Biology subjects

Shefelbine, S. J.

Publications and source records attributed to Shefelbine, S. J..

10 recordsLinked to original sources

bioRxiv2025

TRPV4 Inhibition Reduces Cartilage Growth During Axolotl Limb Regeneration

BackgroundAxolotls can regenerate entire limbs, recapitulating mammalian developmental processes. During development, mechanosensitive ion channels such as TRPV4, PIEZO1, and PIEZO2 regulate tissue morphogenesis by transducing mechanical signals. Their roles in regeneration, however, have yet to be thoroughly explored. To investigate this, we assessed the expression of these channels during limb regeneration using single-cell RNA sequencing dataset, hybridization chain reaction fluorescence in-situ hybridization, and immunofluorescence. Additionally, functional relevance was tested by pharmacological inhibition of TRPV4 and PIEZO1/2 mechanosensitive ion channels during limb regeneration. ResultsWhile PIEZO1 expression was undetected, we observed TRPV4 and PIEZO2 expression in uninjured cartilage and at both the mid-bud and palette blastemal stages of limb regeneration. TRPV4 and PIEZO2 were highly expressed in chondrocytes, with PIEZO2 enriched in fibroblasts. Inhibition with GSK205, a TRPV4 antagonist, significantly reduced calcium influx and humeral length without inhibiting cartilage differentiation. Treatment with gadolinium chloride, a broad spectrum mechanosensitive ion channel inhibitor, had no significant morphological impact. ConclusionsTRPV4 and PIEZO2 are dynamically regulated during axolotl limb regeneration. Selective TRPV4 inhibition altered final limb morphology, but chondrogenesis was unaffected. This suggests a role for these genes in shaping tissue architecture during limb regeneration. These findings underscore the importance of ion channel-mediated mechanotransduction in regenerative patterning. Key findingsO_LITRPV4 and PIEZO2 show distinct, stage- and cell type-specific expression patterns during axolotl limb regeneration C_LIO_LISelective pharmacological inhibition of TRPV4 significantly reduces calcium signaling in chondrocytes and leads to shorter limb and humeral lengths in vivo C_LI

bioengineering↗

bioRxiv2025

Growth and patterning in vertebrate limb developmentA timescale perspective on skeletal specification

1The vertebrate limb provides a powerful system to study how growth and molecular signaling interact to shape complex skeletal patterns. However, how these processes are coordinated across space and time is not fully understood. This study introduces a computational tool to examine how growth interacts with positional cues and self-organizing patterning mechanisms to shape skeletal structures in both mice and axolotl limbs. We developed the Growth-Processing-Propagation (GPP) framework, a reaction-diffusion system within a growing domain, in which the relative contribution of growth, processing (reaction) and propagation (diffusion) is modulated through two non-dimensional parameters, informed by experimental morphogen maps. This formulation normalizes the reaction-diffusion equation relative to growth, enabling investigation of how different spatiotemporal regimes of growth, processing and propagation interact to produce whole limb patterning. The GPP framework captures the progressive formation of limb segments and digit patterns by varying the relative contributions of reaction, diffusion, and growth to the pattern. Our models indicate that in the proximal region (humerus, radius/ulna) the contributions of growth, reaction and diffusion are equally important to patterning, but in the distal elements (hands) the reaction and diffusion contributions are much greater than the contribution of growth to formation of the digits. A single framework predicts whole-limb skeletal patterns in both mice and axolotls, despite their morphological differences, highlighting its potential to explore conserved and divergent features of limb development from an evolutionary perspective through a unified mechanism across species. O_TEXTBOXSignificanceUnderstanding limb skeletal patterning is a central question in developmental biology. Numerical models provide a means to explore this process. We introduce the Growth-Processing-Propagation (GPP) framework, which integrates tissue expansion (growth), gene regulation and cellular specification (processing), and signaling spread (propagation). By normalizing patterning dynamics relative to growth, the framework reveals how the relative contributions of growth, processing, and propagation vary across the limb, influencing the timing and positioning of skeletal elements. The model successfully predicts whole-limb skeletal patterning in both mice and axolotls, supporting the idea of a shared underlying mechanism. This cross-species framework offers a versatile computational tool for studying fundamental processes in morphogenesis, with potential applications in developmental biology, and evolutionary studies. C_TEXTBOX

developmental biology↗

bioRxiv2025

Combined effects of mechanical loading and Piezo1 chemical activation on 22-months-old female mouse bone adaptation

With age, bones mechanosensitivity is reduced, which limits their ability to adapt to loading. The exact mechanism leading to this loss of mechanosensitvity is still unclear, making developing effective treatment challenging. Current treatments mostly focus on preventing bone mass loss (such as bisphosphonates) or promoting bone formation (such as Sclerostin inhibitors) to limit the decline of bones mass. However, treatments do not target the cause of bone mass loss which may be, in part, due to the bones inability to initiate a normal bone mechanapdatation response. In this work, we investigated the effects of 2 weeks of tibia loading, and Piezo1 agonist injection in vivo on 22-month-old mouse bone adaptation response. We used an optimized loading profile, which induced high fluid flow velocity and low strain magnitude in adult mouse tibia. We found that tibia loading and Yoda2 injection have an additive effect on increasing cortical bone parameters in 22-month-old mice. This combination of mechanical and chemical stimulation could be a promising treatment strategy to help promote bone formation in patients who have low bone mass due to aging.

bioengineering↗

bioRxiv2024

3D spatial distribution of Sost mRNA and Sclerostin expression in response to in vivo mechanical loading

Bones adapt to external mechanical loads through a process known as mechanoadaptation. Osteocytes are the bone cells that sense the mechanical environment and initiate a biological response. Investigating the changes in osteocyte molecular expression following mechanical loading has been instrumental in characterizing the regulatory pathways involved in bone adaptation. However, current methods for examining osteocyte molecular expression do not preserve the three-dimensional structure of the bone, which plays a critical role in the mechanical stimuli sensed by the osteocytes and their spatially controlled biological responses. In this study, we used WISH-BONE to investigate the spatial distribution of Sost-mRNA transcripts and its encoded protein, sclerostin, in 3D mouse tibia midshaft following in vivo tibia loading. Our findings showed a decrease in the percentage of Sost-positive osteocytes predominantly at 25% and 37% of the bone length, and in the posterior-lateral side of the tibia after loading. Sclerostin-positive osteocytes in the loaded legs were found to be similar to the contralateral legs after 2 weeks of loading. This work is the first to provide a 3D analysis of Sost and sclerostin distribution in loaded versus contralateral mouse tibia midshafts. It also highlights the importance of the bone region analyzed and the method utilized when interpreting mechanoadaptation results. WISH-BONE represents a powerful tool for further characterization of mechanosensitive genes regulation in bone and holds potential for advancing the development of new treatments targeting mechanosensitivity-related bone disorders.

bioengineering↗

bioRxiv2024

WISH-BONE: Whole-mount In Situ Histology, to label osteocyte mRNA and protein in 3D adult mouse bones

Bone is a three dimensional and highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or only provide two-dimensional spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH-BONE, to label osteocyte mRNA and protein in whole-mount mouse bone. For mRNA labeling, we used Hybridization Chain Reaction - Fluorescence In Situ Hybridization to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy-based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone. However, the relatively dim signal observed from nanobodies staining limited detection. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole-mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH-BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone-related fields of research.

bioengineering↗

bioRxiv2023

Turing pattern prediction in three-dimensional domains: the role of initial conditions and growth

Reaction-diffusion systems have been widely used to model pattern formation in biological systems. However, the emergence of Turing patterns in three-dimensional (3D) domains remains relatively unexplored. A few studies on this topic have shown that extending pattern formation from 2D to 3D is not straightforward. Linear stability analysis, which is commonly used to associate admissible wave modes with predicted patterns in 1D and 2D, has yet to be applied in 3D. We have used this approach, together with finite element modelling of a Turing system with Schnakenberg kinetics, to investigate the effects of initial conditions and growing domains on the competition between admissible modes in 3D Turing pattern emergence. Our results reveal that non-random initial conditions on the activator play a stronger role than those of the inhibitor. We also observe a path dependency of the evolving pattern within a growing domain. Our findings shed new light on the mechanisms ensuring reliable pattern formation in 3D domains and have important implications for the development of more robust models of morphogen patterning in developmental processes.

developmental biology↗

bioRxiv2022

Lamellar Thickness Measurements in Normal and Osteogenesis Imperfecta Human Bone, with development of a method of automated thickness averaging to simplify quantitation.

PurposeLamellar bone that forms in moderate and severe osteogenesis imperfecta (OI) is often composed of structurally irregular lamellae compared to those in normal bone. Polarization light microscopy (PLM) demonstrates lamellar bone well but has rarely been used for quantitative studies; information available on normal bone lamellae tends to be variable and studies specifically assessing OI bone lamellae have not been done. We report on PLM histomorphometry quantifying bright and dark lamellar thicknesses in normal and OI bone. Manual measurements of individual lamellar thicknesses have been made on histologic sections using the cellSens image analysis system; in an effort to augment the number of measurements we also developed a method of automated thickness averaging in quantifying regions of lamellae. MethodsFemoral and tibial cortical bone fragments from 5 individuals 5 - 26 years old (without molecular bone disorders) and 8 individuals 5 - 16 years old with progressively deforming (Sillence III) OI were obtained. The fragments were decalcified, infiltrated in JB4 solution, embedded in JB4 plastic, sectioned at 5 thickness and stained with 1% toluidine blue for light and polarizing microscopy. Manual measurements: Strict criteria for measurement, primarily to eliminate oblique lamellae, included accumulations of 16-20 bright and dark lamellae under PLM with a relatively narrow range of thicknesses, flattened elliptical osteocytes along the longitudinal axis of the lamellae and canaliculi passing from the walls of the osteocyte lacunae at right angles to the lamellae. Histomorphometric measurements of bright and dark lamellae by PLM were made at 20X magnification. Automated measurements: A script for automated measurement of average lamellar thicknesses from PLM images was developed in MATLAB (Mathworks, Natick, MA) to make measurement faster and less subjective. The script isolates a region from an image for measurement and marks each pixel as either bright or dark based on a local average intensity threshold. It then takes multiple pixel measurements along the length of the lamellae in the image and returns the average thickness of each in m. Results1. OI bone mean lamellar thickness values are always less than those in normal bone. The mean value for all OI bright and dark lamellae combined is 1.80 {+/-} 0.72 m and the value in normal bone is 2.54 {+/-} 0.92 m. 2. Mean value for the bright lamellae is less than that for the dark lamellae in both normal and OI bone. The mean value for bright lamellae in OI is 1.47 {+/-} 0.53 m and for dark lamellae 2.18 {+/-} 0.72 m; in normal bone the mean value for bright lamellae is 2.06 {+/-} 0.54 m and for dark lamellae 3.07 {+/-} 0.96 m. The differences are statistically significant: between groups of normal and OI lamellae (p<0.001), normal and OI light bands (p<0.001), and normal and OI dark bands (p<0.001). 3. Ratio of mean values for bright/dark lamellar thicknesses is the same in OI and normal bone. The ratio in OI bone is 0.67 (range: 0.54 - 0.83) and in normal bone 0.67 (range: 0.60 - 0.88). 4. Validation of automated vs. manual datasets: For each lamella in the validation dataset, the percent difference between the automated and manual measurements was calculated. The mean of the absolute values of these percent differences was 18.9%, a statistically non-significant difference (p = 0.0518). Discussion and conclusionsLamellar bone that forms in moderate and severe OI is composed of thinner and less regular lamellae than those in normal bone. i) PLM histomorphometry shows mean lamellar thicknesses (bright and dark merged) are statistically significantly decreased in OI compared to normal bone as are bright and dark lamellar thicknesses measured independently. ii) The automated method can be adapted readily to the assessment process for lamellar thicknesses and is, most likely, more accurate since it averages a greatly increased number of measurements per individual lamella. iii) Lamellar thickness measurements can be helpful in assessing the effect of specific collagen mutations on OI bone synthesis and warrant inclusion in both research and clinical histomorphometric assessments.

bioengineering↗

bioRxiv2021

Local mechanical stimuli shape tissue growth in vertebrate joint morphogenesis

The correct formation of synovial joints is essential for proper motion throughout life. Movement-induced forces are critical to creating correctly shaped joints, but it is unclear how cells sense and respond to these mechanical cues. To determine how mechanical stimuli drive joint morphogenesis, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a TRPV4 agonist to impair chondrocyte mechanosensitivity during joint morphogenesis. We quantified growth and shape in regrown humeri from whole mount light sheet fluorescence images of the regenerated limbs. Results revealed statistically significant differences in morphology and cell proliferation between groups, indicating that mechanical stimuli play a role in the shaping of joints. We simulated local tissue growth in a finite element model with a biological contribution to growth proportional to chondrocyte density, and a mechanical contribution to growth proportional to fluid pressure. Computational predictions agreed with experimental outcomes, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

developmental biology↗

bioRxiv2020

3D Visualization of Macromolecule Synthesis

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here, we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

developmental biology↗

bioRxiv2018

Limb bone scaling in hopping diprotodonts and quadrupedal artiodactyls

Bone adaptation is modulated by the timing, direction, rate, and magnitude of mechanical loads. To investigate whether frequent slow, or infrequent fast, gaits could dominate bone adaptation to load, we compared scaling of the limb bones from two mammalian herbivore clades that use radically different high-speed gaits, bipedal hopping (suborder Macropodiformes; kangaroos and kin) and quadrupedal galloping (order Artiodactyla; goats, deer and kin). Forelimb and hindlimb bones were collected from 20 artiodactyl and 15 macropod species (body mass M 1.05 - 1536 kg) and scanned in computed tomography or X- ray microtomography. Second moment of area (Imax) and bone length (l) were measured. Scaling relations (y = axb) were calculated for l vs M for each bone and for Imax vs M and Imax vs l for every 5% of length. Imax vs M scaling relationships were broadly similar between clades despite the macropod forelimb being nearly unloaded, and the hindlimb highly loaded, during bipedal hopping. Imax vs l and l vs M scaling were related to locomotor and behavioural specialisations. Low-intensity loads may be sufficient to maintain bone mass across a wide range of species. Occasional high-intensity gaits might not break through the load sensitivity saturation engendered by frequent low-intensity gaits.

evolutionary biology↗