After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin ∼10 min after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Wounding increases nuclear ploidy in wound-proximal epidermal cells of the Drosophila pupal notum.

After injury, tissues must replace cell mass and genome copy number. The mitotic cycle is one mechanism for replacement, but non-mitotic strategies have been observed in quiescent tissues to restore tissue ploidy after wounding. Here we report that nuclei of the mitotically capable Drosophila pupal notum enlarged following nearby laser ablation. Measuring DNA content, we determined that nuclei within 100 µm of a laser-wound increased their ploidy to ~8C, consistent with one extra S-phase. These data indicate non-mitotic repair strategies are not exclusively utilized by quiescent tissues and may be an underexplored wound repair strategy in mitotic tissues.

Live imaging basement membrane assembly under the pupal notum epithelium.

Basement membranes are sheet-like extracellular matrices containing Collagen IV, and they are conserved across the animal kingdom. Basement membranes usually line the basal surfaces of epithelia, where they contribute to structure, maintenance, and signaling. Although adult epithelia contact basement membranes, in early embryos the epithelia contact basement membranes only after basement membranes are assembled in embryogenesis. In Drosophila , the pupal notum epithelium is a useful model for live imaging epithelial cell behaviors, yet it is unclear when the basement membrane assembles in the pupa, as pupae are undergoing metamorphosis, similar to embryogenesis. To characterize the basement membrane in the pupal notum, we used spinning disk fluorescent microscopy to visualize Collagen IV subunit Vkg-GFP and adherens junction protein p120ctnRFP. Bright punctae of Vkg-GFP were observed in the X-Y plane, possibly representing Vkg-containing cells. We found that a thin continuous Vkg-containing basement membrane was evident at 14 h APF, which became more enriched with Vkg-GFP over the next 6 h, indicating the basement membrane is still assembling during that time. Live imaging of the pupal notum during this time could provide insight into formation, assembly, and repair of the basement membranes.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension, we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin about 10 minutes after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Wound-Induced Syncytia Outpace Mononucleate Neighbors during Drosophila Wound Repair.

All organisms have evolved to respond to injury. Cell behaviors like proliferation, migration, and invasion replace missing cells and close wounds. However, the role of other wound-induced cell behaviors is not understood, including the formation of syncytia (multinucleated cells). Wound-induced epithelial syncytia were first reported around puncture wounds in post-mitotic Drosophila epidermal tissues, but have more recently been reported in mitotically competent tissues such as the Drosophila pupal epidermis and zebrafish epicardium. The presence of wound-induced syncytia in mitotically active tissues suggests that syncytia offer adaptive benefits, but it is unknown what those benefits are. Here, we use in vivo live imaging to analyze wound-induced syncytia in mitotically competent Drosophila pupae. We find that almost half the epithelial cells near a wound fuse to form large syncytia. These syncytia use several routes to speed wound repair: they outpace diploid cells to complete wound closure; they reduce cell intercalation during wound closure; and they pool the resources of their component cells to concentrate them toward the wound. In addition to wound healing, these properties of syncytia are likely to contribute to their roles in development and pathology.

A mathematical model of calcium signals around laser-induced epithelial wounds.

Cells around epithelial wounds must first become aware of the wound's presence in order to initiate the wound-healing process. An initial response to an epithelial wound is an increase in cytosolic calcium followed by complex calcium-signaling events. While these calcium signals are driven by both physical and chemical wound responses, cells around the wound will all be equipped with the same cellular components to produce and interact with the calcium signals. Here we have developed a mathematical model in the context of laser ablation of the Drosophila pupal notum that integrates tissue-level damage models with a cellular calcium-signaling toolkit. The model replicates experiments in the contexts of control wounds as well as knockdowns of specific cellular components, but it also provides new insights that are not easily accessible experimentally. The model suggests that cell-cell variability is necessary to produce calcium-signaling events observed in experiments; it quantifies calcium concentrations during wound-induced signaling events, and it shows that intercellular transfer of the molecule IP3 is required to coordinate calcium signals across distal cells around the wound. The mathematical model developed here serves as a framework for quantitative studies in both wound signaling and calcium signaling in the Drosophila system.

Piezo initiates transient production of collagen IV to repair damaged basement membranes.

Basement membranes are sheets of extracellular matrix separating tissue layers and providing mechanical support. Their mechanical properties are determined largely by their most abundant protein, Collagen IV (Col4). Although basement membranes are repaired after damage, little is known about how. To wit, since basement membrane is extracellular it is unknown how damage is detected, and since Col4 is long-lived it is unknown how it is regulated to avoid fibrosis. Using the basement membrane of the adult Drosophila midgut as a model, we show that repair is distinct from maintenance. In healthy conditions, midgut Col4 originates from the fat body, but after damage, a subpopulation of enteroblasts we term "matrix menders" transiently express Col4, and Col4 from these cells is required for repair. Activation of the mechanosensitive channel Piezo is required for matrix menders to upregulate Col4, and the signal to initiate repair is a reduction in basement membrane stiffness. Our data suggests that mechanical sensitivity may be a general property of Col4-producing cells.

Mounting Drosophila pupae for laser ablation and live imaging of the dorsal thorax.

This protocol describes the preparation of Drosophilamelanogaster pupae for laser ablation and live imaging of the notum (dorsal thorax). Because the pupa is stationary, it can be continuously live imaged for multiple days if desired, making it ideal for studying wound signaling and repair, from before laser ablation through wound closure. In this protocol, we demonstrate the processes of staging, partially dissecting, mounting, wounding, and live imaging the pupal notum, with the wounding occurring during the live imaging process. For complete details on the use and execution of this protocol, please refer to O'Connor et al. (2021b).

Zones of cellular damage around pulsed-laser wounds.

After a tissue is wounded, cells surrounding the wound adopt distinct wound-healing behaviors to repair the tissue. Considerable effort has been spent on understanding the signaling pathways that regulate immune and tissue-resident cells as they respond to wounds, but these signals must ultimately originate from the physical damage inflicted by the wound. Tissue wounds comprise several types of cellular damage, and recent work indicates that different types of cellular damage initiate different types of signaling. Hence to understand wound signaling, it is important to identify and localize the types of wound-induced cellular damage. Laser ablation is widely used by researchers to create reproducible, aseptic wounds in a tissue that can be live-imaged. Because laser wounding involves a combination of photochemical, photothermal and photomechanical mechanisms, each with distinct spatial dependencies, cells around a pulsed-laser wound will experience a gradient of damage. Here we exploit this gradient to create a map of wound-induced cellular damage. Using genetically-encoded fluorescent proteins, we monitor damaged cellular and sub-cellular components of epithelial cells in living Drosophila pupae in the seconds to minutes following wounding. We hypothesized that the regions of damage would be predictably arrayed around wounds of varying sizes, and subsequent analysis found that all damage radii are linearly related over a 3-fold range of wound size. Thus, around laser wounds, the distinct regions of damage can be estimated after measuring any one. This report identifies several different types of cellular damage within a wounded epithelial tissue in a living animal. By quantitatively mapping the size and placement of these different types of damage, we set the foundation for tracing wound-induced signaling back to the damage that initiates it.

Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection.

The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. Here, we show that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, we show that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. We present experimental and computational evidence that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium.

Elongated Cells Drive Morphogenesis in a Surface-Wrapped Finite-Element Model of Germband Retraction.

During Drosophila embryogenesis, the germband first extends to curl around the posterior end of the embryo and then retracts back; however, retraction is not simply the reversal of extension. At a tissue level, extension is coincident with ventral furrow formation, and at a cellular level, extension occurs via convergent cell neighbor exchanges in the germband, whereas retraction involves only changes in cell shape. To understand how cell shapes, tissue organization, and cellular forces drive germband retraction, we investigate this process using a whole-embryo, surface-wrapped cellular finite-element model. This model represents two key epithelial tissues-amnioserosa and germband-as adjacent sheets of two-dimensional cellular finite elements that are wrapped around an ellipsoidal three-dimensional approximation of an embryo. The model reproduces the detailed kinematics of in vivo retraction by fitting just one free model parameter, the tension along germband cell interfaces; all other cellular forces are constrained to follow ratios inferred from experimental observations. With no additional parameter adjustments, the model also reproduces quantitative assessments of mechanical stress using laser dissection and failures of retraction when amnioserosa cells are removed via mutations or microsurgery. Surprisingly, retraction in the model is robust to changes in cellular force values but is critically dependent on starting from a configuration with highly elongated amnioserosa cells. Their extreme cellular elongation is established during the prior process of germband extension and is then used to drive retraction. The amnioserosa is the one tissue whose cellular morphogenesis is reversed from germband extension to retraction, and this reversal coordinates the forces needed to retract the germband back to its pre-extension position and shape. In this case, cellular force strengths are less important than the carefully established cell shapes that direct them. VIDEO ABSTRACT.

Chemical-PDMS binding kinetics and implications for bioavailability in microfluidic devices.

Microfluidic organ-on-chip devices constructed from polydimethylsiloxane (PDMS) have proven useful in studying both beneficial and adverse effects of drugs, supplements, and potential toxicants. Despite multiple advantages, one clear drawback of PDMS-based devices is binding of hydrophobic chemicals to their exposed surfaces. Chemical binding to PDMS can change the timing and extent of chemical delivery to cells in such devices, potentially altering dose-response curves. Recent efforts have quantified PDMS binding for selected chemicals. Here, we test a wider set of nineteen chemicals using UV-vis or infrared spectroscopy to characterize loss of chemical from solution in two setups with different PDMS-surface-to-solution-volume ratios. We find discernible PDMS binding for eight chemicals and show that PDMS binding is strongest for chemicals with a high octanol-water partition coefficient (log P > 1.85) and low H-bond donor number. Further, by measuring depletion and return of chemical from solution over tens to hundreds of hours and fitting these results to a first order model of binding kinetics, we characterize partitioning into PDMS in terms of binding capacities per unit surface area and both forward and reverse rate constants. These fitted parameters were used to model the impact of PDMS binding on chemical transport and bioavailability under realistic flow conditions and device geometry. The models predict that PDMS binding could alter in-device cellular exposures for both continuous and bolus dosing schemes by up to an order of magnitude compared to nominal input doses.

The Attenuation Distribution Across the Long Axis of Breast Cancer Liver Metastases at CT: A Quantitative Biomarker for Predicting Overall Survival.

OBJECTIVE: The objective of our study was to compare attenuation distribution across the long-axis (ADLA) measurements, Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and Choi criteria for predicting overall survival (OS) in patients with metastatic breast cancer treated with bevacizumab. MATERIALS AND METHODS: We obtained HIPAA-compliant data from a prospective, multisite, phase 3 trial of bevacizumab for the treatment of metastatic breast cancer. For patients with one or more liver metastases measuring 15 mm or larger at baseline, we evaluated up to two target liver lesions using RECIST, Choi criteria, and ADLA measurements, with the latter defined as the SD of the CT attenuation values of each pixel along the tumor long-axis diameter. The optimal percentage change threshold for defining an ADLA response was computed by cross-validation analysis in a Cox model. The log-rank test was applied to evaluate RECIST, Choi criteria, and ADLA for discriminating patients with superior OS. The predictive accuracies of all three techniques were compared using Brier scores and areas under the ROC curve (AUC). All analyses were performed separately using best overall response (BOR) and response at the first follow-up time point (FU1). RESULTS: One hundred sixty-four patients met the inclusion criteria. A 25% decrease in the ADLA measurement from baseline was the optimal ADLA response threshold for BOR and FU1. RECIST, Choi criteria, and ADLA successfully identified patients with superior OS when using BOR (RECIST, p = 0.02; Choi and ADLA, p < 0.001), but only Choi criteria and ADLA measurements were successful when using FU1 (RECIST, p = 0.43; Choi and ADLA, p < 0.001). In a direct comparison, ADLA measurements outperformed both RECIST and Choi criteria using BOR (95% CI for Brier score differences, ADLA-RECIST [-0.58 to -0.08] and ADLA-Choi [-0.55 to -0.06]; 95% CI for AUC differences, ADLA-RECIST [0.16-0.33] and ADLA-Choi [0.17-0.36]) as well as using FU1 (95% CI for Brier score differences, ADLA-RECIST [-0.77 to -0.08] and ADLA-Choi [-0.58 to -0.03]; 95% CI for AUC differences, ADLA-RECIST [0.22-0.39] and ADLA-Choi [0.01-0.22]). CONCLUSION: ADLA measurements may be a useful noninvasive indicator of cancer treatment response. Because ADLA measurements may be extracted relatively easily using existing radiologist workflows, further investigation of the ADLA technique is warranted.

Multiple Mechanisms Drive Calcium Signal Dynamics around Laser-Induced Epithelial Wounds.

Epithelial wound healing is an evolutionarily conserved process that requires coordination across a field of cells. Studies in many organisms have shown that cytosolic calcium levels rise within a field of cells around the wound and spread to neighboring cells, within seconds of wounding. Although calcium is a known potent second messenger and master regulator of wound-healing programs, it is unknown what initiates the rise of cytosolic calcium across the wound field. Here we use laser ablation, a commonly used technique for the precision removal of cells or subcellular components, as a tool to investigate mechanisms of calcium entry upon wounding. Despite its precise ablation capabilities, we find that this technique damages cells outside the primary wound via a laser-induced cavitation bubble, which forms and collapses within microseconds of ablation. This cavitation bubble damages the plasma membranes of cells it contacts, tens of microns away from the wound, allowing direct calcium entry from extracellular fluid into damaged cells. Approximately 45 s after this rapid influx of calcium, we observe a second influx of calcium that spreads to neighboring cells beyond the footprint of cavitation. The occurrence of this second, delayed calcium expansion event is predicted by wound size, indicating that a separate mechanism of calcium entry exists, corresponding to cell loss at the primary wound. Our research demonstrates that the damage profile of laser ablation is more similar to a crush injury than the precision removal of individual cells. The generation of membrane microtears upon ablation is consistent with studies in the field of optoporation, which investigate ablation-induced cellular permeability. We conclude that multiple types of damage, including microtears and cell loss, result in multiple mechanisms of calcium influx around epithelial wounds.

Computational Model of Secondary Palate Fusion and Disruption.

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from chemical-induced cellular alterations, we built a multicellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGFß, BMP, FGF, EGF, and SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, and fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown-a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid) chemical exposures. This computer model adds to similar systems models toward an integrative "virtual embryo" for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental disruption.

Computational modeling and simulation of genital tubercle development.

Hypospadias is a developmental defect of urethral tube closure that has a complex etiology involving genetic and environmental factors, including anti-androgenic and estrogenic disrupting chemicals; however, little is known about the morphoregulatory consequences of androgen/estrogen balance during genital tubercle (GT) development. Computer models that predictively model sexual dimorphism of the GT may provide a useful resource to translate chemical-target bipartite networks and their developmental consequences across the human-relevant chemical universe. Here, we describe a multicellular agent-based model of genital tubercle (GT) development that simulates urethrogenesis from the sexually-indifferent urethral plate stage to urethral tube closure. The prototype model, constructed in CompuCell3D, recapitulates key aspects of GT morphogenesis controlled by SHH, FGF10, and androgen pathways through modulation of stochastic cell behaviors, including differential adhesion, motility, proliferation, and apoptosis. Proper urethral tube closure in the model was shown to depend quantitatively on SHH- and FGF10-induced effects on mesenchymal proliferation and epithelial apoptosis-both ultimately linked to androgen signaling. In the absence of androgen, GT development was feminized and with partial androgen deficiency, the model resolved with incomplete urethral tube closure, thereby providing an in silico platform for probabilistic prediction of hypospadias risk across combinations of minor perturbations to the GT system at various stages of embryonic development.

The Attenuation Distribution Across the Long Axis (ADLA): Preliminary Findings for Assessing Response to Cancer Treatment.

RATIONALE AND OBJECTIVES: Novel image analysis methods may be useful adjuncts to standard cancer treatment response assessment techniques. The attenuation distribution across the long axis (ADLA) is a simple measure of lesion heterogeneity that can be obtained while measuring the long axis diameter of a target lesion. The purpose of this study was to obtain preliminary validation of the ADLA method for predicting treatment response in a small clinical trial. MATERIALS AND METHODS: Under an Institutional Review Board waiver, we obtained de-identified imaging and clinical data from a phase 2 trial of an investigational anticancer therapy at our institution. We retrospectively analyzed all patients with at least one liver metastasis measuring ≥15 mm on baseline contrast-enhanced computed tomography. For each patient at every imaging time point, up to two target liver lesions were evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 and ADLA measurements. The ADLA was obtained as the standard deviation of the post-contrast computed tomography attenuation values in the portal venous phase across a linear function spanning the long-axis diameter. Using Kaplan-Meier survival analysis, the log-rank test was used to evaluate the ability of RECIST 1.1 and ADLA measurements to discriminate patients with longer overall survival (OS). RESULTS: Fifteen patients met inclusion criteria. Median survival was 149 days (range 57-487). Best overall response by the ADLA method successfully separated patients with longer OS (p = .04). Best overall response by RECIST 1.1 did not discriminate patients with longer survival (P > .05). CONCLUSION: In retrospective data analysis from a phase 2 clinical trial, the ADLA method was more predictive of OS than RECIST 1.1. Further studies are needed to explore the utility of this measurement in predicting response to cancer treatment.

Optic nerve sheath fenestration using a Raman-shifted alexandrite laser.

BACKGROUND AND OBJECTIVE: Optic nerve sheath fenestration is an established procedure for relief of potentially damaging overpressure on the optic nerve resulting from idiopathic intracranial hypertension. Prior work showed that a mid-IR free-electron laser could be delivered endoscopically and used to produce an effective fenestration. This study evaluates the efficacy of fenestration using a table-top mid-IR source based on a Raman-shifted alexandrite (RSA) laser. STUDY DESIGN/MATERIALS AND METHODS: Porcine optic nerves were ablated using light from an RSA laser at wavelengths of 6.09, 6.27, and 6.43 µm and pulse energies up to 3 mJ using both free-space and endoscopic beam delivery through 250-µm I.D. hollow-glass waveguides. Waveguide transmission was characterized, ablation thresholds and etch rates were measured, and the efficacy of endoscopic fenestration was evaluated for ex vivo exposures using both optical coherence tomography and histological analysis. RESULTS: Using endoscopic delivery, the RSA laser can effectively fenestrate porcine optic nerves. Performance was optimized at a wavelength of 6.09 µm and delivered pulse energies of 0.5-0.8 mJ (requiring 1.5-2.5 mJ to be incident on the waveguide). Under these conditions, the ablation threshold fluence was 0.8 ± 0.2 J/cm(2) , the ablation rate was 1-4 µm/pulse, and the margins of ablation craters showed little evidence of thermal or mechanical damage. Nonetheless, nominally identical exposures yielded highly variable ablation rates. This led to fenestrations that ranged from too deep to too shallow-either damaging the underlying optic nerve or requiring additional exposure to cut fully through the sheath. Of 48 excised nerves subjected to fenestration at 6.09 µm, 16 ex vivo fenestrations were judged as good, 23 as too deep, and 9 as too shallow. CONCLUSIONS: Mid-IR pulses from the RSA laser, propagated through a flexible hollow waveguide, are capable of cutting through porcine optic nerve sheaths in surgically relevant times with reasonable accuracy and low collateral damage. This can be accomplished at wavelengths of 6.09 or 6.27 µm, with 6.09 µm slightly preferred. The depth of ex vivo fenestrations was difficult to control, but excised nerves lack a sufficient layer of cerebrospinal fluid that would provide an additional margin of safety in actual patients.

Amnioserosa development and function in Drosophila embryogenesis: Critical mechanical roles for an extraembryonic tissue.

Despite being a short-lived, extraembryonic tissue, the amnioserosa plays critical roles in the major morphogenetic events of Drosophila embryogenesis. These roles involve both cellular mechanics and biochemical signaling. Its best-known role is in dorsal closure-well studied by both developmental biologists and biophysicists-but the amnioserosa is also important during earlier developmental stages. Here, we provide an overview of amnioserosa specification and its role in several key developmental stages: germ band extension, germ band retraction, and dorsal closure. We also compare embryonic development in Drosophila and its relative Megaselia to highlight how the amnioserosa and its roles have evolved. Placed in context, the amnioserosa provides a fascinating example of how signaling, mechanics, and morphogen patterns govern cell-type specification and subsequent morphogenetic changes in cell shape, orientation, and movement. Developmental Dynamics 245:558-568, 2016. © 2016 Wiley Periodicals, Inc.

Pathway to a phenocopy: Heat stress effects in early embryogenesis.

BACKGROUND: Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants-having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from nonspecific heat stress to phenocopied abnormalities is unknown. RESULTS: Drosophila embryos subjected to 30-min, 38 °C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 µm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. CONCLUSIONS: The pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity, i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types.