How a reaction-diffusion signal can control spinal cord regeneration in axolotls: A modeling study.

Axolotls are uniquely able to completely regenerate the spinal cord after amputation. The underlying governing mechanisms of this regenerative response have not yet been fully elucidated. We previously found that spinal cord regeneration is mainly driven by cell-cycle acceleration of ependymal cells, recruited by a hypothetical signal propagating from the injury. However, the nature of the signal and its propagation remain unknown. In this theoretical study, we investigated whether the regeneration-inducing signal can follow a reaction-diffusion process. We developed a computational model, validated it with experimental data, and showed that the signal dynamics can be understood in terms of reaction-diffusion mechanism. By developing a theory of the regenerating outgrowth in the limit of fast reaction-diffusion, we demonstrate that control of regenerative response solely relies on cell-to-signal sensitivity and the signal reaction-diffusion characteristic length. This study lays foundations for further identification of the signal controlling regeneration of the spinal cord.

Fluctuations in tissue growth portray homeostasis as a critical state and long-time non-Markovian cell proliferation as Markovian.

Tissue growth is an emerging phenomenon that results from the cell-level interplay between proliferation and apoptosis, which is crucial during embryonic development, tissue regeneration, as well as in pathological conditions such as cancer. In this theoretical article, we address the problem of stochasticity in tissue growth by first considering a minimal Markovian model of tissue size, quantified as the number of cells in a simulated tissue, subjected to both proliferation and apoptosis. We find two dynamic phases, growth and decay, separated by a critical state representing a homeostatic tissue. Since the main limitation of the Markovian model is its neglect of the cell cycle, we incorporated a refractory period that temporarily prevents proliferation immediately following cell division, as a minimal proxy for the cell cycle, and studied the model in the growth phase. Importantly, we obtained from this last model an effective Markovian rate, which accurately describes general trends of tissue size. This study shows that the dynamics of tissue growth can be theoretically conceptualized as a Markovian process where homeostasis is a critical state flanked by decay and growth phases. Notably, in the growing non-Markovian model, a Markovian-like growth process emerges at large time scales.

Quantitative videomicroscopy reveals latent control of cell-pair rotations in vivo.

Collective cell rotations are widely used during animal organogenesis. Theoretical and in vitro studies have conceptualized rotating cells as identical rigid-point objects that stochastically break symmetry to move monotonously and perpetually within an inert environment. However, it is unclear whether this notion can be extrapolated to a natural context, where rotations are ephemeral and heterogeneous cellular cohorts interact with an active epithelium. In zebrafish neuromasts, nascent sibling hair cells invert positions by rotating ≤180° around their geometric center after acquiring different identities via Notch1a-mediated asymmetric repression of Emx2. Here, we show that this multicellular rotation is a three-phasic movement that progresses via coherent homotypic coupling and heterotypic junction remodeling. We found no correlation between rotations and epithelium-wide cellular flow or anisotropic resistive forces. Moreover, the Notch/Emx2 status of the cell dyad does not determine asymmetric interactions with the surrounding epithelium. Aided by computer modeling, we suggest that initial stochastic inhomogeneities generate a metastable state that poises cells to move and spontaneous intercellular coordination of the resulting instabilities enables persistently directional rotations, whereas Notch1a-determined symmetry breaking buffers rotational noise.

Vectorcardiography-derived index allows a robust quantification of ventricular electrical synchrony.

Alteration of muscle activation sequence is a key mechanism in heart failure with reduced ejection fraction. Successful cardiac resynchronization therapy (CRT), which has become standard therapy in these patients, is limited by the lack of precise dyssynchrony quantification. We implemented a computational pipeline that allows assessment of ventricular dyssynchrony by vectorcardiogram reconstruction from the patient's electrocardiogram. We defined a ventricular dyssynchrony index as the distance between the voltage and speed time integrals of an individual observation and the linear fit of these variables obtained from a healthy population. The pipeline was tested in a 1914-patient population. The dyssynchrony index showed minimum values in heathy controls and maximum values in patients with left bundle branch block (LBBB) or with a pacemaker (PM). We established a critical dyssynchrony index value that discriminates electrical dyssynchronous patterns (LBBB and PM) from ventricular synchrony. In 10 patients with PM or CRT devices, dyssynchrony indexes above the critical value were associated with high time to peak strain standard deviation, an echocardiographic measure of mechanical dyssynchrony. Our index proves to be a promising tool to evaluate ventricular activation dyssynchrony, potentially enhancing the selection of candidates for CRT, device configuration during implantation, and post-implant optimization.

Tig1 regulates proximo-distal identity during salamander limb regeneration.

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Size matters: tissue size as a marker for a transition between reaction-diffusion regimes in spatio-temporal distribution of morphogens.

The reaction-diffusion model constitutes one of the most influential mathematical models to study distribution of morphogens in tissues. Despite its widespread use, the effect of finite tissue size on model-predicted spatio-temporal morphogen distributions has not been completely elucidated. In this study, we analytically investigated the spatio-temporal distributions of morphogens predicted by a reaction-diffusion model in a finite one-dimensional domain, as a proxy for a biological tissue, and compared it with the solution of the infinite-domain model. We explored the reduced parameter, the tissue length in units of a characteristic reaction-diffusion length, and identified two reaction-diffusion regimes separated by a crossover tissue size estimated in approximately three characteristic reaction-diffusion lengths. While above this crossover the infinite-domain model constitutes a good approximation, it breaks below this crossover, whereas the finite-domain model faithfully describes the entire parameter space. We evaluated whether the infinite-domain model renders accurate estimations of diffusion coefficients when fitted to finite spatial profiles, a procedure typically followed in fluorescence recovery after photobleaching (FRAP) experiments. We found that the infinite-domain model overestimates diffusion coefficients when the domain is smaller than the crossover tissue size. Thus, the crossover tissue size may be instrumental in selecting the suitable reaction-diffusion model to study tissue morphogenesis.

Photosensitivity and cGAS-Dependent IFN-1 Activation in Patients with Lupus and TREX1 Deficiency.

The exonuclease TREX1 safeguards the cells against DNA accumulation in the cytosol and thereby prevents innate immune activation and autoimmunity. TREX1 mutations lead to chronic DNA damage and cell-intrinsic IFN-1 response. Associated disease phenotypes include Aicardi‒Goutières syndrome, familial chilblain lupus, and systemic lupus erythematosus. Given the role of UV light in lupus pathogenesis, we assessed sensitivity to UV light in patients with lupus and TREX1 mutation by phototesting, which revealed enhanced photosensitivity. TREX1-deficient fibroblasts and keratinocytes generated increased levels of ROS in response to UV irradiation as well as increased levels of 8-oxo-guanine lesions after oxidative stress. Likewise, the primary UV-induced DNA lesions cyclobutane pyrimidine dimers were induced more strongly in TREX1-deficient cells. Further analysis revealed that single-stranded DNA regions, frequently formed during DNA replication and repair, promote cyclobutane pyrimidine dimer formation. Together, this resulted in a strong UV-induced DNA damage response that was associated with a cGAS-dependent IFN-1 activation. In conclusion, these findings link chronic DNA damage to photosensitivity and IFN-1 production in TREX1 deficiency and explain the induction of disease flares on UV exposure in patients with lupus and TREX1 mutation.

Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.

BACKGROUND: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. RESULTS: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. CONCLUSIONS: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.

Spatiotemporal control of cell cycle acceleration during axolotl spinal cord regeneration.

Axolotls are uniquely able to resolve spinal cord injuries, but little is known about the mechanisms underlying spinal cord regeneration. We previously found that tail amputation leads to reactivation of a developmental-like program in spinal cord ependymal cells (Rodrigo Albors et al., 2015), characterized by a high-proliferation zone emerging 4 days post-amputation (Rost et al., 2016). What underlies this spatiotemporal pattern of cell proliferation, however, remained unknown. Here, we use modeling, tightly linked to experimental data, to demonstrate that this regenerative response is consistent with a signal that recruits ependymal cells during ~85 hours after amputation within ~830 µm of the injury. We adapted Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) technology to axolotls (AxFUCCI) to visualize cell cycles in vivo. AxFUCCI axolotls confirmed the predicted appearance time and size of the injury-induced recruitment zone and revealed cell cycle synchrony between ependymal cells. Our modeling and imaging move us closer to understanding bona fide spinal cord regeneration.

A dynamic cell recruitment process drives growth of the Drosophila wing by overscaling the vestigial expression pattern.

Organs mainly attain their size by cell growth and proliferation, but sometimes also grow through recruitment of undifferentiated cells. Here we investigate the participation of cell recruitment in establishing the pattern of Vestigial (Vg), the product of the wing selector gene in Drosophila. We find that the Vg pattern overscales along the dorsal-ventral (DV) axis of the wing imaginal disc, i.e., it expands faster than the DV length of the pouch. The overscaling of the Vg pattern cannot be explained by differential proliferation, apoptosis, or oriented-cell divisions, but can be recapitulated by a mathematical model that explicitly considers cell recruitment. When impairing cell recruitment genetically, we find that the Vg pattern almost perfectly scales and adult wings are approximately 20% smaller. Conversely, impairing cell proliferation results in very small wings, suggesting that cell recruitment and cell proliferation additively contribute to organ growth in this system. Furthermore, using fluorescent reporter tools, we provide direct evidence that cell recruitment is initiated between early and mid third-instar larval development. Altogether, our work quantitatively shows when, how, and by how much cell recruitment shapes the Vg pattern and drives growth of the Drosophila wing.

Signaling-Dependent Control of Apical Membrane Size and Self-Renewal in Rosette-Stage Human Neuroepithelial Stem Cells.

In the developing nervous system, neural stem cells are polarized and maintain an apical domain facing a central lumen. The presence of apical membrane is thought to have a profound influence on maintaining the stem cell state. With the onset of neurogenesis, cells lose their polarization, and the concomitant loss of the apical domain coincides with a loss of the stem cell identity. Little is known about the molecular signals controlling apical membrane size. Here, we use two neuroepithelial cell systems, one derived from regenerating axolotl spinal cord and the other from human embryonic stem cells, to identify a molecular signaling pathway initiated by lysophosphatidic acid that controls apical membrane size and consequently controls and maintains epithelial organization and lumen size in neuroepithelial rosettes. This apical domain size increase occurs independently of effects on proliferation and involves a serum response factor-dependent transcriptional induction of junctional and apical membrane components.

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation.

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.

Familial chilblain lupus due to a gain-of-function mutation in STING.

OBJECTIVES: Familial chilblain lupus is a monogenic form of cutaneous lupus erythematosus caused by loss-of-function mutations in the nucleases TREX1 or SAMHD1. In a family without TREX1 or SAMHD1 mutation, we sought to determine the causative gene and the underlying disease pathology. METHODS: Exome sequencing was used for disease gene identification. Structural analysis was performed by homology modelling and docking simulations. Type I interferon (IFN) activation was assessed in cells transfected with STING cDNA using an IFN-ß reporter and Western blotting. IFN signatures in patient blood in response to tofacitinib treatment were measured by RT-PCR of IFN-stimulated genes. RESULTS: In a multigenerational family with five members affected with chilblain lupus, we identified a heterozygous mutation of STING, a signalling molecule in the cytosolic DNA sensing pathway. Structural and functional analyses indicate that mutant STING enhances homodimerisation in the absence of its ligand cGAMP resulting in constitutive type I IFN activation. Treatment of two affected family members with the Janus kinase (JAK) inhibitor tofacitinib led to a marked suppression of the IFN signature. CONCLUSIONS: A heterozygous gain-of-function mutation in STING can cause familial chilblain lupus. These findings expand the genetic spectrum of type I IFN-dependent disorders and suggest that JAK inhibition may be of therapeutic value.

Accelerated cell divisions drive the outgrowth of the regenerating spinal cord in axolotls.

Axolotls are unique in their ability to regenerate the spinal cord. However, the mechanisms that underlie this phenomenon remain poorly understood. Previously, we showed that regenerating stem cells in the axolotl spinal cord revert to a molecular state resembling embryonic neuroepithelial cells and functionally acquire rapid proliferative divisions (Rodrigo Albors et al., 2015). Here, we refine the analysis of cell proliferation in space and time and identify a high-proliferation zone in the regenerating spinal cord that shifts posteriorly over time. By tracking sparsely-labeled cells, we also quantify cell influx into the regenerate. Taking a mathematical modeling approach, we integrate these quantitative datasets of cell proliferation, neural stem cell activation and cell influx, to predict regenerative tissue outgrowth. Our model shows that while cell influx and neural stem cell activation play a minor role, the acceleration of the cell cycle is the major driver of regenerative spinal cord outgrowth in axolotls.

Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools.

Connective tissues-skeleton, dermis, pericytes, fascia-are a key cell source for regenerating the patterned skeleton during axolotl appendage regeneration. This complexity has made it difficult to identify the cells that regenerate skeletal tissue. Inability to identify these cells has impeded a mechanistic understanding of blastema formation. By tracing cells during digit tip regeneration using brainbow transgenic axolotls, we show that cells from each connective tissue compartment have distinct spatial and temporal profiles of proliferation, migration, and differentiation. Chondrocytes proliferate but do not migrate into the regenerate. In contrast, pericytes proliferate, then migrate into the blastema and give rise solely to pericytes. Periskeletal cells and fibroblasts contribute the bulk of digit blastema cells and acquire diverse fates according to successive waves of migration that choreograph their proximal-distal and tissue contributions. We further show that platelet-derived growth factor signaling is a potent inducer of fibroblast migration, which is required to form the blastema.

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles.

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf ) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf . We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf /Ps ), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements, which very likely involve considerable cell volume changes within seconds to hours.

Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

INTRODUCTION: The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. METHODS AND TREATMENTS: We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. RESULTS: In rbcs 10 µM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 µM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 µM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. ANALYSIS AND DISCUSSION: Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

Cooperativity in Binding Processes: New Insights from Phenomenological Modeling.

Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites.

Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration.

Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.