Fig. 4

Wnt11r Binds to UnpSV1 and Overepressions of wnt11r and unpSV1 Increase Prepatterning

(A) Binding of Wnt11r to the extracellular domain (ECD) of UnpSV1 in vitro. GST-UnpSV1ECD fusion proteins, coupled to glutathione sepharose, were mixed with conditioned media containing secreted Wnt11r-FLAG. Amounts of GST-UnpSV1 and Wnt11r-FLAG used in the analysis were assessed by anti-GST (lower panel) and anti-FLAG (right panel) immunoblotting (IB), respectively. Amounts of Wnt11r-FLAG bound were evaluated by anti-FLAG IB (upper panel).

(B) Coimmunoprecipitation of UnpSV1 with Wnt11r in 293T cells. 293T cells were cotransfected with Wnt11r-FLAG and UnpSV1-myc or its CRD deletion mutant. Whole-cell lysates (WCL) were subjected to anti-FLAG IB to determine the expression of Wnt11r-FLAG (lower panel). The UnpSV1-bound Wnt11r was assessed by IB of the anti-myc immunoprecipitate (upper panel). Schematic diagrams of constructs used in the experiments. SS, signal sequence.

(C and D) Cross-sections of 20 somite stage embryos injected with purified Wnt11r-FLAG protein. (C) In wild-type embryos, Wnt11r binds to adaxial cells as highlighted by the brackets. Binding is abolished in unplugged mutants(asterisks in [D] mark nonspecific staining).

(E–H) Wild-type embryos were injected with mRNAs as indicated. The domain of AChR prepatterning (brackets) was expanded in embryos coinjected with wnt11r and unpSV1myc mRNAs and was dependent on the CRD domain (G and H).

(I) Co-overexpression of wnt11r and unpSV1 significantly increases the number of prepatterned clusters/hemisegment (n = 5–18 hemisegments per bar, average = 10). Results are expressed as the average of different injection experiments (t test, ^**p < 0.01, ^*p < 0.05). Amounts of mRNA (ng/embryo): wnt11r-FLAG, 0.3; SV1myc, 0.5; SV1ΔCRDmyc, 0.5. AChR cluster size distribution was not altered.