|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
| ||||||||||||||||||||
Files in this Data Supplement:
Fig. S1. Regionally restricted enhancer activity of Tbx4 transgenes in E12.5 mouse hindlimbs. lacZ staining of sections of hindlimbs from whole-mount E12.5 mouse embryos carrying (A) pDBM7, (B) pDBM40 or (C) pDBM45. The anterior of each limb is to the left side of each panel.
Fig. S2. Stickleback HLEB can drive hindlimb-specific expression of a lacZ reporter at early stages of mouse development. lacZ staining of whole-mount mouse embryos. (A) An Hsp68LacZ transgene containing a single copy of the stickleback HLEB exhibits staining in the posterior half of the hindlimb field at E9.5. (B) An Hsp68LacZ transgene containing four tandem copies of the stickleback HLEB produces intense staining in the posterior half of the hindlimb bud at E10.5.
Fig. S3. HLEA and HLEB have regionally restricted activity during late embryogenesis. (A,B) lacZ staining of whole-mount hindlimbs of transgenic embryos carrying the pDBM7 HLEA transgene (A) or pDBM5 HLEB transgene (B) at E17.5 or E16.5, respectively. (C,D) Sections of these stained hindlimbs demonstrated that HLEA activity is restricted to the knee and ankle bones (C). HLEB activity was strongest in the femur, tibia and patellar ligament (D). f, femur; tb, tibia; p, patellar ligament. (E,F) Tbx4 mRNA in situ hybridization in sectioned E16.5 hindlimbs showed that Tbx4 is expressed throughout most of the hindlimb. Antisense riboprobe (E) and sense riboprobe (F).
Fig. S4. Alignment of HLEA-768 from eighteen placental mammals, opossum and platypus. Putative Pitx1 binding sites (red) were identified in the mouse sequence and examined for their conservation status among different mammalian species.
Fig. S5. Tbx4 expression levels appear similar in wild-type and ΔHLEA mutant embryos at E9.5. Whole-mount in situ hybridization for Tbx4 on E9.5 wild-type (A,B) and homozygous ΔHLEA (C,D) embryos. (B,D) Dorsal views of the emerging hindlimb buds.
Fig. S6. Fgf8, Hand2 and Alx4 expression in E11.5 wild-type and ΔHLEA mutant hindlimbs. Whole-mount in situ hybridization for Fgf8 (A,B), Hand2 (C,D) and Alx4 (E,F) on wild-type (A,C,E) and ΔHLEA/ΔHLEA (B,D,F) E11.5 embryos.
Fig. S7. Morphological defects in the hindlimbs of homozygous ΔHLEA mice. The skeletons of wild-type and ΔHLEA/ΔHLEA 8-week-old male mice were treated with Alcian Blue and Alizarin Red such that cartilage stained blue and bone stained red. Mutants exhibited a range of different tarsal bone fusions, commonly involving the second, third, and central tarsal bones. Dorsal view of typical wild-type (A) and mutant (B) feet. A side view of wild-type (C) and mutant (D) feet revealed that in most mutants (14 of 15 feet examined), the metatarsal of digit 1 was fused to the first tarsal bone. At a lower frequency, the tibiale was fused to the intermedium. (E) Schematic highlighting three separate fusions observed in D (colored red, green and blue). In mutant feet, the second, third, and central tarsal bones are often fused together to form a single element: compare wild type (F) with mutant (G). A schematic (H) highlights the position of the fused tarsal bones (bones fused in mutant are red). All mutants also lacked the peroneal process, which extends from the head of the fibula. Side view of a wild-type leg (I) and mutant leg (J) demonstrating the absence of the peroneal process. In 33% of mutants, one or both patellae were laterally or medially displaced outside of the trochlear groove. Wild-type knee (K) and a mutant knee (L) with a laterally displaced patella. Black arrowheads (C,D,F,G) mark the position of normal and fused joints in +/+ and ΔHLEA/ΔHLEA, respectively. Red arrowheads (I,J) indicate the expected location of the peroneal process. 1, first tarsal; 2, second tarsal; 3, third tarsal; c, central tarsal; m1, first metatarsal; tb, tibiale; in, intermedium; p, patella.
Adobe PDF
| ||||||||||||||||||||
