Limited column formation in the embryonic growth plate implies divergent growth mechanisms during pre- and postnatal bone development.

Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. In this work, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Finally, correlation between the temporal dynamics of the ratios between clusters and columns and between bone elongation and expansion suggests that clusters may promote expansion, whereas columns support elongation. Overall, our findings support the idea that modulations of division plane rotation of proliferating chondrocytes determines the formation of either clusters or columns, a multifunctional design that regulates morphogenesis throughout pre- and postnatal bone growth. Broadly, this work provides a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.

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.