(L) Measurements of femurs and tibias from microCT analysis of n=3 control (Col2a1cre−ve) and n=3 Yapfl/flTazfl/flCol2a1cre+ve pups. We therefore scanned E17.5 animals using micro computed tomography (microCT) and measured the length of the mineralised component of the hind limb (Fig. 2L,M). . For RNA in situ hybridisation, RNAscope 2.5 HD Manual Assay (ACDBio) was performed according to manufacturer's instructions, using RNAscope probes for Ctgf (314541, ACDBio) and Cyr61 (429001, ACDBio). "— Taz "With Hooligan, Philipp Winkler has shot a flare right in the middle of the other side's block of fans. S3C). Notably, apoptosis was unchanged in all genotypes (Fig. Data were analysed and found to be significant by unpaired t-test (L) and two-way ANOVA with growth plate zone and genotype as the independent variables, relative zone size as the dependent variable (M). Overexpression of Cyr61 in chondrocytes leads to chondrodysplasia (Akiyama et al., 2004; Zhang et al., 2016). (G-J) Lateral cut-away of HREM three-dimensional renderings of control (Col2a1cre−ve) (G) and Yapfl/flTazfl/flCol2a1cre+ve (H-J) E17.5 pups to reveal normal morphology of the cranial base (black arrow) with the tongue (T) sitting below the intact palate (white arrowhead) in the control (G) compared with the abnormally angled cranial base (black arrow, H-J) and the tongue adjacent to the cranial base in the mutants with cleft palate (I,J). To examine the molecular consequence of YAP hyperactivation, we performed RTqPCR analysis on microdissected tibial sections from control (Col2a1cre−ve) and YAP5SAKI/+Col2a1cre+ve E17.5 samples. Cleft palate in Yap/Taz chondrocyte-specific knockout pups. We note that phenotyping at E17.5 does not discern the direct early effects of YAP/TAZ deletion and that, to address this issue, earlier stages of development would need to be examined. Note the elongated growth plate in the Yapfl/flTazfl/flCol2a1cre+ve mutants compared with all other genotypes. We next examined the effect of increased YAP activity in primary chondrocytes, by using the cre-inducible nls-YAP5SA allele. At E13.5, the palatal shelves lie vertically alongside the tongue, and undergo a rapid process of elevation at around E14.0, which requires the tongue to drop by the movement of the lower jaw opening. In contrast, there was an expansion of the round proliferating zone and the pre-hypertrophic/hypertrophic zone in the Yapfl/flTazfl/flCol2a1cre+ve growth plates compared with control, suggesting defects in these stages of chondrogenesis (Fig. 4A,B). (N) Immunostaining for Ki67 in the zones of the proximal growth plate of the tibia. J.G. At E14.5, the palatal shelves of four out of five control (Col2a1cre−ve) foetuses were fully elevated, with the remaining foetus having one palatal shelf unelevated, in contrast to all five Yapfl/flTazfl/flCol2a1cre+ve mutants having unelevated palatal shelves (Fig. 3R). Philipp Winkler was born near Hanover, Germany, in 1986 and has spent time in Japan, Kosovo, Albania, and Serbia. Cells in the pre-hypertrophic/hypertrophic zone were negative for Ki67 staining. 1. Taz 9. Thomas Winkler. Richard Simmons 4. VEGF signaling via VEGF receptor‐2 (VEGFR2) is a major regulator of endothelial cell (EC) functions, including angiogenesis. The peer review history is available online at https://dev.biologists.org/lookup/doi/10.1242/dev.187187.reviewer-comments.pdf. cDNA was synthesised from isolated RNA using the Maxima cDNA Synthesis Kit (Thermo Fisher Scientific). Data are representative of n=3 control (Col2a1cre−ve) and n=3 Lats1/2fl/flCol2a1cre+ve E17.5 pups. Seinen siebten NFL-Titel hat der Veteran auch seinen Mitspielern zu verdanken. n=4 control (Col2a1cre−ve) and n=4 Yapfl/flTazfl/flCol2a1cre+ve tibial growth plates. The effect of genotype on proliferation (D) was not significant (P=0.0550) and effect of genotype on cell density (E) was significant (P=0.0186). Finally, YAP/TAZ also regulate expression of genes that increase cytoskeletal contractility to mechanoactivate and further enforce their signaling. Note the widened sphenoid cartilage (black arrow) in all mutants (L-N) and laterally rotated pterygoid processes (black arrowheads) in the cleft palate mutants (M,N). Ultimately, it is mostly a story about growing the hell up. Interestingly, matrix metalloprotease enzymes are downstream target genes of YAP that can be induced upon integrin binding to stiff matrix substrates (Chakraborty et al., 2017; Nukuda et al., 2015) or dense matrix (Stanton et al., 2019). Total biological replicates analysed were n=7 control (Col2a1cre−ve), n=4 Yapfl/flTazfl/+Col2a1cre+ve and n=4 Yapfl/flTazfl/flCol2a1cre+ve E17.5 pups. Data were analysed by multiple t-tests and adjusted for multiple testing with a 5% false discovery rate. We acknowledge Nic Tapon for providing supervision during the final months of this project. These results show that expression of a nuclear-targeted YAP protein in chondrocytes in vivo does not affect cell proliferation and instead causes severely abnormal cartilage morphogenesis and decreased size of skeletal elements, possibly via altering the ECM. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Thus, although chondrocytes in culture depend strongly on YAP/TAZ for proliferation (which could reflect a programme of tissue regeneration after damage), chondrocytes in vivo employ Hippo-YAP/TAZ signalling primarily to regulate morphogenesis during development. Dewaxed slides were incubated in Alcian Blue solution (1% Alcian Blue in 3% acetic acid) for 5 min before being washed extensively under running water and counterstained. (C,H) Ventral view of skeletal preparations of E17.5 control (Col2a1cre−ve) and Yapfl/flTazfl/flCol2a1cre+ve pups. (A-F) Ventral (A,B), dorsal (C,D) and lateral (E,F) views of control (Col2a1cre−ve) (A,C,E) and Lats1/2fl/flCol2a1cre+ve (B,D,F) E18.5 pups. By E15.5, the four mutants examined had elevated palatal shelves (n=2) or one shelf elevated (n=2), compared to fully elevated palatal shelves in three controls (Col2a1cre−ve; Fig. Submission deadline: 1 September 2021 This implies a reciprocal mechanism in our YAP/TAZ loss- and gain-of function mutants with regards ECM remodelling in the cartilage through modulation of CTGF and CYR61, as well as various matrix remodelling enzymes. To investigate the cellular basis for the YAP/TAZ loss-of-function phenotype in cartilage, we focused on the growth plate of the proximal tibia, a commonly examined cartilage structure for the study of chondrocyte proliferation and differentiation in vivo. In one exception, Goto et al. Severe chondrodysplasia in Lats1/2 chondrocyte-specific knockout pups. We do not capture any email address. Die New York Jets sind die Lachnummer der NFL. We next confirmed the RTqPCR reduction in Ctgf and Cyr61 by in situ hybridisation and saw comparable reductions in signal (Fig. 7F,G). However, in vivo, cartilage-specific knockout of Yap/Taz does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. . Eine kurze Geschichte der Deutschen The quintessence of the German history – expertly portrayed by Heinrich August Winkler Heinrich August Winkler became famous as the author of Data were analysed by multiple t-tests and corrected for multiple testing (I; # indicates adjusted P<0.01) or unpaired t-test (J; P=0.252). Die Cleveland Indians, zweimaliger Meister in den USA, legen nach 105 Jahren und vielen Debatten ihren Namen ab. In parallel with regulation via LATS1/2 kinases, YAP/TAZ can also be regulated by other inputs, such as direct phosphorylation by Src family kinases (Elbediwy et al., 2018; Elbediwy et al., 2016; Li et al., 2016 b; Si et al., 2017). Es ist ein später Schritt zur Gleichberechtigung. Compared with controls (Col2a1cre−ve), in Yapfl/flTazfl/flCol2a1cre+ve growth plates, Yap and Taz expression was reduced by 75% (Fig. 7A). HREM data derived from n=5 control, n=3 non-cleft mutant, n=4 elevated and n=4 non-elevated cleft palate mutants. Philipp Winkler was born near Hanover, Germany, in 1986 and has spent time in Japan, Kosovo, Albania, and Serbia. Black arrow indicates abnormal morphology of the c1 (atlas) vertebra and black arrowheads indicate the flattened rostrum of the skull and the barrel-like ribcage and curved sternum in the Yapfl/flTazfl/flCol2a1cre+ve mutant compared with control. We confirmed increased Yap levels to almost twice that of control and no change in Taz expression (Fig. 7H). (K-N) Frontal cut-away view of the palate (P) at the level of the pterygoid process. Meckel's cartilage was extracted using object thresholding from the E14.5 HREM images, which revealed abnormal morphology and anterior-posterior shortening of Meckel's cartilage in all five Yapfl/flTazfl/flCol2a1cre+ve mutants compared with the five control (Col2a1cre−ve) Meckel's cartilages (Fig. 3S). The effect of genotype on length was significant (P=0.0047). Data represent six technical replicates of primary chondrocytes derived from individual pups (biological replicates) of the indicated genotype and are representative of four independent experiments. CTGF has been shown to bind to ECM components including aggrecan (Aoyama et al., 2009) as well as to cell surface integrins (Nishida et al., 2007) and a range of growth factors. Similarly, we found a decrease in body size when we expressed the nuclear-targeted YAP allele, nls-YAP5SA, in chondrocytes (nls-YAP5SAKI/+Col2a1cre+ve); however, this phenotype was substantially more severe than the previously reported phenotypes and was accompanied by catastrophic chondrodysplasia resembling achondrogenesis (Fig. 5A-F). They describe that YAP/TAZ are activated by VEGF and that their activity is needed for transducing the VEGF signal into a specific transcriptional program, required for a full angiogenic response. Nevertheless, we were able to detect a mild reduction of SOX9- and, to a much lesser extent, COLX-stained area in our Lats1/2fl/flCol2a1cre+ve tibial growth plates (Fig. S5E), similar to that reported for cartilage-specific knockout of Mob1a/b (Goto et al., 2018) – but these effects may be a secondary consequence of the highly abnormal morphology of these growth plates. The models were fitted in R using the function lmer of the R package lme4 (Bates et al., 2015). Directly before laser capture, slides were removed from storage at −80°C and washed twice in ice-cold 95% EtOH for 2 min. Red arrowheads indicate delayed or absent ossification in the vertebrae of the Yapfl/flTazfl/flCol2a1cre+ve mutant compared with control. Haematoxylin and Eosin (H&E) staining of sections of E17.5 control (Col2a1cre−ve), single mutants (Yapfl/flTaz+/+Col2a1cre+ve, Yap+/+Tazfl/flCol2a1cre+ve), animals retaining one intact copy of either Yap or Taz (Yapfl/+Tazfl/flCol2a1cre+ve and Yapfl/flTaz+/flCol2a1cre+ve, respectively) and the double mutant (Yapfl/flTazfl/flCol2a1cre+ve) revealed that only the growth plate of the double mutant was changed in total length (Fig. 4A) and length of each growth plate zone (Fig. 4B) compared with the control. Scale bars: 150 µm (A,C). Binning the number of cells down the longitudinal length of the growth plate in control and Yapfl/flTazfl/flCol2a1cre+ve samples revealed approximately equivalent cell numbers in the proximal two thirds of the growth plate and a slight increase in cells in the mutant in the most distal regions (Fig. 4I); however, the overall total number of cells was not significantly different (Fig. 4J), suggesting no extensive delay in differentiation. Es war eine Saison, die ohne Rücksicht auf Verluste durchgepaukt wurde. Briefly, the ribs and sterna of P0 for wild-type compared with Col2a1cre+ve pups or E17.5 pups for all other genotypes were dissected and excess overlying musculature removed before incubation in 2 mg/ml Pronase (Sigma-Aldrich) for 45 min at 37°C with agitation then 3 mg/ml Collagenase D (Roche) for 45 min at 37°C following extensive rinsing with PBS. Here, the authors show that EK formation depends on αE-catenin, which acts to … Thomas Winkler, Uwe Wiemer repariert Recorder, Receiver und Player – Dinge, die die digitale Wegwerfgesellschaft nicht mehr braucht und an denen sie doch hängt. (A) H&E-stained histological sections of the proximal growth plate of the tibia of E17.5 pups. Seit 2017 lebt die Musikerin Rasha Nahas in Berlin – für die Palästinenserin mit israelischem Pass ist das auch eine Identitätssuche. n=3 nls-YAP5SAKI/+Col2a1creERT+ve E17.5 pups. Community. Schrumpfen im Inland, Wachstum im Ausland: Die NBA startet mehr denn je als globale Marke in die neue Basketballsaison. YAP and TAZ are required to maintain valvular endothelial cells. Conceptualization: H.K.V., B.T. Die Houston Texans haben einen Prediger als Charakter-Coach eingestellt. Hugh Dancy 2. Time: 9:00 (GMT) This research was supported by the Francis Crick Institute (FC001180), the Wellcome Trust (FC001180), Medical Research Council (FC001180), Cancer Research UK (FC001180) and the Australian National University (R4210005). In addition to controlling tissue growth, there is evidence that Hippo-YAP/TAZ signalling has a function in controlling cell fate decisions and cell differentiation during development, including specification of trophectoderm in the early blastocyst (Cockburn et al., 2013; Nishioka et al., 2009) as well as during later patterning of several tissues including the lung (Lange et al., 2015; Mahoney et al., 2014; Szymaniak et al., 2015), neural crest (Manderfield et al., 2015; Wang et al., 2016a), mesenchyme (Cotton et al., 2017), lymphatics (Cho et al., 2019) and pancreas (Rosado-Olivieri et al., 2019). Tara Winner, Mary Kay Independent Beauty Consultant. Data were analysed by one-way ANOVA followed by multiple comparisons, P values as indicated. (P) Isolated mandibles from skeletal preparations of E17.5 control (Col2a1cre−ve) and Yapfl/flTazfl/flCol2a1cre+ve pups. ; Visualization: B.T. Fresh-frozen cryosections (anti-YAP) were fixed in 4% paraformaldehyde for 10 min direct from storage then washed in PBS. This concept is consistent with the known function of integrins in binding to the ECM and transducing signals via Hippo-YAP/TAZ in other tissues such as skin (Elbediwy et al., 2016), pancreas (Mamidi et al., 2018), tooth (Hu et al., 2017), blood vessels (Wang et al., 2016b), as well as in mesenchymal stem cells (Sabra et al., 2017; Tang et al., 2013), osteoblasts (Kaneko et al., 2014) and cancer cells (Kim and Gumbiner, 2015; Wong et al., 2016). Sie klopft sich dafür selbst mächtig auf die Schultern. ... Thomas H. Winkler. A previous report concluded that a major function of YAP/TAZ in cartilage development is to inhibit chondrocyte differentiation by direct transcriptional repression of the Sox9 gene (Deng et al., 2016; Goto et al., 2018). TUNEL staining revealed no apoptosis in either control or mutant samples (Fig. This can occur owing to a failure of the lower jaw to drop during palate elevation because of changes in the morphology of the lower jaw, such as a shortening of the mandibles (Ricks et al., 2002). (F,G) RNA in situ hybridisation to known YAP/TAZ target genes Ctgf (F) and Cyr61 (G). Der deutsche Nationalspieler spielt nun um den Titel. Horizontal black lines demarcate the borders of the round proliferating (RP), columnar proliferating (CP) and pre-hypertrophic/hypertrophic (P-H/H) zones. However, Yapfl/flTazfl/flCol2a1cre+ve animals were not present at weaning, in contrast to the presence of animals carrying all other mutant allele combinations examined (including Yapfl/flTaz+/+Col2a1cre+ve, Yap+/+Tazfl/flCol2a1cre+ve, Yapfl/+Tazfl/flCol2a1cre+ve and Yapfl/flTazfl/+Col2a1cre+ve), or the Col2a1cre+ve allele alone, at expected Mendelian frequencies (Fig. S2A-D). All samples were then permeabilised for 10 min in PBS plus 0.1% Tween-20 then blocked for an hour with 5% normal goat serum in PBS plus 0.1% Triton X-100. Pups were processed as described above for histological analysis. Frank Winkler's 163 research works with 3,920 citations and 8,763 reads, including: Identification and characterization of cancer cells that initiate metastases to the brain and other organs Conflicting reports from previous studies concluded that YAP activity in chondrocytes functions primarily to either repress chondrocyte differentiation, by promoting Sox6 expression and repressing Col10a1 (Deng et al., 2016; Goto et al., 2018) or to promote chondrocyte differentiation by repressing Sox9 (Deng et al., 2016; Goto et al., 2018). Our findings also have important medical relevance, further implicating the Hippo-YAP/TAZ pathway in human birth defects including chondrodysplasia and cleft palate (Williamson et al., 2014). Ex vivo CT scans were acquired using a SkyScan 1176 CT scanner (Bruker MicroCT) with the source voltage set to 50 kV, the source current set to 500 µA, a frame averaging of 5, and a 0.5° step size over a 360° trajectory. (C) Immunostaining for Ki67 in the zones of the proximal growth plate of the tibia in n=3 control (Col2a1cre−ve) and n=3 Yapfl/fl Tazfl/flCol2a1cre+ve E17.5 pups. The Immune System in Development and Regeneration Descriptions of molecular functions of YAP and TAZ in chondrocytes have been derived predominantly from in vitro analyses (Deng et al., 2016; Goto et al., 2018). The Hippo signaling pathway has been implicated in tumor growth, sparking interest in the pathway as a potential therapeutic target. (K) H&E- (left image per genotype) and Alcian Blue- (right image per genotype) stained histological sections of the proximal growth plate of the tibia. (A-F) Ventral (A,B), dorsal (C,D) and lateral (E,F) views of control (Col2a1cre−ve) (A,C,E) and nls-YAP5SAKI/+Col2a1cre+ve (B,D,F) E17.5 pups. However, the signal transduction pathways culminating in the biological consequences of VEGF signaling are only partially understood. (K) Alcian Blue staining of proximal growth plate of the tibia in n=4 control (Col2a1cre−ve) and n=4 Yapfl/flTazfl/flCol2a1cre+ve E17.5 pups. We found that YAP/TAZ are necessary and sufficient to drive chondrocyte proliferation in vitro, but are dispensable for chondrocyte proliferation in vivo. The role of the Hippo-YAP/TAZ pathway during development of the skeleton remains poorly understood. Published by The Company of Biologists Ltd. Then 7-12 growth plate regions per slide (i.e. The hypoxic, stiff, and pro-angiogenic tumor microenvironment induces angiogenesis, giving rise to an immature, proliferative, and permeable vasculature. Along with Skaos from Bavarian Krumbach, No Sports, The Braces from Jülich, El Bosso & die Ping-Pongs from Munster and The Busters from Wiesloch, Blechreiz was one of the pioneers of the German ska scene at the end of the 1980s and the beginning of the 1990s. The effect of genotype on proliferation (O) was not significant (P=0.6278) and effect of genotype on cell density (P) was significant (P=0.0049). ; Investigation: H.K.V., F.P., O.G., B.M., S.B., A.H., R.E.S., T.S., M.H., T.J.M., B.T. Thank you for your interest in spreading the word on Development. YAP/TAZ have been reported widely as positive regulators of cell proliferation in chondrocytes in vitro (Deng et al., 2016; Yang et al., 2016; Zhong et al., 2013) and in the mouse prechondrocytic cell line ATDC5 in vitro (Yang et al., 2017). (D,E,I,J) Isolated femur (D,I) and tibia and fibula (E,J) from skeletal preparations of E17.5 control (Col2a1cre−ve) and Yapfl/flTazfl/flCol2a1cre+ve pups. S1C-E). ... Tara Thomas Body And Skin. Your support helps Urban scholars continue to deliver evidence that can elevate debate, transform communities, and improve lives. The two known direct targets of YAP/TAZ, Ctgf and Cyr61, have well-documented roles in cartilage development (O'Brien and Lau, 1992; Wong et al., 1997). (L) Quantification of percentage area occupied by ECM (Alcian Blue-stained area) per boxed region from K. Data were analysed by two-way ANOVA, with growth plate zone and genotype as the independent variables, percentage area Alcian Blue-stained as the dependent variable. A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. These results confirm that the nls-YAP5SAKI/+Col2a1cre+ve mutants are biologically relevant and demonstrate that suppression of YAP/TAZ activity in cartilage by the Hippo pathway is crucial for cartilage morphogenesis and normal skeletal development. Find link is a tool written by Edward Betts.. searching for Thomas Winkler 25 found (32 total) alternate case: thomas Winkler FC Wels (94 words) exact match in snippet view article find links to article Kramer 2 MF Bayram Gas 3 DF Antonio Ljubas 4 DF Andreas Feichtinger 5 DF Thomas Winkler 6 MF Marco Mittermayr 7 MF Jurgen Scharsching 8 DF Gabriel Schneider The effect of genotype on percentage Alcian Blue-stained area was significant (P=0.0138). . The decrease in Ctgf mRNA levels to ∼25% of control suggests that loss of Ctgf is a major contributor to the Yapfl/flTazfl/flCol2a1cre+ve skeletal defects. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. Join our mailing list to receive news and updates on the series. To determine the developmental origin of the cleft palate phenotype, we performed high resolution episcopic microscopy (HREM) on heads of E17.5 pups and generated three-dimensional models of the head by volume rendering. Growth plate regions were isolated from the centre of each chondrocyte zone and threshold using default settings. Yap and Taz are expressed throughout the growth plate at all stages of chondrogenesis, with both nuclear and cytoplasmic immunostaining apparent throughout (Fig. S3A,B). In vitro, YAP was found to promote proliferation of cartilage-derived chondrocytes, suggesting a possible role of YAP in cartilage growth (Deng et al., 2016; Yang et al., 2016; Zhong et al., 2013). The Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. For timed matings, E0.5 was designated as midday following the morning of finding the vaginal plug. All remaining statistical analyses were performed in Prism 8. n=3 sections per growth plate per genotype. Data were analysed by two-way ANOVA, with skeletal element and genotype as the independent variables, length as dependent variable. Die College-Football-Saison ist ein Superspreader-Event mit geringem sportlichem Wert. Proliferation, as measured by the percentage of Ki67-staining cells (compared with total cells marked by Eosin staining of the nuclei) in each chondrocyte zone of the growth plate, was unchanged in Yapfl/flTazfl/flCol2a1cre+ve samples compared with control (Fig. 4C,D). YAP1 and TAZ control pancreatic cancer initiation in mice by direct up-regulation of JAK–STAT3 signaling. We therefore sought to re-examine the expression of COL10a1 (COLX) and SOX9 in tibial growth plates from our YAP/TAZ loss-of-function and gain-of-function animals. Ki67 immuno-positive and -negative cells were counted manually and the number of Ki67 immuno-positive cells expressed as the percentage total. These experiments show that in the mutants there is lower cell density, but approximately equivalent numbers of cells and equivalent rates of proliferation, across a greater area, with no change in apoptosis. However, we describe here a significant departure in phenotypic outcomes in vitro compared with in vivo. We wondered whether the presence of the YAP homologue TAZ in these animals may be sufficient to compensate for the knockout of the Yap gene and therefore may mask a requirement for Hippo effectors in the positive regulation of proliferation in chondrocytes in vivo. In support of this notion, a recent study has demonstrated that YAP localisation and activity in the embryonic cartilaginous humerus is depended on mechanical stimulation derived from the surrounding muscle (Shea et al., 2020). © 2021   The Company of Biologists Ltd   Registered Charity 277992, Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway. Compared with the lateral emergence of the ribs from the spine in controls, the ribs of Yapfl/flTazfl/flCol2a1cre+ve pups emerged slightly to the anterior before angling sharply down towards the posterior (Fig. 2C,H). In particular, induction of Mmp16 along with Ctgf in the YAP5SAKI/+Col2a1cre+ve samples could contribute to the chondrodysplasia phenotype observed. ; Project administration: B.T. Thus, chondrocytes may also employ the Hippo-YAP/TAZ pathway to sense mechanical forces acting via the ECM and regulate the developmental remodelling response via Ctgf, Cyr61 and several matrix remodelling enzymes. Apoptosis was also unchanged (Fig. After airdrying the slides at room temperature, sections were visualised in brightfield on the Laser Capture Microdissector (LMD7000, Leica) and a 300 µm-wide box drawn down the midline on the tibial growth plate. Meckel's cartilage in the mutants (right panels) was shorter with abnormal morphology (white arrow), including an additional partial rotation at the posterior (arrowhead) before the malleus (M) and incus (I). Severe chondrodysplasia in pups with constitutively nuclear YAP in chondrocytes. Of the ECM component genes examined, Col10a1 was significantly reduced (Fig. 7K) and though neither Mmp2 nor Ctsk showed a significant change in YAP5SAKI/+Col2a1cre+ve samples, there was a substantial increase in Mmp16 expression (Fig. 7L). Deng et al. Apoptosis was assessed by the inclusion of apoptosis marker NucView488 (Biotium). Your Honor (TV Series 2020–2021) cast and crew credits, including actors, actresses, directors, writers and more. Thomas Winkler, Alle Rechte vorbehalten. The effect of genotype on length was not significant (P=0.8110). However, cell density was increased in YAP5SAKI/+Col2a1cre+ve samples (Fig. 5P), suggesting decreased ECM in the intracellular spaces, mirroring the decreased cell density and increased percentage of area occupied by cartilage matrix in Yapfl/flTazfl/flCol2a1cre+ve tibias (Fig. 4E,L). To confirm these results, proliferation was measured by EdU incorporation versus total DAPI-stained nuclei, and again there was no change although cell density was reduced (Fig. 4F-H). Samples were prepared for HREM by incubation in Bouin's fixation for a minimum of 3 days followed by extensive washing in PBS and dehydration, before incubation in JB-4/Dye mix for up to 4 weeks to ensure proper sample penetration and then embedded and imaged as previously described (Mohun and Weninger, 2012; Weninger et al., 2018) (https://dmdd.org.uk/hrem/). I have been a Systems Administrator for over 7 years and I my focus is now on areas such as Puppet, Openstack, Kubernetes, etc. The tongue was tightly wedged in the palatal space in the cleft palate mutants (Fig. 3I,J,M,N), suggesting that the cleft palate phenotype may be a secondary consequence of the tongue physically impeding the elevation and/or closure of the palatal shelves. Blechreiz is a German ska band founded in 1983 in southern Berlin. Stage-specific defects in chondrogenesis can be inferred from the variation in size of growth plate zones, gene expression domains or regions of proliferation or apoptosis (Hallett et al., 2019). These results argue against an essential function for YAP/TAZ in regulating chondrocyte cell differentiation via direct repression of Sox9 or Col10a1 gene expression. Red arrows indicate the ossified (red-stained) portion of the ribs adjacent to the vertebrae emerging towards the anterior before redirecting toward the posterior in the Yapfl/flTazfl/flCol2a1cre+ve mutant compared with the lateral emergence of the ossified rib in the control. Horizontal black lines demarcate the borders of the round proliferating (RP), columnar proliferating (CP) and pre-hypertrophic/hypertrophic (P-H/H) zones. ; Resources: B.T. Ted Mosby 1. Cells were filtered then plated at 3000 cells per well of a black polystyrene flat-bottom (with micro-clear bottom) 96-well plate (Greiner) in DMEM supplemented with 10% fetal calf serum and pen-strep. Kritischer, unabhängiger Journalismus der linken Nachrichtenseite taz: Analysen, Hintergründe, Kommentare, Interviews, Reportagen. We find that, in vitro, Yap/Taz double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear nls-YAP5SA accelerates proliferation, in line with the canonical role of this pathway in most tissues. The Voice of Germany (season 1) is a German reality talent show that premiered on 24 November 2011 on ProSieben and Sat.1.Based on the reality singing competition The Voice of Holland, the series was created by Dutch television producer John de Mol. Whole E17.5 pups were fixed in 10% neutral buffered formalin for two days then transferred to 70% ethanol. (D) Proliferation, measured by percentage cell confluence of field of view, of cultures from C. Data represent mean of biological replicates derived from six technical replicates, treated with or without tamoxifen. Though we could not reliably detect transcripts of Mmp9 or Mmp13 – two key protease regulators of cartilage remodelling (Stickens et al., 2004; Vu et al., 1998) – we saw a reduction in Ctsk and Mmp2 expression in Yapfl/flTazfl/flCol2a1cre+ve, but not Yapfl/flTazfl/+Col2a1cre+ve, mutant samples (Fig. 7E). Thus, the primary role of Hippo-YAP/TAZ signalling in cartilage development is in control of tissue morphogenesis, rather than in control of cell proliferation or cell fate. (C) Primary chondrocyte cultures from ribcages and sterna of nls-YAP5SAKI/+Col2a1creERT+ve E17.5 pups treated with 1 μM 4-hydroxytamoxifen (4-OHT) or ethanol vehicle (EtOH) at 24 h after plating. ... Tara Winkler, US Jaycees NVP 2013. Learn more » (H-L) RTqPCR analysis of laser-microdissected tibial growth plates of four E17.5 control (Col2a1cre−ve) and four nls-YAP5SAKI/+Col2a1cre+ve littermates.

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