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growth arrest specific gene 1 acts as a region-specific mediator of the Fgf10/Fgf8 regulatory loop in the limb

¹ Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210, USA
² Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA

View larger version (88K): [in a new window]   Fig. 1. Gas1 transcripts are detected in developing limbs. Expression of Gas1 was examined by ISH. (A-C) Dorsal view of the forelimbs at E9.25, E9.5 and E10.5. Anterior (a) is at top and posterior (p) at bottom; flb, forelimb bud. Gas1 is expressed in the lateral plate mesoderm and asymmetrically in the limbs with higher levels anteriorly. (D-G) Whole-mount ISH of E11.5-E14.5 forelimbs shows expression in the autopod (dorsal view, anterior at top). White arrowheads indicate the interdigital regions; open arrowheads, perichondorgenic regions; asterisks, joints or prospective joints. (H-K) 35S-ISH of E10.5-E15.5 forelimbs (horizontal sections, anterior at top). (H) Superimposed images from adjacent sections of E10.5 Gas1 expression (red) in anterior two-thirds of the mesenchyme but not in AER (visualized by Fgf8 expression (green) in the adjacent section). At E12.5 (I), Gas1 transcripts (pink granules) are prominent in the prechondrogenic region (black arrowheads) and the interdigits (white arrowheads). At E13.5 (J), Gas1 is expressed in the interdigits (much weaker than at E12.5) and prospective joints (asterisks). At E15.5 (K), Gas1 is localized to perichondrium lining the joint cavities (asterisks).

View larger version (59K): [in a new window]   Fig. 2. Gas1-/- has limb abnormalities. Dorsal view of the newborn forelimb (A,A',C,C') and hindlimb (B,B',D,D') paws of a control (con; A-D) and a Gas1 mutant (-/-; A'-D') animal. Gas1-/- forelimb and hindlimb paws are overall smaller and have syndactyly between digits II and III (A',B'). White arrows indicate soft tissue fusions and white arrowheads, disproportionally reduced digits. (C-D') Skeleton of the paws stained with Alcian Blue and Alizarin red. Black arrowheads indicate the reduction (C') or lack of (D') the second phalange of digit II; black arrows, syndactyly of digits II and III (D'). (E) A table summarizing the frequency of these skeletal defects in the Gas1 mutants.

View larger version (62K): [in a new window]   Fig. 3. Gas1-/- limbs display a delay in chondrogenesis and ossification. Whole-mount Alcian Blue (for cartilage) and Alizarin Red (for calcified bones) staining of control (con; A-F) and Gas1-/- (-/-; A'-F') embryonic limbs (dorsal view) at the indicated stages. In all panels, digit I is on the left, and digit V, on the right, as labeled. Arrowheads indicate delayed cartilage condensation (A',B'); arrows, delayed or lack of phalangeal separation (C',D'); open arrows, delayed ossification of the phalanges (F'); open arrowheads, the normal size of ossification of the metacarpals (F,F'); m, metacarpal; p, phalange.

View larger version (70K): [in a new window]   Fig. 4. Gas1-/- limbs display defective cell proliferation in the AER and distal mesenchyme. In vivo BrdU labeling of control (con; A-C) and Gas1-/- (-/-; A'-C') embryonic forelimbs. (A,A') At E10.5, BrdU-positive cells were markedly reduced in the mutant AER. (B,B') At E11.5, BrdU-positive cells were reduced in the region of distal mesenchyme (asterisk) in Gas1-/-. At E12.5, control (C) and Gas1-/- (C') have similar rates of cell proliferation in the interdigits between digits III/IV and IV/V, but reduced proliferation between digits II/III. Limbs were sectioned horizontally with the anterior at top. Note that the chondrogenic regions of digits III and IV have a lower rate of proliferation in the mutant (C'). (D) A table summarizing the BrdU counts of each stage.

View larger version (150K): [in a new window]   Fig. 5. Gas1-/- limbs have altered programmed cell death (PCD) patterns. PCD during limb development was assessed by TUNEL fluorescent labeling. In Gas1-/- (-/-), the TUNEL assay shows reduced and delayed PCD relative to control (con) at E11.5 (A,A'), E13.5 (B,B') and E14.0 (C,C'). White solid arrows indicate cell death at the opaque patch; white open arrows, the anterior margin; open arrowheads, the interdigital mesenchyme; white arrowheads, the forming joints. The limbs were sectioned horizontally and the digits are as labeled (I-V).

View larger version (76K): [in a new window]   Fig. 6. Molecular analysis of Gas1-/- limbs for patterning genes. Control (con; A-E) and Gas1 mutant (-/-; A'-E') forelimbs were compared for expression of Shh, gremlin, Bmp2, Bmp4, and Ptc1 by ISH at E10.5. (A,A') Shh expression appears normal in Gas1-/- limbs. (C,C') Bmp2, (D,D') Bmp4, and (E,E') Ptc1 expression is not obviously altered. (B,B') gremlin expression appears to be slightly reduced in the limb mesenchyme in the mutant. Hoxd13 expression was examined at E11.5 (F,F') and found to be normal in the mutant — in a smaller domain proportional to the smaller size of the limb. Expression of Ptc1 (dorsal view) and Hoxd13 (lateral view, arrowheads indicate the forelimb expression) was assessed by whole-mount ISH; the others, by 35S-ISH on horizontal sections with anterior at top.

View larger version (63K): [in a new window]   Fig. 7. ISH of Fgf expression in the Gas1-/- AER. (A-C) Fgf4, Fgf9 and Fgf17 gene expression (arrows) is normal in Gas1-/- limb at E10.5 as determined by 35S-ISH on horizontal sections; anterior at top. Control (con; D-G) and Gas-1-/- (-/-; D'-G') collected at E9.5 (D,D'), E10.0 (E,E'), E10.5 (F,F') and E11.5 (G,G') were analyzed for Fgf8 expression by whole-mount ISH. Fgf8 expression is initiated but not maintained. Black arrowheads indicate the location of the AER; white arrowheads, Fgf8 expression in other regions as an internal control (in the tailbud in E and E' and branchial arches in F' and G'); open arrowheads, reappearance of small patches of Fgf8 expression in the posterior AER (G'); flb, forelimb; hlb, hindlimb.

View larger version (74K): [in a new window]   Fig. 8. Analysis of Fgf10 expression in Gas1-/- limbs. Fgf10 expression was detected by 35S-ISH in control (con; A,B,D,F) and Gas1-/- (-/-; A',B',D',F') forelimb buds at E9.5, E10.0, E10.5 and E11.5 in horizontal sections (n=4). Arrowheads indicate the distal tip mesenchyme underneath the AER; brackets, the region of distal tip mesenchyme with reduced Fgf10 expression in the mutant; white arrowhead, moderately recovered Fgf10 expression at the distal region at E11.5 Fgf10 expression was also assessed by whole-mount ISH in control (C,E) and Gas1-/- (C',E') forelimbs at E10.0 and E10.5 (dorsal and dorsal-lateral views, respectively). Only 2 out of 12 E10.0 and 2 out of 13 E10.5 embryos showed clear defects of Fgf10 expression in one of the limbs by whole mount.

View larger version (67K): [in a new window]   Fig. 9. FGF10 restores FgF8 expression in Gas1-/- limbs. Mouse trunk fragments containing forelimbs were isolated from E9.75-E11.5 control (con; B-D) and mutant (-/-; E-G) embryos. (A) Diagram of FGF10 injection in anterior tip domain of the mesenchyme in the right limb. The injected embryo fragments were cultured for 16 hours and then subjected to whole-mount ISH to assay for Fgf8 expression in the AER. (B,E) PBS-injected, (C,D,F,G) injected with recombinant human FGF10 protein: (C,F) 9.2 pg and (D,G) 230 pg. Note that in D and G, the color was developed for 20 minutes to reveal the upregulation of Fgf8 in the injected wild-type limb (open arrowhead in D). The others were developed for 90 minutes. (J) Mutant E9.75 forelimb injected with 560 pg of FGF8 did not show any rescue of Fgf8 expression in the AER. (H,I) Mutant E11.5 forelimbs were injected with PBS (H) or 230 pg of FGF10 (I). Fgf8 expression in the AER was rescued in the FGF10-injected samples (arrowheads in F,G,I). Owing to the angle of the limbs relative to the body at this stage, the image was taken in a ventral view with the anterior pointing down. The control E11.5 limbs retained Fgf8 expression in the AER after culture (not shown).

View larger version (33K): [in a new window]   Fig. 10. Proposed models for Gas1 action and Fgf10 regulation in the developing limb. At the early phase of limb formation, Gas1 is not required for Fgf10/Fgf8 expression in the proximal mesenchyme and the AER, respectively (left). In the wild type (wt), Gas1 is expressed in the anterior two-thirds of the mesenchyme, Fgf10 in a large contiguous domain of the limb mesenchyme composed of a proximal and a distal domain, and Fgf8 in the AER. In the Gas1 mutant, Fgf10 expression is lacking at the distal domain, Fgf8 is not maintained and the AER is thinner (bottom panel). Our FGF10 rescue injection data indicate that FGF10 at the distal mesenchyme is continuously required to maintain Fgf8 expression (black arrow). We propose that Fgf10 expression in the distal mesenchyme is either directly regulated by Gas1 and AER Fgf8, in parallel (red arrows; model a) or by Fgf8 in the AER through a pathway that requires Gas1 activity (converged red arrow; model b). We also propose that the proximal domain of Fgf10 expression can be maintained (purple arrow) by Fgf4, 9 and 17 in the posterior AER in the absence of Fgf8. Fgf8 may also contribute to this regulation normally. The color code for each gene expression domain is at the right bottom corner.