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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02712

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Tbx4 is not required for hindlimb identity or post-bud hindlimb outgrowth

Columbia University, College of Physicians and Surgeons, Department of Genetics and Development, 701 W. 168th St., New York, NY 10032, USA.

View larger version (58K): [in this window] [in a new window]   Fig. 1. Creation and excision characteristics of the Tbx4cond allele.(A) A loxP site and a loxP-flanked selection cassette were inserted into the introns surrounding exon 5 (Naiche and Papaioannou, 2003). The selection cassette was subsequently excised via Cre recombination in vivo (see Materials and methods). Numbered boxes, exons; DT, diptheria toxin gene; neotk, neomycin resistance and thymidine kinase selection cassette; triangles, loxP sites; half-arrows, genotyping primers. (B) Semi-quantitative PCR assessment of Tbx4cond in embryos administered tamoxifen at E7.5 shows no intact Tbx4cond after 24hours. Embryos (two representative samples) are compared with mixed DNA samples of known composition. (C) Dorsal view of the hindlimb field of early E10.5 embryos given tamoxifen at E7.5, hybridized with a probe specific to the deleted region of Tbx4 (deletion probe in A). No expression is detected in embryos carrying ERcre. (D) As in B, with embryos administered tamoxifen at E9.5 and recovered after 24 or 48 hours (two representative embryos from each time point). Tbx4cond isroughly 75-90% excised after 24 hours, and 98-100% excised after 48 hours. (E) No expression of Tbx4 is detected in E10.5 embryos carrying ERcre and given tamoxifen at E9.5, using the same probe as in C.

View larger version (107K): [in this window] [in a new window]   Fig. 2. Phenotype of Tbx4cond/cond embryosadministered tamoxifen at E7.5. (A,B) In ERcre embryos, hindlimb development fails at E11.5. (C,D) Fgf10 is present at low levels in the hindlimb field of E10.0 ERcre embryos. (E,F) Fgf8 is sporadically activated in the AER of ERcre embryos. (F') Dorsal view of hindlimb in F. (G,H) Fgf10 is normally expressed throughout the hindlimb bud at E10.5, but has been lost from E10.5 ERcre hindlimb buds. (I,J) dHand is confined to the posterior third of the hindlimb bud in control embryos but is expanded across the entire ventral margin of E10.5 ERcre hindlimb buds. Brackets indicate margins of the hindlimb bud. (K,L) The anterior hindlimb bud expression of Alx4 is expanded across the hindlimb bud in E10.5 ERcre embryos. (M,N) Shh is not observed in the ERcre hindlimb. (N') Dorsal view of hindlimb buds of embryo in N. (O-T) At E11.5 Fgf8, Shh and dHand are expressed in AER, posterior margin and posterior mesenchyme domains, respectively, in control embryos (O,Q,S), but have been lost from ERcre hindlimbs by this stage (P,R,T). Red arrowheads indicate distal tip of hindlimb bud. Green arrowheads indicate proctodeum. All panels to same scale.

View larger version (99K): [in this window] [in a new window]   Fig. 3. Phenotype of E14.5 Tbx4cond/cond embryos administered tamoxifen at E11.5, 10.5 or 9.5. (A-D) Left hindlimbs have been removed for clarity. (A) E14.5 control embryos have five distinct hindlimb digits. (B) ERcre embryos injected with tamoxifen at E11.5 resemble controls. (C) ERcre embryos injected at E10.5 show soft-tissue fusion of anterior hindlimb digits. (D) ERcre embryos given tamoxifen at E9.5 have only four hindlimb digits. (E-G) Hindlimbs of additional ERcre embryos injected at E9.5, suffering varying degrees of anterior digit fusion. (H-T) Cartilage staining of embryos as in A-D. (H-J) Skeletal development is normal overall, but ERcre embryos injected at E10.5 and 9.5 have aberrant hip attachments (red arrowheads). (K,L) Forelimbs of ERcre embryos given tamoxifen at E9.5 are normal. (M,N) Hindlimbs of embryos carrying the Rosa-ERcre allele but heterozygous for Tbx4 are normal after injection at E9.5. (O-T) Isolated hindlimbs with anterior oriented toward top, except T, which has dorsal oriented toward top. (O,P) Hindlimbs from control embryo and ERcre embryo injected at E11.5 have well-developed skeletal elements and five separated digits emerging from the ankle bones. The limb in P is tilted relative to O. (Q) Hindlimb from ERcre embryo injected at E10.5 shows a hypoplastic fibula, hypoplastic and discontinuous femur, hypoplastic pelvis, and thin anterior digits (bracket). (R) Hindlimb from ERcre embryo administered tamoxifen at E9.5, showing the same defects as in Q. (S) More severely affected hindlimb from an ERcre embryo injected at E9.5 with complete anterior digit fusion. (T) Side view of hindlimb in S shows no articulation between the remnants of the femur and the pelvis. I-V, digit identities from anterior to posterior; fe, femur; fi, fibula; p, pelvis; ti, tibia. Black arrowheads indicate carpal bones (forelimb) or the talus and calcaneus bones (hindlimb).

View larger version (140K): [in this window] [in a new window]   Fig. 4. Limb identity in the hindlimbs of Tbx4cond/cond embryosinjected with tamoxifen at E9.5 and recovered at indicated stage. (A-D) Expression of Tbx4, monitored by a probe 3' of the deleted region (3' probe in Fig. 1A), is normal in ERcre hindlimbs. (E-H) Tbx5 is expressed in the forelimb and excluded from the hindlimb (red arrowheads) in control and ERcre embryos. Pitx1 (I-L) and Hoxc9 (M-P) are expressed normally in the hindlimb (red arrowheads) of control and ERcre embryos.

View larger version (111K): [in this window] [in a new window]   Fig. 5. Expression of limb patterning genes in Tbx4cond/cond embryos injected with tamoxifen at E9.5 and recovered at indicated stage. Posterior is to the right. (A-H') Dorsal views of hindlimbs. (A-B') Shh is expressed in the posterior margin of both control and ERcre hindlimb at E10.5 and 11.5. (C-D') Ptc is expressed in similarly sized domains in the posterior of control and ERcre hindlimbs, but this domain takes up a larger proportion of the smaller ERcre hindlimb at E11.5. (E-F') dHand is expressed along the posterior margin of both control and ERcre hindlimbs, but, as with Ptc, this domain takes up more of the ERcre limb at E11.5. (G-H') Alx4 is expressed in the anterior of control hindlimb buds but is aberrantly upregulated in the posterior limb bud in ERcre embryos. Red arrowheads indicate anteroposterior limits of expression domains. (I-L) Tbx2 and Tbx3 mark anterior and posterior hindlimb margins, but the gap between these margins, indicated by red arrows, is smaller in ERcre embryos. (M,N) Tbx15 is expressed in the center of the limb in control embryos and this domain is not appreciably thinner in ERcre embryos. (O,P) Bmp4 is expressed in the periphery of control and ERcre hindlimbs. The AER has been removed. (Q,R) At E13.5 Bmp2 is expressed in control and ERcre hindlimb interdigital regions but is restricted to a smaller and more distal domain in ERcre anterior digits (black arrowheads). (S) Scatter plot of width of hindlimbs and forelimbs of E11.5 control (blue) and ERcre (red) embryos at widest point.

View larger version (113K): [in this window] [in a new window]   Fig. 6. Expression of FGF pathway genes in Tbx4cond/cond embryos injected with tamoxifen at E9.5 and recovered at indicated stage. Posterior is to the right in all panels. (A-F) Fgf10 is expressed throughout the distal mesenchyme in control (A,C,E) and ERcre (B,D,F) embryos at E10.5-12.5. f, forelimb; h, hindlimb. (G,H) At E13.5 Fgf10 is only seen in four digit tips in the ERcre hindlimbs. (I-L) Fgf8 is expressed in the AER of both control and ERcre embryos. (K') Detail of hindlimb in K. Fgf8 expression normally extends more proximally on the anterior margin (red arrowheads) than the posterior margin (black arrowheads) of the limb bud. (L') Detail of hindlimb in L. Fgf8 expression domain is truncated on the anterior margin. (M,N) FgfR1 is expressed throughout the limb mesenchyme in both control and ERcre embryos. (O,P) The anterior margin of Spry1 expression (red arrowhead) underlying the AER is slightly truncated in ERcre embryos relative to controls. (Q,R) Lef1 is expressed throughout the distal mesenchyme in both control and ERcre embryos.

View larger version (107K): [in this window] [in a new window]   Fig. 7. Phenotype of Tbx4cond/cond embryos with (prx) or without (control) the Prx-cre transgene. (A-D) Expression of intact Tbx4 transcript, monitored with a deletion-specific probe (Fig. 1A). Embryos were also hybridized with Pax1 probe, which marks the somites and anterior forelimb. (A,B) In late E10.5 control embryos Tbx4 is expressed throughout the hindlimb (red arrowheads) and the proctodeum or allantois region (green arrowheads) but is partially lost from the hindlimb of prx-cre embryos. (C,D) Dorsal views of the hindlimbs in A and B, respectively. (E,F) Cartilage staining of E15.5 control and prx-cre hindlimbs. prx-cre embryo has five digits, but severely hypoplastic fibula, femur and pelvis. (G,H) Prx-cre neonates show abnormally turned hindlimbs and small hips. (I-Q) Skeletal preparations of neonates. Red, ossified bone; blue, cartilage. (I,J) prx-cre neonates have small and abnormally turned hindlimbs. (K,L) Ventral views show that in normal embryos the femur is articulated with the pelvis, while in prx-cre embryos there is a large gap between femur and pelvis (black arrowheads). (M,N) Lateral views of hindlimbs. The lower limb elements of prx-cre hindlimbs are abnormally oriented relative to the femur. (O) Dorsal view of a prx-cre hindlimb. The fibula, femur and pelvis are severely hypoplastic and the fibula and femur are not ossified. (P,Q) Dorsal views of left hindfoot. prx-cre hindfoot has partially fused anklebones and mildly reduced second digit (open arrowhead). fe, femur; fi, fibula; p, pelvis; ti, tibia.

View larger version (50K): [in this window] [in a new window]   Fig. 8. A proposed model of Tbx4 and Tbx5 function in the limbs. In the forelimb initiation stages, Tbx5 is sufficient to drive threshold levels of Fgf10, which upregulate Fgf8 in the AER, but in the absence of Tbx5, no Fgf10 expression is seen. In the hindlimb, both Tbx4 and protein X contribute to Fgf10 expression, so in the Tbx4 null, sub-threshold levels of Fgf10 are seen. During the process of limb outgrowth, Tbx4 or Tbx5 contribute to robust expression of Fgf10 in their respective limbs, along with protein X and feedback from the AER, and protein X is sufficient to maintain Fgf10 expression at lower levels in the absence of Tbx4 or Tbx5. Dominant negative alleles block Fgf10 expression by recruiting co-repressor complexes.

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