Structure and diversity of Mexican axolotl lambda light chains

We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl.     

Ontogeny of immunoglobulin expression in the Mexican axolotl

The ontogeny of immunoglobulin (Ig) synthesis was followed at both cellular and serological levels in the Mexican axolotl (Ambystoma mexicanum) using polyclonal antibodies recognizing all Ig molecules and a set of monoclonal antibodies (Mabs) specific for the C mu and Cv heavy Ig chain isotypes and for the light chain constituents shared by IgM and IgY molecules. Clusters of IgM- and of IgY-synthesizing lymphocytes, often located in separate sites, are first present in spleen sections of 7-week-old 25 mm larvae, about one month after differentiation of the spleen anlage (stage 39-40). In 12-week-old 30-35 mm larvae, the relative proportion of IgM- and IgY-synthesizing cells in the spleen is the same as that in adult animals. However, a marked enhancement of the spleen B cell compartment occurs from 5 to 9 months when Ig-positive cells represent about 88% of the lymphocytes population compared to 60% in adults. No structures equivalent to B cell germinal centers were observed at any stage of the spleen differentiation and cells, although often clustered in small groups, remain dispersed in the entire organ. The relative proportions of IgM and IgY B cells throughout the spleen remain constant during development (about 1 IgY+ cell for 5-6 IgM+ cells) and IgM molecules are first detected in the serum of 2.5-month-old larvae. The enhancement of the serum IgM level correlates well with the absolute number of IgM+ cells in the growing spleen. IgY molecules cannot be detected in the serum before the 7th month but their level quickly increases to reach about 60% of the adult value at 10 months. Thyroxine-induced metamorphosis or hyperimmunization of 4- to 6-month-old larvae had no effect upon the temporal expression of the Ig classes in serum.     

Structure and function of gap junctions in the developing brain

Gap-junction-dependent neuronal communication is widespread in the developing brain, and the prevalence of gap-junctional coupling is well correlated with specific developmental events. We summarize here our current knowledge of the contribution of gap junctions to brain development and propose that they carry out this role by taking advantage of the full complement of their functional properties. Thus, hemichannel activation may represent a key step in the initiation of Ca²⁺ waves that coordinate cell cycle events during early prenatal neurogenesis, whereas both hemichannels and/or gap junctions may control the division and migration of cohorts of precusor cells during late prenatal neurogenesis. Finally, the recent discovery that pannexins, a novel group of proteins prominently expressed in the brain, are able to form both hemichannels and gap-junction channels suggests that we need to seek more than just connexins with respect to these junctions.Work in the authors’ laboratories is supported by the Deutsche Forschungsgemeinschaft, SFB 509 (R.D.) and by the Institut Pasteur (R.B.).     

A lethal mutant gene in the Mexican axolotl

Gene ph was discovered in a wild-type axolotl male received from Mexico City. Larvae homozygous for this gene become recognizable by their lighter color at hatching or shortly after. The development of their forelimbs is retarded, and all limbs are of subnormal length because of the reduction in length of their long bones. Many affected larvae die without feeding, and very few survive beyond their third month. At death, older larvae usually show abnormalities of the renal system, edema, ascites, or adhesions of the viscera. The gene is apparently a simple recessive with full penetrance.     

Inter- and intramyotomal gap junctions in the axolotl embryo.

In early tailbud embryos of the axolotl (Ambystoma mexicanum), cells of the anterior myotomes begin to elongate and align along the longitudinal axis of the animal. Soon thereafter, gap junctions appear between the differentiating myotubes. These junctions occur between adjacent cells within a myotome (intramyotomal) and between the cells of adjacent myotomes which are separated from one another by narrow connective tissue septa (intermyotomal). The latter are found at the ends of the elongating cells where muscle-tendon insertion will occur and nerve-muscle synapses will form. The gap junctions are transient: They appear with the onset of myofibrillar formation at the time that nerve fibers enter the intermyotomal septa. The junctions last until the cells have differentiated into mature striated muscle cells and neuromuscular synapses are fully developed.These gap junctions may provide a means for the direct intercellular spread of electrical excitation between the differentiating muscle cells and so account for the observed myogenic contraction of myotomes. We also suggest that these junctions may form a means for cellular communication and interaction during the development of the axial musculature.     

Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits

Rabbits preferentially utilize only one of their multiple functional germline immunoglobulin VH genes. This preferential usage of one gene, VH1, raises the question of how rabbits generate antibody diversity. VDJ diversification was analyzed by cloning and sequencing VH1 gene rearrangements. Comparison of these sequences with that of germline VH1 identified clusters of nucleotide changes, including codon insertions and deletions. To investigate whether gene conversion was involved in this somatic diversification, we searched a data base of rabbit germline VH gene sequences for donor VH genes; potential donors were identified for five diversified regions. We conclude that somatic gene conversion has a major role in generating antibody diversity in rabbits. These studies provide clear evidence for somatic gene conversion of mammalian VDJ genes.     

The developmental genetics of pigment mutants in the Mexican axolotl

The Mexican axolotl (Ambystoma mexicanum) provides a well-defined set of color genes which are useful for various types of analyses. These include the a (albino), m (melanoid), ax (axanthic), and d (white) genes. In addition, various combinations of these genes and a number of as yet undescribed mutants also exist. Three of these mutants (a, ax, and m) have defects associated with specific neural-crest-derived pigment cell types. The fourth mutant (d) appears to provide an unsuitable environment for the migration and maintenance of pigment cells. In one case (m), detailed information concerning the specific nature of the genetic defect is available. The goal of this article is to demonstrate ways in which the existing information on the axolotl color genes can best be utilized in terms of understanding not only the mutant phenotypes, but basic concepts in the cell and developmental biology of pigmentation as well. Thus, an attempt has been made to sort through the genetic and biochemical data relevant to these mutants in order to stimulate renewed interest in a more detailed pursuit of such studies.     

Formation of the pronephros and pronephric duct rudiment in the Mexican axolotl

In the Mexican axolotl (Ambystoma mexicanum), the pronephros begins to form at the four-somite stage. It is initially continuous with the posterior-lateral region of somite 2 and the lateral margin of somites 3 and 4. By the seven-somite stage, the pronephros has become compacted, and the cells are now morphologically distinct from the somitic cells. At this stage, a mass of loosely connected cells, apparently originating from the lateral mesoderm, is seen below somites 4 and 5. By the eight-somite stage, these presumptive duct cells have migrated dorsally to the duct path and are found below somites 5–7. By the nine-somite stage they have begun to migrate caudally.     

Microtubule proteins in axolotl eggs and developing embryos

Tubulin from eggs and embryos of the Mexican axolotl was characterized by electrophoresis and colchicine binding. In urea-polyacrylamide gel electrophoresis, soluble axolotl egg tubulin migrated as two bands, identical to tubulins from sea urchin sperm and Drosophila eggs. However, in SDS-containing gels, on which the α and β subunits of standard tubulins were well resolved, axolotl egg tubulin migrated as a single band with an apparent molecular weight of 53,500. The method of disruption of the eggs affected both yield of tubulin from vinblastine sulfate precipitates and stability of the colchicine binding activity. The colchicine binding activity of soluble tubulin from gently disrupted eggs was specific and of high affinity, with properties similar to those reported for other tubulins. The tubulin pool in unfertilized eggs was determined to be approximately 2 μg/egg; the level decreased 20% after initiation of cleavage and then remained constant through development to postneurula stages. The colchicine binding activity of soluble tubulin from embryos was much less stable than that of unfertilized eggs and decreased further during development. No differences were found in properties of tubulin from eggs of several strains of normally pigmented axolotls; however, tubulin from albino eggs showed slightly different properties in both electrophoresis and colchicine binding. The colchicine binding activity of soluble tubulin accounts for only half the total activity in axolotl eggs; they possess, in addition, a particulate nontubulin colchicine binding activity.     

Development of the sympathetic system in the Mexican axolotl, Ambystoma mexicanum

Migration of trunk neural crest cells in axolotl embryos has been followed by autoradiography using grafts of [³H]thymidine-labeled neural folds. Crest cells form melanocytes, dorsal fin mesenchymal cells, spinal ganglion cells, and reach the sympathetic region. Sympathetic neurons, however, are not identifiable morphologically until about 6 weeks posthatching, in 24-mm larvae. At this stage, neurons, although few, have characteristic ultrastructure and receive synapses. The diffuse ganglia also contain innervated chromaffin cells; these differentiate earlier, a few days posthatching, in 14-mm larvae. A third type of cell is of morphologically indifferent appearance. Catecholamine-specific formaldehyde-induced fluorescence first appears clearly at 14 mm; with growth, the number of fluorescent cells increases. Series of larvae were injected intraperitoneally with nerve growth factor (NGF), six 30-unit injections over 2 weeks. NGF treatment increases the number of neurons apparent in 24-mm larvae. Furthermore, differentiated neurons occur in NGF-treated 20-mm larvae (about 4 weeks posthatching), when there are none in controls. The early appearance of differentiated chromaffin cells and the relatively late appearance of differentiated sympathetic neurons suggest that adrenergic functions during the first few weeks of larval life are controlled humorally by the chromaffin cells, and that at 24 mm, neurons begin to provide faster, finer control.     

The surfactant system of the lung in the Mexican axolotl Ambystoma mexicanum

The broncho-alveolar space of axolotl contains numerous osmiophilic structures, which have been classified morphologically into 3 types of inclusions. Type I inclusions exhibited lattices of square to rectangular grid patterns with membranous elements 6 nm thick. This lattice was crossed by a 2 nm dense line. Type II inclusions were composed of 7 nm dark lines and 15.5 nm light lines in an alternating repeating pattern. Furthermore, the light lines showed an intrapenoid line of 2 nm. Type III inclusions consisted of concentric whorls of lamellae similar in form to spider webs. Only type III inclusions were identified in grand alveolar cells.     

Two actin variants in developing axolotl heart

Heart development in the Mexican salamander, Ambystoma mexicanum has been studied using two-dimensional gel electrophoresis and electron microscopy. At the preheart beat stage two forms of action, α and β, are present in the heart in approximately equal quantity, however, very few definitive thin filaments can be distinguished at this stage. Once the heart beat is initiated and heart development progresses α-actin increases relative to β-actin. This increase in muscle-specific actin is coincidental with the appearance of numerous thin filaments in the myocyte.     

Shark Ig light chain junctions are as diverse as in heavy chains

We have characterized a small family of four genes encoding one of the three nurse shark Ig L chain isotypes, called NS5. All NS5 cDNA sequences are encoded by three loci, of which two are organized as conventional clusters, each consisting of a V and J gene segment that can recombine and one C region exon; the third contains a germline-joined VJ in-frame and the fourth locus is a pseudogene. This is the second nurse shark L chain type where both germline-joined and split V-J organizations have been found. Since there are only two rearranging Ig loci, it was possible for the first time to examine junctional diversity in defined fish Ig genes, comparing productive vs nonproductive rearrangements. N region addition was found to be considerably more extensive in length and in frequency than any other vertebrate L chain so far reported and rivals that in H chain. We put forth the speculation that the unprecedented efficiency of N region addition (87-93% of NS5 sequences) may be a result not only of simultaneous H and L chain rearrangement in the shark but also of processing events that afford greater accessibility of the V or J gene coding ends to terminal deoxynucleotidyltransferase.     

Antibody repertoire development in fetal and neonatal piglets. II. Characterization of heavy chain complementarity-determining region 3 diversity in the developing fetus

Since the actual combinatorial diversity in the V(H) repertoire in fetal piglets represents <1% of the potential in mice and humans, we wondered whether 1) complementarity-determining region 3 (CDR3) diversity was also restricted; 2) CDR3 diversity changed with fetal age; and 3) to what extent CDR3 contributed to the preimmune VDJ repertoire. CDR3 spectratyping and sequence analyses of 213 CDR3s recovered from >30 fetal animals of different ages showed that >95% of VDJ diversity resulted from junctional diversity. Unlike sheep and cattle, somatic hypermutation does not contribute to the repertoire. These studies also revealed that 1) N region additions are as extensive in VDJ rearrangements recovered at 30 days as those in late term fetuses, suggesting that TdT is fully active at the onset of VDJ rearrangement; 2) nearly 90% of all rearrangement are in-frame until late gestation; 3) the oligoclonal CDR3 spectratype of 30-day fetal liver becomes polyclonal by 50 days, while this change occurs much later in spleen; 4) there is little evidence of individual variation in CDR3 spectratype or differences in spectratype among lymphoid tissues with the exception of the thymus; and 4) there is a tendency for usage of the most J(H) proximal D(H) segment (D(H)B) to decrease in older fetuses and for the longer D(H) segment to be trimmed to the same length as the shorter D(H) when used in CDR3. These findings suggest that in the fetal piglet, highly restricted combinatorial diversity and the lack of somatic mutation are compensated by early onset of TdT activity and other mechanisms that contribute to CDR3 junctional diversity.     

Organization of human immunoglobulin heavy chain diversity gene loci

The variable region of immunoglobulin heavy chain is encoded by three separate genes on the germline genome: variable (VH), diversity (DH) and joining (JH) genes. Most human DH genes are encoded in 9-kb repeating sequences. We determined the nucleotide sequence of a 15-kb DNA fragment containing more than one and a half of these repeating units, and identified 12 different DH genes. Based on the sequence similarities of DH coding and the surrounding regions, they can be classified into six different DH gene families (DXP, DA, DK, DN, DM and DLR). Nucleotide sequences of DH genes belonging to different families diverge greatly, while those belonging to the same families are well conserved. Since the 9-kb DNA containing the six DH genes are multiplied at least five times, the total number of DH genes must be approximately 30. These DH genes are sandwiched by 12-nucleotide spacer signals. Most of the somatic DH sequences found in the published VH-DH-JH structures (the somatic DH segment being defined as the region which is not encoded either by germline VH or JH gene) were assigned to one of the germline DH genes. Other than these typical DH genes, however, we found a new kind of DH gene (which we termed DIR) the spacer lengths of whose neighbouring signals were irregular. The DIR gene appears to be involved in DIR-DH or DH-DIR joining by inversion or deletion. Two of the somatic DH sequences were assigned to the DIR genes. Long N segments might, therefore, originate from DIR genes.     

An introduction to the Mexican axolotl (Ambystoma mexicanum)

A number of unusual traits, including a remarkable capacity for wound healing and limb regeneration, make the axolotl an interesting animal model. The author provides an overview of axolotl care and use in biomedical research.