A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila.

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. Here we identify a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (dilp2) and the other extends to adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. We propose that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of dilp2 and suppressing the release of AKH when haemolymph glucose levels are high.

Optogenetic control of YAP cellular localisation and function.

YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Core outcomes for orofacial clefts: reconciling traditional and ICHOM minimum datasets.

OBJECTIVE/DESIGN/SETTING: This retrospective study sought voluntary participation from leading cleft centres from Europe and Brazil regarding core outcome measures. The results of this study would inform the debate on core outcome consensus pertaining to the European Reference Network for rare diseases (ERN CRANIO) and achieve a core outcome set for cleft care providers worldwide. INTERVENTION/METHOD: Five orofacial cleft (OFC) disciplines were identified, within which all of the International Consortium of Health Outcomes Measurement (ICHOM) outcomes fall. One questionnaire was designed for each discipline and comprised 1. the relevant ICHOM's outcomes within that discipline, and 2. a series of questions targeted to clinicians. What core outcomes are currently measured and when, did these align with the ICHOM minimum, if not how did they differ, and would they recommend modified or additional outcomes?. RESULTS: For some disciplines participants agreed with the ICHOM minimums but urged for earlier and more frequent intervention. Some clinicians felt that some of the ICHOM standards were compatible but that different ages were preferred and for others the ICHOM standards were acceptable but developmental stages should be preferred to absolute time points. CONCLUSION/IMPLICATIONS: Core outcomes for OFC were supported in principle but there are differences between the ICHOM recommendations and the 2002 WHO global consensus. The latter are established in many centres with historical archives of OFC outcome data, and it was concluded that with some modifications ICHOM could be moulded into useful core outcomes data for inter-centre comparisons worldwide.

Effects of extended pharmacological disruption of zebrafish embryonic heart biomechanical environment on cardiac function, morphology, and gene expression.

BACKGROUND: Biomechanical stimuli are known to be important to cardiac development, but the mechanisms are not fully understood. Here, we pharmacologically disrupted the biomechanical environment of wild-type zebrafish embryonic hearts for an extended duration and investigated the consequent effects on cardiac function, morphological development, and gene expression. RESULTS: Myocardial contractility was significantly diminished or abolished in zebrafish embryonic hearts treated for 72 hours from 2 dpf with 2,3-butanedione monoxime (BDM). Image-based flow simulations showed that flow wall shear stresses were abolished or significantly reduced with high oscillatory shear indices. At 5 dpf, after removal of BDM, treated embryonic hearts were maldeveloped, having disrupted cardiac looping, smaller ventricles, and poor cardiac function (lower ejected flow, bulboventricular regurgitation, lower contractility, and slower heart rate). RNA sequencing of cardiomyocytes of treated hearts revealed 922 significantly up-regulated genes and 1,698 significantly down-regulated genes. RNA analysis and subsequent qPCR and histology validation suggested that biomechanical disruption led to an up-regulation of inflammatory and apoptotic genes and down-regulation of ECM remodeling and ECM-receptor interaction genes. Biomechanics disruption also prevented the formation of ventricular trabeculation along with notch1 and erbb4a down-regulation. CONCLUSIONS: Extended disruption of biomechanical stimuli caused maldevelopment, and potential genes responsible for this are identified.

Prehospital Ultrasound Use to Guide Resuscitative Thoracotomy in Blunt Traumatic Cardiac Arrest.

Traumatic cardiac arrest is frequently encountered in the air medical transport environment, and resuscitative thoracotomy is a procedure that is sometimes performed in an attempt to salvage these critically injured patients. Focused assessment with sonography for trauma (FAST) is a point-of-care ultrasound protocol commonly used in trauma patients to detect the presence of free fluid in the intraperitoneal and pericardial spaces. The authors present a case of an adult female victim of a motor vehicle collision whose prehospital FAST scan revealed significant hemoperitoneum without hemopericardium. When she developed cardiac arrest, these ultrasound findings aided in the decision to perform resuscitative thoracotomy and helped guide the sequence of maneuvers with prioritization given to cross-clamping the aorta. This case highlights the utility of prehospital ultrasound in yielding timely, actionable diagnostic information that can inform the performance of a high-acuity low-occurrence procedure in the air medical transport environment.

Initiation of Inhaled Nitric Oxide by an Air Transport Team in Adult Coronavirus Disease 2019 Respiratory Failure.

The coronavirus disease 2019 (COVID-19) pandemic has caused a significant increase in the volume of critical care flight transports between outlying referral hospitals and tertiary care facilities. Because of the tropism of severe acute respiratory syndrome coronavirus 2, flight crews are often asked to transport mechanically ventilated patients in refractory hypoxemic respiratory failure. The authors present a case series of 5 patients with COVID-19 acute respiratory distress syndrome (ARDS) who were initiated on inhaled nitric oxide (iNO) by the transport team before rotor wing transport and survived the journey in stable or improved condition upon arrival. Previously, no case reports have described adults with COVID-19 ARDS transported after iNO initiation by the transport team. This case series shows the feasibility of iNO initiation by trained air medical transport teams and suggests a short-term stabilizing effect of iNO in patients with ARDS from COVID-19.

The Hippo pathway effector Wwtr1 regulates cardiac wall maturation in zebrafish.

Cardiac trabeculation is a highly regulated process that starts with the delamination of compact layer cardiomyocytes. The Hippo signaling pathway has been implicated in cardiac development but many questions remain. We have investigated the role of Wwtr1, a nuclear effector of the Hippo pathway, in zebrafish and find that its loss leads to reduced cardiac trabeculation. However, in mosaic animals, wwtr1-/- cardiomyocytes contribute more frequently than wwtr1+/- cardiomyocytes to the trabecular layer of wild-type hearts. To investigate this paradox, we examined the myocardial wall at early stages and found that compact layer cardiomyocytes in wwtr1-/- hearts exhibit disorganized cortical actin structure and abnormal cell-cell junctions. Accordingly, wild-type cardiomyocytes in mosaic mutant hearts contribute less frequently to the trabecular layer than when present in mosaic wild-type hearts, indicating that wwtr1-/- hearts are not able to support trabeculation. We also found that Nrg/Erbb2 signaling, which is required for trabeculation, could promote Wwtr1 nuclear export in cardiomyocytes. Altogether, these data suggest that Wwtr1 establishes the compact wall architecture necessary for trabeculation, and that Nrg/Erbb2 signaling negatively regulates its nuclear localization and therefore its activity.

An autism-linked missense mutation in SHANK3 reveals the modularity of Shank3 function.

Genome sequencing has revealed an increasing number of genetic variations that are associated with neuropsychiatric disorders. Frequently, studies limit their focus to likely gene-disrupting mutations because they are relatively easy to interpret. Missense variants, instead, have often been undervalued. However, some missense variants can be informative for developing a more profound understanding of disease pathogenesis and ultimately targeted therapies. Here we present an example of this by studying a missense variant in a well-known autism spectrum disorder (ASD) causing gene SHANK3. We analyzed Shank3's in vivo phosphorylation profile and identified S685 as one phosphorylation site where one ASD-linked variant has been reported. Detailed analysis of this variant revealed a novel function of Shank3 in recruiting Abelson interactor 1 (ABI1) and the WAVE complex to the post-synaptic density (PSD), which is critical for synapse and dendritic spine development. This function was found to be independent of Shank3's other functions such as binding to GKAP and Homer. Introduction of this human ASD mutation into mice resulted in a small subset of phenotypes seen previously in constitutive Shank3 knockout mice, including increased allogrooming, increased social dominance, and reduced pup USV. Together, these findings demonstrate the modularity of Shank3 function in vivo. This modularity further indicates that there is more than one independent pathogenic pathway downstream of Shank3 and correcting a single downstream pathway is unlikely to be sufficient for clear clinical improvement. In addition, this study illustrates the value of deep biological analysis of select missense mutations in elucidating the pathogenesis of neuropsychiatric phenotypes.

Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.

The phenotypes caused by morpholino-mediated interference of gene function in zebrafish are often not observed in the corresponding mutant(s). We took advantage of the availability of a relatively large collection of transcriptomic datasets to identify common signatures that characterize morpholino-injected animals (morphants). In addition to the previously reported activation of tp53 expression, we observed increased expression of the interferon-stimulated genes (ISGs), isg15 and isg20, the cell death pathway gene casp8, and other cellular stress response genes including phlda3, mdm2 and gadd45aa. Studies involving segmentation stage embryos were more likely to show upregulation of these genes. We also found that the expression of these genes could be upregulated by increasing doses of an egfl7 morpholino, or even high doses of the standard control morpholino. Thus, these data show that morpholinos can induce the expression of ISGs in zebrafish embryos and further our understanding of morpholino effects.

A new parameter-rich structure-aware mechanistic model for amino acid substitution during evolution.

Improvements in the description of amino acid substitution are required to develop better pseudo-energy-based protein structure-aware models for use in phylogenetic studies. These models are used to characterize the probabilities of amino acid substitution and enable better simulation of protein sequences over a phylogeny. A better characterization of amino acid substitution probabilities in turn enables numerous downstream applications, like detecting positive selection, ancestral sequence reconstruction, and evolutionarily-motivated protein engineering. Many existing Markov models for amino acid substitution in molecular evolution disregard molecular structure and describe the amino acid substitution process over longer evolutionary periods poorly. Here, we present a new model upgraded with a site-specific parameterization of pseudo-energy terms in a coarse-grained force field, which describes local heterogeneity in physical constraints on amino acid substitution better than a previous pseudo-energy-based model with minimum cost in runtime. The importance of each weight term parameterization in characterizing underlying features of the site, including contact number, solvent accessibility, and secondary structural elements was evaluated, returning both expected and biologically reasonable relationships between model parameters. This results in the acceptance of proposed amino acid substitutions that more closely resemble those observed site-specific frequencies in gene family alignments. The modular site-specific pseudo-energy function is made available for download through the following website: https://liberles.cst.temple.edu/Software/CASS/index.html.

Sequence, structure, and cooperativity in folding of elementary protein structural motifs.

Residue-level unfolding of two helix-turn-helix proteins--one naturally occurring and one de novo designed--is reconstructed from multiple sets of site-specific (13)C isotopically edited infrared (IR) and circular dichroism (CD) data using Ising-like statistical-mechanical models. Several model variants are parameterized to test the importance of sequence-specific interactions (approximated by Miyazawa-Jernigan statistical potentials), local structural flexibility (derived from the ensemble of NMR structures), interhelical hydrogen bonds, and native contacts separated by intervening disordered regions (through the Wako-Saitô-Muñoz-Eaton scheme, which disallows such configurations). The models are optimized by directly simulating experimental observables: CD ellipticity at 222 nm for model proteins and their fragments and (13)C-amide I' bands for multiple isotopologues of each protein. We find that data can be quantitatively reproduced by the model that allows two interacting segments flanking a disordered loop (double sequence approximation) and incorporates flexibility in the native contact maps, but neither sequence-specific interactions nor hydrogen bonds are required. The near-identical free energy profiles as a function of the global order parameter are consistent with expected similar folding kinetics for nearly identical structures. However, the predicted folding mechanism for the two motifs is different, reflecting the order of local stability. We introduce free energy profiles for "experimental" reaction coordinates--namely, the degree of local folding as sensed by site-specific (13)C-edited IR, which highlight folding heterogeneity and contrast its overall, average description with the detailed, local picture.

Insights into the genetic structure and diversity of 38 South Asian Indians from deep whole-genome sequencing.

South Asia possesses a significant amount of genetic diversity due to considerable intergroup differences in culture and language. There have been numerous reports on the genetic structure of Asian Indians, although these have mostly relied on genotyping microarrays or targeted sequencing of the mitochondria and Y chromosomes. Asian Indians in Singapore are primarily descendants of immigrants from Dravidian-language-speaking states in south India, and 38 individuals from the general population underwent deep whole-genome sequencing with a target coverage of 30X as part of the Singapore Sequencing Indian Project (SSIP). The genetic structure and diversity of these samples were compared against samples from the Singapore Sequencing Malay Project and populations in Phase 1 of the 1,000 Genomes Project (1 KGP). SSIP samples exhibited greater intra-population genetic diversity and possessed higher heterozygous-to-homozygous genotype ratio than other Asian populations. When compared against a panel of well-defined Asian Indians, the genetic makeup of the SSIP samples was closely related to South Indians. However, even though the SSIP samples clustered distinctly from the Europeans in the global population structure analysis with autosomal SNPs, eight samples were assigned to mitochondrial haplogroups that were predominantly present in Europeans and possessed higher European admixture than the remaining samples. An analysis of the relative relatedness between SSIP with two archaic hominins (Denisovan, Neanderthal) identified higher ancient admixture in East Asian populations than in SSIP. The data resource for these samples is publicly available and is expected to serve as a valuable complement to the South Asian samples in Phase 3 of 1 KGP.

Drosophila ORB protein in two mushroom body output neurons is necessary for long-term memory formation.

Memory is initially labile and gradually consolidated over time through new protein synthesis into a long-lasting stable form. Studies of odor-shock associative learning in Drosophila have established the mushroom body (MB) as a key brain structure involved in olfactory long-term memory (LTM) formation. Exactly how early neural activity encoded in thousands of MB neurons is consolidated into protein-synthesis-dependent LTM remains unclear. Here, several independent lines of evidence indicate that changes in two MB vertical lobe V3 (MB-V3) extrinsic neurons are required and contribute to an extended neural network involved in olfactory LTM: (i) inhibiting protein synthesis in MB-V3 neurons impairs LTM; (ii) MB-V3 neurons show enhanced neural activity after spaced but not massed training; (iii) MB-V3 dendrites, synapsing with hundreds of MB α/ß neurons, exhibit dramatic structural plasticity after removal of olfactory inputs; (iv) neurotransmission from MB-V3 neurons is necessary for LTM retrieval; and (v) RNAi-mediated down-regulation of oo18 RNA-binding protein (involved in local regulation of protein translation) in MB-V3 neurons impairs LTM. Our results suggest a model of long-term memory formation that includes a systems-level consolidation process, wherein an early, labile olfactory memory represented by neural activity in a sparse subset of MB neurons is converted into a stable LTM through protein synthesis in dendrites of MB-V3 neurons synapsed onto MB α lobes.

Auditory circuit in the Drosophila brain.

Most animals exhibit innate auditory behaviors driven by genetically hardwired neural circuits. In Drosophila, acoustic information is relayed by Johnston organ neurons from the antenna to the antennal mechanosensory and motor center (AMMC) in the brain. Here, by using structural connectivity analysis, we identified five distinct types of auditory projection neurons (PNs) interconnecting the AMMC, inferior ventrolateral protocerebrum (IVLP), and ventrolateral protocerebrum (VLP) regions of the central brain. These auditory PNs are also functionally distinct; AMMC-B1a, AMMC-B1b, and AMMC-A2 neurons differ in their responses to sound (i.e., they are narrowly tuned or broadly tuned); one type of audioresponsive IVLP commissural PN connecting the two hemispheres is GABAergic; and one type of IVLP-VLP PN acts as a generalist responding to all tested audio frequencies. Our findings delineate an auditory processing pathway involving AMMC→IVLP→VLP in the Drosophila brain.

The phylogenetic distribution and evolution of enzymes within the thymidine kinase 2-like gene family in metazoa.

Deoxyribonucleoside kinases (dNKs) carry out the rate-determining step in the nucleoside salvage pathway within all domains of life where the pathway is present, and, hence, are an indication on whether or not a species/genus retains the ability to salvage deoxyribonucleosides. Here, a phylogenetic tree is constructed for the thymidine kinase 2-like dNK gene family in metazoa. Each enzyme class (deoxycytidine, deoxyguanosine, and deoxythymidine kinases, as well as the multisubstrate dNKs) falls into a monophyletic clade. However, in vertebrates, dCK contains an apparent duplication with one paralog lost in mammals, and a number of crustacean genomes (like Caligus rogercresseyi and Lepeophtheirus salmonis) unexpectedly contain not only the multisubstrate dNKs, related to Drosophila multisubstrate dNK, but also a TK2-like kinase. Additionally, crustaceans (Daphnia, Caligus, and Lepeophtheirus) and some insects (Tribolium, Danaus, Pediculus, and Acyrthosiphon) contain several multisubstrate dNK-like enzymes which group paraphyletically within the arthropod clade. This might suggest that the multisubstrate dNKs underwent multiple rounds of duplications with differential retention of duplicate copies between insect families and more complete retention within some crustaceans and insects. Genomes of several basal animalia contain more than one dNK-like sequence, some of which group outside the remaining eukaryotes (both plants and animals) and/or with bacterial dNKs. Within the vertebrates, the mammalian genomes do not contain the second dCK, while birds, fish, and amphibians do retain it. Phasianidae (chicken and turkey) have lost dGK, while it has been retained in other bird lineages, like zebra finch. Reconstruction of the ancestral sequence between the multisubstrate arthropod dNKs and the TK2 clade of vertebrates followed by homology modeling and discrete molecular dynamics calculations on this sequence were performed to examine the evolutionary path which led to the two different enzyme classes. The structural models showed that the carboxyl terminus of the ancestral sequence is more helical than dNK, in common with TK2, although any implications of this for enzyme specificity will require biochemical validation. Finally, rate-shift and conservation-shift analysis between clades with different specificities uncovered candidate residues outside the active site pocket which may have contributed to differentiation in substrate specificity between enzyme clades.

Stochastic atomistic simulation of polycyclic aromatic hydrocarbon growth in combustion.

Nanoparticles formed in gas phase environments, such as combustion, have an important impact on society both as engineering components and hazardous pollutants. A new software package, the Stochastic Nanoparticle Simulator (SNAPS) was developed, applying a stochastic chemical kinetics methodology, to computationally investigate the growth of nanoparticle precursors through trajectories of chemical reactions. SNAPS was applied to characterize the growth of polycyclic aromatic hydrocarbons (PAHs), important precursors of carbonaceous nanoparticles and soot, in a premixed laminar benzene flame, using a concurrently developed PAH growth chemical reaction mechanism, as well as an existing benzene oxidation mechanism. Simulations of PAH ensembles successfully predicted existing experimentally measured data and provided novel insight into chemical composition and reaction pathways. The most commonly observed PAH isomers in simulations showed the importance of 5-membered rings, which contrasts with traditionally assumed compositions involving primarily pericondensed 6-membered rings. In addition, the chemical growth of PAHs involved complex sequences of highly reversible reactions, rather than relatively direct routes of additions and ring closures. Furthermore, the most common reactions involved 5-membered rings, suggesting their importance to PAH growth. The framework developed in this work will facilitate future investigation of particle inception and soot formation and will benefit engineering of novel combustion technologies to mitigate harmful emissions.

Ab initio investigation of the thermal decomposition of n-butylcyclohexane.

Environmental and energy security concerns have motivated an increased focus on developing clean, efficient combustors, which increasingly relies on insight into the combustion chemistry of fuels. In particular, naphthenes (cycloalkanes and alkylcycloalkanes) are important chemical components of distillate fuels, such as diesel and jet fuels. As such, there is a growing interest in describing napthene reactivity with kinetic mechanisms. Use of these mechanisms in predictive combustion models aids in the development of combustors. This study focuses on the pyrolysis of n-butylcyclohexane (n-BCH), an important representative of naphthenes in jet fuels. Seven different unimolecular decomposition pathways of C-C bond fission were explored utilizing ab initio/DFT methods. Accurate reaction energies were computed using the high-level quantum composite G3B3 method. Variational transition state theory, Rice-Ramsperger-Kassel-Marcus/master equation simulations provided temperature- and pressure-dependent rate constants. Implementation of these pathways into an existing chemical kinetic mechanism improved the prediction of experimental OH radical and H2O speciation in shock tube oxidation. Simulations of this combustion showed a change in the expected decomposition chemistry of n-BCH, predicting increased production of cyclic alkyl radicals instead of straight-chain alkenes. The most prominent reaction pathway for the decomposition of n-BCH is n-BCH = C3H7 + C7H13. The results of this study provide insight into the combustion of n-BCH and will aid in the future development of naphthene kinetic mechanisms.

Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC.

Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization.

Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.

DNA-damage induced cell death in yap1;wwtr1 mutant epidermal basal cells.

In a previous study, it was reported that Yap1 and Wwtr1 in zebrafish regulates the morphogenesis of the posterior body and epidermal fin fold (Kimelman et al., 2017). We report here that DNA damage induces apoptosis of epidermal basal cells (EBCs) in zebrafish yap1-/-;wwtr1-/- embryos. Specifically, these mutant EBCs exhibit active Caspase-3, Caspase-8, and γH2AX, consistent with DNA damage serving as a stimulus of the extrinsic apoptotic pathway in epidermal cells. Live imaging of zebrafish epidermal cells reveals a steady growth of basal cell size in the developing embryo, but this growth is inhibited in mutant basal cells followed by apoptosis, leading to the hypothesis that factors underscoring cell size play a role in this DNA damage-induced apoptosis phenotype. We tested two of these factors using cell stretching and substrate stiffness assays, and found that HaCaT cells cultured on stiff substrates exhibit more numerous γH2AX foci compared to ones cultured on soft substrates. Thus, our experiments suggest that substrate rigidity may modulate genomic stress in epidermal cells, and that Yap1 and Wwtr1 promotes their survival.