Vertebrate organs are derived from epithelial or endothelial sheets of cells that undergo complex morphogenetic transformations. In our lab, we study the embryonic zebrafish heart, a relatively simple organ compared with its mammalian counterpart, to better understand the signaling events that instruct the assembly of the early heart tube. Initially the heart consists of only the outer myocardial and inner endocardial cell layers. The endocardium is a specialized population of endothelial cells that line the interior of the heart. We would like to understand: What are the signals that regulate the morphogenesis of myocardium and endocardium? To what extent do these two tissues communicate during cardiac jogging and looping morphogenesis? What determines the differentiation of endocardium into its different morphological derivatives such as cushion cells? In collaboration with clinical researchers, the group uses developmental genetics combined with cell biological and pharmacological approaches to develop animal models for human cardiovascular diseases. One particular focus of research is to characterize endocardial development. We use highly interdisciplinary approaches to analyze cardiac (both myocardial and endocardial) morphogenesis by combining expression analyses (deep sequencing; microarray studies), functional cell biological and genetic tools for tissue- or single cell level functional studies, in vivo high-resolution 4D-confocal imaging, systems biological approaches, in silico modeling of single cell behaviors during cardiac morphogenesis, and pharmacological studies.
Morphogen signaling during cardiac development
Signaling by morphogens (=form giving molecules) plays an important role in the regulation of cardiac progenitor cell behaviors. In a recent study, we were able to elucidate the molecular crosstalk between two Transforming Growth Factor-β (TGF-β) morphogens, Nodals and Bone morphogenetic proteins (Bmps), and to characterize their impact on left/right asymmetric development of the heart. Our highly-interdisciplinary approach, which included detailed expression analyses combined with functional studies, high-resolution live imaging, and mathematical modeling of this process, suggested that Nodal, via modulating the extracellular matrix within the left cardiac field, dampens the efficiency of Bmp signaling on the left (Figure 1; Veerkamp et al., Dev. Cell 2013).
Figure 1: Reversal of left/right asymmetric Bmp activity within the heart upon expression of Nodal within the right cardiac field. (A) The transgenic reporter line Tg(BRE:dmKO2)mw40 indicates that the activity of “Bmp”-Smads-1/5/8 is higher within the left compared with the right cardiac field upon misexpression of Nodal (Southpaw) within the right cardiac field (asterisks, GFP false-colored in white). Signaling intensities are indicated by color range. (B) Fluorescence two-color in situ hybridization reveals normal lefty1 (lft1, a target gene of Nodal signaling) cardiac expression in clones expressing a dominant-negative Bmp receptor (myl7:dnbmpr2a). L, left; R, right.
Our findings suggest that minor left-right differences in Bmp activity within the cardiac field may determine bi-phasic states: cardiac progenitor cells within the right cardiac cone are slightly more adhesive, and cells on the left side exhibit a slightly more motile character. Thus, cardiac left-right asymmetry may be explained by Nodal modulating an anti-motogenic Bmp activity. In clonal misexpression studies, in which Bmp activity was clonally reduced within the right cardiac field, we observed an inversion of cardiac laterality. Moreover, reducing Bmp activity to below normal levels on the left side even enhanced cardiac jogging towards the left (Figure 2). One implication of these clonal studies is that changes in individual cell motility rates may impact the tissue displacement of larger coherent groups of cells.
Figure 2: (A) Schematic diagram illustrating that the Nodal target Hyaluronan synthase 2 dampens Bmp activity within the left cardiac field which causes lower expression of non-muscle myosin II (NMII) and higher cardiac progenitor cell motility. (B) Cross section through the cardiac field (myocardial cells marked green; F-actin, red)
Molecular control of endocardial chamber morphogenesis
In another study, we have recently analyzed the process of chamber morphogenesis of the endocardium (Dietrich et al., Dev. Cell 2014). During heart development, the onset of heartbeat and blood flow coincides with a ballooning of the cardiac chambers (Figure 3). By combining functional manipulations, fate mapping studies, and high-resolution imaging, we showed that endocardial growth occurs without an influx of external cells. Instead, endocardial cell proliferation is regulated, both by blood flow and by Bmp signaling in a manner independent of Vegf signaling. We also found that during cardiac ballooning stages, endocardial cells obtain distinct chamber- and inner-versus-outer-curvature-specific surface area sizes. Finally, we showed that the hemodynamic-sensitive transcription factor Krüppel-like factor 2a (Klf2a) is an important regulator of endocardial cell morphology. These findings establish the endocardium as the flow-sensitive tissue in the heart with a key role in adapting chamber growth in response to the mechanical stimulus of blood flow.
Taken together, our findings are indicative of dynamic interactions between the myocardium and endocardium that constantly attune cellular sizes and shapes, and thus cardiac chamber dimensions in response to physiological changes, the most important of which is blood flow. Understanding this dynamic crosstalk during development may have implications for understanding how cardiac morphology changes later in life, in response to physiological adaptations or as pathophysiological conditions arise.
Figure 3: Frontal view shows the two distinct endocardial chambers, atrium (A) and ventricle (V), during cardiac ballooning stages in a 48 hour old zebrafish embryo. The two endocardial chambers are visualized with the transgenic reporter lines [Tg(flt1:YFP), green; Tg(kdrl:mcherry), red].
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