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 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.

A specific synthetic RNA promotes cardiac myofibrillogenesis in the Mexican axolotl.

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. However, the defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or RNA from endoderm mesoderm-conditioned medium. We constructed a cDNA library from total conditioned medium RNA in a pcDNAII expression vector. We screened the cDNA library by an organ culture bioassay and isolated a single clone (Cl#4), the synthetic RNA from which corrects the heart defect by promoting myofibrillogenesis. The insert size of the active clone is 166 nt in length with a unique nucleotide sequence. The anti-sense RNA from Cl#4 using SP6 RNA polymerase failed to rescue mutant hearts. The ability of this small RNA to correct the mutant heart defect suggests that the RNA probably does not act as an mRNA. While the precise mechanism of action is not yet known, on the basis of our studies to date it is very clear that the sense strand of Cl#4 RNA has the ability to promote myofibrillogenesis and rescue the mutant hearts both in vitro and in vivo.

Nucleotide sequence and expression of ribosomal protein S3 mRNA during embryogenesis in the Mexican axolotl (Ambystoma mexicanum).

We have isolated and sequenced a full-length (0.9 kb) cDNA clone of ribosomal protein S3 by subtraction hybridization using a single-stranded cDNA library from stage 25-27 (tracer) and the mRNA from stage 15-17 (driver) of embryonic Mexican axolotl (Ambystoma mexicanum). The axolotl is a unique animal model for studying heart development as well as myofibrillogenesis because it carries a mutation in gene c. The deduced amino acid sequence of axolotl S3 protein shows about 93.9% identity with human S3 protein over a 243 amino acid residue overlap. When compared with mouse and Xenopus laevis ribosomal S3 proteins, the axolotl sequenc shows 94.3 and 93.9% identity respectively. Interestingly, the axolotl S3 sequence shows higher identity at the nucleic acid level with human and/or other mammals than with Xenopus. The S3 transcript, as determined by RT-PCR, is present at stage 2-4 in a lower amount and the onset of transcription is most likely at the beginning of gastrulation (10-12). The expression level of S3 transcripts reaches a maximum by mid gastrulation (stages 13-14) and then follows a biphasic pattern being lower at stages 16-17 with subsequently steady increases until the mid tailbud stages (25-27).