Sec24D-dependent transport of extracellular matrix proteins is requ...
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2010 Apr 28;5(4):e10367. doi: 10.1371/journal.pone.0010367.Sec24D-dependent transport of extracellular matrix proteins is required for zebrafish skeletal morphogenesis.1, , , , , .1Department of Medicine, Division of Genetic Medicine and Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.AbstractProtein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor beta1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C(6)-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.PMID:
[PubMed - indexed for MEDLINE] PMCID: PMC2860987 (A) The bulldog mutations map to chromosome 7 between markers Z13880 and Z13936. The number of recombinants and the corresponding distances in centiMorgans (cM) are indicated below. The genes in the critical region are listed. (B) Electropherograms of wild-type +/+, heterozygous +/– and bulm421 –/– genomic DNA shows the C→T transition (arrows) that results in an amber stop codon (TAG) in place of glutamine at position 811 (Q811X). (C) Schematic diagram of the Sec24D primary protein structure of wild-type and bulldog mutants. (D) bulm606 and sec24d morphant embryos (3.5 ng sec24d-MO) have reduced head size (red arrows), shorter body length and pectoral fins (blue arrows). Alcian blue staining of the head skeleton in the right panels shows short Meckel's (red arrows), deformed ceratohyals (black arrows) and kinked pectoral fins (blue arrows) in bulm606 and sec24d morphant embryos compared to wild types. Toluidine blue stained coronal sections of craniofacial cartilage in bulm606 and sec24d morphant embryos demonstrate very low amount of ECM material (purple staining) and abnormal shape and packing density of chondrocytes as compared to wild types, all at 4 dpf. Abbreviation: ch, ceratohyal.PLoS One. ):e10367.(A) RT-PCR assay shows maternally deposited sec24d transcripts at 1 and 2 hpf and increasing levels thereafter. β-actin served as control. (B–H) Expression patterns of sec24d during zebrafish development using whole mount in situ hybridization staining of wild-type embryos. The cross section through the palatoquadrate (pq, arrowheads) at 3 dpf (I) shows robust riboprobe staining (h: hours, d: days). (J–O) Expression patterns of col2α1 (J,K) and sox9a (L,M) at 3 dpf. The expression is slightly upregulated in the posterior pharyngeal cartilages of bulm606 embryos. The Collagen-specific chaperone hsp47 (N,O) transcript is elevated in craniofacial skeleton (arrowheads), fins and the notochord. Arrowheads point to the jaw region in panels (G) through (O). (P) Relative sec24d sec24c, bip and sil1 mRNA levels were assessed by quantitative real-time PCR and adjusted against β-actin using total RNA samples from 4 dpf embryos. In bulm494, mRNA levels for bip and sil1 are induced 5-fold, whereas sec24d mRNA levels are reduced 0.7-fold. *, p&0.02; **, p&0.005. Abbreviation: b, brain, o, optic nerve, ot, otic capsule, r, retina. Anterior (D–O) is to the left.PLoS One. ):e10367.(A–H) Electron micrographs show mature wild-type chondrocytes (cells 1–3 in A) are separated by ECM and contain dense ER membranes (A, E, arrows). In bulm494 mutants (B,F,H), cartilage matrix is sparse and large amounts of electron-dense material accumulate in the rough ER (asterisks, cells marked 1–6 in B). Wild-type chondrocytes (C) are devoid of cell-cell contacts, whereas the majority of bulm494 mutant cells (D) contain adherens junctions (arrowheads). bulm494 chondrocytes (F) contain both normal ER cisternae (arrows) and swollen, ribosome studded, ER (asterisks). sec24d-deficient cells (H) contain smaller and disorganized Golgi complexes (blue arrows) compared to wild-types (G). (I,J) Single-pass confocal images of C6-NBD ceramide stained chondrocytes of wild-type (I) and sec24d/bulm606 mutant (J) embryos at 4 dpf. (K,L) Adherens junctions are marked by β-catenin (red) in bulldog Meckel's cartilage (arrowheads), but are mostly absent in wild types (K). (M,N) Phalloidin (red) shows regular cortical distribution of polymerized actin in wild types (M), but uneven cellular distribution in bulm606 cartilage (arrowheads) (N). Nuclei are stained with TOPRO-3 (blue). Abbreviations: m, n, ECM, extracellular matrix. Scale bars: 2 uM (A,B,E); 500 nm (F–H); 5 uM (I–N).PLoS One. ):e10367.(A) The number of cells in the ceratohyals at 5 dpf is not significantly different between wild-type and bulldog embryos (counted in single optical plane of Alcian blue stained preparations, six different animals each). (B) In contrast, the number of cells spanning the entire width of the ceratohyal at 5 dpf is notably higher in bulldog. (C,D) Single-pass confocal images of the Meckel's cartilage in live embryos marked with membrane tethered GFP tracer. Bulldog mutants (D) show multiple stacked chondrocytes as compared to a single spanning cell in wild-types (C). (E) The average chondrocyte width is comparable between wild-type and bulldog, whereas the length and the length-to-width ratio are significantly lower in the mutants. Cellular dimensions were counted in Meckel's (m) and ceratohyal (ch) cartilages in three different live embryos at 3 dpf. The number of cells used for measurements is indicated in the right graph (E). * denotes p&0.0001; **, p&0.0001; $$, p&0.003; ##, p&0.0001.PLoS One. ):e10367.(A–F) Mesenchymal condensations, stained with Peanut Agglutinin (PNA, green) show similar distribution of Fibronectin (red) in wild-type (A–C) and bulldog hyosymplectic cartilages (D–F). (G–L) Cadherin expression in chondrocytes (marked by a Pan-cadherin antibody in red) is indistinguishable between wild-type and bulldog ceratohyals as shown by PNA staining. The right panels represent merged images of the left and middle panels. Scale bars are 5 uM.PLoS One. ):e10367.(A–F) Collagen2α1 (red) and Wheat Germ Agglutinin (WGA, green) signals are concentrated at juxtanuclear compartments and in the extracellular space of wild-type chondrocytes (A–C). In bulldog, the signals are primarily localized in cytoplasmic vesicular compartments, although weak WGA signal is also detectable at the plasma membrane and the extracellular space (D–F). (G–L) Single pass confocal images of wild-type and bulm606 Meckel's cartilages labeled with the β1-integrin (red) recognizing antibody and counterstained with WGA (green). Merged channels show co-localization of the two labels in the cell boundaries in both wild-type and bulldog chondrocytes. The right panels represent merged images of the left and middle panels. Scale bars are 5 uM.PLoS One. ):e10367.(A,B) sec24c is ubiquitously expressed at cleavage (8-cell, A) and gastrulation (shield, B) stages. (C–F) Sec24c expression at consecutive embryonic days is concentrated in the head and gut regions. Expression in the head overlaps with developing craniofacial structures.PLoS One. ):e10367.Analysis of wild types (A, E, I), sec24c-UTR-MO (B,F,J), bulldog (C, G, K) and double sec24c-UTR-MO+ sec24d-UTR-MO (D, H, L) injected embryos. (A–D) Images of live wild types (A), sec24c-UTR-MO, (2.0 ng injected, B), bulldog (C) and sec24c-UTR-MO+ sec24d-UTR-MO (2.0 ng each, D). The sec24c morphants have reduced body length (blue arrows), but the head appears normal. Double morphants for sec24d+sec24c are significantly shorter than bulldog larvae, almost completely lack fin-folds and have reduced jaw region (red arrow) and pronounced heart edema (D). (E–H) Alcian blue preparations of the head skeleton. The Alcian blue staining confirms normal craniofacial cartilages in sec24c morphants. (I–L) Histological sections counter-stained with toluidine blue at 4 dpf. Insets indicate the plane of the sections. (I,J) Histological, sagittal plastic sections stained with toluidine blue show normal jaw opening and surrounding cartilages in wild types (I) and sec24c morphants (J). Purple staining patterns for glycosaminoglycans of the ECM are also comparable. In contrast, the pharyngeal skeleton of sec24d+sec24c double mutants fails to stain with Alcian blue (H). Histological sections reveal patterned arches that are largely devoid of metachromatically stained cells (L). Abbreviations: cb, ch, ep, ethmoid plate, m, Meckel's cartilage.PLoS One. ):e10367.(A–H) Images of transgenic sox10:mRFP embryos at 30 hpf (lateral view A–D) and 48 hpf (ventral view E–H) after injection with sec24c-UTR-MO, (2.0 ng), sec24d-UTR-MO (3.5 ng), and sec24c-UTR-MO+ sec24d-UTR-MO. Neural crest streams are numbered (1, 2, 3, 4, 5, 6 in A–D). Arrows point to migrating craniofacial primordia (E–H).PLoS One. ):e10367.Publication TypesMeSH TermsSubstancesSecondary Source IDGrant SupportFull Text SourcesOther Literature SourcesMiscellaneous
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希赛网 版权所有 & &&&&湘教QS2-164&&增值电信业务经营许可证湘B2-英语翻译asking for information or help is very common and necessary esoecially when we visit a foreign country so knowing how to ask for information politely is important In English Where are the restrooms and Could you please tel me where the restroomns are .are similar requ_百度作业帮
英语翻译asking for information or help is very common and necessary esoecially when we visit a foreign country so knowing how to ask for information politely is important In English Where are the restrooms and Could you please tel me where the restroomns are .are similar requests-both are corect English but the first could sound rude .It's important to use correct language but sometimes this alone is not enough - we need to learn how to be polite when we make requests
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您可能关注的推广回答者：Morphogenetic movements driving neural tube closure in Xenopus requ...
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2009 Mar 15;327(2):327-38. doi: 10.1016/j.ydbio.. Epub
2008 Dec 24.Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB.1, , .1Department of Biology, University of Virginia, Charlottesville, VA 22903, USA. a.rolo@ucl.ac.ukAbstractVertebrate neural tube formation involves two distinct morphogenetic events--convergent extension (CE) driven by mediolateral cell intercalation, and bending of the neural plate driven largely by cellular apical constriction. However, the cellular and molecular biomechanics of these processes are not understood. Here, using tissue-targeting techniques, we show that the myosin IIB motor protein complex is essential for both these processes, as well as for conferring resistance to deformation to the neural plate tissue. We show that myosin IIB is required for actin-cytoskeletal organization in both superficial and deep layers of the Xenopus neural plate. In the superficial layer, myosin IIB is needed for apical actin accumulation, which underlies constriction of the neuroepithelial cells, and that ultimately drive neural plate bending, whereas in the deep neural cells myosin IIB organizes a cortical actin cytoskeleton, which we describe for the first time, and that is necessary for both normal neural cell cortical tension and shape and for autonomous CE of the neural tissue. We also show that myosin IIB is required for resistance to deformation ("stiffness") in the neural plate, indicating that the cytoskeleton-organizing roles of this protein translate in regulation of the biomechanical properties of the neural plate at the tissue-level.PMID:
[PubMed - indexed for MEDLINE] PMCID: PMC2820227 (A–B) Transverse sections through the mid-trunk regions of embryos stained for MHC-B at stages 12.5 (A) and 16–17 (B) show MHC-B on both the deep and the superficial layers of the neural plate (NP), with strongest labeling in the basal ends of deep neural cells. MHC-B is also present in the mesoderm tissues.(C–K) A stage 24 embryo (C–E) and a stage 17 neural-deep-over-mesoderm explant (F–K) co-stained for MHC-B (C, F, I) and fibronectin (D, G, H), and the respective merged images (E, H, K), coded in red for MHC-B and green for fibronectin are shown.(C–D) A transverse section through the mid-trunk region of a stage 24 embryo shows high MHC-B in the outer basal ends of neural tube (NT) cells, in the neural crest region (NC) and in the deep layer of the epidermis (C), and these areas are coincidental with areas of fibronectin deposition (D, E). MHC-B levels are very high in the notochord (No) (C) (cf. Fig. 2-1) and MHC-B staining is stronger in scattered cells of the outer epidermis (C, marked with asterisks *).(F–H) A transverse section through a neural-deep-over-mesoderm explant (obtained by Z-projection of confocal series) shows that, as in whole-embryos, MHC-B is present in the neural and mesoderm tissues, with stronger staining at tissue boundaries (F), which coincide with fibronectin localization (G, H).(I–K) An en face view (obtained by XY projections of confocal series) of the boundary between NP and mesoderm in a neural-deep-over-mesoderm explant is shown. Some MHC-B has a fibrilar pattern (I) that coincides with fibronectin fibrils (J, K).(L–M) Transverse sections through the mid-trunk regions of stage 22 embryos injected in one cell at the 2-cell stage with CoMO (L) or MHC-B MO (M) together with fluorescein-dextran and stained for MHC-B show that MHC-B MO effectively reduces protein levels, whereas CoMO has no discernible effect. Insets are overlapped images of MHC-B staining and fluorescein-dextran lineage label.NP – neural plate, No – notochord, PS – pre-somitic mesoderm, E – endoderm, S – somitic mesoderm, NT – neural tube, NC – neural crest.Dev Biol. ;327(2):327.(A) Time-lapse video-recording frames during neurulation of un-injected embryos and of embryos injected with 2.5μM of either CoMO or MHC-B MO targeted to the neural plated show delayed neural tube closure in MHC-B morphants (dorsal view, anterior is at the top). Rhodamine fluorescence of injected embryos in has been superimposed to the first frame (t=0h), showing successful targeting to the neural plate. The degree of blastopore closure at the onset of neurulation is similar in all three groups, showing that targeting to the mesoderm was avoided. By stages 16/17 (t=3–5h), neural tube closure in MHC-B morphant embryos is delayed throughout the length neural tissue but is more pronounced in the anterior region, where the dark pigmentation of apically constricting bottle cells can be seen in both un-injected and CoMO injected embryos (white arrows), but are never observed in MHC-B morphant embryos. By stage 19/20 (t=8h) the neural tubes have closed in un-injected and CoMO injected embryos, whereas the anterior neural plates remain open in MHC-B MO injected embryos.(B–C) Tailbud stage embryos co-injected with either CoMO (B) or MHC-B MO (C) and rhodamine-dextran targeted to the neural plate (fluorescence can be seen in B′ and C′) show that MHC-B MO causes dorsal flexure of the trunk.Dev Biol. ;327(2):327.Movements of colored tracking dots on frames of a time-lapse recording of an anterior view of neural plate (A) show that points on the control (left) side move towards the midline faster than points on morphant (right) side of the embryo. A transverse section through the prospective brain region of a stage 18 embryo stained with rhodamine phalloidin (B) shows strong apical actin accumulation on the control (left) side, but no actin accumulation on the morphant (right) side. Transverse sections through the prospective brain (C) and spinal cord (D) regions of a stage 17 embryo show background green fluorescent dextran used to determine cell shape, and red fluorescent dextran co-injected with the MHC-B MO used to identify morphant cells (right sides). Corresponding tracings of cell outlines (C′–D′) show that uninjected cells adopt typical bottle cell morphologies, with long thin necks and constricted apices, whereas morphant cells (*) are short and have broad apices.Dev Biol. ;327(2):327.Keller sandwich explants made from control (A) and morphant (B) embryos are shown, with arrows indicating the boundaries between neural (top) and mesodermal (bottom) tissues. A fluorescence micrograph (B′) shows red fluorescent dextran co-injected with the MO to identify morphant tissues. The extension of neural and mesodermal tissues in control and morphant Keller sandwiches is measured by the final length of tissues in arbitrary units (AU) (C). A partial rescue of neural tissue length is achieved by co-injection of MHC-B and mouse MHC-B mRNA (D). Error bars are s.e.m., p-values were calculated using Student’s t-test.Dev Biol. ;327(2):327.Laser scanning confocal microscopy shows the cortical regions (&2 μm inside the cytoplasm) of neural deep cells from embryos injected at the two-cell stage with mRNA encoding membrane targeted RFP and at the 32-cell stage with mRNA encoding F-actin targeted GFP alone (A) or F-actin targeted GFP with MHC-B MO at 2μM (B) or at 2.5μM (C, D). Control cells have well-organized cortical network of actin bundles that radiate from foci within the cell (A). At 2μM MO, elongated morphant cells have longer actin cables that do not connect foci and they run parallel to direction of elongation (B). At 2.5μM MO, morphant cells either have a few coarse, thick actin cables (C) or the actin cables are almost absent (D). Yolk granules (asterisks * in C, D) are present in the cortex of cells with higher concentration of MHC-B MO, whereas they are excluded from the cortices of control cells (A) and low-dose morphant cells (B). Neural deep cells from embryos injected at the two-cell stage with mRNAs encoding membrane-targeted GFP and at the 32-cell stage with nuclear-targeted GFP (NLS-GFP) alone (E) or NLS- GFP and MHC-B MO (F) are shown. Morphant cells (having nuclear GFP in F) are more elongated than control cells (E). Frames from a time-lapse video-recording (G) show a cell undergoing mitosis and cytokinesis. This cell is initially elongated (0′), and it rounds up to undergo cell division (33′). The NLS-GFP becomes invisible upon nuclear envelope breakdown (23′) and reappears after cytokinesis and re-formation of nuclei in the daughter cells (58′). The scale bar in A applies to B–D. Scale bar in E applies to F.Dev Biol. ;327(2):327.Measurements of negative pressure (expressed as height of a water column in cm) required to produce a bulge of a fixed size in the neural plate using an elastimeter are plotted across different developmental stages. The amount of negative pressure in un-injected embryos increases throughout development, showing that these become increasingly resistant to deformation. MHC-B morphant neural plates are significantly more pliable than wild-type ones, whereas CoMO embryos are similar to controls. Neural plates injected with mRNA encoding for Xdd1 or GFP show no difference in deformability, either compared to one another or compared to un-injected embryos. Error bars are s.e.m., p-values were calculated using Student’s t-test.Dev Biol. ;327(2):327.Publication TypesMeSH TermsSubstancesGrant SupportFull Text SourcesOther Literature SourcesMolecular Biology Databases
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