Stem cell proliferation and differentiation during larval metamorphosis of the model tapeworm Hymenolepis microstoma.

Background: Tapeworm larvae cause important diseases in humans and domestic animals. During infection, the first larval stage undergoes a metamorphosis where tissues are formed de novo from a population of stem cells called germinative cells. This process is difficult to study for human pathogens, as these larvae are infectious and difficult to obtain in the laboratory. Methods: In this work, we analyzed cell proliferation and differentiation during larval metamorphosis in the model tapeworm Hymenolepis microstoma, by in vivo labelling of proliferating cells with the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU), tracing their differentiation with a suite of specific molecular markers for different cell types. Results: Proliferating cells are very abundant and fast-cycling during early metamorphosis: the total number of cells duplicates every ten hours, and the length of G2 is only 75 minutes. New tegumental, muscle and nerve cells differentiate from this pool of proliferating germinative cells, and these processes are very fast, as differentiation markers for neurons and muscle cells appear within 24 hours after exiting the cell cycle, and fusion of new cells to the tegumental syncytium can be detected after only 4 hours. Tegumental and muscle cells appear from early stages of metamorphosis (24 to 48 hours post-infection); in contrast, most markers for differentiating neurons appear later, and the detection of synapsin and neuropeptides correlates with scolex retraction. Finally, we identified populations of proliferating cells that express conserved genes associated with neuronal progenitors and precursors, suggesting the existence of tissue-specific lineages among germinative cells. Discussion: These results provide for the first time a comprehensive view of the development of new tissues during tapeworm larval metamorphosis, providing a framework for similar studies in human and veterinary pathogens.

Transcriptome analysis of the response to thyroid hormone in Xenopus neural stem and progenitor cells.

BACKGROUND: The thyroid hormones-thyroxine (T4) and 3,5,3'triiodothyronine (T3)-regulate the development of the central nervous system (CNS) in vertebrates by acting in different cell types. Although several T3 target genes have been identified in the brain, the changes in the transcriptome in response to T3 specifically in neural stem and progenitor cells (NSPCs) during the early steps of NSPCs activation and neurogenesis have not been studied in vivo. Here, we characterized the transcriptome of FACS-sorted NSPCs in response to T3 during Xenopus laevis metamorphosis. RESULTS: We identified 1252 upregulated and 726 downregulated genes after 16 hours of T3 exposure. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that T3-upregulated genes were significantly enriched in rRNA processing and maturation, protein folding, ribosome biogenesis, translation, mitochondrial function, and proteasome. These results suggest that NSPCs activation induced by T3 is characterized by an early proteome remodeling through the synthesis of the translation machinery and the degradation of proteins by the proteasome. CONCLUSION: This work provides new insights into the dynamics of activation of NPSCs in vivo in response to T3 during a critical period of neurogenesis in the metamorphosis.

Cornifelin expression during Xenopus laevis metamorphosis and in response to spinal cord injury.

BACKGROUND: In a high-throughput RNA sequencing analysis, comparing the transcriptional response between Xenopus laevis regenerative and non-regenerative stages to spinal cord injury, cornifelin was found among the most highly differentially expressed genes. Cornifelin is mainly expressed in stratified squamous epithelia, but its expression in the spinal cord and other central nervous structures has only been described during early development. RESULTS: Here, we report cornifelin expression in the spinal cord, retina, and cornea throughout metamorphosis and in the spinal cord after injury. Cornifelin was detected in the grey matter and meninges of the spinal cord from NF-50 to NF-66, with decreased expression in the grey matter during metamorphosis. In the retina, cornifelin was expressed in the ganglion cell layer, the inner and outer nuclear layer, and the outer segment from NF-50 to NF-66. After spinal cord injury, we only observed cornifelin upregulation in NF-66 but no significant changes in NF-50. However, we found cornifelin positive cells in NF-50 meninges closing the spinal cord stumps 1 day after injury and delineating the borders of the spinal cord following the continuity of tissue regeneration in the following days after injury. Instead, in NF-66, cornifelin positive cells were distributed to the ventral side of the spinal cord at 6 days after injury, and at the injury gap at 10 days after injury. CONCLUSIONS: Cornifelin is expressed in the Xenopus laevis spinal cord and eye during metamorphosis and plays a role in the meningeal response to spinal cord injury.

Transcriptome dynamics during metamorphosis of imaginal discs into wings and thoracic dorsum in Apis mellifera castes.

BACKGROUND: Much of the complex anatomy of a holometabolous insect is built from disc-shaped epithelial structures found inside the larva, i.e., the imaginal discs, which undergo a rapid differentiation during metamorphosis. Imaginal discs-derived structures, like wings, are built through the action of genes under precise regulation. RESULTS: We analyzed 30 honeybee transcriptomes in the search for the gene expression needed for wings and thoracic dorsum construction from the larval wing discs primordia. Analyses were carried out before, during, and after the metamorphic molt and using worker and queen castes. Our RNA-seq libraries revealed 13,202 genes, representing 86.2% of the honeybee annotated genes. Gene Ontology analysis revealed functional terms that were caste-specific or shared by workers and queens. Genes expressed in wing discs and descendant structures showed differential expression profiles dynamics in premetamorphic, metamorphic and postmetamorphic developmental phases, and also between castes. At the metamorphic molt, when ecdysteroids peak, the wing buds of workers showed maximal gene upregulation comparatively to queens, thus underscoring differences in gene expression between castes at the height of the larval-pupal transition. Analysis of small RNA libraries of wing buds allowed us to build miRNA-mRNA interaction networks to predict the regulation of genes expressed during wing discs development. CONCLUSION: Together, these data reveal gene expression dynamics leading to wings and thoracic dorsum formation from the wing discs, besides highlighting caste-specific differences during wing discs metamorphosis.

A role for Lin-28 in growth and metamorphosis in Drosophila melanogaster.

Insect metamorphosis has been a classic model to understand the role of hormones in growth and timing of developmental transitions. In addition to hormones, transitions in some species are regulated by genetic programs, such as the heterochronic gene network discovered in C. elegans. However, the functional link between hormones and heterochronic genes is not clear. The heterochronic gene lin-28 is involved in the maintenance of stem cells, growth and developmental timing in vertebrates. In this work, we used gain-of-function and loss-of-function experiments to study the role of Lin-28 in larval growth and the timing of metamorphosis of Drosophila melanogaster. During the late third instar stage, Lin-28 is mainly expressed in neurons of the central nervous system and in the intestine. Loss-of-function lin-28 mutant larvae are smaller and the larval-to-pupal transition is accelerated. This faster transition correlates with increased levels of ecdysone direct target genes such as Broad-Complex (BR-C) and Ecdysone Receptor (EcR). Overexpression of Lin-28 does not affect the timing of pupariation but most animals are not able to eclose, suggesting defects in metamorphosis. Overexpression of human Lin-28 results in delayed pupariation and the death of animals during metamorphosis. Altogether, these results suggest that Lin-28 is involved in the control of growth during larval development and in the timing and progression of metamorphosis.

Transcriptional regulation of genes involved in retinoic acid metabolism in Senegalese sole larvae.

The aim of this study was the characterization of transcriptional regulatory pathways mediated by retinoic acid (RA) in Senegalese sole larvae. For this purpose, pre-metamorphic larvae were treated with a low concentration of DEAB, an inhibitor of RALDH enzyme, until the end of metamorphosis. No differences in growth, eye migration or survival were observed. Nevertheless, gene expression analysis revealed a total of 20 transcripts differentially expressed during larval development and only six related with DEAB treatments directly involved in RA metabolism and actions (rdh10a, aldh1a2, crbp1, igf2r, rarg and cyp26a1) to adapt to a low-RA environment. In a second experiment, post-metamorphic larvae were exposed to the all-trans RA (atRA) observing an opposite regulation for those genes involved in RA synthesis and degradation (rdh10a, aldh1a2, crbp1 and cyp26a1) as well as other related with thyroid- (dio2) and IGF-axes (igfbp1, igf2r and igfbp5) to balance RA levels. In a third experiment, DEAB-pretreated post-metamorphic larvae were exposed to atRA and TTNPB (a specific RAR agonist). Both drugs down-regulated rdh10a and aldh1a2 and up-regulated cyp26a1 expression demonstrating their important role in RA homeostasis. Moreover, five retinoic receptors that mediate RA actions, the thyroid receptor thrb, and five IGF binding proteins changed differentially their expression. Overall, this study demonstrates that exogenous RA modulates the expression of some genes involved in the RA synthesis, degradation and cellular transport through RAR-mediated regulatory pathways establishing a negative feedback regulatory mechanism necessary to balance endogenous RA levels and gradients.

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera.

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs ß bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs ß and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs ß and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

Molecular cloning and expression analysis of Fem1b from oriental river prawn Macrobrachium nipponense.

Feminization-1 homolog b (Fem1b) is one of the genes essential for male development and play central roles in sex determination of Caenorhabditis elegans. In this study, we cloned and characterized the full-length Fem1b cDNA from the freshwater prawn Macrobrachium nipponense (MnFem1b) in different tissues and at different developmental stages. Real-time quantitative reverse polymerase chain reaction (RT-qPCR) showed that the MnFem1b gene was expressed in all investigated tissues, with the highest expression level found in the testes. The results revealed that the MnFem1b gene might play roles in aspects of development of the male prawn phenotype. The RT-qPCR also revealed that MnFem1b mRNA expression was significantly increased at 10 days after metamorphosis. The expression levels in all investigated tissues showed a certain degree of sexually dimorphism, the expression levels in males were significantly higher than those in females (P < 0.05). Notably, the highest expression of MnFem1b was found in the testes. The expression of MnFem1b in different tissues indicates that it plays multiple biological functions in M. nipponense.

Molecular cloning, characterization, and expression analysis of a Broad-Complex homolog during development in the oriental river prawn Macrobrachium nipponense.

Broad-Complex (BR-C) is an early ecdysone-responsive gene encoding a family of zinc-finger transcription factors. In this study, we isolated the full-length cDNA of a BR-C homolog from the testes of the oriental river prawn (Macrobrachium nipponense), according to established expressed sequence tag information, using the rapid amplification of cDNA ends technique. The homolog was designated as MnBR-C. The full-length cDNA of MnBR-C contained a 1095-bp open reading frame encoding a precursor protein of 365 amino acid residues. Comparative and bioinformatic analyses revealed that MnBR-C exhibited a high degree of homology with BR-C proteins, and contained the BTB and Zf-H2C2-2 domains. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed that the MnBR-C expression level varied significantly in the developing embryo, postembryonic larva, and adult tissue. Real-time qPCR showed that the MnBR-C gene was expressed in all of the tissues investigated, with the highest level of expression in the brain. In addition, MnBR-C was more abundantly expressed in the testes than in the ovaries.

Genomic and functional characterization of a methoprene-tolerant gene in the kissing-bug Rhodnius prolixus.

Metamorphosis, which depends upon a fine balance between two groups of lipid-soluble hormones such as juvenile hormones (JHs) and ecdysteroids, is an important feature in insect evolution. While it is clear that the onset of metamorphosis depends on the decrease of JH levels, the way in which these hormones exert their activities is not fully understood in Triatominae species. The discovery of a Drosophila melanogaster mutant resistant to the treatment with the JH analog methoprene, led finally to the description of the methoprene-tolerant gene in Tribolium castaneum (TcMet) as a putative JH receptor. Here we present the genomic and functional characterization of an ortholog of the methoprene-tolerant gene in the hemimetabolous insect Rhodnius prolixus (RpMet). The analysis of the R. prolixus gene showed that the exonic structure is different from that described for holometabolous species, although all the critical protein motifs are well conserved. Expression analysis showed the presence of RpMet mRNA in all the tested tissues: ovary, testis, rectum, Malpighian tubules and salivary glands. When juvenile individuals were treated with RpMet specific double strand RNA (dsRNA), we observed abnormal molting events that resulted in individuals with morphological alterations (adultoids). Similarly, treatment of newly emerged fed females with dsRNA resulted in an abnormal development of the ovaries, with eggs revealing anomalies in size and accumulation of yolk, as well as a decrease in the amount of heme-binding protein. Altogether, our results validate that RpMet is involved in the transduction of JH signaling, controlling metamorphosis and reproduction in R. prolixus.

Characterization of the male-specific lethal 3 gene in the oriental river prawn, Macrobrachium nipponense.

In this study, male-specific lethal 3 homolog (Mnmsl3) was cloned and characterized from the freshwater prawn Macrobrachium nipponense (Crustacea: Decapoda: Palaemonidae) by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnmsl3 showed high-sequence homology to the insect Msl3 and contained a conserved chromatin organization modifier domain and an MORF4-related gene domain. Real-time quantitative reverse transcription-polymerase chain reaction showed that the Mnmsl3 gene was expressed in all the investigated tissues, with the highest level of expression in the testis. The expression level of Mnmsl3 between males and females was different in the gonad (testis or ovary), abdominal ganglion, and heart. The results revealed that the Mnmsl3 gene might play roles in regulating chromatin and in dosage compensation of M. nipponense. Real-time quantitative reverse transcription-polymerase chain reaction also revealed that Mnmsl3 mRNA expression was significantly increased in both 5 and 20 days post-larvae after metamorphosis, suggesting that Mnmsl3 plays complex and important roles in the early embryonic development and sex differentiation of M. nipponense.

A C-type lectin fold gene from Japanese scallop Mizuhopecten yessoensis, involved with immunity and metamorphosis.

C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins that are well known for their participation in pathogen recognition and clearance. In this study, a putative C-type lectin fold (MyCLF) gene was identified from the Japanese scallop Mizuhopecten yessoensis. The full-length of MyCLF was 645 bp, encoding a polypeptide of 167 amino acids. MyCLF carried a signal peptide of 20 amino acid residues, and a single carbohydrate recognition domain, having relatively high amino acid sequence conservation with C-type lectins reported for other bivalves. The expression of MyCLF mRNA transcripts in adult tissues, after bacterial challenge and during different developmental stages was determined using real-time quantitative RT-PCR. MyCLF was mainly distributed in the mantle, gill, and kidney. The expression of MyCLF clearly increased 3 h after Vibrio anguillarum challenge, and dropped to a minimum level after 9 h compared to the control group. During embryonic development, the expression level increased in the gastrulae, trochophore and early D-shaped larvae, decreased in D-shaped larvae, and then increased hundreds of times in metamorphosing larvae. The results suggested that MyCLF was involved in an immune response and it may play important roles during the metamorphosis phase of M. yessoensis.

Health status of tadpoles and metamorphs of Rhinella arenarum (Anura, Bufonidae) that inhabit agroecosystems and its implications for land use.

Perturbations of water bodies near agricultural and livestock systems can affect embryonic and larval stages of anurans and negatively impact adult populations and structure of amphibian communities. This study is focused on early development of Rhinella arenarum, for which body growth, abnormalities in the oral disc and genetic damage on erythrocytes were analyzed to establish the impact of agroecosystems on local populations of amphibians. Tadpoles and metamorphs of R. arenarum were collected in three agroecosystems (namely, C1, C2, and C3) and in a site without agricultural and livestock activities (SM) from central Argentina. Egg masses of C1 were extracted for breeding tadpoles under laboratory conditions (Lab). Tadpoles were in small size and lighter in weight in C1 and C2. Metamorphs were shorter and lighter in weight in C1 and C3. In SM and Lab samples, no tadpoles with abnormal LTRF (labial tooth row formula) or without labial teeth were observed. In C1, the highest frequency of abnormal LTRF was recorded and was the only site in which tadpoles without labial teeth were found. In C1 and C2 the tadpoles had highest micronucleus frequencies and nuclear abnormalities. C1 can be considered as the site with the highest anthropogenic perturbation and with less healthy tadpoles. Livestock practices such as alternating cattle between parcel and keeping a buffer between crops and water bodies, would allow a better development of the first aquatic stages that are essential for the conservation of the anuran populations.

Global gene expression shift during the transition from early neural development to late neuronal differentiation in Drosophila melanogaster.

Regulation of transcription is one of the mechanisms involved in animal development, directing changes in patterning and cell fate specification. Large temporal data series, based on microarrays across the life cycle of the fly Drosophila melanogaster, revealed the existence of groups of genes which expression increases or decreases temporally correlated during the life cycle. These groups of genes are enriched in different biological functions. Here, instead of searching for temporal coincidence in gene expression using the entire genome expression data, we searched for temporal coincidence in gene expression only within predefined catalogues of functionally related genes and investigated whether a catalogue's expression profile can be used to generate larger catalogues, enriched in genes necessary for the same function. We analyzed the expression profiles from genes already associated with early neurodevelopment and late neurodifferentiation, at embryonic stages 16 and 17 of Drosophila life cycle. We hypothesized that during this interval we would find global downregulation of genes important for early neuronal development together with global upregulation of genes necessary for the final differentiation of neurons. Our results were consistent with this hypothesis. We then investigated if the expression profile of gene catalogues representing particular processes of neural development matched the temporal sequence along which these processes occur. The profiles of genes involved in patterning, neurogenesis, axogenesis or synaptic transmission matched the prediction, with largest transcript values at the time when the corresponding biological process takes place in the embryo. Furthermore, we obtained catalogues enriched in genes involved in temporally matching functions by performing a genome-wide systematic search for genes with their highest expression levels at the corresponding embryonic intervals. These findings imply the use of gene expression data in combination with known biological information to predict the involvement of functionally uncharacterized genes in particular biological events.

Exoskeleton formation in Apis mellifera: cuticular hydrocarbons profiles and expression of desaturase and elongase genes during pupal and adult development.

Cuticular hydrocarbons (CHCs) are abundant in the superficial cuticular layer (envelope) of insects where they play roles as structural, anti-desiccation and semiochemical compounds. Many studies have investigated the CHC composition in the adult insects. However, studies on the profiles of these compounds during cuticle formation and differentiation are scarce and restrict to specific stages of a few insect species. We characterized the CHCs developmental profiles in the honeybee workers during an entire molting cycle (from pupal-to-adult ecdyses) and in mature adults (forager bees). Gas chromatography/mass spectrometry (GC/MS) analysis revealed remarkable differences in the relative quantities of CHCs, thus discriminating pupae, developing and newly-ecdysed adults, and foragers from each other. In parallel, the honeybee genome database was searched for predicted gene models using known amino acid sequences of insect enzymes catalyzing lipid desaturation (desaturases) or elongation (elongases) as queries in BLASTP analysis. The expression levels of six desaturase genes and ten elongase genes potentially involved in CHC biosynthesis were determined by reverse transcription and real time polymerase chain reaction (RT-qPCR) in the developing integument (cuticle and subjacent epidermis). Aiming to predict roles for these genes in CHC biosynthesis, the developmental profiles of CHCs and desaturase/elongase transcript levels were evaluated using Spearman correlation coefficient. This analysis pointed to differential roles for these gene products in the biosynthesis of certain CHC classes. Based on the assumption that homologous proteins may share a similar function, phylogenetic trees were reconstructed as an additional strategy to predict functions and evolutionary relationships of the honeybee desaturases and elongases. Together, these approaches highlighted the molecular complexity underlying the formation of the lesser known layer of the cuticular exoskeleton, the envelope.

Fhos encodes a Drosophila Formin-like protein participating in autophagic programmed cell death.

Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila.

Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma).

BACKGROUND: Thyroid hormones (TH) induce gene expression programs that orchestrate amphibian metamorphosis. In contrast to anurans, many salamanders do not undergo metamorphosis in nature. However, they can be induced to undergo metamorphosis via exposure to thyroxine (T4). We induced metamorphosis in juvenile Mexican axolotls (Ambystoma mexicanum) using 5 and 50 nM T4, collected epidermal tissue from the head at four time points (Days 0, 2, 12, 28), and used microarray analysis to quantify mRNA abundances. RESULTS: Individuals reared in the higher T4 concentration initiated morphological and transcriptional changes earlier and completed metamorphosis by Day 28. In contrast, initiation of metamorphosis was delayed in the lower T4 concentration and none of the individuals completed metamorphosis by Day 28. We identified 402 genes that were statistically differentially expressed by > or = two-fold between T4 treatments at one or more non-Day 0 sampling times. To complement this analysis, we used linear and quadratic regression to identify 542 and 709 genes that were differentially expressed by > or = two-fold in the 5 and 50 nM T4 treatments, respectively. CONCLUSION: We found that T4 concentration affected the timing of gene expression and the shape of temporal gene expression profiles. However, essentially all of the identified genes were similarly affected by 5 and 50 nM T4. We discuss genes and biological processes that appear to be common to salamander and anuran metamorphosis, and also highlight clear transcriptional differences. Our results show that gene expression in axolotls is diverse and precise, and that axolotls provide new insights about amphibian metamorphosis.

A comprehensive expressed sequence tag linkage map for tiger salamander and Mexican axolotl: enabling gene mapping and comparative genomics in Ambystoma.

Expressed sequence tag (EST) markers were developed for Ambystoma tigrinum tigrinum (Eastern tiger salamander) and for A. mexicanum (Mexican axolotl) to generate the first comprehensive linkage map for these model amphibians. We identified 14 large linkage groups (125.5-836.7 cM) that presumably correspond to the 14 haploid chromosomes in the Ambystoma genome. The extent of genome coverage for these linkage groups is apparently high because the total map size (5251 cM) falls within the range of theoretical estimates and is consistent with independent empirical estimates. Unlike most vertebrate species, linkage map size in Ambystoma is not strongly correlated with chromosome arm number. Presumably, the large physical genome size ( approximately 30 Gbp) is a major determinant of map size in Ambystoma. To demonstrate the utility of this resource, we mapped the position of two historically significant A. mexicanum mutants, white and melanoid, and also met, a quantitative trait locus (QTL) that contributes to variation in metamorphic timing. This new collection of EST-based PCR markers will better enable the Ambystoma system by facilitating development of new molecular probes, and the linkage map will allow comparative studies of this important vertebrate group.

Expression of HoxA5 in the heart is upregulated during thyroxin-induced metamorphosis of the Mexican axolotl (Ambystoma mexicanum).

Widespread external and internal changes in body morphology have long been known to be hallmarks of the process of metamorphosis. However, more subtle changes, particularly at the molecular level, are only now beginning to be understood. A number of transcription factors have recently been shown to alter expression either in levels of message or in isoforms expressed. In this article, we describe a dramatic increase in the expression of the homeobox gene HoxA5 in the heart and aorta of the Mexican axolotl Ambystoma mexicanum during the process of thyroxin-induced metamorphosis. Immunohistochemical analysis with anti-HoxA5 antibody in thyroxin-induced metamorphosing animals showed a pattern of expression of HoxA5 comparable to that in spontaneously metamorphosing animals. Further, by in situ hybridization, we were able to show significant qualitative differences in the expression of this gene within the heart. Maximum HoxA5 expression occurred at the midpoint of metamorphosis in the myocardium, whereas the hearts of completely metamorphosed animals had the highest levels of expression in the epicardium and endocardium. In the aorta, smooth-muscle cells of the tunica media as well as cells of the tunica adventitia had an increase in expression of HoxA5 with thyroxin-induced metamorphosis. HoxA5 expression significantly changed in cells of the aorta and ventricle with treatment by thyroid hormone. HoxA5, a positive regulator of p53, may be involved with the apoptotic pathway in heart remodeling during amphibian metamorphosis.

Evolutionary genetics of metamorphic failure using wild-caught vs. laboratory axolotls (Ambystoma mexicanum).

In many organisms metamorphosis allows for an ecologically important habitat-shift from water to land. However, in some salamanders an adaptive life cycle mode has evolved that is characterized by metamorphic failure (paedomorphosis); these species remain in the aquatic habitat throughout the life cycle. Perhaps the most famous example of metamorphic failure is the Mexican axolotl (Ambystoma mexicanum), which has become a focal species for developmental biology since it was introduced into laboratory culture in the 1800s. Our previous genetic linkage mapping analysis, using an interspecific crossing design, demonstrated that a major gene effect underlies the expression of metamorphic failure in laboratory stocks of the Mexican axolotl. Here, we repeated this experiment using A. mexicanum that were sampled directly from their natural habitat at Lake Xochimilco, Mexico. We found no significant association between the major gene and metamorphic failure when wild-caught axolotls were used in the experimental design, although there is evidence of a smaller genetic effect. Thus, there appears to be genetic variation among Mexican axolotls (and possibly A. tigrinum tigrinum) at loci that contribute to metamorphic failure. This result suggests a role for more than one mutation and possibly artificial selection in the evolution of the major gene effect in the laboratory Mexican axolotl.