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First published online 30 November 2006
doi: 10.1242/dev.02622

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Tbx5 is dispensable for forelimb outgrowth

Division of Developmental Biology, MRC-National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

View larger version (51K): [in this window] [in a new window]   Fig. 1. Tbx5 is expressed throughout the limb mesenchyme, including the precursors of cartilage, tendons and muscles. Serial section in situ hybridisation using probes for Tbx5 (A,E), chondrocyte markers Sox9 (B) and Col2a (F), tendon progenitor marker Scx (C,G), and myoblast markers Pax3 (D) and Myod (H). (A-D), E11.5; (E-H), E12.5.

View larger version (118K): [in this window] [in a new window]   Fig. 2. Comparison of Cre recombinase activity in the developing forelimbs of Prx1Cre and Prx1CreERT2 transgenic embryos using the Rosa26RlacZ reporter line. CreERT2 was activated by gavaging the pregnant females at E8.5. Activation of the reporter by Cre recombination is detected by lacZ staining (blue). In Prx1Cre embryos, Cre activity was detected throughout the FL-forming region at the 14-somite stage (A), and was induced in all cells of the FL of 19- and 21-somite stage embryos (B,C). In Prx1CreERT2 embryos, Cre activity was also first detected at the 14-somite stage, but only in a small number of cells (D,G). Low level activity was still apparent in 16- to 17-somite stage embryos (E,H), and it was not until the 21-somite stage that Cre activity was detected throughout the limb bud (F). By E10.5, the Cre reporter was uniformly activated throughout the limb bud (I-K). When the Prx1CreERT2 transgenic was activated in a background of the Tbx5 conditional allele (Tbx5lox/lox) and Cre reporter, a bud formed despite uniform Cre activity throughout the limb (L,M). PCR was used to quantitate the levels of Tbx5 ex3 loss through recombination following TM gavage at E8.5. Recombination of 87%-97% was observed in two independent E10.5 Prx1CreERT2; Tbx5lox/lox FLs (Mut1 and Mut2) (N). The graph shows the mean values and s.d. obtained from three replicates, normalised to a control; the mean value obtained from the wild-type limb is shown as 100%.

View larger version (118K): [in this window] [in a new window]   Fig. 3. Following TM administration at E8.5, the limbs of Prx1CreERT2;Tbx5lox/lox embryos are normally patterned at E10.5. Tbx5 heterozygous (control) (A,C,E,G,I,K,M,O) and mutant (B,D,F,H,J,L,N,P) embryos from pregnant females gavaged with TM at E8.5, were harvested at E10.5 and analysed by in situ hybridisation for: Tbx5 ex3 (A,B); Fgf10 (C,D); Fgf8 (E,F); Sall4 (G,H); Tbx3 (I,J); Shh (K,L); Tbx15 (M,N); Fgf8 and Myod (O,P). (B,H) Arrows indicate the heart and the anterior domain of the limb, respectively. (O, P) Somites are identified based on Myod expression and are numbered in a rostral to caudal direction.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Outgrowth and skeletal patterning is not impaired in limbs devoid of Tbx5. Pregnant females were gavaged with TM at E8.5 (A,D,E), E9.5 (B,F,G) or E10.5 (C,H,I) and harvested 2 days later (A-C), at E14.5 (D,E) or at E16.5 (F-I). Embryos were stained for lacZ (A-C), or with Alizarin Red and Alcian Blue (D-I). (E,G) Skeletal preparations of FL from Prx1CreERT2;Tbx5lox/lox embryos gavaged at E8.5 or E9.5 showed abnormalities comprising a hole in the scapula, lack of the deltoid tuberosity (E,E'',G,G''; arrows) and pronounced elongation of digit 1 (E',G'; arrowheads). By contrast, skeletal preparations of Prx1CreERT2;Tbx5lox/lox embryos gavaged at E10.5 showed normal development (I).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Model depicting two phases of limb development. An initial Tbx5-dependent limb bud initiation phase (top) is followed by a Tbx5-independent limb bud outgrowth phase (bottom). See text for details.