Drosophila Germband Extension Research Articles

Actomyosin cables impart spatiotemporally resolved fluidity to regulate epithelial tissue flow during Drosophila germ band extension

Actomyosin cables impart spatiotemporally resolved fluidity to regulate epithelial tissue flow during Drosophila germ band extension

Contractile actomyosin cables and pulling forces from adjacent tissue cooperate to drive Drosophila germ-band extension.

Germ-band extension in Drosophila is a major embryogenesis event, when convergent-extension dramatically reshapes the germ-band. Fluid-like cell motions narrow the germ-band in one direction (dorsal-ventral axis, DV), elongating in the other (anterior-posterior axis, AP). What forces drive germ-band extension? This is controversial. (1) One force is from planar polarized myosin at cell-cell junctions parallel to DV. Blocking myosin activation with optogenetic RhoGAP (optoGAP) activation reduced germ-band elongation ∼1/3 and suppressed cell intercalation (Herrera-Perez et al., 2021). (2) Pulling forces from the invaginating posterior midgut, adjacent to the germ-band posterior end, also contribute. Blocking invagination with the torso mutation reduced germ-band elongation ∼1/3 (Collinet et al., 2015). Further, DV cell elongation is reduced with the torso-like mutation blocking pulling forces (Lye et al., 2015), but little-affected by optoGAP-mediated myosin blocking. Thus, experiment implicates both myosin cables and pulling forces from adjacent tissue, but their contributions and the mechanisms are poorly understood. Here, we developed a biophysical model of Drosophila germ-band extension, with junctional actomyosin contractility, elastic restoring forces, internal viscous forces, extracellular drag, forces from adjacent tissues and cell intercalation (T1 transitions or rosette formation). Our results quantitatively reveal that planar polarized myosin and posterior midgut pulling are driving forces for convergent extension in the DV and AP directions, respectively. Actomyosin cables shrink junctions and intercalate cells, but cannot elongate tissue at wild-type rates, consistent with the phenotype of torso mutants (Collinet et al., 2015). Normal tissue elongation required both myosin cables and posterior midgut pulling, and in simulations with both, the profile of cell lengths reproduced the experimental monotonic increase in the AP direction (Lye et al., 2015). These results suggest convergent extension is cooperatively driven by myosin contractility and active pulling from adjacent tissue.

Modeling cell intercalation during Drosophila germband extension

Germband extension during Drosophila development is primarily driven by cell intercalation, which involves three key components: planar cell polarity, anisotropic myosin contractile forces on cellular junctions, and cellular deformation and movement. Prior experimental work probed each of these factors in depth, but the connection between them remains unclear. This paper presents an integrated chemomechanical model that combines the three factors into a coherent mathematical framework for studying cell intercalation in the germband tissue. The model produces the planar cell polarization of key proteins, including Rho-kinase, Bazooka and myosin, the development of anisotropic contractile forces, and subsequent cell deformation and rearrangement. Cell intercalation occurs through T1 transitions among four neighboring cells and rosettes involving six cells. Such six-cell rosettes entail stronger myosin-based contractile forces, and on average produce a moderately larger amount of germband extension than the T1 transitions. The resolution of T1 and rosettes is driven by contractile forces on junctions anterior and posterior to the assembly as well as the pulling force of the medial myosin in the anterior and posterior cells. The global stretching due to posterior midgut invagination also plays a minor role. These model predictions are in reasonable agreement with experimental observations.

Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension.

Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell-cell interfaces, while the function of tricellular vertices has remained unaddressed. Here, we identify a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, we find that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. Our results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation.

Basolateral protrusion and apical contraction cooperatively drive Drosophila germband extension

Basolateral protrusion and apical contraction cooperatively drive Drosophila germband extension

Basolateral protrusion and apical contraction cooperatively drive Drosophila germ-band extension.

Throughout development, tissues undergo complex morphological changes, resulting from cellular mechanics that evolve over time and in three-dimensional space. During Drosophila germ-band extension (GBE), cell intercalation is the key mechanism for tissue extension, and the associated apical junction remodelling is driven by polarized myosin-II-dependent contraction. However, the contribution of the basolateral cellular mechanics to GBE remains poorly understood. Here, we characterize how cells coordinate their shape from the apical to the basal side during rosette formation, a hallmark of cell intercalation. Basolateral rosette formation is driven by cells mostly located at the dorsal/ventral part of the rosette (D/V cells). These cells exhibit actin-rich wedge-shaped basolateral protrusions and migrate towards each other. Surprisingly, the formation of basolateral rosettes precedes that of the apical rosettes. Basolateral rosette formation is independent of apical contractility, but requires Rac1-dependent protrusive motility. Furthermore, we identified Src42A as a regulator of basolateral rosette formation. Our data show that in addition to apical contraction, active cell migration driven by basolateral protrusions plays a pivotal role in rosette formation and contributes toGBE.

Unipolar distributions of junctional Myosin II identify cell stripe boundaries that drive cell intercalation throughout Drosophila axis extension.

Convergence and extension movements elongate tissues during development. Drosophila germ-band extension (GBE) is one example, which requires active cell rearrangements driven by Myosin II planar polarisation. Here, we develop novel computational methods to analyse the spatiotemporal dynamics of Myosin II during GBE, at the scale of the tissue. We show that initial Myosin II bipolar cell polarization gives way to unipolar enrichment at parasegmental boundaries and two further boundaries within each parasegment, concomitant with a doubling of cell number as the tissue elongates. These boundaries are the primary sites of cell intercalation, behaving as mechanical barriers and providing a mechanism for how cells remain ordered during GBE. Enrichment at parasegment boundaries during GBE is independent of Wingless signaling, suggesting pair-rule gene control. Our results are consistent with recent work showing that a combinatorial code of Toll-like receptors downstream of pair-rule genes contributes to Myosin II polarization via local cell-cell interactions. We propose an updated cell-cell interaction model for Myosin II polarization that we tested in a vertex-based simulation.

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.

A biomechanical model for cell polarization and intercalation during Drosophila germband extension

Germband extension during Drosophila development features the merging of cells along the dorsal–ventral (DV) axis and their separation along the anterior–posterior (AP) axis. This intercalation process involves planar cell polarity, anisotropic contractile forces along cell edges, and concerted cell deformation and movement. Although prior experiments have probed each of these factors separately, the connection among them remains unclear. This paper presents a chemo-mechanical model that integrates the three factors into a coherent framework. The model predicts the polarization of Rho-kinase, myosin and Bazooka downstream of an anisotropic Shroom distribution. In particular, myosin accumulates on cell edges along the DV axis, causing them to contract into a vertex. Subsequently, medial myosin in the cells anterior and posterior to the vertex helps to elongate it into a new edge parallel to the body axis. Thus, the tissue extends along the AP axis and narrows in the transverse direction through neighbor exchange. Model predictions of the polarity of the proteins and cell and tissue deformation are in good agreement with experimental observations.

Patterns in Space: Coordinating Adhesion and Actomyosin Contractility at E-cadherin Junctions

Cadherin adhesion receptors are fundamental determinants of tissue organization in health and disease. Increasingly, we have come to appreciate that classical cadherins exert their biological actions through active cooperation with the contractile actin cytoskeleton. Rather than being passive resistors of detachment forces, cadherins can regulate the assembly and mechanics of the contractile apparatus itself. Moreover, coordinate spatial patterning of adhesion and contractility is emerging as a determinant of morphogenesis. Here we review recent developments in cadherins and actin cytoskeleton cooperativity, by focusing on E-cadherin adhesive patterning in the epithelia. Next, we discuss the underlying principles of cellular rearrangement during Drosophila germband extension and epithelial cell extrusion, as models of how planar and apical-lateral patterns of contractility organizes tissue architecture.

A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension.

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. We explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. We found surprising parallels between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change--cell extension along the anterior-posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. We propose a model in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension.

Tip-Cell Migration Controls Stalk-Cell Intercalation during Drosophila Tracheal Tube Elongation

Branching morphogenesis remodels epithelial tissues into tubular networks. This process is crucial to many organs, from the insect trachea to the vertebrate vasculature. Although Drosophila tracheal development has been well characterized morphologically and genetically, very little is known about the forces involved during morphogenesis. The repertoire of cell behaviors underlying tracheal primary branch remodeling is limited to cell migration, cell-shape changes, and stalk-cell intercalation (SCI), a process in which cells insert in between cells previously in contact with each other. Here, we identify the major forces that contribute to tracheal primary branch remodeling by using genetic and microsurgery experiments. As the tip cells migrate, they elongate the branches and create a tensile stress. This tensile stress triggers SCI, which, in turn, allows the branches to further elongate. The mechanism that we describe contrasts with "convergent extension by cell intercalation" acting during Drosophila germ band extension (GBE), where cell intercalation is the cause of epithelium elongation. Surprisingly, in tracheal branches, one or two leading cells produce enough mechanical power to intercalate many lagging cells. This may apply to other tubular networks.

Two different mechanisms of planar cell intercalation leading to tissue elongation

During development, certain cells intercalate with each other towards tissue-elongation, exemplified in sea-urchin gut-elongation, amphibian gastrulation, and Drosophila germ-band extension. Their mechanism is not universal among intercalation events. To clarify the minimal cellular properties required for cell-intercalation, we computer-simulated the process using three-dimensional geometrical cell-models. We identified two different mechanisms: (1) cell-junction-remodeling by cell-junction contraction along a specific direction, as observed in Drosophila germ-band extension, and (2) cell-shuffling by orientated cell-extension of bipolar cells, as observed in amphibian gastrulation. The cell-junction-remodeling was characterized by well-defined accumulation of contractile molecules along a specific direction of cell-junctions. Length contraction of approximately one cell-junction per cell is enough for the entire tissue-elongation. The cell-shuffling was characterised by rhythmic cell-extension and orientated movement of cytoskeleton within the elongated cells. Furthermore, tissue-elongation along a polarized axis was limited to a 2.5-fold increase in the cell-junction-remodeling, while no limit was defined for the cell-shuffling.

Cell movement during chick primitive streak formation

Gastrulation in amniotes begins with extensive re-arrangements of cells in the epiblast resulting in the formation of the primitive streak. We have developed a transfection method that enables us to transfect randomly distributed epiblast cells in the Stage XI–XIII chick blastoderms with GFP fusion proteins. This allows us to use time-lapse microscopy for detailed analysis of the movements and proliferation of epiblast cells during streak formation. Cells in the posterior two thirds of the embryo move in two striking counter-rotating flows that meet at the site of streak formation at the posterior end of the embryo. Cells divide during this rotational movement with a cell cycle time of 6–7 h. Daughter cells remain together, forming small clusters and as result of the flow patterns line up in the streak. Expression of the cyclin-dependent kinase inhibitor, P21/Waf inhibits cell division and severely limits embryo growth, but does not inhibit streak formation or associated flows. To investigate the role off cell–cell intercalation in streak formation we have inhibited the Wnt planar-polarity signalling pathway by expression of a dominant negative Wnt11 and a Dishevelled mutant Xdd1. Both treatments do not result in an inhibition of streak formation, but both severely affect extension of the embryo in later development. Likewise inhibition of myosin II which as been shown to drive cell–cell intercalation during Drosophila germ band extension, has no effect on streak formation, but also effectively blocks elongation after regression has started. These experiments make it unlikely that streak formation involves known cell–cell intercalation mechanisms. Expression of a dominant negative FGFR1c receptor construct as well as the soluble extracellular domain of the FGFR1c receptor both effectively block the cell movements associated with streak formation and mesoderm differentiation, showing the importance of FGF signalling in these processes.