Developmental Biology 211, 133–143 (1999) Article ID dbio.1999.9290, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE FGF7 and FGF10 Directly Induce the Apical provided by Elsevier - Publisher Connector Ectodermal Ridge in Chick Embryos Sayuri Yonei-Tamura,*,† Tetsuya Endo,* Hiroshi Yajima,* Hideyo Ohuchi,‡ Hiroyuki Ide,* and Koji Tamura*,†,1 *Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan; †Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037-1099; and ‡Department of Genetic Biochemistry, Faculty of Pharmaceutical Sciences, Kyoto University Graduate School of Pharmaceutical Sciences, Kyoto 606-8501, Japan During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors. © 1999 Academic Press Key Words: apical ectodermal ridge (AER); FGF7; FGF10; limb morphogenesis. INTRODUCTION developing limb bud (Saunders, 1948; Saunders et al., 1976), while mesenchymal factor(s) are responsible for maintain- Tetrapod limbs evolved from paired fins (pectoral and ing the AER as limb outgrowth proceeds (Zwilling and pelvic fins) of fish and appear to inherit embryonic devel- Hansborough, 1956). opment from fins (Akimenko et al., 1995; Akimenko and These interactions between the AER and limb mesen- Ekker, 1995). For example, a common specialized ectoderm, chyme are fundamental to the pattern formation of the which is required for skeletogenesis of the appendage, limb bud. Some molecules involved in these epithelial– appears in the distal margin of the limb and fin primordia mesenchymal tissue interactions have been reported. (Hall, 1991). One such structure is in the tetrapod limb bud Fibroblast growth factor (FGF) family members FGF2, -4, which appears as a ridge-shaped thickening in the apical and -8 have been reported to be the best candidates for the epidermis known as the apical ectodermal ridge (AER). In AER-derived signal since their transcripts are expressed the early limb bud stages, the AER is induced by the lateral in the AER, thus replacing the role of the AER (Fallon et plate mesoderm of the limb field (Saunders and Reuss, 1974; al., 1994; Niswander et al., 1994; Laufer et al., 1994; Carrington and Fallon, 1984). Experiments using avian Mahmood et al., 1995; Vogel et al., 1996). Exogenous embryos have revealed that the AER promotes proliferation insulin and insulin-like growth factor I (IGF-I) maintain and directed outgrowth of subridge mesodermal cells of the the thickness and activity of the AER (Dealy and Kosher, 1995) and, therefore, are candidates for the AER mainte- 1 To whom correspondence should be addressed at Biologi- nance factor. Moreover, recent studies showed that cal Institute, Graduate School of Science, Tohoku Univer- FGF10 is expressed in the limb mesenchyme and sug- sity, Sendai 980-8578, Japan. Fax: (022)-217-6691. E-mail: gested a role of FGF8 and FGF10 in the reciprocal [email protected]. interaction between the AER and limb mesenchyme 0012-1606/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 133 134 Yonei-Tamura et al. (Ohuchi et al., 1997; Xu et al., 1998). On the other hand, Observation of Skeletal Pattern although it is known that applications of several FGFs (FGF1, -2, -4, -8, and -10) to the flank can induce an For skeletal pattern observation, embryos were incubated for 7 additional limb bud with an ectopic AER (Cohn et al., days after operation. Embryos were fixed in 10% formalin, stained 1995; Crossley et al., 1996; Vogel et al., 1996; Ohuchi et in 0.1% Alcian blue in 70% acid alcohol, dehydrated in ethanol, and then cleared in methyl salicylate. al., 1997), there is little evidence about the direct inducer of the AER derived from the presumptive limb bud mesenchyme. In the present study, in order to identify the direct Whole-Mount in Situ Hybridization and inducer of the AER, we investigated the role of several FGFs Immunofluorescent Staining in AER induction. Particularly, we focused on FGF7 and FGF10. FGF7 is known as a diffusible mesenchymal media- Whole-mount in situ hybridization was performed as described (Yonei et al., 1995, for chick embryos; Endo et al., 1997, for tor of epithelial–mesenchymal tissue interactions in several Xenopus embryos; Schulte-Merker et al., 1992, for zebrafish em- organs (Rubin et al., 1995; Post et al., 1996). FGF10, which bryos). Antisense RNA probes for chick Fgf8 (a kind gift from Dr. is expressed in the presumptive limb mesenchyme, is Sumihare Noji) and Msx1 and Msx2 (gifts from Dr. Tsutomu thought to be most similar to FGF7 in both amino acid Nohno) were described previously (Yonei et al., 1995; Hara et al., sequence and functions (Yamasaki et al., 1996; Ohuchi et 1997; Ohuchi et al., 1997). Probes for zebrafish Fgf8 and Xenopus al., 1997; Igarashi et al., 1998). Our results demonstrate that Msx2 were synthesized from plasmids containing an 800-bp frag- FGF7 induces the AER in the flank in a different manner ment of zebrafish Fgf8 (Furthauer et al., 1997) and a 447-bp from FGF1, -2, and -4 and that FGF7 and FGF10 can induce fragment of Xenopus Msx2 (Su et al., 1991). After in situ hybrid- the formation of an additional AER in the ectoderm of the ization, some chick embryos were processed for embedding in OCT m dorsal midline where there is no underlying cell of lateral compound (Miles) and sectioned (10 m). For observation of the mesoderm. FGF7 can activate the expression of Fgf8 in the neural crest cells, frozen sections were immunostained using HNK-1 monoclonal antibody (Becton–Dickinson). ectoderm isolated from the underlying mesoderm in vitro but FGF1, -2, and -4 cannot. Thus, it is likely that FGF7 and FGF10 induce the AER directly while AER induction by FGF1, -2, and -4 may be mediated by mesodermal cells. Our Culture of Flank Ectoderm data also suggest that there may be a correlation between the dorsal median AER induced by FGF7 and FGF10 and the The procedure for ectoderm culture is described in the legend to Fig. 2A. Flank ectoderm was cultured using the floating collagen dorsal median fin in amphibian and fish larva. gel culture method (Emerman et al., 1977). The flank ectoderm including the lateral plate/paraxial mesoderm was dissected from stage 16 chick embryos. The ectoderm was isolated from the mesoderm using 0.5% trypsin in Tyrode at 4°C. Culture dishes MATERIALS AND METHODS were coated with collagen gel according to the accompanying procedure of Cellmatrix I-A (Nitta Gelatin). Explants were placed on collagen gel in Dulbecco’s modified Eagle’s MEM containing Experimental Manipulation 10% FCS, 10 mg/ml insulin, and 5 mg/ml transferrin. The media level was lowered until a thin layer of the ectoderm covered the gel. Chick embryos were staged according to Hamburger and Ham- Two hours after incubation at 37°C in the presence of 5% CO2, ilton (1951). Stage 13/14 chick embryos were used as hosts. The collagen gel was allowed to float in fresh medium and then ectoderm at somite 20 level was cut and partially peeled to be cultured with 10 ng/ml of FGF1, -2, -4, or -7 for 22 h (24 h in total). separated from a mesodermal layer toward somite 23/24 level in the flank. Affi-gel heparin beads (Bio-Rad) soaked in 0.5 mg/ml of several FGFs as described below were inserted in the cavity and placed between the respective regions of the ectoderm and meso- RT-PCR Analysis derm. FGF1 (recombinant human, Boehringer-Mannheim), FGF2 (bovine,R&DSystem), FGF4 (recombinant human,R&D Total RNA was isolated from explants using an RNeasy total System), and FGF7 (recombinant human, Promega) were used for RNA kit (Qiagen). RT-PCR was performed with Fgf8 specific this experiment. This operation was performed with meticulous primers (forward primer, 59-GAT GTG CAC GCC AAG CTC ATC care to prevent making a scrape on the ectoderm around the bead. GTC GAG ACC-39; reverse primer, 59-GCT TCA TGA AGT GCA When grafting the presumptive limb bud mesoderm of stage 16 CCT CGC GTT GGT GCT-39) and bactin specific primers (forward chick embryos, the transplants were isolated using 0.5% trypsin in primer, 59-TCT GAC TGA CCG CGT TAC TC-39; reverse primer, Tyrode at 4°C and inserted in the back using the same procedure as 59-CCA TCA CAC CCT GAT GTC TG-39). These primer sets were bead application (Fig. 4A). For retroviral infection, RCASBP(A) based on the chick Fgf8 mRNA sequence (GenBank No.