Characterization of axolotl lampbrush chromosomes by fluorescence in situ hybridization and immunostaining.

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

Identification of immune and non-immune cells in regenerating axolotl limbs by single-cell sequencing.

Immune cells are known to be critical for successful limb regeneration in the axolotl (Ambystoma mexicanum), but many details regarding their identity, behavior, and function are yet to be resolved. We isolated peripheral leukocytes from the blood of adult axolotls and then created two samples for single-cell sequencing: 1) peripheral leukocytes (N = 7889) and 2) peripheral leukocytes with presumptive macrophages from the intraperitoneal cavity (N = 4998). Using k-means clustering, we identified 6 cell populations from each sample that presented gene expression patterns indicative of erythrocyte, thrombocyte, neutrophil, B-cell, T-cell, and myeloid cell populations. A seventh, presumptive macrophage cell population was identified uniquely from sample 2. We then isolated cells from amputated axolotl limbs at 1 and 6 days post-amputation (DPA) and performed single cell sequencing (N = 8272 and 9906 cells respectively) to identify immune and non-immune cell populations. Using k-means clustering, we identified 8 cell populations overall, with the majority of cells expressing erythrocyte-specific genes. Even though erythrocytes predominated, we used an unbiased approach to identify infiltrating neutrophil, macrophage, and lymphocyte populations at both time points. Additionally, populations expressing genes for epidermal cells, fibroblast-like cells, and endothelial cells were also identified. Consistent with results from previous experimental studies, neutrophils were more abundant at 1 DPA than 6 DPA, while macrophages and non-immune cells exhibited inverse abundance patterns. Of note, we identified a small population of fibroblast-like cells at 1 DPA that was represented by considerably more cells at 6 DPA. We hypothesize that these are early progenitor cells that give rise to the blastema. The enriched gene sets from our work will aid future single-cell investigations of immune cell diversity and function during axolotl limb regeneration.

Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl.

The axolotl (Ambystoma mexicanum) salamander, a urodele amphibian, has an exceptional regenerative capacity to fully restore an amputated limb throughout the life-long lasting neoteny. By contrast, when axolotls are experimentally induced to metamorphosis, attenuation of the limb's regenerative competence is noticeable. Here, we sought to discern the proteomic profiles of the early stages of blastema formation of neotenic and metamorphic axolotls after limb amputation by employing LC-MS/MS technology. We quantified a total of 714 proteins and qRT-PCR for selected genes was performed to validate the proteomics results and provide evidence for the putative link between immune system activity and regenerative potential. This study provides new insights for examination of common and distinct molecular mechanisms in regeneration-permissive neotenic and regeneration-deficient metamorphic stages at the proteome level.

Molecular and biochemical characterization of the Mexican axolotl CD3 (CD3ε and CD3γ/δ).

In mammals, the T-cell receptor (TCR) complex expressed on mature T-cells consists of α/ß or γ/δ clonotypic heterodimers non-covalently associated with four invariant chains forming the CD3 complex (CD3γ, CD3δ, CD3ε and CD3ζ). The TCR is the unit implicated in the antigenic peptide recognition whereas the CD3 subunits present as three different dimers (δ-ε, γ-ε and ζ-ζ) in the receptor complex participate to the signal transduction and are indispensable for the expression of the TCR at the cell surface. We report the cloning, characterization and expression analysis of CD3γ/δ and CD3ε genes in an amphibian urodele, the Mexican axolotl. Amino acid comparisons show that important motifs and residues were preserved between the axolotl CD3 chains and various vertebrate CD3ε, CD3γ, CD3δ and CD3γ/δ chains. During ontogeny, CD3ε transcripts are first detected in the dorsal region of tail-bud embryos before thymus organogenesis. CD3γ/δ transcripts are first detected in the head of 4-week-old larvae. A cross-reactive polyclonal anti-CD3ε antibody was used for the co-immunoprecipitation of the two CD3 proteins of 25 and 29 kDa, respectively, associated with the 90-kDa αß TCR heterodimer.

Meningeal Foam Cells and Ependymal Cells in Axolotl Spinal Cord Regeneration.

A previously unreported population of foam cells (foamy macrophages) accumulates in the invasive fibrotic meninges during gap regeneration of transected adult Axolotl spinal cord (salamander Ambystoma mexicanum) and may act beneficially. Multinucleated giant cells (MNGCs) also occurred in the fibrotic meninges. Actin-label localization and transmission electron microscopy showed characteristic foam cell and MNGC podosome and ruffled border-containing sealing ring structures involved in substratum attachment, with characteristic intermediate filament accumulations surrounding nuclei. These cells co-localized with regenerating cord ependymal cell (ependymoglial) outgrowth. Phase contrast-bright droplets labeled with Oil Red O, DiI, and DyRect polar lipid live cell label showed accumulated foamy macrophages to be heavily lipid-laden, while reactive ependymoglia contained smaller lipid droplets. Both cell types contained both neutral and polar lipids in lipid droplets. Foamy macrophages and ependymoglia expressed the lipid scavenger receptor CD36 (fatty acid translocase) and the co-transporter toll-like receptor-4 (TLR4). Competitive inhibitor treatment using the modified fatty acid Sulfo-N-succinimidyl Oleate verified the role of the lipid scavenger receptor CD36 in lipid uptake studies in vitro. Fluoromyelin staining showed both cell types took up myelin fragments in situ during the regeneration process. Foam cells took up DiI-Ox-LDL and DiI-myelin fragments in vitro while ependymoglia took up only DiI-myelin in vitro. Both cell types expressed the cysteine proteinase cathepsin K, with foam cells sequestering cathepsin K within the sealing ring adjacent to the culture substratum. The two cell types act as sinks for Ox-LDL and myelin fragments within the lesion site, with foamy macrophages showing more Ox-LDL uptake activity. Cathepsin K activity and cellular localization suggested that foamy macrophages digest ECM within reactive meninges, while ependymal cells act from within the spinal cord tissue during outgrowth into the lesion site, acting in complementary fashion. Small MNGCs also expressed lipid transporters and showed cathepsin K activity. Comparison of 3H-glucosamine uptake in ependymal cells and foam cells showed that only ependymal cells produce glycosaminoglycan and proteoglycan-containing ECM, while the cathepsin studies showed both cell types remove ECM. Interaction of foam cells and ependymoglia in vitro supported the dispersion of ependymal outgrowth associated with tissue reconstruction in Axolotl spinal cord regeneration.

Transcriptional response of Mexican axolotls to Ambystoma tigrinum virus (ATV) infection.

BACKGROUND: Very little is known about the immunological responses of amphibians to pathogens that are causing global population declines. We used a custom microarray gene chip to characterize gene expression responses of axolotls (Ambystoma mexicanum) to an emerging viral pathogen, Ambystoma tigrinum virus (ATV). RESULT: At 0, 24, 72, and 144 hours post-infection, spleen and lung samples were removed for estimation of host mRNA abundance and viral load. A total of 158 up-regulated and 105 down-regulated genes were identified across all time points using statistical and fold level criteria. The presumptive functions of these genes suggest a robust innate immune and antiviral gene expression response is initiated by A. mexicanum as early as 24 hours after ATV infection. At 24 hours, we observed transcript abundance changes for genes that are associated with phagocytosis and cytokine signaling, complement, and other general immune and defense responses. By 144 hours, we observed gene expression changes indicating host-mediated cell death, inflammation, and cytotoxicity. CONCLUSION: Although A. mexicanum appears to mount a robust innate immune response, we did not observe gene expression changes indicative of lymphocyte proliferation in the spleen, which is associated with clearance of Frog 3 iridovirus in adult Xenopus. We speculate that ATV may be especially lethal to A. mexicanum and related tiger salamanders because they lack proliferative lymphocyte responses that are needed to clear highly virulent iridoviruses. Genes identified from this study provide important new resources to investigate ATV disease pathology and host-pathogen dynamics in natural populations.

IgX antibodies in the urodele amphibian Ambystoma mexicanum.

Until recently, it was believed that urodele amphibians are able to synthesize only two immunoglobulin isotypes, IgM and IgY. We reinvestigated this issue in the Iberian ribbed newt Pleurodeles waltl and reported recently that this urodele expresses at least three isotypes: IgM, IgP and IgY. In this study, we demonstrate that another urodele, Ambystoma mexicanum, has also a third isotype whose amino acid sequence presents the highest homology with the amino acid sequence of Xenopus IgX. This isotype has typical Ig H-chain characteristics, could form multimers and is mainly expressed in mucosal tissues thereby indicating that it is likely the physiological counterpart of Xenopus IgX and mammalian IgA. Interestingly, no IgP could be found in A. mexicanum, in contrast to P. waltl, in which IgX was not found in previous investigations. These data indicate, for the first time, that different families of urodeles can express different immunoglobulin isotypes.

Role of the immune response in initiating central nervous system regeneration in vertebrates: learning from the fish.

The mammalian central nervous system is not able to regenerate neurons lost upon injury. In contrast, anamniote vertebrates show a remarkable regenerative capacity and are able to replace damaged cells and restore function. Recent studies have shown that in naturally regenerating vertebrates, such as zebrafish, inflammation is a key processes required for the initiation of regeneration. These findings are in contrast to many studies in mammals, where the central nervous system has long been viewed as an immune-privileged organ with inflammation considered one of the key negative factors causing lack of neuronal regeneration. In this review, we discuss similarities and differences between naturally regenerating vertebrates, and those with very limited to non-existing regenerative capacity. We will introduce neural stem and progenitor cells in different species and explain how they differ in their reaction to acute injury of the central nervous system. Next, we illustrate how different organisms respond to injuries by activation of their immune system. Important immune cell types will be discussed in relation to their effects on neural stem cell behavior. Finally, we will give an overview on key inflammatory mediators secreted upon injury that have been linked to activation of neural stem cells and regeneration. Overall, understanding how species with regenerative potential couple inflammation and successful regeneration will help to identify potential targets to stimulate proliferation of neural stem cells and subsequent neurogenesis in mammals and may provide targets for therapeutic intervention strategies for neurodegenerative diseases.

Characterization of an IgY-like low molecular weight immunoglobulin class in the Mexican axolotl.

The general thinking about the phylogenic distribution of vertebrate Ig classes is that fish and urodele amphibians are only able to synthesize polymeric IgM-like molecules and that the emergence of a new class of LMW Ig occurs for the first time in anouran species. Following immunization of the Mexican axolotl (Ambystoma mexicanum, Amphibia, Urodela) with TNP-SRBC, HMW anti-TNP antibody molecules are only detected. We have previously shown that these polymeric Ig are constituted of 76 kDa H-chains associated to 27-30 kDa L-chains, respectively recognized by MAbs 33.45.1 and 33.101.2. However, the euglobulin fraction purified from normal axolotl serum contains, beside HMW Ig, abundant 172 kDa molecules which are recognized by MAb 33.101.2 in Western blotting in non-reducing conditions but are not labelled with MAb 33.45.1. In the present work, we characterize this 172 kDa molecule as a LMW Ig which differs from the HMW Ig both at the level of the physicochemical and antigenic properties of their H-chain components. This new 11.9 S axolotl Ig presents some similarities with anouran IgY. The detection of IgY-like molecules in urodele amphibian extends the occurrence of at least two antigenically different H-chain isotypes to all the representative modern classes of the Tetrapoda superclass.

Monoclonal antibodies to axolotl immunoglobulins specific for different heavy chains isotypes expressed by independent lymphocyte subpopulations.

An immunoblotting analysis of purified axolotl immunoglobulins (Ig) separated by SDS-PAGE reveals two heavy (H) chains isotypes: a 76 kDA chain recognized by the monoclonal antibody (mAb) 33.45.1 and a 66-68 kDa doublet recognized by the mAb 33.39.2. The 76 kDa chain is associated to high molecular weight (HMW) Ig molecules and the 66-68 kDa H chains are associated to low molecular weight (LMW) Ig of 172 kDa. Both H chains isotypes are linked to identical light (L) chains, labelled in immunoblotting by the mAb 33.101.2. Two different axolotl lymphocyte subpopulations are characterized by these two distinct H chains isotypes. One population of splenic lymphocytes (approximately 40%) is labeled by indirect immunofluorescence with mAb 33.45.1, specific for the 76 kDa H chain isotype. Another population (approximately 20%) is labeled by mAb 33.39.2 specific for the 66-68 kDa H chain isotype. Both populations of splenic lymphocytes are stained by mAb 33.101.2 specific for the axolotl L chains. Therefore, the presence of at least two independent Ig classes is now confirmed in a urodele amphibian species at the humoral and cellular levels.

Evidence for low-molecular weight antibodies in the serum of a urodele amphibian, Ambystoma mexicanum.

Adult axolotls (A. mexicanum) were hyperimmunized with the haptenic determinant, azobenzene-arsonate (ARS). Specific antibodies were isolated from their serum by immune-affinity chromatography on immobilized ARS columns. Analysis of the specific ARS-binding molecules by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that these animals were capable of producing both high molecular-weight (presumably IgM) and low molecular-weight antibodies to the ARS hapten. The low molecular-weight anti-ARS antibody produced by one inbred colony of axolotls did not show any restricted heterogeneity, as assessed by isoelectric focusing. Our results suggest that regulatory events, not the absence of genetic information, may be responsible for the apparent lack of Ig isotype diversity in this species.

Maturation of transplantation antigens in Ambystoma mexicanum.

Both juvenile (14-16 week) and adult (18 month) Ambystoma mexicanum reject skin allografts from adult Ambystoma more speedily than they reject such grafts from juvenile axolotls. Donor-specific histocompatibility antigen, prepared from splenocytes, is more effective in inhibiting adult host splenocyte migration when the antigen is prepared from spleen cells from adult, rather than from juvenile Ambystoma. The thymus is fully developed in juvenile Ambystoma, suggesting that the delayed kinetics of rejection of juvenile allografts reflects immaturity in the expression of histocompatibility antigens to donor skin cells.

Mitogen-activated axolotl (Ambystoma mexicanum) splenocytes produce a cytokine that promotes growth of homologous lymphoblasts.

Culture supernatants (PHA-SNs) from axolotl splenocytes cultured with phytohemagglutinin-P (PHA) in medium supplemented with bovine serum albumin (BSA) were collected after 1, 2, and 3 days, pooled, treated to remove residual PHA, precipitated with saturated ammonium sulfate, dialyzed, aliquoted, and stored at -20 degrees C. PHA-SNs stimulated proliferation of homologous lymphoblasts, but not resting splenocytes. SDS-PAGE of metabolically labeled PHA-SNs revealed a band between 14 and 21 kDa. This corresponds to the M(r) of the gel fractions with biological stimulatory activity eluted from PHA-SNs. Blasts absorbed significant bioactivity of PHA-SNs whereas freshly harvested splenocytes did not. Although splenocytes cultured in medium supplemented with 1% fetal bovine serum (FBS) did not proliferate in response to PHA, they did secrete a cytokine with lymphoblast growth-promoting activity. Furthermore, PHA-induced lymphoblasts, initially cultured in medium supplemented with 0.25% BSA, could proliferate in response to PHA-SNs in 1% FBS-supplemented medium.

Characterization of axolotl heavy and light immunoglobulin chains by monoclonal antibodies.

Axolotl specific antibodies to 2,4-dinitrophenyl (DNP) were purified by affinity chromatography from the sera of animals immunized with 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC). The purified anti-TNP/DNP antibodies, when analyzed by SDS-PAGE, were constituted of high molecular weight molecules, which in reducing conditions, were separated into heavy 72-88 kD and light 27-30 kD polypeptides. The axolotl heavy antibody chains strongly bound Concanavalin-A and migrate faster in SDS-PAGE after endoglycosidase-F (Endo-F) treatment. Using the same techniques, no carbohydrate components were detected onto light chains. Monoclonal antibodies (MAbs) were obtained against these purified axolotl immunoglobulins (Ig) and their specificities were studied by immunoblotting. MAbs 33.45.1 and 33.101.2 respectively recognized heavy and light chains determinants of the Ig molecule. These determinants were resistant to Endo-F digestion, suggesting that the two MAbs were not directed to polypeptide-associated N-linked high mannose or complex oligosaccharides. MAbs 33.45.1 and 33.101.2 were compared to 11.5.2, an anti-axolotl thymocytes MAb which was reactive for both axolotl leucocytes and soluble Ig. MAb 11.5.2 reacted in immunoblotting against several high molecular weight axolotl serum proteins, including heavy Ig chains. Light chains were not recognized. However, 11.5.2 did not further recognize Endo-F treated Ig, suggesting its specificity for a carbohydrate determinant of the heavy chain, and link to a large diversity of soluble or membrane glycoproteins.

CXCL14-like immunoreactivity in growth hormone-containing cells of urodele pituitaries.

Immunohistochemical techniques were employed to investigate the distribution of a chemokine, namely, CXCL14-like immunoreactivity in the axolotl (Ambystoma mexicanum) and Japanese black salamander (Hynobius nigrescens) pituitaries. CXCL14-immunoreactive cells concentrated at an area of the pars distalis adjacent to the pars intermedia. We found that these cells correspond to the cells immunoreactive to an antibody against rat growth hormone (GH). Immunoelectron microscopy indicated that the CXCL14-like substance and GH coexisted on the secretory granules in the axolotl pituitary. Western blot analysis of axolotl pituitary extracts revealed the anti-human CXCL14 antibody labeled an approximately 16.6-kDa band that was not labeled by the anti-GH antibody. The CXCL14-like substance in the pars distalis may participate in GH functions in these species.

Evolution of T cell receptor genes. Extensive diversity of V beta families in the Mexican axolotl.

We have cloned 36 different rearranged variable regions (V beta) genes encoding the beta-chain of the T cell receptor in an amphibian species, Ambystoma mexicanum (the Mexican axolotl). Eleven different V beta segments were identified, which can be classified into 9 families on the basis of a minimum of 75% nucleotide identity. All the cloned V beta segments have the canonical features of known mammalian and avian V beta, including conserved residues Cys23, Trp34, Arg69, Tyr90, and Cys92. There seems to be a greater genetic distance between the axolotl V beta families than between the different V beta families of any mammalian species examined to date: most of the axolotl V beta s have fewer than 35% identical nucleotides and the less related families (V beta 4 and V beta 8) have no more than 23.2% identity (13.5% at the amino acid level). Despite their great mutual divergence, several axolotl V beta are sequence-related to some mammalian V beta genes, like the human V beta 13 and V beta 20 segments and their murine V beta 8 and V beta 14 homologues. However, the axolotl V beta 8 and V beta 9 families are not significantly related to any other V beta sequence at the nucleotide level and show limited amino acid similarity to mammalian V alpha, V kappa III, or VH sequences. The detection of nine V beta families among 35 randomly cloned V beta segments suggests that the V beta gene repertoire in the axolotl is probably larger than presently estimated.

Activation by mitogens and superantigens of axolotl lymphocytes: functional characterization and ontogenic study.

Urodele amphibians have weak and slow immune responses compared to mammals and anuran amphibians. Using new culture conditions, we tested the ability of lymphocytes of a well-studied salamander, the Mexican axolotl (Ambystoma mexicanum) to proliferate in vitro with diverse mitogenic agents. We demonstrated that the axolotl has a population of B lymphocytes that proliferate specifically and with a high stimulation index to the lipopolysaccharide (LPS) known as a B-cell mitogen in mammals. This proliferative capacity is observed without significant changes throughout ontogenesis. In the presence of LPS, axolotl B lymphocytes are able to synthesize and secrete both isotopes of immunoglobulin described in this species, IgM and IgY. Moreover, a distinct lymphocyte subpopulation is able to poliferate significantly in response to the mitogens usually known as T-cell specific in mammals, phytohaemagglutinin (PHA) and concanavalin A (Con A). The activated cells are T lymphocytes, as shown by depletion experiments performed in vitro with monoclonal antibodies, and in vivo by thymectomy. Splenic T lymphocytes of young axolotls (before 10 months) do not have this functional ability, which suggests maturation and/or migration phenomena during T-cell ontogenesis in this species. Axolotl lymphocytes are able to proliferate in vitro with a significant stimulation index to staphylococcal enterotoxins A and B (SEA and SEB). These products act on mammalian lymphocytes as superantigens: in combination with products of the major histocompatibility complex (MHC), they bind T-cell receptors with particular V beta elements. The fact that these superantigens are able to activate lymphocytes of a primitive vertebrate suggests a striking conservation of molecular structures implied in superantigen presentation and recognition.