliffe, 2006 and our unpublished data). 3.2. The germline, recombinatorial, and post-recombinatorial diversity of the heavy and light chains At the chicken IgH and Igk loci, there is a unique functional VH (or Vk) gene that can rearrange to form a unique JH (or Jk) segment.

106

Y. Sun et al. / Developmental and Comparative Immunology 39 (2013) 103–109

The remaining VH (or Vk) genes, which are located upstream of the functional VH gene, are all pseudogenes (wVH or wVk, most of which lack the functional RSS and/or leader sequence (Ratcliffe, 2006; Reynaud et al., 1987, 1989). A similar organization of the Igk locus has been verified in the duck and zebra finch genomes (Das et al., 2010; Magor et al., 1994a). Moreover, the Igk loci of the quail, turkey, pigeon, cormorant (Pelecaniformes), hawk (Falconiformes), and mallard duck are also likely to contain only one functional Vk gene segment. Muscovy duck, however, is an exception, containing at least two potentially functional Vk genes in the Igk locus (McCormack et al., 1989). Compared to the mouse and the human, an extremely limited V(D)J recombination diversity has been found in birds B cells. In the IgH locus of the chicken, a restricted rearrangement diversity is produced not only because of the unique V and J segments (Reynaud et al., 1989), but also because of the limited sequence variation among the 15 of 16 germline DH segments and the lack of N nucleotide insertions at either the DJ or VD junctions (Reynaud et al., 1991a). The further diversification of rearranged Ig genes depends on the somatic GC mechanism in the B cells that colonize the embryonic bursa of Fabricius, which provides the optimal microenvironment for B cell development (Reynaud et al., 1989). Serving as sequence donors, the intrachromosomal wVk (or wVH gene segments unidirectionally exchange part of their sequences with the rearranged functional VJ (or VDJ genes to greatly diversify the latter (Carlson et al., 1990). The usage preference of the pseudogenes depends on the distance, sequence similarity and relative orientation between the pseudo-Vk and the rearranged Vk1 segments (McCormack and Thompson, 1990). This has been thoroughly reviewed by McCormack et al. (1991). In chickens, somatic gene conversion continues generating the primary repertoire until several weeks after hatching, and continues to cooperate with the somatic hypermutation in the splenic germinal centers during secondary immune responses (Arakawa et al., 1996; Carlson et al., 1990; Reynaud et al., 1987, 1989).

4. Reptiles 4.1. The heavy- and light-chain isotypes The living reptiles have been classified into three main lineages: turtles, archosaurs, and lepidosaurs. Archosaurs include crocodilians and the progenitors of birds, whereas the living lepidosaurs include snakes, lizards, and tuataras (George et al., 2001). Each of the reptile lineages has its own unique characteristics and has evolved via different pathways (George et al., 2001). To date, the genomic organization of the heavy and light loci has only been characterized in one reptile, the green anole lizard (Anolis carolinensis), which expresses three heavy-chain isotypes – IgM, IgD and IgY – and two light chain isotypes –j and k (Wei et al., 2009; Wu et al., 2010). The germline d sequences of both the lizard and the leopard gecko (Eublepharis macularius) maintain multiple domains, as in primitive vertebrates, but in the lizard, only the first four of 11 Cd exons have been detected in the mRNA of the transmembrane form of IgD, which is even shorter in mammals (Gambon-Deza and Espinel, 2008; Wei et al., 2009). No IgA-encoded gene has been found in any lizard genome or cDNA library, but an IgA-like gene has been cloned from the leopard gecko. Most importantly, this four-CHs IgA gene, together with the Xenopus IgX and duck IgA, is likely a chimeric gene produced by a recombination between IgM and IgY, with CH1–CH2 and CH3–CH4 similar to IgY and IgM, respectively (Deza et al., 2007; Wei et al., 2009). Our unpublished data also suggest that the presence or absence of IgA is quite different among the turtle, snake, and crocodile. Similar to the duck, the IgY(DFc) is also found in lizards and some tur-

tles, but it seems to be absent in other reptile lineages (Benedict and Pollard, 1972; Leslie and Clem, 1972; Wei et al., 2009 and our unpublished data). Despite the absence of DNA or RNA sequences, the presence of IgY(DFc)-like antibodies has been reported in an amphibian, the bullfrog (Rana catesbeiana), and even in an African lungfish species, Protopterus annectens (Fellah et al., 1993; Marchalonis and Edelman, 1966). 4.2. The germline and recombinatorial diversity of the heavy and light chains The germline V gene repertoire and patterns of diversification have not been extensively studied in reptiles. Only a few germline VH segments have been reported from the caiman (Caiman crocodylus crocodylus) and turtle (Litman et al., 1985; Turchin and Hsu, 1996). According to our unpublished data, the turtle, snake, and crocodile express many VH segments from multiple subgroups, some of which are clustered with VHs from bony and cartilaginous fish. In the lizard, multiple Vj and Vk segments are identified in both the germline and expressed Vj and Vk repertoires. Moreover, microhomology-dependent V–J junctions (lacking N and P nucleotides) are common in both expressed j and k chains, suggesting that the combinatorial joining but not the junctional flexibility contributes largely to Ig diversity (Wu et al., 2010). 5. Amphibians 5.1. The heavy- and light-chain isotypes Xenopus, also called clawed frogs, are among the best models for studying the evolution of the immune system and immune genes in cold-blooded vertebrates (Du Pasquier et al., 2000; Robert and Ohta, 2009). As in the mammals, birds, and reptiles, the IgH locus of X. tropicalis is organized as a typical translocon configuration in the order of 50 VH–DH–JH–Cl–Cd–Cv–Ct–Cu 30 (Zhao et al., 2006). The cDNA sequences of IgM, IgY, and IgX were first cloned in X. laevis and then in two urodeles, the Mexican axolotl (Ambystoma mexicanum) and the Iberian ribbed newt (Pleurodeles waltl) (not including IgX) (Amemiya et al., 1989; Fellah et al., 1993, 1992b; Haire et al., 1989; Schaerlinger et al., 2008; Schaerlinger and Frippiat, 2008; Schwager et al., 1988). The IgX in both Xenopus and the axolotl is composed of four CH domains and is mainly expressed as a multimer in mucosal tissues. It is now considered that IgX is orthologous to IgA, although the structure of IgX is quite different from mammalian IgA but similar to birds IgA (Haire et al., 1989; Mansikka, 1992; Mussmann et al., 1996; Schaerlinger and Frippiat, 2008). The four-domain structure of IgY is similar in these three amphibian taxa, reptiles and birds. In the axolotl, the expression profile of IgY varies during development, progressively changing from being expressed in the mucosa of the digestive tract of the young to the serum of adults. In contrast to the axolotl, after metamorphosis, the expression level of IgY in P. waltl quickly increases in the intestine and is maintained at a high level during adulthood, and this expression pattern of IgY seems to compensate for the lack of IgA in P. waltl (Fellah et al., 1992a; Schaerlinger et al., 2008). The IgF of X. tropicalis, encoded by Cu, is the only IgH isotype known to contain a separately encoded genetic hinge in a nonmammalian vertebrate, and this discovery invalidated the proposition that the hinge region was independently generated in Cd, Cc, and Ca after the mammalian radiation. Further analysis of the nucleotide sequence of IgF hinge exon supported the splice-siteincorporation hypothesis of hinge formation (Zhao et al., 2006). Moreover, IgF has only two CH domains, which are homologous to the first and fourth constant domains of IgY, suggesting that the Cu gene might have evolved from the Ct gene (Zhao et al.,

Y. Sun et al. / Developmental and Comparative Immunology 39 (2013) 103–109

2006). IgD has been found in Xenopus and P. waltl, in which it is called IgP. The genomic organization and expressed mRNA of Xenopus IgD contain multiple exons, similar to its counterparts in reptiles and fishes, and the expressed IgP contains four domains (Ohta and Flajnik, 2006; Schaerlinger et al., 2008; Zhao et al., 2006). Three light-chain gene isotypes –q, r, and type III – and their genomic organization have been characterized in X. tropicalis (Qin et al., 2008). The q and type III are orthologous isotypes to the j and k in higher tetrapods, and the r is a more ancient isotype in the lower vertebrates (Qin et al., 2008). 5.2. The germline and recombinatorial diversity of the heavy and light chains Eleven known VH subgroups containing more than 80 VH genes, at least 10 DH and 8 to 9 JH segments are characterized in the IgH loci of both X. laevis and X. tropicalis (Haire et al., 1990; Hsu et al., 1989; Schwager et al., 1991a). A large number of IgH cDNA and DNA clones obtained from different-stage tadpoles and adults have been analyzed in Xenopus, and the results indicate that most of the VH subgroups (10 of 11) and two main JH elements (JH3 and JH5) are similarly used, but that the diversification of CDR3 is significantly different between adults and tadpoles (Schwager et al., 1991a). The diversification of DH usage (including inversion, fusion and reading in different reading frames) and the addition of N nucleotides are common in adults, but tadpoles strongly favor only a few DH elements, which are read in exclusively one frame and lack N diversity (Schwager et al., 1991a). As observed in Xenopus, the axolotl has a relatively large number of VH segments within 11 subgroups, and its most frequently expressed VH subgroups are VH7, VH8, and VH9 (Golub and Charlemagne, 1998). Moreover, the active selection of the productively rearranged IgH chains occurs during the ontogeny of the axolotl, with the percentage of abortive rearrangement decreasing from 25% to almost zero, but this does not appear to occur in Xenopus (with approximately 50% abortive rearrangement in both tadpoles and adults) (Golub et al., 1997; Schwager et al., 1991a). Based on the genomic sequence of X. tropicalis, at least 11, 8, and 17 potentially functional V gene segments and 9, 4, and 7 J gene segments were identified in the q, r, and type III loci, respectively (Qin et al., 2008). Differences in the expression proportions of the three IgL isotypes and their associations with heavy-chain isotypes have not been documented in Xenopus. On the basis of the current evidence, the recombination diversity of the type III (k) light chain is greater than that of the q and r light chains in Xenopus (Haire et al., 1996; Schwager et al., 1991b; Stewart et al., 1993). Finally, most (if not all) of the expressed light chains in the axolotl are also of the k type (Andre et al., 2000). 6. Summary In this paper, we have reviewed the literature on the tetrapod Ig genes and their diversity that has been published in the past several decades, and it is clear that there are still many unanswered questions. Whereas some new Ig isotypes have been continually identified in different tetrapod lineages, such as IgO in the platypus, IgF in Xenopus and IgY(DFc) in the duck and turtle, relatively little is known about the immunological functions of these isotypes. Moreover, the mechanisms used to generate Ig diversity in many tetrapod species have not been characterized. The reasons for this include a lack of proper reagents and, more importantly, an insufficient understanding of non-mammalian (more accurately, non-human and non-mouse) immunity at the physiological, cellular and molecular levels. It will be necessary to comprehensively study the immune systems of several tetrapod species,

107

particularly those located near the branching points of evolutionary trees. Multiple completed, ongoing, and upcoming tetrapod genomic sequences will facilitate the characterization of their complex Ig gene loci as well as their immune-related genes. Acknowledgements This work was supported by the National Science Fund for Distinguished Young Scholars (30725029), the National Basic Research Program of China (973 Program-2010CB945300), and the Taishan Scholar Foundation of Shandong Province. References Almagro, J.C., Hernandez, I., Ramirez, M.C., Vargas-Madrazo, E., 1998. Structural differences between the repertoires of mouse and human germline genes and their evolutionary implications. Immunogenetics 47, 355–363. Almagro, J.C., Martinez, L., Smith, S.L., Alagon, A., Estevez, J., Paniagua, J., 2006. Analysis of the horse V(H) repertoire and comparison with the human IGHV germline genes, and sheep, cattle and pig V(H) sequences. Mol. Immunol. 43, 1836–1845. Amemiya, C.T., Haire, R.N., Litman, G.W., 1989. Nucleotide sequence of a cDNA encoding a third distinct Xenopus immunoglobulin heavy chain isotype. Nucleic Acids Res. 17, 5388. Andre, S., Guillet, F., Charlemagne, J., Fellah, J.S., 2000. Structure and diversity of Mexican axolotl lambda light chains. Immunogenetics 52, 137–144. Arakawa, H., Furusawa, S., Ekino, S., Yamagishi, H., 1996. Immunoglobulin gene hyperconversion ongoing in chicken splenic germinal centers. EMBO J. 15, 2540–2546. Aveskogh, M., Hellman, L., 1998. Evidence for an early appearance of modern postswitch isotypes in mammalian evolution; cloning of IgE, IgG and IgA from the marsupial Monodelphis domestica. Eur. J. Immunol. 28, 2738–2750. Aveskogh, M., Pilstrom, L., Hellman, L., 1999. Cloning and structural analysis of IgM (mu chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica. Dev. Comp. Immunol. 23, 597–606. Baker, M.L., Belov, K., Miller, R.D., 2005. Unusually similar patterns of antibody V segment diversity in distantly related marsupials. J. Immunol. 174, 5665–5671. Bao, Y., Guo, Y., Xiao, S., Zhao, Z., 2010. Molecular characterization of the VH repertoire in Canis familiaris. Vet. Immunol. Immunopathol. 137, 64–75. Becker, R.S., Knight, K.L., 1990. Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits. Cell 63, 987– 997. Belov, K., Hellman, L., 2003. Immunoglobulin genetics of Ornithorhynchus anatinus (platypus) and Tachyglossus aculeatus (short-beaked echidna). Comp. Biochem. Physiol. Part A: Mol. Integr. Physiol. 136, 811–819. Belov, K., Hellman, L., Cooper, D.W., 2002a. Characterisation of echidna IgM provides insights into the time of divergence of extant mammals. Dev. Comp. Immunol. 26, 831–839. Belov, K., Hellman, L., Cooper, D.W., 2002b. Characterization of immunoglobulin gamma 1 from a monotreme, Tachyglossus aculeatus. Immunogenetics 53, 1065–1071. Belov, K., Zenger, K.R., Hellman, L., Cooper, D.W., 2002c. Echidna IgA supports mammalian unity and traditional Therian relationship. Mamm. Genome 13, 656–663. Benedict, A.A., Pollard, L.W., 1972. Three classes of immunoglobulins found in the sea turtle, Chelonia mydas. Folia Microbiol. (Praha) 17, 75–78. Berens, S.J., Wylie, D.E., Lopez, O.J., 1997. Use of a single VH family and long CDR3s in the variable region of cattle Ig heavy chains. Int. Immunol. 9, 189–199. Bininda-Emonds, O.R., Cardillo, M., Jones, K.E., MacPhee, R.D., Beck, R.M., Grenyer, R., Price, S.A., Vos, R.A., Gittleman, J.L., Purvis, A., 2007. The delayed rise of presentday mammals. Nature 446, 507–512. Burnett, R.C., Hanly, W.C., Zhai, S.K., Knight, K.L., 1989. The IgA heavy-chain gene family in rabbit: cloning and sequence analysis of 13 C alpha genes. EMBO J. 8, 4041–4047. Burton, D.R., Woof, J.M., 1992. Human antibody effector function. Adv. Immunol. 51, 1–84. Butler, J.E., Sun, J., Wertz, N., Sinkora, M., 2006. Antibody repertoire development in swine. Dev. Comp. Immunol. 30, 199–221. Butler, J.E., Wertz, N., Sun, J., Wang, H., Chardon, P., Piumi, F., Wells, K., 2004. Antibody repertoire development in fetal and neonatal pigs. VII. Characterization of the preimmune kappa light chain repertoire. J. Immunol. 173, 6794–6805. Carlson, L.M., McCormack, W.T., Postema, C.E., Humphries, E.H., Thompson, C.B., 1990. Templated insertions in the rearranged chicken IgL V gene segment arise by intrachromosomal gene conversion. Genes Dev. 4, 536–547. Charlton, K.A., Moyle, S., Porter, A.J., Harris, W.J., 2000. Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display library. J. Immunol. 164, 6221–6229. Choi, J.W., Kim, J.K., Seo, H.W., Cho, B.W., Song, G., Han, J.Y., 2010. Molecular cloning and comparative analysis of immunoglobulin heavy chain genes from Phasianus colchicus, Meleagris gallopavo, and Coturnix japonica. Vet. Immunol. Immunopathol. 136, 248–256.

108

Y. Sun et al. / Developmental and Comparative Immunology 39 (2013) 103–109

Dahan, A., Reynaud, C.A., Weill, J.C., 1983. Nucleotide sequence of the constant region of a chicken mu heavy chain immunoglobulin mRNA. Nucleic Acids Res. 11, 5381–5389. Das, S., Mohamedy, U., Hirano, M., Nei, M., Nikolaidis, N., 2010. Analysis of the immunoglobulin light chain genes in zebra finch: evolutionary implications. Mol. Biol. Evol. 27, 113–120. De Genst, E., Saerens, D., Muyldermans, S., Conrath, K., 2006. Antibody repertoire development in camelids. Dev. Comp. Immunol. 30, 187–198. Deza, F.G., Espinel, C.S., Beneitez, J.V., 2007. A novel IgA-like immunoglobulin in the reptile Eublepharis macularius. Dev. Comp. Immunol. 31, 596–605. Du Pasquier, L., Robert, J., Courtet, M., Mussmann, R., 2000. B-cell development in the amphibian Xenopus. Immunol. Rev. 175, 201–213. Dufour, V., Malinge, S., Nau, F., 1996. The sheep Ig variable region repertoire consists of a single VH family. J. Immunol. 156, 2163–2170. Fellah, J.S., Iscaki, S., Vaerman, J.P., Charlemagne, J., 1992a. Transient developmental expression of IgY and secretory component like protein in the gut of the axolotl (Ambystoma mexicanum). Dev. Immunol. 2, 181–190. Fellah, J.S., Kerfourn, F., Wiles, M.V., Schwager, J., Charlemagne, J., 1993. Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum upsilon chain deduced from cDNA sequence. Immunogenetics 38, 311–317. Fellah, J.S., Wiles, M.V., Charlemagne, J., Schwager, J., 1992b. Evolution of vertebrate IgM: complete amino acid sequence of the constant region of Ambystoma mexicanum mu chain deduced from cDNA sequence. Eur. J. Immunol. 22, 2595– 2601. Gambon-Deza, F., Espinel, C.S., 2008. IgD in the reptile leopard gecko. Mol. Immunol. 45, 3470–3476. George, R.Z., Janalee, P., Laurie, J.V., 2001. Herpetology: an introductory biology of amphibians and reptiles, second ed. Academic Press, San Diego [u.a.], p. 630. Golub, R., Charlemagne, J., 1998. Structure, diversity, and repertoire of VH families in the Mexican axolotl. J. Immunol. 160, 1233–1239. Golub, R., Fellah, J.S., Charlemagne, J., 1997. Structure and diversity of the heavy chain VDJ junctions in the developing Mexican axolotl. Immunogenetics 46, 402–409. Grey, H.M., 1967. Duck immunoglobulins. II. Biologic and immunochemical studies.. J. Immunol. 98, 820–826. Guo, Y., Bao, Y., Wang, H., Hu, X., Zhao, Z., Li, N., Zhao, Y., 2011. A preliminary analysis of the immunoglobulin genes in the African elephant (Loxodonta africana). PLoS One 6, e16889. Haire, R.N., Amemiya, C.T., Suzuki, D., Litman, G.W., 1990. Eleven distinct VH gene families and additional patterns of sequence variation suggest a high degree of immunoglobulin gene complexity in a lower vertebrate, Xenopus laevis. J. Exp. Med. 171, 1721–1737. Haire, R.N., Ota, T., Rast, J.P., Litman, R.T., Chan, F.Y., Zon, L.I., Litman, G.W., 1996. A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes. J. Immunol. 157, 1544– 1550. Haire, R.N., Shamblott, M.J., Amemiya, C.T., Litman, G.W., 1989. A second Xenopus immunoglobulin heavy chain constant region isotype gene. Nucleic Acids Res. 17, 1776. Hayes, H.C., Petit, E.J., 1993. Mapping of the beta-lactoglobulin gene and of an immunoglobulin M heavy chain-like sequence to homoeologous cattle, sheep, and goat chromosomes. Mamm. Genome 4, 207–210. Heidmann, O., Rougeon, F., 1983. Multiplicity of constant kappa light chain genes in the rabbit genome: a b4b4 homozygous rabbit contains a kappa-bas gene. EMBO J. 2, 437–441. Hole, N.J., Young-Cooper, G.O., Mage, R.G., 1991. Mapping of the duplicated rabbit immunoglobulin kappa light chain locus. Eur. J. Immunol. 21, 403–409. Hsu, E., Schwager, J., Alt, F.W., 1989. Evolution of immunoglobulin genes: VH families in the amphibian Xenopus. Proc. Natl. Acad. Sci. USA 86, 8010– 8014. Humphrey, B.D., Calvert, C.C., Klasing, K.C., 2004. The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos). Dev. Comp. Immunol. 28, 665–672. Johansson, J., Aveskogh, M., Munday, B., Hellman, L., 2002. Heavy chain V region diversity in the duck-billed platypus (Ornithorhynchus anatinus): long and highly variable complementarity-determining region 3 compensates for limited germline diversity. J. Immunol. 168, 5155–5162. Johansson, J., Salazar, J.N., Aveskogh, M., Munday, B., Miller, R.D., Hellman, L., 2005. High variability in complementarity-determining regions compensates for a low number of V gene families in the lambda light chain locus of the platypus. Eur. J. Immunol. 35, 3008–3019. Lanning, D.K., Zhai, S.K., Knight, K.L., 2003. Analysis of the 30 Cmu region of the rabbit Ig heavy chain locus. Gene 309, 135–144. Lefranc, M.P., 2003. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 31, 307–310. Leslie, G.A., Clem, L.W., 1972. Phylogeny of immunoglobulin structure and function. VI. 17S, 7.5S and 5.7S anti-DNP of the turtle, Pseudamys scripta. J. Immunol. 108, 1656–1664. Litman, G.W., Murphy, K., Berger, L., Litman, R., Hinds, K., Erickson, B.W., 1985. Complete nucleotide sequences of three VH genes in Caiman, a phylogenetically ancient reptile: evolutionary diversification in coding segments and variation in the structure and organization of recombination elements. Proc. Natl. Acad. Sci. USA 82, 844–848.

Lucero, J.E., Rosenberg, G.H., Miller, R.D., 1998. Marsupial light chains: complexity and conservation of lambda in the opossum Monodelphis domestica. J. Immunol. 161, 6724–6732. Lucier, M.R., Thompson, R.E., Waire, J., Lin, A.W., Osborne, B.A., Goldsby, R.A., 1998. Multiple sites of V lambda diversification in cattle. J. Immunol. 161, 5438–5444. Lundqvist, M.L., Middleton, D.L., Hazard, S., Warr, G.W., 2001. The immunoglobulin heavy chain locus of the duck. Genomic organization and expression of D, J, and C region genes. J. Biol. Chem. 276, 46729–46736. Magor, K.E., 2011. Immunoglobulin genetics and antibody responses to influenza in ducks. Dev. Comp. Immunol. 35, 1008–1016. Magor, K.E., Higgins, D.A., Middleton, D.L., Warr, G.W., 1994a. CDNA sequence and organization of the immunoglobulin light chain gene of the duck, Anas platyrhynchos. Dev. Comp. Immunol. 18, 523–531. Magor, K.E., Higgins, D.A., Middleton, D.L., Warr, G.W., 1994b. One gene encodes the heavy chains for three different forms of IgY in the duck. J. Immunol. 153, 5549– 5555. Magor, K.E., Warr, G.W., Bando, Y., Middleton, D.L., Higgins, D.A., 1998. Secretory immune system of the duck (Anas platyrhynchos). Identification and expression of the genes encoding IgA and IgM heavy chains. Eur. J. Immunol. 28, 1063– 1068. Mansikka, A., 1992. Chicken IgA H chains. Implications concerning the evolution of H chain genes. J. Immunol. 149, 855–861. Marchalonis, J., Edelman, G.M., 1966. Phylogenetic origins of antibody structure. II. Immunoglobulins in the primary immune response of the bullfrog, Rana catesbiana. J. Exp. Med. 124, 901–913. McCormack, W.T., Carlson, L.M., Tjoelker, L.W., Thompson, C.B., 1989. Evolutionary comparison of the avian IgL locus: combinatorial diversity plays a role in the generation of the antibody repertoire in some avian species. Int. Immunol. 1, 332–341. McCormack, W.T., Thompson, C.B., 1990. Chicken IgL variable region gene conversions display pseudogene donor preference and 50 to 30 polarity. Genes Dev. 4, 548–558. McCormack, W.T., Tjoelker, L.W., Thompson, C.B., 1991. Avian B-cell development: generation of an immunoglobulin repertoire by gene conversion. Annu. Rev. Immunol. 9, 219–241. Miller, R.D., Bergemann, E.R., Rosenberg, G.H., 1999. Marsupial light chains: IGK with four V families in the opossum Monodelphis domestica. Immunogenetics 50, 329–335. Miller, R.D., Grabe, H., Rosenberg, G.H., 1998. V(H) repertoire of a marsupial (Monodelphis domestica). J. Immunol. 160, 259–265. Mussmann, R., Du Pasquier, L., Hsu, E., 1996. Is Xenopus IgX an analog of IgA? Eur. J. Immunol. 26, 2823–2830. Nimmerjahn, F., Ravetch, J.V., 2008. Fcgamma receptors as regulators of immune responses. Nat. Rev. Immunol. 8, 34–47. Nowak, M.A., Parra, Z.E., Hellman, L., Miller, R.D., 2004. The complexity of expressed kappa light chains in egg-laying mammals. Immunogenetics 56, 555–563. Ohta, Y., Flajnik, M., 2006. IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates. Proc. Natl. Acad. Sci. USA 103, 10723– 10728. Ota, T., Nei, M., 1994. Divergent evolution and evolution by the birth-and-death process in the immunoglobulin VH gene family. Mol. Biol. Evol. 11, 469– 482. Parvari, R., Avivi, A., Lentner, F., Ziv, E., Tel-Or, S., Burstein, Y., Schechter, I., 1988. Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus. EMBO J. 7, 739–744. Qin, T., Ren, L., Hu, X., Guo, Y., Fei, J., Zhu, Q., Butler, J.E., Wu, C., Li, N., Hammarstrom, L., Zhao, Y., 2008. Genomic organization of the immunoglobulin light chain gene loci in Xenopus tropicalis: evolutionary implications. Dev. Comp. Immunol. 32, 156–165. Ratcliffe, M.J., 2006. Antibodies, immunoglobulin genes and the bursa of Fabricius in chicken B cell development. Dev. Comp. Immunol. 30, 101–118. Ravetch, J.V., Bolland, S., 2001. IgG Fc receptors. Annu. Rev. Immunol. 19, 275–290. Reynaud, C.A., Anquez, V., Grimal, H., Weill, J.C., 1987. A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48, 379–388. Reynaud, C.A., Anquez, V., Weill, J.C., 1991a. The chicken D locus and its contribution to the immunoglobulin heavy chain repertoire. Eur. J. Immunol. 21, 2661–2670. Reynaud, C.A., Dahan, A., Anquez, V., Weill, J.C., 1989. Somatic hyperconversion diversifies the single Vh gene of the chicken with a high incidence in the D region. Cell 59, 171–183. Reynaud, C.A., Mackay, C.R., Muller, R.G., Weill, J.C., 1991b. Somatic generation of diversity in a mammalian primary lymphoid organ: the sheep ileal Peyer’s patches. Cell 64, 995–1005. Robert, J., Ohta, Y., 2009. Comparative and developmental study of the immune system in Xenopus. Dev. Dyn. 238, 1249–1270. Ros, F., Puels, J., Reichenberger, N., van Schooten, W., Buelow, R., Platzer, J., 2004. Sequence analysis of 0.5 Mb of the rabbit germline immunoglobulin heavy chain locus. Gene 330, 49–59. Ros, F., Reichenberger, N., Dragicevic, T., van Schooten, W.C., Buelow, R., Platzer, J., 2005. Sequence analysis of 0.4 megabases of the rabbit germline immunoglobulin kapp a1 light chain locus. Anim. Genet. 36, 51–57. Saini, S.S., Farrugia, W., Ramsland, P.A., Kaushik, A.K., 2003. Bovine IgM antibodies with exceptionally long complementarity-determining region 3 of the heavy chain share unique structural properties conferring restricted VH + Vlambda pairings. Int. Immunol. 15, 845–853.

Y. Sun et al. / Developmental and Comparative Immunology 39 (2013) 103–109 Schaerlinger, B., Bascove, M., Frippiat, J.P., 2008. A new isotype of immunoglobulin heavy chain in the urodele amphibian Pleurodeles waltl predominantly expressed in larvae. Mol. Immunol. 45, 776–786. Schaerlinger, B., Frippiat, J.P., 2008. IgX antibodies in the urodele amphibian Ambystoma mexicanum. Dev. Comp. Immunol. 32, 908–915. Schiaffella, E., Sehgal, D., Anderson, A.O., Mage, R.G., 1999. Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits. J. Immunol. 162, 3984– 3995. Schroeder Jr., H.W., 2006. Similarity and divergence in the development and expression of the mouse and human antibody repertoires. Dev. Comp. Immunol. 30, 119–135. Schroeder Jr., H.W., Hillson, J.L., Perlmutter, R.M., 1990. Structure and evolution of mammalian VH families. Int. Immunol. 2, 41–50. Schwager, J., Burckert, N., Courtet, M., Du Pasquier, L., 1991a. The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus. EMBO J. 10, 2461–2470. Schwager, J., Burckert, N., Schwager, M., Wilson, M., 1991b. Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity. EMBO J. 10, 505–511. Schwager, J., Mikoryak, C.A., Steiner, L.A., 1988. Amino acid sequence of heavy chain from Xenopus laevis IgM deduced from cDNA sequence. implications for evolution of immunoglobulin domains. Proc. Natl. Acad. Sci. USA 85, 2245– 2249. Sehgal, D., Johnson, G., Wu, T.T., Mage, R.G., 1999. Generation of the primary antibody repertoire in rabbits: expression of a diverse set of Igk-V genes may compensate for limited combinatorial diversity at the heavy chain locus. Immunogenetics 50, 31–42. Sitnikova, T., Nei, M., 1998. Evolution of immunoglobulin kappa chain variable region genes in vertebrates. Mol. Biol. Evol. 15, 50–60. Sitnikova, T., Su, C., 1998. Coevolution of immunoglobulin heavy- and light-chain variable-region gene families. Mol. Biol. Evol. 15, 617–625. Stewart, S.E., Du Pasquier, L., Steiner, L.A., 1993. Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis. Eur. J. Immunol. 23, 1980–1986. Sun, J., Kacskovics, I., Brown, W.R., Butler, J.E., 1994. Expressed swine VH genes belong to a small VH gene family homologous to human VHIII. J. Immunol. 153, 5618–5627. Sun, Y., Wang, C., Wang, Y., Zhang, T., Ren, L., Hu, X., Zhang, R., Meng, Q., Guo, Y., Fei, J., Li, N., Zhao, Y., 2010. A comprehensive analysis of germline and expressed immunoglobulin repertoire in the horse. Dev. Comp. Immunol. 34, 1009–1020. Tobin-Janzen, T.C., Womack, J.E., 1992. Comparative mapping of IGHG1, IGHM, FES, and FOS in domestic cattle. Immunogenetics 36, 157–165. Turchin, A., Hsu, E., 1996. The generation of antibody diversity in the turtle. J. Immunol. 156, 3797–3805.

109

Vajdy, M., Sethupathi, P., Knight, K.L., 1998. Dependence of antibody somatic diversification on gut-associated lymphoid tissue in rabbits. J. Immunol. 160, 2725–2729. Vernersson, M., Aveskogh, M., Hellman, L., 2004. Cloning of IgE from the echidna (Tachyglossus aculeatus) and a comparative analysis of epsilon chains from all three extant mammalian lineages. Dev. Comp. Immunol. 28, 61–75. Vernersson, M., Aveskogh, M., Munday, B., Hellman, L., 2002. Evidence for an early appearance of modern post-switch immunoglobulin isotypes in mammalian evolution (II); cloning of IgE, IgG1 and IgG2 from a monotreme, the duck-billed platypus, Ornithorhynchus anatinus. Eur. J. Immunol. 32, 2145–2155. Wagner, B., Miller, D.C., Lear, T.L., Antczak, D.F., 2004. The complete map of the Ig heavy chain constant gene region reveals evidence for seven IgG isotypes and for IgD in the horse. J. Immunol. 173, 3230–3242. Wang, X., Olp, J.J., Miller, R.D., 2009. On the genomics of immunoglobulins in the gray, short-tailed opossum Monodelphis domestica. Immunogenetics 61, 581– 596. Warr, G.W., Magor, K.E., Higgins, D.A., 1995. IgY: clues to the origins of modern antibodies. Immunol. Today 16, 392–398. Wei, Z., Wu, Q., Ren, L., Hu, X., Guo, Y., Warr, G.W., Hammarstrom, L., Li, N., Zhao, Y., 2009. Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis. J. Immunol. 183, 3858–3864. Weinstein, P.D., Anderson, A.O., Mage, R.G., 1994. Rabbit IgH sequences in appendix germinal centers: VH diversification by gene conversion-like and hypermutation mechanisms. Immunity 1, 647–659. Winstead, C.R., Zhai, S.K., Sethupathi, P., Knight, K.L., 1999. Antigen-induced somatic diversification of rabbit IgH genes: gene conversion and point mutation. J. Immunol. 162, 6602–6612. Wu, Q., Wei, Z., Yang, Z., Wang, T., Ren, L., Hu, X., Meng, Q., Guo, Y., Zhu, Q., Robert, J., Hammarstrom, L., Li, N., Zhao, Y., 2010. Phylogeny, genomic organization and expression of lambda and kappa immunoglobulin light chain genes in a reptile, Anolis carolinensis. Dev. Comp. Immunol. 34, 579–589. Zhao, Y., Cui, H., Whittington, C.M., Wei, Z., Zhang, X., Zhang, Z., Yu, L., Ren, L., Hu, X., Zhang, Y., Hellman, L., Belov, K., Li, N., Hammarstrom, L., 2009. Ornithorhynchus anatinus (platypus) links the evolution of immunoglobulin genes in eutherian mammals and nonmammalian tetrapods. J. Immunol. 183, 3285–3293. Zhao, Y., Kacskovics, I., Rabbani, H., Hammarstrom, L., 2003. Physical mapping of the bovine immunoglobulin heavy chain constant region gene locus. J. Biol. Chem. 278, 35024–35032. Zhao, Y., Pan-Hammarstrom, Q., Yu, S., Wertz, N., Zhang, X., Li, N., Butler, J.E., Hammarstrom, L., 2006. Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis. Proc. Natl. Acad. Sci. USA 103, 12087–12092. Zhao, Y., Rabbani, H., Shimizu, A., Hammarstrom, L., 2000. Mapping of the chicken immunoglobulin heavy-chain constant region gene locus reveals an inverted alpha gene upstream of a condensed upsilon gene. Immunology 101, 348–353.