Sox, Fox, and Lmx1b binding sites differentially regulate a Gdf5-Associated regulatory region during elbow development.

Introduction: The articulating ends of limb bones have precise morphology and asymmetry that ensures proper joint function. Growth differentiation factor 5 (Gdf5) is a secreted morphogen involved in cartilage and bone development that contributes to the architecture of developing joints. Dysregulation of Gdf5 results in joint dysmorphogenesis often leading to progressive joint degeneration or osteoarthritis (OA). The transcription factors and cis-regulatory modules (CRMs) that regulate Gdf5 expression are not well characterized. We previously identified a Gdf5-associated regulatory region (GARR) that contains predicted binding sites for Lmx1b, Osr2, Fox, and the Sox transcription factors. These transcription factors are recognized factors involved in joint morphogenesis and skeletal development. Methods: We used in situ hybridization to Gdf5, Col2A1, and the transcription factors of interest in developing chicken limbs to determine potential overlap in expression. We further analyzed scRNA-seq data derived from limbs and knees in published mouse and chicken datasets, identifying cells with coexpression of Gdf5 and the transcription factors of interest. We also performed site-directed mutatgenesis of the predicted transcription factor binding sites in a GARR-reporter construct and determined any change in activity using targeted regional electroporation (TREP) in micromass and embryonic chicken wing bioassays. Results: Gdf5 expression overlapped the expression of these transcription factors during joint development both by in situ hybridization (ISH) and scRNA-seq analyses. Within the GARR CRM, mutation of two binding sites common to Fox and Sox transcripstion factors reduced enhancer activity to background levels in micromass cultures and in ovo embryonic chicken wing bioassays, whereas mutation of two Sox-only binding sites caused a significant increase in activity. These results indicate that the Fox/Sox binding sites are required for activity, while the Sox-only sites are involved in repression of activity. Mutation of Lmx1b binding sites in GARR caused an overall reduction in enhancer activity in vitro and a dorsal reduction in ovo. Despite a recognized role for Osr2 in joint development, disruption of the predicted Osr2 site did not alter GARR activity. Conclusion: Taken together, our data indicates that GARR integrates positive, repressive, and asymmetrical inputs to fine-tune the expression of Gdf5 during elbow joint development.

Regional bedrock geochemistry associated with podoconiosis evaluated by multivariate analysis.

Podoconiosis is a disease whose etiology remains murky. Currently, the disease is attributed to particles that are believed to move through the skin and into the lymphatic system causing swelling of the lower legs. Identity of these particles or their composition remains unclear, though the presence of silicon and/or aluminum is often noted and frequently cited as causal agents. We applied multivariate analyses to the bedrock compositions of a large set of cases from an online database in an effort to identify underlying patterns or combinations of relative element abundances associated with podoconiosis-endemic regions. Using a combination of principal component analysis, discriminant function analysis, and ANOVA, we analyzed ten oxides from five regions on the African continent known to be associated with podoconiosis. The Hawaiian Islands were included as a control group since they are not known to have cases of podoconiosis despite similarity in geology and agricultural practices. Our analyses suggest that a unique alkaline- and silicon-rich geochemistry underlies regions associated with podoconiosis. Our results also imply that minerals enriched in incompatible elements, such as Ca, K, Mg, and Na, may be stronger predictors of the presence of the disease than either silicon or aluminum.

The dilemma of choosing a reference character for measuring sexual size dimorphism, sexual body component dimorphism, and character scaling: cryptic dimorphism and allometry in the scorpion Hadrurus arizonensis.

Sexual differences in morphology, ranging from subtle to extravagant, occur commonly in many animal species. These differences can encompass overall body size (sexual size dimorphism, SSD) or the size and/or shape of specific body parts (sexual body component dimorphism, SBCD). Interacting forces of natural and sexual selection shape much of the expression of dimorphism we see, though non-adaptive processes may be involved. Differential scaling of individual features can result when selection favors either exaggerated (positive allometry) or reduced (negative allometry) size during growth. Studies of sexual dimorphism and character scaling rely on multivariate models that ideally use an unbiased reference character as an overall measure of body size. We explored several candidate reference characters in a cryptically dimorphic taxon, Hadrurus arizonensis. In this scorpion, essentially every body component among the 16 we examined could be interpreted as dimorphic, but identification of SSD and SBCD depended on which character was used as the reference (prosoma length, prosoma area, total length, principal component 1, or metasoma segment 1 width). Of these characters, discriminant function analysis suggested that metasoma segment 1 width was the most appropriate. The pattern of dimorphism in H. arizonensis mirrored that seen in other more obviously dimorphic scorpions, with static allometry trending towards isometry in most characters. Our findings are consistent with the conclusions of others that fecundity selection likely favors a larger prosoma in female scorpions, whereas sexual selection may favor other body parts being larger in males, especially the metasoma, pectines, and possibly the chela. For this scorpion and probably most other organisms, the choice of reference character profoundly affects interpretations of SSD, SBCD, and allometry. Thus, researchers need to broaden their consideration of an appropriate reference and exercise caution in interpreting findings. We highly recommend use of discriminant function analysis to identify the least-biased reference character.

Venom regeneration in the centipede Scolopendra polymorpha: evidence for asynchronous venom component synthesis.

Venom regeneration comprises a vital process in animals that rely on venom for prey capture and defense. Venom regeneration in scolopendromorph centipedes likely influences their ability to subdue prey and defend themselves, and may influence the quantity and quality of venom extracted by researchers investigating the venom's biochemistry. We investigated venom volume and total protein regeneration during the 14-day period subsequent to venom extraction in the North American centipede Scolopendra polymorpha. We further tested the hypothesis that venom protein components, separated by reversed-phase fast protein liquid chromatography (RP-FPLC), undergo asynchronous (non-parallel) synthesis. During the first 48 h, volume and protein mass increased linearly. Protein regeneration lagged behind volume regeneration, with 65­86% of venom volume and 29­47% of protein mass regenerated during the first 2 days. No additional regeneration occurred over the subsequent 12 days, and neither volume nor protein mass reached initial levels 7 months later (93% and 76%, respectively). Centipede body length was negatively associated with rate of venom regeneration. Analysis of chromatograms of individual venom samples revealed that 5 of 10 chromatographic regions and 12 of 28 peaks demonstrated changes in percent of total peak area (i.e., percent of total protein) among milking intervals, indicating that venom proteins are regenerated asynchronously. Moreover, specimens from Arizona and California differed in relative amounts of some venom components. The considerable regeneration of venom occurring within the first 48 h, despite the reduced protein content, suggests that predatory and defensive capacities are minimally constrained by the timing of venom replacement.

Variation in venom yield and protein concentration of the centipedes Scolopendra polymorpha and Scolopendra subspinipes.

Venom generally comprises a complex mixture of compounds representing a non-trivial metabolic expense. Accordingly, natural selection should fine-tune the amount of venom carried within an animal's venom gland(s). The venom supply of scolopendromorph centipedes likely influences their venom use and has implications for the severity of human envenomations, yet we understand very little about their venom yields and the factors influencing them. We investigated how size, specifically body length, influenced volume yield and protein concentration of electrically extracted venom in Scolopendra polymorpha and Scolopendra subspinipes. We also examined additional potential influences on yield in S. polymorpha, including relative forcipule size, relative mass, geographic origin (Arizona vs. California), sex, time in captivity, and milking history. Volume yield was linearly related to body length, and S. subspinipes yielded a larger length-specific volume than S. polymorpha. Body length and protein concentration were uncorrelated. When considering multiple influences on volume yield in S. polymorpha, the most important factor was body length, but yield was also positively associated with relative forcipule length and relative body mass. S. polymorpha from California yielded a greater volume of venom with a higher protein concentration than conspecifics from Arizona, all else being equal. Previously milked animals yielded less venom with a lower protein concentration. For both species, approximately two-thirds of extractable venom was expressed in the first two pulses, with remaining pulses yielding declining amounts, but venom protein concentration did not vary across pulses. Further study is necessary to ascertain the ecological significance of the factors influencing venom yield and how availability may influence venom use.

Poisons, toxungens, and venoms: redefining and classifying toxic biological secretions and the organisms that employ them.

Despite extensive study of poisonous and venomous organisms and the toxins they produce, a review of the literature reveals inconsistency and ambiguity in the definitions of 'poison' and 'venom'. These two terms are frequently conflated with one another, and with the more general term, 'toxin.' We therefore clarify distinctions among three major classes of toxins (biological, environmental, and anthropogenic or man-made), evaluate prior definitions of venom which differentiate it from poison, and propose more rigorous definitions for poison and venom based on differences in mechanism of delivery. We also introduce a new term, 'toxungen', thereby partitioning toxic biological secretions into three categories: poisons lacking a delivery mechanism, i.e. ingested, inhaled, or absorbed across the body surface; toxungens delivered to the body surface without an accompanying wound; and venoms, delivered to internal tissues via creation of a wound. We further propose a system to classify toxic organisms with respect to delivery mechanism (absent versus present), source (autogenous versus heterogenous), and storage of toxins (aglandular versus glandular). As examples, a frog that acquires toxins from its diet, stores the secretion within cutaneous glands, and transfers the secretion upon contact or ingestion would be heteroglandular-poisonous; an ant that produces its own toxins, stores the secretion in a gland, and sprays it for defence would be autoglandular-toxungenous; and an anemone that produces its own toxins within specialized cells that deliver the secretion via a penetrating wound would be autoaglandular-venomous. Adoption of our scheme should benefit our understanding of both proximate and ultimate causes in the evolution of these toxins.