Figure 1.
Pknox2 is expressed in the zeugopod and partial stylopod domains during limb bud outgrowth.
A: Comparative expression of Meis1 and Pknox2 in limb bud at E10.5. B: Comparative expression of Meis1, Pknox2, Hoxa11 and Hoxd13 in limb bud at E11.5. Pknox2 is mainly detected in the zeugopod domain of both the forelimb and hindlimb at E10.5 and E11.5 and has a similar and partially overlapping pattern with Hoxa11. Meanwhile, Pknox2 expression is partially present in the stylopod domain of the forelimb at E10.5 and E11.5. The domains between two vertical guidelines are presumptive zeugopod. C: Pknox2 expression exists in the zeugopod domains and joint regions of the autopod at E12.5 and E13.5.
Figure 2.
Skeletal preparations for Prx1-Pknox2 transgenic mice.
A: Schematic diagram for Prx1-Pknox2 construct. B: WT embryo. C-D: Prx1-Pkonx2 transgenic mice with mild (C) and severe phenotypes (D). Transgenic mice display a shortened radius and ulna with impaired ossification (arrows, n = 3). The deformity in the forelimb zeugopod is much more severe than in the hindlimb (n = 3). All samples are collected at P0. FL, forelimb; HL, hindlimb.
Figure 3.
Alterations in Hox gene expression in the limb of Prx1-Pknox2 embryos.
A-F’: Expression of Hoxd9 (A, A’), Hoxa10 (B, B’), Hoxd10 (C, C’), Hoxa11 (D, D’), Hoxd11 (E, E’) and Hoxd13 (F, F’) in WT (A-F) and Prx1-Pknox2 embryos (A’-F’) in E11.5 forelimbs. The Hoxa10, Hoxd10 and Hoxd11 expression domains are anteriorly shifted (double arrows in B-C’, E, E’), whereas the Hoxa11 expression domain is shortened in the central zeugopod region (double arrows in D, D’) (n = 3). The limb is oriented so that the anterior is on the top and the posterior is on the bottom.
Figure 4.
Skeletal preparations of Col2-Pknox2 transgenic mice.
A: Schematic diagram for Col2-Pknox2 construct. B: Whole skeletons of WT and Col2-Pknox2 transgenic mice at P0. C: High-power view of the forelimb, carpal bones, elbow and hindlimb at P0. In comparison to WT, Col2-Pknox2 transgenic embryos exhibit defects including a bent radius and ulna with impaired ossification (black arrows), partially fused carpal bones (star) in the wrist, deformed elbow (black arrow: missed olecranon; red arrow: ectopic cartilage) and ectopic rib formation at lumbar vertebrae (L1, black arrow). N = 3.
Figure 5.
Disruption of chondrocyte differentiation and ossification in the zeugopod elements of Col2-Pknox2 mice.
A: Chondrocyte differentiation is blocked at an early stage in Col2-Pknox2 transgenic embryos compared to WT, as revealed by Safranin O staining and in situ hybridization of Col2, Ihh and ColX in the ulna at E15.5 (50X, n = 2). B: HE staining showed that the outline of ulna and radius skeleton in the transgenic embryo was not as clear as that of WT (left panel). The expression levels of Col2 and p-Smad1/5/8, but not Sox9, were down-regulated in the ulna and radius skeletons of transgenic mice compared to WT controls at E12.5 (100X, n = 3), indicating that the chondrocyte condensation and differentiation is impaired upon Pknox2 overexpression.
Figure 6.
Col1-Pknox2 transgenic mice lack deltoid crest in the forelimb.
A: Schematic diagram for Col1-Pknox2 construct. B: Skeletal preparations for Col1-Pknox2 transgenic mice at P0. A missing deltoid crest is observed in transgenic mice (arrows in b) compared to WT (a). C: HE staining of the deltoid crest (DC) in the forelimb. The right panel is a high-power view of the boxed regions in the left panel (50X, n = 3). D: IHC analyses for deltoid crest formation in the forelimb. IHC with antibodies against Col1 and MyoD revealed that elongated muscle cells (white arrow in lower panel) existed at the presumptive deltoid crest (DC) of the Col1-Pknox2 transgenic mice, which was not detected in the WT controls (upper panel). Dashed lines outline the deltoid crest tissues (50X, n = 3). DAPI was used to mark cell nuclei.