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Asymmetric behaviors of myocardial cells drive zebrafish heart tube formation
Many vertebrate organs are derived from monolayered epithelia that undergo morphogenesis to acquire their shape. Whereas asymmetric left-right gene expression within the zebrafish heart field has been well documented, little was known about the tissue movements and cellular changes underlying early cardiac morphogenesis. Heart development in zebrafish involves the fusion of two myocardial progenitor fields at the embryonic midline. These heart fields derive from the left and right lateral plate mesoderm. Fusion of the two heart fields forms the heart cone, a central flat disc which is subsequently transformed into the primary heart tube. Morphogenetic processes and tissue dynamics required for heart cone-to-tube transition are not well understood (Figure 1).
Figure 1: Zebrafish heart tube assembly. All images represent reconstructions of confocal Z-stack sections imaged on whole-mounts. cardiac myosin light chain2:GFP, green (nuclear GFP within myocardial cells); PRKC/aPKC, red; ZO-1, blue. PRKC and ZO-1 were used as a counterstain to visualize the embryonic midline (see also dotted line in D). (A) At the 16-somite stage, wildtype myocardial cells are organized as two bilateral sheets of cells. (B) Both sheets converge onto the midline where they fuse to form the heart cone around the 20-somite stage. (C) Heart cone tilting places the heart into the anterior-posterior orientation by the 28-somite stage. The atrium (arrowhead) is located to the left and anterior whereas the ventricle (arrow) is oriented towards the midline and posterior.
We have recently described the transition of the flat heart field into the primary linear heart tube in zebrafish. We could demonstrate that asymmetric involution of the myocardial epithelium from the right side of the heart field initiates a complex tissue inversion which creates the ventral floor and medial side of the primary heart tube whereas the non-involuting left heart field gives rise to the dorsal roof of the primary heart tube (Figure 2). During heart tube formation, asymmetric left-right gene expression of lefty2 within the myocardium correlates with asymmetric tissue morphogenesis. Disruption of left-right gene expression caused randomized myocardial tissue involution. Time-lapse analysis combined with genetic analyses revealed that motility of the myocardial epithelium is a tissue migration process. Our results demonstrate that asymmetric morphogenetic movements of the two bilateral myocardial cell populations generate different dorso-ventral regions of the zebrafish heart tube. Together, these results provide the framework for the integration of single cell behaviors during the formation of the vertebrate primary heart tube.
Figure 2: Myocardial involution during heart tube formation. (A) Scheme representing the flat heart cone and the primary heart tube. The rapid remodeling of the myocardium during cone-to-tube transition is an unknown morphogenetic process. (B-D) Section along the anterior-posterior axis of the developing heart tube (45° angle away from the embryonic midline) at the 20-somite stage (19 hpf). Myocardial GFP-positive cells (green), immunohistochemical staining of aPKC (red), and zn5 (blue). Central heart cone opening marked with asterisk. (E) Schematic representation displaying asymmetric myocardial involution of the heart cone. (F-H) Similar section plane of a 25-somite stage (21.5 hpf) heart demonstrates the progression of the myocardial involution process. (J-L) At 27 hpf, ventral closure is still in progress. (N) Model representing key stages of heart tube formation due to asymmetric involution of the myocardial field. The color code demonstrates the gradual involution starting with yellow tissue at the border of the right-posterior heart cone opening followed by blue and finally the red area of the posterior myocardium.
Cell polarity and zebrafish cardiac morphogenesis
The zebrafish mutation heart and soul (has) causes severe defects in cardiac morphogenesis in which tilting of the heart cone is blocked and heart tube elongation is impaired. The has gene encodes Protein Kinase C iota (PRKCi) which is required for the establishment of apical-basal polarity of epithelial cells. PRKCs are components of the apical Par3 protein complex which has been linked to the apical Crumbs-Pals1 protein complex. Consistent with a function in apical-basal cell polarity, has mutants show defective formation and maintenance of several embryonic epithelia including the myocardium. The zebrafish mutation nagie oko (nok) also causes severe epithelial defects similar to has. nok encodes a membrane associated guanylate kinase (MAGUK) family protein with functional homology to mammalian Protein associated with Lin-seven 1 (Pals1)/ MAGUK p55 subfamily member 5 (Mpp5). nok/mpp5 mutants have myocardial defects including an incomplete heart tube elongation corresponding with a failure of myocardial cells to correctly expand in size (Figure 3).
Figure 3: Heart morphogenesis defect in zebrafish epithelial mutant. (A) The zebrafish wild-type embryonic heart at 30 hours of development visualized with cardiac myosin light chain 2 probe. The heart is an elongated two-chambered tube with an anterior atrium and (a) and posterior ventricle (v). (B) In comparison, the heart tube is not elongated in nagie oko mutant embryos.
Na,K ATPase interacts with nagie oko/mpp5 in maintaining myocardial polarity
In another study, we demonstrated the importance of correct ion balance for junctional maintenance and epithelial character of epithelial cells. Na,K ATPase, or Na pump, is an essential ion pump involved in regulating ionic concentrations within epithelial cells. We investigated the developmental function and regulatory mechanisms of this ion pump. The zebrafish α1B1 subunit of Na,K ATPase is encoded by the heart and mind (had) locus and had mutants show delayed heart tube elongation (Figure 4). This phenotype is reminiscent of the heart and soul/aPKCi and nagie oko (nok) mutant phenotypes which are characterized by a lack of epithelial cell polarity. In genetic interaction studies, Had/Na,K ATPase and Nok/Mpp5 interacted in the maintenance of apical myocardial junctions raising the intriguing possibility that the ion balance produced by the Na pump is critical in this process. To functionally characterize the role of the ion pump function, we produced a mutant form of Had/Na,K ATPase which specifically affects the ATPase activity that is essential for pumping sodium across the plasma membrane and found that it could not rescue the heart tube elongation phenotype. Our study suggests that the osmotic balance produced by the Na pump contributes to the maintenance of apical junction belts, a function that is uncovered upon loss of Nok/Mpp5.
Figure 4: Heart tube elongation requires the Na pump function. (A) Expression of the myocardial marker cmlc2 in a wild type embryo marks the elongated heart tube. (B) had mutant embryos lacking the Na pump show impaired heart tube elongation. (C) Heart tube elongation defects in had mutant embryos that were injected with a mutant mRNA that encodes a Na pump lacking an important regulatory residue (Serine 25)
Future directions
Research in our laboratory is currently directed towards identifying and characterizing the direct downstream phosphorylation targets of Has/aPKCi in the context of cell polarity and organ morphogenesis. Furthermore, we are interested in the morphogenetic events that drive cardiogenesis. We would like to describe the repertoire of cellular behaviors that underlie cardiac tube elongation and myocardial differentiation. Currently, we are generating the tools necessary to visualize in vivo the development of the zebrafish myocardial and endocardial tissues. In our analysis, we will initially focus on those genes that are involved in directed migratory behavior, control of planar or apical-basal cell polarity, tissue adhesion and cellular remodeling. The identification of the molecular pathways involved in vertebrate epithelial morphogenesis may lead to relevant animal models for human epithelial pathologies and to the development of novel therapeutic approaches.