Project description:The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Delta and cdc11Delta mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Delta mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.
Project description:Mitral valve prolapse (MVP) is a common form of valve disease and can lead to serious secondary complications. The recent identification of MVP causal mutations in primary cilia-related genes has prompted the investigation of cilia-mediated mechanisms of disease inception. Here, we investigate the role of platelet-derived growth factor receptor-alpha (PDGFRα), a receptor known to be present on the primary cilium, during valve development using genetically modified mice, biochemical assays, and high-resolution microscopy. While PDGFRα is expressed throughout the ciliated valve interstitium early in development, its expression becomes restricted on the valve endocardium by birth and through adulthood. Conditional ablation of Pdgfra with Nfatc1-enhancer Cre led to significantly enlarged and hypercellular anterior leaflets with disrupted endothelial adhesions, activated ERK1/2, and a dysregulated extracellular matrix. In vitro culture experiments confirmed a role in suppressing ERK1/2 activation while promoting AKT phosphorylation. These data suggest that PDGFRα functions to suppress mesenchymal transformation and disease phenotypes by stabilizing the valve endocardium through an AKT/ERK pathway.
Project description:In Drosophila, Pumilio (Pum) is important for neuronal homeostasis as well as learning and memory. We have recently characterized a mammalian homolog of Pum, Pum2, which is found in discrete RNA-containing particles in the somatodendritic compartment of polarized neurons. In this study, we investigated the role of Pum2 in developing and mature neurons by RNA interference. In immature neurons, loss of Pum2 led to enhanced dendritic outgrowth and arborization. In mature neurons, Pum2 down-regulation resulted in a significant reduction in dendritic spines and an increase in elongated dendritic filopodia. Furthermore, we observed an increase in excitatory synapse markers along dendritic shafts. Electrophysiological analysis of synaptic function of neurons lacking Pum2 revealed an increased miniature excitatory postsynaptic current frequency. We then identified two specific mRNAs coding for a known translational regulator, eIF4E, and for a voltage-gated sodium channel, Scn1a, which interacts with Pum2 in immunoprecipitations from brain lysates. Finally, we show that Pum2 regulates translation of the eIF4E mRNA. Taken together, our data reveal a previously undescribed role for Pum2 in dendrite morphogenesis, synapse function, and translational control.
Project description:Convergent extension (CE) is a fundamental morphogenetic mechanism that underlies numerous processes in vertebrate development, and its disruption can lead to human congenital disorders such as neural tube closure defects. The dynamic, oriented cell intercalation during CE is regulated by a group of core proteins identified originally in flies to coordinate epithelial planar cell polarity (PCP). The existing model explains how core PCP proteins, including Van Gogh (Vang) and Dishevelled (Dvl), segregate into distinct complexes on opposing cell cortex to coordinate polarity among static epithelial cells. The action of core PCP proteins in the dynamic process of CE, however, remains an enigma. In this report, we show that Vangl2 (Vang-like 2) exerts dual positive and negative regulation on Dvl during CE in both the mouse and Xenopus. We find that Vangl2 binds to Dvl to cell-autonomously promote efficient Dvl plasma membrane recruitment, a pre-requisite for PCP activation. At the same time, Vangl2 inhibits Dvl from interacting with its downstream effector Daam1 (Dishevelled associated activator of morphogenesis 1), and functionally suppresses Dvl ? Daam1 cascade during CE. Our finding uncovers Vangl2-Dvl interaction as a key bi-functional switch that underlies the central logic of PCP signaling during morphogenesis, and provides new insight into PCP-related disorders in humans.
Project description:Over the last decade, scientists have begun to model CNS development, function, and disease in vitro using human pluripotent stem cell (hPSC)-derived organoids. Using traditional protocols, these 3D tissues are generated by combining the innate emergent properties of differentiating hPSC aggregates with a bioreactor environment that induces interstitial transport of oxygen and nutrients and an optional supportive hydrogel extracellular matrix (ECM). During extended culture, the hPSC-derived neural organoids (hNOs) obtain millimeter scale sizes with internal microscale cytoarchitectures, cellular phenotypes, and neuronal circuit behaviors mimetic of those observed in the developing brain, eye, or spinal cord. Early studies evaluated the cytoarchitectural and phenotypical character of these organoids and provided unprecedented insight into the morphogenetic processes that govern CNS development. Comparisons to human fetal tissues revealed their significant similarities and differences. While hNOs have current disease modeling applications and significant future promise, their value as anatomical and physiological models is limited because they fail to form reproducibly and recapitulate more mature in vivo features. These include biomimetic macroscale tissue morphology, positioning of morphogen signaling centers to orchestrate appropriate spatial organization and intra- and inter-connectivity of discrete tissue regions, maturation of physiologically relevant neural circuits, and formation of vascular networks that can support sustained in vitro tissue growth. To address these inadequacies scientists have begun to integrate organoid culture with bioengineering techniques and methodologies including genome editing, biomaterials, and microfabricated and microfluidic platforms that enable spatiotemporal control of cellular differentiation or the biochemical and biophysical cues that orchestrate organoid morphogenesis. This review will examine recent advances in hNO technologies and culture strategies that promote reproducible in vitro morphogenesis and greater biomimicry in structure and function.
Project description:The high mobility group transcription factor SOX9 is expressed in stem cells, progenitor cells, and differentiated cell-types in developing and mature organs. Exposure to a variety of toxicants including dioxin, di(2-ethylhexyl) phthalate, 6:2 chlorinated polyfluorinated ether sulfonate, and chlorpyrifos results in the downregulation of tetrapod Sox9 and/or zebrafish sox9b. Disruption of Sox9/sox9b function through environmental exposures or genetic mutations produce a wide range of phenotypes and adversely affect organ development and health. We generated a dominant-negative sox9b (dnsox9b) to inhibit sox9b target gene expression and used the Gal4/UAS system to drive dnsox9b specifically in cardiomyocytes. Cardiomyocyte-specific inhibition of sox9b function resulted in a decrease in ventricular cardiomyocytes, an increase in atrial cardiomyocytes, hypoplastic endothelial cushions, and impaired epicardial development, ultimately culminating in heart failure. Cardiomyocyte-specific dnsox9b expression significantly reduced end diastolic volume, which corresponded with a decrease in stroke volume, ejection fraction, and cardiac output. Further analysis of isolated cardiac tissue by RT-qPCR revealed cardiomyocyte-specific inhibition of sox9b function significantly decreased the expression of the critical cardiac development genes nkx2.5, nkx2.7, and myl7, as well as c-fos, an immediate early gene necessary for cardiomyocyte progenitor differentiation. Together our studies indicate sox9b transcriptional regulation is necessary for cardiomyocyte development and function.
Project description:All vertebrates possess a thymus, whose epithelial microenvironment is essential for T cell development and maturation. Despite the importance of the thymus for cellular immune defense, many questions surrounding its morphogenesis remain unanswered. Here, we demonstrate that, in contrast to the situation in many other epithelial cell types, differentiation of thymic epithelial cells (TECs) proceeds normally in the absence of canonical Wnt signaling and the classical adhesion molecule E-cadherin. By contrast, TEC-intrinsic activation of β-catenin-dependent Wnt signaling blocks the morphogenesis of the thymus, and overexpression of a secreted Wnt ligand by TECs dominantly modifies the morphogenesis not only of the thymus, but also of the parathyroid and thyroid. These observations indicate that Wnt signaling activity in the thymus needs to be precisely controlled to support normal TEC differentiation, and suggest possible mechanisms underlying anatomical variations of the thymus, parathyroid and thyroid in humans.
Project description:Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAFV600E. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAFV600E. Adult zebrafish expressing BRAFV600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAFV600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAFV600E-mediated transformation.
Project description:Integrin beta1 is critical for basement membrane organization and hair follicle morphogenesis in the skin epidermis; however, less is known about its function in the developing oral epithelium. Since the skin and oral epithelia share structural similarity, we hypothesized that beta1 integrin function would be critical for the normal development of oral epithelium and tooth buds. The conditional (oral mucosa-specific) beta1 integrin knockout (KO) mice displayed severe disruption of the basement membrane of the tongue epithelium and developing tooth buds. Interestingly, unlike the developing hair follicles, early morphological development of the KO molar tooth buds was normal. However, subsequent morphogenetic events, such as cusp formation, cervical loop down-growth, and ameloblast polarization, did not occur normally. Primary KO oral keratinocytes showed defective cell spreading and robust focal adhesions. Our studies indicate that beta1 integrin plays an essential role in the normal development of the oral epithelium and its appendages.
Project description:Extracellular matrix (ECM) assembly and remodelling is critical during development and organ morphogenesis. Dysregulation of ECM is implicated in many pathogenic conditions, including cancer. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. Here, we show by rescue experiments that the Drosophila proteases Notopleural (Np) and Tracheal-prostasin (Tpr) are functional homologues of matriptase and prostasin, respectively. Np mediates morphogenesis and remodelling of apical ECM during tracheal system development and is essential for maintenance of the transepithelial barrier function. Both Np and Tpr degrade the zona pellucida-domain (ZP-domain) protein Dumpy, a component of the transient tracheal apical ECM. Furthermore, we demonstrate that Tpr zymogen and the ZP domain of the ECM protein Piopio are cleaved by Np and matriptase in vitro. Our data indicate that the evolutionarily conserved ZP domain, present in many ECM proteins of vertebrates and invertebrates, is a novel target of the conserved matriptase-prostasin proteolytic cascade.