BACKGROUND: The tunicates form a group of filter-feeding marine animals closely related to vertebrates. They share with them a number of features such as a notochord and a dorsal neural tube in the tadpole larvae of ascidians, one of the three groups that make tunicates. However, a number of typical chordate characters have been lost in different branches of tunicates, a diverse and fast-evolving phylum. Consequently, the tunic, a sort of exoskeleton made of extracellular material including cellulose secreted by the epidermis, is the unifying character defining the tunicate phylum. In the larva of ascidians, the tunic differentiates in the tail into a median fin (with dorsal and ventral extended blades) and a caudal fin.
RESULTS: Here we have performed experiments in the ascidian Phallusia mammillata to address the molecular control of tunic 3D morphogenesis. We have demonstrated that the tail epidermis medio-lateral patterning essential for peripheral nervous system specification also controls tunic elongation into fins. More specifically, when tail epidermis midline identity was abolished by BMP signaling inhibition, or CRISPR/Cas9 inactivation of the transcription factor coding genes Msx or Klf1/2/4/17, median fin did not form. We postulated that this genetic program should regulate effectors of tunic secretion. We thus analyzed the expression and regulation in different ascidian species of two genes acquired by horizontal gene transfer (HGT) from bacteria, CesA coding for a cellulose synthase and Gh6 coding for a cellulase. We have uncovered an unexpected dynamic history of these genes in tunicates and high levels of variability in gene expression and regulation among ascidians. Although, in Phallusia, Gh6 has a regionalized expression in the epidermis compatible with an involvement in fin elongation, our functional studies indicate a minor function during caudal fin formation only.
CONCLUSIONS: Our study constitutes an important step in the study of the integration of HGT-acquired genes into developmental networks and a cellulose-based morphogenesis of extracellular material in animals.

Despite extensive study, the morphogenetic mechanisms of heart looping remain controversial because of a lack of information concerning precise tissue-level deformation and the quantitative relationship between tissue and cellular dynamics; this lack of information causes difficulties in evaluating previously proposed models. To overcome these limitations, we perform four-dimensional (4D) high-resolution imaging to reconstruct a tissue deformation map, which reveals that, at the tissue scale, initial heart looping is achieved by left-right (LR) asymmetry in the direction of deformation within the myocardial tube. We further identify F-actin-dependent directional cell rearrangement in the right myocardium as a major contributor to LR asymmetric tissue deformation. Our findings demonstrate that heart looping involves dynamic and intrinsic cellular behaviors within the tubular tissue and provide a significantly different viewpoint from current models that are based on LR asymmetry of growth and/or stress at the tube boundaries. Finally, we propose a minimally sufficient model for initial heart looping that is also supported by mechanical simulations.

The transcription coactivator, Yes-associated protein (YAP), which is a nuclear effector of the Hippo signaling pathway, has been shown to be a mechano-transducer. By using mutant fish and human 3D spheroids, we have recently demonstrated that YAP is also a mechano-effector. YAP functions in three-dimensional (3D) morphogenesis of organ and global body shape by controlling actomyosin-mediated tissue tension. In this chapter, we present a platform that links the findings in fish embryos with human cells. The protocols for analyzing tissue tension-mediated global body shape/organ morphogenesis in vivo and ex vivo using medaka fish embryos and in vitro using human cell spheroids represent useful tools for unraveling the molecular mechanisms by which YAP functions in regulating global body/organ morphogenesis.

Four-dimensional (4D) biofabrication techniques aim to dynamically produce and control three-dimensional (3D) biological structures that would transform their shapes or functionalities with time, when a stimulus is imposed or cell post-printing self-assembly occurs. The evolution of 3D branching patterns via self-assembly of cells is critical for the 4D biofabrication of artificial organs or tissues with branched geometry. However, it is still unclear how the formation and evolution of these branching patterns are biologically encoded. Here, we study the biofabrication of lung branching structures utilizing a simulation model based on Turing instability that raises a dynamic reaction⁻diffusion (RD) process of the biomolecules and cells. The simulation model incorporates partial differential equations of four variables, describing the tempo-spatial distribution of the variables in 3D over time. The simulation results present the formation and evolution process of 3D branching patterns over time and also interpret both the behaviors of side-branching and tip-splitting as the stalk grows and the fabrication style under an external concentration gradient of morphogen, through 3D visualization. This provides a theoretical framework for rationally guiding the 4D biofabrication of lung airway grafts via cellular self-organization, which would potentially reduce the complexity of future experimental research and number of trials.