Figure 5: Hindlimb-specific functions of Tbx4 correlate with repressor activity that is lost in human small-patella mutation TBX4Q531R.

(a) Transcriptional activities of mouse Tbx5, Tbx4, Tbx4Q538R and amphioxus Tbx4/5 in CV-1 cells assessed with reporter containing T-box-binding regulatory element (TBE) in one or two copies (2× TBE). Error bars indicate s.e.m. (n=5) (b) Deletion analysis of Tbx4/Tbx5 C-terminal domains using Gal4DBD fusions. Deletion end points are indicated in diagrams. Green box indicates the activator domain, whereas magenta box represents the repressor domain. Error bars indicate s.e.m. (n=5). (c) Tbx4 is a transcription activator in e11.5 FL primary cells and a repressor in HL primary cells. The activity of full-length Tbx4 fused to Gal4DBD was assessed by transfection in primary cells of indicated limb buds. Data are represented as means ± s.e.m. (n=3). (d) Full-length amphioxus Tbx4/5 and its C-terminus have activator properties in CV-1 cells transfected as in b above. Error bars indicate s.e.m. (n=3). (e–g) The Tbx4Q538R transgene is unable to rescue (e) ilium development and HL anterior displacement (dotted line) (f) to suppress ectopic tuberosity (asterisk) or (g) change pelvic angle, but rescues femur length (± s.e.m., n=3). Alizarin red and alcian blue staining was carried out as shown in Figure 2.