FGF signaling induces the regeneration of collagen fiber structure during skin wound healing in axolotls.

Axolotls have been considered to be able to regenerate their skin completely. Our recent study updated this theory with the finding that the lattice structure of dermal collagen fibers was not fully regenerated after skin injury. We also discovered that nerves induce the regeneration of collagen fibers. The mechanism of collagen fiber regeneration remains unknown, however. In this study, we focused on the structure of collagen fibers with collagen braiding cells, and cell origin in axolotl skin regeneration. In the wounded dermis, cells involved in skin repair/regeneration were derived from both the surrounding dermis and the subcutaneous tissue. Regardless of cell origin, cells acquired the proper cell morphology to braid collagen fiber with nerve presence. We also found that FGF signaling could substitute for the nerve roles in the conversion of subcutaneous fibroblasts to lattice-shaped dermal fibroblasts. Our findings contribute to the elucidation of the fundamental mechanisms of true skin regeneration and provide useful insights for pioneering new skin treatments.

Automated contour extraction for light-sheet microscopy images of zebrafish embryos based on object edge detection algorithm.

Embryo contour extraction is the initial step in the quantitative analysis of embryo morphology, and it is essential for understanding the developmental process. Recent developments in light-sheet microscopy have enabled the in toto time-lapse imaging of embryos, including zebrafish. However, embryo contour extraction from images generated via light-sheet microscopy is challenging owing to the large amount of data and the variable sizes, shapes, and textures of objects. In this report, we provide a workflow for extracting the contours of zebrafish blastula and gastrula without contour labeling of an embryo. This workflow is based on the edge detection method using a change point detection approach. We assessed the performance of the edge detection method and compared it with widely used edge detection and segmentation methods. The results showed that the edge detection accuracy of the proposed method was superior to those of the Sobel, Laplacian of Gaussian, adaptive threshold, Multi Otsu, and k-means clustering-based methods, and the noise robustness of the proposed method was superior to those of the Multi Otsu and k-means clustering-based methods. The proposed workflow was shown to be useful for automating small-scale contour extractions of zebrafish embryos that cannot be specifically labeled owing to constraints, such as the availability of microscopic channels. This workflow may offer an option for contour extraction when deep learning-based approaches or existing non-deep learning-based methods cannot be applied.

Developmental analyses of mouse embryos and adults using a non-overlapping tracing system for all three germ layers.

Genetic lineage-tracing techniques are powerful tools for studying specific cell populations in development and pathogenesis. Previous techniques have mainly involved systems for tracing a single gene, which are limited in their ability to facilitate direct comparisons of the contributions of different cell lineages. We have developed a new combinatorial system for tracing all three germ layers using self-cleaving 2A peptides and multiple site-specific recombinases (SSRs). In the resulting TRiCK (TRiple Coloured germ layer Knock-in) mice, the three germ layers are conditionally and simultaneously labelled with distinct fluorescent proteins via embryogenesis. We show that previously reported ectopic expressions of lineage markers are the outcome of secondary gene expression. The results presented here also indicate that the commitment of caudal axial stem cells to neural or mesodermal fate proceeds without lineage fluctuations, contrary to the notion of their bi-potency. Moreover, we developed IMES, an optimized tissue clearing method that is highly compatible with a variety of fluorescent proteins and immunostaining, and the combined use of TRiCK mice and IMES can facilitate comprehensive analyses of dynamic contributions of all three germ layers.

Simple mechanosense and response of cilia motion reveal the intrinsic habits of ciliates.

An important habit of ciliates, namely, their behavioral preference for walls, is revealed through experiments and hydrodynamic simulations. A simple mechanical response of individual ciliary beating (i.e., the beating is stalled by the cilium contacting a wall) can solely determine the sliding motion of the ciliate along the wall and result in a wall-preferring behavior. Considering ciliate ethology, this mechanosensing system is likely an advantage in the single cell's ability to locate nutrition. In other words, ciliates can skillfully use both the sliding motion to feed on a surface and the traveling motion in bulk water to locate new surfaces according to the single "swimming" mission.

Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development.

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos.

Skeleton construction upon local regression of the sponge body.

We previously revealed that the mechanism of demosponge skeleton construction is self-organization by multiple rounds of sequential mechanical reactions of player cells. In these reactions, "transport cells" dynamically carry fine skeletal elements (spicules) on epithelia surrounding the inner body space of sponges (basal epithelium (basopinacoderm) and the endodermal epithelium (ENCM)). Once spicules pierce ENCM and apical pinacoderm, subsequently they are cemented to the substratum under the sponge body, or connected to other skeleton-constructing spicules. Thus, the "pierce" step is the key to holding up spicules in the temporary periphery of growing sponges' bodies. Since sponges can regress as well as grow, here we asked how skeleton construction occurs during local regression of the body. We found that prior to local basopinacoderm retraction (and thus body regression), the body became thinner. Some spicules that were originally carried outward stagnated for a while, and were then carried inwards either on ENCM or basopinacoderm. Spicules that were carried inwards on ENCM pierced epithelia after a short transport, and thus became held up at relatively inward positions compared to spicules carried on outwardly extending basopinacoderm. The switch of epithelia on which transport cells migrate efficiently occurred in thinner body spaces where basopinacoderm and ENCM became close to each other. Thus, the mechanisms underlying this phenomenon are rather mechanical: the combination of sequential reactions of skeleton construction and the narrowed body space upon local retraction of basopinacoderm cause spicules to be held up at more-inward positions, which might strengthen the basopinacoderm's attachment to substratum.

System level analysis of motor-related neural activities in larval Drosophila.

The way in which the central nervous system (CNS) governs animal movement is complex and difficult to solve solely by the analyses of muscle movement patterns. We tackle this problem by observing the activity of a large population of neurons in the CNS of larval Drosophila. We focused on two major behaviors of the larvae - forward and backward locomotion - and analyzed the neuronal activity related to these behaviors during the fictive locomotion that occurs spontaneously in the isolated CNS. We expressed a genetically-encoded calcium indicator, GCaMP and a nuclear marker in all neurons and then used digitally scanned light-sheet microscopy to record (at a fast frame rate) neural activities in the entire ventral nerve cord (VNC). We developed image processing tools that automatically detected the cell position based on the nuclear staining and allocate the activity signals to each detected cell. We also applied a machine learning-based method that we recently developed to assign motor status in each time frame. Our experimental procedures and computational pipeline enabled systematic identification of neurons that showed characteristic motor activities in larval Drosophila. We found cells whose activity was biased toward forward locomotion and others biased toward backward locomotion. In particular, we identified neurons near the boundary of the subesophageal zone (SEZ) and thoracic neuromeres, which were strongly active during an early phase of backward but not forward fictive locomotion.

Evolutionary transformation of mouthparts from particle-feeding to piercing carnivory in Viper copepods: Review and 3D analyses of a key innovation using advanced imaging techniques.

BACKGROUND: Novel feeding adaptations often facilitate adaptive radiation and diversification. But the evolutionary origins of such feeding adaptations can be puzzling if they require concordant change in multiple component parts. Pelagic, heterorhabdid copepods (Calanoida) exhibit diverse feeding behaviors that range from simple particle feeding to a highly specialized form of carnivory involving piercing mouthparts that likely inject venom. We review the evolutionary history of heterorhabdid copepods and add new high-resolution, 3D anatomical analyses of the muscular system, glands and gland openings associated with this remarkable evolutionary transformation. RESULTS: We examined four heterorhabdid copepods with different feeding modes: one primitive particle-feeder (Disseta palumbii), one derived and specialized carnivore (Heterorhabdus subspinifrons), and two intermediate taxa (Mesorhabdus gracilis and Heterostylites longicornis). We used two advanced, high-resolution microscopic techniques - serial block-face scanning electron microscopy and two-photon excitation microscopy - to visualize mouthpart form and internal anatomy at unprecedented nanometer resolution. Interactive 3D graphical visualizations allowed putative homologues of muscles and gland cells to be identified with confidence and traced across the evolutionary transformation from particle feeding to piercing carnivory. Notable changes included: a) addition of new gland cells, b) enlargement of some (venom producing?) glands, c) repositioning of gland openings associated with hollow piercing fangs on the mandibles, d) repurposing of some mandibular-muscle function to include gland-squeezing, and e) addition of new muscles that may aid venom injection exclusively in the most specialized piercing species. In addition, live video recording of all four species revealed mandibular blade movements coupled to cyclic contraction of some muscles connected to the esophagus. These behavioral and 3D morphological observations revealed a novel injection system in H. subspinifrons associated with piercing (envenomating?) carnivory. CONCLUSIONS: Collectively, these results suggest that subtle changes in mandibular tooth form, and muscle and gland form and location, facilitated the evolution of a novel, piercing mode of feeding that accelerated diversification of the genus Heterorhabdus. They also highlight the value of interactive 3D animations for understanding evolutionary transformations of complex, multicomponent morphological systems.

Axially-confined in vivo single-cell labeling by primed conversion using blue and red lasers with conventional confocal microscopes.

Green-to-red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave-length illumination with blue and red/near-infrared light. By modifying a confocal laser-scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide-spread use of this method. Here, we investigated whether spatially-restricted primed conversion could be achieved with CLSM without any hardware modification. We found that the primed conversion of Dendra2 using a conventional CLSM with two visible lasers (473 nm and 635 nm) and a high NA objective lens (NA, 1.30) resulted in dramatic restriction of photoconversion volume: half-width half-maximum for the axial dimension was below 5 µm, which is comparable to the outcome of the original method that used the microscope modification. As a proof of this method's effectiveness, we used this technique in living zebrafish embryos and succeeded in revealing the complex anatomy of individual neurons packed between neighboring cells. Because unmodified CLSMs are widely available, this method can be widely applicable for labeling cells with single-cell resolution.

Cell movements of the deep layer of non-neural ectoderm underlie complete neural tube closure in Xenopus.

In developing vertebrates, the neural tube forms from a sheet of neural ectoderm by complex cell movements and morphogenesis. Convergent extension movements and the apical constriction along with apical-basal elongation of cells in the neural ectoderm are thought to be essential for the neural tube closure (NTC) process. In addition, it is known that non-neural ectoderm also plays a crucial role in this process, as the neural tube fails to close in the absence of this tissue in chick and axolotl. However, the cellular and molecular mechanisms by which it functions in NTC are as yet unclear. We demonstrate here that the non-neural superficial epithelium moves in the direction of tensile forces applied along the dorsal-ventral axis during NTC. We found that this force is partly attributable to the deep layer of non-neural ectoderm cells, which moved collectively towards the dorsal midline along with the superficial layer. Moreover, inhibition of this movement by deleting integrin ß1 function resulted in incomplete NTC. Furthermore, we demonstrated that other proposed mechanisms, such as oriented cell division, cell rearrangement and cell-shape changes have no or only minor roles in the non-neural movement. This study is the first to demonstrate dorsally oriented deep-cell migration in non-neural ectoderm, and suggests that a global reorganization of embryo tissues is involved in NTC.

Asymmetric distribution of dynamic calcium signals in the node of mouse embryo during left-right axis formation.

In the node of mouse embryo, rotational movements of cilia generate an external liquid flow known as nodal flow, which determines left-right asymmetric gene expression. How nodal flow is converted into asymmetric gene expression is still controversial, but the increase of Ca(2+) levels in endodermal cells to the left of the node has been proposed to play a role. However, Ca(2+) signals inside the node itself have not yet been described. By our optimized Ca(2+) imaging method, we were able to observe dynamic Ca(2+) signals in the node in live mouse embryos. Pharmacological disruption of Ca(2+) signals did not affect ciliary movements or nodal flow, but did alter the expression patterns of the Nodal and Cerl-2 genes. Quantitative analyses of Ca(2+) signal frequencies and distributions showed that during left-right axis establishment, formerly symmetric Ca(2+) signals became biased to the left side. In iv/iv mutant embryos that showed randomized laterality due to ciliary immotility, Ca(2+) signals were found to be variously left-sided, right-sided, or bilateral, and thus symmetric on average. In Pkd2 mutant embryos, which lacked polycystin-2, a Ca(2+)-permeable cation channel necessary for left-right axis formation, the Ca(2+) signal frequency was lower than in wild-type embryos. Our data support a model in which dynamic Ca(2+) signals in the node are involved in left-right patterning.

Ultrasensitive imaging of Ca2+ dynamics in pancreatic acinar cells of yellow cameleon-nano transgenic mice.

Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo.

Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II.

Motile cilia generate constant fluid flow over epithelial tissue, and thereby influence diverse physiological processes. Such functions of ciliated cells depend on the planar polarity of the cilia and on their basal bodies being oriented in the downstream direction of fluid flow. Recently, another type of basal body planar polarity, characterized by the anterior localization of the basal bodies in individual cells, was reported in the multiciliated ependymal cells that line the surface of brain ventricles. However, little is known about the cellular and molecular mechanisms by which this polarity is established. Here, we report in mice that basal bodies move in the apical cell membrane during differentiation to accumulate in the anterior region of ependymal cells. The planar cell polarity signaling pathway influences basal body orientation, but not their anterior migration, in the neonatal brain. Moreover, we show by pharmacological and genetic studies that non-muscle myosin II is a key regulator of this distribution of basal bodies. This study demonstrates that the orientation and distribution of basal bodies occur by distinct mechanisms.

Epigenetically distinct synaptic architecture in clonal compartments in the teleostean dorsal pallium.

The dorsal telencephalon (i.e. the pallium) exhibits high anatomical diversity across vertebrate classes. The non-mammalian dorsal pallium accommodates various compartmentalized structures among species. The developmental, functional, and evolutional diversity of the dorsal pallium remain unillustrated. Here, we analyzed the structure and epigenetic landscapes of cell lineages in the telencephalon of medaka fish (Oryzias latipes) that possesses a clearly delineated dorsal pallium (Dd2). We found that pallial anatomical regions, including Dd2, are formed by mutually exclusive clonal units, and that each pallium compartment exhibits a distinct epigenetic landscape. In particular, Dd2 possesses a unique open chromatin pattern that preferentially targets synaptic genes. Indeed, Dd2 shows a high density of synapses. Finally, we identified several transcription factors as candidate regulators. Taken together, we suggest that cell lineages are the basic components for the functional regionalization in the pallial anatomical compartments and that their changes have been the driving force for evolutionary diversity.

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector.

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion, which are critical for a holistic understanding of the Leishmania life cycle.

Light-sheet microscopy reveals dorsoventral asymmetric membrane dynamics of Amoeba proteus during pressure-driven locomotion.

Amoebae are found all around the world and play an essential role in the carbon cycle in the environment. Therefore, the behavior of amoebae is a crucial factor when considering the global environment. Amoebae change their distribution through amoeboid locomotion, which are classified into several modes. In the pressure-driven mode, intracellular hydrostatic pressure generated by the contraction of cellular cortex actomyosin causes the pseudopod to extend. During amoeboid locomotion, the cellular surface exhibits dynamic deformation. Therefore, to understand the mechanism of amoeboid locomotion, it is important to characterize cellular membrane dynamics. Here, to clarify membrane dynamics during pressure-driven amoeboid locomotion, we developed a polkadot membrane staining method and performed light-sheet microscopy in Amoeba proteus, which exhibits typical pressure-driven amoeboid locomotion. It was observed that the whole cell membrane moved in the direction of movement, and the dorsal cell membrane in the posterior part of the cell moved more slowly than the other membrane. In addition, membrane complexity varied depending on the focused characteristic size of the membrane structure, and in general, the dorsal side was more complex than the ventral side. In summary, the membrane dynamics of Amoeba proteus during pressure-driven locomotion are asymmetric between the dorsal and ventral sides. This article has an associated interview with the co-first authors of the paper.

Depletion of Ift88 in thymic epithelial cells affects thymic synapse and T-cell differentiation in aged mice.

Primary cilia are ubiquitous hair-like organelles, usually projecting from the cell surface. They are essential for the organogenesis and homeostasis of various physiological functions, and their dysfunction leads to a plethora of human diseases. However, there are few reports on the role of primary cilia in the immune system; therefore, we focused on their role in the thymus that nurtures immature lymphocytes to full-fledged T cells. We detected primary cilia on the thymic epithelial cell (TEC) expressing transforming growth factor ß (TGF-ß) receptor in the basal body, and established a line of an intraflagellar transport protein 88 (Ift88) knockout mice lacking primary cilia in TECs (Ift88-TEC null mutant) to clarify their precise role in thymic organogenesis and T-cell differentiation. The Ift88-TEC null mutant mice showed stunted cilia or lack of cilia in TECs. The intercellular contact between T cells and the "thymic synapse" of medullary TECs was slightly disorganized in Ift88-TEC null mutants. Notably, the CD4- and CD8-single positive thymocyte subsets increased significantly. The absence or disorganization of thymic cilia downregulated the TGF-ß signaling cascade, increasing the number of single positive thymocytes. To our knowledge, this is the first study reporting the physiological role of primary cilia and Ift88 in regulating the differentiation of the thymus and T cells.

Lattice-patterned collagen fibers and their dynamics in axolotl skin regeneration.

The morphology of collagen-producing cells and the structure of produced collagen in the dermis have not been well-described. This lack of insights has been a serious obstacle in the evaluation of skin regeneration. We succeeded in visualizing collagen-producing cells and produced collagen using the axolotl skin, which is highly transparent. The visualized dermal collagen had a lattice-like structure. The collagen-producing fibroblasts consistently possessed the lattice-patterned filopodia along with the lattice-patterned collagen network. The dynamics of this lattice-like structure were also verified in the skin regeneration process of axolotls, and it was found that the correct lattice-like structure was not reorganized after simple skin wounding but was reorganized in the presence of nerves. These findings are not only fundamental insights in dermatology but also valuable insights into the mechanism of skin regeneration.

Near-wall rheotaxis of the ciliate Tetrahymena induced by the kinesthetic sensing of cilia.

To survive in harsh environments, single-celled microorganisms autonomously respond to external stimuli, such as light, heat, and flow. Here, we elucidate the flow response of Tetrahymena, a well-known single-celled freshwater microorganism. Tetrahymena moves upstream against an external flow via a behavior called rheotaxis. While micrometer-sized particles are swept away downstream in a viscous flow, what dynamics underlie the rheotaxis of the ciliate? Our experiments reveal that Tetrahymena slides along walls during upstream movement, which indicates that the cells receive rotational torque from shear flow to control cell orientation. To evaluate the effects of the shear torque and propelling speed, we perform a numerical simulation with a hydrodynamic model swimmer adopting cilia dynamics in a shear flow. The swimmer orientations converge to an upstream alignment, and the swimmer slides upstream along a boundary wall. The results suggest that Tetrahymena automatically responds to shear flow by performing rheotaxis using cilia-stalling mechanics.

Colocalization Analysis of Lipo-Deoxyribozyme Consisting of DNA and Protic Catalysts in a Vesicle-Based Protocellular Membrane Investigated by Confocal Microscopy.

The linkage between the self-reproduction of compartments and the replication of DNA in a compartment is a crucial requirement for cellular life. In our giant vesicle (GV)-based model protocell, this linkage is achieved through the action of a supramolecular catalyst composed of membrane-intruded DNA and amphiphilic acid catalysts (C@DNA) in a GV membrane. In this study, we examined colocalization analysis for the formation of the supramolecular catalyst using a confocal laser scanning fluorescence microscope with high sensitivity and resolution. Red fluorescence spots emitted from DNA tagged with Texas Red (Texas Red-DNA) were observed in a GV membrane stained with phospholipid tagged with BODIPY (BODIPY-HPC). To our knowledge, this is the first direct observation of DNA embedded in a GV-based model protocellular membrane containing cationic lipids. Colocalization analysis based on a histogram of frequencies of "normalized mean deviation product" revealed that the frequencies of positively correlated [lipophilic catalyst tagged with BODIPY (BODIPY-C) and Texas Red-DNA] were significantly higher than those of [BODIPY-HPC and Texas Red-DNA]. This result demonstrates the spontaneous formation of C@DNA in the GV membrane, which serves as a lipo-deoxyribozyme for producing membrane lipids from its precursor.