Multifunctional role of GPCR signaling in epithelial tube formation

Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the apicomedial region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data supports a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.

Cell polarity determinant Dlg1 facilitates epithelial invagination by promoting tissue-scale mechanical coordination

Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains elusive whether additional mechanical inputs are required for folding mediated by apical constriction. Using Drosophila mesoderm invagination as a model, we identified an important role for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ("flanking cells") in facilitating epithelial folding. We found that depletion of the basolateral determinant, Dlg1, disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wildtype embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. Our findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.

Embryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation

Cell apical constriction driven by actomyosin contraction forces is a conserved mechanism during tissue folding in embryo development. While much effort has been made to better understand the molecular mechanisms responsible for apical constriction, it is still not clear if apical actomyosin contraction forces are necessary or sufficient per se to drive tissue folding. To tackle this question, we use the Drosophila embryo model system that forms a furrow on the ventral side, initiating mesoderm internalization. Past computational models support the idea that cell apical contraction forces may not be sufficient and that active or passive cell apico-basal forces may be necessary to drive cell wedging and tissue furrowing. By using 3D computational modelling and in toto embryo image analysis and manipulation, we now challenge this idea and show that embryo-scale force balance of the tissue surface, rather than cell-autonomous shape changes, is necessary and sufficient to drive a buckling of the epithelial surface forming a furrow which propagates and initiates embryo gastrulation.

Evaluation of the effect of irrigants on the accuracy and repeatability of three electronic apex locators: an ex vivo study

Objective: To evaluate the effect of 2% chlorhexidine (CHX) and 2.5% sodium hypochlorite (NaOCl) on the accuracy and repeatability of three Electronic Apex Locators (EALs). Methodology: Thirty one human teeth were connected to a platform, irrigated with 2.0% CHX, and obtained two readings, again irrigated with 2.5% NaOCl and obtained two more readings. The distance between the tip of the file and the apical constriction was measured. Repeatability was calculated using the intraclass correlation coefficient (ICC) and repeatability coefficient (CR). Results: For CHX 2%, with the Mini Apex Locator, 25 of 31 electronic measurements were accurate (80.6%), 28 of 31 measurements for iPex (90.3%) and for Root ZX II 26 (83, 9%). For 2.5% NaOCl, with the Mini Apex Locator, 25 of the 31 measurements were accurate (80.6%), 28 of the 31 measurements for the iPex (90.3%) and for the Root ZX II, 24 (77, 4%). There were no differences between the two irrigants (p>0.05). The values ​​(ICC) were 0.975 and 0.994 for the Mini Apex Locator, 0.981 and 0.971 for the i Pex and 0.994 and 0.995 for the Root ZX II. When using CHX, the (CR) was 0.196 for the Mini Apex Locator, 0.152 for the iPex and 0.088 for the Root ZX II. When using 2.5% NaOCl, the (CR) was 0.088 for the Mini Apex Locator, 0.196 for the iPex and 0.088 for the Root ZX II. Conclusions: The use of 2% CHX and 2.5% NaOCl did not affect the accuracy and repeatability of the three devices.

Microtubule disassembly by caspases is the rate-limiting step of cell extrusion

Epithelial cell death is essential for tissue homeostasis, robustness and morphogenesis. The expulsion of epithelial cells following caspase activation requires well-orchestrated remodeling steps leading to cell elimination without impairing tissue sealing. While numerous studies have provided insight about the process of cell extrusion, we still know very little about the relationship between caspase activation and the remodeling steps of cell extrusion. Moreover, most studies of cell extrusion focused on the regulation of actomyosin and steps leading to the formation of a supracellular contractile ring. However, the contribution of other cellular factors to cell extrusion has been poorly explored. Using the Drosophila pupal notum, a single layer epithelium where most extrusion events are caspase-dependent, we first showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration/dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. We confirmed that local and rapid increase/decrease of microtubules is sufficient to respectively expand/constrict cell apical area. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting steps of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

Morphological Changes of The Root Apex In Anterior Teeth With Periapical Periodontitis: An In-Vivo Study

Introduction: The aim was to analyze the morphological changes of root apex in anterior teeth with periapical periodontitis. Methods: 32 untreated anterior teeth with periapical periodontitis were enrolled, compared with the healthy contralateral teeth. Cone-beam computed tomography was used to measure diameter of the apical constriction. 3D reconstruction technique was used to reconstruct the teeth, analysis the constriction forms, and measure the distances of constriction to apical foramen and anatomical apex respectively. Results: The difference value between buccolingual and mesiodistal diameter was (0.06±0.09) mm in periapical periodontitis and (0.04±0.04) mm in healthy teeth (p<0.05). The mean distances between apical constriction and anatomical apex were (0.97±0.25) mm and (1.59±0.48) mm in periapical periodontitis and healthy teeth. The mean distances of apical constriction to apical foramen were (0.39±0.12) mm and (0.70±0.18) mm in periapical periodontitis and healthy teeth. The most common form of apical construction was flaring (65.6%) in periapical periodontitis. Conclusions: The anterior teeth with periapical periodontitis had shorter distances of apical constriction to anatomical apex and apical foramen, bigger disparities between the diameters of buccolingual and mesiodistal, and higher proportion of flaring apical construction.

Single Visit Apexification Using MTA: A Case Report

Trauma to the dentition during the period of root formation may cause incomplete development of root resulting in open apex. In order to eliminate infection from root canal endodontic treatment is a tooth-saving treatment modality. The absence of a natural apical constriction in a nonvital permanent tooth makes endodontic treatment a challenge. Therefore, it is necessary to induce or create an apical barrier against, which the obturating material can be condensed. Traditionally, calcium hydroxide is considered as the gold standard to induce apexification. Due to certain drawbacks such as very long treatment period, possibility of tooth fracture, and incomplete apical barrier formation, it is being replaced by materials, which have a more predictable outcome like mineral trioxide aggregate (MTA). One-step apexification with MTA reduces the treatment time when compared with traditional calcium hydroxide apexification, which requires an average time of 12–19 months. Also, MTA has various other superior properties compared to calcium hydroxide that are discussed below in detail along with successful treatment of an immature permanent tooth with open apex wherein MTA was used for one-step apexification.

Mechanical competition alters the cellular interpretation of an endogenous genetic program

The intrinsic genetic program of a cell is not sufficient to explain all of the cell’s activities. External mechanical stimuli are increasingly recognized as determinants of cell behavior. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic program of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells and thereby tissue folding. However, some cells do not constrict but instead stretch, even though they share the same genetic program as their constricting neighbors. We show here that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress–strain responses do not reproduce experimental observations, but simulations in which cells behave as ductile materials with nonlinear mechanical properties do. Our models show that this behavior is a general emergent property of actomyosin networks in a supracellular context, in accordance with our experimental observations of actin reorganization within stretching cells.

Apical contacts stemming from incomplete delamination guide progenitor cell allocation through a dragging mechanism

The developmental strategies used by progenitor cells to allow a safe journey from their induction place towards the site of terminal differentiation are still poorly understood. Here, we uncovered a mechanism of progenitor cell allocation that stems from an incomplete process of epithelial delamination that allows progenitors to coordinate their movement with adjacent extra-embryonic tissues. Progenitors of the zebrafish laterality organ originate from the superficial epithelial enveloping layer by an apical constriction process of cell delamination. During this process, progenitors retain long-lasting apical contacts that enable the epithelial layer to pull a subset of progenitors on their way to the vegetal pole. The remaining delaminated cells follow the movement of apically attached progenitors by a protrusion-dependent cell-cell contact mechanism, avoiding sequestration by the adjacent endoderm, ensuring their collective fate and allocation at the site of differentiation. Thus, we reveal that incomplete delamination serves as a cellular platform for coordinated tissue movements during development.

Measuring the Canal Length - A Review

The objectives of root canal treatment are elimination of microorganisms, removal of pulpal remnants, removal of debris, and shaping of the root canal system so that it may be obturated. The most important step in endodontic therapy is canal preparation which can be achieved by accurate working length determination. Working length determines the extent of placing the instruments into the canal, it affects the degree of pain and discomfort which the patient will experience post treatment and it plays an important role in the success of the treatment if placed within correct limits. The cementodentinal junction, where the pulp tissue changes into the apical tissue, is the ideal physiologic apical limit of WL because at this point healing is supposed to be optimal, and the wound to the periapical tissues is minimal.The apical constriction is however, histological and is impossible to locate clinically or radiographically. There are several methods of determining working length which include radiographical methods, digital tactile sense, apical periodontal sensitivity, paper point method and electronic apex locators. The requirements of an ideal method for determining working length include rapid location of the apical constriction in all pulpal conditions, easy measurement, rapid periodic monitoring and confirmation, patient and clinician comfort, minimal radiation to the patient; ease of use in special patients; and cost effectiveness. To achieve the highest degree of accuracy in working length determination, a combination of several methods should be used. This article reviews the different methods to determine WL and their clinical implications. KEY WORDS Working Length, Apex Locator, Radiographic Method.