Expression of fibroblast growth factors 4, 8, and 10 in limbs, flanks, and blastemas of Ambystoma

Abstract

Members of the fibroblast growth factor (FGF) family of molecules are critical to limb outgrowth. Here, we examine the expression of Fgfs in three types of limbs-embryonic (developing), mature (differentiated), and regenerating-as well as in the surrounding non-limb tissues in the Mexican axolotl, Ambystoma mexicanum. We have previously cloned partial cDNAs of Fgf4, 8, and 10 from the axolotl (Christensen et al., 2001); the complete Fgf10 cDNA sequence is presented here. Axolotl Fgf10 showed deduced amino acid sequence identity with all other vertebrate Fgf10 coding sequences of >62%, and also included conserved 5 and 3 untranslated regions in nucleotide sequence comparisons. Semiquantitative reverse transcriptase-polymerase chain reaction showed that fibroblast growth factors are differentially expressed in axolotl limbs. Only Fgf8 and 10 were highly expressed during axolotl limb development, although Fgf4, 8, and 10 are all highly expressed during limb development of other vertebrates. Fgf4 expression, however, was highly expressed in the differentiated salamander limb, whereas expression levels of Fgf8 and 10 decreased. Expression levels of Fgf8 and 10 then increased during limb regeneration, whereas Fgf4 expression was completely absent. In addition, axolotl limb regeneration contrasted to limb development of other vertebrates in that Fgf8 did not seem to be as highly expressed in the distal epithelium; rather, its highest expression was found in the blastema mesenchyme. Finally, we investigated the expression of these Fgfs in non-limb tissues. The Fgfs were clearly expressed in developing flank tissue and then severely downregulated in mature flank tissue. Differential Fgf expression levels in the limb and shoulder (limb field) versus in the flank (nonlimb field) suggest that FGFs may be instrumental during limb field specification as well as instrumental in maintaining the salamander limb in a state of preparation for regeneration.

Global analysis of gene expression inXenopushindlimbs during stage‐dependent complete and incomplete regeneration

“…FGF8 and FGF4 are also expressed in the intermediate mesoderm, where they are involved in regulating limb bud initiation and outgrowth (Boulet et al, 2004). FGF8 is expressed in axolotl limb blastemas (Christensen et al, 2002) and in Xenopus blastemas at the regenerationcomplete stage as well in pseudoblastemas at the regeneration-incomplete stage (Endo et al, 2000). Consistent with the published data, FGF8 was expressed in both regeneration-complete and -incomplete 1dPA and 5dPA blastemas.…”

Mechanisms of urodele limb regeneration

“…Many growth factors are glycoproteins, several of which are expressed by the AEC and stimulate blastema cell proliferation in vitro and in vivo. Fgf1, Fgf2, Fgf8, and the anterior gradient protein (AG) are expressed by the AEC in vivo (Christensen, Weinstein, & Tassava, 2001, 2002; Han, An, & Kim, 2001; Kumar & Brockes, 2007). Blastema cells express the bek (FGFR2) receptor for Fgfs (Poulin & Chiu, 1995; Poulin, Patrie, Botelho, Tassava, & Chiu, 1993) and the AG receptor Prod1 (Kumar & Brockes, 2007).…”

Looking proximally and distally: 100 years of limb regeneration and beyond

“…Factors other than AGP that promote blastema cell proliferation in vitro have been detected in the wound epidermis of the regenerating limb, including Fgf-1, 2, and 8 (Chew and Cameron, 1983;Boilly et al, 1991;Zenjari et al, 1997;Han et al, 2001;Christensen et al, 2001Christensen et al, , 2002Dungan et al, 2002;Giampaoli et al, 2003). Likewise, nerves produce other mitogens including transferrin, Fgf-2, Ggf-2 (neuregulin), and substance P (Munaim and Mescher, 1986;Mescher and Kiffmeyer, 1992;Mescher et al, 1997;Globus and Alles, 1990;Anand et al, 1987;Mullen et al, 1996;Wang et al, 2000).…”