Tetrodotoxin-resistant mechanosensitivity and L-type calcium channel-mediated spontaneous calcium activity in enteric neurons.

Gut motility undergoes a switch from myogenic to neurogenic control in late embryonic development. Here, we report on the electrical events that underlie this transition in the enteric nervous system, using the GCaMP6f reporter in neural crest cell derivatives. We found that spontaneous calcium activity is tetrodotoxin (TTX) resistant at stage E11.5, but not at E18.5. Motility at E18.5 was characterized by periodic, alternating high- and low-frequency contractions of the circular smooth muscle; this frequency modulation was inhibited by TTX. Calcium imaging at the neurogenic-motility stages E18.5-P3 showed that CaV1.2-positive neurons exhibited spontaneous calcium activity, which was inhibited by nicardipine and 2-aminoethoxydiphenyl borate (2-APB). Our protocol locally prevented muscle tone relaxation, arguing for a direct effect of nicardipine on enteric neurons, rather than indirectly by its relaxing effect on muscle. We demonstrated that the ENS was mechanosensitive from early stages on (E14.5) and that this behaviour was TTX and 2-APB resistant. We extended our results on L-type channel-dependent spontaneous activity and TTX-resistant mechanosensitivity to the adult colon. Our results shed light on the critical transition from myogenic to neurogenic motility in the developing gut, as well as on the intriguing pathways mediating electro-mechanical sensitivity in the enteric nervous system. HIGHLIGHTS: What is the central question of this study? What are the first neural electric events underlying the transition from myogenic to neurogenic motility in the developing gut, what channels do they depend on, and does the enteric nervous system already exhibit mechanosensitivity? What is the main finding and its importance? ENS calcium activity is sensitive to tetrodotoxin at stage E18.5 but not E11.5. Spontaneous electric activity at fetal and adult stages is crucially dependent on L-type calcium channels and IP3R receptors, and the enteric nervous system exhibits a tetrodotoxin-resistant mechanosensitive response. Abstract figure legend Tetrodotoxin-resistant Ca2+ rise induced by mechanical stimulation in the E18.5 mouse duodenum.

Embryonic Development of Motility: Lessons from the Chicken.

I outline here the development of intestinal motility in the chicken embryo. The first contractile events are circular smooth muscle driven calcium waves (E6), that gain a clock-like regularity when interstitial cells of Cajal become electrically active (E14). Soon after longitudinal smooth muscle contractions become prominent (E14), the enteric nervous system starts controlling motility (E16) by coupling the longitudinal and circular contractions via inhibitory neurotransmission. It gives rise to circular-longitudinal antagonism, to the migrating motor complex, and to the polarized ascending contraction-descending relaxation pressure response known as the "law of the intestine". The kinetics of gut development in the chicken appears to follow faithfully that of humans by simply converting embryonic days of chicken development into embryonic weeks of human development.

Physical organogenesis of the gut.

The gut has been a central subject of organogenesis since Caspar Friedrich Wolff's seminal 1769 work 'De Formatione Intestinorum'. Today, we are moving from a purely genetic understanding of cell specification to a model in which genetics codes for layers of physical-mechanical and electrical properties that drive organogenesis such that organ function and morphogenesis are deeply intertwined. This Review provides an up-to-date survey of the extrinsic and intrinsic mechanical forces acting on the embryonic vertebrate gut during development and of their role in all aspects of intestinal morphogenesis: enteric nervous system formation, epithelium structuring, muscle orientation and differentiation, anisotropic growth and the development of myogenic and neurogenic motility. I outline numerous implications of this biomechanical perspective in the etiology and treatment of pathologies, such as short bowel syndrome, dysmotility, interstitial cells of Cajal-related disorders and Hirschsprung disease.

A neural crest cell isotropic-to-nematic phase transition in the developing mammalian gut.

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of ß1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.

How Smooth Muscle Contractions Shape the Developing Enteric Nervous System.

Neurons and glia of the enteric nervous system (ENS) are constantly subject to mechanical stress stemming from contractions of the gut wall or pressure of the bolus, both in adulthood and during embryonic development. Because it is known that mechanical forces can have long reaching effects on neural growth, we investigate here how contractions of the circular smooth muscle of the gut impact morphogenesis of the developing fetal ENS, in chicken and mouse embryos. We find that the number of enteric ganglia is fixed early in development and that subsequent ENS morphogenesis consists in the anisotropic expansion of a hexagonal honeycomb (chicken) or a square (mouse) lattice, without de-novo ganglion formation. We image the deformations of the ENS during spontaneous myogenic motility and show that circular smooth muscle contractile waves induce longitudinal strain on the ENS network; we rationalize this behavior by mechanical finite element modeling of the incompressible gut wall. We find that the longitudinal anisotropy of the ENS vanishes when contractile waves are suppressed in organ culture, showing that these contractile forces play a key role in sculpting the developing ENS. We conclude by summarizing different key events in the fetal development of the ENS and the role played by mechanics in the morphogenesis of this unique nerve network.

Dual Functional States of R406W-Desmin Assembly Complexes Cause Cardiomyopathy With Severe Intercalated Disc Derangement in Humans and in Knock-In Mice.

BACKGROUND: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype. METHODS: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin. RESULTS: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype. CONCLUSIONS: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.

Shifting into high gear: how interstitial cells of Cajal change the motility pattern of the developing intestine.

The first contractile waves in the developing embryonic gut are purely myogenic; they only involve smooth muscle. Here, we provide evidence for a transition from smooth muscle to interstitial cell of Cajal (ICC)-driven contractile waves in the developing chicken gut. In situ hybridization staining for anoctamin-1 (ANO1), a known ICC marker, shows that ICCs are already present throughout the gut, as from embryonic day (E)7. We devised a protocol to reveal ICC oscillatory and propagative calcium activity in embryonic gut whole mount and found that the first steady calcium oscillations in ICCs occur on (E14). We show that the activation of ICCs leads to an increase in contractile wave frequency, regularity, directionality, and velocity between E12 and E14. We finally demonstrate that application of the c-KIT antagonist imatinib mesylate in organ culture specifically depletes the ICC network and inhibits the transition to a regular rhythmic wave pattern. We compare our findings to existing results in the mouse and predict that a similar transition should take place in the human fetus between 12 and 14 wk of development. Together, our results point to an abrupt physiological transition from smooth muscle mesenchyme self-initiating waves to ICC-driven motility in the fetus and clarify the contribution of ICCs to the contractile wave pattern.NEW & NOTEWORTHY We reveal a sharp transition from smooth muscle to interstitial cell of Cajal (ICC)-driven motility in the chicken embryo, leading to higher-frequency, more rhythmic contractile waves. We predict the transition to happen between 12 and 14 embryonic wk in humans. We image for the first time the onset of ICC activity in an embryonic gut by calcium imaging. We show the first KIT and anoctamin-1 (ANO1) in situ hybridization micrographs in the embryonic chicken gut.

Smooth muscle contractility causes the gut to grow anisotropically.

The intestine is the most anisotropically shaped organ, but, when grown in culture, embryonic intestinal stem cells form star- or sphere-shaped organoids. Here, we present evidence that spontaneous tonic and phasic contractions of the circular smooth muscle of the embryonic gut cause short-timescale elongation of the organ by a purely mechanical, self-squeezing effect. We present an innovative culture set-up to achieve embryonic gut growth in culture and demonstrate by three different methods (embryological, pharmacological and microsurgical) that gut elongational growth is compromised when smooth muscle contractions are inhibited. We conclude that the cumulated short-term mechanical deformations induced by circular smooth muscle lead to long-term anisotropic growth of the gut, thus demonstrating a self-consistent way by which the function of this organ (peristalsis) directs its shape (morphogenesis). Our model correctly predicts that longitudinal smooth muscle differentiation later in embryogenesis slows down elongation, and that several mice models with defective gut smooth muscle contractility also exhibit gut growth defects. We lay out a comprehensive scheme of forces acting on the gut during embryogenesis and of their role in the morphogenesis of this organ. This knowledge will help design efficient in vitro organ growth protocols and handle gut growth pathologies such as short bowel syndrome.

Embryogenesis of the peristaltic reflex.

KEY POINTS: Neurogenic gut movements start after longitudinal smooth muscle differentiation in three species (mouse, zebrafish, chicken), and at E16 in the chicken embryo. The first activity of the chicken enteric nervous system is dominated by inhibitory neurons. The embryonic enteric nervous system electromechanically couples circular and longitudinal spontaneous myogenic contractions, thereby producing a new, rostro-caudally directed bolus transport pattern: the migrating motor complex. The response of the embryonic gut to mechanical stimulation evolves from a symmetric, myogenic response at E12, to a neurally mediated, polarized, descending inhibitory, 'law of the intestine'-like response at E16. High resolution, whole-mount 3D reconstructions are presented of the enteric nervous system of the chicken embryo at the neural-control stage E16 with the iDISCO+ tissue clarification technique. ABSTRACT: Gut motility is a complex transport phenomenon involving smooth muscle, enteric neurons, glia and interstitial cells of Cajal. Because these different cells differentiate and become active at different times during embryo development, studying the ontogenesis of motility offers a unique opportunity to 'time-reverse-engineer' the peristaltic reflex. Working on chicken embryo intestinal explants in vitro, we found by spatio-temporal mapping and signal processing of diameter and position changes that motility follows a characteristic sequence of increasing complexity: (1) myogenic circular smooth muscle contractions from E6 to E12 that propagate as waves along the intestine, (2) overlapping and independent, myogenic, low-frequency, bulk longitudinal smooth muscle contractions around E14, and (3) tetrodotoxin-sensitive coupling of longitudinal and circular contractions by the enteric nervous system as from E16. Inhibition of nitric oxide synthase neurons shows that the coupling consists in nitric oxide-mediated relaxation of circular smooth muscle when the longitudinal muscle layer is contracted. This mechanosensitive coupling gives rise to a directional, cyclical, propagating bolus transport pattern: the migrating motor complex. We further reveal a transition to a polarized, descending, inhibitory reflex response to mechanical stimulation after neuronal activity sets in at E16. This asymmetric response is the elementary mechanism responsible for peristaltic transport. We finally present unique high-resolution 3D reconstructions of the chicken enteric nervous system at the neural-control stage based on confocal imaging of iDISCO+ clarified tissues. Our study shows that the enteric nervous system gives rise to new peristaltic transport patterns during development by coupling spontaneous circular and longitudinal smooth muscle contraction waves.

The first digestive movements in the embryo are mediated by mechanosensitive smooth muscle calcium waves.

Peristalsis enables transport of the food bolus in the gut. Here, I show by dynamic ex vivo intra-cellular calcium imaging on living embryonic gut explants that the most primitive form of peristalsis that occurs in the embryo is the result of inter-cellular, gap-junction-dependent calcium waves that propagate in the circular smooth muscle layer. I show that the embryonic gut is an intrinsically mechanosensitive organ, as the slightest externally applied mechanical stimulus triggers contractile waves. This dynamic response is an embryonic precursor of the 'law of the intestine' (peristaltic reflex). I show how characteristic features of early peristalsis such as counter-propagating wave annihilation, mechanosensitivity and nucleation after wounding all result from known properties of calcium waves. I finally demonstrate that inter-cellular mechanical tension does not play a role in the propagation mechanism of gut contractile waves, unlike what has been recently shown for the embryonic heartbeat. Calcium waves are a ubiquitous dynamic signalling mechanism in biology: here I show that they are the foundation of digestive movements in the developing embryo.This article is part of the Theo Murphy meeting issue on 'Mechanics of development'.

Mechanical Tension Drives Elongational Growth of the Embryonic Gut.

During embryonic development, most organs are in a state of mechanical compression because they grow in a confined and limited amount of space within the embryo's body; the early gut is an exception because it physiologically herniates out of the coelom. We demonstrate here that physiological hernia is caused by a tensile force transmitted by the vitelline duct on the early gut loop at its attachment point at the umbilicus. We quantify this tensile force and show that applying tension for 48 h induces stress-dependent elongational growth of the embryonic gut in culture, with an average 90% length increase (max: 200%), 65% volume increase (max: 160%), 50% dry mass increase (max: 100%), and 165% cell number increase (max: 300%); this mechanical cue is required for organ growth as guts not subject to tension do not grow. We demonstrate that growth results from increased cell proliferation when tension is applied. These results outline the essential role played by mechanical forces in shaping and driving the proliferation of embryonic organs.

Hair-on-hair static friction coefficient can be determined by tying a knot.

Characterizing the tribological properties of the hair-hair interface is important to quantify the manageability of hair and to assess the performance of hair care products. Audoly et al. (Phys. Rev. Lett. 99, 164301, 2007) derived an equation relating the self-friction coefficient of an elastic fiber to the dimensions of a simple, relaxed overhand knot made from this fiber. I experimentally tested and validated their equation using nylon thread and an independent measurement of its self-friction coefficient. I show that this methodology can be applied to provide high-throughput data on the static self-friction coefficient of single hair fibers in various conditions and to quantitatively assess how hair care treatments (conditioner, relaxant) alter frictional properties. I find that treatment of hair with 1M sodium hydroxide leads to a quick, irreversible self-friction coefficient increase; the resulting fine frictional fibers can be used to form very small knots for microsurgical vessel and organ ligature in medicine or embryology. The relaxed overhand knot method can more generally be used to measure the self-friction coefficients of a wide range of elastic fibers from the nano- (e.g. proteins, nanotubes) to the macro-scale (e.g. textile fiber, fiberglass).

Physics of amniote formation.

We present a detailed study of the formation of the amniotic sac in the avian embryo, and a comparison with the crocodile amniotic sac. We show that the amniotic sac forms at a circular line of stiffness contrast, separating rings of cell domains. Cells align at this boundary, and this in turn orients and concentrates the tension forces. The tissue fold which forms the amniotic sac is locked exactly along this line due to the colocalization of the stiffness contrast and of the tensile force. In addition, the tensile force plays a regenerative role when the amniotic sac is cut. The fold forming the ventral side of the embryo displays the same characteristics. This work shows that amniote embryogenesis consists of a cascade of buckling events taking place at the boundaries between regions of differing mechanical properties. Hence, amniote embryogenesis relies on a simple and robust biomechanical scheme used repeatedly, and selected ancestrally.

Surface Tension Drives the Orientation of Crystals at the Air-Water Interface.

The fabrication of oriented crystalline thin films is essential for a range of applications ranging from semiconductors to optical components, sensors, and catalysis. Here we show by depositing micrometric crystal particles on a liquid interface from an aerosol phase that the surface tension of the liquid alone can drive the crystallographic orientation of initially randomly oriented particles. The X-ray diffraction patterns of the particles at the interface are identical to those of a monocrystalline sample cleaved along the {104} (CaCO3) or {111} (CaF2) face. We show how this orientation effect can be used to produce thin coatings of oriented crystals on a solid substrate. These results also have important implications for our understanding of heterogeneous crystal growth beneath amphiphile monolayers and for 2D self-assembly processes at the air-liquid interface.

Measuring the micromechanical properties of embryonic tissues.

Local mechanical properties play an important role in directing embryogenesis, both at the cell (differentiation, migration) and tissue level (force transmission, organ formation, morphogenesis). Measuring them is a challenge as embryonic tissues are small (µm to mm) and soft (0.1-10 kPa). We describe here how glass fiber cantilevers can be fabricated, calibrated and used to apply small forces (0.1-10 µN), measure contractile activity and assess the bulk tensile elasticity of embryonic tissue. We outline how pressure (hydrostatic or osmotic) can be applied to embryonic tissue to quantify stiffness anisotropy. These techniques can be assembled at low cost and with a minimal amount of equipment. We then present a protocol to prepare tissue sections for local elasticity and adhesion measurements using the atomic force microscope (AFM). We compare AFM nanoindentation maps of native and formaldehyde fixed embryonic tissue sections and discuss how the local elastic modulus obtained by AFM compares to that obtained with other bulk measurement methods. We illustrate all of the techniques presented on the specific example of the chick embryonic digestive tract, emphasizing technical issues and common pitfalls. The main purpose of this report is to make these micromechanical measurement techniques accessible to a wide community of biologists and biophysicists.

Buckling along boundaries of elastic contrast as a mechanism for early vertebrate morphogenesis.

We have investigated the mechanism of formation of the body of a typical vertebrate, the chicken. We find that the body forms initially by folding at boundaries of stiffness contrast. These boundaries are dynamic lines, separating domains of different cell sizes, that are advected in a deterministic thin-film visco-elastic flow. While initially roughly circular, the lines of elastic contrast form large "peanut" shapes evoking a slender figure-8 at the moment of formation of the animal body, due to deformation and flow in a quadrupolar stretch caused by mesoderm migration. Folding of these "peanut" or "figure-8" motives along the lines of stiffness contrast creates the global pattern of the animal, and segregates several important territories. The main result is that the pattern of cell texture in the embryo serves simultaneously two seemingly different purposes: it regionalizes territories that will differentiate to different cell types and it also locks the folds that physically segregate these territories. This explains how the different cellular types segregate in physically separated domains.

When and Why Like-Sized, Oppositely Charged Particles Assemble into Diamond-like Crystals.

Like-sized, oppositely charged nanoparticles are known to assemble into large crystals with diamond-like (ZnS) ordering, in sharp contrast to analogous molecular ions and micrometer-scale colloids, which invariably favor more closely packed structures (NaCl or CsCl). Here, we show that these experimental observations can be understood as a consequence of ionic screening and the slight asymmetry in surface charge present on the assembling particles. With this asymmetry taken into account, free-energy calculations predict that the diamond-like ZnS lattice is more favorable than other 1:1 ionic structures, namely, NaCl or CsCl, when the Debye screening length is considerably larger than the particle size. A thermodynamic model describes how the presence of neutralizing counterions within the interstitial regions of the crystal acts to bias the formation of low-volume-fraction structures. The results provide general insights into the self-assembly of non-close-packed structures via electrostatic interactions.

Monovalent cations trigger inverted bilayer formation of surfactant films.

We monitored single-layer Langmuir-Blodgett films of behenic acid deposited on silanized glass or silicon substrates by atomic force microscopy (AFM) in liquid. We observed the in situ transformation of the monolayer to a bilayer when the surrounding solution was NaOH or KOH with pH > 8.3. The final state is that of an inverted bilayer, in which both the hydrophobic OTS (octadecyltrichlorosilane) and the alkane chains are exposed to the surrounding solution, defying common intuition based on hydrophobic-hydrophilic energy considerations. Strong sodium-containing carboxylic dimers formed between the headgroups are shown to be responsible for the stabilization of this configuration; calcium ions slow down/inhibit the transformation.