Hydrocoel Morphogenesis Forming the Pentaradial Body Plan in a Sea Cucumber, Apostichopus Japonicus

Echinoderms constitute an animal phylum characterized by the pentaradial body plan. During the development from bilateral larvae to pentaradial adults, the formation of the multiple of five hydrocoel lobes, i.e., the buddings from the mesodermal coelom, is the firstly emerging pentameral character. The developmental mechanism underlying the hydrocoel-lobe formation should be revealed to understand the evolutionary process of this unique and highly derived body plan of echinoderms, although the morphogenetic mechanisms of hydrocoel lobes is largely uninvestigated. In this study, using the sea cucumber Apostichopus japonicus, in which the hydrocoel is easily observable, the developmental process of hydrocoel lobes was described in detail, focusing on the cell proliferation and rearrangement. Cell proliferation was not specifically distributed in the growing tips of the hydrocoel lobes and inhibition of the cell proliferation did not affect the lobe formation. During lobe formation, epithelium of the hydrocoel lobes were firstly stratified and then transformed into single-layered, suggesting that radial cell intercalation contributes to hydrocoel-lobe formation.

Mechanics of cell integration in vivo

During embryonic development, regeneration and homeostasis, cells have to physically integrate into their target tissues, where they ultimately execute their function. Despite a significant body of research on how mechanical forces instruct cellular behaviors within the plane of an epithelium, very little is known about the mechanical interplay at the interface between migrating cells and their surrounding tissue, which has its own dynamics, architecture and identity. Here, using quantitative in vivo imaging and molecular perturbations, together with a theoretical model, we reveal that multiciliated cell (MCC) precursors in the Xenopus embryo form dynamic filopodia that pull at the vertices of the overlying epithelial sheet to probe their stiffness and identify the preferred positions for their integration into the tissue. Moreover, we report a novel function for a structural component of vertices, the lipolysis-stimulated lipoprotein receptor (LSR), in filopodia dynamics and show its critical role in cell intercalation. Remarkably, we find that pulling forces equip the MCCs to remodel the epithelial junctions of the neighboring tissue, enabling them to generate a permissive environment for their integration. Our findings reveal the intricate physical crosstalk at the cell-tissue interface and uncover previously unknown functions for mechanical forces in orchestrating cell integration.

Correct regionalisation of a tissue primordium is essential for coordinated morphogenesis

During organ development, tubular organs often form from flat epithelial primordia. In the placodes of the forming tubes of the salivary glands in the Drosophila embryo, we previously identified spatially defined cell behaviours of cell wedging, tilting and cell intercalation that are key to the initial stages of tube formation. Here we address what the requirements are that ensure the continuous formation of a narrow symmetrical tube from an initially asymmetrical primordium whilst overall tissue geometry is constantly changing. We are using live-imaging and quantitative methods to compare wild-type placodes and mutants that either show disrupted cell behaviours or an initial symmetrical placode organisation, with both resulting in severe impairment of the invagination. We find that early transcriptional patterning of key morphogenetic transcription factors drives the selective activation of downstream morphogenetic modules, such as GPCR signalling that activates apical-medial actomyosin activity to drive cell wedging at the future asymmetrically-placed invagination point. Over time, transcription of key factors expands across the rest of the placode and cells switch their behaviour from predominantly intercalating to predominantly apically constricting as their position approaches the invagination pit. Misplacement or enlargement of the initial invagination pit leads to early problems in cell behaviours that eventually result in a defective organ shape. Our work illustrates that the dynamic patterning of the expression of transcription factors and downstream morphogenetic effectors ensures positionally fixed areas of cell behaviour with regards to the invagination point. This patterning in combination with the asymmetric geometrical set-up ensures functional organ formation.

Admp regulates tail bending by controlling the intercalation of the ventral epidermis through myosin phosphorylation

Chordate tailbud embryos have similar morphological features, including a bending tail. A recent study revealed that the actomyosin of the notochord changes the contractility and drive tail bending of the early Ciona tailbud embryo. Yet, the upstream regulator of tail bending remains unknown. In this study, we find that Admp regulates tail bending of Ciona mid-tailbud embryos. Anti-pSmad antibody signal was detected at the ventral midline tail epidermis. Admp knock-down embryo completely inhibited the ventral tail bending and reduced the number of the triangular-shaped cells, which has the apical accumulation of the myosin phosphorylation and inhibited specifically the cell-cell intercalation of the ventral epidermis. The degree of myosin phosphorylation of the ventral cells and tail bending were correlated. Finally, the laser cutter experiments demonstrated the myosin-phosphorylation-dependent tension of the ventral midline epidermis during tail bending. We conclude that Admp is an upstream regulator of the tail bending by controlling myosin phosphorylation and its localization of ventral epidermal cells. These data reveal a new aspect of the function of the Admp that might be evolutionarily conserved in bilaterian animals.Summary StatementAdmp is an upstream regulator of the bending of the tail in the tailbud embryo regulating tissue polarity of the ventral midline epidermis by phosphorylation of myosin.

A combination of notch signaling, preferential adhesion and endocytosis induces a slow mode of cell intercalation in the Drosophila retina

Movement of epithelial cells in a tissue occurs through neighbor exchange and drives tissue shape changes. It requires intercellular junction remodeling, a process typically powered by the contractile actomyosin cytoskeleton. This has mostly been investigated in homogeneous epithelia where intercalation takes minutes. However, in some tissues, intercalation involves different cell types and can take hours. Whether slow and fast intercalation share the same mechanisms remains to be examined. To address this issue, we use the fly eye, where the cone cells exchange neighbors over approximately 10 hours to shape the lens. We uncover three pathways regulating this slow mode of cell intercalation. Firstly, we find a limited requirement for MyosinII. In this case, mathematical modeling predicts an adhesion dominant intercalation mechanism. Genetic experiments support this prediction and reveal a role for adhesion through the Nephrin proteins Roughest and Hibris. Secondly, we find cone cell intercalation is regulated by the Notch-signaling pathway. Thirdly, we show endocytosis is required for membrane removal and Notch activation. Altogether, our work indicates that adhesion, endocytosis and Notch can induce junction remodeling over long-time scales.

Adhesion dynamics regulate cell intercalation behaviour in an active tissue

Cell intercalation is a key cell behaviour of morphogenesis and wound healing, where local cell neighbour exchanges can cause dramatic tissue deformations such as body axis extension. Here, we develop a mechanical model to understand active cell intercalation behaviours in the context of an epithelial tissue. Extending existing descriptions, such as vertex models, the junctional actomyosin cortex of every cell is modelled as a continuum morphoelastic rod, explicitly representing cortices facing each other at bicellular junctions. Cells are described directly in terms of the key subcellular constituents that drive dynamics, with localised stresses from the contractile actomyosin cortex and adhesion molecules coupling apposed cortices. This multi-scale apposed-cortex formulation reveals key behaviours that drive tissue dynamics, such as cell-cell shearing and flow of junctional material past cell vertices. We show that cell neighbour exchanges can be driven by purely junctional mechanisms. Active contractility and viscous turnover in a single bicellular junction are sufficient to shrink and remove a junction. Next, the 4-way vertex is resolved and a new, orthogonal junction extends passively. The adhesion timescale defines a frictional viscosity that is an important regulator of these dynamics, modulating tension transmission in the tissue as well as the speeds of junction shrinkage and growth. The model additionally predicts that rosettes, which form when a vertex becomes common to many cells, are likely to occur in active tissues with high adhesive friction.

In vivo regulation of fluorescent fusion proteins by engineered kinases

Reversible protein phosphorylation by kinases in extensively used to control a plethora of processes essential for proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases for direct, robust and tissue-specific phosphorylation of target fluorescent protein fusions in vivo. We show that synthetic Rok kinases, based on the Drosophila ortholog of Rho-associated protein kinase (ROCK), are functional enzymes and can activate myosin II through phosphorylation of Sqh::GFP or Sqh::mCherry in different morphogenetic processes in a developing fly embryo. We next use the system to study the impact of actomyosin activation specifically in the developing tracheal branches and showed that ectopic activation of actomyosin with engineered Rok kinase did not prevent cell intercalation nor the formation of autocellular junctions. We assume that this approach can be adapted to other kinases and targets in various eukaryotic genetic systems.

Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension

ABSTRACTDistinct spatiotemporal patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar polarized pattern of myosin II localization regulated by Rho1 signaling duringDrosophilabody axis elongation is thought to drive the cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin localization pattern influence the multiple cell behaviors—including cell intercalation, cell shape changes, and apical cell area fluctuations—that simultaneously occur within a tissue during morphogenesis. Here, we use optogenetic activation (optoGEF) and deactivation (optoGAP) of Rho1 signaling to perturb the myosin pattern in the germband epithelium duringDrosophilaaxis elongation and analyze the effects on contractile cell behaviors within the tissue. We find that uniform photoactivation of optoGEF or optoGAP is sufficient to rapidly override the endogenous myosin pattern, abolishing myosin planar polarity and reducing cell intercalation and convergent extension. However, these two perturbations have distinct effects on junctional and medial myosin localization, apical cell area fluctuations, and cell packings within the germband. Activation of Rho1 signaling in optoGEF embryos increases myosin accumulation in the medial-apical domain of germband cells, leading to increased amplitudes of apical cell area fluctuations. This enhanced contractility is translated into heterogeneous reductions in apical cell areas across the tissue, disrupting cellular packings within the germband. Conversely, inactivation of Rho1 signaling in optoGAP embryos decreases both medial and junctional myosin accumulation, leading to a dramatic reduction in cell area fluctuations. These results demonstrate that the level of Rho1 activity and the balance between junctional and medial myosin regulate apical cell area fluctuations and cellular packings in the germband, which have been proposed to influence the biophysics of cell rearrangements and tissue fluidity.STATEMENT OF SIGNIFICANCETissues are shaped by forces produced by dynamic patterns of actomyosin contractility. However, the mechanisms underlying these myosin patterns and their translation into cell behavior and tissue-level movements are not understood. Here, we show that optogenetic tools designed to control upstream regulators of myosin II can be used to rapidly manipulate myosin patterns and analyze the effects on cell behaviors during tissue morphogenesis. Combining optogenetics with live imaging in the developing fruit fly embryo, we show that acute perturbations to upstream myosin regulators are sufficient to rapidly perturb existing myosin patterns and alter cell movements and shapes during axis elongation, resulting in abnormalities in embryo shape. These results directly link myosin contractility patterns to cell behaviors that shape tissues, providing new insights into the mechanisms that generate functional tissues.