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First published online 24 November 2005
doi: 10.1242/dev.02172

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Increasing Fgf4 expression in the mouse limb bud causes polysyndactyly and rescues the skeletal defects that result from loss of Fgf8 function

Pengfei Lu¹, George Minowada^1,2,* and Gail R. Martin^1,

¹ Department of Anatomy and Program in Developmental Biology, School of Medicinè University of California at San Francisco San Francisco, CA 94143-2711, USA.
² Department of Medicine, Division of Pulmonary and Critical Care Medicine, Case Western Reserve University, School of Medicine, University Hospitals of Cleveland, Cleveland, OH 44106, USA.

View larger version (68K): [in a new window]   Fig. 1. Fgf4 expression in limb buds following Cre-mediated activation of a conditional Fgf4 gain-of-function allele. (A) Schematic representation of the Fgf4GOF transgene, in which the CAGG promoter drives expression of the ß-Geo gene, and the triple polyadenylation sequence (3x pA) prevents expression of sequences downstream of the ß-Geo gene. Cre-mediated recombination deletes the ß-Geo gene and the 3x pA, and as a result, the previously silent downstream mouse Fgf4 cDNA is expressed from the `activated' transgene (A-Tg). (B) An assay for lacZ expression [ß-galactosidase (ß-gal) activity] in whole mount shows that the CAGG promoter drives expression of the Fgf4GOF transgene in a wide variety of cell types at E9.5. The Fgf4GOF embryo shown is hemizygous for the transgene. (C-S) Fgf4 expression is compared in limb buds from normal (n) and Msx2-cre;Fgf4GOF embryos, in which the transgene has been activated (A-Tg). (C) Relative levels of Fgf4 expression as assessed by quantitative RT-PCR (see Materials and methods). (D-S) Fgf4 expression as detected by whole-mount RNA in situ hybridization at the stages indicated. In the Msx2-cre;Fgf4GOF limb buds, the in situ hybridization signal represents the sum of Fgf4 RNA produced by the endogenous Fgf4 locus and the activated transgene. All panels show dorsal views, except H,I, which show distal views of the limb buds with dorsal at the top. In D-G,J,K anterior is towards the top; in all other panels, anterior is towards the left. The arrows in I indicate the domain of Fgf4 expression in the ventral ectoderm. The filled and open arrowheads in L,M indicate the anterior and posterior ends, respectively, of the Fgf4 expression domain in the AER. At E13.5, Fgf4 expression in the Msx2-cre;Fgf4GOF limb bud is restricted to the AER covering the distal tips of the digit primordia (asterisks). Abbreviations: FL, forelimbs; HL, hindlimbs; s, somite stage.

View larger version (71K): [in a new window]   Fig. 2. Fgf8, Shh and Grem1 expression in Msx2-cre;Fgf4GOF limb buds. (A-L) Whole-mount RNA in situ hybridization assays for the genes indicated in normal (n) and Msx2-cre;Fgf4GOF (A-Tg) limb buds. (A,B) Individual limb buds at E10.5 are shown in dorsal (anterior towards the left) view. In A' and B', the same limb buds are shown in distal view (dorsal towards the left). The AER, as marked by Fgf8 expression, is of similar AP length and DV width in the normal and Msx2-cre;Fgf4GOF limb buds. (C,D) Hindlimbs at E13.5 are shown in dorsal view (anterior towards the left). In both normal and Msx2-cre;Fgf4GOF limb buds, the AER, as marked by Fgf8 expression, is still present in regions covering the developing digits, but is no longer present over the regions covering the interdigital mesenchyme (arrowheads). This shows that AER regression is not delayed in Msx2-cre;Fgf4GOF limb buds. (E-H) Dorsal views at E10.5, with anterior towards the left. (I-L) Posterior views at E11.5, with dorsal leftwards, distal towards the top. Arrows indicate the regions in which the Shh and Grem1 expression domains are expanded on the ventral side of the Msx2-cre;Fgf4GOF limb buds.

View larger version (67K): [in a new window]   Fig. 3. Limb skeletal abnormalities in Msx2-cre;Fgf4GOF mice. (A-F) Skeletal preparations of newborn mouse limbs, with cartilage stained blue and bone stained red. Individual digits are numbered I-V, from anterior to posterior. (A-E) Dorsal views of autopods from normal and Msx2-cre;Fgf4GOF mice. The supernumerary posterior digit present in all animals that expressed the Msx2-cre-activated Fgf4GOF transgene is indicated by a filled arrowhead. The arrow in C indicates additional supernumerary phalanges found in some of these animals. The calcaneus (Ca) is enlarged in the Msx2-cre;Fgf4GOF hindlimb (open arrowhead in E). (F) Anterior view of an Msx2-cre;Fgf4GOF hindlimb containing a thin digit-like structure ventral to digit I (broken line). (G-J) Whole-mount in situ hybridization for Sox9 expression in normal and Msx2-cre;Fgf4GOF (A-Tg) limb buds at E12.5. The condensations that prefigure the supernumerary posterior digit are evident at this stage (arrowheads in H and J).

View larger version (75K): [in a new window]   Fig. 4. Persistence of interdigital tissue in Msx2-cre;Fgf4GOF limbs and effects on the expression of genes involved in BMP signaling. (A-D) Limbs of normal (n) and Msx2-cre;Fgf4GOF (A-Tg) mice on postnatal day (P) 14, with the digits spread to illustrate the cutaneous syndactyly (white arrowheads). The supernumerary posterior digit is not visible in intact limbs. (E,F) Hindlimbs at E13.5 stained with Lysotracker to label regions with dying cells. The boxes in E and F indicate the regions shown at higher magnification in E' and F', respectively. (G-J) Hindlimb buds of normal and Msx2-cre;Fgf4GOF (A-Tg) mice at E12.5, hybridized in whole mount with probes for the genes indicated.

View larger version (38K): [in a new window]   Fig. 5. Rescue of the skeletal defects in Msx2-cre;Fgf8/fl mice by expression of the Msx2-cre-activated Fgf4GOF transgene. (A-L) Skeletal preparations of newborn mouse limbs, with cartilage stained blue and bone stained red. In Msx2-cre;Fgf8/flox mice, the deltoid tuberosity, a feature of the normal humerus (arrows in A and I), is absent (red circle in E), the femur is extremely hypoplastic (red asterisk in F), one digit is missing and phalanges are absent from some of the remaining digits (red circles in G,H). All of these defects are rescued in Msx2-cre;Fgf8/flox;Fgf4GOF limbs (I-L). Solid and open arrowheads indicate the posterior supernumerary digit and enlarged calcaneus, respectively, in Msx2-cre;Fgf8/flox;Fgf4GOF limbs. Individual digits are numbered I-V, from anterior to posterior; phalanges (p) are numbered 1-3, from proximal to distal. Abbreviations: Au, autopod; Fe, femur; Fi, fibula; Hu, humerus; Ra, radius; St, stylopod; Ti, tibia; Ul, ulna; Ze, zeugopod.