Mechanosensory and command contributions to the Drosophila grooming sequence.

Flies groom in response to competing mechanosensory cues in an anterior-to-posterior order using specific legs. From behavior screens, we identified a pair of cholinergic command-like neurons, Mago-no-Te (MGT), whose optogenetic activation elicits thoracic grooming by the back legs. Thoracic grooming is typically composed of body sweeps and leg rubs in alternation, but clonal analysis coupled with amputation experiments revealed that MGT activation only commands the body sweeps: initiation of leg rubbing requires contact between the leg and thorax. With new electron microscopy (EM) connectome data for the ventral nerve cord (VNC), we uncovered a circuit-based explanation for why stimulation of posterior thoracic mechanosensory bristles initiates cleaning by the back legs. Our previous work showed that flies weigh mechanosensory inputs across the body to select which part to groom, but we did not know why the thorax was always cleaned last. Here, the connectome for the VNC enabled us to identify a pair of GABAergic inhibitory neurons, UMGT1, that receives diverse sensory inputs and synapses onto both MGT and components of its downstream circuits. Optogenetic activation of UMGT1 suppresses thoracic cleaning, representing a mechanism by which mechanosensory stimuli on other body parts could take precedence in the grooming hierarchy. We also anatomically mapped the pre-motor circuit downstream of MGT, including inhibitory feedback connections that may enable rhythmicity and coordination of limb movement during thoracic grooming. The combination of behavioral screens and connectome analysis allowed us to identify a neural circuit connecting sensory-to-motor neurons that contributes to thoracic grooming.

Epithelial-mesenchymal transition contrast in the amputated tail and limb of the northern house gecko, Hemidactylus flaviviridis.

The northern house gecko Hemidactylus flaviviridis exhibits appendage-specific responses to injuries. The autotomized tail regenerates, whereas the severed limb fails to regrow. Many site-specific cellular processes influence tail regeneration. Herein, we analyzed the epithelial-mesenchymal transition contrast in the lizard's amputated appendages (tail and limb). Morphological observations in the healing frame indicated the formation of regeneration blastema in the tail and scar formation in limb. Histology of the tail showed that epithelial cells closer to mesenchyme appeared less columnar and loosely packed, with little intercellular matrix. Whereas in the limb, the columnar epithelial cells remained tightly packed. Collagen deposition was seen in the limb at the intersection of wound epithelium and mesenchyme, favoring scarring by blocking the epithelial-mesenchymal transition. Markers for epithelial-mesenchymal transition were assessed at transcript and protein levels. The regenerating tail showed upregulation of N-cadherin, vimentin, and PCNA, favoring epithelial-mesenchymal transition, cell migration, and proliferation, respectively. In contrast, the scarring limb showed persistently elevated levels of E-cadherin and EpCAM, indicating retention of epithelial characteristics. An attempt was made to screen the resident epithelial stem cell population in both appendages to check their potential role in the epithelial-mesenchymal transition (EMT), hence the differential wound healing. Upregulation in transcript and protein levels of Nanog and Sox2 was observed in the regenerating tail. Fluorescence-activated cell sorting (FACS) provided supporting evidence that the epithelial stem cell population in tail remained significantly higher than in limb. Thus, this study focuses on the mechanistic role of the epithelial-mesenchymal transition in wound healing, highlighting the molecular details of regeneration and scarring events.

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.

Mechanosensory encoding of forces in walking uphill and downhill: force feedback can stabilize leg movements in stick insects.

Force feedback could be valuable in adapting walking to diverse terrains, but the effects of changes in substrate inclination on discharges of sensory receptors that encode forces have rarely been examined. In insects, force feedback is provided by campaniform sensilla, mechanoreceptors that monitor forces as cuticular strains. We neurographically recorded responses of stick insect tibial campaniform sensilla to "naturalistic" forces (joint torques) that occur at the hind leg femur-tibia (FT) joint in uphill, downhill, and level walking. The FT joint torques, obtained in a previous study that used inverse dynamics to analyze data from freely moving stick insects, are quite variable during level walking (including changes in sign) but are larger in magnitude and more consistent when traversing sloped surfaces. Similar to vertebrates, insects used predominantly extension torque in propulsion on uphill slopes and flexion torques to brake forward motion when going downhill. Sensory discharges to joint torques reflected the torque direction but, unexpectedly, often occurred as multiple bursts that encoded the rate of change of positive forces (dF/dt) even when force levels were high. All discharges also showed hysteresis (history dependence), as firing substantially decreased or ceased during transient force decrements. These findings have been tested in simulation in a mathematical model of the sensilla (Szczecinski NS, Dallmann CJ, Quinn RD, Zill SN. Bioinspir Biomim 16: 065001, 2021) that accurately reproduced the biological data. Our results suggest the hypothesis that sensory feedback from the femoro-tibial joint indicating force dynamics (dF/dt) can be used to counter the instability in traversing sloped surfaces in animals and, potentially, in walking machines.NEW & NOTEWORTHY Discharges of sensory receptors (campaniform sensilla) in the hind legs of stick insects can differentially signal forces that occur in walking uphill versus walking downhill. Unexpectedly, sensory firing most closely reflects the rate of change of force (dF/dt) even when the force levels are high. These signals have been replicated in a mathematical model of the receptors and could be used to stabilize leg movements both in the animal and in a walking robot.

Mechanical modeling of mechanosensitive insect strain sensors as a tool to investigate exoskeletal interfaces.

During walking, sensory information is measured and monitored by sensory organs that can be found on and within various limb segments. Strain can be monitored by insect load sensors, campaniform sensilla (CS), which have components embedded within the exoskeleton. CS vary in eccentricity, size, and orientation, which can affect their sensitivity to specific strains. Directly investigating the mechanical interfaces that these sensors utilize to encode changes in load bears various obstacles, such as modeling of viscoelastic properties. To circumvent the difficulties of modeling and performing biological experiments in small insects, we developed 3-dimensional printed resin models based on high-resolution imaging of CS. Through the utilization of strain gauges and a motorized tensile tester, physiologically plausible strain can be mimicked while investigating the compression and tension forces that CS experience; here, this was performed for a field of femoral CS inDrosophila melanogaster. Different loading scenarios differentially affected CS compression and the likely neuronal activity of these sensors and elucidate population coding of stresses acting on the cuticle.

Protein-protein interaction network module changes associated with the vertebrate fin-to-limb transition.

Evolutionary phenotypic transitions, such as the fin-to-limb transition in vertebrates, result from modifications in related proteins and their interactions, often in response to changing environment. Identifying these alterations in protein networks is crucial for a more comprehensive understanding of these transitions. However, previous research has not attempted to compare protein-protein interaction (PPI) networks associated with evolutionary transitions, and most experimental studies concentrate on a limited set of proteins. Therefore, the goal of this work was to develop a network-based platform for investigating the fin-to-limb transition using PPI networks. Quality-enhanced protein networks, constructed by integrating PPI networks with anatomy ontology data, were leveraged to compare protein modules for paired fins (pectoral fin and pelvic fin) of fishes (zebrafish) to those of the paired limbs (forelimb and hindlimb) of mammals (mouse). This also included prediction of novel protein candidates and their validation by enrichment and homology analyses. Hub proteins such as shh and bmp4, which are crucial for module stability, were identified, and their changing roles throughout the transition were examined. Proteins with preserved roles during the fin-to-limb transition were more likely to be hub proteins. This study also addressed hypotheses regarding the role of non-preserved proteins associated with the transition.

Evolutionarily divergent mTOR remodels translatome for tissue regeneration.

An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

Independent and combined associations of upper and lower limb strength with all-cause mortality in community-based older adults: findings from the Chinese Longitudinal Healthy Longevity Survey.

OBJECTIVES: Life expectancy is increasing around the world, and it has been projected that China will have the largest elderly population globally by 2033. This study aimed to examine the association of upper limb strength (ULS) and lower limb strength (LLS) with all-cause mortality based on data from the Chinese Longitudinal Healthy Longevity Survey (2012-2018). STUDY DESIGN: This is a prospective cohort study. METHODS: Participants were 2442 older adults (aged 84.98 ± 11.94 years) recruited from eight regions with a high elderly population in China. Limb muscle strength was evaluated using handgrip strength and objective physical examinations. Cox proportional hazards regression was used to analyse the association of limb muscle strength with all-cause mortality. Demographic characteristics, health status and biological markers were included as confounders. RESULTS: Over a median follow-up period of 42.2 months, 993 participants died. After adjusting for all covariates, low ULS was associated with a higher mortality risk (hazard ratio [HR] = 1.51, 95% confidence interval [CI] = 1.25-1.84), and the association of low LLS with all-cause mortality was only significant for men (HR = 1.36, 95% CI = 1.04-1.79). Participants with combined low ULS and low LLS had the highest risk of mortality compared with participants with normal limb muscle strength (HR = 2.06, 95% CI = 1.61-2.63). The combined association of ULS and LLS with mortality was robust in subgroup and sensitivity analyses. CONCLUSION: Low ULS and low LLS were independently and synergistically associated with a higher all-cause mortality risk. Considering the high prevalence of limb muscle weakness among older adults in China, especially in those aged ≥80 years, limb strength could be considered as an easy-to-perform potential mortality predictor in community health care.

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.

Biomechanics, motor control and dynamic models of the soft limbs of the octopus and other cephalopods.

Muscular hydrostats are organs composed entirely of packed arrays of incompressible muscles and lacking any skeletal support. Found in both vertebrates and invertebrates, they are of great interest for comparative biomechanics from engineering and evolutionary perspectives. The arms of cephalopods (e.g. octopus and squid) are particularly interesting muscular hydrostats because of their flexibility and ability to generate complex behaviors exploiting elaborate nervous systems. Several lines of evidence from octopus studies point to the use of both brain and arm-embedded motor control strategies that have evolved to simplify the complexities associated with the control of flexible and hyper-redundant limbs and bodies. Here, we review earlier and more recent experimental studies on octopus arm biomechanics and neural motor control. We review several dynamic models used to predict the kinematic characteristics of several basic motion primitives, noting the shortcomings of the current models in accounting for behavioral observations. We also discuss the significance of impedance (stiffness and viscosity) in controlling the octopus's motor behavior. These factors are considered in light of several new models of muscle biomechanics that could be used in future research to gain a better understanding of motor control in the octopus. There is also a need for updated models that encompass stiffness and viscosity for designing and controlling soft robotic arms. The field of soft robotics has boomed over the past 15 years and would benefit significantly from further progress in biomechanical and motor control studies on octopus and other muscular hydrostats.

A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.

Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.

Bmp-signaling and the finfold size in zebrafish: implications for the fin-to-limb transition.

In tetrapods, BMP-signaling coordinates limb outgrowth, skeleton patterning, and apoptosis during the formation of their typical autopod structures, the digits. In addition, inhibition of BMP signaling during mouse limb development leads to the persistence and enlargement of an important signaling center, the apical ectodermal ridge (AER), and consequent digit defects. Interestingly, during fish fin development there is a natural elongation of the AER, rapidly converted into an apical finfold (FF), in which osteoblasts differentiate into dermal fin-rays used in aquatic locomotion. Previous reports have led us to suggest that upregulation of Hox13 genes in the distal fin mesenchyme, caused by the origin of novel enhancer modules, may have caused an increment of the BMP signaling potentially leading to the apoptosis of these osteoblasts precursors of the fin-rays. To explore this hypothesis, we characterized the expression of several components of the BMP signaling in zebrafish lines with distinct FF sizes (bmp2b, smad1, smoc1, smoc2, grem1a, msx1b, msx2b, Psamd1/5/9). Our data suggest that the BMP signaling is enhanced in shorter FFs and inhibited in longer FFs, as implied by the differential expression of several components of this network. In addition, we detected an earlier expression of several of these BMP-signaling components associated with the development of short FFs and the opposite tendency during the development of longer FFs. Thus, our results suggest that a heterochronic shift, involving enhanced Hox13 expression and BMP signaling may have caused the reduction of the FF size during the evolutionary transition from fish fins to tetrapod limbs.

Unravelling the limb regeneration mechanisms of Polypedates maculatus, a sub-tropical frog, by transcriptomics.

BACKGROUND: Regeneration studies help to understand the strategies that replace a lost or damaged organ and provide insights into approaches followed in regenerative medicine and engineering. Amphibians regenerate their limbs effortlessly and are indispensable models to study limb regeneration. Xenopus and axolotl are the key models for studying limb regeneration but recent studies on non-model amphibians have revealed species specific differences in regeneration mechanisms. RESULTS: The present study describes the de novo transcriptome of intact limbs and three-day post-amputation blastemas of tadpoles and froglets of the Asian tree frog Polypedates maculatus, a non-model amphibian species commonly found in India. Differential gene expression analysis between early tadpole and froglet limb blastemas discovered species-specific novel regulators of limb regeneration. The present study reports upregulation of proteoglycans, such as epiphycan, chondroadherin, hyaluronan and proteoglycan link protein 1, collagens 2,5,6, 9 and 11, several tumour suppressors and methyltransferases in the P. maculatus tadpole blastemas. Differential gene expression analysis between tadpole and froglet limbs revealed that in addition to the expression of larval-specific haemoglobin and glycoproteins, an upregulation of cysteine and serine protease inhibitors and downregulation of serine proteases, antioxidants, collagenases and inflammatory genes in the tadpole limbs were essential for creating an environment that would support regeneration. Dermal myeloid cells were GAG+, EPYC+, INMT+, LEF1+ and SALL4+ and seemed to migrate from the unamputated regions of the tadpole limb to the blastema. On the other hand, the myeloid cells of the froglet limb blastemas were few and probably contributed to sustained inflammation resulting in healing. CONCLUSIONS: Studies on non-model amphibians give insights into alternate tactics for limb regeneration which can help devise a plethora of methods in regenerative medicine and engineering.

Adapting the pantograph limb: Differential robustness of fore- and hindlimb kinematics against genetically induced perturbation in the neural control networks and its evolutionary implications.

The evolutionary transformation of limb morphology to the four-segmented pantograph of therians is among the milestones of mammalian evolution. But, it is still unknown if changes of the mechanical limb function were accompanied by corresponding changes in development and sensorimotor control. The impressive locomotor performance of mammals leaves no doubt about the high integration of pattern formation, neural control and mechanics. But, deviations from normal intra- and interlimb coordination (spatial and temporal) become evident in the presence of perturbations. We induced a perturbation in the development of the neural circuits of the spinal cord of mice (Mus musculus) using a deletion of the Wilms tumor suppressor gene Wt1 in a subpopulation of dI6 interneurons. These interneurons are assumed to participate in the intermuscular coordination within the limb and in left-right-coordination between the limbs. We describe the locomotor kinematics in mice with conditional Wt1 knockout and compare them to mice without Wt1 deletion. Unlike knockout neonates, knockout adult mice do not display severe deviations from normal (=control group) interlimb coordination, but the coordinated protraction and retraction of the limbs is altered. The forelimbs are more affected by deviations from the control than the hindlimbs. This observation appears to reflect a different degree of integration and resistance against the induced perturbation between the limbs. Interestingly, the observed effects are similar to locomotor deficits reported to arise when sensory feedback from proprioceptors or cutaneous receptors is impaired. A putative participation of Wt1 positive dI6 interneurons in sensorimotor integration is therefore considered.

Limb blastema formation: How much do we know at a genetic and epigenetic level?

Regeneration of missing body parts is an incredible ability which is present in a wide number of species. However, this regenerative capability varies among different organisms. Urodeles (salamanders) are able to completely regenerate limbs after amputation through the essential process of blastema formation. The blastema is a collection of relatively undifferentiated progenitor cells that proliferate and repattern to form the internal tissues of a regenerated limb. Understanding blastema formation in salamanders may enable comparative studies with other animals, including mammals, with more limited regenerative abilities and may inspire future therapeutic approaches in humans. This review focuses on the current state of knowledge about how limb blastemas form in salamanders, highlighting both the possible roles of epigenetic controls in this process as well as limitations to scientific understanding that present opportunities for research.

Bead Implantation and Delivery of Exogenous Growth Factors.

Genetic methods in axolotls (Ambystoma mexicanum) remain in their infancy which has hampered the study of limb regeneration. There is much room for advancement, especially with respect to spatiotemporal regulation of gene expression. Secreted growth factors play a major role in each stage of regeneration. The use of slow-release beads is one of the most effective methods to control the spatiotemporal expression of secretory gene products. The topical administration of secreted factors by slow-release beads may also prove effective for future applications in non-regenerative animals and for medical applications in humans, in which genetic methods are not available. In this chapter, we describe a methodology for using and implanting slow-release beads to deliver exogenous growth factors to salamanders.

The Accessory Limb Model Regenerative Assay and Its Derivatives.

When the Accessory Limb Model (ALM) regenerative assay was first published by Endo, Bryant, and Gardiner in 2004, it provided a robust system for testing the cellular and molecular contributions during each of the basic steps of regeneration: the formation of the wound epithelium, neural induction of the apical epithelial cap, and the formation of a positional disparity between blastema cells. The basic ALM procedure was developed in the axolotl and involves deviating a limb nerve into a lateral wound and grafting skin from the opposing side of the limb axis into the site of injury. In this chapter, we will review the studies that lead to the conception of the ALM, as well as the studies that have followed the development of this assay. We will additionally describe in detail the standard ALM surgery and how to perform this surgery on different limb positions.

Embryonic Tissue and Blastema Transplantations.

Embryo grafts have been an experimental pillar in developmental biology, and particularly, in amphibian biology. Grafts have been essential in constructing fate maps of different cell populations and migratory patterns. Likewise, autografts and allografts in older larvae or adult salamanders have been widely used to disentangle mechanisms of regeneration. The combination of transgenesis and grafting has widened even more the application of this technique.In this chapter, we provide a detailed protocol for embryo transplants in the axolotl (Ambystoma mexicanum ). The location and stages to label connective tissue, muscle, or blood vessels in the limb and blood cells in the whole animal. However, the potential of embryo transplants is enormous and impossible to cover in one chapter. Furthermore, we provide a protocol for blastema transplantation as an example of allograft in older larvae.

Gripping performance in the stick insect Sungaya inexpectata in dependence on the pretarsal architecture.

Insect attachment devices and capabilities have been subject to research efforts for decades, and even though during that time considerable progress has been made, numerous questions remain. Different types of attachment devices are known, alongside most of their working principles, however, some details have yet to be understood. For instance, it is not clear why insects for the most part developed pairs of claws, instead of either three or a single one. In this paper, we investigated the gripping forces generated by the stick insect Sungaya inexpectata, in dependence on the number of available claws. The gripping force experiments were carried out on multiple, standardized substrates of known roughness, and conducted in directions both perpendicular and parallel to the substrate. This was repeated two times: first with a single claw being amputated from each of the animals' legs, then with both claws removed, prior to the measurement. The adhesive pads (arolia) and frictional pads (euplantulae) remained intact. It was discovered that the removal of claws had a detractive effect on the gripping forces in both directions, and on all substrates. Notably, this also included the control of smooth surfaces on which the claws were unable to find any asperities to grip on. The results show that there is a direct connection between the adhesive performance of the distal adhesive pad (arolium) and the presence of intact claws. These observations show collective effects between different attachment devices that work in concert during locomotion, and grant insight into why most insects possess two claws.

Behavioural function and development of body-to-limb proportions and active movement ranges in three stick insect species.

Overall body proportions and relative limb length are highly characteristic for most insect taxa. In case of the legs, limb length has mostly been discussed with regard to parameters of locomotor performance and, in particular cases, as an adaptation to environmental factors or to the mating system. Here, we compare three species of stick and leaf insects (Phasmatodea) that differ strongly in the length ratio between antennae and walking legs, with the antennae of Medauroidea extradentata being much shorter than its legs, nearly equal length of antennae and legs in Carausius morosus, and considerably longer antennae than front legs in Aretaon asperrimus. We show that that relative limb length is directly related to the near-range exploration effort, with complementary function of the antennae and front legs irrespective of their length ratio. Assuming that these inter-species differences hold for both sexes and all developmental stages, we further explore how relative limb length differs between sexes and how it changes throughout postembryonic development. We show that the pattern of limb-to-body proportions is species-characteristic despite sexual dimorphism, and find that the change in sexual dimorphism is strongest during the last two moults. Finally, we show that antennal growth rate is consistently higher than that of front legs, but differs categorically between the species investigated. Whereas antennal growth rate is constant in Carausius, the antennae grow exponentially in Medauroidea and with a sudden boost during the last moult in Aretaon.