Figure 2. Spatiotemporal patterns of gene expression for heart looping may be engineered in vitro.

(A) Select signaling pathways and factors involved in the development of the looping heart tube that can potentially be controlled using existing techniques. The looping heart tube can be broadly divided into four areas based on what part of the heart they will form: outflow tract, future ventricles, future atria, and future atrioventricular canal. Cells in each of those areas have unique gene and protein expression profiles that allow them to specialize in performing their specific tasks. While previously published reviews described these expression profiles in detail [6,34–36,38– 41], here we have identified the few that can potentially be controlled using currently existing techniques (specific reference numbers provided in figure image). (B) Techniques for controlling heart tube looping-associated pathways. We have identified three main existing approaches to modulating signaling pathways in engineered or explanted native tissues: controlled diffusion chambers for creating gradients of soluble factors, functionalized hydrogels, and optogenetic methods. Diffusion-based techniques rely on controlling incubation chamber geometry to prevent convective flows, controlling medium flow in a microfluidic chamber, or both. Functional hydrogels allow for photopatterning and photorelease of a variety of factors with micron-level resolution. Additionally, they allow for control of the mechanical properties of local cell environment via photomediated gel cross-linking or degradation. Finally, optogenetic methods rely on genetic modifications of the cells allowing for spatiotemporal up- and downregulation of specific expression factors in those cells using light.