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.

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.

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.

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.

The minimal gap-junction network among melanophores and xanthophores required for stripe pattern formation in zebrafish.

Connexin 39.4 (Cx39.4) and connexin 41.8 (Cx41.8), two gap-junction proteins expressed in both melanophores and xanthophores, are crucial for the intercellular communication among pigment cells that is necessary for generating the stripe pigment pattern of zebrafish. We have previously characterized the gap-junction properties of Cx39.4 and Cx41.8, but how these proteins contribute to stripe formation remains unclear; this is because distinct types of connexins potentially form heteromeric gap junctions, which precludes accurate elucidation of individual connexin functions in vivo Here, by arranging Cx39.4 and Cx41.8 expression in pigment cells, we have identified the simplest gap-junction network required for stripe generation: Cx39.4 expression in melanophores is required but expression in xanthophores is not necessary for stripe patterning, whereas Cx41.8 expression in xanthophores is sufficient for the patterning, and Cx41.8 expression in melanophores might stabilize the stripes. Moreover, patch-clamp recordings revealed that Cx39.4 gap junctions exhibit spermidine-dependent rectification property. Our results suggest that Cx39.4 facilitates the crucial cell-cell interactions between melanophores and xanthophores that mediate a unidirectional activation-signal transfer from xanthophores to melanophores, which is essential for melanophore survival.

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.

Method for disarranging the pigment pattern of zebrafish by optogenetics.

To investigate the spatiotemporal dynamics of skin pattern formation, we developed a simple method for artificially disarranging the placement of all three pigment cell types in the body trunk of zebrafish (Danio rerio). We generated transgenic fish with melanophores that ectopically expressed a variant of channelrhodopsin-2 (ChR2). Blue light (BL) irradiation induced melanophore depolarization and random migration; the latter resulted in the disarrangement of the two other pigment cell types (xanthophores and iridophores). This BL disarrangement (BLD) method was effective in both young and adult fish, but it did not affect the initial placement of pigment cells in juvenile fish (approximately 5 weeks post-fertilization). Irradiation with BL was not harmful to cells, and the patterning process immediately resumed when BL was switched off. Using the BLD method, we demonstrated that interactions between pigment cells determined stripe width in the absence of any pre-set positional cues, while the initial horizontal alignment of iridophores determined their directionality. The BLD method can be adapted to any zebrafish skin-pattern mutant, providing a novel tool for analyzing pattern formation mechanisms under a variety of conditions and facilitating further study in this field.

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.

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'.

Flexibility of pigment cell behavior permits the robustness of skin pattern formation.

The striped pigmentation pattern of zebrafish is determined by the interaction between pigment cells with different colors. Recent studies show the behaviors of pigment cells are substantially different according to the environment. Interestingly, the resulting patterns are almost identical, suggesting a robustness of the patterning mechanism. To know how this robustness originates, we investigated the behavior of melanophores in various environments including different developmental stages, different body positions, and different genetic backgrounds. Normally, when embryonic melanophores are excluded from the yellow stripe region in the body trunk, two different cellular behaviors are observed. Melanophores migrate to join the black stripe or disappear (die) in the position. In environments where melanophore migration was restricted, we observed that most melanophores disappeared in their position, resulting in the complete exclusion of melanophores from the yellow stripe. In environments where melanophore cell death was restricted, most melanophores migrated to join the black stripes, also resulting in complete exclusion. When both migration and cell death were restricted, melanophores remained alive in the yellow stripes. These results show that migration and cell death complement each other to achieve the exclusion of melanophores. This flexibility may be the basis of the mechanistic robustness of skin pattern formation.

Is pigment patterning in fish skin determined by the Turing mechanism?

More than half a century ago, Alan Turing postulated that pigment patterns may arise from a mechanism that could be mathematically modeled based on the diffusion of two substances that interact with each other. Over the past 15 years, the molecular and genetic tools to verify this prediction have become available. Here, we review experimental studies aimed at identifying the mechanism underlying pigment pattern formation in zebrafish. Extensive molecular genetic studies in this model organism have revealed the interactions between the pigment cells that are responsible for the patterns. The mechanism discovered is substantially different from that predicted by the mathematical model, but it retains the property of 'local activation and long-range inhibition', a necessary condition for Turing pattern formation. Although some of the molecular details of pattern formation remain to be elucidated, current evidence confirms that the underlying mechanism is mathematically equivalent to the Turing mechanism.

Melanophore multinucleation pathways in zebrafish.

In zebrafish, apart from mononuclear melanophores, bi- and trinuclear melanophores are frequently observed; however, the manner in which multinucleation of these cells occurs during fish development remains unknown. Here, we analyzed the processes underlying multinucleation of zebrafish melanophores. Transgenic zebrafish in which melanophore nuclei were labeled with a histone H2B-red fluorescent reporter protein were used to evaluate the distribution of mono-, bi-, and trinuclear melanophores in both the trunk and fin. Half of the melanophores examined were binuclear and approximately 1% were trinuclear. We compared cell size, cell motility, and survival rate between mono- and binuclear melanophores grown in a culture dish, and we found that cell size and survival rate were significantly larger in binuclear melanophores. We then analyzed the behavior of melanoblasts and melanophores from transgenic zebrafish using in vivo and in vitro live-cell imaging. We detected division and differentiation of melanoblasts, as well as melanoblast nuclear division without subsequent cellular division. In addition, we observed cellular and nuclear division in melanophores, although these events were very infrequent in vitro. On the basis of our findings, we present a scheme for melanophore multinucleation in zebrafish.

The Physiological Characterization of Connexin41.8 and Connexin39.4, Which Are Involved in the Striped Pattern Formation of Zebrafish.

The zebrafish has a striped skin pattern on its body, and Connexin41.8 (Cx41.8) and Cx39.4 are involved in striped pattern formation. Mutations in these connexins change the striped pattern to a spot or labyrinth pattern. In this study, we characterized Cx41.8 and Cx39.4 after expression in Xenopus oocytes. In addition, we analyzed Cx41.8 mutants Cx41.8I203F and Cx41.8M7, which caused spot or labyrinth skin patterns, respectively, in transgenic zebrafish. In the electrophysiological analysis, the gap junctions formed by Cx41.8 and Cx39.4 showed distinct sensitivity to transjunctional voltage. Analysis of non-junctional (hemichannel) currents revealed a large voltage-dependent current in Cx39.4-expressing oocytes that was absent in cells expressing Cx41.8. Junctional currents induced by both Cx41.8 and Cx39.4 were reduced by co-expression of Cx41.8I203F and abolished by co-expression of Cx41.8M7. In the transgenic experiment, Cx41.8I203F partially rescued the Cx41.8 null mutant phenotype, whereas Cx41.8M7 failed to rescue the null mutant, and it elicited a more severe phenotype than the Cx41.8 null mutant, as evidenced by a smaller spot pattern. Our results provide evidence that gap junctions formed by Cx41.8 play an important role in stripe/spot patterning and suggest that mutations in Cx41.8 can effect patterning by way of reduced function (I203F) and dominant negative effects (M7). Our results suggest that functional differences in Cx41.8 and Cx39.4 relate to spot or labyrinth mutant phenotypes and also provide evidence that these two connexins interact in vivo and in vitro.

Two Different Functions of Connexin43 Confer Two Different Bone Phenotypes in Zebrafish.

Fish remain nearly the same shape as they grow, but there are two different modes of bone growth. Bones in the tail fin (fin ray segments) are added distally at the tips of the fins and do not elongate once produced. On the other hand, vertebrae enlarge in proportion to body growth. To elucidate how bone growth is controlled, we investigated a zebrafish mutant, steopsel (stp(tl28d)). Vertebrae of stp(tl28d) (/+) fish look normal in larvae (∼30 days) but are distinctly shorter (59-81%) than vertebrae of wild type fish in adults. In contrast, the lengths of fin rays are only slightly shorter (∼95%) than those of the wild type in both larvae and adults. Positional cloning revealed that stp encodes Connexin43 (Cx43), a connexin that functions as a gap junction and hemichannel. Interestingly, cx43 was also identified as the gene causing the short-of-fin (sof) phenotype, in which the fin ray segments are shorter but the vertebrae are normal. To identify the cause of this difference between the alleles, we expressed Cx43 exogenously in Xenopus oocytes and performed electrophysiological analysis of the mutant proteins. Gap junction coupling induced by Cx43(stp) or Cx43(sof) was reduced compared with Cx43-WT. On the other hand, only Cx43(stp) induced abnormally high (50× wild type) transmembrane currents through hemichannels. Our results suggest that Cx43 plays critical and diverse roles in zebrafish bone growth.

Involvement of Delta/Notch signaling in zebrafish adult pigment stripe patterning.

The skin pigment pattern of zebrafish is a good model system in which to study the mechanism of biological pattern formation. Although it is known that interactions between melanophores and xanthophores play a key role in the formation of adult pigment stripes, molecular mechanisms for these interactions remain largely unknown. Here, we show that Delta/Notch signaling contributes to these interactions. Ablation of xanthophores in yellow stripes induced the death of melanophores in black stripes, suggesting that melanophores require a survival signal from distant xanthophores. We found that deltaC and notch1a were expressed by xanthophores and melanophores, respectively. Moreover, inhibition of Delta/Notch signaling killed melanophores, whereas activation of Delta/Notch signaling ectopically in melanophores rescued the survival of these cells, both in the context of pharmacological inhibition of Delta/Notch signaling and after ablation of xanthophores. Finally, we showed by in vivo imaging of cell membranes that melanophores extend long projections towards xanthophores in the yellow stripes. These data suggest that Delta/Notch signaling is responsible for a survival signal provided by xanthophores to melanophores. As cellular projections can enable long-range interaction between membrane-bound ligands and their receptors, we propose that such projections, combined with direct cell-cell contacts, can substitute for the effect of a diffusible factor that would be expected by the conventional reaction-diffusion (Turing) model.

An updated kernel-based Turing model for studying the mechanisms of biological pattern formation.

The reaction-diffusion model presented by Alan Turing has recently been supported by experimental data and accepted by most biologists. However, scientists have recognized shortcomings when the model is used as the working hypothesis in biological experiments, particularly in studies in which the underlying molecular network is not fully understood. To address some such problems, this report proposes a new version of the Turing model. This alternative model is not represented by partial differential equations, but rather by the shape of an activation-inhibition kernel. Therefore, it is named the kernel-based Turing model (KT model). Simulation of the KT model with kernels of various shapes showed that it can generate all standard variations of the stable 2D patterns (spot, stripes and network), as well as some complex patterns that are difficult to generate with conventional mathematical models. The KT model can be used even when the detailed mechanism is poorly known, as the interaction kernel can often be detected by a simple experiment and the KT model simulation can be performed based on that experimental data. These properties of the KT model complement the shortcomings of conventional models and will contribute to the understanding of biological pattern formation.

In vitro analysis suggests that difference in cell movement during direct interaction can generate various pigment patterns in vivo.

Pigment patterns of organisms have invoked strong interest from not only biologists but also, scientists in many other fields. Zebrafish is a useful model animal for studying the mechanism of pigment pattern formation. The zebrafish stripe pattern is primarily two types of pigment cells: melanophores and xanthophores. Previous studies have reported that interactions among these pigment cells are important for pattern formation. In the recent report, we found that the direct contact by xanthophores induces the membrane depolarization of melanophores. From analysis of jaguar mutants, it is suggested that the depolarization affects the movements of melanophores. To analyze the cell movement in detail, we established a unique in vitro system. It allowed us to find that WT xanthophores induced repulsive movement of melanophores through direct contact. The xanthophores also chased the melanophores. As a result, they showed run-and-chase movements. We also analyzed the cell movement of pigment cells from jaguar and leopard mutants, which have fuzzy stripes and spot patterns, respectively. jaguar cells showed inhibited run-and-chase movements, and leopard melanophores scarcely showed repulsive response. Furthermore, we paired mutant and WT cells and showed which of the melanophores and xanthophores have responsibility for the altered cell movements. These results suggested that there is a correspondence relationship between the cell movements and pigment patterns. The correspondence relationship highlighted the importance of the cell movements in the pattern formation and showed that our system is a quite useful system for future study in this field.

Rotating pigment cells exhibit an intrinsic chirality.

In multicellular organisms, cell properties, such as shape, size and function are important in morphogenesis and physiological functions. Recently, 'cellular chirality' has attracted attention as a cellular property because it can cause asymmetry in the bodies of animals. In recent in vitro studies, the left-right bias of cellular migration and of autonomous arrangement of cells under some specific culture conditions were discovered. However, it is difficult to identify the molecular mechanism underlying their intrinsic chirality because the left-right bias observed to date is subtle or is manifested in the stable orientation of cells. Here, we report that zebrafish (Danio rerio) melanophores exhibit clear cellular chirality by unidirectional counterclockwise rotational movement under isolated conditions without any special settings. The chirality is intrinsic to melanophores because the direction of the cellular rotation was not affected by the type of extracellular matrix. We further found that the cellular rotation was generated as a counter action of the clockwise movement of actin cytoskeleton. It suggested that the mechanism that directs actin cytoskeleton in the clockwise direction is pivotal for determining cellular chirality.

Synthesis and evaluation of hedgehog signaling inhibitor with novel core system.

As we previously reported, N-methylpyrrolo[3,2-c]pyridine derivatives 1 (TAK-441) was discovered as a clinical candidate of hedgehog (Hh) signaling inhibitor by modification of the upper part. We next focused on modification of the lower part including core skeletons to discover new Hh signaling inhibitors with novel core rings. Efforts to find novel chemotypes by using X-ray single crystal structure analysis led to some potent Hh signaling inhibitors (2c, 2d, 2e, 2f) with novel core ring systems, which had benzamide moiety at the 5-position as a key component for potent activity. The suppression of Gli1 expression with these new Hh signaling inhibitors were weaker than that of compound 1 (TAK-441) because of low pharmacokinetic property. We recognized again TAK-441 is a good compound as clinical candidate with good structural and pharmacokinetic advantages.

Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.