Xenopus Embryo Manipulation and Analysis

X. tropicalis frogs were obtained from Nasco, the National Xenopus Resource (NXR) and the European Xenopus Resource Centre (EXRC). All animal experiments were approved by the state review board of Baden-Württemberg, Regierungspräsidium Karlsruhe, Germany (permit number 35-9185.81/G-141/18). Federal and institutional guidelines and regulations were followed. Developmental stages were determined according to Nieuwkoop and Faber (Xenbase). Statistical analysis to determine sample size, sex- and gender-based analyses, and randomization of injection were not applicable in this context. In vitro fertilization and culture of embryos were performed as previously described^{62 (link)}. Antisense morpholino oligonucleotides (Mo) were obtained from GeneTools and microinjected using the Harvard Apparatus microinjection system. Morpholinos targeting ccny (10 ng per embryo^{14 (link)}), ccny^SPL (10 ng per embryo; 5’-ATGTTTCCACAGTACGAGAAAAACG-3’), ccnyl1 (10 ng per embryo^{14 (link)}), ccnyl1^SPL (10 ng per embryo; 5’-TTCATTGCAGATTTTCACGATGAGC-3’), lrp6 (5 ng per embryo^{20 (link)}), lrp^UTR (5 ng per embryo; 5’-GCTCAATGCTCCCCCGTAAGCCAGC-3’), β-catenin (10 ng per embryo^{21 (link)}), ppp1r11 (30 ng per embryo; 5’-AGAGCGGTGTTCCTGTTACGGGTAA-3’), ccdc108 (10 ng per embryo^{43 (link)}), dlg5 (10 ng per embryo^{44 (link)}), gas2l2 (5 ng per embryo^{45 (link)}), hyls-1 (10 ng per embryo^{46 (link)}) and standard control (up to 30 ng per embryo) as well as wnt8^DN mRNA (500 pg per embryo) and membraneRFP (mbRFP) mRNA (300 pg per embryo) were microinjected animally 5 nl per each embryonic blastomere. In ccny/l1 DKD, 10 ng of each Mo were co-injected either alone, or with human CCNY-Flag (250 pg per embryo)^{63 (link)} and mouse Ccnyl1-Flag mRNA (250 pg per embryo)^{14 (link)} for rescue experiments. BB marker pCS2-gfp-drCentrin 2 (100 pg per embryo) was co-injected with VF10-RFP-Clamp (150 pg per embryo) and indicated morpholinos. pCS2-gfp-drCentrin 2 was a kind gift from Wieland Huttner, and VF10-RFP-Clamp was generously provided by Gerd Walz. X. tropicalis Flag-ppp1r11 mRNA was injected at a concentration of 200 pg per embryo to investigate ciliary localization, while human ppp1r11 mRNA without Flag-Tag was injected at 25 pg and 50 pg for ppp1r11 Mo rescue experiments. In Fig. 2j, ccny/l1 morphants were rescued with 50 pg of Flag-ppp1r11 DNA. Human LRP6 WT, LRP6^(VA)P(PA) and LRP6^VA DNA were injected at a concentration of 50 pg per embryo. For GSK3 ciliary biosensor experiments, 100 pg biosensor DNA was co-injected with 100 pg of Arl13b-mKate2 DNA. Animal cap explants were dissected at St. 8 and cultured until St. 30 for live cell imaging. WNT3A recombinant protein (R&D Systems; Cat#5036-WN-010) was added at a concentration of 2 µg/ml. In Wnt stimulation of whole embryos, WNT3A was added for 30 min before fixation. Embryos were cultured until St. 28-32 unless indicated otherwise. BIO (Cayman Chemical, Cat#13123) rescue was performed from St. 8 to St. 28 by adding 60 µM BIO to the embryo culture medium. Two different modes were applied for treatment with OA (Cell signaling technology, Cat#5934): 10 nM OA in DMSO (Sigma; Cat#D2650-100ML) was added from St. 26 to St. 32 to analyze movements of epidermal MCCs and for ciliopathy Mo rescue experiments the embryos were analyzed after 1 h, at St. 28. Cilia morphology was analyzed after treatment with 10 nM OA from St. 8 to St. 26. Control groups were treated with DMSO.