Heart development in normal and cardiac-lethal mutant axolotls: a model for the control of vertebrate cardiogenesis

The mechanisms which regulate myocardial differentiation are poorly understood. The cardiac-lethal (c) mutant of Ambystoma mexicanum, in which the heart never begins to beat, provides a valuable model system for studying this process. Using an in vitro assay, we examine the nature of the defect in c/c embryos and find (contrary to previous reports) that the inductive endoderm is not affected by the mutation. Rather, the pre-cardiac mesoderm is directly affected by the c gene and is incapable of responding to normal inductive influences. Furthermore, we find that mutant mesoderm can complete its differentiation into functional cardiomyocytes when co-cultured with wild-type heart mesoderm. With this evidence, we propose a model for the regulation of heart differentiation based on the migration of the heart mesoderm over a gradient of inducer, and the subsequent establishment of a two-component reaction-diffusion system within the mesoderm itself. This model has the potential to explain several poorly understood aspects of cardiogenesis, including the gradual nature of heart induction, the restriction of the heart field, and possibly the early morphogenesis of the heart tube.     

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. © 1994 Wiley-Liss, Inc.     

Cell Interactions in Cardiac Development

During early heart formation, the pre-cardiac mesoderm becomes regionally differentiated into segments destined to form ventricle, atria and sinoatrial tissue. Each region develops a characteristic beatrate and form of action potential, shaped by current through specific ion channels and membrane pumps. Fragments of pre-sinoatrial mesoderm that would normally have a rapid intrinsic beatrate, develop into beating heart tissue with a slow beatrate, characteristic of the ventricle, when transplanted into the prospective conoventricular region at stage 6–7. These transplants also express the ventricular isoform of myosin heavy chain, suggesting that regional commintment of the precardiac mesoderm is influenced by local cues. Application of the patch-clamp technique to single cells isolated from the ventricle of hearts at different ages during the first week of embryonic development has revealed changes in four currents that underlie the shaping of the ventricular action potential: the excitatory sodium current, the inward rectified K current, the delayed rectifier K current, and the T-type Ca current.     

Heart induction in wild-type and cardiac mutant axolotls (Ambystoma mexicanum).

We have re-examined some of the factors affecting the induction of heart-forming mesoderm in the axolotl. The formation of functional, rhythmically contracting myocardial tissue was used as an assay. We have found that heart-forming mesoderm is fully induced and capable of completing its developmental repertoire by the end of neurulation. As has been previously reported, pharyngeal endoderm appears to be the major inductor of heart mesoderm. Unlike previous workers, we have found that the inducing activity appears to be highly localized in the mid-ventral pharyngeal endoderm. The endoderm retains its inductive properties, and the mesoderm retains at least some capacity to respond, long after the heart-forming mesoderm is apparently fully induced. We have also found that RNA extracts from pharyngeal endoderm, which are capable of causing cardiac-lethal (c/c) mutant axolotl hearts to begin beating, are not capable of inducing early wild-type heart-forming mesoderm. Based on these results, we speculate that induction of heart-forming mesoderm is a two-step process. The first signal, occurring during neurulation, directs the mesoderm to begin differentiating into cardiomyocytes, and the second, beginning in mid- to late neurulation and continuing until just prior to the onset of heartbeat, causes myofibrillogenesis and the initiation of rhythmic contractions. The latter signal, which is lacking in c/c mutant embryos, appears to be necessary to override an inhibition present in the embryonic milieu.     

Studies of muscle proteins in embryonic myocardial cells of cardiac lethal mutant mexican axolotls (Ambystoma mexicanum) by use of heavy meromyosin binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis

In the Mexican axolotl Ambystoma mexicanum recessive mutant gene c, by way of abnormal inductive processes from surrounding tissues, results in an absence of embryonic heart function. The lack of contractions in mutant heart cells apparently results from their inability to form normally organized myofibrils, even though a few actin-like (60-A) and myosin-like (150-A) filaments are present. Amorphous "proteinaceous" collections are often visible. In the present study, heavy meromyosin (HMM) treatment of mutant heart tissue greatly increases the number of thin filaments and decorates them in the usual fashion, confirming that they are actin. The amorphous collections disappear with the addition of HMM. In addition, an analysis of the constituent proteins of normal and mutant embryonic hearts and other tissues is made by sodium dodecyl sulfate (SDS) gel electrophoresis. These experiments are in full agreement with the morphological and HMM binding studies. The gels show distinct 42,000-dalton bands for both normal and mutant hearts, supporting the presence of normal actin. During early developmental stages (Harrison's stage 34) the cardiac tissues in normal and mutant siblings have indistinguishable banding patterns, but with increasing development several differences appear. Myosin heavy chain (200,000 daltons) increases substantially in normal hearts during development but very little in mutants. Even so the quantity of 200,000-dalton protein in mutant hearts is significantly more than in any of the nonmuscle tissues studied (i.e. gut, liver, brain). Unlike normal hearts, the mutant hearts lack a prominent 34,000-dalton band, indicating that if mutants contain muscle tropomyosin at all, it is present in drastically reduced amounts. Also, mutant hearts retain large amounts of yolk proteins at stages when the platelets have virtually disappeared from normal hearts. The morphologies and electrophoresis patterns of skeletal muscle from normal and mutant siblings are identical, confirming that gene c affects only heart muscle differentiation and not skeletal muscle. The results of the study suggest that the precardiac mesoderm in cardiac lethal mutant axolotl embryos initiates but then fails to complete its differentiation into functional muscle tissue. It appears that this single gene mutation, by way of abnormal inductive processes, affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.     

A conditional mutant allele for analysis of Mixl1 function in the mouse

Mixl1 is the only member of the Mix/Bix homeobox gene family identified in mammals. During mouse embryogenesis, Mixl1 is first expressed at embryonic day (E)5.5 in cells of the visceral endoderm (VE). At the time of gastrulation, Mixl1 expression is detected in the vicinity of the primitive streak. Mixl1 is expressed in cells located within the primitive streak, in nascent mesoderm cells exiting the primitive streak, and in posterior VE overlying the primitive streak. Genetic ablation of Mixl1 in mice has revealed its crucial role in mesoderm and endoderm cell specification and tissue morphogenesis during early embryonic development. However, the early lethality of the constitutive Mixl1^?/? mutant precludes the study of its role at later stages of embryogenesis and in adult mice. To circumvent this limitation, we have generated a conditional Mixl1 allele (Mixl1^cKO) that permits temporal as well as spatial control of gene ablation. Animals homozygous for the Mixl1^cKO conditional allele were viable and fertile. Mixl1^KO/KO embryos generated by crossing of Mixl1^cKO/cKO mice with Sox2‐Cre or EIIa‐Cre transgenic mice were embryonic lethal at early somite stages. By contrast to wild‐type embryos, Mixl1^KO/KO embryos contained no detectable Mixl1, validating the Mixl1^cKO as a protein null after Cre‐mediated excision. Mixl1^KO/KO embryos resembled the previously reported Mixl1^?/? mutant phenotype. Therefore, the Mixl1 cKO allele provides a tool for investigating the temporal and tissue‐specific requirements for Mixl1 in the mouse. genesis 52:417–423, 2014. © 2014 Wiley Periodicals, Inc.     

The Cardiac Lethal Mutant of Ambystoma mexicanum: A Re-examination

SYNOPSIS. Hearts of embryonic axolotls (Ambystoma mexicanum)homozygous for gene c do not beat in situ. Under appropriateculture conditions they rapidly commence beating, albeit lessvigorously than similarly explanted hearts of comparably stagednormal siblings. As part of this symposium, we have shown acine record demonstrating heartbeat in mutant hearts and comparingit to normal heartbeat. Myocardia of normal embryos exhibita characteristic pattern of birefringent, striated myofibrils.Mutants of the same stage contain hearts with birefringent fibrilsorganized in a pattern similar to that found in normal myocardia.A striking difference is that obvious striations are lackingin fibrils of the mutant. Electrophoresis of normal and mutanthearts in SDS-polyacrylamide gels shows that the major myofibrillarproteins are present. We conclude that induction of c/c heartshas been relatively normal and suggest that, due to the rapidityof recovery, the observed phenomena are perhaps due to somethingas simple as a defect in ionic conditions.     

Morphology of developing heart in cardiac lethal mutant Mexican axolotls, Ambystoma mexicanum

In Ambystoma mexicanum, recessive mutant gene c results in an absence of embryonic heart function because of altered influences from surrounding tissues (Humphrey, 1972). The present light and electron microscope study compares heart development in normal and mutant embryos from Harrison stage 34 or 6 days (at which normal heart beat initiates) through stage 41 or 25 days (at which mutant embryos die). The hearts display increasing differences as development progresses, and by stage 41 mutant abnormalities are striking. The normal myocardium shows organized sarcomeres at stage 34 and numerous intercalated discs subsequently appear. By stage 41, the normal myocardium is composed of highly differentiated muscle cells and shows extensive trabeculation. The mutant myocardium throughout development remains only one cell layer thick with no indication of developing trabeculae. Mutant cells at stage 34 have a few 140 Å and 60 Å filaments along with what appear to be Z bodies. A partial organization of myofibrillar components is occasionally noted at stages 38–41; however, distinct sarcomeres are not apparent and intercalated discs are rarely seen. In general the mutant cells appear less differentiated than usual and in many respects are reminiscent of pre-heart-beat normal cells. Although most mutant cells show images characteristic of pathological conditions (e.g., pleomorphic mitochondria, membranous whorls, and numerous autophagic vacuoles), selective myocardial cell death, a phenomenon associated with normal trabeculation, is not evident. It is clear that gene c, in homozygous condition, results in an altered pattern of heart cell differentiation. The mutation, by way of abnormal inductive processes, appears to affect the synthesis and organization of heart contractile proteins.     

Activin A and transforming growth factor-β stimulate heart formation in axolotls but do 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-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-1 or 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-1 and 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 mesodermal explants, only normal explants contained contractile myocardial tissue. We hypothesize that transforming growth factor- 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.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.F.J. Mangiacapra and M.E. Fransen contributed equally to this work     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.     

Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm

In this study we describe the morphological and genetic analysis of the Drosophila mutant gürtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein^gürtelchen (hkb^gurt) mutant embryos.It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. Our analyses of the visceral phenotype of hkb^gurt mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion.     

Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm

A strain of axolotl, Ambystoma mexicanum, that carries the cardiac lethal or c gene presents an excellent model system in which to study inductive interactions during heart development. Embryos homozygous for gene c contain hearts that fail to beat and do not form sarcomeric myofibrils even though muscle proteins are present. Although they can survive for approximately three weeks, mutant embryos inevitably die due to lack of circulation. Embryonic axolotl hearts can be maintained easily in organ culture using only Holtfreter's solution as a culture medium. Mutant hearts can be induced to differentiate in vitro into functional cardiac muscle containing sarcomeric myofibrils by coculturing the mutant heart tube with anterior endoderm from a normal embryo. The induction of muscle differentiation can also be mediated through organ culture of mutant heart tubes in medium 'conditioned' by normal anterior endoderm. Ribonuclease was shown to abolish the ability of endoderm-conditioned medium to induce cardiac muscle differentiation. The addition of RNA extracted from normal early embryonic anterior endoderm to organ cultures of mutant hearts stimulated the differentiation of these tissues into contractile cardiac muscle containing well-organized sarcomeric myofibrils, while RNA extracted from early embryonic liver or neural tube did not induce either muscular contraction or myofibrillogenesis. Thus, RNA from anterior endoderm of normal embryos induces myofibrillogenesis and the development of contractile activity in mutant hearts, thereby correcting the genetic defect.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Impaired intermediate formation in mouse embryos expressing reduced levels of Tbx6

Intermediate mesoderm (IM) is the strip of tissue lying between the paraxial mesoderm (PAM) and the lateral plate mesoderm that gives rise to the kidneys and gonads. Chick fate mapping studies suggest that IM is specified shortly after cells leave the primitive streak and that these cells do not require external signals to express IM‐specific genes. Surgical manipulations of the chick embryo, however, revealed that PAM‐specific signals are required for IM differentiation into pronephros—the first kidney. Here, we use a genetic approach in mice to examine the dependency of IM on proper PAM formation. In Tbx6 null mutant embryos, which form 7–9 improperly patterned anterior somites, IM formation is severely compromised, while in Tbx6 hypomorphic embryos, where somites form but are improperly patterned along the axis, the impact to IM formation is lessened. These results suggest that IM and its derivatives, the kidneys and the gonads, are directly or indirectly dependent on proper PAM formation. This has implications for humans harboring Tbx6 mutations which are known to have somite‐derived defects including congenital scoliosis.     

Phenotypic consequences and genetic interactions of a null mutation in the Drosophila posterior Sex Combs gene

The Posterior Sex Combs (Psc) gene of Drosophila is a member of the Polycomb (Pc) group of transregulatory genes. Previous analyses of the function of this gene in Drosophila em-bryogenesis have been hampered by the lack of a null mutation. We recently isolated a mutation that deletes the 5′ end of the Psc gene. This allele appears to be a null mutation, and we have used it to determine the Psc zygotic null phenotype and to look at the interactions of a null allele of Psc with five other Pc group mutations. We find evidence for transformations along both the anterior-posterior and dorsal-ventral axes in embryos of a variety of genotypes that include a null mutation in Psc. The phenotypes of embryos that are doubly mutant for a null allele of Psc and a mutation in a second Pc group gene show dramatic synergistic effects, but in their specifics they are dependent on the identify of the second Pc group gene. This is different from the relatively uniform phenotypes seen among double mutants that contained the allele Psc¹, which has both gain and loss of function properties. The differences in the phenotypes of the doubly mutant embryos allow us to eliminate one class of molecular models to explain the dramatic synergism seen with mutations in this group of genes.     

Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm

Using Cre/loxP, we conditionally inactivated the beta-catenin gene in cells of structures that exhibit important embryonic organizer functions: the visceral endoderm, the node, the notochord, and the definitive endoderm. Mesoderm formation was not affected in the mutant embryos, but the node was missing, patterning of the head and trunk was affected, and no notochord or somites were formed. Surprisingly, deletion of beta-catenin in the definitive endoderm led to the formation of multiple hearts all along the anterior-posterior (A/P) axis of the embryo. Ectopic hearts developed in parallel with the normal heart in regions of ectopic Bmp2 expression. We provide evidence that ablation of beta-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm, consistent with the existence of bipotential mesendodermal progenitors in mouse embryos.     

Myofibril-Inducing RNA (MIR) is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts

The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c) embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR), is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c) axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM) expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR) are similar to untreated normal. However, at the protein level, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.     

Absence of TGFβ signaling in embryonic vascular smooth muscle leads to reduced lysyl oxidase expression,impaired elastogenesis,and aneurysm

To address the requirement for TGFβ signaling in the formation and maintenance of the vascular matrix, we employed lineage‐specific mutation of the type II TGFβ receptor gene (Tgfbr2) in vascular smooth muscle precursors in mice. In both neural crest‐ and mesoderm‐derived smooth muscle, absence of TGFβ receptor function resulted in a poorly organized vascular elastic matrix in late‐stage embryos which was prone to dilation and aneurysm. This defect represents a failure to initiate formation of the elastic matrix, rather than a failure to maintain a preexisting matrix. In mutant tissue, lysyl oxidase expression was substantially reduced, which may contribute to the observed pathology. genesis 47:115–121, 2009. © 2009 Wiley‐Liss, Inc.