doi: 10.1242/10.1242/dev.00191
Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo
Pooja Agarwal1,2,*,
John N. Wylie1,*,
Juan Galceran3,
Oksana Arkhitko1,
Cuiling Li4,
Chuxia Deng4,
Rudolf Grosschedl3 and
Benoit G. Bruneau1,2,
1 Programmes in Cardiovascular Research and Developmental Biology, The Hospital
for Sick Children, Toronto, ON M5G 1X8, Canada
2 Department of Molecular and Medical Genetics, University of Toronto, Toronto,
ON M5S 1A8, Canada
3 Gene Center and Institute of Biochemistry, University of Munich, 81377 Munich,
Germany
4 Laboratory of Genetics of Development and Diseases Branch, National Institute
of Diabetes, Digestive and Kidney Diseases, 10/9N105, National Institutes of
Health, Bethesda, MD 20892, USA
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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.
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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).
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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.
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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).
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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 ß-catenin
(ß-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 ß-catenin expression
construct (black bars). Deletion of region II (del II) or a point mutation of
TBEa1 (muta1) did not affect activation by activated ß-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
ß-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.
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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.
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© The Company of Biologists Ltd 2003
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