Morpholinos for splice modificatio

Morpholinos for splice modification


  1. Fig. 1.

    Scanning electron micrographs of wild-type (A,C) and Tbx5del/del (B,D) embryos at E9. Limb bud outgrowth is apparent in wild-type but not in Tbx5del/del embryos (arrows in A,B, asterisks in C,D). Lateral plate mesoderm is seen in embryo of either genotype as a ridge along the side of the embryo.

  2. Fig. 3.

    Tbx5 is upstream of FGF signaling in the forelimb bud. Wild-type embryos express Fgf10 (A,C,E), Fgf8 (B,D) and Snai1 (F) in the nascent limb buds (red arrowheads). These transcripts are absent in the forelimb field of Tbx5del/del embryos, but expression at other sites in Tbx5del/del embryos is intact, including intermediate mesoderm and hindlimb expression of Fgf10 (yellow and blue arrowheads, respectively, in A,C,E).

  3. Fig. 2.

    Intact patterning of the LPM in Tbx5del/del embryos, as demonstrated by expression of Tbx5 mRNA (A,B, arrows) from the wild-type or deleted alleles. LPM differentiation, as shown by Hand2 expression (dHand in figure), is intact in embryos of either genotype (C). Normal patterning of the forelimb field in Tbx5del/del embryos is also apparent as demonstrated by expression of Hoxb8 and Pea3 mRNA in the LPM up to the posterior region of the forelimb field (arrowheads) in both wild-type and Tbx5del/del embryos (D-F). After the initiation of limb bud outgrowth at E9.5, Pea3 expression is expressed in the forelimb mesenchyme of wild-type embryos, but is not detectable in the forelimb field of Tbx5del/del embryos (F, asterisks). Hindlimb markers Pitx1 (G) and Tbx4 (H) are expressed normally in the hindlimb field (hl) but not in the forelimb field (asterisks) of wild-type or Tbx5del/del embryos.

  4. Fig. 4.

    Tbx5 expression precedes Fgf8 expression in the limb field. Expression of Tbx5 (A-C) is shown at E8.5 (five-somite stage, A), E8.5 (eight-somite stage, B) and E9.25 (C). Expression of Fgf8 (D-F) is shown at E8.5 (eight-somite stage, D,E) and E9.25 (F). Yellow arrowheads indicate Tbx5 expression in the forelimb precursors, beginning at the eight-somite stage (B). This expression was readily detectable in all embryos examined at this stage and beyond (B,C). Red arrowheads indicate weak Fgf8 expression in the intermediate mesoderm, beginning at the eight-somite stage. Note that this weak expression was observable in only a few embryos at this stage (compare D with E), but was reproducibly detected at E9.25 and beyond (F).

  5. Fig. 5.

    Tbx5 is upstream of Wnt and FGF signaling. Lef1 and Tcf1 expression is undetectable in the forelimb field of Tbx5del/del embryos (arrowheads in A,C,E), but is intact elsewhere in the embryo, including the hindlimb field (yellow arrows in A). Expression of Tbx5 is intact in Lef1-/-;Tcf1-/- embryos (B). Fgf10 expression is initiated, but expressed at lower levels than in wild type in the forelimb field of Lef1-/-;Tcf1-/- embryos at E9 (D) and E9.5 (F). Tbx5 (G) and Lef1 (H) expression is unaffected in Fgfr2ΔIgIII/ΔIgIII embryos. Arrowheads in B,D-H indicate the forelimb field.

  6. Fig. 6.

    TBX5 activates the Fgf10 promoter. (A) Schematic representation of the upstream regulatory sequences of mouse and human Fgf10. Regions of homology are indicated in red, and are numbered I, II and III; percent nucleotide identity is indicated between the two sequences. Potential TBX5-binding sites (TBEs) are shown as a, b or c, based on the different types of TBEs (see B). A conserved putative TBE is shown by the asterisk. A conserved putative Lef1/Tcf1-binding site is indicated by `L'. (B) Delineation of the three types of TBEs in the Fgf10 promoter. (C) Transactivation by TBX5 of the Fgf10-luciferase reporter construct in COS-7 cells. The reporter construct was transfected with increasing concentrations (0, 100, 500 or 1000 ng) of a Tbx5 expression construct. Mean fold activation is indicated above each bar. (D) Transactivation of the Fgf10-luciferase reporter construct by an activated β-catenin (β-cat) construct alone (black bars) or with a Tbx5 expression construct (white bars) or both (hatched bars). The amount of each plasmid transfected is indicated below the graph. (E) Fgf10 promoter deletion analysis. Deletion constructs were co-transfected with the Tbx5 expression construct (white bars) or the activated β-catenin expression construct (black bars). Deletion of region II (del II) or a point mutation of TBEa1 (muta1) did not affect activation by activated β-catenin, but greatly reduced (1.5 times less versus 18 times less) activation by TBX5. Deletion of region III (del III) results in decreased activation by β -catenin, but did not significantly affect activation by TBX5. Deletion of both regions (del II/III) eliminated activation by either construct. All results are expressed as fold increase in luciferase activity compared with the reporter construct alone. Data are shown as mean±s.d. for one representative experiment performed in triplicate.

  7. Fig. 7.

    A model for early stages of limb bud growth. (A) Schematic representation of genetic interactions in early limb bud initiation. The thicker the arrow, the more crucial the interaction. See text for details. (B) Major steps in early limb bud formation. TBX5 in the lateral plate mesoderm (LPM) activates Fgf10, which in turn signals to the surface ectoderm (SE) to activate WNTs and FGFs, which then signal back to maintain Fgf10 levels, and subsequently Tbx5 expression. Lef1 and Tcf1 cooperate with TBX5 to sustain Fgf10 levels. SO, somites; IM, intermediate mesoderm.