Fig 1.
Deletion of Sufu in the mesenchyme of limb buds with Dermo1-Cre mouse.
A-D: Whole mount in situ hybridization shows the expression of Sufu in fore- (A, B) and hind- (C, D) limb buds at E10.5. Noted the significantly reduced Sufu transcript in the SufuDermo1Cre mutant limb buds (B, D), in comparison with the wild type (A, C). E-F: Immunohistochemical staining of the limb bud section using antibody against Sufu protein in wild type (E) and SufuDermo1Cre(F). G-L: X-Gal staining of R26R/Dermo1-Cre. Whole-mount samples show Cre activity in the limb buds (G-I). J and K are sections of H and I, respectively. L is a horizontal section of I. Note the weaker staining in the core mesenchyme of the forelimb bud (arrowhead in J), compared to the hind limb bud (K). No Cre activity was detected in the limb bud epithelium (arrows in J and K). Mes: mesenchyme; Epi: epithelium; FL: forelimb bud; HL: hind limb bud.
Fig 2.
Sufu deletion results in the loss of digit identity and polydactyly.
A-D: Abnormalities of autopods in SufuDermo1Cre. E-T: Skeletal preparations of Alcian blue/Alizarin Red staining show the alteration of digit number and identity. Note the varying abnormalities in the fore- and hind limbs. Split of the anterior-most digit occurred in forelimb of the Sufu mutant (F, J versus E, I and arrowheads). Mutant hind limb developed 7 digits with complete loss of identity (H, L versus G, K). Phalangeal numbers distal to the metatarsals/metacarpals are not distinguishable in mutants (J, L versus I, K in wild type). Both fore- and hind limbs developed supernumerary bone elements in the wrist and the ankle of the mutant (N, P versus M, O in wild type). Note that the forelimb developed thicker radius (R versus Q), while the hind limb contained splitting of both the fibula and tibia (T versus S).
Fig 3.
Expression of 5’ HoxD genes in Sufu deletion.
A-X: Whole-mount in situ hybridization of limb buds showing the alteration of HoxD11, HoxD12, and HoxD13 in SufuDermo1Cre at E11.5 (A-D; I-L; Q-T) and E12.5 (E-H; M-P; U-X). Note the anteriorly expanded expression for all three genes.
Fig 4.
Expression of AER molecular markers relevant to the activation of the Shh/Fgf loop.
A-H: Whole-mount in situ hybridization of limb buds showing the expression of Fgf4 in the AER was anteriorly expanded in SufuDermo1Cre. The Fgf4 transcript in the anterior AER was detected at E10.5 (arrowheads in C, G versus A, E in the wild type), which became evident at E11. 5 (arrowheads in D, H versus B, F in the wild type). I-P: Fgf8 expression along the AER was intensified and expanded in SufuDermo1Cre (K, O, L, P versus I, M, J, N in the wild type). I’-P': Front views of AER (inserts in I-P, respectively).
Fig 5.
Expression of Shh regulated posterior genes.
A-H: Whole mount in situ hybridization of hind limb buds showing the expression of Shh targeted genes. The expressions of Gli1 (A, B and arrowheads in C, D) and Ptc1 (E, F and arrowheads in G, H) were activated in the anterior limb bud mesenchyme in SufuDermo1Cre. I-P: Whole-mount in situ hybridization of limb buds showing that Gremlin expression was anteriorly expanded in both the fore- (K, L and arrowheads versus I, J) and hind limb buds (O, P and arrowheads versus M, N).
Fig 6.
Sufu deletion cause alteration of Gli2R, Gli3R and Hand2 expression.
A-P: Whole-mount in situ hybridization showing the expression of Gli3 and Hand2. Expression of Gli3 in limb buds of the Sufu mutant (C, D, G, H) was comparable to the wild type (A, B, E, F). Anterior expansion of Hand2 transcript was exhibited at E11.5 (L, P and arrowheads), compared to the wild type (J, N). Note the global distribution of Hand2 transcripts in the entire hind limb bud (P versus L for forelimb bud). Q: Western blots show the decreased Gli2R protein (arrowhead) and Gli3R protein (arrowhead) in limb bud lysates at E10.25. R: Semi-quantitative analysis of the expression of Gli3R in Sufu mutant (M) limb buds compared with wild type (Wt). Student’s t test of significance with *P<0.05, **P<0.01.