Monitoring dynamically the gelation phase transition of agarose with T2 qMRI as a function of concentration at 3T

The purpose of this experiment is to study as a model the gelation phase transition of agarose solutions with transverse relaxation (T2) quantitative magnetic resonance imaging (qMRI). The focus is on the reduction of T2 of agarose solution upon gelation. The sol-to-gel phase transition of agarose may provide a useful and controllable experimental model of tissue formation. Furthermore, it may provide the basis for exact mathematical models useful for understanding the much reduced transverse relaxation times (T2) observed in solid tissues relative to simple liquids. In this context, the purpose of this work was to monitor dynamically with T2 quantitative MRI the liquid-to-gel phase transition of pure agarose as a function of gel concentration. Samples of agarose at various concentrations were allowed to cool down while scanning dynamically with T2 qMRI, 32 x 10milliseconds (ms) echoes, CarrPurcell-Meiboom-Gill (CPMG), 3Tesla.T2 versus; (temperature).curves of each agarose solution show a distinct phase transition region characterized by a sharp T2 reduction. Four agarose solutions were sequentially prepared by dissolving agarose powder in distilled water at concentrations of 1%, 2%, 3%, and 4% by weight/volume. Immediately after preparation and boiling at 98°C, each liquid agarose solution was poured into a plastic container and scanned dynamically at 3.0T as it cooled down with a whole body MRI scanner (Achieva, Philips Medical Systems, Cleveland, OH). A single axial slice multi spin echo CPMG pulse sequence with the following parameters was used: 32 echoes, 10ms echo spacing, 1.5s repetition time (TR), 160 x 160 matrix size, and 2 SENSE factor. The time per dynamic scan was 1minute. The DICOM images were further processed with an adaptive T2 qMRI algorithm programed in Mathcad (Parametric Technology Corporation, Needham, MA) whereby the number of echoes used in the semi-logarithmic linear regression varies automatically from pixel to pixel depending on noise level. The T2 values of agarose gels have been measured during the entire gelation phase transition process at four different concentrations. The T2 versus time (temperature) curves of all the four concentrations shows a rapid drop at about 24 minutes (T~40°C) at which time the gelation phase transition begins. At all temperatures, T2 decreases as a function of increasing agarose concentration. The data shows similar behaviors for all concentrations with a phase transition characterized by a drastic drop in T2 occurring while the temperature drops by approximately 8°C. These results may be useful for testing theoretical models of the Nuclear Magnetic Resonance (NMR) T2 relaxation properties during tissue formation. Quantitative magnetic resonance imaging (qMRI) differs sharply from conventional directly acquired MRI in that objective measures [such as the trio of the basis MR properties: longitudinal relaxation (T1), T2 and Proton Density (PD)] are used for analysis as well as further post-processing rather than relative signal intensities. Q-MRI portrays the spatial distribution of absolute biophysical parameter measurements on a pixel-by-pixel basis; Kevin J. Chang et al 200

Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration

Order and Stochastic Dynamics in Drosophila Planar Cell Polarity

Cells in the wing blade of Drosophila melanogaster exhibit an in-plane polarization causing distal orientation of hairs. Establishment of the Planar Cell Polarity (PCP) involves intercellular interactions as well as a global orienting signal. Many of the genetic and molecular components underlying this process have been experimentally identified and a recently advanced system-level model has suggested that the observed mutant phenotypes can be understood in terms of intercellular interactions involving asymmetric localization of membrane bound proteins. Among key open questions in understanding the emergence of ordered polarization is the effect of stochasticity and the role of the global orienting signal. These issues relate closely to our understanding of ferromagnetism in physical systems. Here we pursue this analogy to understand the emergence of PCP order. To this end we develop a semi-phenomenological representation of the underlying molecular processes and define a “phase diagram” of the model which provides a global view of the dependence of the phenotype on parameters. We show that the dynamics of PCP has two regimes: rapid growth in the amplitude of local polarization followed by a slower process of alignment which progresses from small to large scales. We discuss the response of the tissue to various types of orienting signals and show that global PCP order can be achieved with a weak orienting signal provided that it acts during the early phase of the process. Finally we define and discuss some of the experimental predictions of the model

Zyxin Links Fat Signaling to the Hippo Pathway

Using genetic and molecular analyses, the authors identify Zyx as a positive regulator of Hippo signaling and characterize its role within the pathway

Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands

Serrano (Sano) Functions with the Planar Cell Polarity Genes to Control Tracheal Tube Length

Epithelial tubes are the functional units of many organs, and proper tube geometry is crucial for organ function. Here, we characterize serrano (sano), a novel cytoplasmic protein that is apically enriched in several tube-forming epithelia in Drosophila, including the tracheal system. Loss of sano results in elongated tracheae, whereas Sano overexpression causes shortened tracheae with reduced apical boundaries. Sano overexpression during larval and pupal stages causes planar cell polarity (PCP) defects in several adult tissues. In Sano-overexpressing pupal wing cells, core PCP proteins are mislocalized and prehairs are misoriented; sano loss or overexpression in the eye disrupts ommatidial polarity and rotation. Importantly, Sano binds the PCP regulator Dishevelled (Dsh), and loss or ectopic expression of many known PCP proteins in the trachea gives rise to similar defects observed with loss or gain of sano, revealing a previously unrecognized role for PCP pathway components in tube size control

Ciliopathies: an expanding disease spectrum

Ciliopathies comprise a group of disorders associated with genetic mutations encoding defective proteins, which result in either abnormal formation or function of cilia. As cilia are a component of almost all vertebrate cells, cilia dysfunction can manifest as a constellation of features that include characteristically, retinal degeneration, renal disease and cerebral anomalies. Additional manifestations include congenital fibrocystic diseases of the liver, diabetes, obesity and skeletal dysplasias. Ciliopathic features have been associated with mutations in over 40 genes to date. However, with over 1,000 polypeptides currently identified within the ciliary proteome, several other disorders associated with this constellation of clinical features will likely be ascribed to mutations in other ciliary genes. The mechanisms underlying many of the disease phenotypes associated with ciliary dysfunction have yet to be fully elucidated. Several elegant studies have crucially demonstrated the dynamic ciliary localisation of components of the Hedgehog and Wnt signalling pathways during signal transduction. Given the critical role of the cilium in transducing “outside-in” signals, it is not surprising therefore, that the disease phenotypes consequent to ciliary dysfunction are a manifestation of aberrant signal transduction. Further investigation is now needed to explore the developmental and physiological roles of aberrant signal transduction in the manifestation of ciliopathy phenotypes. Utilisation of conditional and inducible murine models to delete or overexpress individual ciliary genes in a spatiotemporal and organ/cell-specific manner should help clarify some of the functional roles of ciliary proteins in the manifestation of phenotypic features

Emerging evidence of a link between the polycystins and the mTOR pathways

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by the formation of renal cysts. This disease can be caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC-1) and -2 (PC-2), respectively