Supplementary Figure 3: Characterization of Dzip1l^wpy/wpy mice.

(a) Sanger sequencing scans of the wild-type (top) and homozygous Dzip1l^wpy/wpy mutation (bottom), and the location of the stop codon in the predicted protein (on the right). Asterisks mark the mutated base, arrow marks location of the stop codon. (b) Dzip1l^wpy/wpy mutant embryos show evidence of altered hedgehog signalling in the limb. Indicated markers analysed by wholemount in situ hybridisation in 11.5 dpc limbs show expanded ectopic expression of Hoxd13, Grem1 and Gli1 into the anterior of the Dzip1l^wpy/wpy mutant forelimb bud (arrows in b). Anterior is to the top in all limb images. Similar data were obtained in hindlimbs (not shown). n = limbs from 3 independent embryos. (c) IF analysis with a battery of markers to hedgehog-dependent neural populations reveals no consistent difference between wild-type control and Dzip1l^wpy/wpy mutant sections through the neural tube of 10.5 dpc embryos at the level of the forelimb. Similar data were obtained with sections at hindlimb level (not shown). n = multiple sections from 3 independent embryos for all markers except Shh (n=2). Scale bar in b = 200μm; c = 100μm. (d) qRT-PCR analysis of Gli1 transcript levels shows that, compared to Dzip1l^+/+ MEFs, Dzip1l^wpy/wpy MEFs fail to significantly upregulate HH signalling in response to SAG treatment (n=4 cell lines derived from independent embryos, cells treated in three separate experiments; each data point is the average of 3 separate culture wells). Statistical analysis based on separate unpaired two-tailed Student’s t-tests for Dzip1l^+/+ t(6)=14.91, ****p<0.0001, and Dzip1l^wpy/wpy t(6)=0.6943, p=0.5135, ns, not significant, +/-SAG treatment. Error bars, s.e.m. All data presented are from embryos on a C57BL/6:C3H mixed background.