Not just inductive: a crucial mechanical role for the endoderm during heart tube assembly

Abstract

SUMMARYThe heart is the first functioning organ to form during development. During gastrulation, the cardiac progenitors reside in the lateral plate mesoderm but maintain close contact with the underlying endoderm. In amniotes, these bilateral heart fields are initially organized as a pair of flat epithelia that move towards the embryonic midline and fuse above the anterior intestinal portal (AIP) to form the heart tube. This medial motion is typically attributed to active mesodermal migration over the underlying endoderm. In this model, the role of the endoderm is twofold: to serve as a mechanically passive substrate for the crawling mesoderm and to secrete various growth factors necessary for cardiac specification and differentiation. Here, using computational modeling and experiments on chick embryos, we present evidence supporting an active mechanical role for the endoderm during heart tube assembly. Label-tracking experiments suggest that active endodermal shortening around the AIP accounts for most of the heart field motion towards the midline. Results indicate that this shortening is driven by cytoskeletal contraction, as exposure to the myosin-II inhibitor blebbistatin arrested any shortening and also decreased both tissue stiffness (measured by microindentation) and mechanical tension (measured by cutting experiments). In addition, blebbistatin treatment often resulted in cardia bifida and abnormal foregut morphogenesis. Moreover, finite element simulations of our cutting experiments suggest that the endoderm (not the mesoderm) is the primary contractile tissue layer during this process. Taken together, these results indicate that contraction of the endoderm actively pulls the heart fields towards the embryonic midline, where they fuse to form the heart tube. KEY WORDS: Biomechanics, Chick embryo, Computational modeling, Endoderm, Heart development, MorphogenesisNot just inductive: a crucial mechanical role for the endoderm during heart tube assembly Victor D. Varner and Larry A. Taber* DEVELOPMENT of liquid culture media and incubated at 38°C in 95% O 2 and 5% CO 2 . This method prevents artifacts caused by fluid surface tension, which alter the mechanical stresses in the embryo (Voronov and Taber, 2002).In some experiments, embryos were cultured in 100 M (-)-blebbistatin (Sigma, St Louis, MO, USA) to broadly suppress any cytoskeletal contraction dependent on myosin II. The inhibitor could be washed out by rinsing the embryo several times in PBS and then continuing the culture with new blebbistatin-free media. Injection labeling and trackingTo measure tissue motion in both the endoderm and mesoderm around the AIP, small groups of cells (in both germ layers) were labeled at HH stage 7+/8-with the lipophilic fluorescent dye DiI (Molecular Probes, Eugene, OR, USA) mixed in a 20% sucrose solution. DiI injections were made using pulled glass micropipettes and a pneumatic pump (PicoPump PV830, World Precision Instruments). To label cardiogenic mesoderm, the tip of the injection pipette was first pierced thr...

Endoderm convergence controls subduction of the myocardial precursors during heart-tube formation

“…This finding is consistent with observations in other animal model systems, showing that the migration of myocardial cells is influenced by the movements of adjacent tissues [e.g. the endoderm in chick and mouse (Madabhushi and Lacy, 2011;Maretto et al, 2008;Varner and Taber, 2012), and the ectoderm in Drosophila (Haack et al, 2014)]. …”

“…Thus, the subduction and medial migration of myocardial cells after 14 s is probably due to their innate migration potential. Earlier studies in chick suggested that myocardial cells have little ability to migrate, and that endoderm movement drives their migration (Cui et al, 2009;Varner and Taber, 2012). However, a new study showed that chick myocardial cells instead exhibit active displacement and autonomous ventral movement, and that these changes are independent of the endoderm (Aleksandrova et al, 2015).…”

“…The endoderm is also thought to provide a physical substrate over which cardiac precursors migrate, because of the close anatomic proximity of these tissues and the finding that in most animal models, myocardial migration fails in the absence of endoderm (David and Rosa, 2001;Lough and Sugi, 2000). Recent work in chick, however, suggests that endodermal movement directs migration of the heart field by providing mechanical forces that pull cardiac mesoderm to the midline (Varner and Taber, 2012), and that active migration by the cardiac cells is minimal (Cui et al, 2009). Similarly, in mouse, invagination of the endoderm-derived foregut is directly linked to migration of the pericardial mesoderm (Madabhushi and Lacy, 2011;Maretto et al, 2008).…”

S1pr2/Gα13 signaling controls myocardial migration by regulating endoderm convergence

“…It has been proposed that the endoderm serves as a substrate that allows cardiac mesoderm to migrate, and that it releases either cues that guide the migration of cardiac progenitor cells or signaling molecules that promote cardiac differentiation (Schultheiss et al, 1997;Andree et al, 1998;Lough and Sugi, 2000;David and Rosa, 2001;Alsan and Schultheiss, 2002;Nijmeijer et al, 2009). Additionally, the endoderm might provide a mechanical force that drives the migration of myocardial cells (Varner and Taber, 2012). Thus, despite the extensive evidence that the endoderm is important for cardiomyocyte movement during heart tube formation, its exact roles remain to be determined.…”

Extracellular matrix motion and early morphogenesis

“…regression of the anterior intestinal portal) ( Fig. 2A) (Aleksandrova et al, 2015a;Varner and Taber, 2012). Time-lapse recordings show that as the myocardial progenitor fields approach the midline, the cells autonomously exert mechanical stresses within the tissue (Aleksandrova et al, 2015a).…”