Allometry in limb regeneration and scale-invariant patterning as the basis of normal morphogenesis from different sizes of blastemas.

Axolotl (Ambystoma mexicanum) limb regeneration begins with blastemas of various sizes, in contrast to the limb developmental process. Despite this size variation, normal limb morphology, consistent with a limb stump size, is regenerated. This outcome suggests the existence of underlying scale-invariant mechanisms. To identify such mechanisms, we examined the allometric relationships between blastema size, and Sonic Hedgehog (Shh) and Fibroblast Growth Factor 8 (Fgf8) expression patterns against limb stump size. We found that all factors showed allometric rather than isometric scaling; specifically, their relative sizes decrease with an increase in limb stump size. However, the ratio of Shh/Fgf8 signaling dominant region was nearly constant, independent of blastema/body size. Furthermore, the relative spatial patterns of cell density and proliferation activity and the relative position of first digit formation were scale-invariant in the summed Shh/Fgf8 crosstalk region. This scale-invariant nature may underlie the morphogenesis of normal limbs from different sizes of blastemas.

Two types of critical cell density for mechanical elimination of abnormal cell clusters from epithelial tissue.

Recent technological advances in high-resolution imaging and artificial modulation of genetic functions at different times and regions have enabled direct observations of the formation and elimination of abnormal cell populations. A recent trend in cell competition research is the incorporation of cell mechanics. In different tissues and species, abnormal cells developing in epithelial tissues are mechanically eliminated by cell contraction via actomyosin accumulation at the interface between normal and abnormal cells. This mechanical cell elimination process has attracted attention as a potential universal defense mechanism. Here, we theoretically examined the conditions for mechanical elimination of growing abnormal cell populations. Simulations and mathematical analyses using a vertex dynamics model revealed two types of critical cell density associated with mechanical elimination of abnormal cell clusters. One is a subtype of homeostatic density, in which the frequencies of spontaneous mechanical cell elimination and proliferation are balanced, even if no explicit dependence of proliferation or apoptosis on the cell density is assumed. This density is related to the mechanical stability of a single cell. The other is density related to mechanical stability as a cell population under external pressure. Both density types are determined by tissue mechanical properties. In solid tissues, the former type is reached first as the intensity of interfacial contraction increases, and it functions as a critical density. On the other hand, the latter type becomes critical when tissues are highly fluid. The derived analytical solution explicitly reveals the dependence of critical contractile force and density on different parameters. We also found a negative correlation between the proliferation rate of abnormal cells and the likelihood of the abnormal cell population expanding by escaping elimination. This is counterintuitive because in the context of cell competition, fast-growing cell populations generally win. These findings provide new insight into, and interpretation of, the results from experimental studies.

Variant PRC1 competes with retinoic acid-related signals to repress Meis2 in the mouse distal forelimb bud.

Suppression of Meis genes in the distal limb bud is required for proximal-distal (PD) specification of the forelimb. Polycomb group (PcG) factors play a role in downregulation of retinoic acid (RA)-related signals in the distal forelimb bud, causing Meis repression. It is, however, not known whether downregulation of RA-related signals and PcG-mediated proximal gene repression are functionally linked. Here, we reveal that PcG factors and RA-related signals antagonize each other to polarize Meis2 expression along the PD axis in mouse. Supported by mathematical modeling and simulation, we propose that PcG factors are required to adjust the threshold for RA-related signaling to regulate Meis2 expression. Finally, we show that a variant Polycomb repressive complex 1 (PRC1), incorporating PCGF3 and PCGF5, represses Meis2 expression in the distal limb bud. Taken together, we reveal a previously unknown link between PcG proteins and downregulation of RA-related signals to mediate the phase transition of Meis2 transcriptional status during forelimb patterning.

Light-induced local gene expression in primary chick cell culture system.

The ability to manipulate gene expression at a specific region in a tissue or cell culture system is critical for analysis of target gene function. For chick embryos/cells, several gene introduction/induction methods have been established such as those involving retrovirus, electroporation, sonoporation, and lipofection. However, these methods have limitations in the accurate induction of localized gene expression. Here we demonstrate the effective application of a recently developed light-dependent gene expression induction system (LightOn system) using the Neurospora crassa photoreceptor Vivid fused with a Gal4 DNA binding domain and p65 activation domain (GAVPO) that alters its activity in response to light stimulus in a primary chicken cell culture system. We show that the gene expression level and induction specificity in this system are strongly dependent on the light irradiation conditions. Especially, the irradiation interval is an important parameter for modulating gene expression; for shorter time intervals, higher induction specificity can be achieved. Further, by adjusting light irradiation conditions, the expression level in primary chicken cells can be regulated in a multiple step manner, in contrast to the binary expression seen for gene disruption or introduction (i.e., null or overexpression). This result indicates that the light-dependent expression control method can be a useful technique in chick models to examine how gene function is affected by gradual changes in gene expression levels. We applied this light induction system to regulate Sox9 expression in cultures of chick limb mesenchyme cells and showed that induced SOX9 protein could modulate expression of downstream genes.

Dynamic Polymer Networks: A New Avenue towards Sustainable and Advanced Soft Machines.

While the fascinating field of soft machines has grown rapidly over the last two decades, the materials they are constructed from have remained largely unchanged during this time. Parallel activities have led to significant advances in the field of dynamic polymer networks, leading to the design of three-dimensionally cross-linked polymeric materials that are able to adapt and transform through stimuli-induced bond exchange. Recent work has begun to merge these two fields of research by incorporating the stimuli-responsive properties of dynamic polymer networks into soft machine components. These include dielectric elastomers, stretchable electrodes, nanogenerators, and energy storage devices. In this Minireview, we outline recent progress made in this emerging research area and discuss future directions for the field.

Quantitative analysis of tissue deformation dynamics reveals three characteristic growth modes and globally aligned anisotropic tissue deformation during chick limb development.

Tissue-level characterization of deformation dynamics is crucial for understanding organ morphogenetic mechanisms, especially the interhierarchical links among molecular activities, cellular behaviors and tissue/organ morphogenetic processes. Limb development is a well-studied topic in vertebrate organogenesis. Nevertheless, there is still little understanding of tissue-level deformation relative to molecular and cellular dynamics. This is mainly because live recording of detailed cell behaviors in whole tissues is technically difficult. To overcome this limitation, by applying a recently developed Bayesian approach, we here constructed tissue deformation maps for chick limb development with high precision, based on snapshot lineage tracing using dye injection. The precision of the constructed maps was validated with a clear statistical criterion. From the geometrical analysis of the map, we identified three characteristic tissue growth modes in the limb and showed that they are consistent with local growth factor activity and cell cycle length. In particular, we report that SHH signaling activity changes dynamically with developmental stage and strongly correlates with the dynamic shift in the tissue growth mode. We also found anisotropic tissue deformation along the proximal-distal axis. Morphogenetic simulation and experimental studies suggested that this directional tissue elongation, and not local growth, has the greatest impact on limb shaping. This result was supported by the novel finding that anisotropic tissue elongation along the proximal-distal axis occurs independently of cell proliferation. Our study marks a pivotal point for multi-scale system understanding in vertebrate development.

Morphometric staging of organ development based on cross sectional images.

An objective, continuous, and robust method for staging developing embryos or organs is essential for providing a common measure of time when studying quantitative/systems developmental biology. However, classical methods based on factors such as somite number or qualitative visual attributes are discrete and/or ambiguous due to observers' subjectivity. Thus, an alternative staging method based on an explicit and continuous description of developmental states over time, such as anatomy/morphology, is needed. Here, we briefly propose a novel staging method as a natural extension of classical staging based on cross sectional images of organs, which are more accessible than full 3D structures. The contours are represented as 2D closed curves and quantified using elliptic Fourier descriptors. Treating the ambiguity in classical staging as a statistical model, the relationship between the novel morphometric staging and classical staging can be determined. This method was validated by applying it to two different sets of data: chick forebrain and Xenopus hindlimb development. Using this method, it is also possible to reconstruct the time evolution of the average morphology, which would be useful for quantitatively comparing morphologies between embryos or between normal and abnormal conditions.

Possible roles of mechanical cell elimination intrinsic to growing tissues from the perspective of tissue growth efficiency and homeostasis.

Cell competition is a phenomenon originally described as the competition between cell populations with different genetic backgrounds; losing cells with lower fitness are eliminated. With the progress in identification of related molecules, some reports described the relevance of cell mechanics during elimination. Furthermore, recent live imaging studies have shown that even in tissues composed of genetically identical cells, a non-negligible number of cells are eliminated during growth. Thus, mechanical cell elimination (MCE) as a consequence of mechanical cellular interactions is an unavoidable event in growing tissues and a commonly observed phenomenon. Here, we studied MCE in a genetically-homogeneous tissue from the perspective of tissue growth efficiency and homeostasis. First, we propose two quantitative measures, cell and tissue fitness, to evaluate cellular competitiveness and tissue growth efficiency, respectively. By mechanical tissue simulation in a pure population where all cells have the same mechanical traits, we clarified the dependence of cell elimination rate or cell fitness on different mechanical/growth parameters. In particular, we found that geometrical (specifically, cell size) and mechanical (stress magnitude) heterogeneities are common determinants of the elimination rate. Based on these results, we propose possible mechanical feedback mechanisms that could improve tissue growth efficiency and density/stress homeostasis. Moreover, when cells with different mechanical traits are mixed (e.g., in the presence of phenotypic variation), we show that MCE could drive a drastic shift in cell trait distribution, thereby improving tissue growth efficiency through the selection of cellular traits, i.e. intra-tissue "evolution". Along with the improvement of growth efficiency, cell density, stress state, and phenotype (mechanical traits) were also shown to be homogenized through growth. More theoretically, we propose a mathematical model that approximates cell competition dynamics, by which the time evolution of tissue fitness and cellular trait distribution can be predicted without directly simulating a cell-based mechanical model.

Adaptive significance of critical weight for metamorphosis in holometabolous insects.

Holometabolous insect larvae become committed to metamorphosis when they reach a critical weight. Although the physiological mechanisms involved in this process have been well-studied, the adaptive significance of the critical weight remains unclear. Here, we developed a life history model for holometabolous insects and evaluated it from the viewpoint of optimal energy allocation. We found that, without a priori assumptions about critical weight, the optimal growth schedule is always biphasic: larval tissues grow predominately until they reach a certain threshold, after which the imaginal tissues begin rapid growth, suggesting that the emergence of a critical weight as a phase-transition point is a natural consequence of optimal growth scheduling. Our model predicts the optimal timing of critical-weight attainment, in agreement with observations in phylogenetically-distinct species. Furthermore, it also predicts the scaling of growth scheduling against environmental change, i.e., the relative value and timing of the critical weight should be constant, thus providing a general interpretation of observed phenotypic plasticity. This scaling relationship allows the classification of adaptive responses in critical weight into five possible types that reflect the ecological features of focal insects. In this manner, our theory and its consistency with experimental observations demonstrate the adaptive significance of critical weight.

Novel features of the Mullins effect in filled elastomers revealed by stretching measurements in various geometries.

Stretching experiments with various geometries are performed using a custom-built tensile tester to reveal the intriguing features of the mechanical softening phenomena of filled elastomers in loading-unloading cycles, commonly known as the Mullins effect. The dissipated energy (D), residual strain (εr), and dissipation factor (Δ; the ratio of D to input strain energy) in the loading-unloading cycles are evaluated as a function of the maximum stretch in cyclic loading (λm) using three types of extension, i.e., uniaxial, planar, and equibiaxial extension, for silica-filled elastomers with various filler contents, and with or without a silane coupling agent. The dissipated energy D and εr increase with an increase in λm, and they depend on the type of extension when compared at the same λm: D and εr increase in the order of equibiaxial, planar, and uniaxial extension. In contrast, the values of Δ obtained for various degrees and types of extension are collapsed into a single curve when the first invariant of the deformation tensor (I1,m) corresponding to λm is employed as a variable: Δ steeply increases with an increase in I1,m in the small deformation regime of I1,m < 3.2, while Δ levels off in the large deformation regime of I1,m > 3.5. The plateau values of Δ increase with an increase in filler content. The characteristic dependence of Δ on I1,m in each of the small and large deformation regimes is expected to reflect the destruction process of the inherent structures, including filler networks and the filler-polymer interface, and the friction between the fillers and the rubber matrix, respectively.

Wnt11 gene therapy with adeno-associated virus 9 improves the survival of mice with myocarditis induced by coxsackievirus B3 through the suppression of the inflammatory reaction.

The wnt signaling pathway plays important roles in development and in many diseases. Recently several reports suggest that non-canonical Wnt proteins contribute to the inflammatory response in adult animals. However, the effects of Wnt proteins on virus-induced myocarditis have not been explored. Here, we investigated the effect of Wnt11 protein in a model of myocarditis induced by coxsackievirus B3 (CVB3) using recombinant adeno-associated virus 9 (rAAV9). The effect of Wnt11 gene therapy on a CVB3-induced myocarditis model was examined using male BALB/c mice. Mice received a single intravenous injection of either rAAV9-Wnt11 or rAAV9-LacZ 2 weeks before intraperitoneal administration of CVB3. Intravenous injection of the rAAV9 vector resulted in efficient, durable, and relatively cardiac-specific transgene expression. Survival was significantly greater among rAAV9-Wnt11 treated mice than among mice treated with rAAV9-LacZ (87.5% vs. 54.1%, P < 0.05). Wnt11 expression also reduced the infiltration of inflammatory cells, necrosis of the myocardium, and suppressed the mRNA expression of inflammatory cytokines. This is the first report to show that Wnt11 expression improves the survival of mice with CVB3-induced myocarditis. AAV9-mediated Wnt11 gene therapy produces beneficial effects on cardiac function and increases the survival of mice with CVB3-induced myocarditis through the suppression of both infiltration of inflammatory cells and gene expression of inflammatory cytokines.

Application of local gene induction by infrared laser-mediated microscope and temperature stimulator to amphibian regeneration study.

Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three-dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single-cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR-LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature-controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR-LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre-LoxP system as well as to analyze gene function in a target region or cells with less off-target effects in the study of amphibian regeneration.

Cellular sensory mechanisms for detecting specific fold-changes in extracellular cues.

Cellular sensory systems often respond not to the absolute levels of inputs but to the fold-changes in inputs. Such a property is called fold-change detection (FCD) and is important for accurately sensing dynamic changes in environmental signals in the presence of fluctuations in their absolute levels. Previous studies defined FCD as input-scale invariance and proposed several biochemical models that achieve such a condition. Here, we prove that the previous FCD models can be approximated by a log-differentiator. Although the log-differentiator satisfies the input-scale invariance requirement, its response amplitude and response duration strongly depend on the input timescale. This creates limitations in the specificity and repeatability of detecting fold-changes in inputs. Nevertheless, FCD with specificity and repeatability by cells has been reported in the context of Drosophila wing development. Motivated by this fact and by extending previous FCD models, we here propose two possible mechanisms to achieve FCD with specificity and repeatability. One is the integrate-and-fire type: a system integrates the rate of temporal change in input and makes a response when the integrated value reaches a constant threshold, and this is followed by the reset of the integrated value. The other is the dynamic threshold type: a system response occurs when the input level reaches a threshold, whose value is multiplied by a certain constant after each response. These two mechanisms can be implemented biochemically by appropriately combining feed-forward and feedback loops. The main difference between the two models is their memory of input history; we discuss possible ways to distinguish between the two models experimentally.

Bayesian inference of whole-organ deformation dynamics from limited space-time point data.

To understand the morphogenetic mechanisms of organ development and regeneration, it is essential to clarify the inter-hierarchical relationship between microscopic, molecular/cellular activities and organ-level tissue deformation dynamics. While the former have been studied for several decades, the latter - macroscopic geometrical information about physical tissue deformation - is often missing, especially for many vertebrates. This is mainly because live recording of detailed cell behaviors in whole tissues during vertebrate organogenesis is technically difficult. In this study, we have developed a novel method that combines snapshot lineage tracing with Bayesian statistical estimation to construct whole-organ deformation maps from landmark data on limited numbers of space-time points. Following the validation of the method using artificially generated data sets, we applied it to the analysis of tissue deformation dynamics in chick limb development. A quantitative tissue deformation map for St.23-St.24 has been constructed, and its precision has been proven by evaluating its predictive performance. Geometrical analyses of the map have revealed a spatially heterogeneous volume growth pattern that is consistent with the expression pattern of a major morphogen and anisotropic tissue deformation along an axis. Thus, our method enables deformation dynamics analysis in organogenesis using practical lineage marking techniques.

Canalization-based vein formation in a growing leaf.

Vein formation is an important process in plant leaf development. The phytohormone auxin is known as the most important molecule for the control of venation patterning; and the canalization model, in which cells experiencing higher auxin flux differentiate into specific cells for auxin transportation, is widely accepted. To date, several mathematical models based on the canalization hypothesis have been proposed that have succeeded in reproducing vein patterns similar to those observed in actual leaves. However, most previous studies focused on patterning in fixed domains, and, in a few exceptional studies, limited tissue growth - such as cell proliferation at leaf margins and small deformations without large changes in cell number - were dealt with. Considering that, in actual leaf development, venation patterning occurs in an exponentially growing tissue, whether the canalization hypothesis still applies is an important issue to be addressed. In this study, we first show through a pilot simulation that the coupling of chemical dynamics for canalization and tissue growth as independent models cannot reproduce normal venation patterning. We then examine conditions sufficient for achieving normal patterning in a growing leaf by introducing various constraints on chemical dynamics, tissue growth, and cell mechanics; in doing so, we found that auxin flux- or differentiation-dependent modification of the cell cycle and elasticity of cell edges are essential. The predictions given by our simulation study will serve as guideposts in experiments aimed at finding the key factors for achieving normal venation patterning in developing plant leaves.

hoxc12/c13 as key regulators for rebooting the developmental program in Xenopus limb regeneration.

During organ regeneration, after the initial responses to injury, gene expression patterns similar to those in normal development are reestablished during subsequent morphogenesis phases. This supports the idea that regeneration recapitulates development and predicts the existence of genes that reboot the developmental program after the initial responses. However, such rebooting mechanisms are largely unknown. Here, we explore core rebooting factors that operate during Xenopus limb regeneration. Transcriptomic analysis of larval limb blastema reveals that hoxc12/c13 show the highest regeneration specificity in expression. Knocking out each of them through genome editing inhibits cell proliferation and expression of a group of genes that are essential for development, resulting in autopod regeneration failure, while limb development and initial blastema formation are not affected. Furthermore, the induction of hoxc12/c13 expression partially restores froglet regenerative capacity which is normally very limited compared to larval regeneration. Thus, we demonstrate the existence of genes that have a profound impact alone on rebooting of the developmental program in a regeneration-specific manner.

Random cell movement promotes synchronization of the segmentation clock.

In vertebrate somitogenesis, the expression of segmentation clock genes oscillates and the oscillation is synchronized over nearby cells. Both experimental and theoretical studies have shown that the synchronization among cells is realized by intercellular interaction via Delta-Notch signaling. However, the following questions emerge: (i) During somitogenesis, dynamic rearrangement of relative cell positions is observed in the posterior presomitic mesoderm. Can a synchronized state be stably sustained under random cell movement? (ii) Experimental studies have reported that the synchronization of cells can be recovered in about 10 or fewer oscillation cycles after the complete loss of synchrony. However, such a quick recovery of synchronization is not possible according to previous theoretical models. In this paper, we first show by numerical modeling that synchronized oscillation can be sustained under random cell movement. We also find that for initial perturbation, the synchronization of cells is recovered much faster and it is for a wider range of reaction parameters than the case without cell movement. When the posterior presomitic mesoderm is rectangular, faster synchronization is achieved if cells exchange their locations more with neighbors located along the longer side of the domain. Finally, we discuss that the enhancement of synchronization by random cell movement occurs in several different models for the oscillation of segmentation clock genes.

Endoscopic Aortic Valve Replacement: Initial Outcomes of Isolated and Concomitant Surgery.

BACKGROUND: The applicability of totally endoscopic surgical aortic valve replacement (AVR) in multivalve operations is unknown. This study describes an approach and perioperative outcomes of totally endoscopic isolated and concomitant AVR using various valve types. METHODS: A total of 216 patients (114 male; mean age, 71.3 ± 11.3 years) underwent totally endoscopic AVR from May 2017 to October 2022 in a tertiary care center. The 3-port technique was used: a 3- to 4-cm main port without rib spreading, a 10-mm 3-dimensional endoscopic port, and a 5-mm left-hand port with femoral cannulations. Sutures were hand tied with a knot pusher. Descriptive analyses compared perioperative outcomes between patients with or without concomitant procedures. RESULTS: Of 216 patients, concomitant surgery was performed in 33 (15.2%) patients. Of the 33, 21 (63.6%) had a concomitant mitral procedure. A stented bioprosthesis was implanted in 165 (76.3%) patients, a mechanical valve in 22 (10.2%) patients, and a rapid deployment or sutureless valve in 29 (13.4%) patients. Median operation time and aortic cross-clamp time were 175 minutes (interquartile range; 150-194 minutes) and 78 minutes (interquartile range; 67-92 minutes) for isolated AVR, respectively. Thirty-day mortality occurred in 1 patient (0.5%). Two patients (0.9%) had conversion to sternotomy. Major neurologic events occurred in 3 patients (1.4%). The major adverse event rate was similar between patients with or without concomitant procedures. CONCLUSIONS: Endoscopic AVR can safely address concomitant valve diseases.

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.