Specific heparan sulfate structures involved in retinal axon targeting

Development ◽  
2002 ◽  
Vol 129 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Atsushi Irie ◽  
Edwin A. Yates ◽  
Jeremy E. Turnbull ◽  
Christine E. Holt
Keyword(s):  
Heparan Sulfate ◽  
Axon Guidance ◽  
Structural Characteristics ◽  
In Situ Hybridisation ◽  
Optic Tract ◽  
Structural Diversity ◽  
Axon Targeting ◽  
Retinal Axon

Heparan sulfate (HS), a structurally diverse molecule comprising distinct sequences of sulfated disaccharide units, is abundant in the developing brain and binds to axon guidance molecules. Addition of HS to the developing Xenopus optic pathway causes severe targeting errors yet it is not known how the structural diversity of this molecule relates to its role in axon guidance. We have used an in vivo brain assay to identify the structural characteristics of HS that induce aberrant axon targeting. Inhibiting sulfation of endogenous HS with chlorate causes axons to bypass their target, the tectum, and treatment with chemically modified heparins reveals that 2-O- and 6-O-sulfate groups have potent bypass-inducing activity. Experiments with purified heparin saccharides show that bypass-inducing activity correlates with distinct structures, particularly those containing a combination of 2-O- and 6-O-sulfate groups. Taken together the results indicate that specific sequences, rather than gross structural composition, are critical for activity. In situ hybridisation revealed that HS 6-O-sulfotransferase is regionally expressed along the border of the dorsal optic tract whereas 2-O-sulfotransferase is expressed broadly. Our results demonstrate that specific HS sequences are essential for regulating retinotectal axon targeting and suggest that regionalised biosynthesis of specific HS structures is important for guiding axons into the tectum.

The metalloproteinase ADAM10 is required for retinal ganglion cell axon guidance in the developing visual systems

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Y. Y. Chen ◽  
C. L. Hehr ◽  
K. Atkinson-Leadbeater ◽  
J. C. Hocking ◽  
S. McFarlane
Keyword(s):  
Ganglion Cell ◽  
Axon Guidance ◽  
Retinal Ganglion Cell ◽  
Growth Cone ◽  
Expression Patterns ◽  
Optic Tract ◽  
Axon Growth ◽  
Proteolytic Processing ◽  
Retinal Ganglion

Background: The growth cone interprets cues in its environment in order to reach its target. We want to identify molecules that regulate growth cone behaviour in the developing embryo. We investigated the role of A disintegrin and metalloproteinase 10 (ADAM10) in axon guidance in the developing visual system of African frog, Xenopus laevis. Methods: We first examined the expression patterns of adam10 mRNA by in situ hybridization. We then exposed the developing optic tract to an ADAM10 inhibitor, GI254023X, in vivo. Lastly, we inhibited ADAM10 function in diencephalic neuroepithelial cells (through which retinal ganglion cell (RGC) axons extend) or RGCs by electroporating or transfecting an ADAM10 dominant negative (dn-adam10). Results: We show that adam10 mRNA is expressed in the dorsal neuroepithelium over the time RGC axons extend towards their target, the optic tectum. Second, pharmacological inhibition of ADAM10 in an in vivo exposed brain preparation causes the failure of RGC axons to recognize their target at low concentrations (0.5, 1 μM), and the failure of the axons to make a caudal turn in the mid-diencephalon at higher concentration (5 μM). Thus, ADAM10 function is required for RGC axon guidance at two key guidance decisions. Finally, molecular inhibition of ADAM10 function by electroporating dn-adam10 in the brain neuroepithelium causes defects in RGC axon target recognition (57%) and/or defects in caudal turn (12%), as seen with the pharmacological inhibitor. In contrast, molecular inhibition of ADAM10 within the RGC axons has no effect. Conclusions: These data argue strongly that ADAM10 acts cell non-autonomously within the neuroepithelium to regulate the guidance of RGC axons. This study shows for the first time that a metalloproteinase acts in a cell non-autonomous fashion to direct vertebrate axon growth. It will provide important insights into candidate molecules that could be used to reform nerve connections if destroyed because of injury or disease. References Hattori M, Osterfield M, Flanagan JG. Regulated cleavage of a contact-mediated axon repellent. Science 2000; 289(5483):1360-5. Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123(2):291-304. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997; 90(2):271-80.

scholarly journals Cloning and regulation of the rat activin betaE subunit

2000 ◽  
Vol 24 (3) ◽  
pp. 409-418 ◽  
Author(s):  
MK O'Bryan ◽  
KL Sebire ◽  
O Gerdprasert ◽  
MP Hedger ◽  
MT Hearn ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Alternative Polyadenylation ◽  
Northern Blotting ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Amino Acid Residues ◽  
Mrna Transcripts ◽  
Polymerase Chain

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

Incidence of chromosomal abnormalities in bovine blastocysts derived from unsorted and sex-sorted spermatozoa

2010 ◽  
Vol 22 (8) ◽  
pp. 1272 ◽  
Author(s):  
M. Garcia-Herreros ◽  
T. F. Carter ◽  
D. A. F. Villagómez ◽  
A. D. MacAulay ◽  
D. Rath ◽  
...  
Keyword(s):  
Chromosomal Abnormalities ◽  
In Situ Hybridisation ◽  
Fluorescent In Situ Hybridisation

The aim of the present study was to examine the incidence of chromosomal abnormalities in bovine blastocysts produced by IVF with unsorted, X-sorted or Y-sorted spermatozoa. In Experiment 1, individual blastocysts were processed to examine the incidence of mixoploidy using fluorescent in situ hybridisation. Overall, 80% (44/55) of blastocysts were mixoploid (10/15, 14/15 and 20/25 for X-sorted, Y-sorted and unsorted spermatozoa, respectively; P > 0.05). However, the prevalence of abnormal XY chromosome complements was relatively low in all groups; on average, only a small fraction of the total nuclei per embryo appeared polyploid (1.64%, 5.62% and 6.0% for X-sorted, Y-sorted and unsorted spermatozoa, respectively). Interestingly, 20% (5/25) of blastocysts derived from unsorted spermatozoa were found to be chimeric (XX/XY). In Experiment 2, chimeric embryos were detected among the blastocysts derived from two of five sires tested. In addition, one chimeric blastocyst was detected among nine in vivo-derived blastocysts obtained following AI. In conclusion, based on the results of the present study, the incidence of chromosomal abnormalities did not different between blastocysts derived from sex-sorted or unsorted spermatozoa. In addition, the occurrence of mixed sex chimeras was not limited to a single sire and was not unique to blastocysts derived from IVF.

scholarly journals Novel lncRNA UPLA1 mediates tumorigenesis and prognosis in lung adenocarcinoma

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Xiaoyang Han ◽  
Hua Jiang ◽  
Jianni Qi ◽  
Jiamei Li ◽  
Jinghan Yang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
In Situ Hybridisation ◽  
Vital Role ◽  
Luciferase Reporter ◽  
Fluorescence In Situ Hybridisation ◽  
Biological Functions ◽  
Pulldown Assay

AbstractWith the development of molecular biotechnology and sequencing techniques, long non-coding RNAs (lncRNAs) have been shown to play a vital role in a variety of cancers including lung cancer. In our previous study, we used RNA sequencing and high-content screening proliferation screening data to identify lncRNAs that were significantly associated with tumour biological functions such as LINC01426. Herein, based on previous work, we report a novel lncRNA UPLA1 (upregulation promoting LUAD-associated transcript-1), which has not been explored or reported in any previous studies. Our results showed that UPLA1 is highly expressed and regulates important biological functions in lung adenocarcinoma. In vitro experiments revealed that UPLA1 promoted the migration, invasion, and proliferation abilities, and is related to cell cycle arrest, in lung adenocarcinoma cells. Moreover, the upregulation of UPLA1 significantly improved the growth of tumours in vivo. We identified that UPLA1 was mainly located in the nucleus using fluorescence in situ hybridisation, and that it promoted Wnt/β-catenin signalling by binding to desmoplakin using RNA pulldown assay and mass spectrometry. Additionally, luciferase reporter assay revealed that YY1 is the transcription factor of UPLA1 and suppressed the expression of UPLA1 as a transcriptional inhibitor. This finding provides important evidence regarding the two roles of YY1 in cancer. Furthermore, in situ hybridisation assay results showed that UPLA1 was closely related to the prognosis and tumour, node, metastasis (TNM) stage of lung adenocarcinoma. In summary, our results suggest that the novel lncRNA UPLA1 promotes the progression of lung adenocarcinoma and may be used as a prognostic marker, and thus, has considerable clinical significance.

scholarly journals Topographically Specific Effects of ELF-1 on Retinal Axon Guidance In Vitro and Retinal Axon Mapping In Vivo

Cell ◽  
1996 ◽  
Vol 86 (5) ◽  
pp. 755-766 ◽  
Author(s):  
Masaru Nakamoto ◽  
Hwai-Jong Cheng ◽  
Glenn C Friedman ◽  
Todd McLaughlin ◽  
Michael J Hansen ◽  
...  
Keyword(s):  
Axon Guidance ◽  
Specific Effects ◽  
Retinal Axon

Abstract 3437: Integrin-linked-kinase Binding Proteins Are Essential Components Of The Cardiac Mechanical Strech Sensor

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Andreas Hartkopf ◽  
Inken Huttner ◽  
Tillman Dahme ◽  
Benjamin Meder ◽  
Britta Vogel ◽  
...  
Keyword(s):  
Binding Proteins ◽  
In Situ Hybridisation ◽  
Cardiac Contractility ◽  
Down Regulation ◽  
Knock Down ◽  
Essential Components ◽  
Integrin Linked Kinase ◽  
Heart Contractility

The cardiac strech sensor enables the heart to adapt its force of contraction to continually changing demands. We recently identified in the zebrafish mutant main squeeze (msq) Integrin-Linked-Kinase (ILK) as a component of this strech sensor. Mutations in zebrafish ilk lead to down-regulation of stretch responsive cardiac genes and cause progressive heart failure. Antisense-mediated abrogation of zebrafish β-parvin, which forms complexes with ILK, phenocopies ilk mutant zebrafish. We here describe two novel ILK-binding-proteins, acting in concert with ILK to control cardiac contractility. In a yeast-to-hybrid-screen with a cardiac specific library we identified two ILK-binding proteins (ILK-bp-1 and ILK-bp-2). Just as we have recently shown for ILK, ILK-bp-1/2 are highly expressed in the zebrafish heart and localize at the sarcomeric Z-disc. To reveal the in vivo function of ILK-bp-1/2 we performed knock-down studies on zebrafish with antisense oligonucleotide injection. Similar to the phenotype of ILK-deficient zebrafish, the knock down of ILK-bp-1/2 leads to loss of cardiac contractility and, as shown by in-situ-hybridisation, to down-regulation of the stretch responsive gene anf. We analysed the ILK-bp-1/2 morphant hearts structurally and ultra-structurally. Just as we have recently shown for ilk deficient hearts, we found that their morphogenesis and myofibrillogenesis are normal, indicating that knock-down of ILK-binding-proteins does not induce a structural but rather a functional heart defect. Taken together, these results indicate, that next to β-parvin other ILK-binding proteins play important roles in the control of heart contractility. ILK and ILK-bp-1/2 act in concert to enable the heart to adapt itself to continually changing demands.

Embryonic gene expression patterns of TGF beta 1, beta 2 and beta 3 suggest different developmental functions in vivo

Development ◽  
1991 ◽  
Vol 111 (1) ◽  
pp. 131-143 ◽  
Author(s):  
F.A. Millan ◽  
F. Denhez ◽  
P. Kondaiah ◽  
R.J. Akhurst
Keyword(s):  
Growth Factors ◽  
Developmental Process ◽  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Tgf Beta ◽  
Genes Encoding ◽  
Beta 2 ◽  
Valve Formation

We have compared the expression of the genes encoding transforming growth factors beta 1, beta 2 and beta 3 during mouse embryogenesis from 9.5 to 16.5 days p.c. using in situ hybridisation to cellular RNAs. Each gene has a different expression pattern, which gives some indication of possible biological function in vivo. All three genes appear to be involved in chondroossification, though each is expressed in a different cell type. Transcripts of each gene are also present in embryonic epithelia. Epithelial expression of TGF beta 1, beta 2 and beta 3 RNA is associated with regions of active morphogenesis involving epithelial-mesenchymal interactions. In addition, widespread epithelial expression of TGF beta 2 RNA can be correlated with epithelial differentiation per se. The localisation of TGF beta 2 RNA in neuronal tissue might also be correlated with differentiation. Finally both TGF beta 1 and beta 2 transcripts are seen in regions actively undergoing cardiac septation and valve formation, suggesting some interaction of these growth factors in this developmental process.

scholarly journals P107 The expression and regulation of Oncostatin M and its receptor in intestinal inflammation

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S189-S189
Author(s):  
R Cineus ◽  
D Boesel ◽  
S Hainbuch ◽  
C Jukes ◽  
Y H Hsieh ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Intestinal Inflammation ◽  
In Situ Hybridisation ◽  
Oncostatin M ◽  
Transcriptional Responses ◽  
The Impact ◽  
Pro Inflammatory Cytokines

Abstract Background Intestinal homeostasis depends on the interplay between the gut microbiota, epithelium and immune cells. A novel role of Oncostatin M (OSM), a pro-inflammatory cytokine has recently been identified in mouse and human intestinal inflammation. Previous studies have shown OSM as a key driver of chronic inflammation in anti-TNF-α-refractory colitis. A single-nucleotide polymorphism in the human OSM genetic locus is strongly associated with risk of developing inflammatory bowel disease (IBD), thus, biological therapies targeting OSM could have therapeutic potential. Our project aims to explore the impact of OSM on intestinal barrier function in health and disease. Methods To evaluate the role of OSM in intestinal inflammation, we utilized a combination of in vitro and in vivo techniques. This included the generation of 3D intestinal organoids from mice and patients. Organoids were stimulated with a repertoire of different cytokines to determine the responsiveness of OSM receptor (OSMR) to different cytokine signals using a quantitative-PCR-based approach. For in vivo modelling of disease, the Helicobacter hepaticus colitis model was used, as it combines both immune and dysbiosis-driven aspects of disease. This allowed us to measure OSM and OSMR expression in response to inflammation and within specific organs and cell subsets. Furthermore, RNAscope in situ hybridisation was used to determine the localisation of OSM- and OSMR-expressing cells in inflamed mucosal tissue from colitic mice and IBD patients. Results RNAscope in situ hybridisation as well as gene expression analysis have shown that the OSM and OSMR were highly expressed in C57BL/6 mice upon induction of colitis in the H. hepaticus model of disease and in mucosal tissues of IBD patients. In addition, a plethora of pro-inflammatory cytokines were upregulated during colitis, with colitic mice showing increased tissue pathology. Furthermore, FACS analysis shows excessive immune cell infiltration in the spleen, colon and mesenteric lymph nodes of colitic mice. Conclusion Our preliminary results have shown that different gut-resident hematopoietic and non-hematopoietic cell types express OSM and OSMR and this expression was modulated by pro-inflammatory cytokines. We therefore hypothesis that OSM might drive distinct transcriptional responses in various gut-resident cell populations. Thus, differential targeting of the OSM receptor might be a potential therapeutic approach in IBD.

scholarly journals MiR130b from Schlafen4+ MDSCs stimulates epithelial proliferation and correlates with preneoplastic changes prior to gastric cancer

Gut ◽  
2020 ◽  
Vol 69 (10) ◽  
pp. 1750-1761 ◽  
Author(s):  
Lin Ding ◽  
Qian Li ◽  
Jayati Chakrabarti ◽  
Andres Munoz ◽  
Emmanuelle Faure-Kumar ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cell ◽  
In Situ Hybridisation ◽  
Suppressor Cells ◽  
Western Blots ◽  
Gastric Cancers ◽  
Spasmolytic Polypeptide ◽  
Myeloid Differentiation Factor

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM).ObjectiveTo identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM.DesignWe performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4– cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b.ResultsMicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth.ConclusionTaken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.

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