Genetic interference with HvNotch provides new insights into the role of the Notch-signalling pathway for developmental pattern formation in Hydra.

The Notch-signalling pathway plays an important role in pattern formation in Hydra. Using pharmacological Notch inhibitors (DAPT and SAHM1), it has been demonstrated that HvNotch is required for head regeneration and tentacle patterning in Hydra. HvNotch is also involved in establishing the parent-bud boundary and instructing buds to develop feet and detach from the parent. To further investigate the functions of HvNotch, we successfully constructed NICD (HvNotch intracellular domain)-overexpressing and HvNotch-knockdown transgenic Hydra strains. NICD-overexpressing transgenic Hydra showed a pronounced inhibition on the expression of predicted HvNotch-target genes, suggesting a dominant negative effect of ectopic NICD. This resulted in a "Y-shaped" phenotype, which arises from the parent-bud boundary defect seen in polyps treated with DAPT. Additionally, "multiple heads", "two-headed" and "ectopic tentacles" phenotypes were observed. The HvNotch-knockdown transgenic Hydra with reduced expression of HvNotch exhibited similar, but not identical phenotypes, with the addition of a "two feet" phenotype. Furthermore, we observed regeneration defects in both, overexpression and knockdown strains. We integrated these findings into a mathematical model based on long-range gradients of signalling molecules underlying sharply defined positions of HvNotch-signalling cells at the Hydra tentacle and bud boundaries.

Behavioral responses to offshore windfarms during migration of a declining shorebird species revealed by GPS-telemetry.

EU member countries and the UK are currently installing numerous offshore windfarms (OWFs) in the Baltic and North Seas to achieve decarbonization of their energy systems. OWFs may have adverse effects on birds; however, estimates of collision risks and barrier effects for migratory species are notably lacking, but are essential to inform marine spatial planning. We therefore compiled an international dataset consisting of 259 migration tracks for 143 Global Positioning System-tagged Eurasian curlews (Numenius arquata arquata) from seven European countries recorded over 6 years, to assess individual response behaviors when approaching OWFs in the North and Baltic Seas at two different spatial scales (i.e. up to 3.5 km and up to 30 km distance). Generalized additive mixed models revealed a significant small-scale increase in flight altitudes, which was strongest at 0-500 m from the OWF and which was more pronounced during autumn than during spring, due to higher proportions of time spent migrating at rotor level. Furthermore, four different small-scale integrated step selection models consistently detected horizontal avoidance responses in about 70% of approaching curlews, which was strongest at approximately 450 m from the OWFs. No distinct, large-scale avoidance effects were observed on the horizontal plane, although they could possibly have been confounded by changes in flight altitudes close to land. Overall, 28.8% of the flight tracks crossed OWFs at least once during migration. Flight altitudes within the OWFs overlapped with the rotor level to a high degree in autumn (50%) but to a significantly lesser extent in spring (18.5%). Approximately 15.8% and 5.8% of the entire curlew population were estimated to be at increased risk during autumn and spring migration, respectively. Our data clearly show strong small-scale avoidance responses, which are likely to reduce collision risk, but simultaneously highlight the substantial barrier effect of OWFs for migrating species. Although alterations in flight paths of curlews due to OWFs seem to be moderate with respect to the overall migration route, there is an urgent need to quantify the respective energetic costs, given the massive ongoing construction of OWFs in both sea areas.

Large-scale effects of offshore wind farms on seabirds of high conservation concern.

The North Sea is a key area worldwide for the installation of offshore wind farms (OWFs). We analysed data from multiple sources to quantify the effects of OWFs on seabirds from the family Gaviidae (loons) in the German North Sea. The distribution and abundance of loons changed substantially from the period before to the period after OWF construction. Densities of loons were significantly reduced at distances of up to 9-12 km from the OWF footprints. Abundance declined by 94% within the OWF + 1 km zone and by 52% within the OWF + 10 km zone. The observed redistribution was a large-scale effect, with birds aggregating within the study area at large distances from the OWFs. Although renewable energies will be needed to provide a large share of our energy demands in the future, it is necessary to minimize the costs in terms of less-adaptable species, to avoid amplifying the biodiversity crisis.

Non-muscle myosin II drives critical steps of nematocyst morphogenesis.

Nematocysts are generated by secretion of proteins into a post-Golgi compartment. They consist of a capsule that elongates into a long tube, which is coiled inside the capsule matrix and expelled during its nano-second discharge deployed for prey capture. The driving force for discharge is an extreme osmotic pressure of 150 bar. The complex processes of tube elongation and invagination under these biomechanical constraints have so far been elusive. Here, we show that a non-muscle myosin II homolog (HyNMII) is essential for nematocyst formation in Hydra. In early nematocysts, HyNMII assembles to a collar around the neck of the protruding tube. HyNMII then facilitates tube outgrowth by compressing it along the longitudinal axis as evidenced by inhibitor treatment and genetic knockdown. In addition, live imaging of a NOWA::NOWA-GFP transgenic line, which re-defined NOWA as a tube component facilitating invagination, allowed us to analyze the impact of HyNMII on tube maturation.

Injury-induced MAPK activation triggers body axis formation in Hydra by default Wnt signaling.

Hydra's almost unlimited regenerative potential is based on Wnt signaling, but so far it is unknown how the injury stimulus is transmitted to discrete patterning fates in head and foot regenerates. We previously identified mitogen-activated protein kinases (MAPKs) among the earliest injury response molecules in Hydra head regeneration. Here, we show that three MAPKs-p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs)-are essential to initiate regeneration in Hydra, independent of the wound position. Their activation occurs in response to any injury and requires calcium and reactive oxygen species (ROS) signaling. Phosphorylated MAPKs hereby exhibit cross talk with mutual antagonism between the ERK pathway and stress-induced MAPKs, orchestrating a balance between cell survival and apoptosis. Importantly, Wnt3 and Wnt9/10c, which are induced by MAPK signaling, can partially rescue regeneration in tissues treated with MAPK inhibitors. Also, foot regenerates can be reverted to form head tissue by a pharmacological increase of ß-catenin signaling or the application of recombinant Wnts. We propose a model in which a ß-catenin-based stable gradient of head-forming capacity along the primary body axis, by differentially integrating an indiscriminate injury response, determines the fate of the regenerating tissue. Hereby, Wnt signaling acquires sustained activation in the head regenerate, while it is transient in the presumptive foot tissue. Given the high level of evolutionary conservation of MAPKs and Wnts, we assume that this mechanism is deeply embedded in our genome.

Pattern formation features might explain homoplasy: fertile surfaces in higher fungi as an example.

Fungi show a high degree of morphological convergence. Regarded for a long time as an obstacle for phylogenetic studies, homoplasy has also been proposed as a source of information about underlying morphogenetic patterning mechanisms. The "local-activation and long-range inhibition principle" (LALIP), underlying the famous reaction-diffusion model proposed by Alan Turing in 1952, appears to be one of the universal phenomena that can explain the ontogenetic origin of seriate patterns in living organisms. Reproductive structures of fungi in the class Agaricomycetes show a highly periodic structure resulting in, for example, poroid, odontoid, lamellate or labyrinthic hymenophores. In this paper, we claim that self-organized patterns might underlie the basic ontogenetic processes of these structures. Simulations based on LALIP-driven models and covering a wide range of parameters show an absolute mutual correspondence with the morphospace explored by extant agaricomycetes. This could not only explain geometric particularities but could also account for the limited possibilities displayed by hymenial configurations, thus making homoplasy a direct consequence of the limited morphospace resulting from the proposed patterning dynamics.

Computational modelling of nephron progenitor cell movement and aggregation during kidney organogenesis.

During early kidney organogenesis, nephron progenitor (NP) cells move from the tip to the corner region of the ureteric bud (UB) branches in order to form the pretubular aggregate, the early structure giving rise to nephron formation. NP cells derive from metanephric mesenchymal cells and physically interact with them during the movement. Chemotaxis and cell-cell adhesion differences are believed to drive the cell patterning during this critical period of organogenesis. However, the effect of these forces to the cell patterns and their respective movements are known in limited details. We applied a Cellular Potts Model to explore how these forces and organizations contribute to directed cell movement and aggregation. Model parameters were estimated based on fitting to experimental data obtained in ex vivo kidney explant and dissociation-reaggregation organoid culture studies. Our simulations indicated that optimal enrichment and aggregation of NP cells in the UB corner niche requires chemoattractant secretion from both the UB epithelial cells and the NP cells themselves, as well as differences in cell-cell adhesion energies. Furthermore, NP cells were observed, both experimentally and by modelling, to move at higher speed in the UB corner as compared to the tip region where they originated. The existence of different cell speed domains along the UB was confirmed using self-organizing map analysis. In summary, we saw faster NP cell movements near aggregation. The applicability of Cellular Potts Model approach to simulate cell movement and patterning was found to be good during for this early nephrogenesis process. Further refinement of the model should allow us to recapitulate the effects of developmental changes of cell phenotypes and molecular crosstalk during further organ development.

Beyond BACI: Offsetting carcass numbers with flight intensity to improve risk assessments of bird collisions with power lines.

The continuing global expansion of electricity networks increases the risk of bird collisions with power lines. Several field studies have demonstrated that this risk can be reduced by marking lines with flight diverters. A before-after control-impact (BACI) design is currently the suggested approach for evaluating the effectiveness of these diverters and is generally assumed to give unbiased results.Using systematic flight survey data, we demonstrate that the assumptions underlying the BACI approach are frequently violated, leading to biased effectiveness estimates. We present an alternative field and statistical design in which the number of bird strike victims is directly related to bird flight intensity ("fusion design"), instead of estimating it indirectly using a control site. The presented design is validated based on simulations.We demonstrate that the presented method is unbiased and shows an approximately 3-fold higher statistical power compared with BACI, even under ideal/unbiased data conditions, with similar field-experimental effort. Moreover, this approach can provide a direct analysis of bird reactions/collisions, estimation of collision rates, and the possibility of conducting the required fieldwork within a single season.Our presented method can be used to standardize and improve future studies on diverter effectiveness, for example, by supporting the acquisition of a more detailed picture of species-, diverter type-, and habitat-specific estimates.

Beyond Turing: far-from-equilibrium patterns and mechano-chemical feedback.

Turing patterns are commonly understood as specific instabilities of a spatially homogeneous steady state, resulting from activator-inhibitor interaction destabilized by diffusion. We argue that this view is restrictive and its agreement with biological observations is problematic. We present two alternatives to the classical Turing analysis of patterns. First, we employ the abstract framework of evolution equations to enable the study of far-from-equilibrium patterns. Second, we introduce a mechano-chemical model, with the surface on which the pattern forms being dynamic and playing an active role in the pattern formation, effectively replacing the inhibitor. We highlight the advantages of these two alternatives vis-à-vis the classical Turing analysis, and give an overview of recent results and future challenges for both approaches. This article is part of the theme issue 'Recent progress and open frontiers in Turing's theory of morphogenesis'.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra.

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Analysis of local habitat selection and large-scale attraction/avoidance based on animal tracking data: is there a single best method?

BACKGROUND: New wildlife telemetry and tracking technologies have become available in the last decade, leading to a large increase in the volume and resolution of animal tracking data. These technical developments have been accompanied by various statistical tools aimed at analysing the data obtained by these methods. METHODS: We used simulated habitat and tracking data to compare some of the different statistical methods frequently used to infer local resource selection and large-scale attraction/avoidance from tracking data. Notably, we compared spatial logistic regression models (SLRMs), spatio-temporal point process models (ST-PPMs), step selection models (SSMs), and integrated step selection models (iSSMs) and their interplay with habitat and animal movement properties in terms of statistical hypothesis testing. RESULTS: We demonstrated that only iSSMs and ST-PPMs showed nominal type I error rates in all studied cases, whereas SSMs may slightly and SLRMs may frequently and strongly exceed these levels. iSSMs appeared to have on average a more robust and higher statistical power than ST-PPMs. CONCLUSIONS: Based on our results, we recommend the use of iSSMs to infer habitat selection or large-scale attraction/avoidance from animal tracking data. Further advantages over other approaches include short computation times, predictive capacity, and the possibility of deriving mechanistic movement models.

Migrating curlews on schedule: departure and arrival patterns of a long-distance migrant depend on time and breeding location rather than on wind conditions.

BACKGROUND: Departure decisions in long-distance migratory bird species may depend on favourable weather conditions and beneficial resources at the destination location, overarched by genetic triggers. However, few studies have tried to validate the significance of these three concepts simultaneously, and long-term, high-resolution tagging datasets recording individual movements across consecutive years are scarce. We used such a dataset to explore intraspecific and intra-individual variabilities in departure and arrival decisions from/to wintering grounds in relation to these three different concepts in bird migration. METHODS: We equipped 23 curlews (Numenius arquata) wintering in the Wadden Sea with Global Positioning System data loggers to record their spatio-temporal patterns of departure from and arrival at their wintering site, and the first part of their spring migration. We obtained data for 42 migrations over 6 years, with 12 individuals performing repeat migrations in consecutive years. Day of year of departure and arrival was related to 38 meteorological and bird-related predictors using the least absolute shrinkage and selection operator (LASSO) to identify drivers of departure and arrival decisions. RESULTS: Curlews migrated almost exclusively to Arctic and sub-Arctic Russia for breeding. They left their wintering site mainly during the evening from mid- to late April and returned between the end of June and mid-July. There was no difference in departure times between the sexes. Weather parameters did not impact departure decisions; if departure days coincided with headwind conditions, the birds accounted for this by flying at higher altitudes of up to several kilometres. Curlews breeding further away in areas with late snowmelt departed later. Departures dates varied by only < 4 days in individual curlews tagged over consecutive years. CONCLUSIONS: These results suggest that the trigger for migration in this long-distance migrant is largely independent of weather conditions but is subject to resource availability in breeding areas. The high intra-individual repeatability of departure days among subsequent years and the lack of relationship to weather parameters suggest the importance of genetic triggers in prompting the start of migration. Further insights into the timing of migration in immatures and closely related birds might help to further unravel the genetic mechanisms triggering migration patterns.

Northern gannets (Morus bassanus) are strongly affected by operating offshore wind farms during the breeding season.

Northern gannets (Morus bassanus) have been ranked as one of the most vulnerable species in terms of collision with offshore wind farm (OWF) turbines, and strong avoidance of OWFs has been documented for this species. Gannets increasingly encounter OWFs within the ranges of their largest breeding colonies along the European coasts. However, information on their actual reactions to OWFs during the breeding season is lacking. We investigated the possible effects of OWFs located 23-35 km north of the colony on Helgoland in the southern North Sea on breeding gannets. GPS tags were applied to 28 adult gannets breeding on Helgoland for several weeks over 2 years. Most gannets (89%) predominantly avoided the OWFs in both years, but 11% frequently entered them when foraging or commuting between the colony and foraging areas. Flight heights inside the OWFs were close to the rotor-blade zone, especially for individuals predominantly avoiding the OWFs. Gannets preferred distances of 250-450 m to the turbines when being inside the OWF. A point process modelling approach revealed that the gannets resource selection of the OWF area compared with the surroundings (outside OWF = up to 15 km from the OWF border) was reduced by 21% in 2015 and 37% in 2016. This study provides the first detailed characterisation of individual reactions of gannets to OWFs during the breeding season and one of the first comprehensive studies of OWF effects on this species based on telemetry data. The documented effects need to be considered during the planning processes for future OWFs, especially those located close to large seabird breeding colonies.

Effects of offshore windfarms on seabird abundance: Strong effects in spring and in the breeding season.

The increasing development of offshore wind farms (OWFs) worldwide leads to possible conflicts with the ecological requirements of top predators that largely depend on offshore areas. Seabird species exhibit different behavioural reactions to OWFs, ranging from avoidance resulting in habitat loss, to attraction causing an increased risk of colliding with the turbines. We investigated how OWFs affected the densities and distributions of guillemots and kittiwakes breeding in the southern North Sea and if the effects varied among seasons using a 'before-after control impact' (BACI) analysis approach based on a large-scale and long-term dataset covering 14 years before and 3 years after the construction of OWFs. Guillemot relative density in the OWF decreased by 63% in spring, and by 44% in the breeding season. Kittiwake relative density in the OWF decreased by 45% in the breeding season, and not significantly by 10% in spring. We furthermore estimated the response radii to the OWF for both species and seasons, finding that guillemots showed a response radius of ~9 km in spring and kittiwakes a radius of ~20 km in the breeding season. The results underline the value of large-scale and long-term assessments considering seasonal variation throughout the yearly cycle. The here provided information on the seasonally different reactions of seabirds to OWFs adds substantially to our current knowledge and provides the necessary basis for reliable estimations of OWF effects on guillemots and kittiwakes. Such evaluations are urgently needed for future planning and management recommendations to decision-makers.

Population size estimates based on the frequency of genetically assigned parent-offspring pairs within a subsample.

Estimating population density as precise as possible is a key premise for managing wild animal species. This can be a challenging task if the species in question is elusive or, due to high quantities, hard to count. We present a new, mathematically derived estimator for population size, where the estimation is based solely on the frequency of genetically assigned parent-offspring pairs within a subsample of an ungulate population. By use of molecular markers like microsatellites, the number of these parent-offspring pairs can be determined. The study's aim was to clarify whether a classical capture-mark-recapture (CMR) method can be adapted or extended by this genetic element to a genetic-based capture-mark-recapture (g-CMR). We numerically validate the presented estimator (and corresponding variance estimates) and provide the R-code for the computation of estimates of population size including confidence intervals. The presented method provides a new framework to precisely estimate population size based on the genetic analysis of a one-time subsample. This is especially of value where traditional CMR methods or other DNA-based (fecal or hair) capture-recapture methods fail or are too difficult to apply. The DNA source used is basically irrelevant, but in the present case the sampling of an annual hunting bag is to serve as data basis. In addition to the high quality of muscle tissue samples, hunting bags provide additional and essential information for wildlife management practices, such as age, weight, or sex. In cases where a g-CMR method is ecologically and hunting-wise appropriate, it enables a wide applicability, also through its species-independent use.

Operational offshore wind farms and associated ship traffic cause profound changes in distribution patterns of Loons (Gavia spp.).

Seabirds select suitable habitats at sea, but these habitats may be strongly impacted by marine spatial planning, including the construction of offshore wind farms (OWFs) and the associated ship traffic. Loons (Gavia spp.) are particularly vulnerable to anthropogenic activities and are also of high conservation status, making them particularly relevant to marine planning processes. We investigated the effects of OWF construction and ship traffic on Loon distributions in the German North Sea on a large spatial scale, using a 'before-after' control impact analysis approach and a long-term data set. Many OWFs were built in or close to core areas of Loon distributions. Loons showed significant shifts in their distribution in the 'after' period and subsequently aggregated between two OWF clusters, indicating the remaining suitable habitat. The decrease in Loon abundance became significant as far as about 16 km from the closest OWF. Ship traffic also had a significant negative impact on Loons, indicating that OWFs deterred Loons through the combined effect of ship traffic and the wind turbines themselves. This study provides the first analysis of the extensive effects of OWFs and ships on Loons on a large spatial scale. The results provide an essential baseline for future marine spatial planning processes in the German North Sea and elsewhere.

Decreasing δ13C and δ15N values in four coastal species at different trophic levels indicate a fundamental food-web shift in the southern North and Baltic Seas between 1988 and 2016.

Marine ecosystems are exposed to increasing human pressures and climatic change worldwide. It has therefore become essential to describe ecosystem statuses with respect to multinational protection schemes, often necessitating long-term monitoring programmes. Changes in the food-web structure, which can be monitored via stable isotope measurements, represent an important descriptor of the status of marine ecosystems. We investigated long-term changes (29 years) in isotopic values (δ13C and δ15N) in four indicative organisms at different trophic levels in the southern North and Baltic Seas: bladderwrack (Fucus vesiculosus), blue mussel (Mytilus ssp.), eelpout (Zoarces viviparus), and herring gull (Larus argentatus). Time series analyses using generalised additive models revealed largely consistent declines in δ13C and δ15N throughout all trophic levels of the coastal food web at all study sites, indicating a clear change in these coastal regions from 1988 to 2016. There were no clear long-term patterns in egg biometrics for herring gulls, except for a consistent increase in eggshell thickness. The declines in stable isotope values were in line with the results of previous long-term studies of single higher-trophic-level species, which suggested that the noted changes were mainly caused by altered foraging patterns of the studied species. The current results demonstrate that declines in δ13C and δ15N have occurred throughout the whole food web, not just in particular species. We discuss the possible reasons for the decrease in stable isotope values, including decreasing eutrophication and an increase in terrestrial carbon sources.

Post-Turing tissue pattern formation: Advent of mechanochemistry.

Chemical and mechanical pattern formation is fundamental during embryogenesis and tissue development. Yet, the underlying molecular and cellular mechanisms are still elusive in many cases. Most current theories assume that tissue development is driven by chemical processes: either as a sequence of chemical patterns each depending on the previous one, or by patterns spontaneously arising from specific chemical interactions (such as "Turing-patterns"). Within both theories, mechanical patterns are usually regarded as passive by-products of chemical pre-patters. However, several experiments question these theories, and an increasing number of studies shows that tissue mechanics can actively influence chemical patterns during development. In this study, we thus focus on the interplay between chemical and mechanical processes during tissue development. On one hand, based on recent experimental data, we develop new mechanochemical simulation models of evolving tissues, in which the full 3D representation of the tissue appears to be critical for obtaining a realistic mechanochemical behaviour. The presented modelling approach is flexible and numerically studied using state of the art finite element methods. Thus, it may serve as a basis to combine simulations with new experimental methods in tissue development. On the other hand, we apply the developed approach and demonstrate that even simple interactions between tissue mechanics and chemistry spontaneously lead to robust and complex mechanochemical patterns. Especially, we demonstrate that the main contradictions arising in the framework of purely chemical theories are naturally and automatically resolved using the mechanochemical patterning theory.

Beyond Turing: mechanochemical pattern formation in biological tissues.

BACKGROUND: During embryogenesis, chemical (morphogen) and mechanical patterns develop within tissues in a self-organized way. More than 60 years ago, Turing proposed his famous reaction-diffusion model for such processes, assuming chemical interactions as the main driving force in tissue patterning. However, experimental identification of corresponding molecular candidates is still incomplete. Recent results suggest that beside morphogens, also tissue mechanics play a significant role in these patterning processes. RESULTS: Combining continuous finite strain with discrete cellular tissue models, we present and numerically investigate mechanochemical processes, in which morphogen dynamics and tissue mechanics are coupled by feedback loops. We consider three different mechanical cues involved in such feedbacks: strain, stress, and compression. Based on experimental results, for each case, we present a feedback loop spontaneously creating robust mechanochemical patterns. In contrast to Turing-type models, simple mechanochemical interaction terms are sufficient to create de novo patterns. CONCLUSIONS: Our results emphasize mechanochemical processes as possible candidates controlling different steps of embryogenesis. To motivate further experimental research discovering related mechanisms in living tissues, we also present predictive in silicio experiments. REVIEWERS: Reviewer 1 - Marek Kimmel; Reviewer 2 - Konstantin Doubrovinski (nominated by Ned Wingreen); Reviewer 3 - Jun Allard (nominated by William Hlavacek).