Pinching and pushing: fold formation in the Drosophila dorsal epidermis

Epithelial folding is a fundamental morphogenetic process that shapes planar epithelial sheets into complex three-dimensional structures. Multiple mechanisms can generate epithelial folds, including apical constriction, which acts locally at the cellular level, differential growth on the tissue scale, or buckling because of compression from neighboring tissues. Here, we investigate the formation of dorsally located epithelial folds at segment boundaries during the late stages of Drosophila embryogenesis. We found that the fold formation at the segment boundaries occurs through the juxtaposition of two key morphogenetic processes: local apical constriction and tissue-level compressive forces from posterior segments. Further, we found that epidermal spreading and fold formation are accompanied by spatiotemporal pulses of Hedgehog (Hh) signaling. A computational model that incorporates the local forces generated from the differential tensions of the apical, basal, and lateral sides of the cell and active forces generated within the whole tissue recapitulates the overall fold formation process in wild-type and Hh overexpression conditions. In sum, this work demonstrates how epithelial folding depends on multiple, separable physical mechanisms to generate the final morphology of the dorsal epidermis. This work illustrates the modularity of morphogenetic unit operations that occur during epithelial morphogenesis.     

Identification of emergent motion compartments in the amniote embryo

Abstract

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ～400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen's node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen's node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

A Force Balance Can Explain Local and Global Cell Movements during Early Zebrafish Development

Embryonic morphogenesis takes place via a series of dramatic collective cell movements. The mechanisms that coordinate these intricate structural transformations across an entire organism are not well understood. In this study, we used gentle mechanical deformation of developing zebrafish embryos to probe the role of physical forces in generating long-range intercellular coordination during epiboly, the process in which the blastoderm spreads over the yolk cell. Geometric distortion of the embryo resulted in nonuniform blastoderm migration and realignment of the anterior-posterior (AP) axis, as defined by the locations at which the head and tail form, toward the new long axis of the embryo and away from the initial animal-vegetal axis defined by the starting location of the blastoderm. We found that local alterations in the rate of blastoderm migration correlated with the local geometry of the embryo. Chemical disruption of the contractile ring of actin and myosin immediately vegetal to the blastoderm margin via Ca²⁺ reduction or treatment with blebbistatin restored uniform migration and eliminated AP axis reorientation in mechanically deformed embryos; it also resulted in cellular disorganization at the blastoderm margin. Our results support a model in which tension generated by the contractile actomyosin ring coordinates epiboly on both the organismal and cellular scales. Our observations likewise suggest that the AP axis is distinct from the initial animal-vegetal axis in zebrafish.     

Identification of emergent motion compartments in the amniote embryo

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ∼400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen''s node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen''s node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

cis-acting control elements for Krüppel expression in the Drosophila embryo.

Krüppel (Kr), a gap gene of Drosophila, shows complex spatial patterns of expression during the different stages of embryogenesis. In order to identify cis-acting sequences required for normal Kr gene expression, we analysed the expression patterns of fusion gene constructs in transgenic embryos. In these constructs, bacterial lacZ expression was placed under the control of Kr sequences in front of a basal promoter. We identified cis-acting Kr control units which drive beta-galactosidase expression in 10 known locations of Kr expression in early and late embryos. More than one cis-regulatory element drives the expression in the anterior domain at the blastoderm stage, in the nervous system, the midline precursor cells and in the amino-serosa. In addition, two cis-acting elements direct the first zygotic expression of Kr in a striped subpattern within the central region of the blastoderm embryo. Both elements respond to alterations in the activities of maternal organizer genes known to be required for Kr expression in establishing the thoracic and anterior abdominal segments in the wild-type embryo.     

Contractility,differential tension and membrane removal lead zebrafish epiboly biomechanics

Precise tissue remodeling during development is essential for shaping embryos and optimal organ function. Epiboly is an early gastrulation event by which the blastoderm expands around the yolk to engulf it. Three different layers are involved in this process, an epithelial layer (the enveloping layer, EVL), the embryo proper, constituted by the deep cells (DCs), and the yolk cell. Although teleost epiboly has been studied for many years, a clear understanding of its mechanics was still missing. Here we present new information on the cellular, molecular and mechanical elements involved in epiboly that, together with some other recent data and upon comparison with previous biomechanical models, lets conclude that the expansion of the epithelia is passive and driven by active cortical contraction and membrane removal in the adjacent layer, the External Yolk Syncytial Layer (E-YSL). The isotropic actomyosin contraction of the E-YSL cortex generates an anisotropic stress pattern and a directional net movement consequence of the differences in the deformation response of the 2 opposites adjacent domains (EVL and the Yolk Cytoplasmic Layer - YCL). Contractility is accompanied by the local formation of membrane folds and its removal by Rab5ab dependent macropinocytosis. The increase in area of the epithelia during the expansion is achieved by cell-shape changes (flattening) responding to spherical geometrical cues. The counterbalance between the geometry of the embryo and forces dissipation among different elements is therefore essential for epiboly global coordination.     

Dynamic topology of the cephalochordate to amniote morphological transition: a self-organized system of Russian dolls

This note presents a mechanistic explanation of the transition between the morphology of cephalochordates to that of amniotes. By a careful study of the morphogenetic movements which occur during the early stages of development of a typical amniote (a chicken embryo), we are able to show that the formation of a vertebrate body follows a sequence: first, formation of dorsal folds, then head and heart as dorsal and ventral folds, and finally another dorsal fold, which eventually builds up the chorion. This order has a physical origin linked to the velocity field of the tissue flow. These folds form at right angles to the flow direction, and the topology of the chordates flow is hyperbolic. This mechanism explains the differences between the successive bauplans, by the cumulate forward and backward movement of the flow. Eventually, the entire phenomenon can be described as a self-organized system of Russian dolls, by which the heart finds itself inside the embryo, and the embryo itself inside the chorion. In addition, the phenomenon has a mirror symmetry in the anterior and in the posterior part, thereby explaining naturally the existence of animals having a caudal heart.     

Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus

The segmental plan of the Drosophila embryo is already established at the blastoderm stage through the action of maternal effect genes which determine the polarity of the embryo and zygotically active genes involved in segmentation. We have analyzed the first example of a group of maternally acting genes which are necessary for establishing the developmental potential of the posterior 25% of the blastoderm. Females, homozygous for the X-linked maternal-effect mutation female sterile(1)Nasrat211 [fs(1)N211], produce embryos, characterized as torso-like, which lack all posterior endodermal derivatives as well as structures characteristic of abdominal segments 8 to 10. In addition, anterior endodermal derivatives are deficient and the absence of pharyngeal musculature causes a collapse of the cephalopharyngeal apparatus. The columnar blastoderm cell layer is defective at the posterior tip below the pole cells in these embryos. This defect, however, is presumably secondary to some abnormal feature of pole cell formation since in double mutants of fs(1)Nasrat211; tudor3 the blastoderm is normal but the embryos still show the torso-like phenotype. In situ hybridization with RNA probes derived from the fushi tarazu gene establishes that the cellular determination of the posterior blastoderm of embryos produced by fs(1)N211 is changed. This represents the first direct demonstration that a maternal-effect mutation alters the spatial distribution of a zygotic gene product involved in the segmental patterning of the embryo.     

Tracking morphogenetic tissue deformations in the early chick embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to study the physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.     

Do lamellipodia have the mechanical capacity to drive convergent extension?

Convergent extension (CE), a kinematic motif associated with several important morphogenetic movements in embryos, entails narrowing of a tissue in one in-plane direction and elongation in the other. Although the cell elongation and intercalation which accompany this process have been investigated and relevant genes and biochemical pathways have been studied in multiple organisms, a fundamental question that has not yet been answered is "Do the lamellipodia thought to drive these motions actually have the mechanical capacity to do so?" Here, we address this and a number of related issues using a state-of-the-art computational model which can replicate cell motions, changes in cell shape and tissue deformations. The model is based on the cell-level finite element approach of Chen and Brodland, but has additional features which allow it to model lamellipodium formation and contraction. In studying CE, computational models provide an important complement to molecular approaches because they reveal the "mechanical pathways" through which gene products must ultimately act in order to produce physical movements. The model shows that lamellipodia can drive CE, that they do so through cell intercalations and that the elongated cells characteristic of CE arise only when adjacent tissues resist convergence, a result which we confirm experimentally.     

short gastrulation, a mutation causing delays in stage-specific cell shape changes during gastrulation in Drosophila melanogaster

Mutations at the short gastrulation locus affect the timing of certain early morphogenetic events occurring during gastrulation in Drosophila melanogaster. Specifically, the invagination and subsequent closing of the posterior midgut and the anterior midgut appear to be delayed in these embryos. In addition, their germbands do not extent the full distance anteriorly on the dorsal side of the embryo. The dorsal cells are abnormally thick and fall into extremely deep dorsal folds as the germband extends. sog embryos continue development, but form disorganized first instar larvae. Normal sog expression is required in the zygote, but not in the mother for normal embryonic development and viability. Analysis of adult and larval gynandromorphs indicates that sog expression is required only in the ventral and/or anterior and posterior ends of the embryo, arguing that the dorsal abnormalities caused by the mutation are secondary consequences of defects elsewhere in mutant embryos.     

Shaping and bending of the avian neural plate as analysed with a fluorescent-histochemical marker

Shaping and bending of the neural plate are cardinal events of neurulation. These processes are initiated in avian embryos shortly after the onset of gastrulation and concluded concomitantly with the completion of gastrulation. The epiblast undergoes extensive morphogenetic movements during gastrulation and neurulation, but the directions, distances, rates, mechanisms and roles of such rearrangements are largely unknown. To begin to understand these morphogenetic movements, we have mapped regional displacements of the epiblast by injecting a fluorescent-histochemical marker into selected prenodal, nodal and postnodal levels of the blastoderm. Lateral epiblast regions (600 microns lateral to the midline and consisting primarily of surface epithelium) are displaced craniomedially, medial regions (300 microns lateral to the midline and consisting of neural plate and preingressed mesoderm) predominantly medially, and midline regions (consisting of neural plate and primitive streak) predominantly caudally. Displacements within the avian neural plate parallel those previously described for the amphibian neural plate. Furthermore, similar tissue displacements occur within the prenodal and postnodal levels of the avian epiblast despite the fact that neurulation is occurring in the former and gastrulation in the latter. Finally, our results show that ectodermal rudiments contained within a single cross-sectional level of the embryo are a composite of cells derived from multiple craniocaudal and mediolateral levels. Thus, regional tissue displacements are important events to consider in the analysis of the early morphogenesis of axial and paraxial organ rudiments derived from the epiblast.     

Tuning cell shape change with contractile ratchets

Throughout the lifespan of an organism, shape changes are necessary for cells to carry out their essential functions. Nowhere is this more dramatic than embryonic development and gastrulation, when cell shape changes drive large-scale rearrangements in tissue architecture to establish the body plan of the organism. A longstanding question for both cell and developmental biologists has been how are forces generated to change cell shape? Recent studies in both cell culture and developing embryos have combined live imaging, computational analysis, genetics, and biophysics to identify ratchet-like behaviors in actomyosin networks that operate to incrementally change cell shape, drive cell movement, and deform tissues. Our analysis of several cell shape changes leads us to propose four regulatory modules associated with ratchet-like deformations that are tuned to generate diverse cell behaviors, coordinating cell shape change across a tissue.     

A Preferred Curvature-Based Continuum Mechanics Framework for Modeling Embryogenesis

Mechanics plays a key role in the development of higher organisms. However, understanding this relationship is complicated by the difficulty of modeling the link between local forces generated at the subcellular level and deformations observed at the tissue and whole-embryo levels. Here we propose an approach first developed for lipid bilayers and cell membranes, in which force-generation by cytoskeletal elements enters a continuum mechanics formulation for the full system in the form of local changes in preferred curvature. This allows us to express and solve the system using only tissue strains. Locations of preferred curvature are simply related to products of gene expression. A solution, in that context, means relaxing the system’s mechanical energy to yield global morphogenetic predictions that accommodate a tendency toward the local preferred curvature, without a need to explicitly model force-generation mechanisms at the molecular level. Our computational framework, which we call SPHARM-MECH, extends a 3D spherical harmonics parameterization known as SPHARM to combine this level of abstraction with a sparse shape representation. The integration of these two principles allows computer simulations to be performed in three dimensions on highly complex shapes, gene expression patterns, and mechanical constraints. We demonstrate our approach by modeling mesoderm invagination in the fruit-fly embryo, where local forces generated by the acto-myosin meshwork in the region of the future mesoderm lead to formation of a ventral tissue fold. The process is accompanied by substantial changes in cell shape and long-range cell movements. Applying SPHARM-MECH to whole-embryo live imaging data acquired with light-sheet microscopy reveals significant correlation between calculated and observed tissue movements. Our analysis predicts the observed cell shape anisotropy on the ventral side of the embryo and suggests an active mechanical role of mesoderm invagination in supporting the onset of germ-band extension.     

Regional requirements for Dishevelled signaling during Xenopus gastrulation: separable effects on blastopore closure, mesendoderm internalization and archenteron formation

During amphibian gastrulation, the embryo is transformed by the combined actions of several different tissues. Paradoxically, many of these morphogenetic processes can occur autonomously in tissue explants, yet the tissues in intact embryos must interact and be coordinated with one another in order to accomplish the major goals of gastrulation: closure of the blastopore to bring the endoderm and mesoderm fully inside the ectoderm, and generation of the archenteron. Here, we present high-resolution 3D digital datasets of frog gastrulae, and morphometrics that allow simultaneous assessment of the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To examine how the diverse morphogenetic engines work together to accomplish gastrulation, we combined these tools with time-lapse analysis of gastrulation, and examined both wild-type embryos and embryos in which gastrulation was disrupted by the manipulation of Dishevelled (Xdsh) signaling. Remarkably, although inhibition of Xdsh signaling disrupted both convergent extension and blastopore closure, mesendoderm internalization proceeded very effectively in these embryos. In addition, much of archenteron elongation was found to be independent of Xdsh signaling, especially during the second half of gastrulation. Finally, even in normal embryos, we found a surprising degree of dissociability between the various morphogenetic processes that occur during gastrulation. Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, and suggest that the loose relationship between morphogenetic processes may have facilitated the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species.     

Determination in Drosophila Embryos

SYNOPSIS. In this review we describe data of experiments whichinterfere with the formation of the metameric pattern duringembryogenesis. Ligating embryos before blastoderm stage leadsto a gap in the segmentation pattern of the differentiated embryo.The gap can extend up to 6 segments but terminal segments arealways recognizable. In posterior but not in anterior fragmentswe find abnormally large but fewer segments. This increase insegment size results from a different determination of blastodermcells after ligation. During nuclear multiplication stages whena gap can be produced, the zygotic genome is not yet active.Information to develop the metameric pattern in ligated embryosmust therefore have been made during oogenesis. Recently Nüisslein-Volhard and Wieschaus (1980) have describedthree zygotic mutations which form embryos with a gap of segmentssimilar to our ligated embryos. We have discussed these mutantphenotypes in connection with our experimental data. Segmentation is controlled at several levels. During oogenesisthe anterior-posterior and dorsal-ventral axes become established(Nüsslein-Volhard, 1979). Also during oogenesis, but extendinginto early embryonic life, information is generated to subdividethe embryo into blocks of cells forming the metameric pattern.At blastoderm the identity of segments becomes established.     

Mechanics of blastocyst morphogenesis

During pre‐implantation development, the mammalian zygote transforms into the blastocyst, the structure that will implant the embryo in the maternal uterus. Consisting of a squamous epithelium enveloping a fluid‐filled cavity and the inner cell mass, the blastocyst is sculpted by a succession of morphogenetic events. These deformations result from the changes in the forces and mechanical properties of the tissue composing the embryo. Here, I review the recent studies, which, for the first time, informed us on the mechanics of blastocyst morphogenesis.