Supporting Figures

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Fig. 7. Dnchc2 alleles. (A) In a mapping cross of »1,000 recombination opportunities, Dnchc2^lln was mapped to an estimated 7.9-Mb interval between D9Ski101 (http://mouse.ski.mskcc.org) and the centromere of mouse chromosome 9, an interval that included the Dnchc2 gene. (B) A T-to-C mutation associated with Dnchc2^lln causes a phenylalanine-to-serine (F3890S) substitution (shaded in yellow) in a conserved region of the protein. The mouse Dnchc2 protein is 98% identical to rat (Rn) Dnchc2 (GenBank: NP_075413), 40% identical to Caenorhabditis elegans (Ce) CHE-3 (NP_492221), and 40% identical to Chlamydomonas (Cr) Dhc1b (CAB56748). (C) The amount of wild-type Dnchc2 protein was analyzed in whole embryo extracts from an e10.5 Dnchc2^GT litter (nine embryos, labeled as 1-9) and used Focal Adhesion Kinase (FAK) as a loading control. Embryos nos. 1, 2, and 3 are Dnchc2^GT homozygous mutants (m); embryos nos. 5, 7, and 8 are Dnchc2^GT heterozygotes (h); and embryos nos. 4, 6, and 9 are wild-type homozygotes (w). The filled arrowhead indicates the presence of Dnchc2 wild-type protein in both wild-type and Dnchc2^GT heterozygous embryos, and the unfilled arrowhead indicates the presence of Dnchc2-b-geo fusion protein in both Dnchc2^GT heterozygous and homozygous embryos. Dnchc2 wild-type protein was absent in Dnchc2^GT homozygous mutants, indicating that Dnchc2^GT was a null allele.

>Fig. 8. (A) In situ hybridization of Olig2 in wild-type and Dnchc2^lln mutant embryos showed lack of Olig2 expression in the rostral but not the caudal Dnchc2^lln neural tube. The arrow indicates the rostral limit of Olig2 expression. (B) As monitored by Dnchc2-lacZ staining, Dnchc2 was expressed more strongly in rostral than in caudal regions of embryonic day (e)9.5 embryos (Dnchc2^GT/+). Dnchc2-lacZ was also detected at higher levels in the limb bud, branchial arches, and the midbrain-hindbrain junction.

Fig. 9. Dnchc2^lln Ptch1 double-mutant analysis. Whole-mount (Upper) and neural tube sections (Lower) of Ptch1-lacZ staining of e9.5 wild-type (with one copy of Ptch1-lacZ), Ptch1, and Dnchc2^lln single (with one copy of Ptch1-lacZ), and Dnchc2^lln Ptch1 double mutants. In wild-type embryos, Ptch1-lacZ was detected in the ventral neural tube; in Ptch1 mutants, Ptch1-lacZ was expressed at high levels throughout the neural tube and in most other tissues of the embryo; and in both Dnchc2^lln single and Dnchc2^lln Ptch1 double mutants, Ptch1-lacZ was expressed at low levels in the neural tube. No ectopic expression of Ptch1-lacZ was detected in Dnchc2^lln Ptch1 double mutants in contrast to the Ptch1 single mutant.