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Sexually dimorphic breeding tubercles (BTs) are keratinized epidermal structures that form clusters on the dorsal surface of the anterior rays of zebrafish male pectoral fins. BTs appear during sexual maturation and are maintained through regular shedding and renewal of the keratinized surface. Following pectoral fin amputation, BT clusters regenerate after the initiation of revascularization, but concomitantly with a second wave of angiogenesis. This second wave of regeneration forms a web-like blood vessel network that penetrates the supportive epidermis of BTs. Upon analyzing the effects of sex steroids and their inhibitors, we show that androgens induce and estrogens inhibit BT cluster formation in intact and regenerating pectoral fins. Androgen-induced BT formation in females is accompanied by the formation of a male-like blood vessel network. Treatment of females with both androgens and an angiogenesis inhibitor results in the formation of undersized BT clusters when compared with females treated with androgens alone. Overall, the growth and regeneration of large BTs requires a hormonal stimulus and the presence of an additional blood vessel network that is naturally found in males.

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 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.