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.

Expression of axolotl RNA-binding protein during development of the Mexican axolotl.

Amphibians occupy a central position in phylogeny between aquatic and terrestrial vertebrates and are widely used as model systems for studying vertebrate development. We have undertaken a comprehensive molecular approach to understand the early events related to embryonic development in the Mexican axolotl, Ambystoma mexicanum, which is an exquisite animal model for such explorations. Axolotl RBP is a RNA-binding protein which was isolated from the embryonic Mexican axolotl by subtraction hybridization and was found to show highest similarity with human, mouse, and Xenopus cold-inducible RNA-binding protein (CIRP). The reverse transcriptase polymerase chain reaction (RT-PCR) analysis suggests that it is expressed in most of the axolotl tissues except liver; the expression level appears to be highest in adult brain. We have also determined the temporal and spatial pattern of its expression at various stages of development. RT-PCR and in situ hybridization analyses indicate that expression of the AxRBP gene starts at stage 10-12 (gastrula), reaches a maxima around stage 15-20 (early tailbud), and then gradually declines through stage 40 (hatching). In situ hybridization suggests that the expression is at a maximum in neural plate and neural fold at stage 15 (neurula) of embryonic development.

Cloning, sequencing and expression of a novel homeobox gene AxNox-1 from the Mexican axolotl.

We have cloned and sequenced a cDNA containing a homeobox gene, AxNox-1, from a stage 18 axolotl embryonic cDNA library which shows only moderate levels of similarity to other known homeobox genes. The nucleotide sequence of the cDNA has an open reading frame for 335 amino acids and besides the homeodomain, there is an acidic domain and a proline-rich domain present in the protein. The transcripts for this gene are detectable at stage 4 of embryonic development and, hence, there is a good possibility that the transcripts are maternally contributed. Expression levels for AxNox-1 reach maximum levels by stage 12 of development and thereafter decline to very low levels by stage 25. High levels of the transcript for AxNox-1 are later found in the brains of both neotenous and metamorphosing adult axolotls. Low amounts of the message are also found to be present in a number of other organs that were tested. In situ hybridization studies on whole mounts and sections suggest that this gene is expressed predominantly in neural tissue during development.

Cloning and sequencing of the cDNA for an RNA-binding protein from the Mexican axolotl: binding affinity of the in vitro synthesized protein.

A full length cDNA for an RNA-binding protein (axolotl RBP) with consensus sequence (RNP-CS) from the Mexican axolotl, Ambystoma mexicanum, has been cloned from a subtraction library. In vitro translation with synthetic mRNA and subsequent hybrid-arrested translation with a specific antisense oligonucleotide confirms that the axolotl RBP cDNA encodes an approx. 16 kDa polypeptide. Computer-assisted analyses revealed amino acid similarities of 58-60% to various RNA-binding proteins and a 90 amino acid region at the amino-terminal end constituting the putative RNA-binding domain (RNP-CS) with two highly conserved motifs, RNP2 and RNP1. Phylogenetic analysis suggests that the putative RNA-binding protein from axolotl is unique. A binding assay with radiolabeled axolotl RBP showed that this RNA-binding protein bound strongly with poly(A) and to a lesser degree with poly(U), but not at all with poly(G), poly(C), or DNA.

The heart of metamorphosing Mexican axolotl but not that of the cardiac mutant is associated with the upregulation of Hox A5.

The Mexican axolotl (Ambystoma mexicanum) is a facultative neotene which rarely undergoes metamorphosis in the wild. We now report for the first time a dramatic increase in the expression of HoxA5 in axolotl hearts as determined by RT-PCR and in situ hybridization analyses during spontaneous metamorphosis. The Mexican axolotl has a naturally occurring mutation called gene c which allows hearts in homozygous (c/c) embryos to form but never to beat. RT-PCR analysis has not shown any significant differences of HoxA5 expression in normal and mutant hearts. The predicted open reading frame of our already published partial cDNA clone of HoxA5 was confirmed by expressing it as a fusion protein with Glutathione transferase (GST fusion protein). Phylogenetic analysis with the deduced amino acid sequence of the isolated cDNA of the axolotl homolog of the murine HoxA5 shows that the axolotl sequence clusters more closely with the human and mouse HoxA5 homologs than with axolotl sequence. Western blot analysis revealed that anti-mouse HoxA5 antibody recognizes the axolotl HoxA5 protein.

Expression of myosin heavy chain transcripts in normal and cardiac mutant Mexican axolotls.

In this study, we have cloned a 1.0 kb myosin heavy chain (MHC) cDNA by screening an axolotl heart cDNA library with the monoclonal antibody MF20 against a light meromyosin (LMM) region of MHC. The nucleotide sequence analysis shows 85-86% homology at the amino acid and 78-81% homology at the nucleic acid level with MHC from other vertebrates. Phylogenetic analyses suggest that axolotl beta-MHC forms a cluster with the myosin II group of vertebrate striated muscles. Within the myosin II cluster, axolotl beta-MHC forms a distinct subclade from avian MHC and is instead closer to mammalian MHC. RT-PCR analyses show that transcripts of beta-MHC are present at stage 2 and the onset of the MHC gene expression is at stage 8-10 (gastrulation). Expression increases with embryonic development and reaches a maximum at stage 20. Beyond stage 35, the heart-beat initiation stage, the expression level of beta-MHC is higher in cardiac muscle than in skeletal muscle. We could not detect significant differences in the levels of expression of MHC transcripts in normal and cardiac lethal mutant (c/c) axolotls (Ambystoma mexicanum).

Identification and expression of a homologue of the murine HoxA5 gene in the Mexican axolotl (Ambystoma mexicanum).

An excellent model for studying heart development in vertebrates is the cardiac non-function lethal mutant (gene c) Mexican axolotl, Ambystoma mexicanum. In order to facilitate our analyses of the mutant system, we have undertaken a search for stage-specific molecular markers during embryonic development of the axolotl. We have concentrated on homeobox genes as suitable candidates for monitoring molecular changes during development. A 270-bp probe encoding a portion of the axolotl homeobox gene Ahox-1 was generated by PCR from a stage-18 axolotl embryonic cDNA library. 32P-labelled PCR-amplified Ahox-1 DNA was used as the probe for screening a lambda AM18 cDNA library using moderately stringent conditions. We isolated six clones and determined their partial nucleotide (nt) sequences. One of the clones, which has very high homology to human, mouse and rat Hox A5 (83 and 99% at the nt and amino-acid levels, respectively, in the homeodomain region), was analyzed further. RT-PCR analyses show that the level of expression of HoxA5 is very low at stage 11 of embryonic development (gastrula). The level of expression reaches maximum at stage 25 (tailbud) and then plateaus at stages 30 and 35 (heartbeat onset). Although the expression of Ahox-1 was also found to start at stage 11, it reaches a maximum level at stage 25 and declines at stage 35. We have also studied, using RT-PCR, the tissue-specific expression of HoxA5 and Ahox-1 in juvenile axolotl.