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First published online 25 June 2008
doi: 10.1242/dev.017384

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Dual hindlimb control elements in the Tbx4 gene and region-specific control of bone size in vertebrate limbs

Howard Hughes Medical Institute and Department of Developmental Biology, Stanford University, Stanford, CA 94305-5329, USA.

View larger version (95K): [in this window] [in a new window]   Fig. 1. A BAC enhancer scan across the mouse Tbx4 region. (A) Schematic of genomic region encompassing Tbx4. Arrows denote the direction of transcription of Tbx4 and flanking genes. Gray bars show BAC locations with the inserted IRES-βGeo cassette in blue. Mapped cis-regulatory intervals are shaded: yellow, hindlimb interval I; green, lung interval; pink, hindlimb interval II. (B,C) Whole-mount in situ hybridization for Tbx4 mRNA in E12.5 mouse embryos. (D-I) Whole-mount lacZ staining of transgenic embryos carrying RP24-376P4, RP24-84E15 or RP23-136J3. Dotted lines denote boundaries of limbs, umbilical cord (uc) and genital tubercle (gt). lu, lung.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Comparative genomics reveals locations of candidate cis-regulatory elements. (A) Sequence comparison of mouse with human, opossum, chicken and zebrafish Tbx4. Colored bars (top) denote location of cis-regulatory intervals; blue boxes indicate position of Tbx2 and Tbx4 exons. All regions with >70% identity over a 100 bp window are colored: blue, exons; red, conserved intergenic sequence; pink, conserved intronic sequences). Block-shaded portions of plots indicate positions of confirmed enhancers (see B): yellow, hindlimb enhancer A (HLEA); green, lung enhancer; pink, hindlimb enhancer B (HLEB); gray, umbilical cord. (B) Genomic regions assayed for enhancer activity. Black bars represent fragments that did not drive consistent expression patterns at E12.5. (C-G) Whole-mount lacZ staining of E12.5 transgenic mouse embryos illustrating expression patterns seen with the different constructs. tr, trachea; lu, lung; uc, umbilical cord.

View larger version (60K): [in this window] [in a new window]   Fig. 3. HLEB is conserved from mammals to cartilaginous fish. (A) VISTA plot (Frazer et al., 2004) comparing the 654 bp mouse HLEB sequence against sequences from human, opossum, platypus, chicken, lizard, frog, teleost fish (stickleback) and cartilaginous fish. Percentage sequence identity [y-axis, ranging from 40% to 100% (lower to upper thick line in each panel)] is shown in a sliding window across the region (x-axis, bp). Pink shading indicates regions of 20 bp or more that are 65% identical. (B,C) Whole-mount lacZ staining of E12.5 transgenic mouse embryos. (B) Hsp68LacZ transgene pDBM5 with four copies of mouse HLEB. (C) Hsp68LacZ transgene pDBM20 with four tandem copies of stickleback HLEB.

View larger version (134K): [in this window] [in a new window]   Fig. 4. Developmental time course of HLEA and HLEB activity compared with the endogenous expression of Tbx4. (A-F) Whole-mount lacZ staining of mouse embryos from stable transgenic lines carrying HLEA transgene pDBM7 (A-C) or HLEB transgene pDBM5 (D-F) show that these enhancers are active in the early hindlimb field (A,D) and developing hindlimb bud (B,C,E,F). (G-I) Tbx4 mRNA as detected by whole-mount in situ hybridization shows similar patterns of expression. Embryonic stages as indicated.

View larger version (87K): [in this window] [in a new window]   Fig. 5. HLEA and HLEB are both required for robust hindlimb expression of Tbx4. (A) Schematic of mouse Tbx4 genomic region. BAC transgene RP24-84E15 and derivatives are represented by gray bars. Yellow ovals, HLEA; green circles, lung enhancer; gray circles, umbilical cord element; pink ovals, HLEB; blue box, IRES-βGeo. (B-I) Lateral (B,D,F,H) and ventral-posterior (C,E,G,I) views of whole-mount lacZ staining of E12.5 transgenic mouse embryos carrying BAC transgenes RP24-84E15 (B,C), HLEA (D,E), HLEB (F,G) or HLEAHLEB (H,I).

View larger version (81K): [in this window] [in a new window]   Fig. 6. Mutation of a conserved putative Pitx1 binding site in HLEA results in reduced hindlimb enhancer activity. (A) VISTA plot comparing 768 bp subregion of mouse HLEA against human, cat, dog, cow, armadillo, elephant, opossum and platypus sequences. Horizontal panels show sequence identity across the region, ranging from 50-100%. Pink shading indicates regions of 20 bp or more that are 65% identical. Arrowheads indicate positions of putative Pitx1 binding sites, including one that is perfectly conserved (red arrowhead). The accompanying sequence alignment (beneath) shows perfectly conserved bases (asterisks), the sequence of the wild-type Pitx1 site (red), and the sequence of the mutated site in Pitx1Mut. (B) Plot of the area of lacZ staining in forelimbs and hindlimbs of transgenic mouse embryos carrying wild-type (HLEA-768) or mutant (Pitx1Mut) enhancers. Each data point represents an individual transgenic embryo. (C,D) Whole-mount lacZ staining of E12.5 transgenic mouse embryos carrying wild-type (C) and mutant (D) enhancers.

View larger version (55K): [in this window] [in a new window]   Fig. 7. Targeted knockout of HLEA reduces expression of Tbx4 in hindlimbs. (A) Schematic showing homologous targeting of the Tbx4 locus in mouse ES cells to create Tbx4floxNeoHLEA, a floxed HLEA knock-in allele, and Tbx4HLEA, where the floxed HLEA allele has been deleted using Cre recombinase. Gray bars, first and second exons of Tbx4; yellow ovals, HLEA; triangles, loxP sites; red arrows, genotyping primers; Neo, neomycin resistance cassette. (B) Southern blot of ES cell clones digested with MfeI shows a wild-type (Wt) band of 24 kb and a targeted Tbx4floxNeoHLEA band of 9.1 kb with an external 3' probe (see A). (C) Mice carrying the Tbx4floxNeoHLEA allele were crossed to a Cre deleter strain to generate the Tbx4HLEA allele. Genotyping by PCR confirmed the presence of heterozygous and homozygous animals. (D) The relative expression levels of wild-type 129P2 and HLEA 129P2 Tbx4 alleles were compared with a wild-type DBA Tbx4 allele in E11.5 hindlimbs and lungs. Black bars, expression ratio of wild-type 129P2 to DBA; gray bars, ratio of HLEA 129P2 to DBA. (E-J) Whole-mount in situ hybridization for Tbx4 mRNA in wild-type and homozygous HLEA knockout embryos. Lateral views of E10.5 embryos (E,F) and dorsal views of E10.5 hindlimb buds (G,H) and E12.5 hindlimbs (I,J). Arrowheads indicate anterior side of hindlimb buds.

View larger version (28K): [in this window] [in a new window]   Fig. 8. Homozygous HLEA mice show reduced size of hindlimb bones. (A,B) Bar charts showing mean sizes of wild-type (black bars) and homozygous HLEA mutant (gray bars) bones in hindlimbs (A) and forelimbs (B) of adult mice. Error bars indicate s.e.m. Red brackets in diagrams to right show positions of measurements. (C) Metatarsals and phalanges of digit rays 1 (red), 3 (blue) and 5 (gray) were measured in the feet of the wild type and homozygous mutants. Shown are percent decreases in the sizes of individual bones, or in the combined length of digit rays, in mutants compared with wild type. *P<0.05, **P<0.005, ***P<5x10-5.

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