Why is cytoskeletal contraction required for cardiac fusion before but not after looping begins?

Abstract

Cytoskeletal contraction is crucial to numerous morphogenetic processes, but its role in early heart development is poorly understood. Studies in chick embryos have shown that inhibiting myosin-II-based contraction prior to Hamburger-Hamilton (HH) stage 10 (33 hr incubation) impedes fusion of the mesodermal heart fields that create the primitive heart tube (HT), as well as the ensuing process of cardiac looping. If contraction is inhibited at or after looping begins at HH10, however, fusion and looping proceed relatively normally. To explore the mechanisms behind this seemingly fundamental change in behavior, we measured spatiotemporal distributions of tissue stiffness, stress, and strain around the anterior intestinal portal (AIP), the opening to the foregut where contraction and cardiac fusion occur. The results indicate that stiffness and tangential tension decreased bilaterally along the AIP with distance from the embryonic midline. The gradients in stiffness and tension, as well as strain rate, increased to peaks at HH9 (30 hr) and decreased afterward. Exposure to the myosin II inhibitor blebbistatin reduced these effects, suggesting that they are mainly generated by active cytoskeletal contraction, and finite-element modeling indicates that the measured mechanical gradients are consistent with a relatively uniform contraction of the endodermal layer in conjunction with constraints imposed by the attached mesoderm. Taken together, our results suggest that, before HH10, endodermal contraction pulls the bilateral heart fields toward the midline where they fuse to create the HT. By HH10, however, the fusion process is far enough along to enable apposing cardiac progenitor cells to keep “zipping” together during looping without the need for continued high contractile forces. These findings should shed new light on a perplexing question in early heart development.

On the role of mechanics in driving mesenchymal-to-epithelial transitions

“…MET is required for proper heart function [57] and shortly after epithelialization is when the first cardiac electrical activity is recorded [58, 59]. During their movement to the ventral midline, HPCs move through a dynamically changing mechanical micro-environment, which includes extensive fibronectin remodeling [60, 61] increases in tissue stiffness [62] and tension originating from the convergent extension of the endoderm [63–65]. While knocking down actomyosin based cell contractility prior to heart tube formation and epithelialization consistently causes cardiac defects [63, 66], perturbing cell contractility near the onset of looping, post-MET does not, which implicates a crucial role for cell mechanics in providing cues to guide the early organization of HPCs.…”

A new hypothesis for foregut and heart tube formation based on differential growth and actomyosin contraction

“…3Aa-Ad,B). Previous studies have found that contraction around the AIP also pulls pre-cardiac mesoderm towards the midline during these early stages (Varner and Taber, 2012a;Aleksandrova et al, 2015;Shi et al, 2015).…”

“…10D), consistent with our model and supporting the view that growth helps reduce iSPL tension as the AIP descends. Varner and Taber (2012a) and Shi et al (2015) used linear incisions to examine tension in the AIP during stages HH8-10 (Fig. 11A).…”

Spatiotemporally Controlled Mechanical Cues Drive Progenitor Mesenchymal-to-Epithelial Transition Enabling Proper Heart Formation and Function

“…On their way to the ventral midline HPCs traverse a dynamically changing mechanical microenvironment marked by fibronectin remodeling [10, 16, 20], changing tissue stiffness [19, 36], and tension from ventral convergent extension [15, 19, 29]. While MET is not a prerequisite for HPC differentiation or arrival at the ventral midline, the proper timing of MET is both sensitive to the mechanical environment of the HFR and required for proper heart structure (Figure 2H–I, Figure 5G, Figure S2) and function (Figure 7C and Figure S6D–H).…”

“…Cardiac defects can also arise from defects in early HPC polarity [10], actomyosin contractility [19], and the microenvironment of the heart forming region (HFR; [20]). Even though these processes are likely involved in establishing the mechanical microenvironment of the HFR, the exact role of physical mechanics in early heart formation remains unclear.…”