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The cDNA equivalent to 40?ng total RNA was used for qRT-PCR. DNA sequences of the primers used in qRT-PCR are listed in Table S1 in supplementary materials. Palatal shelf explant culture was performed as described previously (Jin and Ding, 2006). Briefly, mouse palatal shelves at E13.5�CE14.0 were dissected out in cold A-1210477 cell line ��-MEM containing 25?mM HEPES. The dissected palatal shelves were paired and placed in an organ culture plate containing ��-MEM medium with 0.1?��g/ml ascorbic acid and cultured at 37?��C with 5% CO2 for up to 48?hours. Branchial arch explant culture was performed as a hanging drop culture. Branchial arch explants were isolated from E9.5�CE10.5 embryos. Ectoderm and mesenchyme explants were prepared from branchial arch explants by enzymatic digestion with dispase (2.4?U/ml, Roche Applied Science) followed by dissection. The explants were cultured in ��-MEM for up to 48?hours and stained for ��-gal enzyme activity or subjected to RNA isolation for qRT-PCR analysis. Recombinant human RSPO2 (rhRSPO2) and mouse Wnt3a (rmWnt3a) proteins and human Endothelin1 (hEDN1) peptides were purchased from R&D systems (Minneapolis, MN) and added to the explant culture as indicated. The experimental data were analyzed by Student's t-test for two group comparison or one-way analysis of variance (ANOVA) for multiple diglyceride comparisons followed by Dunnett's test. Statistical significance was set at p?this website The average of the technical duplicate was used as a sample value to calculate the mean and standard mean error (SEM) of each group. We and others previously reported that Rspo2 null mice develop multiple defects in limb, lung, and craniofacial structures ( Aoki et al., 2008, Bell et al., 2008, Nam et al., 2007a?and?Yamada et al., 2009). Approximately 71% of Rspo2?/? mice (n?=?15/21) collected at E18.5 exhibited a cleft phenotype of secondary palate without cleft lip ( Fig.?1A and B). We performed a systematic analysis to determine the nature of craniofacial defects including cleft palate in Rspo2?/? mice. Cleft palate is caused by various defects at different steps of palatogenesis (Chai and Maxson, 2006?and?Stanier and Moore, 2004). To investigate the stage(s) affected during palatogenesis in Rspo2?/? mice, we performed histological analyses of Rspo2?/? embryos from E12.5 to E15.5. Outgrowth and initial downward growth of the palatal shelves occurred normally in Rspo2?/? mice at E12.5 ( Fig.?1C and D). The tongue of Rspo2?/? mice was smaller than that of wild type mice ( Fig.?1C and F). Between E13.5 and E14.5, the tongue in Rspo2?/? mice failed to move downward, which resulted in an obstruction and delay of bilateral, horizontal elevation of the palatal shelves ( Fig.?1G and H). Because the timing of palatal shelf elevation is critical to complete palate formation ( Chai and Maxson, 2006?and?Jiang et al.