At day 5, the EBS were stained for Nkx2.2. in the corresponding O4I1 gray scale image (A-F) DAPI nuclear stain is usually blue. Scale bar: 50?m. For members of the RND family to act as dominant negatives, they must retain the ability to form trimers (Nikaido and Takatsuka, 2009). It remains a possibility that this electroporated mouse Ptch1 cannot form trimers with endogenous chicken Ptch1. We therefore tested whether chicken Ptch1 lacking antiporter activity was able to induce the Shh response, after misexpression in the developing neural tube. Again, we observed little effect on neural tube patterning (supplementary material Fig. S1), indicating that suppressing the proton-driven antiporter activity of Ptch1 has little effect on the Shh response. The inability of Ptch1D499A to act as a dominant-negative inhibitor of endogenous Ptch1 raises the issue of whether its proton-driven antiporter activity is usually important in regulating the Shh response at these stages of development. Ptch1loop2, a deletion mutant of Ptch1 that is unable to bind Shh is usually a potent inhibitor of the Shh response. Consistent with an earlier observation (Briscoe et al., 2001), we found that expression of Ptch1loop2 had a strong cell-autonomous inhibitory effect on the Shh response (Fig.?1C,D). To assess whether this effect is usually mediated by its antiporter activity, we expressed O4I1 a Ptch1 allele that was unable to bind Shh but also lacks antiporter activity: Ptch1loop2/D499A. Ptch1loop2/D499A had no effect on Shh activity, based on the lack of ectopic cell-autonomous Pax7 induction, and only mildly inhibited motor neuron induction, as determined by Isl1/2 expression (Fig.?1E,F). The dramatic difference between the strong inhibition of the Shh response by Ptch1loop2 and the mild effects of Ptch1loop2/D499A demonstrates that this proton-driven antiporter activity is crucial for Smo inhibition by Ptch1loop2. Importantly, the loss of repressive activity of Ptch1 did not automatically result in the cell-autonomous activation of the Shh response, indicating that Ptch1loop2/D499A is not a strong inhibitor of endogenous Ptch1 function. To assess the activities of the Ptch1 mutants in the absence of endogenous Ptch1 activity, we expressed them in immortalized mouse embryonic fibroblasts (MEFs). MEFs are devoid of functional Ptch1 protein (Rohatgi et al., 2007) and have an autonomously upregulated Shh response (Taipale et al., 2000) that can be measured by measuring the integration of the gene into the locus (Goodrich et al., 1997). We found that SAG, a Smo agonist, further induced Shh pathway activation in MEFs, whereas cyclopamine reduced Shh pathway activity (Chen et al., 2002; Taipale et al., 2000) (Fig.?2A). This indicates that, despite the absence of Ptch1, Smo can be activated or inhibited in these cells. The addition of ShhN (a truncated and soluble form of Shh) also increased the Shh response, indicating that there is a Ptch1-impartial response to Shh. Open in a separate windows Fig. 2. The Shh-binding loop 2 of Ptch1 can mediate the Shh response in fibroblasts independently of the proton-driven antiporter activity. (A) After MEFs were produced to confluence, cells were cultured overnight in low-serum medium and treated with ShhN-conditioned medium, 200?M SAG or 1?M cyclopamine. Cells were lysed and activity was assessed by determining -galactosidase levels. Data show means.e.m. from three experiments performed UCHL2 in triplicate. (B,C) O4I1 MEFs were co-transfected with Ptch1, Ptch1 mutants or Disp1 as control vector, and a reporter and cells expressing Ptch1 were responsive to ShhN, cells expressing Ptch1loop2 were unresponsive (Fig.?2B), consistent with the O4I1 inability of Ptch1loop2 to bind Shh, mirroring our observations (Fig.?1C,D). For comparison, cells (Fig.?2B). We expanded this experiment using different.