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The shape of the epithelial monolayer can be depicted as a curved tissue in three-dimensional (3D) space, where individual cells are tightly adhered to one another. The 3D morphogenesis of these tissues is governed by cell dynamics, and a variety of mathematical modeling and simulation studies have been conducted to investigate this process. One promising approach is the cell-center model, which can account for the discreteness of cells. The cell nucleus, which is considered to correspond to the cell center, can be observed experimentally. However, there has been a shortage of cell-center models specifically tailored for simulating 3D monolayer tissue deformation. In this study, we developed a mathematical model based on the cell-center model to simulate 3D monolayer tissue deformation. Our model was confirmed by simulating the in-plane deformation, out-of-plane deformation, and invagination due to apical constriction.

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.

The clinicians face various challenges in trying to find the optimum working length during root canal treatment. Unless proper biomechanical preparation up to the apical limit is done, reinfection of the canal occurs. Apical reference point must be measured with at most care as to avoid any damage to the surrounding periodontal tissues during the treatment.

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

The internalization of multi-cellular tissues is a key morphogenetic process during animal development and organ formation. A good example of this is the initial stages of vertebrate central nervous system formation whereby a transient embryonic structure called the neural plate is able to undergo collective cell rearrangements within the dorsal midline. Despite the fact that defects in neural plate midline internalization may result in a series of severe clinical conditions, such as spina bifida and anencephaly, the biochemical and biomechanical details of this process remain only partially characterized. Here we review the main cellular and molecular mechanisms underlying midline cell and tissue internalization during vertebrate neural tube formation. We discuss the contribution of collective cell mechanisms including convergence and extension, as well as apical constriction facilitating midline neural plate shaping. Furthermore, we summarize recent studies that shed light on how the interplay of signaling pathways and cell biomechanics modulate neural plate internalization. In addition, we discuss how adhesion-dependent cell-cell contact appears to be a critical component during midline cell convergence and surface cell contraction via cell-cell mechanical coupling. We envision that more detailed high-resolution quantitative data at both cell and tissue levels will be required to properly model the mechanisms of vertebrate neural plate internalization with the hope of preventing human neural tube defects.

International audience; In Cnidaria, modes of gastrulation to produce the two body layers vary greatly between species. In the hydrozoan species Clytia hemisphaerica gastrulation involves unipolar ingression of presumptive endoderm cells from an oral domain of the blastula, followed by migration of these cells to fill the blastocoel with concomitant narrowing of the gastrula and elongation along the oral-aboral axis. We developed a 2D computational boundary model capable of simulating the morphogenetic changes during embryonic development from early blastula stage to the end of gastrulation. Cells are modeled as polygons with elastic membranes and cytoplasm, colliding and adhering to other cells, and capable of forming filopodia. With this model we could simulate compaction of the embryo preceding gastrulation, bottle cell formation, ingression, and intercalation between cells of the ingressing pre-sumptive endoderm. We show that embryo elongation is dependent on the number of endodermal cells, low endodermal cell-cell adhesion, and planar cell polarity (PCP). When the strength of PCP is reduced in our model, resultant embryo morphologies closely resemble those reported previously following morpholino-mediated knockdown of the core PCP proteins Strabismus and Frizzled. Based on our results, we postulate that cellular processes of apical constriction, compaction, ingression, and then reduced cell-cell adhesion and mediolateral intercalation in the presumptive endoderm, are required and when combined, sufficient for Clytia gastrulation.

Studied 62 teeth removed for medical reasons in 54 adults (32 men and 22 women) of different ages to determine age-related changes in the root tips of teeth.Were used methods of odontoscope, x-ray examination, and prepared and studied thin sections of tooth roots. It is established that with age, in the absence of resorption of the apex and gipertireoze in the apex of the tooth root, in adults there is a reduction of the diameter of the root canal in the apical third of the root, due to the formation of secondary dentine, as well as a slight thickening of the cement in the region of apical hole. Thus, increases with age the distance between apical constriction, apical foramen and the radiographic apex of the tooth root. People young and middle age obliteration of root canal teeth are studied according to x-rays does not occur. In elderly and senile obliteration of the root canals of the teeth in the area of the tops are marked at 75% and 79%, respectively, but in thin sections of the roots, which according to x-ray examination is determined by the obliteration of the cavity of the tooth root, the channels typically have a lumen, that is, they are passable for endodontic instruments.

A critical juncture in early development is the partitioning of cells that will adopt different fates into three germ layers: the ectoderm, the mesoderm, and the endoderm. This step is achieved through the internalization of specified cells from the outermost surface layer, through a process called gastrulation. In Drosophila, gastrulation is achieved through cell shape changes (i.e., apical constriction) that change tissue curvature and lead to the folding of a surface epithelium. Folding of embryonic tissue results in mesoderm and endoderm invagination, not as individual cells, but as collective tissue units. The tractability of Drosophila as a model system is best exemplified by how much we know about Drosophila gastrulation, from the signals that pattern the embryo to the molecular components that generate force, and how these components are organized to promote cell and tissue shape changes. For mesoderm invagination, graded signaling by the morphogen, Spätzle, sets up a gradient in transcriptional activity that leads to the expression of a secreted ligand (Folded gastrulation) and a transmembrane protein (T48). Together with the GPCR Mist, which is expressed in the mesoderm, and the GPCR Smog, which is expressed uniformly, these signals activate heterotrimeric G-protein and small Rho-family G-protein signaling to promote apical contractility and changes in cell and tissue shape. A notable feature of this signaling pathway is its intricate organization in both space and time. At the cellular level, signaling components and the cytoskeleton exhibit striking polarity, not only along the apical–basal cell axis, but also within the apical domain. Furthermore, gene expression controls a highly choreographed chain of events, the dynamics of which are critical for primordium invagination; it does not simply throw the cytoskeletal “on” switch. Finally, studies of Drosophila gastrulation have provided insight into how global tissue mechanics and movements are intertwined as multiple tissues simultaneously change shape. Overall, these studies have contributed to the view that cells respond to forces that propagate over great distances, demonstrating that cellular decisions, and, ultimately, tissue shape changes, proceed by integrating cues across an entire embryo.

The present study aimed at the development of an endodontic simulation model, able to implement the electronic method of working length determination (EAL) in a setting of undergraduate learning. The development of the simulation model was accompanied by several investigations, helping to assess products’ potential and aiming to create the sound scientific basis for its future implementation as a learning tool. This investigation was conducted in the framework of the regular phantom-lab course. During the initial implementation 450 student have been trained. The models’ capability to imitate clinical conditions was investigated by means of radiographic, macrophotograpic and CAD/CAM techniques. The EAL-features of the present model allowed not only for precise determination of apical constriction (R²=0,0198) versus radiographic method (R² = 0,0019), but also granted a precise preparation of the apical constriction in the vast majority of cases (up to 83%).

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.

The initial step in bile duct development is the formation of a biliary lumen, a process which involves several cellular mechanisms, such as cell division and polarization, and secretion of fluid. However, how these mechanisms are orchestrated in time and space is difficult to understand. Here, we built a computational model of biliary lumen formation which represents every cell and its function in detail. With the model we can simulate the effect of biophysical aspects that affect duct formation. We have tested the individual and combined effects of directed cell division, apical constriction, and osmotic effects on lumen expansion by varying the parameters that control their relative strength. Our simulations suggest that successful bile duct lumen formation requires the simultaneous contribution of directed cell division of cholangiocytes, local osmotic effects generated by salt excretion in the lumen, and temporally-controlled differentiation of hepatoblasts to cholangiocytes, with apical constriction of cholangiocytes only moderately affecting luminal size. Biliary ducts collect bile from liver lobules, the smallest functional and anatomical units of liver, and carry it to the gallbladder. Disruptions in this process caused by defective embryonic development, or through ductal reaction in liver disease have a major impact on life quality and survival of patients. A deep understanding of the processes underlying bile duct lumen formation is crucial to identify intervention points to avoid or treat the appearance of defective bile ducts. Several hypotheses have been proposed to characterize the biophysical mechanisms driving initial bile duct lumen formation during embryogenesis. Here, guided by the quantification of morphological features and expression of genes in bile ducts from embryonic mouse liver, we sharpened these hypotheses and collected data to develop a high resolution individual cell-based computational model that enables to test alternative hypotheses in silico. This model permits realistic simulations of tissue and cell mechanics at sub-cellular scale. Our simulations suggest that successful bile duct lumen formation requires a simultaneous contribution of directed cell division of cholangiocytes, local osmotic effects generated by salt excretion in the lumen, and temporally-controlled differentiation of hepatoblasts to cholangiocytes, with apical constriction of cholangiocytes only moderately affecting luminal size.

During organ development, tubular organs often form from flat epithelial primordia. In the placodes of the forming tubes of the salivary glands in the Drosophila embryo, we previously identified spatially defined cell behaviors of cell wedging, tilting, and cell intercalation that are key to the initial stages of tube formation. Here, we address what the requirements are that ensure the continuous formation of a narrow symmetrical tube from an initially asymmetrical primordium whilst overall tissue geometry is constantly changing. We are using live-imaging and quantitative methods to compare wild-type placodes and mutants that either show disrupted cell behaviors or an initial symmetrical placode organization, with both resulting in severe impairment of the invagination. We find that early transcriptional patterning of key morphogenetic transcription factors drives the selective activation of downstream morphogenetic modules, such as GPCR signaling that activates apical-medial actomyosin activity to drive cell wedging at the future asymmetrically placed invagination point. Over time, transcription of key factors expands across the rest of the placode and cells switch their behavior from predominantly intercalating to predominantly apically constricting as their position approaches the invagination pit. Misplacement or enlargement of the initial invagination pit leads to early problems in cell behaviors that eventually result in a defective organ shape. Our work illustrates that the dynamic patterning of the expression of transcription factors and downstream morphogenetic effectors ensures positionally fixed areas of cell behavior with regards to the invagination point. This patterning in combination with the asymmetric geometrical setup ensures functional organ formation.

Epithelial folding is a fundamental morphogenetic process that shapes planar epithelial sheets into complex three-dimensional structures. Multiple mechanisms can generate epithelial folds, including apical constriction, which acts locally at the cellular level, differential growth on the tissue scale, or buckling because of compression from neighboring tissues. Here, we investigate the formation of dorsally located epithelial folds at segment boundaries during the late stages of Drosophila embryogenesis. We found that the fold formation at the segment boundaries occurs through the juxtaposition of two key morphogenetic processes: local apical constriction and tissue-level compressive forces from posterior segments. Further, we found that epidermal spreading and fold formation are accompanied by spatiotemporal pulses of Hedgehog (Hh) signaling. A computational model that incorporates the local forces generated from the differential tensions of the apical, basal, and lateral sides of the cell and active forces generated within the whole tissue recapitulates the overall fold formation process in wild-type and Hh overexpression conditions. In sum, this work demonstrates how epithelial folding depends on multiple, separable physical mechanisms to generate the final morphology of the dorsal epidermis. This work illustrates the modularity of morphogenetic unit operations that occur during epithelial morphogenesis.

The developmental strategies used by progenitor cells to endure a safe journey from their induction place towards the site of terminal differentiation are still poorly understood. Here we uncovered a progenitor cell allocation mechanism 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 surface epithelial enveloping layer by an apical constriction process of cell delamination. During this process, progenitors retain long-term apical contacts that enable the epithelial layer to pull a subset of progenitors along their way towards the vegetal pole. The remaining delaminated progenitors follow apically-attached progenitors’ movement by a co-attraction mechanism, avoiding sequestration by the adjacent endoderm, ensuring their fate and collective allocation at the differentiation site. Thus, we reveal that incomplete delamination serves as a cellular platform for coordinated tissue movements during development.Impact StatementIncomplete delamination serves as a cellular platform for coordinated tissue movements during development, guiding newly formed progenitor cell groups to the differentiation site.

During development, coordinated cell shape changes and cell divisions sculpt tissues. While these individual cell behaviors have been extensively studied, how cell shape changes and cell divisions that occur concurrently in epithelia influence tissue shape is less understood. We addressed this question in two contexts of the earlyDrosophilaembryo: premature cell division during mesoderm invagination, and native ectodermal cell divisions with ectopic activation of apical contractility. Using quantitative live-cell imaging, we demonstrated that mitotic entry reverses apical contractility by interfering with medioapical RhoA signaling. While premature mitotic entry inhibits mesoderm invagination, which relies on apical constriction, mitotic entry in an artificially contractile ectoderm induced ectopic tissue invaginations. Ectopic invaginations resulted from medioapical myosin loss in neighboring mitotic cells. This myosin loss enabled non-mitotic cells to apically constrict through mitotic cell stretching. Thus, the spatial pattern of mitotic entry can differentially regulate tissue shape through signal interference between apical contractility and mitosis.

Miao et al. show that a membrane trafficking pathway centered on Sbf and Rab35 is essential for the irreversibility of pulsed contractile events during apical constriction. Sbf/Rab35 disruption leads to a convoluted cell surface, suggesting that membrane remodeling is essential for the construction of effective actomyosin networks., During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface.

This work provides direct evidence that heterotrimeric G proteins can be activated in vivo by a cytoplasmic factor instead of by a GPCR. Specifically, DAPLE, a nonreceptor protein bearing an evolutionarily conserved Gα-binding and -activating (GBA) motif, triggers apical cell constriction during neurulation in Xenopus laevis embryos via G protein–dependent signaling., Heterotrimeric G proteins are signaling switches that control organismal morphogenesis across metazoans. In invertebrates, specific GPCRs instruct G proteins to promote collective apical cell constriction in the context of epithelial tissue morphogenesis. In contrast, tissue-specific factors that instruct G proteins during analogous processes in vertebrates are largely unknown. Here, we show that DAPLE, a non-GPCR protein linked to human neurodevelopmental disorders, is expressed specifically in the neural plate of Xenopus laevis embryos to trigger a G protein signaling pathway that promotes apical cell constriction during neurulation. DAPLE localizes to apical cell–cell junctions in the neuroepithelium, where it activates G protein signaling to drive actomyosin-dependent apical constriction and subsequent bending of the neural plate. This function is mediated by a Gα-binding-and-activating (GBA) motif that was acquired by DAPLE in vertebrates during evolution. These findings reveal that regulation of tissue remodeling during vertebrate development can be driven by an unconventional mechanism of heterotrimeric G protein activation that operates in lieu of GPCRs.

Despite the achievements of modern dentistry, the question of choosing the apical termination point of root canals for endodontic treatment remains opened. It is known, that the inflammatory processes of the tooth pulp do not make a destructive effect on the apical constriction, when this inflammation is limited to the root canal. At the same time, the safety of the apical constriction in the tooth’s root canal with chronic apical periodontitis remains questionable because of the possibility of spreading destructive processes from the periapical tissues to the inner and outer surface of the tooth root. The article presents the results of studying the variability of apical constriction in healthy teeth, and also in teeth with apical periodontitis. A demonstration of an apical constriction on the "transparent tooth" model was made.

Neural tube closure requires apical constriction during which contraction of the apical F-actin network forces the cell into a wedged shape, facilitating the folding of the neural plate into a tube. However, how F-actin assembly at the apical surface is regulated in mammalian neurulation remains largely unknown. We report here that formin homology 2 domain-containing 3 (Fhod3), a formin protein that mediates F-actin assembly, is essential for cranial neural tube closure in mouse embryos. We found that Fhod3 is expressed in the lateral neural plate but not in the floor region of the closing neural plate at the hindbrain. Consistently, in Fhod3-null embryos, neural plate bending at the midline occurred normally, but lateral plates seemed floppy and failed to flex dorsomedially. Because the apical accumulation of F-actin and constriction were impaired specifically at the lateral plates in Fhod3-null embryos, we concluded that Fhod3-mediated actin assembly contributes to lateral plate-specific apical constriction to advance closure. Intriguingly, Fhod3 expression at the hindbrain was restricted to neuromeric segments called rhombomeres. The rhombomere-specific accumulation of apical F-actin induced by the rhombomere-restricted expression of Fhod3 was responsible for the outward bulging of rhombomeres involving apical constriction along the anteroposterior axis, as rhombomeric bulging was less prominent in Fhod3-null embryos than in the wild type. Fhod3 thus plays a crucial role in the morphological changes associated with neural tube closure at the hindbrain by mediating apical constriction not only in the mediolateral but also in the anteroposterior direction, thereby contributing to tube closure and rhombomere segmentation, respectively.

Non-centrosomal microtubule arrays serve crucial functions in cells, yet the mechanisms of their generation are poorly understood. During budding of the epithelial tubes of the salivary glands in the Drosophila embryo, we previously demonstrated that the activity of pulsatile apical-medial actomyosin depends on a longitudinal non-centrosomal microtubule array. Here we uncover that the exit from the last embryonic division cycle of the epidermal cells of the salivary gland placode leads to one centrosome in the cells losing all microtubule-nucleation capacity. This restriction of nucleation activity to the second, Centrobin-enriched, centrosome is key for proper morphogenesis. Furthermore, the microtubule-severing protein Katanin and the minus-end-binding protein Patronin accumulate in an apical-medial position only in placodal cells. Loss of either in the placode prevents formation of the longitudinal microtubule array and leads to loss of apical-medial actomyosin and impaired apical constriction. We thus propose a mechanism whereby Katanin-severing at the single active centrosome releases microtubule minus-ends that are then anchored by apical-medial Patronin to promote formation of the longitudinal microtubule array crucial for apical constriction and tube formation., Non-centrosomal microtubules provide essential functions in many cells, but the mechanisms of their formation are poorly understood. Here, the authors show that during tube formation of the Drosophila salivary glands, microtubules are released from a single active centrosome via katanin, triggering recruitment of Patronin, and leading to formation of a non-centrosomal network key to the tube invagination process.

The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1– PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1 and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

Introducción: Son numerosos los factores que pueden influenciar el éxito o fracaso del tratamiento de conductos, entre ellos, es imprescindible un buen conocimiento anatómico y manejo de las técnicas de instrumentación, irrigación y obturación. El calibrado apical es una técnica que permite determinar el diámetro de la constricción apical durante la instrumentación Esta revisión pretende esclarecer qué limite se debe establecer para realizar la instrumentación y el calibrado, evaluar la precisión de las técnicas de localización de ápices, determinar el diámetro adecuado para el calibrado apical mediante las innovadoras técnicas de agrandamiento en dientes con patologías periapicales y el manejo clínico de dientes cona anomalías que afectan a la constricción. Metodología: se realizaron diferentes búsquedas según los parámetros que se querían analizar. Se obtuvieron un total de 242, 20 de los cuales se ajustaron a los criterios de inclusión y exclusión. También se realizó una búsqueda manual en revistas de gran factor de impacto, obteniendo 7 estudios. Resultados y conclusiones: en esta revisión bibliográfica, se han expuesto estudios experimentales in vivo e in vitro. Estos estudios establecen la constricción apical como límite de la instrumentación. La combinación del localizador de ápices y las radiografías aportan mayor precisión en la determinación de la longitud de trabajo. Preflaring y limas sin ahusamiento proporcionan mayor precisión en el calibrado del diámetro de la constricción. Estudios recientes muestran casos de periodontitis apicales, donde el alargamiento apical junto con la limpieza mecánica, proporciona buenos resultados reduciendo los tratamientos a una sola visita. En reportes de casos recientes, se ha visto como en anomalías que afectan a la constricción apical como dientes taurodónticos o dens invaginatus, el uso de CBCT, microscopía, irrigación ultrasónica a concentraciones bajas y sistema de obturación termoplástica favorecen el éxito de estos tratamientos complejos. Introduction: There are many factors that can influence on the success or failure of root canal treatment, among them, a good anatomical knowledge and a management of instrumentation, irrigation and obturation techniques. Apical gauging is a technique use to determine the diameter of apical constriction during instrumentation. This review aims to clarify what limit should be established to perform the instrumentation, to evaluate the precision of apex locators techniques, determine the appropriate diameter to apical gauge using innovative enlargement methods in teeth with periapical pathologies and the clinical management of teeth with anomalies that affect the apical constriction. Methodology: Different searches were carried out according to the parameters to be analyses on Pubmed. A total of 242 were obtained, 20 of which were adjusted to the inclusion and exclusion criteria. A manual search was also carried out in journals with a high impact factors obtaining 7 studies. Results and conclusions: In this literature review, experimental in vivo and in vitro studies have been reported. These studies establish the apical constriction as the limit of the instrumentation. The combination technique of the apex locator and radiography provide greater precision in determining the working length. Preflaring and non-taper files provide better results in the apical gauging of apical constriction`s diameter. Recent studies show cases of apical periodontitis, where apical enlargement with mechanical cleaning provides good results reducing treatment to a single visit. In recent case reports, it has been seen the use of CBCT, microscopy, ultrasonic irrigation at low concentrations and thermoplastic obturation system to the complex treatments of teeth with anomalies. Universidad de Sevilla. Máster Oficial en Odontología Restauradora Estética y Funcional

Neural tube closure (NTC) is a fundamental process during vertebrate embryonic development and is indispensable for the formation of the central nervous system. Here, using Xenopus laevis embryos, live imaging, single cell tracking, optogenetics and loss of function experiments we examine the contribution of convergent extension (CE) and apical constriction (AC) and we define the role of the surface ectoderm (SE) during NTC. We show that NTC is a two-stage process and that CE and AC do not overlap temporally while their spatial activity is distinct. PCP driven CE is restricted to the caudal part of the neural plate (NP) and takes place during the first stage. CE is essential for correct positioning of the NP rostral most region in the midline of the dorsoventral axis. AC occurs after CE throughout the NP and is the sole contributor of anterior NTC. We go on to show that the SE is mechanically coupled with the NP providing resistive forces during NTC. Its movement towards the midline is passive and driven by forces generated through NP morphogenesis. Last, we show that increase of SE resistive forces is detrimental for NP morphogenesis, showing that correct SE development is permissive for NTC.

Made available in DSpace on 2021-07-14T10:31:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-28. Added 1 bitstream(s) on 2021-07-14T11:32:13Z : No. of bitstreams: 1 S1806-83242021000100261.pdf: 928966 bytes, checksum: e6fd16f73fcbd8b0b8ada07b7adb7b0d (MD5) This research evaluated, in vivo, the accuracy of three electronic apex locators - EALs (Root ZXII, E-PEX and FIND) in teeth with vital pulp submitted to biopulpectomy, preserving the periodontal stump. For this study, 90 single-rooted teeth with extraction indication were selected. After positive pulpal cold sensitivity test, pulp chamber access was performed. The cervical and middle thirds of root canals were instrumented with Reciproc R25, and the K#15 file was used as a standard instrument to determine working length, forming 2 groups: Constriction (insertion of the instrument until the apical constriction limit) and Foramen (insertion of the instrument until the foramen and then repositioning at constriction, without removing the file from the canal). The hand file was stabilized with a light-cured flow resin. After extraction, the samples were analyzed through microCT SkyScan 1272, with CTAN software, which evaluated the proximity between the tip of the file to the apical constriction, providing data for comparative analysis using Kruskal-Wallis and Dunn tests (p

Spatial organization of differently fated cells within an organ is essential and needs to be maintained during development. This is largely implemented via compartment boundaries that serve as barriers between distinct cell types. Biased accumulation of junctional non-muscle Myosin II along the interface between differently fated groups of cells contributes to boundary integrity and maintains its shape via increased tension [1-4]. Here we test whether interfacial Myosin driven tension is responsible for the elimination of aberrantly specified cells that would otherwise compromise compartment organization. To this end, we genetically reduce Myosin II levels in three different patterns: in both wild-type and misspecified cells, only in misspecified cells and specifically at the interface between wild-type and aberrantly specified cells. We find that recognition and elimination of aberrantly specified cells do not rely on tensile forces driven by interfacial Myosin cables. Moreover, apical constriction of misspecified cells and their separation from wild type neighbors occurs even when Myosin level is greatly reduced. Thus, we conclude that the forces that drive elimination of aberrantly specified cells are largely independent of Myosin II.

Apical constriction, or reduction of the apical domain, underlies many morphogenetic events during development, such as furrow or tube formation. Actomyosin complexes play an essential role in apical constriction, however the detailed analysis of molecular mechanisms is still pending. Here we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is localized to and promotes the formation of the circumferential actomyosin belt adjacent to apical junctions. We find that Lmo7-dependent apical constriction requires the RhoA-ROCK-Non-muscle myosin II (NMII) pathway. Strikingly, Lmo7 binds and recruits NMII heavy chains to apical junctions. Lmo7 overexpression altered the subcellular distribution of Wtip, a sensor of mechanical tension. Our findings suggest that Lmo7 serves as a scaffold organizing the actomyosin network to regulate contractility at apical junctions.

During embryo development, tissues often undergo multiple concomitant changes in shape. It is unclear which signaling pathways and cellular mechanisms are responsible for multiple simultaneous tissue shape transformations. We focus on the process of concomitant tissue folding and extension that is key during gastrulation and neurulation. We use the Drosophila embryo as model system and focus on the process of mesoderm invagination. Here, we show that the prospective mesoderm simultaneously folds and extends. We report that mesoderm cells, under the control of anterior-posterior and dorsal-ventral gene patterning synergy, establish two sets of adherens junctions at different apical-basal positions with specialized functions: while apical junctions drive apical constriction initiating tissue bending, lateral junctions concomitantly drive polarized cell intercalation, resulting in tissue convergence-extension. Thus, epithelial cells devise multiple specialized junctional sets that drive composite morphogenetic processes under the synergistic control of apparently orthogonal signaling sources.

The journal of cell biology : JCB 220(11), e202104107 (2021). doi:10.1083/jcb.202104107, Published by Rockefeller Univ. Press, New York, NY

Ventral furrow (VF) formation in Drosophila melanogaster is an important model of epithelial folding. Past studies of VF formation focus on the role of apical constriction in driving folding. However, the relative contributions of other forces are largely unexplored. When basal membrane formation is genetically blocked using RNAi-mediated anillin knockdown (scra RNAi), the VF is still capable of folding. scra RNAi and control embryos display quantifiable cell length differences throughout gastrulation, as well as qualitative differences in membrane integrity. To interpret our observations, we developed a computational model of VF formation that explicitly simulates the flows of the viscous cytoplasm. The viscosity included in our model is required for tissue invagination in the complete absence of basal membranes and explains the observed differences in membrane lengths across conditions. In the absence of basal membranes, epithelial folding requires the presence of viscous shear forces from cytoplasm. Our model characterizes folding during VF formation as a swimming phenomenon, where tissue deforms by pushing against the ambient viscous surroundings. Since VF formation is successful in scra RNAi embryos, we propose that models of gastrulation should also be tested for their ability to replicate folding in the absence of basal membranes.

During embryonic development, cellular forces synchronize in space and time to generate functional tissue shapes. Apical constriction is one of these force-generating processes, and it is necessary to modulate epithelial curvature in fundamental morphogenetic events, such as neural tube folding. The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues currently hinders the progress of the field. Here we report the development of “OptoShroom3”, a new optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination of individual cells in an epithelial cell sheet reduced their apical surface while illumination of groups of cells caused deformation in the adjacent regions. By using OptoShroom3, we further manipulated 3D tissue shapes. Light-induced apical constriction provoked the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids led to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

Summary Somitogenesis is often described using the clock-and-wavefront (CW) model, which does not explain how molecular signaling rearranges the pre-somitic mesoderm (PSM) cells into somites. Our scanning electron microscopy analysis of chicken embryos reveals a caudally-progressing epithelialization front in the dorsal PSM that precedes somite formation. Signs of apical constriction and tissue segmentation appear in this layer 3-4 somite lengths caudal to the last-formed somite. We propose a mechanical instability model in which a steady increase of apical contractility leads to periodic failure of adhesion junctions within the dorsal PSM and positions the future inter-somite boundaries. This model produces spatially periodic segments whose size depends on the speed of the activation front of contraction (F), and the buildup rate of contractility (Λ). The Λ/F ratio determines whether this mechanism produces spatially and temporally regular or irregular segments, and whether segment size increases with the front speed., Graphical abstract, Highlights • Dorsal pre-somitic mesoderm of chicken embryos epithelializes before somite formation • Dorsal epithelium shows signs of apical constriction and early segmentation • A mechanical instability model can reproduce sequential segmentation • A single ratio describes spatial and temporal patterns of segmentation, Poultry Embryology; Mechanical Modeling; Developmental Biology

The aim of this study was to investigate the root canal morphology and the thickness of crown and root of mandibular incisors in a Chinese subpopulation by micro-computed tomography (micro-CT). In total, 208 mandibular incisors were scanned using micro-CT. The anatomical features of the canals (canal configuration, apical constriction, foramen-to-apex distance, accessory canal vertical distribution, and canal geometrical parameters) and the thickness of the crown and root 2/3 were evaluated. Three canal categories, labeled as Single (77.88%), Merged (15.87%), and Separated (6.25%), were summarized. The most frequent constriction type in main foramina was single constriction (42.53%). Wide and narrow diameters in a single main foramen were 0.37 ± 0.14 mm and 0.26 ± 0.07 mm, respectively. The distance from the anatomical foramen to the physiological foramen and the anatomical apex was 0.49 ± 0.20 mm and 0.36 ± 0.28 mm, respectively. During the virtual root-end resection, 97.12% of roots underwent successful resection at the 2-mm level, with the foramina visible on the resection surface. During 2-D cross-sectional analyses, the shape parameters of the root and canal showed significant positive correlation (P

Apical constriction driven by non-muscle myosin II (“myosin”) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of myosin, we find that during Drosophila mesoderm invagination, myosin contractility is critical to prevent tissue relaxation during the early, “priming” stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. The binary response suggests that the mesoderm is mechanically bistable during gastrulation. Combined modeling analysis and experimental measurements suggest that the observed mechanical bistability may arise from apicobasal shrinkage of the surrounding ectoderm, which promotes mesoderm invagination by facilitating a buckling transition. Our results suggest that Drosophila mesoderm invagination requires a joint action of local myosin contractility and mechanical bistability of the epithelium to trigger epithelial buckling.

Older age is associated with reduced immune function. Our aim was to study how age affects the development of apical periodontitis (AP). AP was induced in two age groups of mice (young vs. adult). Histological samples were stained by Hematoxylin Eosin, Brown and Brenn, and Tartrate-Resistant Acid Phosphatase. In addition, the samples were scanned by Micro-Computerized-Tomography (micro-CT) to evaluate apical constriction and periapical lesion size. Cell density in the periapical region was computationally assessed. Moreover, lesion immune cell populations were characterized by flow cytometry and immunofluorescence. The young group presented more canals with necrotic radicular pulp compared to the adults. There was no difference in bacteria location in the canals between the groups. Apical constriction size was larger in the young mice compared to the adults. The periapical cell density was higher in the young group, while the dominant immune cells in the lesions were neutrophils, which also exhibited the highest young/adult ratio. Immunofluorescence demonstrated neutrophils in the lesion. More osteoclasts were present in the lesions of the young mice, in correlation to the higher volume of bone resorption in this group. Overall, we conclude that the immune reaction to AP stimuli was attenuated in the adult mice compared to the young.

During tissue morphogenesis, cell shape changes resulting from cell-generated forces often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. In this study, we identify an actomyosin dependent mechanism involving Rab11- mediated trafficking in regulating apical constriction in theDrosophilaembryo. DuringDrosophilamesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures (“Rab11 vesicles”) by promoting a directional bias in dynein mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding.

In the physicists’ perspective, epithelial tissues constitute an exotic type of active matter with non-linear properties reminiscent of amorphous materials. In the context of a circular proliferating epithelium, modeled by the quasistatic vertex model, we identify novel discrete tissue scale rearrangements, i.e. cellular flow avalanches, which are a form of collective cell movement. During the avalanches, the cellular trajectories are radial in the periphery and form a vortex in the core. After the onset of these avalanches, the epithelial area grows discontinuously. The avalanches are found to be stochastic, and their strength is determined by the density of cells in the tissue. Overall, avalanches regularize the spatial tension distribution along tissue. Furthermore, the avalanche distribution is found to obey a power law, with an exponent consistent with sheer induced avalanches in amorphous materials. To decipher the role of avalanches in organ development, we simulate epithelial growth of theDrosophilaeye disc during the third instar using a computational model, which includes both signaling and mechanistic signalling. During the third instar, the morphogenetic furrow (MF), a ∼10 cell wide wave of apical area constriction propagates through the epithelium, making it a system with interesting mechanical properties. These simulations are used to understand the details of the growth process, the effect of the MF on the growth dynamics on the tissue scale, and to make predictions. The avalanches are found to depend on the strength of the apical constriction of cells in the MF, with stronger apical constriction leading to less frequent and more pronounced avalanches. The results herein highlight the dependence of simulated tissue growth dynamics on relaxation timescales, and serve as a guide forin vitroexperiments.

The generation of structurally standardized human pluripotent stem cell (hPSC)‐derived neural embryonic tissues has the potential to model genetic and environmental mediators of early neurodevelopmental defects. Current neural patterning systems have so far focused on directing cell fate specification spatio‐temporally but not morphogenetic processes. Here, the formation of a structurally reproducible and highly‐organized neuroepithelium (NE) tissue is directed from hPSCs, which recapitulates morphogenetic cellular processes relevant to early neurulation. These include having a continuous, polarized epithelium and a distinct invagination‐like folding, where primitive ectodermal cells undergo E‐to‐N‐cadherin switching and apical constriction as they acquire a NE fate. This is accomplished by spatio‐temporal patterning of the mesoendoderm, which guides the development and self‐organization of the adjacent primitive ectoderm into the NE. It is uncovered that TGFβ signaling emanating from endodermal cells support tissue folding of the prospective NE. Evaluation of NE tissue structural dysmorphia, which is uniquely achievable in the model, enables the detection of apical constriction and cell adhesion dysfunctions in patient‐derived hPSCs as well as differentiating between different classes of neural tube defect‐inducing drugs., This paper reports the generation of a reproducible and highly organized neuroepithelial (NE) tissue by combining human pluripotent stem cell (hPSC) micropatterning and a temporally sequenced induction protocol to specify NE cells in spatial juxtaposition to mesoendoderm cells. By evaluating NE tissue structural dysmorphia in the micropatterned NE model, we can successfully model gene‐ and drug‐induced neurodevelopmental defects.

Rab35 is a small GTPase that regulates plasma membrane to early endosome vesicular trafficking and mediates actin remodeling to form actin-rich cellular structures. While the function of Rab35 in the cellular context has been examined, its role during development has not been well studied. In this study, we take advantage of the sea urchin's high fecundity, external fertilization, and transparent embryos to determine the function of Rab35 during development. We found that loss of function of Rab35 results in defects in skeletogenesis and gastrulation, which were rescued by co-injection of sea urchin Rab35. The loss of Rab35's function results in decreased endocytosis and impaired exocytosis, which may be important for skeletogenesis and gastrulation. Skeletal spicules of Rab35 knockdown embryos have reduced organized actin compared to the control, supporting the notion that Rab35 regulates actin dynamics. In addition, the skeletal and gastrulation defects induced by Rab35 knockdown were rescued by co-injection with Fascin, an actin-bundling protein, indicating that proper actin dynamics play a critical role for both skeletogenesis and gastrulation. Overall, results indicate that through its role in mediating vesicular trafficking and actin remodeling, Rab35 is an important regulator of embryonic structure formation in early development.

Cephalization is a major innovation of animal evolution and implies a synchronization of nervous system, mouth, and foregut polarization to align alimentary tract and sensomotoric system for effective foraging. However, the underlying integration of morphogenetic programs is poorly understood. Here, we show that invagination of neuroectoderm through de novo polarization and apical constriction creates the mouth opening in the Caenorhabditis elegans embryo. Simultaneously, all 18 juxta-oral sensory organ dendritic tips become symmetrically positioned around the mouth: While the two bilaterally symmetric amphid sensilla endings are towed to the mouth opening, labial and cephalic sensilla become positioned independently. Dendrite towing is enabled by the pre-polarized sensory amphid pores intercalating into the leading edge of the anteriorly migrating epidermal sheet, while apical constriction-mediated cell–cell re-arrangements mediate positioning of all other sensory organs. These two processes can be separated by gradual inactivation of the 26S proteasome activator, RPN-6.1. Moreover, RPN- 6.1 also shows a dose-dependent requirement for maintenance of coordinated apical polarization of other organs with apical lumen, the pharynx, and the intestine. Thus, our data unveil integration of morphogenetic programs during the coordination of alimentary tract and sensory organ formation and suggest that this process requires tight control of ubiquitin-dependent protein degradation.

Post-zygotic mutations that generate tissue mosaicism are increasingly associated with severe congenital defects, including those arising from failed neural tube closure. Here we report that neural fold elevation during mouse spinal neurulation is vulnerable to deletion of the VANGL planar cell polarity protein 2 (Vangl2) gene in as few as 16% of neuroepithelial cells. Vangl2-deleted cells are typically dispersed throughout the neuroepithelium, and each non-autonomously prevents apical constriction by an average of five Vangl2-replete neighbours. This inhibition of apical constriction involves diminished myosin-II localisation on neighbour cell borders and shortening of basally-extending microtubule tails, which are known to facilitate apical constriction. Vangl2-deleted neuroepithelial cells themselves continue to apically constrict and preferentially recruit myosin-II to their apical cell cortex rather than to apical cap localisations. Such non-autonomous effects can explain how post-zygotic mutations affecting a minority of cells can cause catastrophic failure of morphogenesis leading to clinically important birth defects., Mutations that cause tissue mosaicism have been identified in individuals with severe congenital defects. Here, the authors show that mosaic deletion of Vangl2 in the murine neuroepithlium causes spina bifida by preventing apical constriction via reduced myosin II and tubulin organisation.

Recent studies have revealed that pathogenic mutations in the endocytic receptor LRP2 in humans are associated with severe neural tube closure defects (NTDs) such as anencephaly and spina bifida. Here, we combined analysis of neural tube closure in mouse and in the African Clawed FrogXenopus laevisto elucidate the etiology of Lrp2-related NTDs.Lrp2loss-of-function (LOF) impaired neuroepithelial morphogenesis, culminating in NTDs that impeded anterior neural plate folding and neural tube closure in both model organisms. Loss of Lrp2 severely affected apical constriction as well as proper localization of the core planar cell polarity (PCP) protein Vangl2, demonstrating a highly conserved role of the receptor in these processes essential for neural tube formation. In addition, we identified a novel functional interaction of Lrp2 with the intracellular adaptor proteins Shroom3 and Gipc1 in the developing forebrain. Our data suggest that during neurulation, motifs within the intracellular domain of Lrp2 function as a hub that orchestrates endocytic membrane removal for efficient apical constriction as well as PCP component trafficking in a temporospatial manner.Summary statementAnalysis of neurulation in mouse andXenopusreveals novel roles for Lrp2-mediated endocytosis in orchestrating apical constriction and planar cell polarity essential for neural tube closure.

SummaryThe contraction of the amnioserosa by apical constriction provides the major force for Drosophila dorsal closure. The nucleation, movement and dispersal of apicomedial actomyosin complexes generate pulsed constrictions during early dorsal closure whereas persistent apicomedial and circumapical actomyosin complexes drive the unpulsed constrictions that follow. What governs the spatiotemporal assembly of these distinct complexes, endows them with their pulsatile dynamics, and directs their motility remains unresolved. Here we identify an essential role for microtubule growth in regulating the timely contraction of the amnioserosa. We show that a symmetric cage of apical microtubules forms around the coalescing apicomedial myosin complex. An asymmetric tail of microtubules then trails the moving myosin complex and disperses as the myosin complex dissolves. Perturbing microtubule growth reduced the coalescence and movement of apicomedial myosin complexes and redistributed myosin and its activator, Rho kinase to the circumapical pool and altered the cell constriction and tissue contraction dynamics of the amnioserosa. We show that RhoGEF2, the activator of the Rho1 GTPase, is transiently associated with microtubule plus end binding protein EB1 and the apicomedial actomyosin complex. Our results suggest that microtubule growth from moving patronin platforms modulates actomyosin contractility through the spatiotemporal regulation of Rho1 activity. We propose that microtubule reorganisation enables a self-organising, mechanosensitive feedback loop that buffers the tissue against mechanical stresses by modulating actomyosin contractility.

Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains associates with the actin-binding protein Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. By contrast, the N-terminal domain localizes to apical junctions in the ectoderm and basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Targeted proximity biotinylation (TPB) using anti-GFP antibody fused to the biotin ligase BirA identified SSX2IP as a candidate protein that binds GFP-WtipN. SSX2IP, also known as Msd1 or ADIP, is a component of cell junctions, centriolar satellite protein and a targeting factor for ciliary membrane proteins. WtipN physically associated with SSX2IP and the two proteins readily formed mixed aggregates in overexpressing cells. By contrast, we observed only partial colocalization of full length Wtip and SSX2IP, suggesting that Wtip adopts a ‘closed’ conformation in the cell. Furthermore, the double depletion of Wtip and SSX2IP in early embryos uncovered the functional interaction of the two proteins during neural tube closure. Our results suggest that the association of SSX2IP and Wtip is essential for cell junction remodeling and morphogenetic processes that accompany neurulation. We propose that TPB can be a general approach that is applicable to other GFP-tagged proteins.

The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape changes of HCs are important for the development of auditory epithelia and hearing function. It was previously observed that HCs and inner sulcus cells (ISCs) demonstrate cellular shape changes similar to the apical constriction of the neural epithelia. Apical constriction is induced via actomyosin cable contraction in the apical junctional complex and necessary for the physiological function of the epithelium. Actomyosin cable contraction is mainly regulated by myosin regulatory light chain (MRLC) phosphorylation by myosin light chain kinase (MLCK). However, MRLC and MLCK isoforms expressed in HCs and ISCs are unknown. Hence, we investigated the expression patterns and roles of MRLCs and MLCKs in HCs. Droplet digital PCR revealed that HCs expressed MYL12A/B and MYL9, which are non-muscle MRLC and smooth muscle MLCK (smMLCK), respectively. Immunofluorescence staining throughout the organ of Corti demonstrated that only MYL12 was expressed in the apical portion of HCs, whereas MYL12 and MYL9 were expressed on ISCs. In addition, purified MYL12B was phosphorylated by smMLCK in vitro, and the harvested HCs contained phosphorylated MYL12. Furthermore, accompanied by the expansion of the cell area of outer HCs, MYL12 phosphorylation was reduced by ML-7, which is an inhibitor of smMLCK. In conclusion, MYL12 phosphorylation by smMLCK contributed to the apical constriction-like cellular shape change of HCs possibly relating to the development of auditory epithelia and hearing function.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction., The neural tube‐closure regulator LUZP1 drives apical constriction of epithelial cells by inhibiting myosin phosphatase activity in a microtubule‐dependent manner

Summary Neurons are highly polarized cells with morphologically and functionally distinct dendritic and axonal processes. The molecular mechanisms that establish axon-dendrite polarity in vivo are poorly understood. Here, we describe the initial polarization of posterior deirid (PDE), a ciliated mechanosensory neuron, during development in vivo through 4D live imaging with endogenously tagged proteins. PDE inherits and maintains apicobasal polarity from its epithelial precursor. Its apical domain is directly transformed into the ciliated dendritic tip through apical constriction, which is followed by axonal outgrowth from the opposite basal side of the cell. The apical Par complex and junctional proteins persistently localize at the developing dendritic domain throughout this transition. Consistent with their instructive role in axon-dendrite polarization, conditional depletion of the Par complex and junctional proteins results in robust defects in dendrite and axon formation. During apical constriction, a microtubule-organizing center (MTOC) containing the microtubule nucleator γ-tubulin ring complex (γ-TuRC) forms along the apical junction between PDE and its sister cell in a manner dependent on the Par complex and junctional proteins. This junctional MTOC patterns neuronal microtubule polarity and facilitate the dynein-dependent recruitment of the basal body for ciliogenesis. When non-ciliated neurons are genetically manipulated to obtain ciliated neuronal fate, inherited apicobasal polarity is required for generating ciliated dendritic tips. We propose that inherited apicobasal polarity, together with apical cell-cell interactions drive the morphological and cytoskeletal polarity in early neuronal differentiation.

Shard et al. show that the emergence of neural stem cells from the Drosophila optic lobe neuroepithelium is an EMT-like process involving tissue-wide coordination. Neuralized acts downstream of NSC fate acquisition to downregulate the Crumbs complex to facilitate epithelium remodeling., Many tissues are produced by specialized progenitor cells emanating from epithelia via epithelial-to-mesenchymal transition (EMT). Most studies have so far focused on EMT involving single or isolated groups of cells. Here we describe an EMT-like process that requires tissue-level coordination. This EMT-like process occurs along a continuous front in the Drosophila optic lobe neuroepithelium to produce neural stem cells (NSCs). We find that emerging NSCs remain epithelial and apically constrict before dividing asymmetrically to produce neurons. Apical constriction is associated with contractile myosin pulses and involves RhoGEF3 and down-regulation of the Crumbs complex by the E3 ubiquitin ligase Neuralized. Anisotropy in Crumbs complex levels also results in accumulation of junctional myosin. Disrupting the regulation of Crumbs by Neuralized lowered junctional myosin and led to imprecision in the integration of emerging NSCs into the front. Thus, Neuralized promotes smooth progression of the differentiation front by coupling epithelium remodeling at the tissue level with NSC fate acquisition.