Hierarchical patterning modes orchestrate hair follicle morphogenesis

Two theories address the origin of repeating patterns, such as hair follicles, limb digits, and intestinal villi, during development. The Turing reaction–diffusion system posits that interacting diffusible signals produced by static cells first define a prepattern that then induces cell rearrangements to produce an anatomical structure. The second theory, that of mesenchymal self-organisation, proposes that mobile cells can form periodic patterns of cell aggregates directly, without reference to any prepattern. Early hair follicle development is characterised by the rapid appearance of periodic arrangements of altered gene expression in the epidermis and prominent clustering of the adjacent dermal mesenchymal cells. We assess the contributions and interplay between reaction–diffusion and mesenchymal self-organisation processes in hair follicle patterning, identifying a network of fibroblast growth factor (FGF), wingless-related integration site (WNT), and bone morphogenetic protein (BMP) signalling interactions capable of spontaneously producing a periodic pattern. Using time-lapse imaging, we find that mesenchymal cell condensation at hair follicles is locally directed by an epidermal prepattern. However, imposing this prepattern’s condition of high FGF and low BMP activity across the entire skin reveals a latent dermal capacity to undergo spatially patterned self-organisation in the absence of epithelial direction. This mesenchymal self-organisation relies on restricted transforming growth factor (TGF) β signalling, which serves to drive chemotactic mesenchymal patterning when reaction–diffusion patterning is suppressed, but, in normal conditions, facilitates cell movement to locally prepatterned sources of FGF. This work illustrates a hierarchy of periodic patterning modes operating in organogenesis

Geomorphological mapping with a small unmanned aircraft system (sUAS): feature detection and accuracy assessment of a photogrammetrically-derived digital terrain model

Sherpa Romeo green journal. Permission to archive accepted author manuscript.Small unmanned aircraft systems (sUAS) are a relatively new type of aerial platform for acquiring high-resolution remote sensing measurements of Earth surface processes and landforms. However, despite growing application there has been little quantitative assessment of sUAS performance. Here we present results from a field experiment designed to evaluate the accuracy of a photogrammetrically-derived digital terrain model (DTM) developed from imagery acquired with a low-cost digital camera onboard an sUAS. We also show the utility of the highresolution (0.1 m) sUAS imagery for resolving small-scale biogeomorphic features. The experiment was conducted in an area with active and stabilized aeolian landforms in the southern Canadian Prairies. Images were acquired with a Hawkeye RQ-84Z Aerohawk fixed-wing sUAS. A total of 280 images were acquired along 14 flight lines, covering an area of 1.95 km2. The survey was completed in 4.5 hours, including GPS surveying, sUAS setup and flight time. Standard image processing and photogrammetric techniques were used to produce a 1 m resolution DTM and a 0.1 m resolution orthorectified image mosaic. The latter revealed previously un-mapped bioturbation features. The vertical accuracy of the DTM was evaluated with 99 Real-Time Kinematic GPS points, while 20 of these points were used to quantify horizontal accuracy. The horizontal root mean squared error (RMSE) of the orthoimage was 0.18 m, while the vertical RMSE of the DTM was 0.29 m, which is equivalent to the RMSE of a bare earth LiDAR DTM for the same site. The combined error from both datasets was used to define a threshold of the minimum elevation difference that could be reliably attributed to erosion or deposition in the seven years separating the sUAS and LiDAR datasets. Overall, our results suggest that sUAS-acquired imagery may provide a low-cost, rapid, and flexible alternative to airborne LiDAR for geomorphological mapping.Ye

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Thigh-length compression stockings and DVT after stroke

Controversy exists as to whether neoadjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite randomised controlled trials of more than 3000 patients. We undertook a systematic review and meta-analysis to assess the effect of such treatment on survival in patients with this disease

Dependence of mesenchyme-only patterning on restricted TGFβ availability.

<p>(A) Western blot detection of phospho-SMAD2, total SMAD2 and γ-tubulin in E13.5 skin cultures treated with recombinant transforming growth factor (TGF) β2 (100 ng/ml), the TGFβ receptor inhibitor LY2109761 (25 μM,) or both agents for 8 h. (B) Effects of TGFβ2 supplementation and LY2109761 on normal and mesenchyme-only patterning. Condensates are slow to appear in LY2109761, and expression of the placode marker <i>Dkk4</i> expands through the epidermis. Mesenchyme-only patterning in FGF^HiBMP^Lo conditions is abolished upon either suppression or augmentation of TGFβ signalling. Scale bars: 250 μm. (C) Whole-mount in situ hybridisation (top panel) and corresponding transverse section (bottom panel) detecting spatial arrangement of <i>Tgfb2</i> expression in E14.5 mouse embryos. Expression is most intense at sites of dermal condensate formation. Scale bars: top panel = 1 mm, bottom panel = 50 μm. (D) At E13.5, phospho-SMAD2 immunofluorescence detects signal throughout the dermal mesenchyme (De.) and epidermis (Ep.), with this signal becoming intensified in the nascent dermal condensate at E14.5 (arrowhead). Epidermis is demarcated by dotted lines. Scale bar: 25 μm. (E) Dermal mesenchymal cell attraction (arrows) to sources of TGFβ2. Images of bovine serum albumin (BSA) control and TGFβ2 loaded beads placed on E12.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin for 48 h. Scale bars: 250 μm.</p

TGFβ enhances cell attraction to focal FGF sources.

<p>(A, B) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of E13.5 or E13.75 (with condensates) skins treated with transforming growth factor (TGF) β2, fibroblast growth factor (FGF) 9, or bone morphogenetic protein (BMP) 4 for 8 or 24 h, respectively, followed by assessment of transcript abundance. TGFβ2 upregulates expression of genes associated with cell movement and the extracellular matrix. Statistical significance from control was calculated using a Student <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). Error bars represent SEM from at least 3 independent experiments. (C) Cell aggregation at FGF9 beads in E12.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explants. TGFβ2 (100 ng/ml) or LY2109761 (25 μM) is present in the culture medium as indicated. TGFβ2 enhances aggregation at FGF9 beads, while LY2109761 suppresses cell accumulation. (D) FGF9 presence in culture medium does not detectably increase cell recruitment to TGFβ2 beads. (E, F) Quantification of areas of high cell density around FGF9- or TGFβ2-coated beads under conditions as indicated. Statistical significance was calculated using Student <i>t</i> tests (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). Error bars represent SEM of at least 3 independent experiments. Scale bars: 250 μm. The raw numerical values (for A, B, E, and F) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s016" target="_blank">S4 Data</a>.</p

BMP destabilises mesenchymal aggregates.

<p>(A) Epidermis and dermis isolated from E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explants cultured with LDN193189 (LDN) (10 μM) or fibroblast growth factor (FGF) 9 (1 μg/ml) for 27 h, counterstained with propidium iodide (PI) and imaged using confocal microscopy. Scale bar: 100 μm. (B) Number of condensates per square mm, (C) mean condensate area, and (D) intercondensate cell density measured in E13.5 TCF/Lef::H2B-GFP skin explants cultured with either LDN or FGF. Error bars represent SEM from 5 independent experiments. Significant difference was calculated using a Student paired <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). (E) E13.75 TCF/Lef::H2B-GFP skin explants (with pre-existing dermal condensates) were treated with bone morphogenetic protein (BMP) 4 for 72 h. Skins were counterstained with 4’6-Diamidino-2-phenylindole dihydrochloride (DAPI) and confocal imaged for GFP at 72 h. Scale bars: 100 μm. (F) Quantification of individual condensate area following BMP supplementation. Error bars represent SEM from at least 3 independent experiments. Significant difference was tested using a paired Student <i>t</i> test (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001). The raw numerical values (for B, C, D, and F) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s015" target="_blank">S3 Data</a>.</p

Dermal condensate formation occurs after epidermal patterning through local cell attraction.

<p>(A) Single frames from time-lapse sequences of E13.5 TCF/Lef::H2B-green fluorescent protein (GFP) skin explant culture captured by confocal microscopy. Dashed circles indicate ultimate condensate location. Scale bar: 50 μm. (B) Analysis of tracked cells showing the probability of joining the dermal condensate based upon initial location relative to its centre. Two hundred and forty individual cells were tracked across 8 condensates from 4 independent skins. (C) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue). Tracking was halted on cell entry. (D) Plots showing the mean Euclidean angle (top) and mean level of persistence (bottom) of condensate-entering and intercondensate cells for 6-h windows relative to time of entry into the condensate. Error bars represent SEM (condensate cells <i>n</i> = 9, 14, and 20 and intercondensate <i>n</i> = 263, 245, and 197 for 12, 6, and 0 h before entry, respectively). Statistical significance was calculated using a Kruskal–Wallis test (<i>p</i> < 0.0001 and <i>p</i> < 0.001 for angle and persistence, respectively) followed by Mann–Whitney U tests with Bonferroni’s correction (**<i>p</i> < 0.01). The raw numerical tracking data (for B, C, and D) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s014" target="_blank">S2 Data</a>. (E) Detection of a molecular prepattern prior to dermal condensate formation. TCF/Lef::H2B-GFP skin explants were fixed at intermediate stages of pattern formation, imaged to detect GFP, and <i>Dkk4</i> expression determined in the same skin sample. Asterisk represents an area where <i>Dkk4</i>-positive foci are present but corresponding dermal condensates are absent. Scale bar: 500 μm. (F) Time-lapse images of E12.75 TCF/Lef::H2B-GFP dorsal skin explants cultured with recombinant fibroblast growth factor (FGF) 9- or bovine serum albumin (BSA)-loaded beads. Cells accumulate around FGF9-loaded beads. Scale bar: 250 μm.</p

Cell behaviours underlying mesenchymal self-organisation.

<p>(A) Time-lapse images showing dermal condensate formation in FGF^HiBMP^Lo conditions. Scale bar: 50 μm. (B) Protractor plot showing the distribution of Euclidean angles and Euclidean distances of individual cell movements in 6-h windows for cell tracks that start outside of, but ultimately terminate in, a follicle (condensate = red) and those that remain outside (intercondensate = blue) under FGF^HiBMP^Lo conditions. Tracking was halted on cell entry. Plots showing (C) the mean Euclidean angle and (D) the mean level of persistence of condensate and intercondensate cells for 360-minute windows relative to time of entry into the condensate. From 6 h before entry, the condensate-bound cells show oriented and persistent movement under FGF^HiBMP^Lo conditions. Error bars represent SEM (condensate cells <i>n</i> = 17, 21, and 25 and intercondensate <i>n</i> = 91, 104, and 108 for 12, 6, and 0 h before entry, respectively). Statistical significance was calculated using a Kruskal–Wallis test followed by Mann–Whitney U tests with Bonferroni’s correction (***<i>p</i> < 0.001). (E) Comparison between per track summaries of condensate (Cond.) and intercondensate (Int.) cells under control or FGF^HiBMP^Lo conditions for (top) accumulated velocity, (middle) Euclidian velocity, and (bottom) persistence. Statistical significance was calculated using a Kruskal–Wallis test followed by Mann–Whitney U tests with Bonferroni’s correction (*<i>p</i> < 0.05, ***<i>p</i> < 0.001). Error bars represent SEM (control intercondensate <i>n</i> = 292, control condensate <i>n</i> = 28, FGF^HiBMP^Lo intercondensate <i>n</i> = 137 and FGF^HiBMP^Lo condensate <i>n</i> = 33). Raw tracking data for (B–E) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002117#pbio.2002117.s014" target="_blank">S2 Data</a>. (F) Particle image velocimetry analysis of normal and FGF^HiBMP^Lo condensate formation over 30 h. Coloured tracks show very local cell movement in control conditions but a much broader field of recruitment for the mesenchyme-only patterned condensates. Colour scale shows track length. Scale bar: 100 μm. (G) Simulation of boundary effects on patterning in chemotactic aggregation-driven patterning. (H) Experimental test of pattern behaviours. Distinct pattern behaviours at tissue edges. Under control conditions, primordia align along the edge. FGF^HiBMP^Lo condensates align with but form at a distance from boundaries introduced in skin explants prior to pattern formation. White dotted lines indicate the boundary. Magenta dotted lines indicate the extent of the patterned region where dermal condensates form. Scale bar: 250 μm.</p

Overlaying of reaction-diffusion signalling and dermal condensation mechanisms.

<p>(A) A signalling network including bone morphogenetic protein–fibroblast growth factor–wingless-related integration site (BMP–FGF–WNT) interactions rapidly produces a periodic prepattern of hair placodes with high WNT/β-catenin activity. (B) FGF20 production by placodes leads to local dermal cell attraction and condensate formation, facilitated by widespread transforming growth factor (TGF) β activity. BMP production inhibits expansion of placode gene expression and further dermal cell attraction. (C) In the absence of epidermal patterning, no localised attractant signals from placodes are present, and dermal BMP signalling is uniform. TGFβ stimulates dermal cell–cell attraction, which is inhibited by BMPs. Suppression of BMP signalling permits TGFβ-driven mesenchymal patterning in the absence of a functioning epidermal reaction–diffusion network. Condensate expansion is restricted by mesenchymal cell depletion from the surrounding dermis.</p