PMC:4925210 / 41556-43874
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4925210","sourcedb":"PMC","sourceid":"4925210","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4925210","text":"Xenopus Embryo Manipulations\nXenopus embryos obtained by in vitro fertilization were maintained in 0.1x modified Barth medium 62 and staged according to Nieuwkoop and Faber 63. The sequences of the antisense morpholino oligomers (GeneTools, LLC) used in this study are listed in Suppl. Table 2. The efficacy of Dvl2-MO and Pkd1-sMO1 was demonstrated previously64, 65. The efficacy of Pkd1-sMO2 was verified by RT-PCR of RNA extracted from stage 35 embryos (Supplementary Fig. 7e) using primers spanning exons 17/18 of the predicted Xenopus laevis Polycystin1 (Xenbase Gene #483413, 5’-TGT CCT TGA AGT GCG TGT CA-3’ and 5’-CCC GTA GAT GTG GTG CTT GA-3’).In the case of Wnt9A-MO, it was verified using a Wnt9A-GFP fusion construct injected into Xenopus embryos in the presence or absence of the Wnt9A-MO. For morphological scoring of edema formation embryos were injected at the 2- to 4-cell stage with the indicated amounts of MO and cultured until sibling embryos reaches stage 43. For whole the imaging of the individual pronephric kidneys embryos were injected unilaterally at the 2-cell stage embryo with the indicated amounts of MOs, cultured until stage 40 and fixed in Dent's fixative (4:1 Methanol:DMSO). Whole mount immunofluorescence was performed by incubating the embryos sequentially with the 3G8 and 4A6 antibodies66 and developing them using AlexaFluor-448 and AlexaFluor-555-labeled α-mouse secondary antibodies (Life Technologies). Embryos were analyzed individually by confocal microscopy comparing the injected to the contralateral control side. Pronephric kidneys with dilated and shortened nephron segments were classified as dysplastic. In individual cases 3-dimensional (3D) reconstruction of the kidneys was used to verify differences in tubule diameter. Analysis was performed in a blinded fashion to avoid bias.\nRescue experiments for the Dvl2-MO were performed by injecting 0.5 μg synthetic mRNA encoding wild type human DVL2 or mutant DVL2-E499G into a single blastomere at the 2-cell stage followed by injection of 3.2 pMol Dvl2-MO into all four blastomeres at the 4-cell stage. Embryos were cultured until stage 40 and processed for whole kidney imaging as outlined above. Scoring was based on comparing the kidneys of the Dvl2-MO-injected side to the one of the Dvl2-MO/mRNA-injected side","divisions":[{"label":"Title","span":{"begin":0,"end":28}}],"tracks":[{"project":"2_test","denotations":[{"id":"27214281-12771126-73835281","span":{"begin":364,"end":366},"obj":"12771126"},{"id":"27214281-12771126-73835281","span":{"begin":364,"end":366},"obj":"12771126"},{"id":"27214281-7556934-73835282","span":{"begin":1327,"end":1329},"obj":"7556934"},{"id":"27214281-7556934-73835282","span":{"begin":1327,"end":1329},"obj":"7556934"}],"attributes":[{"subj":"27214281-12771126-73835281","pred":"source","obj":"2_test"},{"subj":"27214281-12771126-73835281","pred":"source","obj":"2_test"},{"subj":"27214281-7556934-73835282","pred":"source","obj":"2_test"},{"subj":"27214281-7556934-73835282","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ec99","default":true}]}]}}