Portal de Periódicos da CAPES

Parsing the Heart: Genetic Modules for Organ Assembly

1997; Cell Press; Volume: 91; Issue: 2 Linguagem: Inglês

10.1016/s0092-8674(00)80397-9

1097-4172

Mark C. Fishman, Eric N. Olson,

Hippo pathway signaling and YAP/TAZ

"parse: resolve … into component parts" —Oxford English Dictionary What are the pathways for the assembly of vertebrate organs, and how have these pathways evolved from those in more primitive organisms? Our goal here is to suggest one logical framework for approaching organ development, based upon recent genetic studies and buttressed by evolutionary observations. We do not presume the model to be as compelling as that established for pattern and polarity in the Drosophila melanogaster embryo, although we draw upon the logic of that system, in proposing that single gene mutations can reveal units of vertebrate organ assembly, particularly the heart. These units we refer to as "modules," by which we mean regions or functions of the heart, such as chamber designation or localized pacemaker activity, which can be selectively perturbed by single gene mutations in mice or fish. The modules appear to be recent evolutionary additions, not present in primitive chordates, in contrast to myogenic cellular activity, which appears to be an ancient metazoan property with molecular pathways conserved from insect to mammal. We suggest, in a heuristic vein, that it may be possible to view vertebrate heart assembly as the sum product of separable genetic modules. The earliest functioning embryonic vertebrate heart is a linear tube (Figure 1), aligned along the ventral midline and formed by the fusion of epithelial plates of cardiogenic cells from the left and right lateral mesoderm. The heart tube forms in the human at about 3 weeks of gestation, in the mouse at about embryonic day 8, and in the zebrafish about 19 hr after fertilization. The contractile cells of the tube constitute the myocardium, which grows to several layers of thickness. The lumen is lined by a single-cell layer of endocardium formed by a type of noncontractile endothelial cell similar to that which lines blood vessels. The tube is subdivided along its length into segmental zones of gene expression that presage the location of the atrial and ventricular chambers. In vertebrates with four-chambered hearts, such as mammals, the segment of the heart tube destined to form the right ventricle lies anterior to the future left ventricular segment. The process of looping (Figure 1) therefore converts anterior–posterior patterning of the heart tube into left–right asymmetry. Until the looping stage, all vertebrate hearts are essentially indistinguishable (5Fishman M.C Chien K.R Development. 1997; 124: 2099-2117Crossref PubMed Google Scholar). The fish retains the two-chamber arrangement as an adult. With the advent of lungs comes the need to separate deoxygenated from oxygenated blood during the passage of blood through the heart. In birds, mammals, and some reptiles, this separation is accomplished by development within the heart tube of complete structural septa between the right and left ventricles and between the right and left atria (Figure 1). In other lower vertebrates, such as the frog, there is a single ventricle, but it is designed so that deoxygenated and oxygenated blood flow through it in fairly separate streams. The evolution of vertebrates from an ancestral chordate was a process challenged by cardiovascular limitations. Growth demanded proportional increases in blood supply. The ancestral chordate is believed to have resembled the larva of current-day tunicates (15Romer A.S Science. 1967; 158: 1629-1637Crossref PubMed Scopus (101) Google Scholar), the heart of which is a single muscle layer analogous to a vessel and resembling the heart of Drosophila. This tunicate heart lacks chambers and endocardium and functions by peristalsis, reversing the direction of pumping every few minutes (14Randall, D.J., and Davie, P.S. (1980). The hearts of urochordates and cephalochordates. In Hearts and Heart-Like Organs, G.H. Bourne, ed. (Academic Press).Google Scholar). In contrast, the vertebrate heart manifests sequential chamber contractions and drives flow in one direction at relatively high pressures. The modules added to generate the vertebrate heart (Figure 2) support these physiological functions. The polarity of blood flow requires proper placement of a single overriding pacemaker, which establishes the origin of the heart beat; a conduction system to synchronize the beat; and a node at the atrioventricular junction to cause a pause and thereby ensure sequential contractions, first of the low-pressure atrium and then of the high-pressure ventricle. Valves are needed to prevent retrograde flow. High-pressure generation is accomplished by concentric growth of the ventricular myocardium. As a consequence of myocardial growth, diffusion no longer suffices to provide nutrients, so the myocardium is invested with its own vessels, the coronary arteries, which grow in from an encasing epicardium. Some of these elements also appear to have arisen by convergent evolution in the heart (or hearts) of particular invertebrates, especially in large ones with high cardiac outputs, such as the octopus. A key participating element in many of these modules is the endothelium, a cell type not present in hearts or vessels of tunicates or reported in invertebrates, to our knowledge. Endocardial cells give rise to the valves (4Eisenberg L.M Markwald R.R Circ. Res. 1995; 77: 1-6Crossref PubMed Scopus (513) Google Scholar); endocardial signals (such as neuregulin) regulate myocardial growth; and conduction tissue forms from myocardial cells adjacent to endothelium. Hence, the advent of endothelium is intimately related to the development of elements needed to generate unidirectional flow at high pressures. The vertebrate heart is asymmetric as a result of looping of the ventricle to the right. The role of looping is not clear. One possibility is that it positions inflow and outflow through the heart to connect seamlessly with flow through the major vessels, which form by local aggregation of angioblasts. In vertebrates with a ventricular septum, the position of the bend in the loop defines the position of the interventricular septum between the right and left venticles (Figure 1). Among the transcription factors that drive the initiation of myogenic cell fate, at least two (Nkx/Tinman and myocyte enhancer factor-2 [MEF2]) are conserved in this role from fly to mouse (Figure 3). Without the homeobox gene tinman (1Bodmer R Development. 1993; 118: 719-729Crossref PubMed Google Scholar), the fly remains heartless. The related Nkx family in vertebrates also is expressed in presumptive cardiogenic regions and in other sites as well (7Harvey R.P Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (477) Google Scholar). Members of the MEF2 family of MADS-box transcription factors directly activate cardiac structural genes and are required for cardioblast differentiation. In fruit flies, there is a single MEF2 gene, which is directly activated by Tinman (Figure 3). In vertebrates, there are four MEF2 genes that are expressed in overlapping patterns in the cardiac as well as smooth and skeletal muscle lineages (9Lin Q Schwarz J Bucana C Olson E.N Science. 1997; 276: 1404-1407Crossref PubMed Scopus (746) Google Scholar). Fruit fly and vertebrate myocyte commitment also appears to be similar in dependence upon signaling by members of the transforming growth factor-β family (dpp in the fly6Frasch M Nature. 1995; 374: 464-467Crossref PubMed Scopus (367) Google ScholarBMP4 in vertebrates). Mutations of Nkx2.5 (11Lyons I Parsons L.M Hartley L Li R Andrews J.E Robb L Harvey R.P Genes Dev. 1995; 9: 1654-1666Crossref PubMed Scopus (926) Google Scholar) or of a single MEF2 gene, MEF2C (9Lin Q Schwarz J Bucana C Olson E.N Science. 1997; 276: 1404-1407Crossref PubMed Scopus (746) Google Scholar), in mice do not stop the establishment of cardiac cell fate. Rather, as described below, they perturb later stages of development, such as looping and chamber designation. Based on the lack of cardioblasts in tinman mutants and the failure of cardioblasts to differentiate in Drosophila mutants lacking MEF2, the loss-of-function phenotypes of Nkx2.5 and MEF2C mutant mice might have been predicted to be more severe. The observation that the major cardiac defects in these mutants occur during relatively late stages of cardiac morphogenesis suggests that the early cardiogenic functions that are confined to single regulators in flies are distributed among several related family members in vertebrates. This redundancy may be the reason that the heart cell fate is reasonably well protected against single gene mutation. Interestingly, some of these same cell fate genes are redeployed in pathways that generate higher-order structures, pathways that are more sensitive to their mutation, perhaps suggesting less redundancy in these later evolutionary functions. Some of the "new" modules of vertebrate hearts are selectively perturbed by single gene mutations. Remarkably similar phenotypes have been noted in mouse and fish mutations. Mutations in zebrafish were discovered in large-scale chemical mutagenesis screens (2Chen J.-N Haffter P Odenthal J Vogelsang E Brand M van-Eeden F.J.M Furutani-Seiki M Granato M Hammerschmidt M Heisenberg C.-P et al.Development. 1996; 123: 293-302PubMed Google Scholar, 18Stainier D.Y.R Fouquet B Chen J.N Warren K Weinstein B Meiler S Mohideen M.A.P.K Neuhauss S.C.F Solnica-Krezel L Schier A.F et al.Development. 1996; 123: 285-292PubMed Google Scholar), so the genes are unknown, but it seems improbable that they match by accident those genes intentionally mutated in the mouse. This suggests a conservation of essential cardiogenic pathways and units of form over 450 million years of evolution. For example, as shown in Table 1, the zebrafish mutant pandora is missing a portion of the ventricle. It resembles mouse mutants lacking the transcription factors MEF2C and dHAND, as well as another mouse mutant called heart defect (hdf), which was obtained by a random transgene insertion into an unknown gene (20Yamamura H Zhang M Markwald R.R Mjaatvedt C.H Dev. Biol. 1997; 186: 58-72Crossref PubMed Scopus (102) Google Scholar). This selective truncation also resembles the effect of retinoic acid on the developing zebrafish heart. Mutation of the cloche gene in zebrafish and of the vascular endothelial growth factor receptor gene, flk, in the mouse prevents the formation of most endothelium, including endocardium. Three zebrafish mutations and targeted mutation of GATA4 in mouse (8Kuo C.T Morrisey E.E Anandappa R Sigrist K Lu M.M Parmacek M.S Soudais C Leiden J.M Genes Dev. 1997; 11: 1048-1060Crossref PubMed Scopus (834) Google Scholar, 12Molkentin J.D Lin Q Duncan S.A Olson E.N Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (919) Google Scholar) cause cardia bifida (failure of the bilateral cardiac primordia to fuse at the midline, with the subsequent formation of two hearts). Concentric thickening of the myocardium of the heart, especially the ventricle, does not occur in three zebrafish mutants and in mice bearing mutations in RXRα (19Sucov H.M Dyson E Gumeringer C.L Price J Chien K.R Evans R.M Genes Dev. 1994; 8: 1007-1018Crossref PubMed Scopus (524) Google Scholar) and other genes.Table 1Modules of Heart Development, Revealed by Mutations in Mouse and FishCategory"Modular" StepMutant PhenotypeMouse MutantFish MutantCell fateEndotheliumNo endotheliumflkclocheAxial formTube fusionCardia bifidaGATA4bonnie and clyde miles apartConcentric growth of myocardiumThin myocardiumRXRαγsantaRARαvalentineNF1heart of glassn-myc TEF-1"Segmentation"Ventricular truncationMEF2CpandoradHANDBlob of heart tissue—heart and soulVentricular septationVentricular septation defectn-myc—Outflow tract septation and alignmentConotruncal defectsNF-1 RXR/RAR—Component partsCoronary arteryAbsence of coronary arteriesα4 integrin VCAM-1—Valve generationAbsence of valves—jekyllIntegrationLateral asymmetryAbnormal loopinginv iviguana cyclops floating head Many other midline mutantsThe genes affected by the fish mutations have not been cloned. We apologize for the omission of several mutations and many references, from the text and this table, due to space constraints. Some may be found in recent reviews (16Rossant J Circ. Res. 1996; 78: 349-353Crossref PubMed Scopus (97) Google Scholar, 5Fishman M.C Chien K.R Development. 1997; 124: 2099-2117Crossref PubMed Google Scholar). Open table in a new tab The genes affected by the fish mutations have not been cloned. We apologize for the omission of several mutations and many references, from the text and this table, due to space constraints. Some may be found in recent reviews (16Rossant J Circ. Res. 1996; 78: 349-353Crossref PubMed Scopus (97) Google Scholar, 5Fishman M.C Chien K.R Development. 1997; 124: 2099-2117Crossref PubMed Google Scholar). Of course, there are no mutations in the fish analogous to those that disrupt septation of the heart or outflow tract, which contribute to separation of blood flow to the lungs. It is interesting that one set of basic–helix-loop-helix transcription factors, dHAND and eHAND, are expressed separately in the future right and left ventricular chambers, respectively, during mouse cardiogenesis, as well as in neural crest cells, which contribute to septation of the outflow tract. Inactivation of dHAND expression in the mouse results both in the absence of the right ventricle and in neural crest–related abnormalities of the outflow tract (17Srivastava D Thomas T Lin Q Kirby M.L Brown D Olson E.N Nature Genet. 1997; 16: 154-160Crossref PubMed Scopus (542) Google Scholar). We suggest, therefore, that the "modular" steps in Table 1 are units of organ assembly, each the end product of a particular biochemical pathway. While there are likely to be many proteins in each pathway, our hope is that with even one or two genes assigned by mutational analysis to each pathway, the rest may be amenable to combined genetic and biochemical analyses. For example, the observation that several mutations have the same phenotype may indicate that their gene products function in the same pathway. How complete is the list of units? There will undoubtedly be more, but we suspect that the list of phenotypes informative in this regard may be limited. Even though the number of genes mutated in mice is small, the zebrafish screens have achieved sufficient saturation so that new phenotypes are being discovered at diminishing rates. Why do these phenotypes crop up repeatedly with different mutations in different species? Perhaps the modules constitute genetic units added relatively independently and late in evolution and have less redundancy than those governing essential establishment of cell fate. Alternatively, it may be that unlike screens of Drosophila body form, in which cuticle pattern was a good surrogate for molecular patterning, gross anatomy will not suffice and the "real" unitary decisions will be discovered in screens based upon more subtle phenotypes, such as changes in patterns of gene expression. Even if the modular hypothesis is true, by no means would we predict genetic analysis to reveal all genes involved in the development of the heart. Targeted mutations of many genes in mice are without evident effect, an observation ascribed to redundancy of function among genes or to subtlety of phenotype; other mutations have effects too pleotropic to be informative. The growth and function of the heart must be coordinated with the rest of the organism. Not only must the venous input connect to the atrium and the arterial outflow to the ventricle, but the heart needs to generate blood flow proper to the animal's size and metabolic needs. There is a linear relationship between the amount pumped with each beat and body weight, assayed in animals varying nearly 2000-fold in weight. What genes provide this coordination of heart size and orientation to overall body plan? The process of size coordination may begin by interaction of neighboring tissues with cardiac progenitors. In Drosophila, dorsal ectoderm supports continued tinman expression (via dpp6Frasch M Nature. 1995; 374: 464-467Crossref PubMed Scopus (367) Google Scholar). In frogs (3Cleaver O.B Patterson K.D Krieg P.A Development. 1996; 122: 3549-3556Crossref PubMed Google Scholar) and fish, Nkx2.5 overexpression causes enlargement of the hearts. It is an interesting possibility that the establishment of anterior–posterior and dorsal–ventral axes in the early embryo regulates the amount of tissue allocated to each organ, thereby tying organ dimensions to animal size. The midline may also position the heart relative to vessels, so as to provide seamless connections. Most major vessels form by local aggregation of angioblasts and so must subsequently be joined with the endocardium of the heart. The midline structures (notochord, hypochord, and neural tube) have at least two influences that could be relevant in this regard. First, the notochord guides assembly of the aorta (the major artery of the body, formed beneath the notochord). The notochordless zebrafish mutant, floating head, has angioblasts and many vessels but no aorta. The aorta can be rescued by exposure to wild-type notochord. Second, the midline tissues are critical to the lateral asymmetry of the heart, reflected in its looping (10Lohr J.L Danos M.C Yost H.J Development. 1997; 124: 1465-1472PubMed Google Scholar). Soon after the vertebrate heart tube forms, the atrial end bends to the left and then returns to the midline, after which the ventricle loops to the right. These processes are disrupted by midline perturbations in the zebrafish. How early laterality deficiencies affect later chamber morphogenesis is not known, although it is believed that they are a cause of malorientations, such as reversal of left or right chambers or misconnections of vessels leading to and from the heart. These are difficult linkages to assess because even looping, the standard assay of cardiac asymmetry, occurs after chamber designation and depends upon proper cellular differentiation. The limited number of informative phenotypes and similarity among species suggest that there are at least a few elements of heart formation that are "modular" and amenable to dissection as distinct genetic and biochemical pathways. We propose that these were added to a primitive chordate heart (Figure 2), a notion similar to that for other modular evolutionary additions (13Raff R.A The Shape of Life. University of Chicago Press, Chicago1996Crossref Google Scholar). Of course, we recognize that it may be overly reductionist to claim that the units we see by standard histology are those for which genetic pathways exist and that those pathways have been added, in a modular way, to a primitive single-chambered peristaltic pump of a presumptive ancestor. The next steps in the evaluation of this hypothesis include identification of the other elements of the modular pathways that regulate morphogenesis and determination of relationships among pathways that define individual cell fate and higher-order form. As mutants that affect other organ systems are characterized, it will also be of interest to determine whether the type of modular logic that appears to underlie cardiac evolution and development reflects a general strategy for organogenesis.