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Dev Dyn
2001 Jan 01;2201:40-8. doi: 10.1002/1097-0177(2000)9999:9999<::AID-DVDY1085>3.0.CO;2-8.
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Expression patterns of Fgf-8 during development and limb regeneration of the axolotl.
Han MJ
,
An JY
,
Kim WS
.
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Fgf-8 is one of the key signaling molecules implicated in the initiation, outgrowth, and patterning of vertebrate limbs. However, it is not clear whether FGF-8 plays similar role in development and regeneration of urodele limbs. We isolated a Fgf-8 cDNA from the Mexican axolotl (Ambystoma mexicanum) through the screening of an embryo cDNA library. The cloned 1.26-kb cDNA contained an open reading frame encoding 212 amino acid residues with 84%, 86%, and 80% amino acid identities to those of Xenopus, chick, and mouse, respectively. By using the above clone as a probe, we examined the temporal and spatial expression patterns of Fgf-8 in developing embryos and in regenerating larval limbs. In developing embryos, Fgf-8 was expressed in the neural fold, midbrain-hindbrain junction, tail and limb buds, pharyngeal clefts, and primordia of maxilla and mandible. In the developing axolotl limb, Fgf-8 began to be expressed in the prospective forelimb region at pre-limb-bud and limb bud stages. Interestingly, strong expression was detected in the mesenchymal tissue of the limb bud before digit forming stages. In the regenerating limb, Fgf-8 expression was noted in the basal layer of the apical epithelial cap (AEC) and the underlying thin layer of mesenchymal tissue during blastema formation stages. These data suggest that Fgf-8 is involved in the organogenesis of various craniofacial structures, the initiation and outgrowth of limb development, and the blastema formation and outgrowth of regenerating limbs. In the developing limb of axolotl, unlike in Xenopus or in amniotes such as chick and mouse, the Fgf-8 expression domain was localized mainly in the mesenchyme rather than epidermis. The unique expression pattern of Fgf-8 in axolotl suggests that the regulatory mechanism of Fgf-8 expression is different between urodeles and other higher species. The expression of Fgf-8 in the deep layer of the AEC and the thin layer of underlying mesenchymal tissue in the regenerating limbs support the previous notion that the amphibian AEC is a functional equivalent of the AER in amniotes.
Fig. 2. The expression profiles of Fgf-8 in whole embryos. All embryos are lateral view except A (dorsal view). A: Stage 15 (neurula stage). Fgf-8 transcript is expressed in the prospective midbrain-hindbrain junction (isthmus; white arrowheads), anterior neural fold (arrowhead), and pos- terior blastopore (asterisk). B: Stage 24 (early tail bud stage). Fgf-8 is expressed lightly in the vaguely recognizable tail bud (asterisk), gill swelling (arrow), facial primodia (arrowhead) and isthmus of brain (white arrowhead). C: Stage 32 (middle tail bud stage). D: Stage 35 (late tail bud stage). Fgf-8 is expressed in gill swelling (arrow), tail bud (asterisks), mandibular arch (arrowheads), and isthmus (white arrowhead). Scale bars ﰈ 500 ﰁm
Fig. 3. The expression profiles of Fgf-8 in the developing limbs of axolotl larvae. A: At 1 day after hatching, when limb buds start to form, Fgf-8 is expressed in the epidermis and mesenchyme of the budding limb (arrowhead). Gills were removed for better viewing of the limb bud. B: Longitudinal section of forelimb bud at 1 day after hatching. The Fgf-8 transcript is detected in the mesenchyme as well as epidermis. Anterior is to the top and distal is to the right in the image. C,D: At 2 days (C) and at 4 days (D) after hatching, a strong expression is detected in the mesenchymal and subepidermal tissue of forelimb bud (arrowheads). The expression at 4 days after hatching is more intense than that at 2 days after hatching, and the expression domain is somewhat anteriorly polarized. E,F: Sections of limb bud at 4 days after hatching in the anterior-posterior plane (E) and dorsal-ventral plane (F). Note the ante- riorly polarized expression of Fgf-8 in the anterior-posterior axis (E), but not as much as pronounced in the dorsal-ventral axis (F). Anterior (in E) and dorsal (in F) are to the top and distal is to the right in both images. G: This expression pattern is continued until 8 days after hatching, but the level of expression is clearly reduced (arrowhead). H,I: At 11 days (H) and 14 days (I) after hatching, when the digits are formed, only the trace level (H) or background level (I) of expression can be detected. C; ventral views except in E and F., Scale bars 200 m in A; 20 m in B; 100 m in C (except E and F in which bars are 50 m)
Fig. 4. The expression profiles of Fgf-8 in the regenerating forelimbs of axolotl larvae. Forelimbs were amputated at the level of distal stylopo- dium (white lines). A: Two days after amputation (dedifferentiation stage). No expression of Fgf-8 is detected until this stage. B: Four days after amputation (early bud stage). Very low level of signal is detected in the blastema. C: Six days after amputation. D: Eight days after amputation. Medium bud stage. Fgf-8 signal is detected mainly in the blastema (white arrowheads). Fgf-8 transcripts are localized in the basal layer of the apical ectodermal cap and underlying thin layer of mesenchymal tissue (refer Fig. 5). E: Ten days after amputation (late bud stage). Weak signal of Fgf-8 is seen in the blastema (white arrowhead). F: Twelve days after amputation (palette stage). Only background level of expression is de- tected. Scale bars 1 mm.
Fig. 5. Histologic examination of Fgf-8 expression in sectioned re- generating tissues. Tissues were prepared by sectioning the 8-days after amputation samples. A,B: Anteroposteriorly sectioned plane. C,D: Dor- soventrally sectioned plane. B and D are magnified images of boxes in A and C, respectively. a: anterior, p: posterior, d: dorsal, v: ventral. The Fgf-8 expression shows somewhat polarized pattern in the anterior- posterior plane. However, in the dorsal-ventral plane, polarized expres- sion pattern is not shown. H, humerus; m, mesenchyme; AEC, apical epithelial cap. Scale bars 100 m.
