Nicole Gorfinkiel

Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation

The dynamic reshaping of tissues during morphogenesis results from a combination of individual cell behaviors and collective cell rearrangements. However, a comprehensive framework to unambiguously measure and link cell behavior to tissue morphogenesis is lacking. Here we introduce such a kinematic framework, bridging cell and tissue behaviors at an intermediate, mesoscopic, level of cell clusters or domains. By measuring domain deformation in terms of the relative motion of cell positions and the evolution of their shapes, we characterized the basic invariant quantities that measure fundamental classes of cell behavior, namely tensorial rates of cell shape change and cell intercalation. In doing so we introduce an explicit definition of cell intercalation as a continuous process. We mapped strain rates spatiotemporally in three models of tissue morphogenesis, gaining insight into morphogenetic mechanisms. Our quantitative approach has broad relevance for the precise characterization and comparison of morphogenetic phenotypes.

Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure

Fluctuations in the shape of amnioserosa (AS) cells during Drosophila dorsal closure (DC) provide an ideal system with which to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behaviour emerges from the activity of individual cells. Using quantitative image analysis we show that apical shape fluctuations are driven by the medial cytoskeleton, with periodic foci of contractile myosin and actin travelling across cell apices. Shape changes were mostly anisotropic and neighbouring cells were often, but transiently, organised into strings with parallel deformations. During the early stages of DC, shape fluctuations with long cycle lengths produced no net tissue contraction. Cycle lengths shortened with the onset of net tissue contraction, followed by a damping of fluctuation amplitude. Eventually, fluctuations became undetectable as AS cells contracted rapidly. These transitions were accompanied by an increase in apical myosin, both at cell-cell junctions and medially, the latter ultimately forming a coherent, but still dynamic, sheet across cells. Mutants with increased myosin activity or actin polymerisation exhibited precocious cell contraction through changes in the subcellular localisation of myosin. thickveins mutant embryos, which exhibited defects in the actin cable at the leading edge, showed similar timings of fluctuation damping to the wild type, suggesting that damping is an autonomous property of the AS. Our results suggest that cell shape fluctuations are a property of cells with low and increasing levels of apical myosin, and that medial and junctional myosin populations combine to contract AS cell apices and drive DC.

Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction

The Hedgehog (Hh) morphogenetic gradient controls multiple developmental patterning events in Drosophila and vertebrates. Patched (Ptc), the Hh receptor, restrains both Hh spreading and Hh signaling. We report how endocytosis regulates the concentration and activity of Hh in the wing imaginal disc. Our studies show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner, and that both Hh and Ptc are targeted to lysosomal degradation. We also found that the ptc14 mutant does not block Hh spreading, as it has a failure in endocytosis. However, this mutant protein is able to control the expression of Hh target genes as the wild-type protein, indicating that the internalization mediated by Ptc is not required for signal transduction. In addition, we noted that both in this mutant and in those not producing Ptc protein, Hh still occurred in the endocytic vesicles of Hh-receiving cells, suggesting the existence of a second, Ptc-independent, mechanism of Hh internalization.

Mechanical control of global cell behaviour during dorsal closure in Drosophila

Halfway through embryonic development, the epidermis of Drosophila exhibits a gap at the dorsal side covered by an extraembryonic epithelium, the amnioserosa (AS). Dorsal closure (DC) is the process whereby interactions between the two epithelia establish epidermal continuity. Although genetic and biomechanical analysis have identified the AS as a force-generating tissue, we do not know how individual cell behaviours are transformed into tissue movements. To approach this question we have applied a novel image-analysis method to measure strain rates in local domains of cells and performed a kinematic analysis of DC. Our study reveals spatial and temporal differences in the rate of apical constriction of AS cells. We find a slow phase of DC, during which apical contraction of cells at the posterior end predominates, and a subsequent fast phase, during which all the cells engage in the contraction, which correlates with the zippering process. There is a radial gradient of AS apical contraction, with marginal cells contracting earlier than more centrally located cells. We have applied this analysis to the study of mutant situations and associated a particular genotype with quantitative and reproducible changes in the rate of cell contraction and hence in the overall rate of the process. Our mutant analysis reveals the contribution of mechanical elements to the rate and pattern of DC.

Quantitative approaches in developmental biology

The tissues of a developing embryo are simultaneously patterned, moved and differentiated according to an exchange of information between their constituent cells. We argue that these complex self-organizing phenomena can only be fully understood with quantitative mathematical frameworks that allow specific hypotheses to be formulated and tested. The quantitative and dynamic imaging of growing embryos at the molecular, cellular and tissue level is the key experimental advance required to achieve this interaction between theory and experiment. Here we describe how mathematical modelling has become an invaluable method to integrate quantitative biological information across temporal and spatial scales, serving to connect the activity of regulatory molecules with the morphological development of organisms.

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.

Dynamics of actomyosin contractile activity during epithelial morphogenesis

In the past few years, advances in microscopy and quantitative image analysis have lead to a completely new understanding of the processes underlying the cell shape changes and cell rearrangements that drive tissue morphogenesis. In a handful of tissues so far, though the number will surely increase rapidly, it has been shown that cell behaviour is not continuous but proceeds in pulses driven by the contractile activity of dynamic cortical actomyosin networks. The patterns and dynamics of temporary subcellular contractile foci, driven by local increases in actin and myosin, are remarkably similar in disparate tissues. Cells in all tissues display a similar range of intervals between contractions, with increasing frequencies associated with stronger tissue morphogenesis. Contractile foci appear to flow within cells with speeds that are consistent across tissues. We highlight the difference between contractile tension and stiffness, the latter being a requirement for any ratchet mechanism that stabilises contraction to produce effective tissue morphogenesis. At least two different types of ratchet mechanism are discussed, with the stiffness conferred either by a more stable actomyosin population at cell-cell junctions or through cortical actomyosin forming a quasi-stable supra-cellular network. Pulsatile contractions, polarized cell organization and various stiffening ratchet mechanisms combine to provide a rich variety of options for robust epithelial tissue remodelling.

Requirements for adherens junction components in the interaction between epithelial tissues during dorsal closure in Drosophila

Dynamic interactions between epithelial sheets are a regular feature of morphogenetic processes. Dorsal closure in Drosophila relies on the coordinated movements of two epithelia, the epidermis and the amnioserosa, and provides an excellent model system for a genetic and cell biological approach. Here, we have analyzed the contribution of junctional organization of these epithelia to dorsal closure. We observe a stringent requirement for adherens junctions at the leading edge, the interface between the amnioserosa and the epidermis, for the transmission of the forces generated during the process. We also find that interactions between Armadillo and E-cadherin play an important role in maintaining the adhesion at the leading edge, revealing the particular dynamics of this interface. Our results show that regulated cell adhesion is a crucial element of the interactions that shape epithelial sheets in morphogenetic processes.

Integrative approaches to morphogenesis: Lessons from dorsal closure

Although developmental biology has been dominated by the genetic analysis of embryonic development, in recent years genetic tools have been combined with new approaches such as imaging of live processes, automated and quantitative image analysis, mechanical perturbation and mathematical modeling, to study the principles underlying the formation of organisms. Here we focus on recent work carried out on Dorsal Closure, a morphogenetic process during Drosophila embryogenesis, to illustrate how this multidisciplinary approach is yielding new and unexpected insights into how cells organize themselves through the activity of their molecular components to give rise to the stereotyped and macroscopic movements observed during development. genesis 49:522–533, 2011.

Drosophila terminalia as an appendage-like structure.

In Drosophila, the homeotic gene Distal-less (Dll) has a fundamental role in the establishment of the identity of ventral appendages such as the leg and antenna. This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). During the development of the two components of the anal primordium - the hindgut and the analia - only the latter is dependent on Dll and hedgehog (hh) functions. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream to Dpp. These results suggest that the terminalia show similar behaviour to the leg disc or the antennal part of the eye-antennal disc consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage.