The distribution of Dishevelled in convergently extending mesoderm

Convergent extension (CE) is a conserved morphogenetic movement that drives axial lengthening of the primary body axis and depends on the planar cell polarity (PCP) pathway. In Drosophila epithelia, a polarised subcellular accumulation of PCP core components, such as Dishevelled (Dvl) protein, is associated with PCP function. Dvl has long been thought to accumulate in the mediolateral protrusions in Xenopus chordamesoderm cells undergoing CE. Here we present a quantitative analysis of Dvl intracellular localisation in Xenopus chordamesoderm cells. We find that, surprisingly, accumulations previously observed at mediolateral protrusions of chordamesodermal cells are not protrusion-specific but reflect yolk-free cytoplasm and are quantitatively matched by the distribution of the cytoplasm-filling lineage marker dextran. However, separating cell cortex-associated from bulk Dvl signal reveals a statistical enrichment of Dvl in notochord–somite boundary-(NSB)-directed protrusions, which is dependent upon NSB proximity. Dvl puncta were also observed, but only upon elevated overexpression. These puncta showed no statistically significant spatial bias, in contrast to the strongly posteriorly-enriched GFP-Dvl puncta previously reported in zebrafish. We propose that Dvl distribution is more subtle and dynamic than previously appreciated and that in vertebrate mesoderm it reflects processes other than protrusion as such

Invagination of Ectodermal Placodes Is Driven by Cell Intercalation-Mediated Contraction of the Suprabasal Tissue Canopy

Ectodermal organs such as teeth, hair follicles, and mammary glands begin their development as placodes. These are local epithelial thickenings that invaginate into mesenchymal space. There is currently little mechanistic understanding of the cellular processes driving the early morphogenesis of these organs and of why they lead to invagination rather than simple tissue thickening. Here, we show that placode invagination depends on horizontal contraction of superficial layers of cells that form a shrinking and thickening canopy over underlying epithelial cells. This contraction occurs by cell intercalation and is mechanically coupled to the basal layer by peripheral basal cells that extend apically and centripetally while remaining attached to the basal lamina. This process is topologically analogous to well-studied apical constriction mechanisms, but very different from them both in scale and molecular mechanism. Mechanical cell-cell coupling is propagated through the tissue via E-cadherin junctions, which in turn depend on tissue-wide tension. We further present evidence that this mechanism is conserved among different ectodermal organs and is, therefore, a novel and fundamental morphogenetic motif widespread in embryonic development

Computational biology:Turing's lessons in simplicity

Biophysical modeling of development started with Alan Turing. His two-morphogen reaction-diffusion model was a radical but powerful simplification. Despite its apparent limitations, the model captured real developmental processes that only recently have been validated at the molecular level in many systems. The precision and robustness of reaction-diffusion patterning, despite boundary condition-dependence, remain active areas of investigation in developmental biology

Cellular mechanisms of reverse epithelial curvature in tissue morphogenesis

Epithelial bending plays an essential role during the multiple stages of organogenesis and can be classified into two types: invagination and evagination. The early stages of invaginating and evaginating organs are often depicted as simple concave and convex curves respectively, but in fact majority of the epithelial organs develop through a more complex pattern of curvature: concave flanked by convex and vice versa respectively. At the cellular level, this is far from a geometrical truism: locally cells must passively adapt to, or actively create such an epithelial structure that is typically composed of opposite and connected folds that form at least one s-shaped curve that we here, based on its appearance, term as “reverse curves.” In recent years, invagination and evagination have been studied in increasing cellular detail. A diversity of mechanisms, including apical/basal constriction, vertical telescoping and extrinsic factors, all orchestrate epithelial bending to give different organs their final shape. However, how cells behave collectively to generate reverse curves remains less well-known. Here we review experimental models that characteristically form reverse curves during organogenesis. These include the circumvallate papillae in the tongue, crypt–villus structure in the intestine, and early tooth germ and describe how, in each case, reverse curves form to connect an invaginated or evaginated placode or opposite epithelial folds. Furthermore, by referring to the multicellular system that occur in the invagination and evagination, we attempt to provide a summary of mechanisms thought to be involved in reverse curvature consisting of apical/basal constriction, and extrinsic factors. Finally, we describe the emerging techniques in the current investigations, such as organoid culture, computational modelling and live imaging technologies that have been utilized to improve our understanding of the cellular mechanisms in early tissue morphogenesis

Early perturbation of Wnt signaling reveals patterning and invagination-evagination control points in molar tooth development

Tooth formation requires complex signaling interactions both within the oral epithelium and between the epithelium and the underlying mesenchyme. Previous studies of the Wnt/β-catenin pathway have shown that tooth formation is partly inhibited in loss-of-function mutants, and gain-of-function mutants have perturbed tooth morphology. However, the stage at which Wnt signaling is first important in tooth formation remains unclear. Here, using an Fgf8-promoter-driven, and therefore early, deletion of β-catenin in mouse molar epithelium, we found that loss of Wnt/β-catenin signaling completely deletes the molar tooth, demonstrating that this pathway is central to the earliest stages of tooth formation. Early expression of a dominant-active β-catenin protein also perturbs tooth formation, producing a large domed evagination at early stages and supernumerary teeth later on. The early evaginations are associated with premature mesenchymal condensation marker, and are reduced by inhibition of condensation-associated collagen synthesis. We propose that invagination versus evagination morphogenesis is regulated by the relative timing of epithelial versus mesenchymal cell convergence regulated by canonical Wnt signaling. Together, these studies reveal new aspects of Wnt/β-catenin signaling in tooth formation and in epithelial morphogenesis more broadly.</p

Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.publishedVersio

Pervasive cortical and white matter anomalies in a mouse model for CHARGE syndrome

CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth, Genital anomalies and Ear abnormalities) syndrome is a disorder caused by mutations in the gene encoding CHD7, an ATP dependent chromatin remodelling factor, and is characterised by a diverse array of congenital anomalies. These include a range of neuroanatomical comorbidities which likely underlie the varied neurodevelopmental disorders associated with CHARGE syndrome, which include intellectual disability, motor coordination deficits, executive dysfunction, and autism spectrum disorder. Cranial imaging studies are challenging in CHARGE syndrome patients, but high-throughput magnetic resonance imaging (MRI) techniques in mouse models allow for the unbiased identification of neuroanatomical defects. Here, we present a comprehensive neuroanatomical survey of a Chd7 haploinsufficient mouse model of CHARGE syndrome. Our study uncovered widespread brain hypoplasia and reductions in white matter volume across the brain. The severity of hypoplasia appeared more pronounced in posterior areas of the neocortex compared to anterior regions. We also perform the first assessment of white matter tract integrity in this model through diffusion tensor imaging (DTI) to assess the potential functional consequences of widespread reductions in myelin, which suggested the presence of white matter integrity defects. To determine if white matter alterations correspond to cellular changes, we quantified oligodendrocyte lineage cells in the postnatal corpus callosum, uncovering reduced numbers of mature oligodendrocytes. Together, these results present a range of promising avenues of focus for future cranial imaging studies in CHARGE syndrome patients.</p

Craniofacial dysmorphology in Down syndrome is caused by increased dosage of Dyrk1a and at least three other genes

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), occurs in 1 in 800 live births and is the most common human aneuploidy. DS results in multiple phenotypes, including craniofacial dysmorphology, which is characterised by midfacial hypoplasia, brachycephaly and micrognathia. The genetic and developmental causes of this are poorly understood. Using morphometric analysis of the Dp1Tyb mouse model of DS and an associated mouse genetic mapping panel, we demonstrate that four Hsa21-orthologous regions of mouse chromosome 16 contain dosage-sensitive genes that cause the DS craniofacial phenotype, and identify one of these causative genes as Dyrk1a. We show that the earliest and most severe defects in Dp1Tyb skulls are in bones of neural crest (NC) origin, and that mineralisation of the Dp1Tyb skull base synchondroses is aberrant. Furthermore, we show that increased dosage of Dyrk1a results in decreased NC cell proliferation and a decrease in size and cellularity of the NC-derived frontal bone primordia. Thus, DS craniofacial dysmorphology is caused by an increased dosage of Dyrk1a and at least three other genes.</p