Molecular biology of heart development in the Mexican axolotl, Ambystoma mexicanum.
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Ambystoma↗
Biomedical subjects
L F Lemanski
Publications and source records attributed to L F Lemanski.
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.
Ambystoma↗
Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls (Ambystoma mexicanum).
Regulated assembly of contractile proteins into sarcomeric structures, such as A- and I-bands, is still currently being defined. The presence of distinct isoforms of several muscle proteins suggests a possible mechanism by which myocytes regulate assembly during myofibrillogenesis. Of several muscle isoforms located within the A-band, myosin binding proteins (MyBP) are reported to be involved in the regulation and stabilization of thick filaments during sarcomere assembly. The present confocal study characterizes the expression of one of these myosin binding proteins, C-protein (MyBP-C) in wild-type and cardiac lethal mutant embryos of the axolotl, Ambystoma mexicanum. C-protein isoforms are also detected in distinct temporal patterns in whole-mounted heart tubes and thoracic skeletal muscles. Confocal analysis of axolotl embryos shows both cardiac and skeletal muscles to regulate the expression of C-protein isoforms over a specific developmental window. Although the CPROAxslow isoform is present during the initial heartbeat stage, its expression is not retained in the adult heart. C-protein isoforms are simultaneously expressed in both cardiac and skeletal muscle during embryogenesis.
Ambystoma↗
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).
Ambystoma mexicanum↗
A method for the isolation and culture of embryonic cardiomyocytes from Mexican axolotl.
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Ambystoma↗
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).
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.
Ambystoma↗
Cloning, sequencing and expression of an isoform of cardiac C-protein from the Mexican axolotl (Ambystoma mexicanum).
C-protein, a myosin binding protein, is thought to regulate and stabilize thick filaments during assembly of sarcomeric A-bands. Multiple isoforms of C-protein have been characterized in avian and mammalian systems. We now report the isolation and the nucleic acid sequence of a partial C-protein cDNA clone from an axolotl heart cDNA expression library in lambda gt11. The clone was isolated by screening the library with a heterologous monoclonal anti-C-protein antibody (MF1). Sequence comparison revealed that CPROAxocard1 has an average sequence identity of 62-68% at the nuclei acid and 72-78% at the amino acid levels respectively to human and chicken sequences. We could not detect any significant differences at the levels of expression of the cardiac isoform of C-protein (CPROAxocard1) in normal and non-beating heart tissues of the double-recessive cardiac lethal mutant (c/c) axolotl, Ambystoma mexicanum. This is the first report of a C-protein sequence from an amphibian species.
Ambystoma mexicanum↗
Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl, Ambystoma mexicanum.
Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and alpha-actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, local- izes only in skeletal muscle of the axolotl. This slow axolotl isoform (Axslow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C315 antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Axcard2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.
Actins↗
Activin A and transforming growth factor-beta stimulate heart formation in axolotls but not rescue cardiac lethal mutants.
In the Mexican axolotl (salamander), Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts lack organized sarcomeric myofibrils and do not contract throughout their lengths. We have previously shown that RNA purified from normal anterior endoderm or from juvenile heart tissue is able to rescue mutant embryonic hearts in an in vitro organ culture system. Under these conditions as many as 55% of formerly quiescent mutant hearts initiate regular contractions within 48 hours. After earlier reports that transforming growth factor-beta 1 and, to a lesser extent, platelet-derived growth factor-BB could substitute for anterior endoderm as a promoter of cardiac mesodermal differentiation in normal axolotl embryos, we decided to examine the effect of growth factors in the cardiac mutant axolotl system. In one type of experiment, stage 35 mutant hearts were incubated in activin A, transforming growth factors-beta 1 or beta 2, platelet-derived growth factor, or epidermal growth factor, but no rescue of mutant hearts was achieved. Considering the possibility that growth factors would only be effective at earlier stages of development, we tested transforming growth factors-beta 1 and beta 5, and activin A on normal and mutant precardiac mesoderm explanted in the absence of endoderm at neurula stage 14. We found that, although these growth factors stimulated heart tube formation in both normal and mutant mesoderm explants, only normal explants contained contractile myocardial tissue. We hypothesize that transforming growth factor-beta superfamily peptides initiate a cascade of responses in mesoderm that result in both changes in cell shape (the basis for heart morphogenesis) and terminal myocardial cytodifferentiation. The cardiac lethal mutation appears to be deficient only in the latter process.
Activins↗
Confocal microscopy of a newly identified protein associated with heart development in the Mexican axolotl.
Recessive mutant gene c for "'cardiac nonfunction" in the mexican axolotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 weeks after fertilization, but eventually die from a lack of circulation. Morphological studies show that mutant hearts lack organized sarcomeric myofibrils. This abnormality can be corrected by co-culturing early mutant hearts with normal anterior endoderm/mesoderm tissues, by culturing them in a medium "conditioned" by this normal tissue, or by RNA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is constructed using this RNA. On the basis of sequence analyses on this cDNA library, it was estimated that 56% of the total RNA present in the conditioned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribosomal RNAs that showed no significant homology with other known sequences in the Genebank was examined further. An RT-PCR analysis showed that this RNA (designated "N1") is expressed in juvenile skeletal muscle, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide antibodies were produced against the most antigenic portion of the polypeptide which was deduced from this RNA. Western blot analyses of adult heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analyzed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investigated to determine its localization in normal isolated hearts at embryonic stages 35, 38, and 41 and on cross-sections through the heart regions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 using immunohistochemical techniques and confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.
Ambystoma mexicanum↗
In vivo protein synthesis in developing hearts of normal and cardiac mutant axolotls (Ambystoma mexicanum).
Recessive mutant gene c in axolotls causes a failure of the hearts of affected embryos to function. The mutant hearts (c/c) lack organized sarcomeric myofibrils. The present study was undertaken to determine the overall pattern of in vivo protein synthesis and subsequent accumulation of the newly synthesized proteins for a 24-h period in normal (+/+ or +/c) and cardiac mutant (c/c) axolotl hearts at various stages of development. Additionally, selected cytoskeletal/myofibrillar proteins were analyzed in detail for their synthesis during heart development. For such analyses, the hearts were radiolabeled with 35S-methionine for 24 h and subjected to SDS-PAGE and autoradiography. Quantitative densitometric analyses of the bands show that, even though the overall protein pattern is similar in normal and mutant heart tissues, a general reduction in the synthesis of the proteins in mutant hearts is observed even at the earlier stages of development (stages 35-36 and 37-38). Synthesis and accumulation of most of the proteins is significantly inhibited in mutant hearts at later stages (stages 41-42). Tropomyosin synthesis in mutant hearts is at a level of only 72.6% of that in normal embryonic hearts at stage 35. The synthesis and the accumulation of the tropomyosin in mutant hearts decreases further with increasing age until the protein essentially stops being synthesized by stage 41.
Ambystoma↗
The cardiac mutant gene c in axolotls: cellular, developmental, and molecular studies.
The cardiac mutant axolotl is an interesting model for studying heart development. The mutant gene results in a failure of heart cells to form organized myofibrils and as a consequence the heart fails to beat. Experiments have shown that mutant hearts can be "rescued" (i.e., turned into normally contracting organs) by the addition of RNA purified from conditioned media produced by normal embryonic anterior endoderm-mesoderm cultures. These corrected hearts form myofibrils of normal morphology. New advances in recombinant DNA technology applied to this system should provide significant insights into the regulatory mechanisms of myofibrillogenesis as well as the inductive processes related to the control of gene expression during embryonic heart development. In a broader biological sense, the use of gene c in axolotls is potentially capable of helping to solve major unanswered questions in modern biology related to the genetic regulation of differentiation in vertebrates.
Ambystoma↗
Molecular cloning, sequencing and expression of an isoform of cardiac alpha-tropomyosin from the Mexican axolotl (Ambystoma mexicanum).
The cDNA for alpha-tropomyosin (TM) was cloned by the polymerase chain reaction (PCR) from a lambda gt11 library constructed with mRNA from juvenile axolotl heart tissues. Subsequently, the nucleotide sequence of the cDNA was determined. This is the first reported cDNA for axolotl alpha-tropomyosin. Comparative analyses of the deduced amino acid sequence of this cDNA with Xenopus skeletal muscle alpha-tropomyosin sequences indicate that the axolotl heart cDNA has 93% and 96% homology in the regions of amino acids 39-80 (exon 2b) and 258-284 (exon 9a), respectively. However, there is about 86.55% homology at the nucleic acid level (coding region) and 97.5% homology at the amino acid level with that of Xenopus skeletal muscle alpha-tropomyosin cDNA. Northern blot analyses with polyA+ RNA from juvenile heart suggest the presence of at least two different transcripts for alpha-tropomyosin in axolotl heart. The results of 3'-RACE concur with those of northern blot analyses.
Ambystoma mexicanum↗
Immunofluorescent studies on titin and myosin in developing hearts of normal and cardiac mutant axolotls.
Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34-35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26-35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35-42. At tailbud stage 26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32-33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages.
Aging↗
Localization and distribution of gap junctions in normal and cardiomyopathic hamster heart.
Gap junctions in mammalian heart function to provide low-resistance channels between adjacent cells for passage of ions and small molecules. It is clear that the almost unrestricted passage of ions between cells, ionic coupling, is required for coordinate and synchronous contraction. This knowledge of gap junction function has made it important to study their properties in normal and abnormal tissues. In the present study, we analyzed gap junction distribution in normal and cardiomyopathic heart tissue utilizing immunofluorescent and electron microscopy techniques. Frozen, unfixed sections of age-matched normal and cardiomyopathic cardiac tissues were immunofluorescently stained using an antibody directed against a specific peptide sequence of the connexin-43 gap junction protein. These studies revealed a characteristic punctate staining pattern for the intercalated discs in normal tissues. Some of the intercalated discs in cardiomyopathic hearts appeared to stain normally; however, others stained diffusely. The pixel intensity distribution of the confocal images demonstrated a marked difference of up to 90% increase in the number of pixels in cardiomyopathic myocardium (CM), yet the pixel intensity of gap junctions had a decrease of approximately 60%. This suggests the possibility that connexin-43 is present in CM cells in significant quantity; however, it does not become localized on the membranes as in normal cells. Electron-microscopic findings corroborate these observations on CM cells by showing an irregular distribution of intercalated discs relatively smaller in size with abnormal orientation and distribution.
Animals↗
Effects of anisosmotic conditions on the cytoskeletal architecture of cultured PC12 cells.
PC12 cells show a classical volume regulatory process when submitted to hypo-osmotic conditions. The present study examined the effects of such osmotic shock on the structural organization of different cytoskeletal elements. Results were obtained by use of different light and electron microscopy techniques combined with immunostaining methods. It appeared that the osmotically induced changes in cell volume were concomitant with important modifications in the organization of the microfilament network. Microfilaments concentrated in the perinuclear area, leaving only radial extensions of poorly organized structures in the cytoplasm. The latter were the only actin structures immunologically stained in the cytoplasm and seemed to anchor to the plasma membrane. Measurements of the fluorescence intensity of PC12 cells treated with FITC-labeled phalloidin indicated a progressive depolymerization, followed by a repolymerization of F-actin. This occurs in parallel with microfilament reorganization and volume regulatory processes. The appearance of microfilament reorganization was a function of both the incubation period and the amplitude of the osmolarity changes. During the first minutes of osmotic shock, a decrease was observed in the density and length of microvilli, which normally cover the PC12 cell surfaces, suggesting an early reorganization of the underlying microfilament network. Microtubules and intermediate filament networks were not affected by the hypo-osmotic conditions.
Actin Cytoskeleton↗
Spectrin in developing normal and cardiomyopathic hamster heart.
In the present study, we compare fetal, newborn and adult normal and cardiomyopathic hearts for the presence of spectrin. Spectrin is a cytoskeletal-membrane protein found associated with the membranes and cortical regions of a variety of cell types. Its function in non-erythroid tissue is not fully understood. We determined previously that spectrin is associated with the membranes, myofibrils, intercalated discs and possibly the T-tubules of developing normal Syrian hamsters. Thus, it is a good candidate for comparison with cardiomyopathic (CM) Syrian hamster hearts since this model system exhibits myofibril and membrane abnormalities. Normal and CM hamsters were analysed at three stages of fetal development, as well as newborn, and adult stages using immunohistochemical, electron microscopic, and electrophoretic techniques. The data presented here indicate that no obvious difference exists in the distribution of spectrin between normal and CM hearts. Thus, the isoform of spectrin recognized by our antibody with the present protocols is not affected by the cardiomyopathic condition and can be ruled out as a contributor to the myofibril disarray and membrane-associated defects seen in these animals.
Animals↗