Mechanism of pectoral fin outgrowth in zebrafish development
2010). Loss of fish actinotrichia proteins and the fin-to-limb transition. Nature 466, 234-237. We apologise to the authors and readers for this mistake. SUMMARY Fins and limbs, which are considered to be homologous paired vertebrate appendages, have obvious morphological differences that arise during development. One major difference in their development is that the AER (apical ectodermal ridge), which organizes fin/limb development, transitions into a different, elongated organizing structure
... rganizing structure in the fin bud, the AF (apical fold). Although the role of AER in limb development has been clarified in many studies, little is known about the role of AF in fin development. Here, we investigated AF-driven morphogenesis in the pectoral fin of zebrafish. After the AER-AF transition at ~36 hours post-fertilization, the AF was identifiable distal to the circumferential blood vessel of the fin bud. Moreover, the AF was divisible into two regions: the proximal AF (pAF) and the distal AF (dAF). Removing the AF caused the AER and a new AF to re-form. Interestingly, repeatedly removing the AF led to excessive elongation of the fin mesenchyme, suggesting that prolonged exposure to AER signals results in elongation of mesenchyme region for endoskeleton. Removal of the dAF affected outgrowth of the pAF region, suggesting that dAF signals act on the pAF. We also found that the elongation of the AF was caused by morphological changes in ectodermal cells. Our results suggest that the timing of the AER-AF transition mediates the differences between fins and limbs, and that the acquisition of a mechanism to maintain the AER was a crucial evolutionary step in the development of tetrapod limbs. DEVELOPMENT 2917 RESEARCH ARTICLE Pectoral fin outgrowth mechanism endoskeletal and fin ray development that support the clock model. We also found that changes in ectodermal cell shape are important for AF outgrowth at later stages of development. MATERIALS AND METHODS Fish stock, maintenance, staging and fin observation Adult zebrafish (Danio rerio) were maintained at 27°C. Embryos obtained by natural crossing were staged by a standard method (Kimmel et al., 1995) , and were raised at 28.5°C until the appropriate stages. Excised pectoral fins and fin buds were put on glass slides filled with Tyrode's solution under coverslips, and analyzed under a BX51 microscope, a DP72 microscope with e-tiling systems (Olympus) and TCS-SP5 confocal microscope (Leica). Immunochemistry and in situ hybridization Embryos were fixed in 4% paraformaldehyde (PFA) in PBS for 10 hours at 4°C, then either dehydrated for preparing paraffin-embedded sections or put into gelatin-embedded solution [gelatin (Sigma, G7041):30% sucrose:DDW3:2:1] for preparing gelatin-frozen sections, which is a more delicate and sensitive technique than other sectioning methods (Fagotto and Gumbiner, 1994; Suzuki et al.