An archetype and scaling of developmental tissue dynamics across species.

Morphometric studies have revealed the existence of simple geometric relationships among various animal shapes. However, we have little knowledge of the mathematical principles behind the morphogenetic dynamics that form the organ/body shapes of different species. Here, we address this issue by focusing on limb morphogenesis in Gallus gallus domesticus (chicken) and Xenopus laevis (African clawed frog). To compare the deformation dynamics between tissues with different sizes/shapes as well as their developmental rates, we introduce a species-specific rescaled spatial coordinate and a common clock necessary for cross-species synchronization of developmental times. We find that tissue dynamics are well conserved across species under this spacetime coordinate system, at least from the early stages of development through the phase when basic digit patterning is established. For this developmental period, we also reveal that the tissue dynamics of both species are mapped with each other through a time-variant linear transformation in real physical space, from which hypotheses on a species-independent archetype of tissue dynamics and morphogenetic scaling are proposed.

Behavioral photosensitivity of multi-color-blind medaka: enhanced response under ultraviolet light in the absence of short-wavelength-sensitive opsins.

BACKGROUND: The behavioral photosensitivity of animals could be quantified via the optomotor response (OMR), for example, and the luminous efficiency function (the range of visible light) should largely rely on the repertoire and expression of light-absorbing proteins in the retina, i.e., the opsins. In fact, the OMR under red light was suppressed in medaka lacking the red (long-wavelength sensitive [LWS]) opsin. RESULTS: We investigated the ultraviolet (UV)- or blue-light sensitivity of medaka lacking the violet (short-wavelength sensitive 1 [SWS1]) and blue (SWS2) opsins. The sws1/sws2 double or sws1/sws2/lws triple mutants were as viable as the wild type. The remaining green (rhodopsin 2 [RH2]) or red opsins were not upregulated. Interestingly, the OMR of the double or triple mutants was equivalent or even increased under UV or blue light (λ = 350, 365, or 450 nm), which demonstrated that the rotating stripes (i.e., changes in luminance) could fully be recognized under UV light using RH2 alone. The OMR test using dichromatic stripes projected onto an RGB display consistently showed that the presence or absence of SWS1 and SWS2 did not affect the equiluminant conditions. CONCLUSIONS: RH2 and LWS, but not SWS1 and SWS2, should predominantly contribute to the postreceptoral processes leading to the OMR or, possibly, to luminance detection in general, as the medium-wavelength-sensitive and LWS cones, but not the SWS cones, are responsible for luminance detection in humans.

Adaptive Optics Microscopy with Wavefront Sensing Based on Neighbor Correlation.

Complex structures in living cells and tissues induce wavefront errors when light waves pass through them, and images observed with optical microscopes are undesirably blurred. This problem is especially serious for living plant cells because images are strikingly degraded even within a single cell. Adaptive optics (AO) is expected to be a solution to this problem by correcting such wavefront errors, thus enabling high-resolution imaging. In particular, scene-based AO involves wavefront sensing based on the image correlation between subapertures in a Shack-Hartmann wavefront sensor and thus does not require an intense point light source. However, the complex 3D structures of living cells often cause low correlation between subimages, leading to loss of accuracy in wavefront sensing. This paper proposes a novel method for scene-based sensing using only image correlations between adjacent subapertures. The method can minimize changes between subimages to be correlated and thus prevent inaccuracy in phase estimation. Using an artificial test target mimicking the optical properties of a layer of living plant cells, an imaging performance with a Strehl ratio of approximately 0.5 was confirmed. Upon observation of chloroplast autofluorescence inside living leaf cells of the moss Physcomitrium patens, recovered resolution images were successfully obtained even with complex biological structures. Under bright-field illumination, the proposed method outperformed the conventional method, demonstrating the future potential of this method for label- and damage-free AO microscopy. Several points for improvement in terms of the effect of AO correction are discussed.

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.

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.

Targeted single-cell gene induction by optimizing the dually regulated CRE/loxP system by a newly defined heat-shock promoter and the steroid hormone in Arabidopsis thaliana.

Multicellular organisms rely on intercellular communication systems to organize their cellular functions. In studies focusing on intercellular communication, the key experimental techniques include the generation of chimeric tissue using transgenic DNA recombination systems represented by the CRE/loxP system. If an experimental system enables the induction of chimeras at highly targeted cell(s), it will facilitate the reproducibility and precision of experiments. However, multiple technical limitations have made this challenging. The stochastic nature of DNA recombination events, especially, hampers reproducible generation of intended chimeric patterns. Infrared laser-evoked gene operator (IR-LEGO), a microscopic system that irradiates targeted cells using an IR laser, can induce heat shock-mediated expression of transgenes, for example, CRE recombinase gene, in the cells. In this study, we developed a method that induces CRE/loxP recombination in the target cell(s) of plant roots and leaves in a highly specific manner. We combined IR-LEGO, an improved heat-shock-specific promoter, and dexamethasone-dependent regulation of CRE. The optimal IR-laser power and irradiation duration were estimated via exhaustive irradiation trials and subsequent statistical modeling. Under optimized conditions, CRE/loxP recombination was efficiently induced without cellular damage. We also found that the induction efficiency varied among tissue types and cellular sizes. The developed method offers an experimental system to generate a precisely designed chimeric tissue, and thus, will be useful for analyzing intercellular communication at high resolution in roots and leaves.

Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection.

BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.

The shh limb enhancer is activated in patterned limb regeneration but not in hypomorphic limb regeneration in Xenopus laevis.

Xenopus young tadpoles regenerate a limb with the anteroposterior (AP) pattern, but metamorphosed froglets regenerate a hypomorphic limb after amputation. The key gene for AP patterning, shh, is expressed in a regenerating limb of the tadpole but not in that of the froglet. Genomic DNA in the shh limb-specific enhancer, MFCS1 (ZRS), is hypermethylated in froglets but hypomethylated in tadpoles: shh expression may be controlled by epigenetic regulation of MFCS1. Is MFCS1 specifically activated for regenerating the AP-patterned limb? We generated transgenic Xenopus laevis lines that visualize the MFCS1 enhancer activity with a GFP reporter. The transgenic tadpoles showed GFP expression in hoxd13-and shh-expressing domains of developing and regenerating limbs, whereas the froglets showed no GFP expression in the regenerating limbs despite having hoxd13 expression. Genome sequence analysis and co-transfection assays using cultured cells revealed that Hoxd13 can activate Xenopus MFCS1. These results suggest that MFCS1 activation correlates with regeneration of AP-patterned limbs and that re-activation of epigenetically inactivated MFCS1 would be crucial to confer the ability to non-regenerative animals for regenerating a properly patterned limb.

Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish.

Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka (Oryzias latipes) mutants deficient in teleost-specific androgen receptor ohnologs (ara and arb). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage.

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.

Uphill energy transfer mechanism for photosynthesis in an Antarctic alga.

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Šresolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

The pentameric hydrocoel lobes organize adult pentameral structures in a sea cucumber, Apostichopus japonicus.

Despite being one of the bilaterians, the body plan of echinoderms shifts from bilateral symmetry to five-fold radial, or pentaradial symmetry during embryogenesis or their metamorphosis. While the clarification of the developmental mechanism behind this transition will be a basis for understanding their unique body plan evolution, it is still poorly understood. With this regard, the hydrocoel, a mesodermal coelom formed on the left side of bilateral larva, would be a clue for understanding the mechanism as it is the first pentaradial structure that appears before metamorphosis and develops into the water vascular system of adults. By analyzing the development of a sea cucumber, Apostichopus japonicus, we found that the hydrocoel expresses genes related in muscle and neural formation such as myosin heavy chain, tropomyosin, soxC, and elav, implying that cells of the hydrocoel contributes to muscle and neural structures in the adult. Furthermore, ablation of one of the hydrocoel lobes led to incomplete development of adult pentameral structures. The ablation of primary hydrocoel lobes resulted in the reduction in tentacles and the ablation of secondary hydrocoel lobes resulted in the reduction in water vascular canals and nerve cords. Our findings suggest that the hydrocoel lobes may serve as a potential organizing center for establishing the pentaradial body plan in echinoderms.

Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz Temperature Imaging with a Genetically Encoded Nanothermometer.

Despite improved sensitivity of nanothermometers, direct observation of heat transport inside single cells has remained challenging for the lack of high-speed temperature imaging techniques. Here, we identified insufficient temperature resolution under short signal integration time and slow sensor kinetics as two major bottlenecks. To overcome the limitations, we developed B-gTEMP, a nanothermometer based on the tandem fusion of mNeonGreen and tdTomato fluorescent proteins. We visualized the propagation of heat inside intracellular space by tracking the temporal variation of local temperature at a time resolution of 155 µs and a temperature resolution 0.042 °C. By comparing the fast in situ temperature dynamics with computer-simulated heat diffusion, we estimated the thermal diffusivity of live HeLa cells. The present thermal diffusivity in cells was about 1/5.3 of that of water and much smaller than the values reported for bulk tissues, which may account for observations of heterogeneous intracellular temperature distributions.

Diaphorin, a Polyketide Produced by a Bacterial Symbiont of the Asian Citrus Psyllid, Inhibits the Growth and Cell Division of Bacillus subtilis but Promotes the Growth and Metabolic Activity of Escherichia coli.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a notorious agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Diaphorin belongs to the pederin family of bioactive agents found in various host-symbiont systems, including beetles, lichens, and sponges, harboring phylogenetically diverse bacterial producers. Previous studies showed that diaphorin, which is present in D. citri at concentrations of 2 to 20 mM, has inhibitory effects on various eukaryotes, including the natural enemies of D. citri. However, little is known about its effects on prokaryotic organisms. To address this issue, the present study assessed the biological activities of diaphorin on two model prokaryotes, Escherichia coli (Gammaproteobacteria: Enterobacterales) and Bacillus subtilis (Firmicutes: Bacilli). Their growth and morphological features were analyzed using spectrophotometry, optical microscopy followed by image analysis, and transmission electron microscopy. The metabolic activity of E. coli was further assessed using the ß-galactosidase assay. The results revealed that physiological concentrations of diaphorin inhibit the growth and cell division of B. subtilis but promote the growth and metabolic activity of E. coli. This finding implies that diaphorin functions as a defensive agent of the holobiont (host plus symbionts) against some bacterial lineages but is metabolically beneficial for others, which potentially include obligate symbionts of D. citri. IMPORTANCE Certain secondary metabolites, including antibiotics, evolve to mediate interactions among organisms. These molecules have distinct spectra for microorganisms and are often more effective against Gram-positive bacteria than Gram-negative ones. However, it is rare that a single molecule has completely opposite activities on distinct bacterial lineages. The present study revealed that a secondary metabolite synthesized by an organelle-like bacterial symbiont of psyllids inhibits the growth of Gram-positive Bacillus subtilis but promotes the growth of Gram-negative Escherichia coli. This finding not only provides insights into the evolution of microbiomes in animal hosts but also may potentially be exploited to promote the effectiveness of industrial material production by microorganisms.

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.

Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids.

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction.

Mosaic gene expression analysis of semaphorin-plexin interactions in Caenorhabditis elegans using the IR-LEGO single-cell gene induction system.

Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism. However, the level of gene induction is not controllable due to the usage of a heat-shock promoter. Here, we applied IR-LEGO to examine the cell-cell interactions mediated by semaphoring-plexin signaling in Caenorhabditis elegans by inducing wild-type semaphorin/plexin in single cells within the population of mutant cells lacking the relevant proteins. We found that the cell contact-dependent termination of the extension of vulval precursor cells is elicited by the forward signaling mediated by the semaphorin receptor, PLX-1, but not by the reverse signaling via the transmembrane semaphorin, SMP-1. By utilizing Cre/loxP recombination coupled with the IR-LEGO system to induce SMP-1 at a physiological level, we found that SMP-1 interacts with PLX-1 only in trans upon contact between vulval precursor cells. In contrast, when overexpressed, SMP-1 exhibits the ability to cis-interact with PLX-1 on a single cell. These results indicate that mosaic analysis with IR-LEGO, especially when combined with an in vivo recombination system, efficiently complements conventional methods.

Discovery of a novel siphonaxanthin biosynthetic precursor in Codium fragile that accumulates only by exposure to blue-green light.

Photosynthetic organisms adapt to a variety of light conditions. Codium fragile, a macrosiphonous green alga, binds a unique carbonyl carotenoid, siphonaxanthin, to its major photosynthetic light-harvesting complexes, allowing it to utilize dim blue-green light for photosynthesis. Here, we describe the absolute chemical structure of a novel siphonaxanthin biosynthetic precursor, 19-deoxysiphonaxanthin, that accumulates specifically in the photosynthetic antenna only when cultivated under blue-green light. The action spectra of pigment accumulation suggest that siphonaxanthin biosynthesis is regulated by a specific wavelength profile. The results provide clues to a new acclimation mechanism to withstand hours of intense light at low tide and why siphonous algae have been growing invasively on the world's coasts for more than a century.

Development of a screening system for agents that modulate taste receptor expression with the CRISPR-Cas9 system in medaka.

Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.