b-cell development in the amphibian xenopus

201

Immunological Reviews 2000Vol. 175: 201–213Printed in Denmark. All rights reserved

Copyright © Munksgaard 2000

Immunological ReviewsISSN 0105-2896

Louis Du PasquierJacques RobertMichèle CourtetRainer Mußmann

B-cell development in the amphibian Xenopus

Authors’ addresses

Louis Du Pasquier1, Jacques Robert2, Michèle Courtet1, Rainer Mußmann3,1Basel Institute for Immunology, Basel, Switzerland.2Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.3The Netherlands Cancer Institute, Division of Molecular Biology, Amsterdam, The Netherlands.

Correspondence to:

Louis Du PasquierBasel Institute for ImmunologyGrenzacherstrasse 487CH-4005 Basel SwitzerlandFax: 41 61 605 1222e-mail: [email protected]

Acknowledgements

We thank Dr M. F. Flajnik and Dr H. Etlinger for suggestions, comments and critical review of the manuscript. We thank Allison Dwileski and Lucy Trippmacher for the figures and the preparation of this manuscript. The Basel Institute for Immunology was founded and supported by Hoffman La Roche Ltd, Basel, Switzerland.

Summary: The amphibian Xenopus and mammals have similar organizationand usage of their immunoglobulin gene loci with combinatorial joiningof V, D and J elements. The differences in B-cell development betweenmammals and this amphibian are due to major differences in developmen-tal kinetics, cell number and lymphoid organ architecture. Unlike mam-mals, the immune system of Xenopus develops early under pressure todevelop quickly and to produce a heterogeneous repertoire before lym-phocyte numbers reach 5,000, thereby imposing a limitation on clonalamplification. In addition, it is submitted to metamorphosis. Thus, duringthe early antigen-independent period, several features of B-cell develop-ment related to immune diversification are under strict genetically prepro-gramed control: 1) D reading frames contribute complementary deter-mining region 3 with features that occur in mammals by somatic selec-tion, 2) the temporal stepwise utilization of VH genes in Xenopus occur infamilies probably because of structural DNA features rather than their posi-tion in the locus. Larval and adult immune responses differ in heterogene-ity. Larval rearrangements lack N diversity. During the course of immuneresponses, somatic mutants are generated at the same rate as in other ver-tebrates but are not optimally selected, probably due to the simpler orga-nization of the lymphoid organs, with neither lymph nodes nor germinalcenters resulting in poor affinity maturation. Switch from IgM to otherisotypes is mediated by loop-excision deletion of the IgM constant regiongene via switch regions which, unlike their mammalian counterpart, areA-T rich and reveal conserved microsites for the breakpoints.

Introduction

One of the best cold blooded vertebrate models for studying the

early development of the immune system is the (anuran)

amphibian Xenopus of the Pipidae family, also known as the

South African frog or the clawed toad (1–3). Since the last com-

mon ancestor to modern amphibians and mammals diverged

and became terrestrial at the end of the Devonian period 370

million years ago, Xenopus is a good model in deciding what is

accessory and what is essential in the immune system of verte-

brates. Indeed, what has been observed in both classes has to be

essential. As in mammals, the B-cell receptor diversity in this

species is generated via somatic rearrangement and combinato-

rial joining of multiple V, D and J elements within immunoglo-

bulin heavy and light chain loci (4). Xenopus development varies

202 Immunological Reviews 175/2000

Du Pasquier et al · B-cell development in Xenopus

partially from that of mammals and these variations appear to

influence B-cell differentiation as follows:

1) The tadpole immune system must develop its repertoire of

B and T-cell receptors (TCRs) as quickly and diversely as

possible since larvae hatch 2 days following fertilization. In

contrast to mammalian embryos, which remain in a rela-

tively antigen-free uterus, Xenopus larvae hatch in the sur-

rounding antigen-rich water.

2) When immunological competence occurs around day 12,

the number of cells is very small, with a few thousand B

cells and probably three times as many T cells. There is no

spleen prior to this stage and a response cannot be induced.

The differentiation events that take place in the immune

system depend either solely or essentially on intrinsic fac-

tors. This system is not in contact with the outside world

except, perhaps, via the blood, where there is no architec-

ture to guide the differentiation of cells. An equivalent stage

is reached in mammals when millions of lymphocytes are

already present.

3) There are few lymphoid organs both in Xenopus larvae and in

adults. The structures of these organs differ from those of

their mammalian counterparts and no germinal centers

have been described. Even in the adult, the total number of

lymphocytes differs in magnitude from that in mice. A

100 g Xenopus has approximately 1×108 lymphocytes (T+B),

whereas a 25 g mouse has twice this number in the spleen

alone.

4) The transition at metamorphosis when new adult-specific

antigens are expressed in a larval environment that is

immunologically competent is accompanied by many vari-

ations in lymphocyte phenotypes and function. This is

another natural experiment on the genesis of self tolerance

that does not exist in the same form in mammals. At this

time, compared with larvae, variations are seen in T–B col-

laboration in alloresponses and in antibody repertoire.

Because of these variations, studying the onset and evolution of

B-cell populations with or without specific immunizations in

Xenopus could assist in identifying essential versus accessory fac-

tors in the development of B cells in mammals.

The players – immunoglobulin genes, B cells and lymphoid

organs – in Xenopus

The Ig loci of Xenopus

Heavy chain – constant region

There are three isotypes of immunoglobulin heavy chain con-

stant region genes: IgM (4–6); IgY, a homolog of IgG (7, 8); and

IgX, preferentially expressed in the gut (9, 10). Their genes seg-

regate together but their order on the chromosome has yet to

be determined. The transmembrane and cytoplasmic domains

of Xenopus membrane-bound IgM (mIgM) are similar to the cor-

responding domains of all known vertebrate mIgM molecules.

The membrane forms of the two other Ig isotypes, mIgX and

mIgY, exhibit the specific structure found in all Ig membrane

exons but are not directly homologous with any specific mam-

malian non-µ Ig isotype; they are most similar to Xenopus mIgM.

Based on the conserved transmembrane domains of Xenopus

mIgX and mIgY, we believe that the heavy chain genes of IgM

and IgX initially arose from the original µ gene by means of

duplication. The transmembrane and cytoplasmic domains of

Xenopus mIgY have some conserved residues found in avian

mIgY and mammalian mIgG and mIgE, suggesting that the

more modern isotypes share a common but ancient ancestor

with amphibian mIgY (8).

Heavy chain – variable regions

Xenopus has approximately 100 VH genes belonging to 11 fami-

lies, more than 10 DH genes and eight to nine JH genes depend-

ing on the species (laevis: eight, gilli: nine) (11–13). The VH

locus is diploidized in the pseudotetraploid X. laevis as shown by

segregation studies (10) and by in situ hybridization with cDNA

probes and tyramide enhancing technology (Renaissance Tyra-

mide Signal Amplification, NEN Life Science Products) (Fig. 1)

(14). The VH genes are interspersed in the region (15) but not

scattered totally at random (4). A partial deletion mapping (L.

Du Pasquier, unpublished) using tumor cell lines that have

undergone different rearrangements of their Ig heavy chain loci

(16) showed that the VH8 and 5 genes are on the outside of the

locus, and a group encompassing most of the VH, i.e. VH1, 2 and

3 (at least 60 genes), is not close to the most 5' DH gene. The

sequence of the whole DH region is incomplete, but two

genomic D elements have been cloned (13). The distance

between the last D and the first JH is greater than 6 kb, the

amount of sequence upstream of JH determined without find-

ing a D element. The DH locus of Xenopus is therefore much fur-

ther upstream of the VH locus than in mammals. This could

explain some differences in the Ig rearrangement profiles of

Xenopus, such as the low incidence of DJ rearrangements (13).

Indeed, Yu et al. argue (17) that the distance between the seg-

ments can affect the kinetics of rearrangements of the different

elements, which could explain the observations in Xenopus.

Light chain

There are three isotypes of light chain, called �, � and �-like,

corresponding to products ranging from 25,000 to 29,000 Da

(18–21). The light chain locus architecture, numbers, and the

genetic linkages of the various loci are not determined with

Immunological Reviews 175/2000 203


precision except for the �-like r isotype. The latter’s architec-

ture is similar to that of mammals containing multiple V, at least

four Js and one C gene or segments (21). Compared with those

of � and �, the structure of the � J is different with the diglycine

bulge being replaced by a diserine (20).

B cells

These days, no cell exists that cannot be detected by flow

cytometry. Markers for Xenopus B cells are unfortunately scarce

except for heavy and light chain Ig (22). CD5, a marker for a

subpopulation of B cells in mammals (23), is a marker for T

cells in Xenopus but it is also detected on activated Xenopus B cells

after stimulation with phorbol 12-myristate 13-acetate (24).

So far, three stages: 1) pre-B cells (expressing heavy chain but

no light chain), 2) B cells, and 3) plasma cells have been chara-

terized in the spleen. The pre-B and plasma cells are of low den-

sity and heterogeneous in size while small sIg+ B lymphocytes

are of high density and much more homogeneous in size (25).

CD45, the leukocyte common antigen, is a structurally heter-

ogenous molecule ranging in molecular weight from 180 to

220 kDa. Xenopus B cells express the 220 kDa variant (26). Allelic

exclusion is the rule in Xenopus B cells, regardless of the number

of Ig loci present, following gene duplication by allopoly-

ploidization (27, 28).

Larval B cells differ from adult B cells in the re-expression

of surface Ig after capping. Whereas all adult liver and splenic B

cells re-express their Ig receptor 96 h after capping, only 30%

of larval B cells do so. Perhaps the cells that do not re-express are

immature B cells. After receiving a negative signal from anti-Ig

antibody these cells die as described for B cells from fetal mice

(29). Between larvae and adults the compartmentalization of B

cells changes. Adult immature B cells could perhaps be found in

adult bone marrow instead of larval spleen and liver (30).

Lymphoid organs of Xenopus

The spleen, which is also hematopoietically active in adults, is

the main site of B-cell differentiation (Fig. 2). The architecture

of the spleen shows a clear thymus-dependent area, with cells

labeled with anti-CD8 reagents (3, 31, 32) surrounding the

B-cell area around the central arteriola.

In accordance with data published so far in Xenopus, and in

general in cold blooded vertebrates (33), no structural equiva-

Fig. 1. Localization of the heavy chain locus by in situ hybridization. The chromosome preparation from the tumor cell line B3 B7 was hybridized with a cDNA probe from a VH1–IgM Xenopus clone labeled with digoxigenin (Dig) and revealed with anti-Dig antibody and enhanced by the tyramide method (L. Du Pasquier, M. Courtet, in preparation). Objective: ×100

Fig. 2. Immunohistochemistry microscopy on frozen spleen section at low magnification from an unimmunized Xenopus juvenile LG-15 double stained for IgM+ (blue) and BrdU+ (brown) cells. The frog was treated overnight by adding 2 mg/ml of 5-bromo-2' deoxyuridine (BrdU) in the water in darkness. Spleen sections were then incubated with anti-IgM 10A9 mAb followed by alkaline-conjugated goat anti-mouse secondary Ab preadsorbed twice on Xenopus blood cells. Following staining with AK substrate, BrdU incorporated in DNA was detected after DNA hydrolysis with hydrochloric acid using anti-BrdU mAb, horseradish peroxidase-conjugated goat anti-mouse secondary Ab and dimethyl-aminoazobenzene as substrate (brown cells). Notice the white pulp nodule in the center with the B-cell areas, stained in blue around the central arteriola, unlike those found in mammals. Objective: ×10



lent to germinal centers has been observed in the white pulp of

the spleen. Following immunization, specific antigen-reactive

cells were seldom found in clusters and were never associated

with structures resembling germinal centers (Fig. 2). Antibody-

producing cells were detected in the spleen as plasma cells but

no cluster of proliferating antigen-specific B cells was identified

by immunochemistry using 5-bromo-2' deoxyuridine (BrdU)

incorporation as an indicator of DNA synthesis (Fig. 3).

B cells are also in the adult gut (10) and thymus (34). In

contrast, the larval gut does not contain any B cells (i.e. Ig sur-

face positive). Even though compartmentalization is not very

pronounced in Xenopus lymphoid organs, thymus B cells seem to

switch more readily to IgY production upon T-cell help than

splenic B cells (34).

There are no lymph nodes in anuran amphibians, although

some lymphoid accumulations are found near the heart (33).

Rearrangement of Ig genes occurs in the liver and presumably

also in the spleen of tadpoles. Bone marrow is absent in tad-

poles. It was thought not to be lymphopoietic in adults (25)

but recombination-activating gene (RAG) activity (RAG trans-

cription) has been detected in it (35). Ig isotype production is

organ dependent, IgM is everywhere while IgG is not produced

in the gut. IgX, in contrast, is preferentially expressed in the gut

in intraepithelial plasma cells (10).

The lack of germinal centers typical of cold blooded verte-

brate lymphoid organs is thought to be responsible for the poor

affinity maturation in Xenopus (see somatic mutation section)

and could be due to the absence of classical follicular dendritic

cells (FDC). In mice, in the absence of FDC, germinal centers

are not formed normally, while affinity maturation, although

delayed, still occurs (36). Large thymus-independent dendritic

cells in the periphery of the splenic white pulp could represent

the Xenopus equivalent of FDC (37).

Further research in immune response regulation is now

required in Xenopus. Such research should involve the study of

antigen-presenting cells (FDC) and chemokines and their recep-

Fig. 3. A. Immunofluorescence microscopy on frozen sections of juvenile Xenopus LG-15 spleen. Detection of splenic B cells producing anti-2,4-dinitrophenol (anti-DNP) antibody, 3 weeks after immunization with DNP–bovine serum albumin (10 µg i.p.). Frozen section was incubated with DNP conjugated to alkaline phosphatase and stained with naphthol-AS-phosphate + fast blue as AK substrate. B. Spleen seen at low magnification (100×) with three white pulp nodules. Arrows point to putative antigen-specific plasma-cell like cells. C. Higher magnification (400×) of antigen reactive lymphocytes.



tors (38) involved in establishing the architecture of lymphoid

organs and the migration of helper cells. The same applies to

interleukins for which studies have just begun (39, 40).

Ontogeny – putting everything together

B-cell appearance and expansion during ontogeny

Xenopus larvae hatch 2 days after fertilization. From this stage

onwards, the young are exposed to the outside world and are

under pressure to develop a diverse functional immune system

rapidly. The liver and the thymus are colonized during the first

week of life by lymphopoietic precursor cells migrating from

the lateral plate mesoderm and ventral blood island. Lym-

phopoiesis occurring in late larval life and after metamorphosis

produces a stable, persistent population of ventral blood island-

derived stem cells as well as dorsally derived stem cells. Larval

stem cells can give rise to adult cell populations as shown by

ploidy marker experiments (41, 42). In Xenopus, developmental

studies of B cells are limited by the lack of markers. The pheno-

type of what is known as a B cell is always linked to the pre-

existence of a rearrangement, since only cells that have either a

heavy chain alone or a complete HL dimer on the surface can

be identified.

The development of the larval immune system in general

and that of B cells in particular can be divided into two periods:

one starts on day 4 when the first RAG message is detected and

ends on day 12 when the spleen can be identified. The second

period extends from day 12 to the end of metamorphosis,

approximately 50 days later. Before day 12, when the animal is

not yet immunologically competent, B cells are likely to

develop exclusively in the liver under the influence of intrinsic

determinants. Since light chain can only be detected from day

10 onwards, it is certain that each development and diversifica-

tion detected at the H chain level in the cytoplasm, prior to

then, is independent of external antigens. The spleen becomes

visible on day 12 and antibody responses can be elicited from

this day on (43). Extrinsic agents can now contribute to shap-

ing the immune repertoire.

Four to five days after fertilization half a dozen IgH chains

expressing immature B-cell precursors are detectable in the

liver of the larvae (Fig. 4). The number of immature B cells dou-

bles approximately once a day. After 2 weeks a few thousand B

cells are in the liver and spleen. At this stage the B-cell reper-

toire is very diverse, and each cell may express a different B-cell

receptor (44). This diversity presumably optimizes the ability

of young larvae to produce as many antibodies as possible

against a wide range of different antigens. This is consistent

with observations in another species of amphibian, the mid-

wife toad Alytes obstetricans, where during this developmental

period, cells specific to an early epitope are progressively

diluted by cells expressing different specificities (45).

During metamorphosis the lymphoid organs undergo a

drastic reduction in size and cell number and at the same time

RAG enzyme activity declines (46). A second histogenesis of

the lymphoid organ takes place during the month and a half

that follows metamorphosis and is most visible in the thymus

but to a certain extent also in the spleen (47). RAG enzymes are

active in adult, where they are detectable in the spleen and in

the bone marrow (35).

Rearrangement throughout ontogeny

RAG1 message is detected 4 days after fertilization at 22°C.

Heavy chain genes rearrange first, starting on day 5, followed

by light chains on day 8 (44). Until day 8 the developing

immune system is not functional because most IgM+ cells are

pre-B cells (48), with no L chain rearrangement having been

observed. VH genes then rearrange in a somewhat stepwise

manner (Fig. 4) (44). For L chain, only two loci out of three, �

and �� were precisely monitored during ontogeny, and � rear-

ranges earlier (18, 20, 44). If what has been observed in

tumors also occurs in normal B cells, rearrangement of � seems

to occur, as in mammals, with the deletion of � (16). No data

exists concerning a putative surrogate L chain in amphibians.

The stepwise rearrangements of H and L chain genes in

Xenopus (44) are different from those reported in mammals.

There is no positional effect. Different members of the VH1 fam-

ily can be used first and they are dispersed throughout the locus

(see previous section). The preferential expression of con-

served VH genes in different species must therefore be due to

common regulatory elements, for instance, recombination sig-

nal sequences. In Xenopus the sequence of events is compressed

over 5–6 days even though it retains the basic features found in

mammals. Those features are likely to be due to intrinsic factors

linked to the mechanisms of rearrangement. Indeed, the spacer

in the recombination signal sequences shows some family

traits, some of them conserved between Xenopus and mammals,

and they have been shown to be important in orienting rear-

rangement (49).

The Xenopus VH1 is homologous to the VH3 clan that rear-

range early in mouse and humans (50–53). However, the con-

served regions are within framework 1 and 3 and not within

the antigen-binding sites. This suggests that these gene seg-

ments are not selected on the basis of a common antigenic

specificity unless the antibodies using such VH bind antigens in

a different manner, for instance via the charge of the amino

acids in the � sheets rather than via the complementarity deter-



mining region (CDR) loops. In addition, VH1 rearrangement in

Xenopus occurs when L chains are not produced. Therefore, the

selective advantage of VH1 is unclear. In mammals, several mod-

els have been proposed to account for the usage of certain VH

during early ontogeny. For example, it has been reported that

fetal human and mouse B-cell repertoires are enriched for mul-

tireactive antibodies that exhibit low affinity binding to self

antigens (e.g. DNA antigens) (54). With the strict developmen-

tal control of the fetal and neonatal Ig repertoire, this led to the

hypothesis that the early generation of self-reactive antibodies

represents a critical feature of mammalian immune repertoire

development. A low level of self reactivity may serve as an

essential stimulant for B-cell selection and proliferation. Alter-

natively, the immune system developmental program, less strict

in Xenopus than in mammals, may reflect evolutionary pressure

to express a plastic set of antibodies, enabling an immunologi-

cally naive individual to respond to a wide range of antigens

but with a low affinity.

Very few D–J rearrangements (<1%) on the abortively rear-

ranged allele were isolated in adult and larva compared with

mammals, either at the cDNA or genomic DNA level (13). This

observation, which could be due to the position of the different

V, D and J loci (see above), does not leave much possibility for

the generation of a Dµ (55) protein in amphibians.

Ig heterogeneity throughout ontogeny

Heterogeneity of the rearrangement has been assessed from day

5 to adulthood (44). In isogenic Xenopus, all VDJ genes contain-

ing VH1 isolated from 5 to 7 day old LG15 larvae (when they

had 5–50 IgH chain but no IgL chain-positive cells) were dif-

ferent, showing the absence of clonal amplification. Diversifi-

cation appears to be maximized at this stage. At the genomic

DNA and cDNA level, the combinatorial and junctional diver-

sity of VDJ genes of 9–10 day old larvae were similar. The rela-

tively high frequency of non-functional out of frame rear-

rangements is typical of a developing population when precur-

Fig. 4. B cells during Xenopus development. Immunofluorescent staining with anti-� (open circles) and anti-L chains (black circles) has been lined up with the sequential rearrangements of VH (µ) and VL (� and �) segments. [Data from (44)]



sor B cells have not rearranged both Ig alleles and where the

ratio of functional to non-functional rearrangements reflects

the three possible reading frames (one in frame and two out of

frame). At a later stage (day 13–15), surface Ig+ B cells accumu-

late in lymphoid organs. Since selected B cells have now rear-

ranged both alleles, the ratio of functional to non-functional

rearranged VDJ genes in surface Ig+ B cells is now 1:1. Around

this period, rearrangements of all VH gene families (except

VH11, which is also rarely used in adults) were found. JH6 and

DH12 and 16 were observed for the first time in rearranged VDJ

genes. Finally, 38 cDNA sequences were obtained from tadpoles

at stages 56–58 (day 30–55). All were different (13). At the

same stage, nine different genomic rearrangements were iso-

lated from a B-cell genomic DNA library.

The diversity of sequences does not clearly account for the

restriction in antibody populations characterized by isoelectric

focusing (56, 57). Perhaps there is a microheterogenity in Xeno-

pus antibody sequences not necessarily linked to a charge differ-

ence which results in an apparently restricted response (58).

Even in the absence of N diversity, the CDR3s of developing

Xenopus are quite diverse at the cDNA level, but it is difficult to

correlate this diversity with that of antibody. Plasma cells pro-

duce an enormous amount of antibody that can mask the het-

erogeneity present in B cells at the protein level.

Secondary rearrangements

Secondary rearrangements leading to V gene replacements have

been found in Xenopus L chain genes within a circular DNA library

(59), although no functional studies have been performed to see

if this results in a selective advantage. In species with low cell

numbers, the selective value of this mechanism appears to be

evident, while in species with ongoing rearrangements the util-

ity is less obvious. The economical argument, that receptor edit-

ing saves B cells, could apply to frogs at metamorphosis, where

there are fewer B cells and the mechanism would allow escape

from autoimmunity. Receptor editing is more likely to result

from the rearranging mechanism that is not tightly regulated

and can go on or be reactivated without a specific need.

Properties of CDR3 in Xenopus

Composition

As mentioned above, CDR3 contributes most to the conforma-

tion of the antigen-combining site, and DH elements are the

source of most of CDR3 diversity. In Xenopus, DH segments can

potentially be used in all six reading frames if no stop codon

occurs. DH elements can be fused, a flexibility not shared with

mammalian DHs (13, 46, 58, 60). At the center of the antigen-

binding site, CDR3 amino acids form solvent exposed loops

rich in glycine and tyrosine. Accordingly, in mammals, IgH

CDR3s encoding glycines and tyrosines are selected for during

B-cell differentiation, whereas highly hydrophobic IgH CDR3s

are not selected (61). Similarly, Xenopus IgH CDR3 sequences

from different developmental stages are rich in hydrophilic and

loop-promoting residues (Fig. 5). IgH CDR3s encoding the gly-

cine-rich DH reading frame 1 of the two most frequently used

DH elements, DH1 and 10, are selected for during B-cell differen-

tiation. Interestingly, not a single strongly hydrophobic CDR3

sequence was found in Xenopus, even in the pre-B-cell popula-

tions of 5 and 7 day old larvae. In contrast to Xenopus, the

human, rabbit, mouse and chicken DH reading frame can pro-

duce highly hydrophobic sequences, but this rarely occurs in

peripheral B cells (62, 63). This indicates species-specific selec-

tion during evolution on the genomic sequences of Xenopus DH

elements. This might be linked to the pressure to rapidly

develop a functional immune system with a small number of

cells. The negative post-rearrangement selection of B cells due

to highly hydrophobic IgH CDR3 sequences, as observed in

mammals, is minimized in Xenopus, thus avoiding wastage, a fac-

tor always thought to play a role in shaping the immune system

of animals with small numbers of cells (64). The selection of a

germ-line DH sequence with a “compatible” sequence indepen-

dent of the reading frame could eliminate the need for early

selection steps such as those effected by the pre-B-cell receptor

in mammals (65). This might be relevant at early larval stages

of development, when the small number of B cells limits the

diversity of the B-cell repertoire.

Length

Most of the larval and young post-metamorphic Ig gene sam-

ples contained a CDR3 of 3–10 codons, two codons shorter

than adult CDR3s which were 5–12 codons long (66); the lat-

ter were shorter on the average than mammalian CDR3S, which

can reach up to 20 residues (58). Perhaps the shorter CDR3

generates a more limited spectrum of antigen-combining sites

(66). The reason for the short CDR3 in tadpole is in part due to

the lack of N diversity (13), which in adults is most likely to be

generated by terminal deoxynucleotidyl transferase (67).

Homology-based junction

In contrast to other DH and JH gene segments, DH1 and JH3 con-

tain overlapping sequences of two or three nucleotides at their

3' and 5' ends, respectively. In mammals it has been argued that

such short overlaps can direct the rearrangement, resulting in

redundant, so-called homology-based, V, D and J junctions (68,

69). Among 12 DH1–VH3 junctions from B-cell genomic DNA of



9–21 day old larvae, only two junctions, which also corre-

sponded to homology-based junctions, were identical. How-

ever, when cDNA of 23 day old larvae was analyzed, 12 out of

16 DH1–JH3 junctions were identical, and the dominant DH1–JH3

junctional sequence corresponded to one of the possible

homology-based junctions. The bias towards homology-based

junctions in cDNA sequences could therefore result from selec-

tion following rearrangement and leading to the presence of a

tyrosine at this position (44).

B cells at work

Antibody production in larvae and adults

Antibody responses can be elicted in Xenopus tadpoles (70, 71),

similar to tadpoles of other anurans such as the midwife toad

Alytes obstetricans (45), the bullfrog Rana catesbeiana (71, 72) and

Rana pipiens (73). From the moment the spleen becomes visible,

antibodies, mostly of the IgM type, can be detected (43). The

isotypes and the molecular nature of adult and larval Ig are

identical. IgM is first detected in the peritoneal fluid of larvae

at stage 49 (12 days) and IgG homolog at 15 days. IgX was seen

in liver plasma cells in tadpole but was not present in the gut,

which at this stage contains very few lymphoid cells and no B

cells. The ability of larvae to produce antibodies increased dur-

ing development from stage 50 to stage 59. The concomitant

injection of primed irradiated adult cells in addition to antigen

increased the amount of antibody made, especially the IgG

type. The anti-2,4-dinitrophenol (anti-DNP) antibodies pro-

duced by larvae were of lower affinity than those produced by

adults. Sibling larvae given additional primed adult cells pro-

duce more antibody but of the larval type (43, 74). Tadpole

and adult Xenopus, manipulated to be of comparable size by

blocking development with sodium perchlorate, exhibited

stage-specific antibody expression. The production of adult-

type higher affinity anti-DNP antibodies is independent of the

age and size of the individual and is concomitant with the com-

pletion of metamorphosis. The appearance of new antibody

specificities at such a time suggests that their expression occurs

with the cell turnover and renewal during a period of morpho-

logical change (47, 75). Specific memory, despite all the

changes, can cross metamorphosis (72). There is a relatively

higher membrane Ig expression in tadpoles compared with

adults. This could mean that plasma cell differentiation is not

optimal at early stages of development (76). Immunized and

A B

C D

Fig. 5. Xenopus IgH CDR3 sequences from different developmental stages. Amino acid sequence deduced from cDNA sequences of 5–7 day old larvae (A), DNA sequences of 15, 21, and 48 day old larvae (B), and cDNA sequences of immunized adults (C, D) (13, 78). The codons between the invariant Cys92 and Trp103 are shown. Residues encoded by nucleotides potentially derived from a DH element are shown in bold.



non-immunized individuals show different profiles of V region

expression. VHIII is expressed largely in non-experimentally

immunized animals. VHI is used preferentially in the anti-DNP

response as seen from protein sequences (77) and from the

cDNA sequences of immunized animals (76, 78).

Selection of somatic mutants during larval and adult immune

responses

Somatic mutations occur in Xenopus larvae or adults at a rate not

significantly lower than that observed in mammals (76, 78).

Why the antibody response is so poor compared with mam-

mals with little affinity maturation remains a mystery. Some

lines of evidence indicate that there is minimal selection of B

cells with higher affinity receptors. One line comes from com-

paring the actual ratio of replacement to silent mutations (R/S)

with the ratio expected from random mutation. R/S in CDR is

somewhat higher but not statistically significant. The other line

of evidence comes from the strong ratio of GC to AT base pairs

altered by mutations both in tadpoles and adults. It is possible

that selection of the mutants is limiting. To assess whether the

effect of selection can be observed in a system not involving Ig

genes we have used a tabulation of the spectra of spontaneous

mutations in seven different systems involving the loss of the

enzymes hypoxanthine guanine phosphoribosyltransferase and

adenine phosphoribosyltransferase in mammals (79). For each

system the log of the GC/AT mutation ratio was plotted against

the percentage of point mutations. In a loss of activity mutation

analysis the fraction of point mutations is a measure of the

stringency of selection. If almost total loss of activity is required

for mutants to get through the screen then the system being

analyzed will be enriched in other types of mutations. The log

of GC/AC mutation ratio correlates positively with the percent-

age of point mutations and hence correlates negatively with the

stringency of selection (79). Similarly, the mammalian lym-

phoid cell line 18.81, which mutates spontaneously without

antigen selection in vitro, also shows a profound GC bias (80,

81). In addition, the pattern of mutation seen in Xenopus is sim-

ilar to that found in mice deficient in the MSH2 mismatch

repair protein (82), as if the hypermutation pathway frequently

mutated G and C or as if the Xenopus MSH2-dependent pathway

that normally correlates G and C mismatches is not operational.

This is compatible with two non-mutually exclusive notions:

1) that hypermutation at the Ig locus is caused by a reduction

(qualitative or quantitative) of the error correction machinery

normally present in all cells rather than by the presence of addi-

tional error-prone repair system in B cells, and 2) that there is

minimal selection of high affinity B cells by antigen in Xenopus

due to the lack of germinal centers (see above) (59, 76, 78).

Switch

Some selection for higher affinity antibody occurs as immune

responses develop and progress in Xenopus. Under the influence

of T cells, heavy chain class switch occurs in Xenopus. cDNA

libraries made from immunized animals have higher ratios of

IgY/IgM clones than those from non-experimentally immu-

nized animals (13). The mechanism appears similar to that in

mammals, with loop excision and disappearance of the m locus

on the switched allele (83). The Xenopus Ig locus contains switch

regions that participate in the somatic transition from the

expression of one isotype to the other. When performed within

the first week of age, thymectomy in Xenopus prevents the switch

from IgM to IgY (84) but not from IgM to IgX (9). Low levels

of switching also occur in tadpoles, with IgX-positive cells hav-

ing been detected in the liver of day 30 tadpoles. IgY in tadpole

can be produced upon repeated immunization or after injec-

tion of adult syngeneic T cells (43). In addition, the switch to

IgY is highly temperature dependent in Xenopus. IgM switching

in animals at 19°C does not occur until 1 month after immuni-

zation. At 22°C, switch is easily detected after 21 days, and IgY

can be analyzed on isoelectric focusing gels. It is even more

readily induced at 27°C (57).

The switch region of IgM, S�, of about 5 kb has been iden-

tified in the JH–C� intron. S� contains 23 repeats each with a

length of approximately 150 bp. Each repeat consists of internal

smaller repeats and palindromic sequences, such as AGCT,

which they share with mammalian switch regions. A deletion

of the �-gene and joining of the S regions of � and occurs in

Xenopus B cells expressing IgX. Although not sequenced in its

totality, the switch region of IgX, S�� shows no sequence homol-

ogy with S� in its 3' portion and is characterized by 16 and 121

bp repeats and a high frequency of CATG, AGCA and TGCA pal-

indromes. Both IgM and IgXS regions are AT rich and not GC rich

like mammalian S regions. The majority of the recombinations

occur at positions (microsites) where a single strand DNA fold-

ing program predicts the transition from a stem to a loop struc-

ture. This feature is conserved in most mammalian switch junc-

tions, which points to the general existence and involvement of

microsites as a component that determines the recombination

breakpoint. The recombinogenic nature of the switch regions

is therefore linked to its structure rather than to its base com-

position, the repetitive occurrence of palindromes being essen-

tial for creating many microsites (83).

Conclusions

In Xenopus, all the major events involved in mammalian B-cell

differentiation occur (Fig. 6), consistent with homogeneity of



the immune system in vertebrates. However, results from

studying B-cell differentiation at very early developmental

stages in Xenopus, when the number of cells is small and perhaps

limiting, reveal differences that can alter interpretations of

events at the molecular level during early ontogeny in mam-

mals.

Among the many questions that remain unanswered is:

why is there minimal selection for antibody-combining site

mutations? This review reveals many gaps in our knowledge

about the genesis of the lymphoid organs and the cellular traffic

responsible for selection. This topic could be the focus of future

studies in Xenopus involving projects on chemokines and inter-

leukins. Research should move away from the major genetic

loci that have been studied so far, such as MHC, Ig and TCR, even

though much remains to be done in this area (for instance TCR

a, g, and d have still not been isolated).

Xenopus is an interesting model because of its ontogeny, but

its pseudotetraploid nature and its relatively long generation

time turn it into a genetic nightmare. With the prospect of fur-

ther developing the genetically simpler X. tropicalis, which also

takes only 8–10 months to reach sexual maturity, things may

soon change. In addition, Xenopus proves a good model for

transgenesis. Before transgenesis, nuclear transplantation mod-

els were once thought to be exploitable for performing single

lymphocyte genetics but were hampered by the fact that tad-

poles derived from lymphocyte nuclei did not live long enough

to allow immunological experiments (85, 86). The introduc-

tion of transgenic animals (87), especially using the genetically

simple X. tropicalis (88), can increase our possibilities and

should allow such experimental projects to regain momentum

in the future. This will probably require a painful and labor-

intensive adaptation to the new species; new libraries and

reagents will need to be created. However, a model will then be

available where molecular genetics and physiological aspects of

the immune system can be studied in a single species. Finally,

B cells do not develop in isolation. T cells and accessory cells

Fig. 6. The ontogeny of the B-cell compartment in Xenopus. [Modified from (2)]



should also be considered simultaneously. With the cloning of

TCR genes and preparation of new monoclonal antibodies,

more tools are available, and the prospect of comprehensively

studying the developing Xenopus immune system becomes more

realistic and feasible. The addition of lymphoid cell lines to the

arsenal of tools for the immunologist is welcome and has

already made possible the isolation and sequencing of two lym-

phocyte cell surface proteins and the elucidation of some

aspects of Ig gene rearrangement (16, 24, 89, 90).

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