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Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFβ-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFβ target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

Induction choices for kidney-after-heart transplant recipients are variable. We examined the impact of kidney induction types on kidney graft and patient survival in heart transplant recipients.We analyzed the Scientific Registry of Transplant Recipient database from inception through the end of 2018 to study kidney and patient outcomes in the United States after heart transplantation. We only included recipients who were discharged on tacrolimus and mycophenolate maintenance. We grouped recipients by induction type into 3 groups: depletional (N = 307), nondepletional (n = 253), and no-induction (steroid only) (n = 57). We studied patients and kidney survival using Cox PH regression, with transplant centers included as a random effect. We adjusted the models for heart induction, recipient and donor age, gender, time between heart and kidney transplant, heart transplant indication, HLA mismatches, payor, live-donor kidney, transplant year, dialysis status, and diabetes mellitus at the time of kidney transplant.The 1-y kidney rejection rates and creatinine levels were similar in all groups. The 1-y rehospitalization rate was higher in the depletional group (51.7%) and nondepletional group (50.7%) than in the no-induction group (39.1%) although this was not statistically significant. There were no differences in recipient or kidney survival by kidney induction type. Live-donor kidney was associated with improved patient (hazard ratio, 0.74; 95% confidence interval, 0.54-1.0; P = 0.05) and kidney survival (hazard ratio, 0.45; 95% confidence interval, 0.24-0.84; P = 0.012].Type of kidney induction did not influence patient or kidney graft survival in heart transplant recipients. No-induction may be the preferred choice due to the lack of clinical benefits associated with induction use.

Objective Human papillomavirus (HPV) is the most common sexually transmitted agent in the world. HPV6b is a low-risk type of HPVs that causes benign verrucous hyperplastic lesions of the skin and anal genital mucosa. Previous research has indicated that HPV genotype 6 is sometimes associated with high-grade lesions and anal cancer. The pathogenesis of low-risk HPV infection and its relationship to high-risk HPV is not clear at present. The E7 protein, which is encoded by HPV early –expressing genes, plays an important role in HPV infection. The aim of this study is to investigate the human gene expression signature of the HPV6b E7-transfected HaCaT stable cell line. The identification of differentially expressed genes might provide a more comprehensive understanding of HPV6b infection and will allow us to explore the specific role of E7 protein. Methods We established a stable cell line transfected with the HPV6b E7 gene and analyzed the line's genome-wide expression profile by microarray. Quantitative real-time PCR (qRT-PCR) was used to verify the differentially expressed genes. GO enrichment analysis was applied for gene annotation according to functions. KEGG analysis, a system for analyzing gene function and genome information, was used to help us integrate differentially expressed genes into pathways. Results A total of 3519 genes were identified to be significantly differentially expressed between the HPV 6bE7-HaCaT stable cell line and a control cell line, among which 1884 genes were up-regulated and 1635 genes were down-regulated with a fold-change > 2.0 between the two groups. The expression profiles of the top 20 up-regulated and the top 20 down-regulated genes in the HPV 6bE7-HaCaT stable cell line as analyzed by qRT-PCR were consistent with the microarray data. The most significantly enhanced genes HPV 6bE7-HaCaT cells were SIMC1, S100A8 and S100A9, whereas PXDN expression was markedly down-regulated. GO analysis showed that HPV 6bE7 primarily affected biological processes and that the most significant difference was in heart induction (GO:0003129). Many differentially expressed genes were linked to histone H4-K20 demethylation (GO:0035574). KEGG analysis showed that the most significant changes in gene expression were related to primary bile acid biosynthesis, and the most diverse biological processes were related to systemic lupus erythematosus pathogenesis. Conclusions The global gene expression profile of the HPV 6bE7-HaCaT stable cell line was analyzed, revealing the genes regulated by E7 protein and providing insight into the pathogenesis of HPV6b infection.

It is theorized that the triggering of some arrhythmic episodes in cardiac-sensitive subjects might be directly linked to the functional uncoupling of gastro myoelectrical activity following the consumption of particular foods or by other influences on the gut. The intent of this research is to reveal the likely adverse mechanism involved through investigative analytical review. The resulting evidence shows there exists a triggering source within the visceral smooth muscle system of the gut and a mode of transmission incorporated in the autonomic nervous system mediated by the central autonomic network that could significantly affect the electrophysiology of the heart. A formal clinical study is indicated to determine the specific types of troublesome foods and other factors associated to better understand the overall process.

The process of heart induction appears to be highly conserved among vertebrates. The endoderm is important for heart induction in vertebrates. The details of the mechanism remain poorly understood. The localized inhibition of canonical Wnt signaling, which is regulated by endogenous Wnt antagonists, is required for heart induction. The size of the developing heart is regulated by inhibitory signals. The bone morphogenetic protein (BMP), Wnt, fibroblast growth factor (FGF) and Notch signaling pathways are important during early heart development. Different transcription factor profiles, in different regions of the original heart field, direct differentiation of the individual subdomains of the developing heart. Using a defined set of transcription factors, it is now possible to reprogram non-cardiac cells to the myocardial lineage.

Background The vertebrate heart makes its first sign of appearance at the gastrulation. Gene regulatory network controlling heart development is still not completely understood. Wnt signaling plays a pivotal role in orchestrating different aspects of early and late embryonic development including heart formation. Wnt ligands through canonical and non-canonical pathways have been shown to be essential for cardiac specification and morphogenesis. For example, Wnt3A and Wnt8 found to be capable of inhibiting endogenous heart induction, antagonizing Dkk-1 and Crescent and sufficient to induce heart formation in non-cardiogenic ventral marginal zone mesoderm [1]. Wnt receptors, Frizzleds, are seven-transmembrane proteins to which Wnts bind to their extracellular part, the cysteine-rich domain (CRD) [2]. Little is still known about the mechanism of Wnt-Frizzled receptors signaling during cardiogenesis. The Wnt receptor, Frizzled-7 (fzd7), has been shown by us and others to play important roles in different embryonic developmental contexts including neural crest induction [3] and convergent extension movements during gastrulation [4]. Xfz7 expression pattern during cardiogenesis in comparison to other early heart markers has not been yet investigated. In the current study, we have analyzed Xfzd7 expression in relation to that of XNkx2.5, XTroponin1-C, XGata-4, -5 and -6 and show its co-localization with these heart markers. We are aiming by this analysis to provide some clues about gene expression profile during cardiogenesis through which we might be able to shed some light on the cause of abnormal cardiac development that are Wnt-related. Materials and methods Xenopus laevis embryos were staged according to the normal time table of Nieuwkoop and Faber [5]. Riboprobes of Xfzd7, XNkx2.5, XTroponin-1C, Gata-4, -5 and -6 were synthesized. Single and double in situ hybridisation was carried out according to the methods described by Harland [6] and Knecht, et al. [7]. Embryos were frozen sectioned and imaged using Leica microscopes with Axiovision imaging software.

Heart induction in Xenopus occurs in paired regions of the dorsoanterior mesoderm in response to signals from the Spemann organizer and underlying dorsoanterior endoderm. These tissues together are sufficient to induce heart formation in noncardiogenic ventral marginal zone mesoderm. Similarly, in avians the underlying definitive endoderm induces cardiogenesis in precardiac mesoderm. Heart-inducing factors in amphibians are not known, and although certain BMPs and FGFs can mimic aspects of cardiogenesis in avians, neither can induce the full range of activities elicited by the inducing tissues. Here we report that the Wnt antagonists Dkk-1 and Crescent can induce heart formation in explants of ventral marginal zone mesoderm. Other Wnt antagonists, including the frizzled domain-containing proteins Frzb and Szl, lacked this activity. Unlike Wnt antagonism, inhibition of BMP signaling did not promote cardiogenesis. Ectopic expression of GSK3β, which inhibits β-catenin-mediated Wnt signaling, also induced cardiogenesis in ventral mesoderm. Analysis of Wnt proteins expressed during gastrulation revealed that Wnt3A and Wnt8, but not Wnt5A or Wnt11, inhibited endogenous heart induction. These results indicate that diffusion of Dkk-1 and Crescent from the organizer initiate cardiogenesis in adjacent mesoderm by establishing a zone of low Wnt3A and Wnt8 activity.

This paper summarizes the development of the heart from the formation of the heart mesoderm to cardiac septation. A brief account of morphological changes is provided, but attention is focused on mechanisms rather than on morphologic descriptions. Heart induction and differentiation, and the expression of cardiac specific proteins, are reviewed. New developments in these areas include the possible role of cell surface proteins and peptide growth factors in the segregation of the splanchnic mesoderm and in cardiac commitment. Past and recent experiments indicate that the heart morphogenetic information is engraved in the precardiac mesoderm. In spite of this, specific differentiative signals can be overriden experimentally demonstrating the unstability of the cardiac phenotype at the early heart tube stage. The relationship between differentiation and morphogenesis is analyzed. While cardiac differentiation appears to be a prerequisite for morphogenesis, a number of experiments indicate that differentiation can proceed in the absence of any morphogenesis. Formation of the heart loop is separated into two different components; looping itself and the acquisition of handedness. Late heart morphogenesis is explained in terms of differential tissue growth and tissue remodeling. This not only includes morphogenetic changes intrinsic to the heart but the addition of new cell types (neural crest, epicardium, vessels, nerves) that become integrated into the developing heart. The contribution of specific mechanisms to our understanding of heart development, such as cell death and hemodynamics is also analyzed.

Heart induction in Xenopus has been thought to be dependent primarily on the interaction of the heart primordia with the Spemann organizer. We demonstrate, however, that signals derived from the deep dorsoanterior endoderm during early gastrulation are also essential for heart formation. The presence of deep endoderm dramatically enhances heart formation in explants of heart primordia, both in the presence and absence of organizer. Likewise, extirpation of the entire endoderm can decrease the frequency of heart formation in embryos that retain organizer activity. Finally, we show that the combined presence of both endoderm and organizer is necessary and sufficient to induce heart in ventral mesoderm explants that would not otherwise form heart tissue. Xenopus heart induction, therefore, may be a multistep process requiring separate dorsalization and cardiogenic signalling events. This is the first demonstration of a heart-inducing role for the endoderm in Xenopus, indicating that the mechanism of heart formation may be similar in most vertebrates.

Publisher Summary This chapter focuses on the long heritage of classical and molecular genetic studies of amphibian heart development and discusses how the inductive, patterning, and morphogenetic processes are part of the broader mechanisms that establish the body plan, including axial pattern, germ layer and organ specification, and morphogenesis. Amphibian cardiogenesis research have been instrumental in providing a coherent picture of how signaling pathways specify and form vertebrate heart tissue during early embryonic development. The past decade in particular has been remarkable because of the advance made in uncovering the nature of embryonic signals, including the complex Wnt, Wnt antagonism, Nodal, FGF, Notch and BMP signal transduction pathways, attesting to the power of amphibian embryology in teasing apart early developmental events. Importantly, these signals have turned out to play evolutionarily conserved roles in heart induction in species with such divergent anatomies as those represented by amphibians, birds and mammals. Improved methods of imaging heart development and the advent of stable transgenesis for conditional, tissue-specific gene modulation should open the door to more detailed morphogenetic studies, as well as research into later stages of heart development.

The β-sulphonic amino acid taurine is synthe-sised in animals from dietary sulphur amino acids. Cats exclusively use taurine to conjugate cholic acid rather than being able to use the alternate glycine conjugation. Since total body synthesis of taurine in cats is limiting, metabolic deficiencies of taurine occur when the dietary intake of taurine is restricted. A deficiency of taurine in cats is expressed by aberrant functions of a wide range of organ systems. Pathological changes occur in the eye, feline central degeneration; reproductive abnormalities occur in the female, a high incidence of fetal resorptions and abortions, low birth weight and survival of live-born young; growth rate in the new born kitten is depressed; heart induction of dilated cardiomyopathy and compromised immune function. All these conditions are prevented or reversed with adequate dietary taurine. While all tissues contain taurine, the concentration varies with the tissue. Generally plasma has been used to assess taurine status in cats, but the concentration of taurine in plasma varies widely. Food deprivation of cats given high taurine diets causes a marked fall in the concentration of taurine in plasma. Major changes in whole blood concentration do not occur as rapidly as plasma and therefore appear to be a superior diagnostic test for taurine status. An inadequate number of samples have been analysed to define a marginal taurine level from whole blood concentration to prevent clinical signs. The minimal dietary concentration of taurine to prevent clinical signs of efficiency is dependent on the type of diet. For commercial expanded (dry) cat foods a concentration of 1200 mg taurine/kg dry matter appears adequate. Higher concentrations are required in canned diets, 2000 to 2500 mg taurine/kg dry matter to supply adequate taurine. The reasons for the higher concentration of taurine required in canned foods is not due to availability of taurine in the classical context. Rather it appears that heating during the canning process produces products which increase enterohepatic loss of taurine.

Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posterior dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.

It is well established that the heart has only a limited ability to repair damage caused by heart disease. However, the observation that human embryonic stem (ES) cells differentiate into beating cardiomyocytes raises the possibility that these cells may be used to regenerate damaged cardiac tissue. Unfortunately, limited progress has been made in developing protocols for the efficient differentiation of human ES cells into ventricular cardiomyocytes. Studies in various animal models have provided significant insights into the signaling molecules that are necessary for heart induction, making it clear that both the spatial and temporal activation and suppression of defined signaling pathways are critical for the development of the myocardial cells in both embryos and ES cell cultures. We propose that there are at least four distinct steps that lead to the development of cardiac lineages in the embryo, namely: (1) establishment of organizing centers, (2) mesendoderm induction, (3) establishment of cardiac precursors, and (4) terminal differentiation of beating cardiomyocytes. Here, we provide a description of each stage, the signaling pathways that dominate each stage and present evidence that those signaling pathways also govern cardiac differentiation in ES cell models. Further identification of soluble growth factors and signaling cascades that promote heart formation is of great importance for an improved understanding of cardiac development and function and may reveal new paradigms for the treatment of cardiovascular disease.

The LIM‐homeodomain transcription factor Islet1 (Isl1) is a marker of pluripotential undifferentiated cardiovascular progenitors that will give rise to myocytes, endothelial cells, and smooth muscle cells within the heart and the vasculature. During development, Isl1 is actively expressed in cardiac progenitors prior to their differentiation. Isl1 function is also required for normal heart development, as Isl1 mutant hearts are completely lacking the outflow tract, right ventricle, and have severely reduced atrial tissue. Isl1 is expressed both in cardiogenic mesoderm and in endoderm, the latter a tissue that is required for heart induction. It will also be important to understand tissue‐specific roles for Isl1 in this context. The early expression of Isl1 within multipotential cardiac progenitors and its essential role in heart development suggest that understanding factors that are both upstream and downstream of Isl1 will give critical insights into genetic pathways required for specification of distinct cardiovascular lineages and for normal heart development.

The TGFβ family member Nodal has been implicated in heart induction through misexpression of a dominant negative version of the type I Nodal receptor (Alk4) and targeted deletion of the co-receptor Cripto in murine ESCs and mouse embryos; however, whether Nodal acts directly or indirectly to induce heart tissue or interacts with other signaling molecules or pathways remained unclear. Here we present Xenopus embryological studies demonstrating an unforeseen role for the DAN family protein Cerberus within presumptive foregut endoderm as essential for differentiation of cardiac mesoderm in response to Nodal. Ectopic activation of Nodal signaling in non-cardiogenic ventroposterior mesendoderm, either by misexpression of the Nodal homologue XNr1 together with Cripto or by a constitutively active Alk4 (caAlk4), induced both cardiac markers and Cerberus. Mosaic lineage tracing studies revealed that Nodal/Cripto and caAlk4 induced cardiac markers cell non-autonomously, thus supporting the idea that Cerberus or another diffusible factor is an essential mediator of Nodal-induced cardiogenesis. Cerberus alone was found sufficient to initiate cardiogenesis at a distance from its site of synthesis. Conversely, morpholino-mediated specific knockdown of Cerberus reduced both endogenous cardiomyogenesis and ectopic heart induction resulting from misactivation of Nodal/Cripto signaling. Since the specific knockdown of Cerberus did not abrogate heart induction by the Wnt antagonist Dkk1, Nodal/Cripto and Wnt antagonists appear to initiate cardiogenesis through distinct pathways. This idea was further supported by the combinatorial effect of morpholino-medicated knockdown of Cerberus and Hex, which is required for Dkk1-induced cardiogenesis, and the differential roles of essential downstream effectors: Nodal pathway activation did not induce the transcriptional repressor Hex while Dkk-1 did not induce Cerberus. These studies demonstrated that cardiogenesis in mesoderm depends on Nodal-mediated induction of Cerberus in underlying endoderm, and that this pathway functions in a pathway parallel to cardiogenesis initiated through the induction of Hex by Wnt antagonists. Both pathways operate in endoderm to initiate cardiogenesis in overlying mesoderm.

Inhibition of canonical Wnt/β-catenin signaling by Dickkopf-1 (Dkk-1) or Crescent initiates cardiogenesis in vertebrate embryos. However, nearly nothing is known about the downstream effectors of these secreted Wnt antagonists or the mechanism by which they activate heart formation. Here we show that Wnt antagonists in Xenopus stimulate cardiogenesis non-cell-autonomously, up to several cells away from those in which canonical Wnt/β-catenin signaling is blocked, indicative of an indirect role in heart induction. A screen for downstream mediators revealed that Dkk-1 and other inhibitors of the canonical Wnt pathway induce the homeodomain transcription factor Hex, which is normally expressed in endoderm underlying the presumptive cardiac mesoderm in amphibian, bird, and mammalian embryos. Loss of Hex function blocks both endogenous heart development and ectopic heart induction by Dkk-1. As with the canonical Wnt pathway antagonists, ectopic Hex induces expression of cardiac markers non-cell-autonomously. Thus, to initiate cardiogenesis, Wnt antagonists act on endoderm to up-regulate Hex, which, in turn, controls production of a diffusible heart-inducing factor. This novel function for Hex suggests an etiology for the cardiac malformations in Hex mutant mice and will make possible the isolation of factors that induce heart directly in the mesoderm.

Developmental biologists have uncovered many of the signaling proteins that are required to recruit early embryonic cells to the myocardial lineage. A detailed understanding of their function should provide insights into the difficult task of inducing embryonic stem cells to develop cardiomyocyte precursors for cell-based therapies. These proteins may also prove to be useful to stimulate progenitor cells to differentiate into cardiomyocytes within the damaged heart.

In the Mexican axolotl, Ambystoma mexicanum, recessive mutant gene c, when homozygous, results in a failure of the heart to form sarcomeric myofibrils and contract normally. Previous studies have shown that purified RNA from normal anterior endoderm or from medium conditioned with anterior endoderm/pre-cardiac mesoderm has the capacity to rescue mutant hearts in organ culture. In the present study, RNA extracted from adult sheep heart was tested for its capacity to promote differentiation in the mutant axolotl hearts. Mutant hearts cultured in the presence of the sheep heart RNA in Steinberg's solution for 48 h displayed rhythmic contractions. Ultrastructural studies showed that the rescued mutant axolotl ventricular myocardial cells contained myofibrils of normal morphology. Mutant hearts cultured in Steinberg's solution alone did not beat throughout their lengths and myofibrils were not observable in the ventricles. Confocal microscopy confirmed the increase of Tropomyosin expression and formation of myofibrils in mutant hearts treated by sheep heart RNA. Thus, sheep heart RNA promotes myofibrillogenesis and the development of contractile function in embryonic cardiac mutant axolotl hearts.

Background: The mouse anterior visceral endoderm, an extraembryonic tissue, expresses several genes essential for normal development of structures rostral to the anterior limit of the notochord and has been termed the head organizer. This tissue also has heart-inducing activity and expresses mCer1 which, like its Xenopus homolog cerberus , can induce markers of cardiac specification and anterior neural tissue when ectopically expressed. We investigated the relationship between head and heart induction in Xenopus embryos, which lack extraembryonic tissues. Results: We found three regions of gene expression in the Xenopus organizer: deep endoderm, which expressed cerberus ; prechordal mesoderm, which showed overlapping but non-identical expression of genes characteristic of the murine head organizer, such as XHex and XANF-1 ; and leading-edge dorsoanterior endoderm, which expressed both cerberus and a subset of the genes expressed by the prechordal mesoderm. Microsurgical ablation of the cerberus -expressing endoderm decreased the incidence of heart, but not head, formation. Removal of prechordal mesoderm, in contrast, caused deficits of anterior head structures. Finally, although misexpression of cerberus induced ectopic heads, it was unable to induce genes thought to participate in head induction. Conclusions: In Xenopus , the cerberus -expressing endoderm is required for heart, but not head, inducing activity. Therefore, this tissue is not the topological equivalent of the murine anterior visceral endoderm. We propose that, in Xenopus, cerberus is redundant to other bone morphogenetic protein (BMP) and Wnt antagonists located in prechordal mesoderm for head induction, but may be necessary for heart induction.

Publisher Summary This chapter reviews the current state of knowledge concerning the tissue interactions that regulate heart determination in vertebrates. The method throughout is comparative, with a given phenomenon examined in several species in order to extract fundamental principles as well as to understand how the cardiac developmental program can be modified in different organisms. It is found that avian and amphibian species have a two-step model of vertebrate cardiac induction. Signals from anterior endoderm induce a cardiac field in the overlying mesoderm. Only a subdomain of this field actually becomes heart in vivo. The restriction of the heart field is affected both by BMP signals from the surrounding ventral/lateral tissues that promote cardiogenesis and by signals from dorsal/medial structures, including neural tissues that inhibit heart formation. The end result is specification of heart tissue in the anterior ventral mesoderm. It is also hoped that discussing cardiac development in several vertebrate species will encourage workers on one species to try to integrate their results with the findings of those who work in other experimental systems.

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.

We have re-examined some of the factors affecting the induction of heart-forming mesodern 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.

Mutant genes have been used in the past as research ‘tools’ for better understanding selected biological phenomena and for solving certain biological problems. It is particularly exciting when these mutations affect specific major organs of vertebrates. One such naturally-occurring recessive mutant gene, designated c for ‘cardiac lethal’ was discovered recently in the Mexican axolotl, Ambystoma mexicanum. Homozygous embryos (c/c) exhibit a lack of heart Function even though initial development of the organ seems normal. Cardiac lethal mutant (c/c) embryos are obtained by mating heterozygous adults (+/c x +/c) and can first be distinguished from their normal (+/+ or +/c) siblings at stage 35 (one week postfertilization) when the normals develop vigorously contracting hearts. Mutant embryos at this stage appear grossly normal, but their hearts fail to contract and cardiovascular circulation is not established. In spite of their nonfunctional hearts, mutant embryos survive through stage 41 (about 20 days beyond the heart-beat stage) and during this time display normal swimming movements indicating that skeletal muscle is not affected by the mutation.

Homozygosity for gene c in Ambystoma mexicanum results in no detectable heartbeat in situ. Alteration of the cardiac environment through organ culture results in rapid initiation of spontaneous heartbeat, indicating that absence of cardiac function in situ is not the result of failure of embryonic induction.

Homozygosity for simple recessive gene c in axolotl embryos results in the absence of a heartbeat. Gene c alters the morphology of the mutant anterior endoderm - the primary heart inductor.

Presumptive heart mesoderm from early neurulae of the newt, Taricha torosa, was combined in explants with endoderm from five different regions of the embryo to study the distribution in the endoderm of the capability to induce heart formation. The percentages of such cultures that ultimately formed beating heart tissue, and the average numbers of days in culture before doing so, are compared with control levels established by explanting presumptive heart mesoderm alone. The ability to induce hearts is found to be broadly distributed and to be strongest in anterior endoderm. The heart-forming response is accelerated only by the endoderm normally underlying the presumptive heart mesoderm, and this endoderm retains its inductive capability into tailbud stages. Other anterior endoderm is a powerful heart inductor but acts more slowly. Posterior endoderm not only did not promote heart formation, but also increased the time in culture before the beginning of contraction in the few responding cases. Mesoderm immediately posterior to the presumptive heart mesoderm can also be induced to form beating tissue. However, compared to presumptive heart mesoderm, a much longer time elapses before contractions begin. Since endoderm near that which normally underlies the heart has substantial ability to induce heart, the mesoderm overlying these regions experiences some measure of heart induction. Normally these regions of affected mesoderm do not contribute to the heart, but under altered conditions they can. This extended mesodermal area of heart potential is the heart “field.” The existence and extent of the heart field appears to be a result of the distribution of heart-inducing activity in the endoderm.

Heart determination is a gradual, cumulative process involving inductive and suppressive interactions between the heart mesoderm and nearby embryonic tissues. Our analysis of heart determination in the California newt, Taricha torosa, includes defect and other in vivo experiments, and explants in epidermal vesicles and into hanging drops. Explants of presumptive heart mesoderm from neurulae into hanging drops of a completely defined salt solution (Niu-Twitty solution) produce beating hearts only infrequently. The addition of various other tissues and fractions of tissue homogenates changes the frequency and the rate of differentiation. These two parameters were used to assess the stimulatory and suppressive effects of tissues and tissue fractions on the differentiation of the presumptive heart mesoderm. At least three different factors are active in eliciting and regulating heart differentiation. A specific heart inductor in anterior endoderm increases the rate and the freqency of heart differentiation. A general stimulatory factor in epidermis and other embryonic tissues increases the frequency, but not the rate, of heart differentiation. An inhibitory agent in cranial fold and neural plate tissues delays or prevents heart differentiation. These three factors operate from intact tissues in the embryo or when explanted in vesicles or hanging drops, and are effectively present in a fraction (from sephadex column chromatography) of homogenates of the appropriate tissues.

Presumptive heart mesoderm from early neurulae of the newt Taricha torosa was isolated alone in salt solution or was combined in explants with ectoderm, or ectoderm and endoderm, from various regions of the embryo. The presumptive heart mesoderm formed a beating heart when isolated alone in only 14% of the cases. When explanted in combination with anterior presumptive epidermis, beating hearts developed in about one-third of the cases. Beating hearts formed in all explants of presumptive heart mesoderm combined with anterior presumptive epidermis and endoderm from the future heart region, and in two-thirds of the cases if the endoderm came from more dorsal or posterior regions. In the series where the endoderm was tested from the dorsal or posterior regions, all explants that failed to produce beating hearts contained neural tissue that had differentiated from the presumptive epidermis whereas each explant that formed a heart lacked neural tissue. In no case did a beating heart develop from presumptive heart mesoderm in an explant that included part of the anterior neural plate and fold as well as anterior presumptive epidermis. Hearts failed to develop in these explants even when endoderm from the future heart region was added. Endodermless animals failed to produce hearts, but endodermless animals also deprived of their neural plate and fold did produce hearts. It is concluded that both ectoderm and endoderm can influence the differentiation of the presumptive heart mesoderm. Heart formation is suppressed by anterior neural plate and fold, but is stimulated by endoderm.