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Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)