A vagal-brainstem interoceptive circuit for cough-like defensive behaviors in mice.

Coughing is a respiratory behavior that plays a crucial role in protecting the respiratory system. Here we show that the nucleus of the solitary tract (NTS) in mice contains heterogenous neuronal populations that differentially control breathing. Within these subtypes, activation of tachykinin 1 (Tac1)-expressing neurons triggers specific respiratory behaviors that, as revealed by our detailed characterization, are cough-like behaviors. Chemogenetic silencing or genetic ablation of Tac1 neurons inhibits cough-like behaviors induced by tussive challenges. These Tac1 neurons receive synaptic inputs from the bronchopulmonary chemosensory and mechanosensory neurons in the vagal ganglion and coordinate medullary regions to control distinct aspects of cough-like defensive behaviors. We propose that these Tac1 neurons in the NTS are a key component of the airway-vagal-brain neural circuit that controls cough-like defensive behaviors in mice and that they coordinate the downstream modular circuits to elicit the sequential motor pattern of forceful expiratory responses.

Intradental mechano-nociceptors serve as sentinels that prevent tooth damage.

Pain is the anticipated output of the trigeminal sensory neurons that innervate the tooth's vital interior 1,2 ; however, the contribution of intradental neurons to healthy tooth sensation has yet to be defined. Here, we employ in vivo Ca 2+ imaging to identify and define a population of myelinated high-threshold mechanoreceptors (intradental HTMRs) that detect superficial structural damage of the tooth and initiate jaw opening to protect teeth from damage. Intradental HTMRs remain inactive when direct forces are applied to the intact tooth but become responsive to forces when the structural integrity of the tooth is compromised, and the dentin or pulp is exposed. Their terminals collectively innervate the inner dentin through overlapping receptive fields, allowing them to monitor the superficial structures of the tooth. Indeed, intradental HTMRs detect superficial enamel damage and encode its degree, and their responses persist in the absence of either PIEZO2 or Na v 1.8 3,4 . Optogenetic activation of intradental HTMRs triggers a rapid, jaw opening reflex via contraction of the digastric muscle. Taken together, our data indicate that intradental HTMRs serve as sentinels that guard against mechanical threats to the tooth, and their activation results in physical tooth separation to minimize irreversible structural damage. Our work provides a new perspective on the role of intradental neurons as protective rather than exclusively pain-inducing and illustrates additional diversity in the functions of interoreceptors.

To be or not to be: orb, the fusome and oocyte specification in Drosophila.

In the fruit fly Drosophila melanogaster, two cells in a cyst of 16 interconnected cells have the potential to become the oocyte, but only one of these will assume an oocyte fate as the cysts transition through regions 2a and 2b of the germarium. The mechanism of specification depends on a polarized microtubule network, a dynein dependent Egl:BicD mRNA cargo complex, a special membranous structure called the fusome and its associated proteins, and the translational regulator orb. In this work, we have investigated the role of orb and the fusome in oocyte specification. We show here that specification is a stepwise process. Initially, orb mRNAs accumulate in the two pro-oocytes in close association with the fusome. This association is accompanied by the activation of the orb autoregulatory loop, generating high levels of Orb. Subsequently, orb mRNAs become enriched in only one of the pro-oocytes, the presumptive oocyte, and this is followed, with a delay, by Orb localization to the oocyte. We find that fusome association of orb mRNAs is essential for oocyte specification in the germarium, is mediated by the orb 3' UTR, and requires Orb protein. We also show that the microtubule minus end binding protein Patronin functions downstream of orb in oocyte specification. Finally, in contrast to a previously proposed model for oocyte selection, we find that the choice of which pro-oocyte becomes the oocyte does not seem to be predetermined by the amount of fusome material in these two cells, but instead depends upon a competition for orb gene products.

Deconstructing gastrulation at single-cell resolution.

Gastrulation movements in all animal embryos start with regulated deformations of patterned epithelial sheets, which are driven by cell divisions, cell shape changes, and cell intercalations. Each of these behaviors has been associated with distinct aspects of gastrulation1-4 and has been a subject of intense research using genetic, cell biological, and more recently, biophysical approaches.5-14 Most of these studies, however, focus either on cellular processes driving gastrulation or on large-scale tissue deformations.15-23 Recent advances in microscopy and image processing create a unique opportunity for integrating these complementary viewpoints.24-28 Here, we take a step toward bridging these complementary strategies and deconstruct the early stages of gastrulation in the entire Drosophila embryo. Our approach relies on an integrated computational framework for cell segmentation and tracking and on efficient algorithms for event detection. The detected events are then mapped back onto the blastoderm shell, providing an intuitive visual means to examine complex cellular activity patterns within the context of their initial anatomic domains. By analyzing these maps, we identified that the loss of nearly half of surface cells to invaginations is compensated primarily by transient mitotic rounding. In addition, by analyzing mapped cell intercalation events, we derived direct quantitative relations between intercalation frequency and the rate of axis elongation. This work is setting the stage for systems-level dissection of a pivotal step in animal development.

Trial of labor after cesarean in women in their second delivery - labor characteristics and postpartum factors which influence a patient's choice.

INTRODUCTION: We aimed to assess trial of labor rates in patients in their second pregnancy following a cesarean delivery (CD), and asses the correlation to delivery and postpartum characteristics of their primary delivery. MATERIALS AND METHODS: This was a retrospective cohort of deliveries at our institution between 2009 and 2016. Files of patients with one past CD and a subsequent second delivery were reviewed. Included were patients with a favorable past indication for CD - malpresentation, nonreassuring fetal heart rate, multiple gestation, or placenta previa/abruption. Cases in which a TOLAC was undertaken were compared to those who did not undergo a TOLAC regarding maternal, obstetric, and neonatal outcomes. RESULTS: Five hundred and thirty-six deliveries matched the inclusion criteria, in which 269 patients attempted a TOLAC (TOLAC group) and 267 patients did not (no TOLAC group). Patient demographics at the time of primary CD were similar, but the rate of preterm deliveries was higher among the no TOLAC group. In their second delivery, patients who attempted a TOLAC were younger, had a lower body mass index, had a lower rate of assisted reproduction, and has less pregnancy-related complications (diabetes, hypertensive disorders). CONCLUSIONS: In patients with a first CD, a history of preterm delivery negatively correlated with a TOLAC, while patient age, body mass index and gestational comorbidities negatively affected TOLAC rates in subsequent delivery.

Yield of Brain Magnetic Resonance Imaging in Epilepsy Diagnosis from 1998 to 2020: A Large Retrospective Cohort Study.

BACKGROUND: We aimed to find the clinical significance of brain abnormalities on magnetic resonance imaging (MRI) in epilepsy and the lateralization of these findings with electroencephalogram (EEG). METHODS: We retrospectively analyzed the results of all EEGs and brain MRIs of 600 consecutive epilepsy patients from 1998 to 2020. RESULTS: Data were available for 563 cases (267 females). Ninety percent of the patients were 18 years old or younger. A total of 345 patients (61.3%) had focal epilepsy, 180 (32%), generalized, and 38 (6.7%), inconclusive. In 187 (33.2%), the first MRI was abnormal and in 81 (out of 108 repeated MRI), the second was pathological. The most frequent brain abnormalities were cortical dysplasia in 41 (18.1%), other structural abnormalities in 25 (11%), various phacomatoses in 23 (10.1%), and mesial temporal sclerosis in 17 (7.5%). Among 226 patients with abnormal MRI, 171 (75.6%) had focal epilepsy when compared with 36 (15.9%) with generalized epilepsy (p <0.001). In 121 patients (53.5%), the result of the abnormal MRI contributed significantly to the understanding of the epilepsy etiology. The side of abnormality was lateralized to the EEG focus in 120 cases (53%); in 10/15 cases with infantile spasms (66%), MRI was significantly abnormal. In 33, in whom the first MRI was normal, a second MRI revealed a significant abnormality. CONCLUSION: Brain MRI is an important tool in epilepsy diagnosis, mainly in focal seizures and infantile spasms. A repeat MRI is mandatory in intractable focal cases to improve the yield of this test.

Active Instability and Nonlinear Dynamics of Cell-Cell Junctions.

Active cell-junction remodeling is important for tissue morphogenesis, yet its underlying physics is not understood. We study a mechanical model that describes junctions as dynamic active force dipoles. Their instability can trigger cell intercalations by a critical collapse. Nonlinearities in tissue's elastic response can stabilize the collapse either by a limit cycle or condensation of junction lengths at cusps of the energy landscape. Furthermore, active junction networks undergo collective instability to drive active in-plane ordering or develop a limit cycle of collective oscillations, which extends over regions of the energy landscape corresponding to distinct network topologies.

Concomitant diagnosis of endometrial and breast cancer - does the sequence matters?

OBJECTIVE: To examine whether patients with both breast cancer (BC) and endometrial cancer (EC) have different features of disease, and whether the sequence of appearance of these tumors is correlated with a more aggressive course. METHODS: A retrospective, multi-center observational cohort study of patients treated in two tertiary medical centers between 2014 and 2020. Files of patients who had a co-diagnosis of BC and EC were reviewed and clinical, epidemiological, pathological and genetic characteristics were collected. RESULTS: 67 patients with a co-diagnosis of both malignances were divided into two groups according to primary tumor diagnosis: BC first group (43/67, 64%) and EC first group (24/67, 36%). The time interval between diagnosis of malignancies was significantly longer in the BC first group (mean 144.5 months vs. 67 months, p < 0.05). BRCA mutations were found in higher numbers in the BC first group (27.5% vs. 9.5%, p = 0.18). A significantly higher number of patients in the BC first group had uterine serous carcinoma (USC) histology (44% vs. 12.5%, p < 0.05). This was independent of tamoxifen usage among patients (OR 0.65, 95% CI 0.17-2.49). CONCLUSIONS: In patients suffering from both BC and EC, the sequence of occurrence of malignancies has relevance: When EC presents as a second primary tumor, it tends to present in a more aggressive form, independent of previous tamoxifen use. The time interval between the diagnosis of malignancies was significantly longer in this group, offering an opportunity to improve preventive measures to decrease the likelihood of a subsequent lethal second cancer.

Application of 3D MAPs pipeline identifies the morphological sequence chondrocytes undergo and the regulatory role of GDF5 in this process.

The activity of epiphyseal growth plates, which drives long bone elongation, depends on extensive changes in chondrocyte size and shape during differentiation. Here, we develop a pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which combines light-sheet microscopy, segmentation algorithms and 3D morphometric analysis to characterize morphogenetic cellular behaviors while maintaining the spatial context of the growth plate. Using 3D MAPs, we create a 3D image database of hundreds of thousands of chondrocytes. Analysis reveals broad repertoire of morphological changes, growth strategies and cell organizations during differentiation. Moreover, identifying a reduction in Smad 1/5/9 activity together with multiple abnormalities in cell growth, shape and organization provides an explanation for the shortening of Gdf5 KO tibias. Overall, our findings provide insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover cellular mechanisms that may regulate this process.

Size scaling in collective cell growth.

Size is a fundamental feature of living entities and is intimately tied to their function. Scaling laws, which can be traced to D'Arcy Thompson and Julian Huxley, have emerged as a powerful tool for studying regulation of the growth dynamics of organisms and their constituent parts. Yet, throughout the 20th century, as scaling laws were established for single cells, quantitative studies of the coordinated growth of multicellular structures have lagged, largely owing to technical challenges associated with imaging and image processing. Here, we present a supervised learning approach for quantifying the growth dynamics of germline cysts during oogenesis. Our analysis uncovers growth patterns induced by the groupwise developmental dynamics among connected cells, and differential growth rates of their organelles. We also identify inter-organelle volumetric scaling laws, finding that nurse cell growth is linear over several orders of magnitude. Our approach leverages the ever-increasing quantity and quality of imaging data, and is readily amenable for studies of collective cell growth in other developmental contexts, including early mammalian embryogenesis and germline development.

Epilepsy and electroencephalogram evolution in YWHAG gene mutation: A new phenotype and review of the literature.

A male patient with a de novo mutation in the YWHAG gene and mild phenotype is presented. He had normal delivery and normal development, with normal speech and social milestones. At the age of 9 months, myoclonic seizures started, with generalized epileptiform discharges. The child responded well to levetiracetam monotherapy with complete seizure resolution. Levetiracetam was stopped and he remained seizure-free for 10 months. His development was appropriate for age according to psychological evaluation and he attended a regular kindergarten. At the age of approximately 4 years, the seizures reappeared with different semiology of staring with eye blinking. Electroencephalogram (EEG) showed multifocal spikes. Brain magnetic resonance imaging did not reveal any structural abnormality. Genetic analysis revealed a de novo likely pathogenic missense variant in the YWHAG gene (c.619G>A p.Glu207Lys). We compared our case to the other cases published in the literature. Our case is unique in its seizure semiology and evolution of EEG. Moreover, in contrast to our case, the majority of cases described in the literature have dysmorphism and intellectual disability or autistic spectrum disorder. This report emphasizes the phenotypic heterogeneity of YWHAG mutation as is the case in other developmental encephalopathies.

Template-based mapping of dynamic motifs in tissue morphogenesis.

Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. Here, we present a "template-based search" approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. We formulated the task of motif mapping as a subsequence matching problem and solved it using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows our algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate our approach, we mapped cell intercalations during germband extension in the early Drosophila embryo. Our framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. Our formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.

Metabolic Regulation of Developmental Cell Cycles and Zygotic Transcription.

The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles [1]. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication [2]. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? We propose that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, we demonstrate that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. Our work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription.

Development of migrating tendon-bone attachments involves replacement of progenitor populations.

Tendon-bone attachment sites, called entheses, are essential for musculoskeletal function. They are formed embryonically by Sox9+ progenitors and continue to develop postnatally, utilizing Gli1 lineage cells. Despite their importance, we lack information on the transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and the Gli1 lineage. Here, by performing a series of lineage tracing experiments in mice, we identify the onset of Gli1 lineage contribution to different entheses. We show that Gli1 expression is regulated embryonically by SHH signaling, whereas postnatally it is maintained by IHH signaling. During bone elongation, some entheses migrate along the bone shaft, whereas others remain stationary. Interestingly, in stationary entheses Sox9+ cells differentiate into the Gli1 lineage, but in migrating entheses this lineage is replaced by Gli1 lineage. These Gli1+ progenitors are defined embryonically to occupy the different domains of the mature enthesis. Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple embryonic tissue, whereas another population contributes to its maturation. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.

The Proprioceptive System Masterminds Spinal Alignment: Insight into the Mechanism of Scoliosis.

Maintaining posture requires tight regulation of the position and orientation of numerous spinal components. Yet, surprisingly little is known about this regulatory mechanism, whose failure may result in spinal deformity as in adolescent idiopathic scoliosis. Here, we use genetic mouse models to demonstrate the involvement of proprioception in regulating spine alignment. Null mutants for Runx3 transcription factor, which lack TrkC neurons connecting between proprioceptive mechanoreceptors and spinal cord, developed peripubertal scoliosis not preceded by vertebral dysplasia or muscle asymmetry. Deletion of Runx3 in the peripheral nervous system or specifically in peripheral sensory neurons, or of enhancer elements driving Runx3 expression in proprioceptive neurons, induced a similar phenotype. Egr3 knockout mice, lacking muscle spindles, but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types may be required for this regulatory mechanism. These findings uncover a central role for the proprioceptive system in maintaining spinal alignment.

The Proprioceptive System Regulates Morphologic Restoration of Fractured Bones.

Successful fracture repair requires restoration of bone morphology and mechanical integrity. Recent evidence shows that fractured bones of neonatal mice undergo spontaneous realignment, dubbed "natural reduction." Here, we show that natural reduction is regulated by the proprioceptive system and improves with age. Comparison among mice of different ages revealed, surprisingly, that 3-month-old mice exhibited more rapid and effective natural reduction than newborns. Fractured bones of null mutants for transcription factor Runx3, lacking functional proprioceptors, failed to realign properly. Blocking Runx3 expression in the peripheral nervous system, but not in limb mesenchyme, recapitulated the null phenotype, as did inactivation of muscles flanking the fracture site. Egr3 knockout mice, which lack muscle spindles but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types, as well as muscle contraction, are required for this regulatory mechanism. These findings uncover a physiological role for proprioception in non-autonomous regulation of skeletal integrity.

Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology.

Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

Isometric Scaling in Developing Long Bones Is Achieved by an Optimal Epiphyseal Growth Balance.

One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.

A mechanical Jack-like Mechanism drives spontaneous fracture healing in neonatal mice.

Treatment of fractured bones involves correction of displacement or angulation, known as reduction. However, angulated long-bone fractures in infants often heal and regain proper morphology spontaneously, without reduction. To study the mechanism underlying spontaneous regeneration of fractured bones, we left humeral fractures induced in newborn mice unstabilized, and rapid realignment of initially angulated bones was seen. This realignment was surprisingly not mediated by bone remodeling, but instead involved substantial movement of the two fragments prior to callus ossification. Analysis of gene expression profiles, cell proliferation, and bone growth revealed the formation of a functional, bidirectional growth plate at the concave side of the fracture. This growth plate acts like a mechanical jack, generating opposing forces that straighten the two fragments. Finally, we show that muscle force is important in this process, as blocking muscle contraction disrupts growth plate formation, leading to premature callus ossification and failed reduction.