Once cardiac specification is complete, substantial morphogenetic changes are necessary to create the three-dimensional form of the functional heart [23
]. First, as myocardial differentiation progresses, the bilateral populations of cardiomyocytes move medially, eventually merging at the embryonic midline. Through a process called cardiac fusion, they form a ring of cells, referred to as the cardiac cone (), that encircles the endocardial precursors. Next, the cardiac cone extends into a linear tube: the cone’s axis gradually lengthens and shifts from a dorsal-ventral plane to an anterior-posterior plane (). Once extension is complete, the heart tube is a muscular cylinder lined with endocardium, and the inner and outer circumferences of the cone have become the arterial and venous apertures of the tube (). Next, localized bulges emerge from the walls of the heart tube: this process, called ballooning, creates the characteristic curvatures of the ventricle and the atrium (). Chambers are further demarcated by the distinctive constriction of the atrioventricular (AV) canal; morphogenetic changes also occur inside this portion of the heart, where endocardial cushions (ECs) form and are then remodeled into AV valve leaflets [24
]. Altogether, cardiac morphogenesis is a dynamic and multifaceted process that must be carefully regulated to insure proper cardiac structure.
Many zebrafish mutations disrupt cardiac morphology, and analysis of these mutants has indicated several important regulators of discrete morphogenetic processes. In the most extreme of these mutant phenotypes, the bilateral populations of cardiomyocytes never travel to the midline, resulting in two separate hearts forming in lateral positions, a condition known as cardia bifida
]. Analysis of cardia bifida
mutants has revealed key requirements for myocardial migration. Mutations in the transcription factor genes casanova (sox32)
, bonnie and clyde
, and faust (gata5)
and the Nodal coreceptor gene one-eyed pinhead
all disrupt endoderm specification and secondarily cause cardia bifida
, suggesting that the endoderm provides an important signal or substrate utilized by migrating cardiomyocytes [17
]. The molecular mechanisms of endodermal-myocardial interactions remain mysterious, although it is intriguing to consider a potential role for sphingolipid signaling, since the sphingosine-1-phosphate receptor gene miles apart
) plays a cell non-autonomous role in promoting myocardial migration [28
]. In addition to putative interactions with the endoderm, migrating cardiomyocytes require interactions with components of the extracellular matrix, particularly Fibronectin, as demonstrated by the cardia bifida
phenotype of natter (fibronectin)
Another class of zebrafish mutations permit myocardial migration to the midline but then affect the morphology of the heart tube or cardiac chambers. Mutations in the genes heart and soul (prcki), snakehead (atp1a1a.1),
and nagie oko (mpp5)
hinder heart tube extension: mutant hearts resemble arrested cardiac cones or stunted heart tubes [12
]. The PRKCi, Atp1a1a.1, and Mpp5 proteins are all known to exhibit apicobasally polarized localization within epithelial cells, suggesting the importance of cell polarity to the process of tube extension.
Mutations in the heart of glass (heg)
, santa (krit1)
, and valentine (ccm2)
genes do not seem to influence the shape of the heart tube, but they do cause severe distortion of chamber shape: the mutant hearts have thin-walled, extremely dilated chambers [35
]. All three of these genes are expressed in endothelial cells, with heg
clearly being expressed in the endocardium, suggesting that endocardial-myocardial signaling plays a key role in regulating chamber wall thickening. Epigenetic influences, such as the biomechanical forces created by blood flow, also contribute to the formation of chamber shape. The zebrafish locus weak atrium
encodes an atrium-specific myosin heavy chain (amhc
) that is required for atrial contractility [13
]. In addition to atrial defects and the consequent reduction in blood flow, weak atrium
mutants also display ventricular defects: the mutant ventricle acquires an unusually small shape without characteristic chamber curvatures. Since amhc
is expressed only in the atrium [13
], this ventricular phenotype represents a secondary consequence of atrial dysfunction, presumably reflecting an impact of normal hemodynamics on chamber ballooning.
AV valve morphogenesis, like chamber morphogenesis, involves interplay between multiple signals, including those generated by biomechanical forces. Several zebrafish mutations interfere with the formation of ECs in the AV canal. For example, jekyll (udp-glucose dehydrogenase)
mutants fail to form ECs [37
]. UDP-glucose dehydrogenase (Ugdh) produces substrates used in the modification of extracellular matrix proteins that are known to facilitate Wnt and Fgf signaling [38
]. Perhaps Ugdh is important for the control of Wnt signaling during EC formation: constitutive activation of Wnt signaling via mutation of the tumor suppressor gene apc
leads to excessive EC formation beyond the boundaries of the AV canal, and overexpression of apc
inhibits EC formation [39
]. Other studies employing pharmacological inhibitors have suggested that Calcineurin/NFAT signaling promotes EC formation, while Notch signaling inhibits EC formation [24
]. Additionally, a number of lines of evidence demonstrate that cardiac function plays a key role in the induction of ECs. Mutation of genes that are important for cardiac contractility, including the cardiac actin gene cardiofunk
) and the cardiac troponin T gene silent heart
), prevents EC formation, suggesting that shear stress, produced by blood flow, or a stretch response, produced by contraction, could be required to initiate valve development [24
]. The notion of shear stress influencing valve morphogenesis has also been suggested by Hove et al.
, who demonstrated that implanting beads to obstruct blood flow at either the inflow or outflow end of the heart can inhibit EC formation [42
Altogether, studies of zebrafish mutations affecting cardiac morphogenesis indicate the vast complexity of its regulation. To reveal the precise way that each pathway regulates a particular morphogenetic process, it is essential to understand the effects that specific genes exert on the dynamic changes to cardiac cytoarchitecture that underlie morphogenesis. How do the shapes and sizes of individual cells dictate the dimensions of the entire organ? How do cell-cell interactions contribute to morphological rearrangements? How do individual cell movements establish larger patterns of tissue movement? The optical accessibility of the zebrafish provides unparalleled opportunities for resolving the mechanisms of morphogenesis on a cellular and subcellular level. Visualization of myocardial and endocardial cells has been greatly enhanced by transgenes that express gfp
in cardiomyocytes (e.g. Tg(cmlc2:egfp)
] or endothelial cells (e.g. Tg(flk1:egfp)
]. In concert with other molecular markers, these transgenes have been instrumental in revealing how key regulatory genes effect changes in cellular polarity, interactions, and morphology that, in turn, control specific steps of morphogenesis.
Any morphogenetic rearrangement of embryonic tissues is likely to begin with changes in the cytoarchitecture of individual cells. For example, Trinh and Stainier have demonstrated that migrating cardiomyocytes begin to form a polarized epithelium as they approach the embryonic midline [29
]. By examining the localization of markers of apicobasal polarity in embryos expressing Tg(cmlc2:egfp)
(), they determined the dynamics of polarity acquisition in differentiating cardiomyocytes. Intriguingly, mutations that disrupt apicobasal polarity, such as natter
and hands off
, also inhibit myocardial migration; these data reveal previously unappreciated roles of Fibronectin and Hand2 in the establishment of apicobasal polarity and suggest that formation of a polarized epithelium is a requirement for the coordination of myocardial migration [29
]. Myocardial epithelial polarity also seems to be integral to the process of heart tube extension. Both PRKCi and Mpp5 are components of apically localized protein complexes, and analysis of prkci
mutants demonstrates that both genes are required cell-autonomously to insure the polarity and integrity of the myocardial epithelium [34
]. As Atp1a1a.1 is basolaterally localized in chick cardiomyocytes [45
], it is interesting to speculate that its function during heart tube extension could also be related to myocardial apicobasal polarity [32
]. Together, these data point to the importance of organization and coherence of the myocardium during the formation of the cardiac cone and its transformation into the heart tube.
Figure 2 Examination of cardiac cytoarchitecture demonstrates that the migrating myocardium is a polarized epithelium. (A,B) Confocal images of transverse sections, dorsal to the top, of Tg(cmlc2:egfp)  embryos at the 20-somite stage, as in . Expression (more ...)
Detailed examination of cytoarchitecture has also shed light on the mechanisms regulating AV valve formation. Using Tg(flk1:egfp)
to visualize endocardium, Beis et al.
determined that EC formation is preceded by striking changes in cell shapes and protein localization within the endocardium of the AV canal [24
]. Specifically, AV endocardial cells, previously squamous in appearance, become more cuboidal and begin to exhibit lateral localization of the adhesion molecule Dm-grasp. Examination of this early aspect of AV endocardial differentiation in embryos with valve defects has demonstrated distinct roles for the implicated pathways. In silent heart
mutants, AV endocardium remains squamous and does not express Dm-grasp, indicating the importance of cardiac function for the initiation of endocardial differentiation [24
]. In contrast, Calcineurin signaling is not required for initial differentiation of the AV canal endocardium, but it is important for the subsequent epithelial to mesenchymal transformation that creates ECs [24
]. Notch signaling, on the other hand, seems to be responsible for the spatial restriction of AV canal differentiation [24
]. In embryos treated with the Notch pathway inhibitor DAPT, ventricular endocardium undergoes differentiation similar to that normally observed only in the AV canal.
Characterization of cardiac cell shape changes and their subcellular patterning adds depth to our understanding of the forces that drive cardiac morphogenesis; mutant analysis complements this approach by bridging the gap between gene function and cellular activities. It will be interesting to see how future studies apply cytoarchitectural analysis to other aspects of cardiac morphogenesis, such as cardiac ballooning or the interactions of myocardial and endocardial cells during tube assembly. Furthermore, future work will add a new point of view of morphogenesis by employing transgenes for timelapse imaging of cardiac cell rearrangements. Eventually, cytoarchitectural and timelapse approaches to morphogenesis are likely to merge, once the reagents are available for monitoring the dynamics of subcellular protein localization while morphogenesis is underway.