Bone is a connective tissue composed of many cell types, including osteoblasts. How bones acquire their unique size and shape during development remains poorly understood. Herein we investigated the ...molecular and cellular mechanisms of bone morphogenesis in the zebrafish scale by using transgenic lines to enable visualization of specific types of osteoblasts. We demonstrate that the zebrafish scale contains three distinct types of osteoblasts: (i) a monolayer of central osteoblasts along the inner surface of scales; (ii) marginal osteoblasts elongated along the scale edge; and (iii) submarginal osteoblasts located between the central and marginal osteoblast populations. The size of the central osteoblasts increases progressively during development, suggesting that scale growth is mediated primarily by cell growth rather than the recruitment of new osteoblasts. In addition, the total number of central osteoblasts increases in regenerated scales and is correlated with scale size, possibly allowing for the rapid growth of regenerating scales. Moreover, osteoblast proliferation is not detected during regeneration, suggesting that the osteoblasts originate from post-mitotic precursor cells. Sonic hedgehog a (shha) is expressed in the epidermal cells that make contact with the marginal osteoblasts. Pharmacological inhibition of Hedgehog (Hh) signaling during regeneration reduces the number of marginal osteoblasts and interferes with scale growth, indicating that epidermis-derived Shh regulates scale regeneration. Finally, genetic inhibition of Wnt/planar cell polarity (PCP) signaling in the epidermis results in misorientation of scales with regard to the body axis. These results reveal a novel role for the epidermis in the regulation of bone patterning, namely the regeneration of osteoblasts and directional bone growth.
•The zebrafish scale contains three distinct types of osteoblasts.•Central osteoblasts increase in size during scale growth.•The total number of regenerated osteoblasts is correlated with scale size.•Epidermis-derived shha is required for the regeneration of osteoblasts.•Wnt/PCP signaling in the epidermis is required for directional scale growth.
Spear-like collagen complexes, known as actinotrichia, underlie the epidermal cell layer in the tip of teleost fins and are known to contribute toward fin formation; however, their specific role ...remains largely unclear. In this study, we investigated of actinotrichia in the role of caudal fin formation by generating collagen9a1c (col9a1c)-knockout zebrafish. Although actinotrichia were initially produced normally and aligned correctly in the knockout fish, the number of actinotrichia decreased as the fish grew and their alignment became disordered. Simultaneously, the fin tip gradually shortened in the dorsal-ventral direction and the entire fin became oval-shaped, while the fin-rays rarely bifurcated and instead underwent fusion, suggesting that actinotrichia are essential for spreading fins dorsoventrally. Furthermore, the epithelial cells that are usually thinly spread in normal fish became spherical in the knockout fish, reducing the area covered by each cell and thus the area of the fin tip. Together, these findings suggest that the tight alignment of actinotrichia provides physical support in the dorsal-ventral direction that allows caudal fins to expand in a triangular-shape.
•The physical role of collagen fibers called actinotrichia in caudal fin shape formation was investigated.•Actinotrichia are arranged in parallel to form a two-dimensional scaffold.•When the scaffold is lost, an extreme contraction of epithelial cells occurs.•The contraction of the epithelial cells is thought to shorten the fins along dorsal-ventral axis.
Differentiation of osteoclasts (OCs) from hematopoietic cells requires cellular interaction with osteoblasts (OBs). Due to the difficulty of live-imaging in the bone, however, the cellular and ...molecular mechanisms underlying intercellular communication involved in OC differentiation are still elusive. Here, we develop a fracture healing model using the scale of trap:GFP; osterix:mCherry transgenic zebrafish to visualize the interaction between OCs and OBs. Transplantation assays followed by flow cytometric analysis reveal that most trap:GFP
OCs in the fractured scale are detected in the osterix:mCherry
fraction because of uptake of OB-derived extracellular vesicles (EVs). In vivo live-imaging shows that immature OCs actively interact with osterix:mCherry
OBs and engulf EVs prior to convergence at the fracture site. In vitro cell culture assays show that OB-derived EVs promote OC differentiation via Rankl signaling. Collectively, these data suggest that EV-mediated intercellular communication with OBs plays an important role in the differentiation of OCs in bone tissue.
Type I collagen plays a pivotal role in shaping bone morphology and determining its physical properties by serving as a template for ossification. Nevertheless, the mechanisms underlying bone ...collagen formation, particularly the principles governing its orientation, remain unknown owing to the lack of a method that enables continuous in vivo observations. To address this challenge, we constructed a method to visualize bone collagen by tagging with green fluorescent protein (GFP) in zebrafish and observed the interactions between osteoblasts and collagen fibers during bone formation in vivo. When collagen type I alpha 2 chain (Col1a2)-GFP was expressed under the control of the osteoblast-specific promoters osx or osc in zebrafish, bone collagen was observed clearly enough to identify its localization, whereas collagen from other organs was not. Therefore, we determined that this method was of sufficient quality for the detailed in vivo observation of bone collagen. Next, bone collagen in the scales, fin rays, and opercular bones of zebrafish was observed in detail, when bone formation is more active. High-magnification imaging showed that Col1a2-GFP can visualize collagen sufficiently to analyze the collagen fiber orientation and microstructure of bones.
Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of the expression promoter. Both promoters (osx and osc) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within the bone were exclusive, with osx region localizing mainly to the outer edge of the bone and osc region localizing to the central area of the bone. This suggests the existence of distinct osteoblast subpopulations with different gene expression profiles, each of which may play a unique role in osteogenesis.
These findings would contribute to a better understanding of the mechanisms governing bone collagen formation by osteoblasts.
•Col1a2-GFP expressed in osteoblasts specifically accumulates in zebrafish bones.•Col1a2-GFP in bones can clearly visualize bone collagen fibers.•Visualization of bone collagen fibers by Col1a2-GFP enables observation of dynamics in the bone growth process.
Stress has been shown to affect brain activity and exert potent and complex modulatory effects on pain. Several behavioral tests have shown that acute stress produces hyperalgesia, depending on the ...stress conditions. In the present study, we investigated the effects of single restraint stress on the tactile threshold and anxiety sensitivity in mice. Mice were evaluated for the tactile threshold using von Frey filaments and for anxiety sensitivity using the elevated plus maze (EPM) test. Tactile thresholds were lowered by both 2 and 4 hour of restraint stress, but anxiety‐like behaviors were observed only after 4 hour of restraint stress in the EPM test. In addition, we found that alfaxalone, which is a positive allosteric modulator of the γ‐aminobutyric acid (GABA)A receptor, prevented restraint stress‐induced hyperalgesia‐like and anxiety‐like behaviors. These results indicate that GABAergic function appears to be critical in the regulation of physical stress‐induced hyperalgesia and anxiety.
Alfaxalone (3 mg/kg, i.p.) reversed the effects of restraint stress, as shown by a significant increase in both the tactile threshold and the percentage of time spent in the open arms of the EPM test.
Fibrous collagen imparts physical strength and flexibility to tissues by forming huge complexes. The density and orientation of collagen fibers must be correctly specified for the optimal physical ...property of the collagen complex. However, little is known about its underlying cellular mechanisms. Actinotrichia are collagen fibers aligned at the fin-tip of bony fish and are easily visible under the microscope due to their thick, linear structure. We used the actinotrichia as a model system to investigate how cells manipulate collagen fibers. The 3D image obtained by focused ion beam scanning electron microscopy (FIB-SEM) showed that the pseudopodia of mesenchymal cells encircle the multiple actinotrichia. We then co-incubated the mesenchymal cells and actinotrichia in vitro, and time-lapse analysis revealed how cells use pseudopods to align collagen fiber orientation. This in vitro behavior is dependent on actin polymerization in mesenchymal cells. Inhibition of actin polymerization in mesenchymal cells results in mis-orientation of actinotrichia in the fin. These results reveal how mesenchymal cells are involved in fin formation and have important implications for the physical interaction between cells and collagen fibers.
We herein describe the case of a 27‐day‐old male infant who was brought to the emergency room for intermittent crying, and swelling of the left scrotum. Based on the clinical findings, necrotizing ...fasciitis was suspected, and surgical intervention was successfully completed within a few hours of admission. Streptococcus agalactiae type Ia was cultured from the drained abscess, and was considered the causative pathogen. To our knowledge, this is the first report of neonatal necrotizing fasciitis caused by S. agalactiae. Prompt diagnosis and immediate surgical debridement are crucial in the initial management of this disease.
Many animals exhibit stereotypical left–right (LR) asymmetry in their internal organs. The mechanisms of LR axis formation required for the subsequent LR asymmetric development are well understood, ...especially in some vertebrates. However, the molecular mechanisms underlying LR asymmetric morphogenesis, particularly how mechanical force is integrated into the LR asymmetric morphogenesis of organs, are poorly understood. Here, we identified
zipper (
zip), encoding a
Drosophila non-muscle myosin II (myosin II) heavy chain, as a gene required for LR asymmetric development of the embryonic anterior midgut (AMG). Myosin II is known to directly generate mechanical force in various types of cells during morphogenesis and cell migration. We found that myosin II was involved in two events in the LR asymmetric development of the AMG. First, it introduced an LR bias to the directional position of circular visceral muscle (CVMU) cells, which externally cover the midgut epithelium. Second, it was required for the LR-biased rotation of the AMG. Our results suggest that myosin II in CVMU cells plays a crucial role in generating the force leading to LR asymmetric morphogenesis. Taken together with previous studies in vertebrates, the involvement of myosin II in LR asymmetric morphogenesis might be conserved evolutionarily.
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