Dynamics of actinotrichia, fibrous collagen structures in zebrafish fin tissues, unveiled by novel fluorescent probes.

Collagen fibers provide physical support to animal tissues by orienting in the correct position and at optimal density. Actinotrichia are thick collagen fibers that are present at the tips of fish fins and serve as scaffolds for bone formation. The arrangement and density of actinotrichia must be constantly maintained with a high degree of regularity to form spatial patterns in the fin bones, but the mechanisms of this process are largely unknown. To address this issue, we first identified two fluorescent probes that can stain actinotrichia clearly in vivo. Using these probes and time-lapse observation of actinotrichia synthesized at different growth stages, we revealed the following previously unknown dynamics of actinotrichia. (i) Actinotrichia do not stay stationary at the place where they are produced; instead, they move towards the dorsal area during the notochord bending and (ii) move towards the distal tip during the fin growth. (iii) Actinotrichia elongate asymmetrically as new collagen is added at the proximal side. (iv) Density is maintained by the insertion of new actinotrichia. (v) Actinotrichia are selectively degraded by osteoclasts. These findings suggest that the regular arrangement of actinotrichia is the outcome of multiple dynamic processes.

Histological Observation of Helmet Development in the Treehopper Poppea capricornis (Insecta: Hemiptera: Membracidae).

The treehoppers (Hemiptera, Membracidae) are known for possessing a large three-dimensional structure called a helmet. Although some ecological functions of the helmet have already been elucidated, the developmental mechanisms underlying the complex and diverse morphology of the helmet are still largely unknown. The process of helmet formation was first described in Antianthe expansa, which possesses a simple roof-shaped helmet. However, the developmental process in species with more complex helmet morphologies remains largely unexplored. Hence, in this study, we used Poppea capricornis, which possesses a more complex helmet structure than A. expansa, to investigate the helmet development using paraffin sections, micro-CT, and scanning electronic microscopy. Our focus was on the overall helmet developmental process common to both species and formation of structures unique to Poppea and its comparison to Antianthe. As a result, we discovered that miniature structures were also formed in Poppea, similar to Antianthe, during the helmet formation. Common structures that were shared between the two species were discernible at this stage. Additionally, we observed that suprahumeral horns and posterior horns, two morphological traits specific to the Poppea helmet that are apparently similar anatomically, are formed through two distinctly different developmental mechanisms. The suprahumeral horns appeared to be formed by utilizing the nymphal suprahumeral bud as a mold, while we could not detect any nymphal structures potentially used for a mold in the posterior horns formation. Our findings suggest that the helmet formation mechanisms of Antianthe and Poppea employ a common mechanism but form species-specific structures by multiple mechanisms.

Adhesion and shrinkage transform the rounded pupal horn into an angular adult horn in Japanese rhinoceros beetle.

Clarifying the mechanisms underlying shape alterations during insect metamorphosis is important for understanding exoskeletal morphogenesis. The large horn of the Japanese rhinoceros beetle Trypoxylus dichotomus is the result of drastic metamorphosis, wherein it appears as a rounded shape during pupation and then undergoes remodeling into an angular adult shape. However, the mechanical mechanisms underlying this remodeling process remain unknown. In this study, we investigated the remodeling mechanisms of the Japanese rhinoceros beetle horn by developing a physical simulation. We identified three factors contributing to remodeling by biological experiments - ventral adhesion, uneven shrinkage, and volume reduction - which were demonstrated to be crucial for transformation using a physical simulation. Furthermore, we corroborated our findings by applying the simulation to the mandibular remodeling of stag beetles. These results indicated that physical simulation applies to pupal remodeling in other beetles, and the morphogenic mechanism could explain various exoskeletal shapes.

In vivo imaging of bone collagen dynamics in zebrafish.

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.

Lateral bone ridge expansion and internal tissue replacement for vertebral body growth in Pacific bluefin tuna Thunnus orientalis.

Vertebral growth is an essential developmental process to support the expansion of the vertebrate body. In teleosts, the lateral side of the vertebral bodies develops to form different structures among species in the late stages of vertebral growth, although lateral structures are not apparent in the early stages. Lateral structures are one of the structural features that determine the diversity of teleost vertebrae. However, explanations for the formation of lateral structures are conflicting because few reports have investigated the growth of teleost vertebral bodies. To clarify the growth process, we analyzed the morphological changes in the vertebral body of Pacific bluefin tuna Thunnus orientalis at different developmental stages using micro-computed tomography (CT) scans. The micro-CT scans showed that the vertebral centrum formed a plate-like ridge on the lateral side along the cranial-caudal direction and extended laterally with increasing thickness. Simultaneously, the proximal region of the lateral ridges became porous as the vertebrae grew to form bone marrow cavities. Furthermore, we used histological observations to describe the relationship between these morphological changes and osteoblast and osteoclast activities. Osteoblasts accumulated on the distal edges of the lateral ridges, whereas osteoclasts were distributed in the bone marrow cavities. These observations suggest that bone resorption occurs proximally to form bone marrow cavities in addition to bone synthesis at the edges of the lateral ridges. The bone marrow cavities were occupied by blood vessels, extracellular matrix, and adipocytes, and the internal tissue composition changed to increase the area of adipose tissue. Because the ratio of bone volume decreases in large vertebrae, bone formation and resorption are regulated to separate the external cortical and internal trabecular bones to support the vertebrae. This study is the first to report the formation of lateral structures and can be applied to similar lateral structures in the vertebrae of other teleost species.

A Case of Syphilis Suspected Preoperatively as a Case of Tongue Cancer.

Syphilis is a sexually transmitted disease caused by Treponema pallidum (TP). We report a case of syphilis that was initially suspected as tongue cancer. An 86-year-old man consulted a neighborhood clinic with an approximately one-month history of pain in the right tongue. The result of scraping cytology of the tongue performed at the clinic was classified as class V, squamous cell carcinoma, and the patient was referred to our hospital. Physical examination revealed a mass on the right side of the tongue and a firm cervical mass. Biopsy revealed no evidence of malignancy; however, the imaging findings led to the suspicion of tongue cancer and lymph node metastasis. The results of blood examination revealed that the patient had syphilis, but since the patient showed few other symptoms, we decided to treat the infection after the planned surgery. We performed right partial glossectomy and neck dissection; however, the postoperative histopathology revealed no evidence of malignancy but nonspecific inflammatory changes with TP spirochetes. The incidence of syphilis has increased dramatically around the world, including Japan, during the last 20 years, and it no longer remains a rare disease. Therefore, syphilis should be included in the differential diagnosis of oral or cervical masses.

Complement factor D targeting protects endotheliopathy in organoid and monkey models of COVID-19.

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes.

Butterfly wing color made of pigmented liquid.

A previously undescribed mechanism underlying butterfly wing coloration patterns was discovered in two distantly related butterfly species, Siproeta stelenes and Philaethria diatonica. These butterflies have bright green wings, but the color pattern is not derived from solid pigments or nanostructures of the scales or from the color of the cuticular membrane but rather from a liquid retained in the wing membrane. Wing structure differs between the green and non-green areas. In the non-green region, the upper and lower cuticular membranes are attached to each other, whereas in the green region, we observed a space of 5-10 µm where green liquid is held and living cells are present. A pigment analysis and tracer experiment revealed that the color of the liquid is derived from hemolymph components, bilin and carotenoid pigments. This discovery broadens our understanding of the diverse ways in which butterfly wings obtain their coloration and patterns.

Collagen9a1c localizes to collagen fibers called actinotrichia in zebrafish fins.

Teleost fish fins are supported by spear-shaped collagen crystals called actinotrichia. Actinotrichia are distributed radially at the distal end of the fins and thought to be necessary for proper formation of the fin and fin-bones. We previously reported that collagen9a1c ( col9a1c ) gene product is essential for the regular arrangement of actinotrichia using col9a1c -knockout zebrafish. Here, we examined the localization pattern of the EGFP-tagged Col9a1c protein in the fins to understand its role in the arrangement of actinotrichia. We found that EGFP-Col9a1c specifically localizes to actinotrichia.

Piezo1 mutant zebrafish as a model of idiopathic scoliosis.

Scoliosis is a condition where the spine curves sideways, unique to humans due to their upright posture. However, the cause of this disease is not well understood because it is challenging to find a model for experimentation. This study aimed to create a model for human idiopathic scoliosis by manipulating the function of mechanosensitive channels called Piezo channels in zebrafish. Zebrafish were chosen because they experience similar biomechanical forces to humans, particularly in relation to the role of mechanical force in scoliosis progression. Here we describe piezo1 and piezo2a are involved in bone formation, with a double knockout resulting in congenital systemic malformations. However, an in-frame mutation of piezo1 led to fully penetrant juvenile-onset scoliosis, bone asymmetry, reduced tissue mineral density, and abnormal intervertebral discs-resembling non-congenital scoliosis symptoms in humans. These findings suggest that functional Piezo channels responding to mechanical forces are crucial for bone formation and maintaining spine integrity, providing insights into skeletal disorders.

The present and future of Turing models in developmental biology.

The Turing model (or reaction-diffusion model), first published in 1952, is a mathematical model that can account for autonomy in the morphogenesis of organisms. Although initially controversial, the model has gradually gained wider acceptance among experimental embryologists due to the accumulation of experimental data to support it. More recently, this model and others based on it have been used not only to explain biological phenomena conceptually but also as working hypotheses for molecular-level experiments and as internal components of more-complex 3D models. In this Spotlight, I will provide a personal perspective from an experimental biologist on some of the recent developments of the Turing model.

Mechanical role of actinotrichia in shaping the caudal fin of zebrafish.

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.

Studies of Turing pattern formation in zebrafish skin.

Skin patterns are the first example of the existence of Turing patterns in living organisms. Extensive research on zebrafish, a model organism with stripes on its skin, has revealed the principles of pattern formation at the molecular and cellular levels. Surprisingly, although the networks of cell-cell interactions have been observed to satisfy the 'short-range activation and long-range inhibition' prerequisites for Turing pattern formation, numerous individual reactions were not envisioned based on the classical reaction-diffusion model. For example, in real skin, it is not an alteration in concentrations of chemicals, but autonomous migration and proliferation of pigment cells that establish patterns, and cell-cell interactions are mediated via direct contact through cell protrusions. Therefore, the classical reaction-diffusion mechanism cannot be used as it is for modelling skin pattern formation. Various studies are underway to adapt mathematical models to the experimental findings on research into skin patterns, and the purpose of this review is to organize and present them. These novel theoretical methods could be applied to autonomous pattern formation phenomena other than skin patterns. This article is part of the theme issue 'Recent progress and open frontiers in Turing's theory of morphogenesis'.

Discovery of Soticlestat, a Potent and Selective Inhibitor for Cholesterol 24-Hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1), a brain-specific cytochrome P450 (CYP) family enzyme, plays a role in the homeostasis of brain cholesterol by converting cholesterol to 24S-hydroxycholesterol (24HC). Despite a wide range of potential of CH24H as a drug target, no potent and selective inhibitors have been identified. Here, we report on the structure-based drug design (SBDD) of novel 4-arylpyridine derivatives based on the X-ray co-crystal structure of hit derivative 1b. Optimization of 4-arylpyridine derivatives led us to identify 3v ((4-benzyl-4-hydroxypiperidin-1-yl)(2,4'-bipyridin-3-yl)methanone, IC50 = 7.4 nM) as a highly potent, selective, and brain-penetrant CH24H inhibitor. Following oral administration to mice, 3v resulted in a dose-dependent reduction of 24HC levels in the brain (1, 3, and 10 mg/kg). Compound 3v (soticlestat, also known as TAK-935) is currently under clinical investigation for the treatment of Dravet syndrome and Lennox-Gastaut syndrome as a novel drug class for epilepsies.

Pivot burrowing of scarab beetle (Trypoxylus dichotomus) larva.

Many organisms live in the soil but only a little is known about their ecology especially movement style. Scarab beetle larvae do not have appendages to shovel soil and their trunk is thick compared to their body length. Hence, their movement through the soil is perplexing. Here, we established the observation and analysis system of larval movement and found that the last larval instars of Trypoxylus dichotomus burrow in two different ways, depending on the hardness of the soil. If the soil is soft, the larvae keep their body in a straight line and use longitudinal expansion and contraction; if the soil is hard, they flex and rotate their body. It is thought that the larvae adapt to diverse soil conditions using two different excavation methods. These results are important for understanding the soil ecology and pose a challenge to engineer of newer excavation technology.

Three-dimensional topology optimization model to simulate the external shapes of bone.

Elucidation of the mechanism by which the shape of bones is formed is essential for understanding vertebrate development. Bones support the body of vertebrates by withstanding external loads, such as those imposed by gravity and muscle tension. Many studies have reported that bone formation varies in response to external loads. An increased external load induces bone synthesis, whereas a decreased external load induces bone resorption. This relationship led to the hypothesis that bone shape adapts to external load. In fact, by simulating this relationship through topology optimization, the internal trabecular structure of bones can be successfully reproduced, thereby facilitating the study of bone diseases. In contrast, there have been few attempts to simulate the external structure of bones, which determines vertebrate morphology. However, the external shape of bones may be reproduced through topology optimization because cells of the same type form both the internal and external structures of bones. Here, we constructed a three-dimensional topology optimization model to attempt the reproduction of the external shape of teleost vertebrae. In teleosts, the internal structure of the vertebral bodies is invariable, exhibiting an hourglass shape, whereas the lateral structure supporting the internal structure differs among species. Based on the anatomical observations, we applied different external loads to the hourglass-shaped part. The simulations produced a variety of three-dimensional structures, some of which exhibited several structural features similar to those of actual teleost vertebrae. In addition, by adjusting the geometric parameters, such as the width of the hourglass shape, we reproduced the variation in the teleost vertebrae shapes. These results suggest that a simulation using topology optimization can successfully reproduce the external shapes of teleost vertebrae. By applying our topology optimization model to various bones of vertebrates, we can understand how the external shape of bones adapts to external loads.

ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes.

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Computational analyses decipher the primordial folding coding the 3D structure of the beetle horn.

The beetle horn primordium is a complex and compactly folded epithelial sheet located beneath the larval cuticle. Only by unfolding the primordium can the complete 3D shape of the horn appear, suggesting that the morphology of beetle horns is encoded in the primordial folding pattern. To decipher the folding pattern, we developed a method to manipulate the primordial local folding on a computer and clarified the contribution of the folding of each primordium region to transformation. We found that the three major morphological changes (branching of distal tips, proximodistal elongation, and angular change) were caused by the folding of different regions, and that the folding mechanism also differs according to the region. The computational methods we used are applicable to the morphological study of other exoskeletal animals.

Effective nonlocal kernels on reaction-diffusion networks.

A new method to derive an essential integral kernel from any given reaction-diffusion network is proposed. Any network describing metabolites or signals with arbitrary many factors can be reduced to a single or a simpler system of integro-differential equations called "effective equation" including the reduced integral kernel (called "effective kernel") in the convolution type. As one typical example, the Mexican hat shaped kernel is theoretically derived from two component activator-inhibitor systems. It is also shown that a three component system with quite different appearance from activator-inhibitor systems is reduced to an effective equation with the Mexican hat shaped kernel. It means that the two different systems have essentially the same effective equations and that they exhibit essentially the same spatial and temporal patterns. Thus, we can identify two different systems with the understanding in unified concept through the reduced effective kernels. Other two applications of this method are also given: Applications to pigment patterns on skins (two factors network with long range interaction) and waves of differentiation (called proneural waves) in visual systems on brains (four factors network with long range interaction). In the applications, we observe the reproduction of the same spatial and temporal patterns as those appearing in pre-existing models through the numerical simulations of the effective equations.