DEV01885 Supplementary Material

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• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1. Prrx1 and Msx2 showed no significant difference in the microarray analysis, and served as real-time RT-PCR controls in the secondary samples. Prrx1 and Msx2 are both expressed in the same limb and genital mesenchymal cells in which Hoxd genes are expressed. Both genes showed no significant difference in their expression, either in limbs (A,C) or in genitalia (B,D).

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2. Papss2 expression is altered in HoxD^Del1–13 mutant limbs and genitals. Papss2 expression marks the digit condensations. (A) At E11.5, HoxD^Del1–13 and wild-type limbs are indistinguishable. By E12.5, Papss2 expression is markedly reduced in mutant limbs as the smaller digits of the mutants become apparent. Subsequently Papss2 expression diminishes in wild-type limbs, such that at E14.5 it is higher in the mutant. (B) Real-time RT-PCR measurements correspond closely to what is seen by WISH. Highly significant (**P<0.01) reduction in expression was also measured in genitalia, although the expression level is less than 15% of that detected in limbs. (C) Papss2 expression in genitalia is barely detectable by WISH.

• Supplemental Figure 3 (Adobe PDF) -

  Fig. S3. Two candidate genes (Aldh1a2 and Foxp1) for which differential expression was found only in limbs show highly dynamic expression patterns. (A) At E12.5, Aldh1a2 is expressed in the interdigital zone. This domain extends more proximally in wild type than in HoxD^Del1–13 autopods. At E13.5, Aldh1a2 expression in the mutant limbs overtakes wild-type expression levels, probably reflecting the altered kinetics and morphology of digit formation in the mutant. By real-time RT-PCR the differences are only statistically different at E12.5 (C). (B) Foxp1 expression increases rapidly in forelimbs from E11.5 to E13.5. In the secondary samples used in C, the change at E12.5 and E13.5 was not statistically significant. However, by WISH analysis, the expression pattern is obviously different in mutant and wild-type digits. (C) Real-time RT-PCR quantification of the changes in expression described above.

• Supplemental Figure 4 (Adobe PDF) -

  Fig. S4. Expression of the five candidate genes for which WISH images were not included in the other figures. Dorsal views of right forelimbs at E12.5 (left), and lateral views of genital buds (right) for which the greatest fold change was measured by real-time RT-PCR, in either E13.5 or E14.5 embryos (see Table 3). (A) Nr2f1 staining is visible in the interdigital zone of wild-type and mutant forelimbs (arrows). The decrease measured by real-time RT-PCR in HoxD^Del1–13 limbs is not obvious by WISH, probably due to weak staining. WISH staining failed at E14.5 (right) and therefore could not confirm the 2-fold increase in E14.5 mutant genitals measured by real-time RT-PCR. Strong staining of E12.5 genitals was observed (not shown), but with no apparent difference by WISH or real-time RT-PCR at that age. (B) Lisch7 expression is more obviously gained in wild-type forelimbs when visualized by WISH than when measured by real-time RT-PCR. Genital staining at E13.5 (right) is prominent but the difference associated with the small fold change measured by real-time RT-PCR (1.25-fold) is not obvious. (C) Shox2 is expressed very strongly in the proximal portion of E12.5 limbs of both genotypes, with no obvious reproducible difference by WISH. Similarly Shox2 is expressed in the proximal portion of the E13.5 genital bud. The gain shown on the right in the mutant genital bud could not always be reproduced by WISH, and was identified only as marginally statistically significant by real-time RT-PCR (P=0.042). (D) Pcdha is expressed most prominently in the developing central nervous system (not shown). However, a spot of expression (arrow) at the base of presumptive digit 5 was reproducibly seen in forelimbs of both genotypes. This domain appears to be slightly darker in the mutant limb bud, which could account for the 1.6-fold increase seen in HoxD^Del1–13 forelimbs by real-time RT-PCR. A loss of expression of Pcdha was observed in E13.5 and E14.5 mutant genitals (in three out of three embryos). Pcdha is one of two candidates in which the differential expression is in an opposite direction in genitals to that which was measured in limbs (with Nr2f1). (E) Stra6 expression is higher in wild type than in mutant limbs. This higher expression was significant by real-time RT-PCR from E11.5-E14.5, and is visible by WISH. No significant difference in the expression of Stra6 was noted in genitals by WISH or real-time RT-PCR, despite prominent expression (data not shown).