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Supplement Figure S1: A, Sequence of Xcadherin-11 Morpholino 1 (Xcad-11MO) and 2 (Xcad-11 MO2) and control morpholino in comparison to the Xcadherin-11 sequence. The Xcad-11MO binding sequence spans the start codon while Xcad-11MO2 binds in the 5’ UTR. B, Function of Xcad-11MO. The function of the chosen Xcad-11MO was demonstrated by in vitro transcription and translation. The TNT was performed according to manufacturer’s description (Promega). For detection of synthesized protein S35-labelled methionine was incorporated. The addition of Xcad-11MO to the reaction mix resulted in a strong decrease of detectable protein whereas the addition of control morpholino had no influence (left panel). Xcad-11MO was not able to suppress protein production of murine cadherin-6 or XB-cadherin (right panel). C, Endogenous function of Xcad-11MO and Xcad-11MO2. After injection of 2 pmol Xcad-11MO and Xcad-11MO2 a strong decrease of protein was detected in embryo lysates in comparison to wildtype embryos. Endogenous XB-cadherin was not affected by Xcad-11Mo and Xcad-11MO2 injection. For detection, αCad-11 and 6D5 αXB-cadherin antibody were used. D, Specificity of Xcadherin-11 function. The impaired invasion of pharyngeal pouches caused by injection of Xcad-11MO could neither be rescued by co-injection of the type1 XB-cadherin nor by type II murine cadherin-6 as demonstrated by whole mount in situ hybridizations with the neural crest marker twist. Instead, human Cadherin-11 was able to rescue twist signal in the pharyngeal pouches (up to 90%). Injected side: middle panel, control side: left panel. Right panel: statistics for rescue experiments. In case of Xcad-11MO injection 28%, and in case of 9 pmol Xcad-11MO2 39% of the embryos showed a normal invasion in the pharyngeal pouches. A rescue with 50 pg XB-cadherin or with 100 pg murine cadherin-6 resulted in only 37% or 31% twist positive cells in the branchial arches, respectively. |
