The role of TGF-β1 was studied in vitro. COPD epithelium in large airways, when compared to controls, showed decreased β-tubulin IV + ciliated cells  and increased MUC5AC + goblet cells . Both features were recapitulated in the ALI-cultured epithelium from COPD patients. Exogenous TGF-β1 reduced mucociliary differentiation while neutralizing TGF-β1 during ALI increased both specialized cell types. The COPD airway epithelium displays altered differentiation for ciliated cells, which recapitulates in vitro, at least in part through TGF-β1.In COPD, epithelial changes are prominent features in the airways, such as goblet cell hyperplasia and squamous metaplasia. In contrast, it remains unclear whether ciliated cells are reduced and which pathways dysregulate epithelial differentiation. We hypothesized that bronchial epithelial cell lineage specification is dysregulated in COPD because of an aberrant reprogramming through transforming growth factor (TGF)-β1. Surgical lung tissue from 81 COPD and 61 control (smokers and non-smokers) patients was assessed for bronchial epithelial cell phenotyping by immunohistochemistry, both  In addition, structural changes in COPD airways occur and involve dysregulation of the epithelial-mesenchymal unit, with epithelial modifications and subepithelial fibrosis3.Chronic obstructive pulmonary disease (COPD) is a frequent and major respiratory disease, characterized by a progressive and poorly reversible airflow limitation in subjects exposed to noxious particles and gases, most frequently from cigarette smoke. The disease is associated with an abnormal chronic inflammatory response of the airways4, namely ciliated cells , mucus-producing goblet cells 5, basal cells (acting as progenitor cells), and CC16-producing club (ex Clara) cells6. Mucociliary clearance represents a fundamental defence mechanism that requires the cooperation between goblet and ciliated cells. Ciliated cell commitment is initially driven through activation of Notch signaling, followed by the upregulation of hundreds of structural and regulatory ciliary genes of the multiciliated cell program. Among the transcriptional network involved in ciliogenesis, Forkhead/winged helix proteins (notably FOXJ1) have a central role by regulating more than 500 genes7. Motile cilium biogenesis involves the replication, trafficking and apical docking of basal bodies in the plasma membrane followed by the elongation of axonemes8. The axoneme of motile cilia, the cylindrical scaffold, contains hundreds of proteins organized in nine peripheral microtubules doublets (consisting of α- and β-tubules), one central pair and dynein arms. Recently, Perotin et al. showed that primary cilia (non-motile) are dysregulated in COPD patients both in remodelled and non-remodelled epithelium9. More broadly, ciliopathies are particularly prone to induce cystic diseases at various sites, notably the lung in primary ciliary dyskinesia (bronchiectasis) or the kidneys (in polycystic kidney disease)11.The airways are covered by a muco-ciliated, pseudostratified epithelium, consisting of four main cell types12. An important hallmark of the disease is the excess of mucus, due to increases in goblet cells and in mucus secretion14 which have been directly related to cigarette smoke exposure15 and to the activation by inflammatory mediators such as interleukin (IL)-4, IL-1316, IL-1717, tumour necrosis factor, neutrophil elastase18 and S100 proteins19. In contrast, a concomitant loss in ciliated cells is less well documented in COPD3, while reduced beat frequency20, changes in the nasal mucosa21 as well as smoke-induced loss22 or shortening24 of the cilia have been reported. Besides IL-13, which may promote goblet cell hyperplasia and loss of cilia in vitro25 with relevance to asthma, transforming growth factor (TGF)-β is upregulated in the bronchial epithelium and in air-liquid interface (ALI)-human bronchial epithelial cells (HEBC) from COPD patients26 and has a central role in the remodeling of the epithelium in this disease27.Several changes of the bronchial epithelium have been reported in COPD. Basal cell hyperplasia is an early abnormality described in smokers and COPD that leads to squamous metaplasia or goblet cell hyperplasiaex vivo as a result of an aberrant reprogramming through TGF-β1.The present study was therefore designed to directly assess bronchial epithelial cell differentiation both in lung tissue and bronchial epithelial cultures differentiated upon ALI, to address the hypothesis that ciliated cell numbers are reduced in conducting airways from COPD and that this feature reflects an epithelial dysregulation that could persist CO)  Fig. ,D. In coin vitro model to study the differentiation process. We found that the bronchial epithelium reconstituted from large airway tissue of COPD patients cultured upon ALI for 2 weeks, recapitulated the epithelial features observed in situ, namely increased MUC5AC and decreased β-tubulin IV expression with no change in p63 expression  and western blot (WB), transcription factors involved in differentiation of goblet cells (SPDEF) and ciliated cells (FOXJ1) were assessed by real time quantitative polymerase chain reaction (RT-qPCR). Axonemal dynein intermediate chain 2  messenger ribonucleic acid (mRNA), was measured to support the decrease in structural component of the cilia (suggested by the β-tubulin IV decrease in IHC and WB). No difference was found in SPDEF mRNA expression between COPD and control-derived HBEC either suggesting post-transcriptional regulation or the implication of other pathways controlling goblet cell differentiation, survival and/or proliferation in COPD. In contrast, DNAI2 and FOXJ1 mRNA were both decreased in HBEC cultures from COPD patients as compared to those from controls Fig. . DNAI2 m26. First, in kinetic experiments on controls HBEC, exogenous TGF-β1 started to decrease MUC5AC+ cells from 24 h and reached significance at 72 hours of treatment  and in vitro data could also be observed for goblet cells (but not for ciliated cells). As this model implies the differentiation of progenitor/stem basal cells, these data could infer that the differentiation programming is intrinsically imprinted in these cells. Besides the possibility that basal cells could display defects in pathways driving selective differentiation towards goblet or ciliated cells, a more global alteration in progenitor competency has been recently showed. Basal cells from smokers and COPD exhibit impaired self-renewal and pluri-potentiality in vitro31. Other in vitro studies showed that basal cells from COPD patients have limited ability to regenerate a fully differentiated epithelium and that DNA methylation alteration could be involved32 whereas the success of performing ALI culture up to 28 days could not be related to the underlying respiratory disease but only to the presence of beating cilia in the initial biopsy33. An abnormal programming of bronchial epithelial cells has also been shown in COPD for epithelial dedifferentiation through mesenchymal transition27, as well as in asthma for goblet cell hyperplasia34.This study shows that the defect in ciliated cells in COPD is recapitulated 31, Lam et al. implied autophagy pathway through an epigenetic histone deacetylase-6 regulation35. The shortening of the cilia and remodeling of the epithelium in smokers and COPD  were linked to EGFR activation through EGF and amphiregulin, further highlighting the complexity of the regulation of epithelial differentiation36. Previous work also showed that exposure of ALI-HBEC to cigarette smoke extract decreased the number of ciliated cells and increased the number of MUC5AC positive cells after 28 days of exposure37. Whereas EGFR activation by EGF mediates squamous metaplasia and EMT38, EGFR ligation by amphiregulin induces goblet cell proliferation and to a lesser extent reduced ciliated cell differentiation36. Goblet cell hyperplasia may also originate from a transdifferentiation of ciliated cells triggered by a dual signaling through EGFR and IL-13/IL-4Rα39. The mechanisms of altered bronchial epithelial programming are thus multifactorial as evidenced in murine and cell culture experimental models. Finally, we did not find the defect in ciliated cells in small airways while goblet cells were increased. This result could be due to technical bias, as the epithelium is thinner, notably in COPD patients, this quantification by IHC could have overestimated the percentage of positive area in COPD patients . This should be ideally confirmed by a study focusing on small airway tissue and ALI-HBEC originating from the same patients. Alternatively, goblet cell metaplasia in small airways could represent a dominant feature in COPD. TGF-β1 is a central cytokine in chronic airways diseases40, notably in COPD through several lines of evidence including a genetic polymorphism42, increased expression in the COPD airway epithelium43, and upregulated production in COPD-derived HBEC cultures. Whereas the role of TGF-β1 for tissue fibrosis through fibroblast activation is well known44, its role in controlling bronchial epithelial differentiation remains controversial. It has been reported that TGF-β1 may induce, in vitro, aberrant differentiation of airway epithelial cells through EMT as well as squamous metaplasia, as evidenced by expression of involucrin or transglutaminase46. Decreased or increased MUC5AC expression through TGF-β2 activation has been described48, while TGF-β1 had no effect in both studies. In contrast, Lazard et al. reported an increase in basal cells and decreases in ciliated and goblet cells upon treatment with TGF-β1 of human nasal epithelial cells in ALI49. Conflicting results regarding TGF-β1 could relate to differences in experimental conditions , as well as in the status of the studied epithelium. In murine experimental asthma, intra-peritoneal treatment with anti-TGF-β1 decreased goblet cell numbers and remodeling50. In our study, exogenous TGF-β1 affected global mucociliary differentiation and subsequently increased the proportion of uncommitted basal cells, both in control and COPD HBEC, suggesting that the defective generation of ciliated cells in the COPD epithelium could at least partly relate to TGF-β1. In contrast, goblet cell hyperplasia occurs through TGF-β-independent mechanisms, which could consist of pro-inflammatory mediators (such as cytokines or neutrophil elastase) released upon cigarette smoking.The mechanisms of cilia dysfunction remain unclear. In parallel to basal cell self-renewal dysfunction and pluri-potentiality described by Ghosh12. Although it cannot be excluded that some degree of epithelial remodeling underlies this finding, our data indicate that a defect in generating ciliated cells could occur in COPD, independently (at least in part) from obvious squamous metaplasia and that the absence of basal cell hyperplasia in our study could relate to the fact that we excluded area with obvious squamous metaplasia, which clearly contain more p63+ cells. In contrast and as previously reported13, enhanced differentiation in goblet cells also occurs in COPD, mainly in relation to smoking history per se. It is tempting to speculate that basal cell hyperplasia reflects a global specification defect of these cells to differentiate into ciliated cells, which normally represent the most abundant specialized cell type. Ultimately, it is likely that the defect in ciliated cells and concomitant goblet cell hyperplasia contribute to impaired muco-ciliary clearance in COPD, along with reduced cilia length23 and beating frequency20.Basal cell hyperplasia is the first abnormality seen at the onset of aberrant bronchial epithelial differentiation in smokers and COPD, probably reflecting impaired progenitor function of these cells that leads to squamous metaplasia or goblet cell hyperplasia33, could have partly influenced our observations. Also, a 2-week time-point was selected for analyses of ALI cultures for changes in differentiation to optimize the chance of capturing signals leading to ciliated cells; however, this may have limited the observation of features of terminal differentiation (including ciliogenesis) usually observed at 4 weeks. Finally, activation of cell death that could occur more selectively in ciliated cells following cigarette smoking52 and/or disease progression, could also contribute to our findings, and should be studied in future studies.Some limitations are part of this study. First, lung samples for controls and mild-to-moderate COPD originated from surgical tissue resected for lung cancer whereas those for very severe COPD consisted of lung explants. It remains possible that this bias affected some findings. Similarly, inhaled corticosteroids, although not influencing the success rate of ALI cultureex vivo at least in part through aberrant activation by TGF-β1 signaling.Altogether, these data show that the large airway epithelium is imprinted in COPD with a defective differentiation for ciliated cells and recapitulates All experiments were performed in accordance with relevant guidelines and regulations. Additional details are provided in the online Supplementary File.One hundred and forty-two patients were enrolled in this study, consisting of 61 controls (24 non-smokers and 37 smokers or ex-smokers) and 81 COPD patients, namely 24 mild  stage I), 23 moderate (GOLD stage II) and 7 severe COPD (GOLD stage III) undergoing lung resection surgery for a solitary tumor and recruited between 2007 and 2018  were obtained from surgical specimens and processed for IHC. One additional large airway sample was obtained for primary epithelial cell culture. Among the 142 enrolled patients  in the kinetic experiment. Finally, recombinant TGF-β1, anti-human TGF-β1 antibody or control mouse immunoglobulin (Ig)G were added (every other day with fresh medium) during the 2 weeks of ALI. No significant cytotoxicity was observed (release of lactate dehydrogenase <10%) in the presented conditions or in COPD patients  represent more than 90% of the total cells after culture in submerged conditions. Cultures were carried out in air/liquid interface (ALI) for 2 weeks, to allow re-differentiation into a pseudo-stratified, mucociliary airway epitheliumts Table .54) and as percentage of positive cells  for p63 and FOXJ1 staining. Ciliated cells (FOXJ1 and β-tubulin IV) were quantified outside of areas of obvious remodeling  were manually delineated for each patient for the quantification. Immunostainings were quantified in these areas using TissueIA software. Color deconvolution was applied to each pixel using hematoxylin and DAB matrices of the software. On the DAB matrice, a threshold was adjusted for DAB detection according to intensity  on representative stained versus not stained tissue areas. In a similar way, a threshold was also adjusted for tissue detection. These parameters were kept constant throughout the study for each immunostaining. Results were expressed as stained area (below threshold)/tissue area (below threshold) for β-tubulin IV and MUC5AC stainings  for β-tubulin IV, MUC5AC, p63 and involucrin. Whole-mount β-tubulin IV, MUC5AC, p63 immunofluorescence were quantified as percentage of positive area. Five optical sections for each stained inserts were acquired from the top to the bottom of the inserts by structured illumination using a Zeiss AxioImager equipped with an ApoTome.z1 module (20x Plan-Apochromat objective). From the acquired image stacks, maximum intensity projections were generated for analysis using the image analysis tool Author version 2017.2 (Visiopharm). p63, beta-tubulin-IV and MUC5AC stained pixels were detected using a thresholding classification method. Results were expressed as percentage of stained area.HBEC were assayed for FOXJ1, β-tubulin IV, phospho-Smad 2/3 and GAPDH  expression by western blot. ECL Prime chemiluminescent substrate was used to develop the immunochemical signal that was captured by a CCD-camera imager avoiding overexposure. Quantity One software was used for analysis. Each band individually selected was quantitated by densitometry and normalized for the corresponding GAPDH band intensity. All blot are shown in the Supplementary File.55.Total RNA was isolated from HBEC and reverse-transcribed. Expression levels of SPDEF, DNAI2, FOXJ1 mRNA were quantified by RT-qPCR and normalized to the geometric mean of three housekeeping genes p value less than 0.05 was considered as statistically significant. Statistical analyses were performed using IBM SPSS Statistics  and figures were done using GraphPad Prism .Results were shown as scatter dot plots with medians and interquartile ranges. All tests used were non-parametric, Mann-Whitney U test  and Friedman test (for paired data) followed by Dunns post hoc test (for multiple comparisons). Correlation coefficients were calculated using Spearman’s rank method. A Supplementary data


The glycocalyx is a highly hydrated, glycoprotein-rich coat shrouding many eukaryotic and prokaryotic cells. The intestinal epithelial glycocalyx, comprising glycosylated transmembrane mucins, is part of the primary host-microbe interface and is essential for nutrient absorption. Its disruption has been implicated in numerous gastrointestinal diseases. Yet, due to challenges in preserving and visualizing its native organization, glycocalyx structure-function relationships remain unclear. Here, we characterize the nanoarchitecture of the murine enteric glycocalyx using freeze-etching and electron tomography. Micrometer-long mucin filaments emerge from microvillar-tips and, through zigzagged lateral interactions form a three-dimensional columnar network with a 30 nm mesh. Filament-termini converge into globular structures ~30 nm apart that are liquid-crystalline packed within a single plane. Finally, we assess glycocalyx deformability and porosity using intravital microscopy. We argue that the columnar network architecture and the liquid-crystalline packing of the filament termini allow the glycocalyx to function as a deformable size-exclusion filter of luminal contents. Sun, Krystofiak et al. show the nanoarchitecture of the murine enteric glycocalyx, glycoprotein-rich coat covering cells and assess its porosity and deformability in mice, providing a comprehensive structural framework. This study suggests that the glycocalyx may function as a deformable size-exclusion filter of luminal contents. In the small intestine, thousands of actin-based membrane protrusions called microvilli on the apical surface of each enterocyte serve as the site of nutrient absorption. At the same time, the gut luminal environment is unique in its inhabitation by a vast and diverse microbial population, including residential symbiotic microbiota, and potentially pathogenic microorganisms that accompany food intake3. The intestinal mucosa is also exposed to mechanical stresses associated with peristalsis and luminal content propulsion onward along the intestinal tract4. To protect the intestinal mucosa from pathogens and mechanical stresses, epithelial cells generate protective layers comprised primarily of secreted and transmembrane glycoproteins that line the entirety of the intestinal tract. In the small intestine, the glycocalyx layer directly covers the entire surface of the epithelial cells7 while the overlaying lubricant mucus layer is thin and discontinuous8. The glycocalyx comprises highly diverse glycoproteins and glycolipids expressed on the epithelial cell membrane, many of which serve as receptors for bacterial adhesion11. The glycocalyx thus act as attachment sites for normal flora to limit colonization by pathogens, in addition to functioning as size-selective diffusion barrier to exclude deleterious bacteria and viruses14. In addition to these protective roles, the intestinal glycocalyx contributes to the lubrication and hydrophobicity of the mucosal surface15, prevents mucosal auto-digestion and ulceration, participates in cellular signaling, and functions as selective diffusion barrier for both endogenous and exogenous substances. Given these multitude roles in intestinal function and homeostasis, it is not surprising that glycocalyx impairment is implicated in a number of diseases of the intestinal tract, including inflammatory bowel disease and cancer16.The intestinal epithelium is the largest interface between our bodies and the external environment18. The extracellular domains of these mucins range from 500 to 5000 amino acids in length consisting largely of mucin tandem repeats19. These repeats are rich in amino acids proline, serine, and threonine with extensive O-glycosylation on the serine and threonine residues20. This heavy sugar modification introduces steric hindrance and a negative charge repulsion, causing the mucins to assume a rod-like conformation21. Strong glycosylation of mucin domains also establishes highly hydrophilic regions along the protein, which may affect structuring of local water molecules, mucin filament interactions, and the overall mechanical properties of the glycocalyx layer23. Indeed, changes in the level of mucin glycosylation have been implicated in various pathological conditions24.Transmembrane mucins MUC1, MUC3, MUC4, MUC12, MUC13, and MUC17 are expressed by the intestinal mucosal epithelial cells and are presumed to be the main components of the glycocalyx26. The glycocalyx in its native state is intrinsically difficult to study because of its highly hydrated nature. Conventional electron microscopy requires removal or replacement of water; this dehydration causes the glycocalyx structure to denature, leading to collapse of the filament network. Previous reports on the structure of the intestinal glycocalyx described its structure as made of densely entangled filaments forming a fuzzy coat covering the brush border27.Despite the wealth of biochemical data on both gel-forming and transmembrane mucins, progress in obtaining molecular-level details of mucin organization has been limited28. An early freeze-etching study of cryoprotected rat intestinal tissue with limited etching showed that the glycocalyx is made of parallel filaments extending toward the lumen29. In this study, we used deep-etching and electron tomography to reveal the detailed three-dimensional (3D) organization of the enteric glycocalyx approximating its native state. We complement this approach with intravital imaging of the glycocalyx within the intestinal lumen of live, anesthetized mice to directly explore the porosity and deformability of this multifunctional layer in vivo, thus providing the most comprehensive structural framework for the enteric glycocalyx to date.Freeze-etching is a replica-based cryogenic technique where frozen water is removed by etching or sublimation keeping the glycocalyx organization near its native state30 and are collectively known as the brush border. The glycocalyx forms a layer over the microvillar surface , which can bind sialic acid and tin Fig. . We alsoder Fig. .n = 53), which was comparable to the length of microvilli  and was composed of densely interwoven columnar filaments  when counted from freeze-etching cross-fractures that run just above the microvilli and 6.7 ± 2 (n = 22 microvilli) by counting the number of filaments that could be visualized emerging from the tips in lateral views of the microvilli. These values are likely to be an underestimate of the number of mucins due to the merging of filaments. Although the glycocalyx network appeared largely devoid of non-filamentous structures, occasionally we observed 20–50 nm granular structures . Qualitatively, it appeared that the inter-filament contacts were more frequent in the middle of the glycocalyx layer than toward the luminal or microvillar ends  in the reconstructed tomograms . We next evaluated the organization of the globular tips and found that their packing varied regionally, ranging from ordered hexagonal packing . We also evaluated the distances between termini using an autocorrelation function and radial distribution function on a two-dimensional projection image of the tomographic volume, which showed distances of 38.5 and 40.3 nm, respectively  immediately after euthanasia. These segments were then exposed to dextran solutions of different sizes, conjugated with a fluorophore. We found that a fixable 3 kDa fluorescent dextran, with Stokes radius ⪅1.2 nmtes Fig. , magentates Fig. , most linal Fig. , green. nal Fig. , green.F42 and ultimately intestinal lumen. We used transgenic mice expressing mTomato43, a fluorescently tagged plasma membrane marker, which delineated enterocyte membranes, including the brush border  or Medium 199 (ThermoFisher) and either directly frozen or fixed for downstream processing. For intravital microscopy, mice were anesthetized by an intraperitoneal (IP) injection of a mixture of ketamine (100 mg kg−1) and xylazine (20 mg kg−1). The intestine was externalized, and the body temperature of the animals was controlled and maintained at 37–38 °C. At least three animals were imaged per condition tested.All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by NIH and approved the Animal Care and use Committees for the National Institute on Deafness and Other Communication Disorders  and the National Cancer Institute . mT/mGFP mice were purchased from Jackson Laboratory. For fixed and frozen sample preparation, C57BL/6 mice and rats (Sprague Dawley) of both sexes and age between 1 month and 3 months were euthanized by CO2O. Samples were fast frozen by rapid contact with the surface of a liquid nitrogen-cooled sapphire block using a Life Cell CF-100 freezing machine. Unfixed intestinal tissues were fine dissected in Medium 199 (ThermoFisher) and were directly frozen as described in the same media or the media was replaced with ddH2O immediately before freezing to reduce the amount of salt frozen with the tissue. Frozen tissues were transferred to a Balzers freeze-fracture apparatus and underwent freeze-fracture at −110 °C followed by freeze-etch at −100 °C for 10 min. Freeze-etched samples were then rotary shadowed with platinum and stabilized with carbon using electron-beam metal-evaporation guns (Cressington Scientific) to create replicas of the exposed surfaces. Samples were subsequently cleaned with sodium hypochlorite and washed with distilled water and then collected onto 300 mesh hexagonal copper grids (Electron Microscopy Sciences). A total of 20 replicas from 6 different mice aged between 1 month and 3 months were produced and examined.Samples were fixed with 2% glutaraldehyde and then washed extensively in ddH50. Montage blending and tomogram reconstruction were done using the IMOD software suite51. Total of 13 sets of double-tilt tilt series and 19 montages were acquired, processed, and analyzed.Replicas were examined using a 200 kV JEOL 2100 electron microscope equipped with an Orius 832 CCD camera (Gatan) or a OneView CMOS camera (Gatan). Single images were captured with DigitalMicrograph (Gatan). SerialEM was used to generate montage images and acquire tilt series from −60° to +60° at 1° increments52. Spacing between glycocalyx filament termini of two-dimensional projection images was determined via autocorrelation function53 (FIJI plugin) and radial distribution function (FIJI plugin). 3D analysis of distance between filament termini was carried out using a custom MATLAB (Mathworks) script. We manually collected 3D coordinates of individual terminal from a large tomographic volume in FIJI. These coordinates were imported into MATLAB where we use the standard distance between two-point formula for Cartesian coordinate system to calculate and sort distances between two termini to obtain nearest neighbor for each terminal and the average and standard deviation. The MATLAB histfit function was used to produce the histogram that shows the distribution of nearest neighbor distances with a Gaussian fit overlay.Fourier analysis of glycocalyx packing was done in DigitalMicrograph (Gatan) or FIJIVisualization and segmentation of tomograms were performed in Amira (ThermoFisher). 3D renderings of filaments were generated by manual thresholding using the Isosurface module.54. WGA  was used to label the glycocalyx layer. Samples were counterstained with fluorophore-conjugated phalloidin.Intestinal tissues were collected from adult C57BL/6 mice and fixed with 4% paraformaldehyde. After fixation, the samples were put through a sucrose gradient (10% >20% >30%) for cryoprotection. Once the tissue sunk in the 30% sucrose solution, it was placed in a mold in Optimal Cutting Temperature compound (O.C.T) and allowed to freeze on dry ice. Cryosections (10–15-µm thick) were then cut and adhered to positively charged glass slides (Electron Microscopy Sciences). Sections were then stored at −80 °C before use. For immunolabeling, sections were first washed with 1× PBS and then permeabilized with 0.5% Triton. For mucin immunostaining, human anti-MUC17  was used to stain for its murine structural homolog MUC3 followed by Alexa-488-conjugated secondary antibody (ThermoFisher). This human anti-MUC17 antibody targets the n-terminal side of the SEA domain−1) and xylazine (20 mg kg−1). The small intestine was surgically externalized, and the epithelium was exposed via a small incision in an area devoid of intestinal content. During the procedure, the epithelial tissue was constantly moistened by applying saline. The anesthetized mouse was placed on the microscopic stage and covered with a heated pad (37 °C) to maintain body temperature. Fixable fluorescent dextran conjugates of 3 or 2000 kDa in size (ThermoFisher D-3305 or D7137) were injected directly into the intestinal lumen via the incision, and the externalized epithelium was then positioned on a coverslip mounted on the stage above the objective and immobilized using custom-made holders. The blood flow was assessed visually by using the eyepiece and only regions close to blood vessels were imaged. The microscope used was a NIKON TiE inverted fluorescence microscope equipped with a Yokogawa CSU-21 spinning disc head and an Andor DU-897 camera. NIKON Elements software was used for image analysis.Mice were anesthetized by an IP injection of a mixture of ketamine (100 mg kgFurther information on research design is available in the Supplementary InformationDescription of additional supplementary filesSupplementary Movie 1Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting SummaryPeer Review File


Hepatitis B virus (HBV) is the leading cause of liver cancer, but the mechanisms by which HBV causes liver cancer are poorly understood and chemotherapeutic strategies to cure liver cancer are not available. A better understanding of how HBV requisitions cellular components in the liver will identify novel therapeutic targets for HBV associated hepatocellular carcinoma (HCC).The development of HCC involves deregulation in several cellular signalling pathways including Wnt/FZD/β-catenin, PI3K/Akt/mTOR, IRS1/IGF, and Ras/Raf/MAPK. HBV is known to dysregulate several hepatocyte pathways and cell cycle regulation resulting in HCC development. A number of these HBV induced changes are also mediated through the Wnt/FZD/β-catenin pathway. The lack of a suitable human liver model for the study of HBV has hampered research into understanding pathogenesis of HBV. Primary human hepatocytes provide one option; however, these cells are prone to losing their hepatic functionality and their ability to support HBV replication. Another approach involves induced-pluripotent stem (iPS) cell-derived hepatocytes. However, iPS technology relies on retroviruses or lentiviruses for effective gene delivery and pose the risk of activating a range of oncogenes. Liver organoids developed from patient-derived liver tissues provide a significant advance in HCC research. Liver organoids retain the characteristics of their original tissue, undergo unlimited expansion, can be differentiated into mature hepatocytes and are susceptible to natural infection with HBV.By utilizing new ex vivo techniques like liver organoids it will become possible to develop improved and personalized therapeutic approaches that will improve HCC outcomes and potentially lead to a cure for HBV. Hepatitis B virus (HBV) is a major health concern in many regions of the world, where chronic carrier rates range from 10 to 20%. Despite the availability of a safe and effective vaccine, 5.2 million cases of acute infection were reported in the year 2000 and there are over 400 million chronic carriers globally. In Australia, 239,000 people are chronically infected, and there are an estimated 90,000 people who have not been diagnosed and are unaware of their infection. Liver cancer is the second most common cause of cancer death after lung cancer. More alarmingly, while the mortality rate for most cancers has decreased significantly over the last decade and is projected to continue this sliding trend over the next 20 years, liver cancer remains one of the common cancers with an increasing death rate. The main type of liver cancer is hepatocellular carcinoma (HCC). The mortality rate from HCC is projected to increase by ~ 40% by 2030 (Cancer UK). Over 90% of cases of liver cancer globally have a viral aetiology, and the vast majority of these is due to chronic hepatitis B infection [Although HBV is a known hepatocarcinogen, the precise mechanism by which it causes HCC is unknown and the optimal therapeutic regimens for the treatment of HBV associated HCC have not yet been established. Treatment options for HCC are limited and currently Sorafenib monotherapy is the standard of care for patients with advanced HCC. However, the overall survival of patients treated with sorafenib is disappointing with the median survival of less than 3 months . HoweverThe mechanism underlying the development of HBV associated HCC is multifactorial, linking changes within cell signalling pathways and cell cycle regulation, together with an inflammatory and cytokine reaction driven by antigen presenting cells in response to degradation products of apoptotic cells and viral antigens , 6. Seve++ pathways [The Wnt cascade has emerged as a critical regulator of stem cells and activation of Wnt signalling has been associated with numerous cancers . Wnt sigpathways , howeverIn the Wnt “off” state, β-catenin is primarily involved in cell-cell adherens junctions and free cytoplasmic β-catenin is targeted for degradation by a cytoplasmic destruction complex that contains Axin, adenomatous polyposis coli (APC), glycogen synthase kinase3-β (GSK3β) and casein kinase 1 (CK1). Sequential phosphorylation of the N-terminus of β-catenin by CK1 and GSK3 leads to recognition of β-catenin by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-trcp). Ubiquitylation of β-catenin targets it for proteasomal degradation and keeps cytoplasmic and nuclear levels of β-catenin low Fig. . The canCTNNB1) that constitutively activate Wnt signalling have been reported in up to 40% of HCC cases [Wnt signalling is involved in several physiological and pathophysiological processes during embryonic development and carcinogenesis , 24. WntCC cases . The mutCC cases .The Wnt/FZD/β-catenin signalling pathway is also linked to cell cycle regulation  and has With the important role of Wnt/FZD/β-catenin signalling in the development of HCC, and other cancers, intense research efforts have been directed to developing new compounds to inhibit this pathway . Inhibitcip1, which is increased in HBV infection of hepatocytes and contributes to G2 cell cycle arrest [Infection of hepatocytes with HBV is associated with the upregulation of PI3K/AKT and Ras/ERK1/2 –10  induced apoptosis . FasL isHuman data show that HBV infected patients, particularly those with detectable serum HBV surface antigen (HBsAg), are at increased risk of HBV reactivation when treated with TNF antagonists. Hepatocyte apoptosis mediated by TNF is an important mechanism by which HBV infected cells are eliminated from the liver. Therefore, therapeutics that augment the mechanisms through which TNF constrains HBV could be of great benefit to patients with chronic HBV infection. Cellular inhibitor of apoptosis proteins (cIAPs) regulate TNF signalling by promoting NF-κB activation downstream of TNF receptor 1 (TNFR1) ligation, and this activation, in turn, promotes cell survival by antagonizing TNF mediated cell death. By attenuating TNF signalling during hepatitis B infection, cIAPs thereby restrict hepatocyte death and allow viral persistence. However, when the function of cIAPs is antagonized, TNF-mediated ligation of TNFR1 causes cell death. Mice deficient in cIAPs in the liver have an enhanced clearance of HBV infection . This maThe cellular mechanism underlying the development of hepatocellular carcinoma in HBV infected patients is likely to be multifactorial. An initiating oncogenic stimulus, which results in a number of changes within cell signalling pathways and cell cycle regulation, is tightly linked with an inflammatory and cytokine reaction driven by Kupffer cells in response to degradation products of apoptotic cells, chemical carcinogens and viral antigens , 13–15.cip1 [cip1 in G2 which appears to serve an important role in delaying cell cycle progression to mitosis in order to protect cells against DNA damaging agents and apoptosis [cip1thereby producing G2 cell cycle arrest [cip1 and apoptotic responses in hepatocytes.Like many DNA viruses, HBV manipulates the cell cycle machinery so that it can replicate more efficiently. Two important components of cell cycle regulation that are affected by HBV include a member of the kinase inhibitor protein family, p21cip1 , 49–52, cip1  and indecip1 , 52 mechcip1 . Activatpoptosis , 52. HBVe arrest –10. Thise arrest . These sThe Wnt/β-catenin pathway has also been shown to regulate G2 cell cycle arrest . Cell cyAlthough HBV is a known hepatocarcinogen, the precise mechanism by which it causes HCC is unknown. An important obstacle to the study of molecular mechanisms underlying HBV associated HCC development has been the lack of in vitro Fig.  and in vHCC is a complex multistage and multifactorial disease. The molecular pathogenesis and host-viral interactions that drive tumourigenesis remain elusive. One of the main challenges is the lack of satisfactory model systems to elucidate the underlying mechanisms. At the same time, there are major unmet needs for tumour characterisation and personalised therapeutic strategies to target driver mutations for better treatment outcome. A wide range of infection models have been used to address these unmet needs.A lack of suitable human liver models for both HBV and HCV has hampered research in to the pathogenesis and developing a cure for HBV and vaccine development for HCV. Although the best source of primary human hepatocytes is fresh resected liver , 60 thesImmortalized continuous liver cancer derived cell lines have been the preferred model system to overcome the limitations of accessing primary human hepatocytes. These cell lines have been crucial to date for both research for pharmacological drug screening and validation Fig. . The mai40 T antigen (SV40 Tag) or human papillomavirus 16 E6/E7 genes [hTERT retrovirus or Cre-loxP with the tet-on and -off system [hTERT is only suitable for a small subset of human hepatic cells, including foetal and neonatal hepatocytes, with mixed results [Attempts have also been made to create immortal continuous human hepatocyte cell lines using viral oncogenes to bypass growth arrest of cultured adult hepatocytes, such as transduction of the simian virus E7 genes –82. HumaE7 genes , 84. Thif system , 85. How results –88.Since the discovery of “Yamanaka” factors  , there hMany alternative technologies have been developed to address these safety concerns. On one hand, mouse iPS cells were generated free from viral vectors through repeated transfection of expression plasmids containing cDNAs for the “Yamanaka” factors . On the In addition, differentiation of iPS-derived hepatocytes also depends heavily on the induction cocktail  as well Macaca fascicularis from Mauritius Island [Among animals available for HBV research, the chimpanzee is the only model that can be infected naturally by patient sera and develop chronic infection closely mimicking human conditions Fig. . Howevers Island  was founFor non-primate animals, HBV can only infect tree shrew tupaia . Many atA full-length HBV genome transgenic mouse has also been shown to produce complete infectious HBV particles . HoweverAlthough HBV transgenic mouse models are very valuable, they are not very useful to investigate host-pathogen interactions, especially with respect to the immunological response and viral clearance. That is not to mention the labour, cost, and time to generate and validate the models. In fact, the most efficient mouse model for studying the immunopathogenesis of HBV infection makes use of hydrodynamic tail-vein injection to introduce HBV into mice. This technique results in a high efficiency of transfection of hepatocytes , 129. HeRecently, it was shown that complementation of primary hepatocytes isolated from cynomolgus macaques, rhesus macaques, and pigs with human NTCP by adenoviral transduction resulted in fully susceptible HBV infection comparable to human hepatocytes . HoweverRecently, Huch and colleagues , 133 shoCompared to iPS-derived hepatocytes, the adult stem-cell-derived liver organoid model possesses many advantages. Firstly, liver organoid cells have been shown to have a stable genome and very low risk of spontaneous mutation or chromosomal alteration . SecondlWhile iPS cells and hepatoma cell lines stop short as a model system for researching HBV infection or drug assay for anti-viral capability, liver organoids offer far more scope. For example, HBV chronic-infected patients who are at high risk for developing cirrhosis or HCC, liver organoid technology can support the development of promising personalised medicine regimens including but not limited to: testing cirrhosis/tumour tissues for precise drug regimes, manufacture potential materials with high susceptibility for transplantation, or generate genome-edited ex vivo tissue free of defects or with boosted immunological response. This is especially important for HCC, where tumour tissues are extensively heterogeneous and only the liver organoid system can closely recapitulate this complexity. Thus, it is tremendously valuable for better diagnosis and treatment. In fact, many translational applications along these lines have been carried out for pancreatic  and coloThe organoid revolution led by the Clevers laboratory has made personalised medicine a tangible reality for solid tumours of the colon, breast and pancreas. Using high throughput screening, tumour cell responses can be tested to drugs in isolation and in combination. Our ability to now grow HCC tumours in tissue culture as patient-derived tumour organoids means we can do the same for liver cancer patients. Furthermore, organoid technology has led to a better understanding of the molecular mechanisms of how a normal colon epithelial cell becomes a cancer cell. We are on the cusp of extending this understanding to liver cancer to curtail the alarming trend in mortality.On the other hand, organoids established from normal human tissues are proving to be invaluable models of natural infection, particularly for pathogens that only infect human cells. Normal liver organoids were first established, again by the Clevers laboratory, in 2013 , and the


Treatments for Hepatitis C virus (HCV) infection have vastly improved over the past few decades with current regimens now offering pangenotypic activity with excellent cure rates reported in clinical trials, including in the HIV-HCV coinfected population. However, there is some concern that stringent inclusion and exclusion criteria in the trials may lead to results that are not achievable in real-world populations.Our study evaluated a real-world HIV-HCV coinfected population and compared them to the eligibility criteria for trials of two of the most recent approved HCV agents; sofosbuvir/velpatasvir and glecaprevir/pibrentasvir.Our study included 219 HIV-HCV coinfected patients and found that 89% met exclusion criteria for the sofosbuvir/velpatasvir trial and 90% met exclusion criteria for the glecaprevir/pibrentasvir trial. The majority of patients met more than one exclusion criteria with the most frequent criteria for exclusion being a non-approved ART regimen (58 and 47% respectively), having a psychiatric disorder (52%), active alcohol or injection drug use (27%), having an HIV viral load > 50 copies/ml (15%), a CrCl < 60 ml/min (13%) and a history of decompensated cirrhosis (13%).Although the newer Hepatitis C treatments are very effective, the real world HIV-HCV coinfected population often have comorbidities and other characteristics that make them ineligible for clinical trials, such that they are barriers to treatment. These barriers need to be recognized and addressed in order to optimize treatment outcomes in the HIV patient population. In the recent decade, antiviral treatments for HCV have improved dramatically with treatment outcomes that were previously thought to be unachievable. Direct acting antivirals (DAA) therapy, unlike precursor therapies, has demonstrated very high cure rates among patients with chronic HCV infection regardless of previously recognized negative predictors of positive treatment response including high viral load, HCV genotype, and HIV infection status . The nexHowever, the treatment cascade for HCV infection in the US shows that there are significant barriers to achieving treatment goals including suboptimal identification of individuals who are chronically infected, poor linkage to care where identified and low treatment and cure rates . Some ofWhile recent clinical trials have shown robust cure rates, their applicability to real world populations remains in question. The necessity of imposing rigid eligibility criteria for clinical trial may limit generalizability of the results. Historically, persons living with HIV (PLWH) were either excluded or underrepresented in earlier HCV DAA trials but more contemporary ones have had trials dedicated to that cohort. However, some studies suggest that the entry criteria for such studies for HIV-HCV infected patients are so restrictive that relatively few individuals are eligible  .This is important because, of the estimated 4 million individuals with chronic HCV infection in the US, about 400,000 are HIV co-infected . ConversThis study sought to identify real-world eligibility of HIV-HCV co-infected individuals for contemporary DAA regimens and to identify modifiable and non-modifiable characteristics that impede eligibility for DAA therapy.This was a cross sectional study of HIV–HCV co-infected patients at the Yale-New Haven Health system HIV clinic (Nathan Smith clinic). The Yale-New Haven HIV clinic is the largest HIV clinic in the state of Connecticut, with over 20 physicians and mid-level providers, who provide specialty care and services to over 1500 patients primarily residing in the Greater New Haven area majority of whom are male (60%) and racial or ethnic minorities (60%). Consultation and follow-up management for HCV therapy in the HIV clinic is typically performed by 2–3 physician providers once a week. Study data was collected between January 1, 2016 and December 31, 2016 and reflects the time point at which patient records were reviewed.HIV infection was defined as patients who had a positive Ab test, Ag/Ab test, and or positive HIV RNA assay historically (lower limit of detection [LLOD] of the lab assay was < 20 copies/ml). HCV infection was defined as a positive HCV antibody test and/or at least one detectable HCV RNA assay (HCV assay detected > 15 IU/ml). HIV and HCV viral load testing was performed using COBAS Ampliprep/COBAS Taqman, HIV and HCV version 2.0, Pleasanton California, USA respectively. HCV genotyping was performed using Versant HCV genotype, 2.0 assay (LiPA), Siemens Healthineers, Erlagen, Germany. Active substance use was defined as any drug use in the past 12 months. Cirrhosis status was defined as per liver biopsy, results of a single non-invasive test algorithm interpreted with recommended cut-offs for each test  or by ICD-9/10 codes in chart. Decompensated cirrhosis was defined as the presence of ascites, history of portosystemic encephalopathy, and/or variceal bleeding. Prior treatment for HCV was captured regardless of agent used, treatment duration or outcome. For patients on cART, the regimen captured was the regimen that the patient was on at the time of data collection. Mental health (psychiatric) disorders were captured by diagnostic codes (DSM 5) based on chart documentation by providers. Other patient characteristics, comorbidities and laboratory values were captured and reflect the most recent values at the time of chart review for all patients.Patients were included in the analysis if they were (1) adults age > 18 years, (2) HIV infected (3) had laboratory evidence of HCV infection (as described previously). Among all patients who were registered at the YNHH HIV clinic, HIV-HCV coinfected patients were identified through an electronic medical records search utilizing the YNHH joint data analytics team (J-DAT team), where cases were identified based on a series of diagnostic codes and/or available laboratory values for both HIV and HCV infection.clinicaltrials.gov website) and utilized to create a database grid to which the criteria were matched to patient characteristics and data including labs.Study protocol eligibility criteria of the following pangenotypic HCV-DAA trials which were either inclusive of or exclusively for HIV-HCV coinfected patients were matched to characteristics of identified eligible patients: sofosbuvir-velpatasvir (SOF-VEL) ASTRAL-1 , glecaprP value of 0.05.Frequency tables were created to assess the proportion of enrolled patients who met all inclusion criteria AND at least one individual exclusion criteria. Simple frequencies were reported for each exclusion criteria as well as the total number of enrolled patients who met one or more of the exclusion criteria. Subgroup analyses were performed to assess for differences in characteristics and proportion of patients meeting each eligibility criteria for patients who were black or non-black as well as male versus female  utilizing N-1 Chi square test or Fishers exact test with significance set at a A total of 219 patients with HIV/HCV co-infection were included in our study. Our study population was predominantly male (67%), with 53% being of black race, 31% white and 14% of Hispanic ethnicity. The median age was 56 years with a range of 28–74 years  based regimens) with 80% having an HIV viral load < 20 copies/ml. Specific ART medications are shown in Table An overwhelming majority (88%) of patients had a history of intravenous drug use with 15% actively injecting, 84% with a history of alcohol use. Over half (52%) of the patients also had a DSM-5 psychiatric diagnosis such as depression, bipolar disorder, anxiety or schizophrenia. About 18% had chronic kidney disease (CKD) stage 3 or greater.When eligibility criteria from the sofosbuvir/velpatasvir ASTRAL-1 trial was applied to our cohort, 89% of met at least one exclusion criteria with the most common reason for exclusion being on a non-approved antiretroviral regimen. When the antiretroviral regimen was removed as an exclusion criterion, 76% of patients remained ineligible. Other major exclusion criteria met by a significant number of our cohort included having a psychiatric disorder 52%), active alcohol or injection drug use (27%), having an HIV viral load > 50 copies/ml (15%), a CrCl < 60 ml/min (13%) and a history of decompensated cirrhosis (13%) [Table %, activeFor the glecaprevir/pibrentasvir Expedition-2 trial, 90% of patients met at least one exclusion criteria and 81% remained ineligible if the antiretroviral regimen was not included as an exclusion criterion. Top exclusion criteria met included: having a psychiatric disorder (52%), a non-approved ART regimen (47%), active alcohol or IDU (27%), having a viral load > 20 copies/ml (20%) and hemoglobin < 12 g/dl for men and < 11 g/dl for women (18%), a CD4 count < 200 cells/mm3 (13%) and a history of decompensated liver cirrhosis (13%) [Table P < 0.001) [Table There were no statistically significant differences in overall proportion of excluded patients when comparing patients by gender or ethnicity Table . However1) Table .Table 3SThe cascade of HCV care in the United States has been shown to be suboptimal with very few individuals with chronic infection who have achieved a cure . While tMultiple studies have shown that HIV infected individuals achieve similar HCV treatment results compared to their uninfected counterparts –14. HoweThe high rates of drug-drug interactions (DDIs) between current cART and DAA therapy have been shown in other studies. A Dutch study which evaluated DDI among 418 HIV/HCV coinfected individuals who were on ART, found that 47% had a category 2/3 DDI between HCV DAA and their cART prior to initiation of DAA treatment. Regimen switches were carried out in 51% of those with a category 2 DDI, while 98% of patients with a category 3 DDI had a regimen switch . AnotherAnother key eligibility criteria that was problematic for our cohort was the presence of mental health disorders. Part of the concern is that it is not clear which specific disorders and what severity preclude HCV treatment such that providers may make subjective and possibly inappropriate decisions to withhold HCV therapies in these patients. Clinical trials have shown that mental health disorders such as depression , and psyRegarding alcohol and other substance use, similar to mental health disorders, study protocols typically state that for individuals with active or recent use  they are left to the discretion of investigators to determine their ability to adhere to study protocols. This typically leads to study exclusion. However, while active injection drug is frequently an exclusionary consideration in HCV trials, studies have shown that such individuals may be successfully treated with DAA therapy , 20. ThiThe requirement for HIV disease control in patients who are candidates for HCV therapy may also be inappropriate. While the current management paradigm of HIV disease is lifelong ART with disease control, HCV can be cured with relatively short term therapy (8–12 weeks) in almost all patients including those with HIV. Therefore, while poor adherence can cut across comorbid conditions in an individual, and poor control of one disease may be a reliable surrogate for risk of poor adherence to the treatment of another comorbidity, individuals may be motivated by a finite and short duration of therapy and the prospect of a cure to preferentially adhere to HCV therapy. Similarly, having a low CD4 count as an exclusion criteria may not be clinically appropriate.Certain exclusion criteria may not be modifiable when identified such as presence of liver cirrhosis with decompensation. However, even in those individuals, referral to or consultation with an HCV expert may facilitate treatment.Our cohort which had a majority of patients of Black race could have faced unique barriers to study eligibility. In our cohort, black individuals had significantly higher rates of active alcohol or injection drug use compared to non-black individuals. Typically, black patients are underrepresented in HCV clinical trials despite being disproportionately impacted by HCV . ContribWe had a small sample size, utilized data from a single center and had a high proportion of patients who were black, male and had decompensated liver cirrhosis which may not reflect characteristics of other HCV-HIV infected cohorts such that it may limit the generalizability of our findings. We used robust definitions of psychiatric disorders, and didn’t quantify severity of alcohol and drug use, which may have led to overestimation of ineligibility rates. Patient characteristics were determined as documented by providers which can be incomplete, biased or inaccurate.While the cure rates of people with HIV-HCV coinfection are impressive, more efforts are needed to address barriers to treatment eligibility as have been identified in this and other studies. Contemporary evidence suggests that historically hard-to-treat patients such as substance users can be successfully treated for hepatitis C such that study designs should be expanded to include such populations. In addition, while not all barriers identified are modifiable, a multidisciplinary care approach may also result in more optimal management outcomes for these patients.


In pappaa mutants, hair cell death is enhanced by stimulation of mitochondrial calcium or ROS production and suppressed by a mitochondrial ROS scavenger. As a secreted metalloprotease, Pappaa stimulates extracellular insulin-like growth factor 1 (IGF1) bioavailability. We found that the pappaa mutants’ enhanced hair cell loss can be suppressed by stimulation of IGF1 availability and that Pappaa-IGF1 signaling acts post-developmentally to support hair cell survival. These results reveal Pappaa as an extracellular regulator of hair cell survival and essential mitochondrial function.To support cell survival, mitochondria must balance energy production with oxidative stress. Inner ear hair cells are particularly vulnerable to oxidative stress; thus require tight mitochondrial regulation. We identified a novel molecular regulator of the hair cells’ mitochondria and survival: Pregnancy-associated plasma protein-aa (Pappaa). Hair cells in zebrafish  Without a sufficient regenerative capacity, a nervous system’s form and function critically depends on the molecular and cellular mechanisms that support its cells’ longevity. Neural cell survival is inherently challenged by the nervous system’s high energy demand, which is required to support basic functions, including maintaining membrane potential, propagating electrical signals, and coordinating the release and uptake of neurotransmitters . MetabolHair cells of the inner ear are a population of neural cells that are particularly susceptible to oxidative stress-induced death  These sppappaa mutants, we reveal a novel role for Pappaa in regulating mitochondrial function to support hair cell survival.IGF1 is synthesized both in the liver for systemic distribution and locally in tissues, including the nervous system . IGF1’s Tg (hsp70:dnIGF1Ra-GFP)] (dnIGF1Ra-GFP expression was induced from 24 hr post fertilization (hpf) to 5 days post fertilization (dpf). At five dpf, larvae were exposed to neomycin for 1 hr and evaluated for hair cell survival 4 hr later. Larvae expressing dnIGF1Ra-GFP showed a greater reduction in hair cell survival compared to heat-shocked wild type and non-heat- shocked Tg (hsp70:dnIGF1Ra-GFP) larvae ] . dnIGF1R) larvae .Next, we evaluated mitochondrial activity in hair cells following attenuation of IGF1R signaling. A mitochondria’s transmembrane potential is closely linked to its functions  and can pappaa expression in lateral line neuromasts that co-localizes with the position of support cells, which surround the hair cell rosette   larvae   larvae (n3c:GFP) . Again,  (pappaa . Our in s reside . As a co) larvae , and con neurons .pappaa mutants (hereafter referred to as p170pappaa). p170pappaa mutants harbor nonsense mutations upstream of Pappaa’s proteolytic domain and show reduced IGF1R activation in other neural regions of pappaa expression ). We found that induced expression of Pappaa, beginning at four dpf and through neomycin treatment at five dpf, resulted in the complete rescue of p170pappaa hair cell sensitivity to neomycin and raised p170pappaa hair cell survival to wild type levels  channels found on the tips of stereocilia . MET chachannels , by hairir cells , suggest+2 transfer from the endoplasmic reticulum to the mitochondria   . pappaapneomycin . Given tneomycin , we aske mitoSOX , again wild type . This inotracker .p170pappaa hair cells predisposed them closer to a cytotoxic threshold of oxidative stress. To test this idea, we asked whether p170pappaa showed reduced hair cell survival following pharmacological stimulation of mitochondrial ROS via exposure to Antimycin A, an inhibitor of the mitochondrial electron transport chain , in which a mitochondria-targeted genetically encoded Ca+2 indicator (GCaMP3) is expressed in hair cells , an inhibitor of the mitochondrial permeability transition pore that causes buildup of mitochondrial Ca2+ and further hyperpolarizes mitochondria    (Weisigend sod2) , but notchondria . These ritoTEMPO  and obseed death . These rExtracellular factors and the regulation of mitochondrial function and oxidative stress have been demonstrated to support the survival of various cell types . For celp170pappaa hair cells showed increased mortality to neomycin , WIK background) were crossed into the following transgenic zebrafish backgrounds: s356tTg(brn3c:GFP), Tg(hsp70:dnIGF1Ra-GFP)zf243, mI5Tg(hsp70:pappaa-GFP),Tg(mnx1:GFP), w78and Tg(myo6b:mitoGCaMP3) and then incrossed. To establish the Tg(hsp70:pappaa-GFP) line, used gateway cloning vectors (Invitrogen) to generate a transgenesis construct under the control of a ubiquitous heat shock promoter (hsp70). The pappaa sequence was generated from cDNA of 120 hpf TLF embryos. Expression was assessed by fluorescence of a co-translated and post-translationally cleaved green GFP after induction by exposure in a heated water bath for 1 hr. The stable Tg(hsp70:pappaa-GFP) line was maintained on a p170/+pappaa background.. Embryonic and larval zebrafish were raised in E3 media  at 29°C on a 14 hr/10 hr light/dark cycle through 5 days post fertilization (dpf) (p170pappaa larvae was performed as previously described (To generate on (dpf) . All expescribed .Tg(brn3c:GFP) larvae through the addition of compounds to the larvae’s E3 media at five dpf unless otherwise noted. Neomycin sulfate solution (Sigma-Aldrich N1142) was added at 1–30 µM for 1 hr. Cyclosporin A  was added at 0.3–3 µM for 1 hr. Antimycin A  was added at 100–500 pM for 24 hr, beginning at four dpf. MitoTEMPO  was added at 10–100 µM 30 min prior to a 1 hr exposure to 10 µM neomycin. To stimulate IGF1 signaling: larvae were pre-treated with either NBI-31772 at 30–100 µM , or recombinant IGF1 at 10–100 ng/mL  for 24 hr prior (beginning at four dpf) and then exposed to 10 µM neomycin for 1 hr on five dpf. To inhibit IGF1 signaling, larvae were treated with NVP-AEW541 at 30 µM  for 24 hr (beginning at four dpf), before live imaging. Following each treatment period, larvae were washed 3 times with E3 and left to recover in E3 for 4 hr at 28°C before fixation with 4% paraformaldehyde . For mitoTEMPO, NBI-31772, and IGF1 treatments, the compounds were re-added to the E3 media for the 4 hr recovery period post neomycin washout. Vehicle-treated controls were exposed to either 0.9% sodium chloride in E3 (neomycin control), 0.1 mg BSA in E3 (IGF1 control) or 0.1% DMSO in E3 for the other compounds.The following treatments were performed on Tg(brn3c:GFP)), TLF, Tg(hsp70:dnIGF1R-GFP), or Tg(hsp70:pappaa-GFP) larvae where hair cells are marked by GFP (brn3c) or anti-myosin VI antibody . For each larva, hair cells were counted from the same three stereotypically positioned neuromasts    and averTg(hsp70:dnIGF1Ra-GFP) larvae, which express dnIGF1Ra-GFP under the control of zebrafish hsp70 promoter  larvae in the p170pappaa background were heat shocked once for 30 min at 37°C at four dpf. To control for possible effects of heat shocking, non-transgenic wild type and p170pappaa larvae were exposed to the same treatment.To induce expression of a dominant negative form of IGF1Ra, we used promoter . dnIGF1RTg(mnx1:GFP) and 200 five dpf Tg(brn3c:GFP) larvae were rinsed for 15 min in Ringer’s solution  (p170pappaa larvae were identified by lack of swim bladder inflation (Tg(mnx1:GFP) larvae and to collect hair cells we used tails dissected from Tg(brn3c:GFP) larvae. Samples were pooled into 1.5 mL tubes containing Ringer’s solution on ice, which was then replaced with 1.3 mL of 0.25% trypsin-EDTA (Sigma-Aldrich) for digestion. Tg(mnx1:GFP) samples were incubated for 90 min and Tg(brn3c:GFP) were incubated for 20 min. Samples were titrated gently by P1000 pipette tip every 15 min for motor neurons and every 5 min for hair cells. To stop cell digestion, 200 µL of 30% FBS and 6 mM CaCl2 in PBS solution  and kept on ice until sorting. Immediately before sorting, cells were filtered through a 40 µm cell strainer and stained with DAPI. A two-gates sorting strategy was employed. DAPI was used to isolate live cells, followed by a forward scatter (FSC) and GFP gate to isolate GFP+ cells. Sorted cells were collected into RNAse-free tubes containing 500 µL of TRIzol reagent (Invitrogen) for RNA extraction.For each genotype, 30 five dpf  pH 7.2) . pappaapnflation . To collsolution  was addesod1, forward: GTCGTCTGGCTTGTGGAGTG and reverse: TGTCAGCGGGCTAGTGCTT; for sod2, forward: CCGGACTATGTTAAGGCCATCT and reverse: ACACTCGGTTGCTCTCTTTTCTCT; for gpx, forward: AGATGTCATTCCTGCACACG and reverse: AAGGAGAAGCTTCCTCAGCC; for catalase, forward: AGGGCAACTGGGATCTTACA and reverse: TTTATGGGACCAGACCTTGG. b-actin was used as an endogenous control with the following primer sequences: forward TACAGCTTCACCACCACAGC and reverse: AAGGAAGGCTGGAAGAGAGC . Real-time Quantitative PCR (RT-qPCR) was performed using Sso fast Eva Green Supermix (Biorad 1725200) in a StepOnePlus Real-Time PCR System (Applied Biosystems) based on manufacture recommendation. Reactions were run in 3–4 technical replicates containing cDNA from 50 ng of total RNA/reaction. The primer sequences for the antioxidant genes were previously described  and are AAGAGAGC . CyclingAAGAGAGC : Relativt method . PCR ampor 1 min . The PCRp170pappaa and +/+pappaa larvae at room temperature. Images were acquired with an Olympus Fluoview confocal laser scanning microscope (FV1000) using Fluoview software (FV10-ASW 4.2). To detect oxidative stress, Tg(brn3c:GFP) larvae were incubated in 10 µM CellROX Deep Red  and 1 µM mitoSOX Red  in E3 for 60 min and 30 min, respectively. To detect mitochondrial transmembrane potential, Tg(brn3c:GFP) larvae were incubated in 25 nM TMRE  for 20 min. To investigate the effects of inhibiting IGF1 signaling on mitochondrial transmembrane potential, larvae were incubated in 25 nM TMRE for 20 min following treatment with NVP-AEW541. To detect MET channel function, Tg(brn3c:GFP) larvae were incubated in 3 µM FM1-43  for 30 s. To measure mitochondrial mass, larvae were incubated in 100 nM mitotracker green FM  for 5 min. Following the incubation period, larvae were washed three times in E3, anesthetized in 0.002% tricaine (Sigma-Aldrich) in E3, and mounted as previously described  was used to subtract background fluorescence from each reporter. The CTCF formula was as follows: Integrated Density - (Area of selected cells X Mean fluorescence of background) (All experiments were done on 5–6 dpf escribed . Fluoresescribed  by drawikground) . The meaAB_10013626) or using Tg(brn3c:GFP) larvae , and support cells  (AB_2576217). After staining, larvae were mounted in 70% glycerol in PBS. Images were acquired with an Olympus Fluoview confocal laser scanning microscope (FV1000) using Fluoview software (FV10-ASW 4.2).For whole-mount immunostaining, larvae at five dpf were fixed in 4% paraformaldehyde for 1 hr at room temperature then rinsed three times with PBS. Larvae were blocked for 1 hr at room temperature in incubation buffer . Larvae were incubated in primary antibodies in IB overnight at 4°C. Primary antibodies were as follows: hair cells  . Followipappaa (For whole-mount in situ hybridization: digoxygenin-UTP-labeled antisense riboprobes for pappaa  were usepappaa . Images SCR_002798). Prior to use of parametric statistics, the assumption of normality was tested using Shapiro-Wilk’s test. Parametric analyses were performed using a two-tailed unpaired t-test with Welch’s correction, multiple t tests with a Holm-Sidak correction, or 2-way ANOVA with a Holm-Sidak correction. Data are presented as means ± standard error of the mean . Significance was set at p<0.05. N for each experiment is detailed in the figure legends. All data presented are from individual experiments except for data in All data were analyzed using GraphPad Prism Software 7.0b .Thank you for submitting your work entitled "Pregnancy-associated plasma protein-aa promotes neuron survival by regulating mitochondrial function" for consideration by eLife at this time. While there was general enthusiasm for the studies, there were a number of concerns raised by the reviewers that we feel would not be able to be addressed in the standard period given for revision. However, given the overall enthusiasm we would consider a revised version of the manuscript if you can meet the referees' concerns. Please note that this would be considered a new submission.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered for publication in Reviewer #1:pappaa mutants are more sensitive to damage. They present evidence that hair cells in mutants have higher levels of ROS production, increases in mitochondrial polarization and increases in mitochondrial calcium, all previously implicated in neomycin-induced hair cell death. They also present evidence that manipulating IGF signaling alters the effects of the pappaa mutation on hair cell survival. Finally, they show that pappaa mutants lose motor neurons over time. Several issues need to be addressed before the manuscript could be considered for publication.Alassaf et al., examine the effects of mutation in the IGF signaling regulator Pappaa on hair cell survival after exposure to neomycin. They find that The authors propose that IGF signaling is important for hair cell survival but do not test this directly. They have used the dn-igfr transgenic line in previous publications – does this method of inhibiting IGF signaling also alter hair cell survival? Does the dn-igfr or the IGF antagonist NVP-AEW541 shift the neomycin dose-response curve? Does SC79 and IGF-1 protect wildtype hair cells against neomycin?Other points:The authors should refer to hair cells in the abstract rather than describing them as neurons.The authors suggest that the failure to add new hair cells is because of degeneration . However, growth of the lateral line depends on the ability of larvae to feed. Are mutants able to feed and grow normally? If not, this phenotype is likely indirect.The analysis to determine whether Pappaa is expressed in hair cells  is inconclusive. Expression centrally in neuromasts (2A) usually corresponds to hair cells. Co-localization with GFP is inconclusive (2B). There is no direct comparison between hair cells and support cells by RT-PCR. Comparison to whole larva is not relevant.pappaa mutants differentially sensitive to this treatment?Experiments shown in 3G are inconclusive. Previous work has shown that mitoTEMPO protects hair cells from neomycin exposure in wildtype animals. Are Additional experiments are needed for the analysis of IGFR signaling . The specificity of the phospho-IGF1R staining should be tested. This staining should be sensitive to NVP-AEW541. It is also important to determine whether staining is actually in hair cells.pappaa mutants. The effect of SC79 (5E) and IGF-1 (5F) on wildtype animals should be shown as discussed above. In particular the authors should test whether these reagents are differentially effective in wildtype and pappaa mutant embryos.Controls are missing from pharmacological experiments . The effect of NVP-AEW541 on hair cells in the absence of neomycin should be shown (5D), and conditions repeated on Reviewer #2:The topic of this study is of great interest to both auditory neuroscientists and to the broader neuroscience community. With that said, hair cells, while they share properties with neurons, are highly specialized and have their own distinct properties. As the data on mitochondrial function and the contribution of IGF1R signaling to survival were collected from hair cells, the emphasis on "neurons" in place of "hair cells" in the title and the manuscript is misleading. Additionally, I have major concerns about how the authors performed and analyzed the live imaging experiments.Point by point concerns are addressed below:- Naturally occurring cell death : Couldn't this observation also indicate a failure to proliferate?- FM1-43 uptake : The dye was diluted with DMSO, which confounds the results as DMSO could be facilitating entry into hair cells.- Image analysis : The method used for analyzing differences in the fluorescence of indicators relative to brn3c:GFP fluorescence (F indicator/ FGFP) is unconventional. I'm concerned that using GFP fluorescence to normalize for variable neuromast size would give misleading results; variances in hair-cell GFP fluorescence appears completely independent of variances in indicator fluorescence . Is this a technique that has been previously used? If not, the authors need to clearly explain their rationale. Additionally, the authors need to provide more detail about how their images were processed and analyzed in ImageJ 2+ uptake and transmembrane potential”: replace "a doubling" with " ~2-fold greater".- Subsection “Pappaa regulates mitochondrial Cap170pappaa mutants relative to their siblings could indicate that the neuromasts are not developing normally.- Subsection “Pappaa-IGF1 receptor signaling is required for neuron survival”: "based on their numbers" – the observation that there are more hair cells in - Subsection “Pappaa-IGF1 receptor signaling is required for neuron survival”: "regulation of mitochondrial function in neurons" – the previous paragraph in the discussion suggests, since Pappaa is expressed in spinal motor neurons but not hair cells, that Pappaa may promote motor neuron survival via mechanisms that differ from hair cells. How do the authors extrapolate that the evidence they show for mitochondrial dysfunction contributing to hair-cell loss in the mutant also applies to loss of neurons?- Pharmacology: 1% DMSO seems unusually high . Why did the authors choose that concentration?Reviewer #3:pappaa mutants, confirming the relationship between mitochondrial ROS and hair cell loss. Overall, this is a thorough description of a novel role for the extracellular factor Pappaa in hair cell and neuron presence. The manuscript is well written and flows nicely. The data presented are solid and offer potential new insights into the role of IGF in maintenance of neurons and hair cells in vivo. However, a few additional experiments would solidify this role, specifically making the connection between cell survival and pappaa mutation and addressing the known role of IGF in mitochondrial biology. Suggestions for modifications to strengthen this work to make it suitable for publication are below.The manuscript "Pregnancy-associated plasma protein-aa promotes neuron survival by regulating mitochondrial function" is a thorough assessment of both hair cell and neuron presence and measures of mitochondrial health in vivo with loss of the extracellular protein Pappaa using the zebrafish system. Using in vivo imaging of mitochondrial/cytoplasmic ROS, mitochondrial calcium, and mitochondrial membrane potential, the authors demonstrate that loss of Pappaa leads to alterations in these measures of mitochondrial health. These abnormal mitochondrial measures correlate with loss of neurons and hair cells in older larvae as well as increased susceptibility of hair cells to calcium and neomycin-mediated death. Strikingly, ROS reduction decreases hair cell loss in Essential revisions:1) The authors argue that loss of Pappaa function causes decreased numbers of hair cell and neurons; however, while number of hair cell and motor neurons is decreased by 9 dpf, no measures of cell death in the absence of chemical ablation are shown. Additionally, no measures of proliferation are shown. To clarify the basic function of Pappaa in neuron presence, a measure of proliferation for hair cells and motor neurons as well as a measure of cell death in these populations should be shown. Numerous methods exist to assay these cellular processes in vivo in zebrafish larvae.pappaa mutants. Essentially sensitizing the system. To determine if defects in these aspects of mitochondrial biology are the primary drivers of the hair cell and neuron loss, the authors should look at mitochondrial size, mitochondrial load, and mitochondrial localization using either a transient transgenic approach or mitotracker.2) IGF signaling has been implicated in the regulation of mitochondrial dynamics, mitophagy and mitochondrial biogenesis. Disrupting these processes could lead to mitochondrial stress, which would then lead to the calcium level, ROS, and matrix potential defects observed in pappaa mutants. Additionally, the ability of exogenous IGF or AKT to rescue motor neurons should be shown to confirm or refute their similarity to hair cells.3) The majority of the work presented was done in hair cells and the results are then assumed to represent what is also happening in motor neurons. To determine if this is indeed the case, at least a subset of the measures of mitochondrial calcium, potential, and ROS should be done in motor neurons of 4) In subsection “Pappaa-IGF1 receptor signaling is required for neuron survival” the authors argue that hair cells and motor neurons develop normally to day 5, indicating a role for Pappaa in maintenance rather than development; however, it is not shown if Pappaa is maternally expressed and, if it is, how long the protein lasts. This would be essential for this argument as it is entirely possible that maternal Pappaa allows for normal development rather than Pappaa not being necessary for development. Alternatively, a maternal zygotic mutant could also answer this question.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by Didier Stainier as the Senior Editor, a Reviewing Editor, and two reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Pregnancy-associated plasma protein-aa supports hair cell survival by regulating mitochondrial function" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The revised manuscript "Pregnancy-associated plasma protein-aa supports hair cell survival by regulating mitochondrial function" carefully describes the function of IGF signaling in hair cell resistance to cell death. The authors have convincingly shown that loss of Pappaa results in increased susceptibility to neomycin-mediated hair cell death and that this is likely through increased susceptibility to mitochondrial stress pathways. Additionally, the authors have strengthened the work by showing that Pappaa functions in the IGF availability pathway through using the IGFBP inhibitor NBI-31772. Together, their work argues for a role for IGF signaling in the maintenance of hair cell survival through supporting mitochondria/inhibiting mitochondrial ROS stress.Essential revisions:1) The reviewers again raised concerns about the numbers of replicates, with no clear way to know how many times experiments were performed. In addition, experiments appear to be often underpowered with small n's. These are of particular concern for imaging experiments presented in Figure 5 and Figure 6. The authors need to clearly state the numbers of samples used for calculations, the numbers of experiments performed, and provide justification.2) In a previous version of this manuscript, exogenous IGF was shown to suppress neomycin-mediated hair cell death. It is unclear why this was taken out as it clearly compliments Figure 3C which shows that enhancing IGF signaling can suppress hair cell death. It should at least be included in the supplement unless there is a specific reason to exclude it.3) Throughout the manuscript, the authors use "% surviving cells" as a measure of hair cell survival. This implies that they counted the hair cells in the same neuromasts before and after treatment. Their methods state that they normalized number of cells after treatment to average number of cells in the vehicle treated control. While this is a suitable way to control for the data, this should be spelled out in the Results section rather than hidden in the Materials and methods section.1) Subsection “Pappaa regulates survival of hair cell sensory neurons”, the authors state: "This live probe (re:TMRE) emits more fluorescence as the membrane potential becomes more negative." This is not true. This vital cationic dye just accumulates to a higher degree in mitochondria that have a more negative matrix potential. The dye does not change the its emission profile to my knowledge. The language should be changed to reflect this.2) In the revised manuscript, the authors provide "absolute fluorescence levels" and refer to these articles . Both studies quantified relative FM1-43 fluorescence by performing background subtraction of fluorescence, selecting the region of interest (e.g. neuromast), and measuring the average fluorescent intensity i.e. total fluorescent intensity/area. If this method is what the authors also used, they should update their Material and Methods section and add citations accordingly. [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:[…] The authors propose that IGF signaling is important for hair cell survival but do not test this directly. They have used the dn-igfr transgenic line in previous publications – does this method of inhibiting IGF signaling also alter hair cell survival? Does the dn-igfr or the IGF antagonist NVP-AEW541 shift the neomycin dose-response curve? Does SC79 and IGF-1 protect wildtype hair cells against neomycin?hsp70:dnIGF1Ra-GFP transgenic line to reduce IGF1R signaling and ask whether IGF1 signaling mediates hair cell survival. As shown in Figure 1B and Figure 4C, we found that induction of dnIGF1Ra increases hair cell death in larvae exposed to neomycin and therefore shifts the neomycin dose-response curve. We have excluded the IGF-1 and SC79 supplementation experiments from the manuscript because we wanted to focus mainly on the influence of endogenous IGF1 signaling and the pathway’s extracellular regulation , rather than exogenous supplementation by IGF1 and SC79.Per this suggestion, we used the The authors should refer to hair cells in the Abstract rather than describing them as neurons.The manuscript has been edited to reflect these changes and now solely focuses on the effect of Pappaa-IGF1 signaling on hair cells.The authors suggest that the failure to add new hair cells is because of degeneration . However, growth of the lateral line depends on the ability of larvae to feed. Are mutants able to feed and grow normally? If not, this phenotype is likely indirect.pappaa mutants do show reduced feeding. Therefore, we have excluded this result and its interpretation from the manuscript.We thank the reviewer for calling our attention to this important point. The analysis to determine whether pappaa is expressed in hair cells  is inconclusive. Expression centrally in neuromasts (2A) usually corresponds to hair cells. Co-localization with GFP is inconclusive (2B). There is no direct comparison between hair cells and support cells by RT-PCR. Comparison to whole larva is not relevant.pappaa in situ expression shows clear expression by the neuromasts. We believe the expression centrally in each neuromast is by the inner support cells, not the hair cells. By analyzing Z-stack confocal images slice by slice in Brn3c-GFP larvae stained for pappaa expression by FISH, we do not observe pappaa mRNA in hair cells. We performed the RT-PCR of pappaa on FAC sorted hair cells to rule out the possibility that hair cells express pappaa, but at a level below FISH detection. RT-PCR on whole larvae and FAC sorted motor neurons provided positive controls. Unfortunately, we do not have a transgenic line that only labels support cells to use in a complementary experiment.We have modified Figure 2 to provide more conclusive expression data. The colorimetric Experiments shown in 3G are inconclusive. Previous work has shown that mitoTEMPO protects hair cells from neomycin exposure in wildtype animals. Are pappaa mutants differentially sensitive to this treatment?pappaa mutants. It is difficult to determine relative sensitivity of the two genotypes to mitoTEMPO because mitoTEMPO produces an all or nothing survival effect in both genotypes at doses ranging from 10-100 μM. Rather than assessing relative sensitivity, the rationale behind our experiment was to determine whether the excessive ROS production by pappaa mutant hair cells was the root cause of their increased death when exposed to neomycin. If mitoTEMPO did not or only partially rescued hair cell loss in pappaa mutants, then this outcome would have suggested the mutant hair cells have additional defects that underlie their increased neomycin sensitivity. Given that mitoTEMPO provided up to complete protection against neomycin, we concluded that ROS was the primary reason.Results of the mitoTEMPO experiment are now in Figure 7B. Consistent with previous work , we find mitoTEMPO protects hair cells from neomycin exposure in wildtype and Additional experiments are needed for the analysis of IGFR signaling . The specificity of the phospho-IGF1R staining should be tested. This staining should be sensitive to NVP-AEW541. It is also important to determine whether staining is actually in hair cells.pappaa mutant hair cells) because it does not significantly add to the manuscript.The anti-pIGF1R antibody used was validated by Chablais and Jazwinska (2010) in zebrafish using NVP-AEW541. However, we decided to exclude the result . The effect of NVP-AEW541 on hair cells in the absence of neomycin should be shown (5D), and conditions repeated on pappaa mutants. The effect of SC79 (5E) and IGF-1 (5F) on wildtype animals should be shown as discussed above. In particular the authors should test whether these reagents are differentially effective in wildtype and pappaa mutant embryos.hsp70:dnIGF1Ra-GFP transgenic line to reduce IGF1R signaling and ask whether IGF1 signaling mediates hair cell survival . Notably, expression of dnIGF1Ra alone (no neomycin) did not affect hair cell survival. Because we decided to focus more on the extracellular regulation of endogenous IGF1 signaling rather than exogenous supplementation of IGF1 and SC79, we included results from pharmacological stimulation of IGF1 bioavailability by NBI-31772 .We did not observe hair cell death when larvae were treated with NVP-AEW541 alone. That said, these controls and experiments are now moot, since we have decided against including the results in the manuscript. As detailed above, we instead used the Reviewer #2:[…] Point by point concerns are addressed below:- Naturally occurring cell death : Couldn't this observation also indicate a failure to proliferate?pappaa mutants develop on time and are capable of regeneration. Therefore, we do not believe that a failure to proliferate is causing the lack of age-related increase in hair cells. However, as reviewer 1 pointed out, growth of lateral line hair cells depends on the larvae’s ability to grow and feed normally. Given that pappaa mutant larvae show reduced ability to feed, we have decided to exclude this result and interpretation from the current manuscript.The hair cells in - FM1-43 uptake : The dye was diluted with DMSO, which confounds the results as DMSO could be facilitating entry into hair cells.FM1-43 was dissolved in DMSO, as per manufacturer recommendation. We used FM1-43 at a low final concentration (3uM) made from a 5 mM stock in DMSO. The final concentration of DMSO would have been at 0.06%. This method has been used previously to demonstrate that FM1-43 (dissolved in DMSO) only enters hair cells via MET channels when exposure time is less than 1 minute .- Image analysis : The method used for analyzing differences in the fluorescence of indicators relative to brn3c:GFP fluorescence (F indicator/ FGFP) is unconventional. I'm concerned that using GFP fluorescence to normalize for variable neuromast size would give misleading results; variances in hair-cell GFP fluorescence appears completely independent of variances in indicator fluorescence . Is this a technique that has been previously used? If not, the authors need to clearly explain their rationale.To provide further analysis, we have now included the absolute fluorescence levels. This follows the field standard . Additionally, we have included the normalized fluorescence (F indicator/F GFP), which we feel is important to control for potential variability in labeling across samples.Additionally, the authors need to provide more detail about how their images were processed and analyzed in ImageJ We have provided more detail on the image processing in subsection “Live imaging”. We have added the text “Fluorescent intensity of the reporter was measured using ImageJ  by drawing a region of interest around brn3c:GFP-labaled hair cell cluster of the neuromast from Z-stack summation projections that included the full depth of the hair cells. Background fluorescent intensity was measured by drawing a ROI away from the neuromast in the same Z-stack summation projection. The corrected total cell fluorescence (CTCF) was used to subtract background fluorescence from each reporter. The CTCF formula was as follows: Integrated Density – (Area of selected cells X Mean fluorescence of background) . Relative fluorescent intensity was reported as the ratio to GFP fluorescence.” No specific plugins were used.2+ uptake and transmembrane potential”: replace "a doubling" with " ~2-fold greater".- Subsection “Pappaa regulates mitochondrial CaThis change has been made.p170 mutants relative to their siblings could indicate that the neuromasts are not developing normally.- Subsection “Pappaa-IGF1 receptor signaling is required for neuron survival”: "based on their numbers" – the observation that there are more hair cells in pappaapappaa affects neuromast development because (1) the hair cell and support cell morphologies are normal , (2) the mutants respond to acoustic stimuli and do not show hallmark behavioral signs of dysfunctional hair cells  and (3) we show that Pappaa is dispensable for hair cell development in the context of hair cell sensitivity to neomycin . The increased number of hair cells in neuromasts of pappaa mutants is very modest: ~2 per neuromast. If we did not account for this difference and used absolute hair cell counts to reflect mortality, then the hair cell loss would appear artificially inflated in the pappaa mutants compared to wild type controls. Therefore, we normalized hair cell loss to vehicle treated control wild type or pappaa mutants to most accurately represent and compare the relative hair cell loss between the two genotypes.We do not believe - Subsection “Pappaa-IGF1 receptor signaling is required for neuron survival”: "regulation of mitochondrial function in neurons" – the previous paragraph in the discussion suggests, since Pappaa is expressed in spinal motor neurons but not hair cells, that Pappaa may promote motor neuron survival via mechanisms that differ from hair cells. How do the authors extrapolate that the evidence they show for mitochondrial dysfunction contributing to hair-cell loss in the mutant also applies to loss of neurons?The current manuscript no longer includes results or discussion of Pappaa’s influence on motor neuron survival. Therefore, this section has been omitted.- Pharmacology: 1% DMSO seems unusually high . Why did the authors choose that concentration?“1% DMSO” was a typo. Changes were made to reflect the real concentration (0.1%).Reviewer #3:[…] Essential revisions:1) The authors argue that loss of Pappaa function causes decreased numbers of hair cell and neurons; however, while number of hair cell and motor neurons is decreased by 9 dpf, no measures of cell death in the absence of chemical ablation are shown. Additionally, no measures of proliferation are shown. To clarify the basic function of Pappaa in neuron presence, a measure of proliferation for hair cells and motor neurons as well as a measure of cell death in these populations should be shown. Numerous methods exist to assay these cellular processes in vivo in zebrafish larvae.pappaa mutants develop on time and are capable of regeneration, we do not believe that failure to proliferate is causing the lack of age-related increase in hair cells. However, as reviewer 1 has pointed out, growth of lateral line hair cells depends on the larvae’s ability to grow and feed normally. Given that pappaa mutant larvae show reduced ability to feed, we have decided to exclude this experiment from our manuscript. We also decided to focus solely on hair cells. Results describing motor neuron degeneration have been excluded from the revised manuscript.Because hair cells in 2) IGF signaling has been implicated in the regulation of mitochondrial dynamics, mitophagy and mitochondrial biogenesis. Disrupting these processes could lead to mitochondrial stress which would then lead to the calcium level, ROS, and matrix potential defects observed in pappaa mutants. Essentially sensitizing the system. To determine if defects in these aspects of mitochondrial biology are the primary drivers of the hair cell and neuron loss, the authors should look at mitochondrial size, mitochondrial load, and mitochondrial localization using either a transient transgenic approach or mitotracker.pappaa mutants by electron microscopy. We believe results from these analyses are beyond the scope of the current study.We analyzed mitochondrial load using mitotracker and found no significant difference . A future direction of this project is to evaluate mitochondrial size, morphology, and localization in 3) The majority of the work presented was done in hair cells and the results are then assumed to represent what is also happening in motor neurons. To determine if this is indeed the case, at least a subset of the measures of mitochondrial calcium, potential, and ROS should be done in motor neurons of pappaa mutants. Additionally, the ability of exogenous IGF or AKT to rescue motor neurons should be shown to confirm or refute their similarity to hair cells.In the manuscript’s revised form, we only show results for hair cells and we have limited our conclusions to hair cells.4) In subsection “Pappaa-IGF1 receptor signaling is required for neuron survival” the authors argue that hair cells and motor neurons develop normally to day 5, indicating a role for pappaa in maintenance rather than development; however, it is not shown if pappaa is maternally expressed and, if it is, how long the protein lasts. This would be essential for this argument as it is entirely possible that maternal pappaa allows for normal development rather than pappaa not being necessary for development. Alternatively, a maternal zygotic mutant could also answer this question.pappaa mutant hair cell sensitivity to neomycin.In our revised manuscript, we have included an experiment in which we induce Pappaa expression at 4 dpf . We found that post-developmental expression of Pappaa is sufficient to rescue [Editors' note: the author responses to the re-review follow.][…] Essential revisions:1) The reviewers again raised concerns about the numbers of replicates, with no clear way to know how many times experiments were performed. In addition, experiments appear to be often underpowered with small n's. These are of particular concern for imaging experiments presented in Figure 5 and Figure 6. The authors need to clearly state the numbers of samples used for calculations, the numbers of experiments performed, and provide justification.We have added more details concerning the specific number of neuromasts and animals for each treatment group in the figures and in the figure legends. Please note that the N in this paper refers to the number of animals per group (shown at base of bars) and not neuromasts. Multiple neuromasts per animal were analyzed and averaged. To address the number of experimental trials, we’ve added the following in the methods “All data presented are from individual experiments except for data in Figure 1B, Figure 3B, and Figure 6E. Data collected from multiple experiments were normalized to their respective controls prior to pooling”. We also included the number of experiments in the relevant figure legends.Because each live imaging experiment was done on the same day to control for any technical variabilities, we were limited in the number of animals tested. Please note that multiple neuromasts were analyzed per animal resulting in an average of 20 neuromasts per genotype (or treatment group). However, we do agree that some experiments had a particularly small number of animals tested (4 or fewer). Therefore, we have repeated experiments presented in Figure 5—figure supplement 1 and Figure 6B and 6D-D’ to include a bigger sample size.2) In a previous version of this manuscript, exogenous IGF was shown to suppress neomycin-mediated hair cell death. It is unclear why this was taken out as it clearly compliments Figure 3C which shows that enhancing IGF signaling can suppress hair cell death. It should at least be included in the supplement unless there is a specific reason to exclude it.Thank you for the suggestion. We’ve incorporated the exogenous IGF1 experiment to the revised manuscript (now figure 3C).3) Throughout the manuscript, the authors use "% surviving cells" as a measure of hair cell survival. This implies that they counted the hair cells in the same neuromasts before and after treatment. Their methods state that they normalized number of cells after treatment to average number of cells in the vehicle treated control. While this is a suitable way to control for the data, this should be spelled out in the Results section rather than hidden in the Materials and methods section.We have changed the Y-axis titles of relevant graphs to better clarify this.