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The fusion of epithelial sheets is an essential and conserved morphogenetic event that requires the maintenance of tissue continuity. This is secured by membranebound or diffusible signals that instruct the epithelial cells, in a coordinated fashion, to change shapes and adhesive properties and when, how and where to move. Here we show that during Dorsal Closure (DC) in Drosophila, the Jun kinase (JNK) signaling pathway modulates integrins expression and ensures tissue endurance. An excess of JNK activity, as an outcome of a failure in the negative feedback implemented by the dual-specificity phosphatase Puckered (Puc), promotes the loss of integrins [the ß-subunit Myospheroid (Mys)] and amnioserosa detachment. Likewise, integrins signal back to the pathway to regulate the duration and strength of JNK activity. Mys is necessary for the regulation of JNK activity levels and in its absence, puc expression is downregulated and JNK activity increases. [ABSTRACT FROM AUTHOR]

The fusion of epithelial sheets is an essential and conserved morphogenetic event that requires the maintenance of tissue continuity. This is secured by membrane-bound or diffusible signals that instruct the epithelial cells, in a coordinated fashion, to change shapes and adhesive properties and when, how and where to move. Here we show that during Dorsal Closure (DC) in Drosophila, the Jun kinase (JNK) signaling pathway modulates integrins expression and ensures tissue endurance. An excess of JNK activity, as an outcome of a failure in the negative feedback implemented by the dual-specificity phosphatase Puckered (Puc), promotes the loss of integrins [the ß-subunit Myospheroid (Mys)] and amnioserosa detachment. Likewise, integrins signal back to the pathway to regulate the duration and strength of JNK activity. Mys is necessary for the regulation of JNK activity levels and in its absence, puc expression is downregulated and JNK activity increases.

Rab11, a small Ras like GTPase marking the recycling endosomes, plays instrumental roles in Drosophila embryonic epithelial morphogenesis where an array of reports testify its importance in the maintenance of cyto-architectural as well as functional attributes of the concerned cells. Proper Rab11 functions ensure a precise regulation of developmentally active cell signaling pathways which in turn promote the expression of morphogens and other physico-chemical cues which finally forge an embryo out of a single layer of cells. Earlier reports have established that Rab11 functions are vital for fly embryonic development where amorphic mutants such as EP3017 homozygotes show a fair degree of epithelial defects along with incomplete dorsal closure. Here, we present a detailed account of the effects of Rab11 loss of function in the dorso-lateral epithelium which resulted in severe dorsal closure defects along with an elevated JNK-Dpp expression. We further observed that the dorso-lateral epithelial cells undergo epithelial to mesenchymal transition as well as apoptosis in Rab11 mutants with elevated expression levels of MMP1 and Caspase-3, where Caspase-3 contributes to the Rab11 knockout phenotype contrary to the knockdown mutants or hypomorphs. Interestingly, the elevated expressions of the core JNK-Dpp signaling could be rescued with a simultaneous knockdown of wingless in the Rab11 knockout mutants suggesting a genetic interaction of Rab11 with the Wingless pathway during dorsal closure, an ideal model of epithelial wound healing. [ABSTRACT FROM AUTHOR]

The conservation of gene networks that specify and differentiate distinct tissues has long been a subject of great interest to evolutionary developmental biologists, but the question of how pre-existing tissue-specific developmental trajectories merge is rarely asked. During the radiation of flies, two extraembryonic epithelia, known as serosa and amnion, evolved into one, called amnioserosa. This unique extraembryonic epithelium is found in fly species of the group Schizophora, including the genetic model organism Drosophila melanogaster, and has been studied in depth. Close relatives of this group develop a serosa and a rudimentary amnion. The scuttle fly Megaselia abdita has emerged as an excellent model organism to study this extraembryonic tissue organization. In this review, development and functions of the extraembryonic tissue complements of Drosophila and Megaselia are compared. It is concluded that the amnioserosa combines cells, genetic pathway components and functions that were previously associated either with serosa development or amnion development. The composite developmental trajectory of the amnioserosa raises the question of whether merging tissue-specific gene networks is a common evolutionary process. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'. [ABSTRACT FROM AUTHOR]

Septate junctions (SJs) serve as occluding barriers in invertebrate epithelia. In Drosophila, at least 30 genes are required for the formation or maintenance of SJs. Interestingly, loss-of-function mutations in core SJ components are embryonic lethal, with defects in developmental events such as head involution and dorsal closure (DC) that occur prior to the formation of a mature SJ, indicating a role for these proteins in mid-embryogenesis independent of their occluding function. To understand this novel function in development, we examined loss-of-function mutations in three core SJ proteins during the process of DC. DC occurs during mid-embryogenesis to seal a dorsal gap in the epidermis following germ band retraction. Closure is driven by contraction of the extraembryonic amnioserosa cells that temporarily cover the dorsal surface and by cell shape changes (elongation) of lateral epidermal cells that bring the contralateral sheets together at the dorsal midline. Using live imaging and examination of fixed tissues, we show that early events in DC occur normally in SJ mutant embryos, but during later closure, coracle, Macroglobulin complement-related and Neurexin-IV mutant embryos exhibit slower rates of closure and display aberrant cells shapes in the dorsolateral epidermis, including dorsoventral length and apical surface area. SJ mutant embryos also show mild defects in actomyosin structures along the leading edge, but laser cutting experiments suggest similar tension and viscoelastic properties in SJ mutant versus wild type epidermis. In a high percentage of SJ mutant embryos, the epidermis tears free from the amnioserosa near the end of DC and live imaging and immunostaining reveal reduced levels of E-cadherin, suggesting that defective adhesion may be responsible for these tears. Supporting this notion, reducing E-cadherin by half significantly enhances the penetrance of DC defects in coracle mutant embryos.

Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal side of the Drosophila embryo. This process involves the coordination of several forces generated in the epidermal cell layer and in the amnioserosa cells, covering the hole. Ultimately, these forces arise due to cytoskeletal rearrangements that induce changes in cell shape and result in tissue movement. While a number of cytoskeleton regulatory proteins have already been linked to dorsal closure, here we expand this list by demonstrating that four of the six Drosophila formin type actin assembly factors are needed to bring about the proper fusion of the epithelia. An analysis of the morphological and dynamic properties of dorsal closure in formin mutants revealed a differential contribution for each formin, although we found evidence for functional redundancies as well. Therefore, we propose that the four formins promote the formation of several, and only partly identical, actin structures each with a specific role in the mechanics of dorsal closure. [ABSTRACT FROM AUTHOR]

Dorsal closure is a prominent morphogenetic process during Drosophila embryogenesis, which involves two epithelial tissues, that is, the squamous amnioserosa and the columnar lateral epidermis. Non-muscle myosin II-driven constriction in the amnioserosa leads to a decrease in the apical surface area and pulls on the adjacent lateral epidermis, which subsequently moves dorsally. The pull by the amnioserosa becomes obvious in an elongation of the epidermal cells, especially of those in the first row. The contribution of the epidermal cell elongation has remained unclear to dorsal closure. Cell elongation may be a mere passive consequence or an active response to the pulling by the amnioserosa. Here, we found that the lateral epidermis actively responds. We analyzed tensions within tissues and cell junctions by laser ablation before and during dorsal closure, the elliptical and dorsal closure stages, respectively. Furthermore, we genetically and optochemically induced chronic and acute cell contraction, respectively. In this way, we found that tension in the epidermis increased during dorsal closure. A correspondingly increased tension was not observed at individual junctions, however. Junctional tension even decreased during dorsal closure in the epidermis. We strikingly observed a strong increase of the microtubule amount in the epidermis, while non-muscle myosin II increased in both tissues. Our data suggest that the epidermis actively antagonizes the pull from the amnioserosa during dorsal closure and the increased microtubules might help the epidermis bear part of the mechanical force.

Tissues build complex structures like lumens and microvilli to carry out their functions. Most of the mechanisms used to build these structures rely on cells remodelling their apical plasma membranes, which ultimately constitute the specialised compartments. In addition to apical remodelling, these shape changes also depend on the proper attachment of the basal plasma membrane to the extracellular matrix (ECM). The ECM provides cues to establish apicobasal polarity, and it also transduces forces that allow apical remodelling. However, physical crosstalk mechanisms between basal ECM attachment and the apical plasma membrane remain understudied, and the ones described so far are very diverse, which highlights the importance of identifying the general principles. Here, we review apicobasal crosstalk of two well-established models of membrane remodelling taking place during Drosophila melanogaster embryogenesis: amnioserosa cell shape oscillations during dorsal closure and subcellular tube formation in tracheal cells. We discuss how anchoring to the basal ECM affects apical architecture and the mechanisms that mediate these interactions. We analyse this knowledge under the scope of other morphogenetic processes and discuss what aspects of apicobasal crosstalk may represent widespread phenomena and which ones are used to build subsets of specialised compartments.

Steroid hormones influence diverse biological processes throughout the animal life cycle, including metabolism, stress resistance, reproduction, and lifespan. In insects, the steroid hormone, 20-hydroxyecdysone (20E), is the central hormone regulator of molting and metamorphosis, and plays roles in tissue morphogenesis. For example, amnioserosa contraction, which is a major driving force in Drosophila dorsal closure (DC), is defective in embryos mutant for 20E biosynthesis. Here, we show that 20E signaling modulates the transcription of several DC participants in the amnioserosa and other dorsal tissues during late embryonic development, including zipper, which encodes for non-muscle myosin. Canonical ecdysone signaling typically involves the binding of Ecdysone receptor (EcR) and Ultraspiracle heterodimers to ecdysone-response elements (EcREs) within the promoters of responsive genes to drive expression. During DC, however, we provide evidence that 20E signaling instead acts in parallel to the JNK cascade via a direct interaction between EcR and the AP-1 transcription factor subunit, Jun, which together binds to genomic regions containing AP-1 binding sites but no EcREs to control gene expression. Our work demonstrates a novel mode of action for 20E signaling in Drosophila that likely functions beyond DC, and may provide further insights into mammalian steroid hormone receptor interactions with AP-1.

Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal side of the Drosophila embryo. This process involves the coordination of several forces generated in the epidermal cell layer and in the amnioserosa cells, covering the hole. Ultimately, these forces arise due to cytoskeletal rearrangements that induce changes in cell shape and result in tissue movement. While a number of cytoskeleton regulatory proteins have already been linked to dorsal closure, here we expand this list by demonstrating that four of the six Drosophila formin type actin assembly factors are needed to bring about the proper fusion of the epithelia. An analysis of the morphological and dynamic properties of dorsal closure in formin mutants revealed a differential contribution for each formin, although we found evidence for functional redundancies as well. Therefore, we propose that the four formins promote the formation of several, and only partly identical, actin structures each with a specific role in the mechanics of dorsal closure.

The leading edge-to-cadherin contact transitions that occur during metazoan developmental processes and disease states require fine coordination of Rac and Rho pathways. Recently the elmo-mbc complex, a Rac GEF and RhoGAP19D, a Rho GAP were identified as key, conserved regulators that link Rac and Rho during these transitions. The corresponding Rho GEF and Rac GAP remain hidden amongst the large family of GEF and GAP proteins. Identification of these regulators is essential to understand GTPase coordination during these transitions. Here we find two candidates based on the mammalian literature and use RNAi to explore the fly ortholog effects on the dorsal closure epidermis. RhoGEF64C and RhoGAP92B are strong contenders to couple Rac and Rho during mesenchymal-to-epithelial-like transitions. [ABSTRACT FROM AUTHOR]

Cell sheet morphogenesis characterizes key developmental transitions and homeostasis, in vertebrates and throughout phylogeny, including gastrulation, neural tube formation and wound healing. Dorsal closure, a process during Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. ∼140 genes are currently known to affect dorsal closure and new genes are identified each year. Many of these genes were identified in screens that resulted in arrested development. Dorsal closure is remarkably robust and many questions regarding the molecular mechanisms involved in this complex biological process remain. Thus, it is important to identify all genes that contribute to the kinematics and dynamics of closure. Here, we used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of Drosophila melanogaster’s 2nd chromosome to identify “dorsal closure deficiencies”. Through two crosses, we unambiguously identified embryos homozygous for each deficiency and time-lapse imaged them for the duration of closure. Images were analyzed for defects in cell shapes and tissue movements. Embryos homozygous for 47 deficiencies have notable, diverse defects in closure, demonstrating that a number of discrete processes comprise closure and are susceptible to mutational disruption. Further analysis of these deficiencies will lead to the identification of at least 30 novel “dorsal closure genes”. We expect that many of these novel genes will identify links to pathways and structures already known to coordinate various aspects of closure. We also expect to identify new processes and pathways that contribute to closure.

Cell sheet morphogenesis is essential for metazoan development and homeostasis of animal form -- it contributes to developmental milestones including gastrulation, neural tube closure, heart and palate formation and to tissue maintenance during wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis and a model for cell sheet morphogenesis, is a remarkably robust process during which coordination of conserved gene expression patterns and signaling cascades regulate the cellular shape changes and movements. New 'dorsal closure genes' continue to be discovered due to advances in imaging and genetics. Here, we extend our previous study of the right arm of the 2nd chromosome to the left arm of the 2nd chromosome using the Bloomington deficiency kit's set of large deletions, which collectively remove 98.9% of the genes on the left arm of chromosome two (2L) to identify 'dorsal closure deficiencies'. We successfully screened 87.2% of the genes and identified diverse dorsal closure defects in embryos homozygous for 49 deficiencies, 27 of which delete no known dorsal closure gene. These homozygous deficiencies cause defects in cell shape, canthus formation and tissue dynamics. Within these deficiencies, we have identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on 2L that affect lateral epidermal cells. We will continue to identify novel 'dorsal closure genes' with further analysis. These forward genetic screens are expected to identify new processes and pathways that contribute to closure and links between pathways and structures already known to coordinate various aspects of closure. [ABSTRACT FROM AUTHOR]

Dorsal closure during Drosophila embryogenesis provides a robust genetic platform to study the basic cellular mechanisms that govern epithelial wound healing and morphogenesis. As dorsal closure proceeds, the lateral epithelial tissue (LE) adjacent to the dorsal opening advance contra-laterally, with a simultaneous retraction of the amnioserosa. The process involves a fair degree of coordinated cell shape changes in the dorsal most epithelial (DME) cells as well as a few penultimate rows of lateral epithelial (LE) cells (collectively referred here as Dorsolateral Epithelial (DLE) cells), lining the periphery of the amnioserosa, which in due course of time extend contra-laterally and ultimately fuse over the dorsal hole, giving rise to a dorsal epithelial continuum. The JNK-Dpp signaling in the dorsolateral epidermis, plays an instrumental role in guiding their fate during this process. A large array of genes have been reported to be involved in the regulation of this core signaling pathway, yet the mechanisms by which they do so is hitherto unclear, which forms the objective of our present study. Here we show a probable mechanism via which lgl , a conserved tumour suppressor gene, regulates the JNK–Dpp pathway during dorsal closure and epithelial morphogenesis. A conditional/targeted knock-down of lgl in the dorsolateral epithelium of embryos results in failure of dorsal closure. Interestingly, we also observed a similar phenotype in a Rab11 knockdown condition. Our experiment suggests Rab11 to be interacting with lgl as they seem to synergize in order to regulate the core JNK-Dpp signaling pathway during dorsal closure and also during adult thorax closure process. • Rab11 or lgl knockdown in the dorso-lateral epidermis results in the failure of dorsal closure in Drosophila embryos. • A targeted knockdown of lgl or Rab11 in the dorsolateral epidermal cells results in an improper morphology. • Rab11 expression increases significantly in the dorso-lateral embryonic epidermis where lgl is knocked down. • A simultaneous knockdown of Rab11 in an lgl depleted background in the dorsolateral epidermis results in a rescue. • Rab11 and lgl interact to regulate the JNK-Dpp pathway by maintenance of cell junctions and cortical cytoskeleton. [ABSTRACT FROM AUTHOR]

In segmented tissues, anterior and posterior compartments represent independent morphogenetic domains, which are made of distinct lineages separated by boundaries. During dorsal closure of the Drosophila embryo, specific “mixer cells” (MCs) are reprogrammed in a JNK-dependent manner to express the posterior determinant engrailed (en) and cross the segment boundary. Here, we show that JNK signaling induces de novo expression of en in the MCs through repression of Polycomb (Pc) and release of the en locus from the silencing PcG bodies. Whereas reprogramming occurs in MCs from all thoracic and abdominal segments, cell mixing is restricted to the central abdominal region. We demonstrate that this spatial control of MC remodeling depends on the antagonist activity of the Hox genes abdominal-A and Abdominal-B. Together, these results reveal an essential JNK/en/Pc/Hox gene regulatory network important in controlling both the plasticity of segment boundaries and developmental reprogramming.

A simple machine is a basic of device that takes mechanical advantage to apply force. Animals and plants self-assemble through the operation of a wide variety of simple machines. Embryos of different species actuate these simple machines to drive the geometric transformations that convert a disordered mass of cells into organized structures with discrete identities and function. These transformations are intrinsically coupled to sequential and overlapping steps of self-organization and self-assembly. The processes of self-organization have been explored through the molecular composition of cells and tissues and their information networks. By contrast, efforts to understand the simple machines underlying self-assembly must integrate molecular composition with the physical principles of mechanics. This primer is concerned with effort to elucidate the operation of these machines, focusing on the "problem" of morphogenesis. Advances in understanding self-assembly will ultimately connect molecular-, subcellular-, cellular- and meso-scale functions of plants and animals and their ability to interact with larger ecologies and environmental influences., (Copyright © 2024. Published by Elsevier Inc.)

Summary: Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high‐pressure freezing and freeze‐substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze‐substitution with per‐mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time‐efficient FIB‐SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM‐TEM workflows to comprehensively analyse biological structures. Lay Description: Focused ion beam scanning electron microscopy is becoming a widely used technique for the three‐dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy‐based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub‐cellular and sub‐organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl‐embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures. [ABSTRACT FROM AUTHOR]

The septate junction (SJ) is the occluding junction found in the ectodermal epithelia of invertebrate organisms, and is essential to maintain chemically distinct compartments in epithelial organs, to provide the blood–brain barrier in the nervous system, and to provide an important line of defense against invading pathogens. More than 20 genes have been identified to function in the establishment or maintenance of SJs in Drosophila melanogaster. Numerous studies have demonstrated the cell biological function of these proteins in establishing the occluding junction, whereas very few studies have examined further developmental roles for them. Here we examined embryos with mutations in nine different core SJ genes and found that all nine result in defects in embryonic development as early as germ band retraction, with the most penetrant defect observed in head involution. SJ genes are also required for cell shape changes and cell rearrangements that drive the elongation of the salivary gland during midembryogenesis. Interestingly, these developmental events occur at a time prior to the formation of the occluding junction, when SJ proteins localize along the lateral membrane and have not yet coalesced into the region of the SJ. Together, these observations reveal an underappreciated role for a large group of SJ genes in essential developmental events during embryogenesis, and suggest that the function of these proteins in facilitating cell shape changes and rearrangements is independent of their role in the occluding junction.

Evolution of morphogenesis is generally associated with changes in genetic regulation. Here, we report evidence indicating that dorsal closure, a conserved morphogenetic process in dipterans, evolved as the consequence of rearrangements in epithelial organization rather than signaling regulation. In Drosophila melanogaster, dorsal closure consists of a two-tissue system where the contraction of extraembryonic amnioserosa and a JNK/Dpp-dependent epidermal actomyosin cable result in microtubule-dependent seaming of the epidermis. We find that dorsal closure in Megaselia abdita, a three-tissue system comprising serosa, amnion and epidermis, differs in morphogenetic rearrangements despite conservation of JNK/Dpp signaling. In addition to an actomyosin cable, M. abdita dorsal closure is driven by the rupture and contraction of the serosa and the consecutive microtubule-dependent seaming of amnion and epidermis. Our study indicates that the evolutionary transition to a reduced system of dorsal closure involves simplification of the seaming process without changing the signaling pathways of closure progression.

Abstract Intercellular endosomes (IEs) are endocytosed vesicles shuttled through the adherens junctions (AJs) between two neighboring epidermal cells during Drosophila dorsal closure. The cell-to-cell transport of IEs requires DE-cadherin (DE-cad), microtubules (MTs) and kinesin. However, the mechanisms by which IEs can be transported through the AJs are unknown. Here, we demonstrate the presence of AJ-associated pores with MTs traversing through the pores. Live imaging allows direct visualization of IEs being transported through the AJ-associated pores. By using an optogenetic dimerization system, we observe that the dimerized IE-kinesin complexes move across AJs into the neighboring cell. The AJ-associated pores also allow intercellular movement of soluble proteins. Importantly, most epidermal cells form dorsoventral-oriented two-cell syncytia. Together, we present a model in which an AJ-associated pore mediates the intercellular transport of IEs and proteins between two cells in direct contact. Highlights • We demonstrated the presence of AJ-associated pores with MTs traversing through the pores. • Live imaging allows direct visualization of vesicles being transported through the AJ-associated pores. • The dimerized vesicle-kinesin complexes move across AJs into the neighboring cell. • Most epidermal cells form dorsoventral-oriented two-cell syncytia. [ABSTRACT FROM AUTHOR]

Abstract In late Drosophila embryos, the epidermis exhibits a dorsal hole as a consequence of germ band retraction. It is sealed during dorsal closure (DC), a morphogenetic process in which the two lateral epidermal layers converge towards the dorsal midline and fuse. We previously demonstrated the involvement of the Cbt transcription factor in Drosophila DC. However its molecular role in the process remained obscure. In this study, we used genomic approaches to identify genes regulated by Cbt as well as its direct targets during late embryogenesis. Our results reveal a complex transcriptional circuit downstream of Cbt and evidence that it is functionally related with the Insulin/insulin-like growth factor signaling pathway. In this context, Cbt may act as a positive regulator of the pathway, leading to the repression of Foxo activity. Our results also suggest that the DC defects observed in cbt embryos could be partially due to Foxo overactivation and that a regulatory feedback loop between Foxo and Cbt may be operating in the DC context. Highlights • Cabut is a Drosophila transcription factor involved in dorsal closure. • Genomic approaches reveal a complex transcriptional circuit downstream of Cabut. • Cabut is a positive regulator of insulin pathway activity during late embryogenesis. • Foxo overactivation contributes to the dorsal closure phenotype of cabut embryos. • A regulatory feedback loop between Foxo and Cabut may occur during dorsal closure. [ABSTRACT FROM AUTHOR]

Cell sheet morphogenesis characterizes key developmental transitions and homeostasis, in vertebrates and throughout phylogeny, including gastrulation, neural tube formation and wound healing. Dorsal closure, a process during Drosophila embryogenesis, has emerged as a model for cell sheet morphogenesis. ~140 genes are currently known to affect dorsal closure and new genes are identified each year. Many of these genes were identified in screens that resulted in arrested development. Dorsal closure is remarkably robust and many questions regarding the molecular mechanisms involved in this complex biological process remain. Thus, it is important to identify all genes that contribute to the kinematics and dynamics of closure. Here, we used a set of large deletions (deficiencies), which collectively remove 98.5% of the genes on the right arm of Drosophila melanogaster's 2nd chromosome to identify "dorsal closure deficiencies". Through two crosses, we unambiguously identified embryos homozygous for each deficiency and time-lapse imaged them for the duration of closure. Images were analyzed for defects in cell shapes and tissue movements. Embryos homozygous for 47 deficiencies have notable, diverse defects in closure, demonstrating that a number of discrete processes comprise closure and are susceptible to mutational disruption. Further analysis of these deficiencies will lead to the identification of at least 30 novel "dorsal closure genes". We expect that many of these novel genes will identify links to pathways and structures already known to coordinate various aspects of closure. We also expect to identify new processes and pathways that contribute to closure. [ABSTRACT FROM AUTHOR]

Dorsal closure is a morphogenetic process similar to wound healing, whereby a gap in the epithelium is closed through the coordination of the elongation of epidermal cells and contraction of Amnioserosa cells (AS). Throughout the early stages of this process, the AS cells exhibit fluctuations of their apical area which provides an ideal system to understand contractile epithelia, both in terms of the cellular mechanisms and how tissue behavior emerges from the activity of individual cells. In this work, we present a vertex-based model to explore the dynamics of Amnioserosa cells and provide insights into the regulation of dorsal closure. Towards this end, moving horizon estimation is used to predict the mechanics of AS cell oscillations and contractions modeled as a quasi-linear parameter-varying system with bounded unknown parameters. We perform estimation through the minimization of the least-squares moving horizon cost with respect to both state variables and unknown parameters simultaneously. For simulation purposes, data from the work published in [13] are used to evaluate the performance of moving-horizon estimation as compared to the extended Kalman filter. The comparative analysis reveals that the moving-horizon approach performs better than those provided by the Kalman filtering and with an increased robustness to measurement and process noises. [ABSTRACT FROM AUTHOR]

Drosophila embryogenesis has proven to be an extremely powerful system for developmental gene discovery and characterization. We isolated five new EMS-induced alleles that do not complement the l(3R)5G83 lethal line isolated in the Nüsslein-Volhard and Wieschaus screens. We have named this locus chem. Lethality of the new alleles as homozygous zygotic mutants is not completely penetrant, and they have an extended phenocritical period. Like the original allele, a fraction of mutant embryos die with cuticular defects, notably head involution and dorsal closure defects. Embryonic defects are much more extreme in germline clones, where the majority of mutant embryos die during embryogenesis and do not form cuticle, implying a strong chem maternal contribution. chem mutations genetically interact with mutations in cytoskeletal genes (arm) and with mutations in the epithelial polarity genes coracle, crumbs, and yurt. chem mutants dorsal open defects are similar to those present in yurt mutants, and, likewise, they have epithelial polarity defects. chem1 and chem3 mutations suppress yurt3, and chem3 mutants suppress crumbs1 mutations. In contrast, chem1 and coracle2 mutations enhance each other. Compared to controls, in chem mutants in embryonic lateral epithelia Crumbs expression is mislocalized and reduced, Coracle is increased and mislocalized basally at embryonic stages 13–14, then reduced at stage 16. Arm expression has a similar pattern but levels are reduced.

Various mechanical models of live amnioserosa cells during Drosophila melanogaster’s dorsal closure are proposed. Such models account for specific biomechanical oscillating behaviors and depend on a different set of parameters. The identification of the parameters for each of the proposed models is accomplished according to a least-squares approach in such a way to best fit the cellular dynamics extracted from live images. For the purpose of comparison, the resulting models after identification are validated to allow for the selection of the most appropriate description of such a cell dynamics. The proposed methodology is general and it may be applied to other planar biological processes. [ABSTRACT FROM PUBLISHER]

Drosophila dorsal closure is a morphogenetic movement that involves flanking epidermal cells, assembling actomyosin cables, and migrating dorsally over the underlying amnioserosa to seal at the dorsal midline. Echinoid (Ed)--a cell adhesion molecule of adherens junctions (AJs)--participates in several developmental processes. The disappearance of Ed from the amnioserosa is required to define the epidermal leading edge for actomyosin cable assembly and coordinated cell migration. However, the mechanism by which Ed is cleared from amnioserosa is unknown. Here, we show that Ed is cleared in amnioserosa by both transcriptional and posttranslational mechanisms. First, Ed mRNA transcription was repressed in amnioserosa prior to the onset of dorsal closure. Second, the ubiquitin ligase Smurf downregulated pretranslated Ed by binding to the PPXY motif of Ed. During dorsal closure, Smurf colocalized with Ed at AJs, and Smurf overexpression prematurely degraded Ed in the amnioserosa. Conversely, Ed persisted in the amnioserosa of Smurf mutant embryos, which, in turn, affected actomyosin cable formation. Together, our results demonstrate that transcriptional repression of Ed followed by Smurf-mediated downregulation of pretranslated Ed in amnioserosa regulates the establishment of a taut leading edge during dorsal closure. [ABSTRACT FROM AUTHOR]

We isolated and characterized embryonic lethal mutations in piragua ( prg ). The prg locus encodes a protein with an amino terminus Zinc Finger-Associated-Domain (ZAD) and nine C 2 H 2 zinc fingers (ZF). prg mRNA and protein expression during embryogenesis is dynamic with widespread maternal contribution, and subsequent expression in epithelial precursors. About a quarter of prg mutant embryos do not develop cuticle, and from those that do a small fraction have cuticular defects. Roughly half of prg mutants die during embryogenesis. prg mutants have an extended phenocritical period encompassing embryogenesis and first instar larval stage, since the other half of prg mutants die as first or second instar larvae. During dorsal closure, time-lapse high-resolution imaging shows defects arising out of sluggishness in closure, resolving at times in failures of closure. prg is expressed in imaginal discs, and is required for imaginal development. prg was identified in imaginal tissue in a cell super competition screen, together with other genes, like flower . We find that flower mutations are also embryonic lethal with a similar phenocritical period and strong embryonic mutant phenotypes (head involution defects, primarily). The two loci interact genetically in the embryo, as they increase embryonic mortality to close to 90% with the same embryonic phenotypes (dorsal closure and head involution defects, plus lack of cuticle). Mutant prg clones generated in developing dorsal thorax and eye imaginal tissue have strong developmental defects (lack of bristles and ommatidial malformations). prg is required in several developmental morphogenetic processes. [ABSTRACT FROM AUTHOR]

Summary In segmented tissues, anterior and posterior compartments represent independent morphogenetic domains, which are made of distinct lineages separated by boundaries. During dorsal closure of the Drosophila embryo, specific “mixer cells” (MCs) are reprogrammed in a JNK-dependent manner to express the posterior determinant engrailed ( en ) and cross the segment boundary. Here, we show that JNK signaling induces de novo expression of en in the MCs through repression of Polycomb ( Pc ) and release of the en locus from the silencing PcG bodies. Whereas reprogramming occurs in MCs from all thoracic and abdominal segments, cell mixing is restricted to the central abdominal region. We demonstrate that this spatial control of MC remodeling depends on the antagonist activity of the Hox genes abdominal-A and Abdominal-B . Together, these results reveal an essential JNK/ en / Pc /Hox gene regulatory network important in controlling both the plasticity of segment boundaries and developmental reprogramming. [ABSTRACT FROM AUTHOR]

Dorsal closure of the Drosophila embryonic epithelium provides an excellent model system for the in vivo analysis of molecularmechanisms regulating cytoskeletal rearrangements. In this study, we investigated the function of the Drosophila spectraplakin Short stop (Shot), a conserved cytoskeletal structural protein, during closure of the dorsal embryonic epithelium. We show that Shot is essential for the efficient final zippering of the opposing epithelial margins. By using isoform-specific mutant alleles and genetic rescue experiments with truncated Shot variants, we demonstrate that Shot functions as an actin-microtubule cross-linker in mediating zippering. At the leading edge of epithelial cells, Shot regulates protrusion dynamics by promoting filopodia formation. Fluorescence recovery after photobleaching (FRAP) analysis and in vivo imaging of microtubule growth revealed that Shot stabilizes dynamic microtubules. The actin- and microtubule-binding activities of Shot are simultaneously required in the same molecule, indicating that Shot is engaged as a physical crosslinker in this process. We propose that Shot-mediated interactions between microtubules and actin filaments facilitate filopodia formation, which promotes zippering by initiating contact between opposing epithelial cells. [ABSTRACT FROM AUTHOR]

Summary Tissue morphogenesis requires force-generating mechanisms to organize cells into complex structures. Although many such mechanisms have been characterized, we know little about how forces are integrated across developing tissues. We provide evidence that integrin-mediated cell-extracellular matrix (ECM) adhesion modulates the transmission of apically generated tension during dorsal closure (DC) in Drosophila . Integrin-containing adhesive structures resembling focal adhesions were identified on the basal surface of the amnioserosa (AS), an extraembryonic epithelium essential for DC. Genetic modulation of integrin-mediated adhesion results in defective DC. Quantitative image analysis and laser ablation experiments reveal that basal cell-ECM adhesions provide resistance to apical cell displacements and force transmission between neighboring cells in the AS. Finally, we provide evidence for integrin-dependent force transmission to the AS substrate. Overall, we find that integrins regulate force transmission within and between cells, thereby playing an essential role in transmitting tension in developing tissues. [ABSTRACT FROM AUTHOR]

Summary Insect embryos complete the outer form of the body via dorsal closure (DC) of the epidermal flanks, replacing the transient extraembryonic (EE) tissue. Cell shape changes and morphogenetic behavior are well characterized for DC in Drosophila, but these data represent a single species with a secondarily reduced EE component (the amnioserosa) that is not representative across the insects. Here, we examine DC in the red flour beetle, Tribolium castaneum, providing the first detailed, functional analysis of DC in an insect with complete EE tissues (distinct amnion and serosa). Surprisingly, we find that differences between Drosophila and Tribolium DC are not restricted to the EE tissue, but also encompass the dorsal epidermis, which differs in cellular architecture and method of final closure (zippering). We then experimentally manipulated EE tissue complement via RNAi for Tc-zen1, allowing us to eliminate the serosa and still examine viable DC in a system with a single EE tissue (the amnion). We find that the EE domain is particularly plastic in morphogenetic behavior and tissue structure. In contrast, embryonic features and overall kinetics are robust to Tc-zen1RNAi manipulation in Tribolium and conserved with a more distantly related insect, but remain substantially different from Drosophila. Although correct DC is essential, plasticity and regulative, compensatory capacity have permitted DC to evolve within the insects. Thus, DC does not represent a strong developmental constraint on the nature of EE development, a property that may have contributed to the reduction of the EE component in the fly lineage.

Summary Dorsal closure (DC) is an essential step during Drosophila development whereby a hole is sealed in the dorsal epidermis and serves as a model for cell sheet morphogenesis and wound healing. It involves the orchestrated interplay of transcriptional networks and dynamic regulation of cell machinery to bring about shape changes, mechanical forces, and emergent properties. Here we provide insight into the regulation of dorsal closure by describing novel autonomous and non-autonomous roles for U-shaped (Ush) in the amnioserosa, the epidermis, and in mediation of communication between the tissues. We identified Ush by gene expression microarray analysis of Dpp signaling targets and show that Ush mediates some DC functions of Dpp. By selectively restoring Ush function in either the AS or the epidermis in ush mutants, we show that the AS makes a greater (Ush-dependent) contribution to closure than the epidermis. A signal from the AS induces epidermal cell elongation and JNK activation in the DME, while cable formation requires Ush on both sides of the leading edge, i.e. in both the AS and epidermis. Our study demonstrates that the amnioserosa and epidermis communicate at several steps during the process: sometimes the epidermis instructs the amnioserosa, other times the AS instructs the epidermis, and still other times they appear to collaborate.