Melody Harper,Yinan Hu,Joan Donahue,Benjamin Acosta,Flora Dievenich Braes,Stacy Nguyen,Jenny Zeng,Julianna Barbaro,Hyungwoo Lee,Hoa Bui,Sarah K. McMenamin
Thyroid hormone regulates proximodistal patterning in fin rays
zebrafish fins,fin rays,regeneration,proximodistal patterning,thyroid hormone
Significance While pathways regulating overall fin size in zebrafish have come to light, the signals coordinating patterning of the fin rays along the proximodistal axis have remained less clear. We demonstrate that thyroid hormone (TH) regulates proximodistal patterning in the rays: TH is necessary and sufficient to induce distal identities during both development and regeneration. These effects are mediated by the nuclear hormone receptor Thrab, which represses distal features when TH is absent. We show that the processes regulating proximodistal patterning and fin length can be decoupled, with major morphological effects. Further, this role of TH in distalizing rays is shown to be conserved between multiple teleosts.
Thyroid hormone regulates proximodistal patterning in fin rays

While pathways regulating overall fin size in zebrafish have come to light, the signals coordinating patterning of the fin rays along the proximodistal axis have remained less clear. We demonstrate that thyroid hormone (TH) regulates proximodistal patterning in the rays: TH is necessary and sufficient to induce distal identities during both development and regeneration. These effects are mediated by the nuclear hormone receptor Thrab, which represses distal features when TH is absent. We show that the processes regulating proximodistal patterning and fin length can be decoupled, with major morphological effects. Further, this role of TH in distalizing rays is shown to be conserved between multiple teleosts.

How organisms generate complex shapes is a central question in biology. To produce a robust phenotype, cells in a developing organ must interpret positional information and execute growth and pattern morphogenesis in a regionally appropriate and highly coordinated manner (1). Along an extending axis, even subtle shifts in coordinated patterning processes relative to outgrowth can profoundly alter the ultimate shape, e.g., by altering the number of somites along the anteroposterior axis (2). Appendages such as limbs show specific proximodistal patterning, with different character states expressed at different locations along the axis. Such patterning shows incredible diversity between lineages, and relative shifts in proximodistal identity likely facilitated major evolutionary transitions (3). Developing tissues create patterns through morphogenetic processes that may be cued by regional gradients of morphogens, bioelectric or mechanical signals (reviewed in refs. 4 and 5). In addition to these local processes, global factors such as hormones can coordinate morphogenesis in disparate tissues (e.g., refs. 6–8); such systemic signals could theoretically be leveraged to establish or reinforce patterns during outgrowth of an axis. Zebrafish fins are a premier model for studying growth and regeneration, and can serve as informative models of proximodistal patterning. Fin length mutants have allowed great progress toward understanding the mechanisms that regulate growth of rays (9–11). In contrast, the signals underlying proximodistal patterning of the rays have remained largely elusive. Although more subtle than the polarity of tetrapod limbs, individual fin rays show distinct patterning along the axis: Ray segments taper and shorten distally, and periodically bifurcate into branches (Fig. 1A). Ray segments are added distally during fin outgrowth, in a manner resembling somitogenesis (12). Likewise, bifurcations are added terminally during development or regeneration, with most rays eventually forming one or more branches (e.g., SI Appendix, Fig. S1). Changes in proximodistal patterning are important in evolutionary transformations and adaptations. Notably, the fin-to-limb transition involved major changes in patterning and proportion along the proximodistal axis (3, 14, 15). Among teleosts, fin ray patterning varies considerably between species, from uniformly thick rays with no bifurcations (e.g., syngnathids, sculpins), to tapering rays with multiple bifurcations along each ray (guppies, killifish). These differences in proximodistal morphology contribute to the functional and biomechanical properties of the appendages (see ref. 16). The local mechanisms governing aspects of certain proximodistal features have been elucidated. Formation of bifurcations requires precise Shh signaling (17–19) as well as coordinated and opposing activities of osteoblasts and osteoclasts (20). Nonetheless, it remains unknown what positional identity cues direct bifurcation machinery to be deployed at correct locations along growing rays. Furthermore, while certain mechanisms governing overall segment length have been discovered (21, 22), pathways regulating the progressive distal shortening of segments remain entirely unknown (although they can be modeled computationally; ref. 23). Thyroid hormone (TH) is an essential regulator of cellular metabolism and homeostasis. This endocrine factor coordinates postembryonic developmental processes in diverse vertebrates, including zebrafish (6, 24–28). In zebrafish reared under hypothyroid (HypoTH) or hyperthyroid (HyperTH) conditions, we observed changes in the fin skeleton consistent with coordinated shifts along the proximodistal axis. We hypothesized that TH regulates aspects of proximodistal patterning, and that these processes operate independently from size-instructive signals controlling fin ray length.

We discovered that hallmarks of skeletal distalization are induced and coordinated by developmental TH titer (Fig. 1). Transgenic ablation of the thyroid follicles during larval development results in permanently HypoTH fish (24); fins of adults reared under these HypoTH conditions showed markedly proximalized fin rays (Fig. 1B and SI Appendix, Fig. S1B). Conversely, a congenitally HyperTH mutant (24) showed moderate distalization (Fig. 1C). Bifurcations are the most discrete indicators of proximodistal patterning, and in caudal fins of WT adult fish, the majority of internal rays form at least one bifurcation (peripheral rays never bifurcate). In the absence of TH (HypoTH conditions), far fewer bifurcations formed (Fig. 1D). To examine the patterning along the length of the axis, we focused on the 2nd dorsal ray. We found that bifurcations that did form in a HypoTH context were positioned considerably farther from the body, suggesting proximalization of the axis (Fig. 1E). The shifted position of the bifurcation was also reflected in the number of ray segments proximal to the bifurcation (SI Appendix, Fig. S2B) as well as the proportion of the total ray length at the bifurcation (SI Appendix, Fig. S2C). A mutant with reduced TH production (29) also showed fewer bifurcations that were shifted farther from the body (SI Appendix, Fig. S3). Despite the differing pattern of branching, HypoTH fish maintained overall similar rates of growth during development compared to WT, euthyroid siblings (SI Appendix, Fig. S1 B–D). Contrasting with the proximalized patterning of HypoTH fin rays, conditions of excess TH (HyperTH) caused bifurcations to develop somewhat closer to the body, suggesting distalization of the axis (Fig. 1 C and I and SI Appendix, Fig. S2 G–I). All of the paired and median fins showed corresponding shifts in the relative location of bifurcations (SI Appendix, Fig. S4). In WT control groups, sex did not appear to influence ray patterning (SI Appendix, Fig. S5). Fin rays are composed of segments that become progressively shorter and thinner along the axis. TH regulates the progressive shortening of rays: In the absence of the hormone, longer segments continued to be built farther from the body (Fig. 1 F–G and SI Appendix, Fig. S2 E and F). In contrast, HyperTH conditions caused segments to shorten precociously along the axis (Fig. 1K and SI Appendix, Fig. S2K), resulting in slightly shorter distal segments (Fig. 1J). Rays from HypoTH fish also showed proximalized trends in bone density (SI Appendix, Fig. S6); the dense distal segments in the HypoTH context are particularly striking since HypoTH conditions tend to decrease overall mineralization and bone density in other skeletal elements (26). We asked whether treatment with TH during development could rescue proximodistal patterning in HypoTH fish. Early treatments (5 to 30 or 30 to 60 days post fertilization, dpf) did not change patterning. However, treating HypoTH fish with exogenous TH during juvenile development (60 to 90 dpf) was sufficient to rescue the formation of bifurcations (SI Appendix, Fig. S7). In a WT background, continuous treatment with exogenous TH during development did not significantly alter bifurcations (SI Appendix, Fig. S8).

If TH alters axis identity, we hypothesized that in addition to the proximalized aspects of patterning, HypoTH fins would show proximalized patterns of gene expression. Examining transcriptomes from three proximodistal regions of uninjured fins, we found 233 genes expressed differentially along the proximodistal axis in a control WT background (Fig. 1L, SI Appendix, Fig. S9 A–C, and Dataset S2), showing strong correspondence with a previously published dataset (13). Of these proximally or distally enriched genes, the majority lost differential expression in a HypoTH context (128/233, 55%; Fig. 1L and SI Appendix, Fig. S9B). In particular, characteristically distal expression patterns were attenuated in the distal regions of fins from HypoTH backgrounds (Fig. 1L). Multidimensional scaling suggested that expression patterns were shifted proximally in a HypoTH context (Fig. 1M). Moreover, several transcripts known to characterize proximal or distal identities (13) showed proximalized patterns of expression in HypoTH conditions (SI Appendix, Fig. S9 D–G). Consistent with previous analyses (13), we did not detect a signal of TH-related genes being expressed in a proximodistal gradient in WT, although we did find that dio2 was expressed more strongly in the proximal regions of WT fins (SI Appendix, Fig. S9H and Dataset S2). Retinoic acid (RA) mediates axis patterning in tetrapods (e.g., ref. 30), and many RA-pathway genes are expressed in a proximodistal gradient in uninjured WT fins (13). Many genes associated with RA metabolism show gradients along the proximodistal axis, and we asked whether TH was required for this graded expression. Of the six RA-associated transcripts expressed in a proximodistal differential, only one showed altered expression in a HypoTH context (SI Appendix, Fig. S9 B–G and Dataset S2), suggesting that TH does not regulate distal identity by altering regional production of RA transcripts. Other genes associated with proximodistal identity in tetrapod limbs (e.g., meis, pbx, hox) did not show expression differences between proximal and distal regions in WT, and also did not show dependence on TH (Dataset S2). The relative activities of osteoblasts and osteoclasts may mediate the formation of bifurcations (20). We asked whether markers for these two skeletogenic cell types might be relatively altered in a HypoTH context, but none were differentially expressed between TH conditions above the significance threshold (SI Appendix, Fig. S9 I and J and Dataset S2). In all, these transcriptomic data suggest that TH is essential in establishing distal patterns of gene expression in mature, intact fins. We did not detect evidence that the hormone functions by regulating the expression of a specific molecular pathway or cell type (SI Appendix, Fig. S9).

Fin length mutants show dramatic changes in the relative lengths of fins, but the overall patterning of the rays is comparable to that in WT (11) (Fig. 2 and SI Appendix, Fig. S10). We predicted that altering TH in the context of a lengthened or shortened fin would alter ray patterning along the axis. Indeed, HypoTH conditions proximalized both lof and sof rays (Fig. 2 and SI Appendix, Fig. S10), comparable to the proximalization seen in fins of nonmutant HypoTH. This apparent lack of genetic interaction suggests that TH regulation of proximodistal patterning operates independently from size-regulating pathways.

Canonical TH signaling functions through dual-action nuclear TH receptors, which repress transcription when the hormonal ligand is absent and promote expression when the hormone is present (31). We hypothesized that TH functions through one or more of the three zebrafish TH receptors—Thraa, Thrab, and Thrb—to modulate fin ray patterning (27). Rays of euthyroid thrab mutants showed patterning comparable to that of WT siblings; in contrast, HypoTH thrab mutants showed a striking rescue relative to HypoTH nonmutants (Fig. 3 A–F and SI Appendix, Fig. S11). While HypoTH nonmutant siblings (and HypoTH thrab +/− heterozygotes, see SI Appendix, Fig. S11) rarely formed bifurcations, thrab knockout (−/−) permitted formation of bifurcations even in a HypoTH context (Fig. 3C). We infer that unliganded Thrab actively represses distalization. Nonetheless, the absence of Thrab did not completely rescue a WT morphology in the HypoTH background (Fig. 3 C–E and SI Appendix, Fig. S11), suggesting that Thrab-independent TH mechanisms also positively regulate distalization of the rays. Contrasting with the rescued patterning evident in HypoTH thrab mutants, HypoTH thraa and thrb mutants showed typical HypoTH phenotypes with no evidence of rescue (SI Appendix, Fig. S12). We found that thrab is expressed at the tips of rays during regeneration, including in both blastema and epidermis (Fig. 3 G and H), along with many other genes actively expressed at the growing edge (e.g., see refs. 32 and 33). We asked whether regional differences in thrab expression along the proximodistal axis might mediate distalization by modulating regional responses to circulating TH. However, the level of thrab expression remained comparable as different regions of the proximodistal axis regenerated (SI Appendix, Fig. S13 A and B). Thus, although the receptor is expressed during regeneration, regional differences in Thrab expression along the axis do not correlate with different stages of proximodistal morphogenesis.

We asked whether thrab expression domains might correspond to regions of high TH activity. A TH activity reporter (34) showed that TH is indeed active at the distal tips of both uninjured (SI Appendix, Fig. S13C) and regenerating rays (Fig. 3 I and J and SI Appendix, Figs. S13E and S14). TH activity was detectable in the presumptive blastema as well as in the epidermis (Fig. 3J and SI Appendix, Fig. S13F). TH also appeared to be active in the preosteoblast lineage and showed activity in regions where osteoblasts were present (Fig. 3J and SI Appendix, Fig. S13F). The TH activity reporter showed reduced activity in regenerating thrab mutants, suggesting that the hormone acts via Thrab during regeneration (SI Appendix, Fig. S13 G–I). We asked whether TH activity showed a gradient along the proximodistal axis or any shifts in activity coincident with bifurcation. However, TH activity showed no clear trends over the course of regeneration (Fig. 3K and SI Appendix, Fig. S14 A–C).

We tested whether patterning could be rescued in fins from HypoTH fish during regeneration. Indeed, treating HypoTH fish with exogenous TH throughout 3 wk of regeneration was sufficient to robustly rescue distalization (Fig. 4C and SI Appendix, Fig. S15). Positional information in the early blastema dictates regenerate length (35, 36), and we asked whether our rescue of distal morphology was due to effects of TH on the early blastema. To test this, we treated HypoTH fish with TH for 24 h during each of the first 3 dpa, but none of these treatments were sufficient to rescue distal patterning in regenerates (SI Appendix, Fig. S16). This led us to hypothesize that TH is acutely required to create distal morphology, and we tested this possibility by delaying TH treatments until later stages of regenerative outgrowth. Even when the onset of treatment was delayed to 7 or 14 dpa, exogenous TH was still sufficient to rescue the formation of bifurcations (Fig. 4 D and E and SI Appendix, Fig. S15). These data suggest that fin rays remain receptive to TH-induced distalization throughout the regenerative process.

Our rescue experiments demonstrated that previously proximalized HypoTH patterning can be overridden during regeneration by treatment with TH. We next asked whether normal distal patterning could be remembered and rebuilt during regeneration even in the absence of TH, and we tested this in two different ways. First, we treated WT fish with either of two TH-blocking drugs during regeneration; these indeed inhibited bifurcations from forming (SI Appendix, Fig. S17). Second, we used HypoTH fish that had been rescued with TH during development (as in SI Appendix, Fig. S7), then regenerated the fins in the absence of TH. Despite developing with a WT-like pattern, regeneration in the absence of additional TH caused fins to revert to a proximalized HypoTH pattern (SI Appendix, Fig. S18). These experiments demonstrate that proximodistal patterning is not remembered by tissues, but is generated in direct response to TH during outgrowth.

Since Shh signaling is essential for the establishment of bifurcations (17–19), and since thrab and TH activity are detectable in the epidermis where Shh is active, we hypothesized that the hormone might be required for Shh activity. We found that HypoTH regenerates maintained shha expression, although the overall expression level was somewhat decreased relative to WT (Fig. 5 A–C). Notably, shha domains failed to distinctly separate in the absence of TH (Fig. 5B″). This finding contrasts markedly with experimental treatments that can functionally inhibit or delay bifurcations, but nonetheless show clear separation of shha expression domains (17, 20, 37). We asked whether TH was required for the integrity of downstream Shh machinery and the migration of Shh-responsive cells. As fins regenerate, Shh/Smo signaling is essential for establishing bifurcations (17, 18), and as in ref. 17, we used a photoconvertable ptch2 reporter (39) to assess the dynamic activity of the pathway. Even in a HypoTH context, new ptch2 was continuously expressed during regeneration. Previously, Shh/Smo responsive cells are shown to be displaced to the distal tips of growing rays in WT regenerates (17, 18). This process was not dependent on TH, and previously ptch2-expressing cells showed displacement even in HypoTH (compare arrows, Fig. 5 D and E). In all, the Shh-dependent machinery underlying branch formation appears to be intact in regenerating fins of HypoTH fish; yet bifurcation processes are not initiated at the appropriate locations.

We asked whether a requirement for TH in ray patterning was limited to zebrafish. TH was required for distal patterning in a closely related Danio species (40): Transgenic thyroid ablation in Danio albolineatus resulted in proximalized fin rays as it did in D. rerio (Fig. 6 A and B and SI Appendix, Fig. S19 A–F). Medaka (Oryzias latipes) are separated from zebrafish by ~200 million years of evolution (41, 42), and even in this distant relative, pharmacologically blocking TH inhibited bifurcation during regeneration (Fig. 6 C and D and SI Appendix, Fig. S19 H–J).

Collectively, our results demonstrate that TH is both necessary and sufficient to induce distal patterning of fin rays during developmental and regenerative outgrowth. Numerous indicators of proximodistal identity showed coordinated responses to developmental TH titer: The location of bifurcations, ray segment morphology, bone density, and expression patterns along the axis are all influenced by TH. The requirement for TH to build bifurcations in an appropriate proximodistal location is shared across all the fins of the zebrafish as well as across multiple teleost species. Our findings suggest that the processes regulating size and processes regulating patterning are governed by distinct developmental modules that may be decoupled. Previous work established that growth of the rays is regulated by bioelectricity pathways (see ref. 43), and we have shown that even when fin length is disrupted, there remains a requirement for TH in patterning the rays (Fig. 2 and SI Appendix, Fig. S10). In principle, modulating proximodistal patterning can produce a spectrum of different ray phenotypes like those found in nature, from nonbranching, uniformly thick, proximalized rays (e.g., those of sculpin) to highly distalized, multibranched rays (e.g., guppy). These types of modular changes to patterning could underlie evolutionary diversity across fins and other appendages. Patterning shifts in the fins could be mediated by changes in local or global TH metabolism, or could be entirely independent of TH, acting on downstream or parallel mechanisms that regulate axis patterning. In addition to the location of bifurcations, our study considers the morphology of ray segments as a readout of proximodistal patterning. Certain mutations cause fins to develop with overall shorter segments (e.g., refs. 44 and 45) or overall lengthened segments along the entire length of the ray (e.g., refs. 9, 11, and 46). In contrast, fins from HypoTH and HyperTH backgrounds showed proximal segments comparable in length to those of WT siblings, but which exhibited shifts in the slope of segment length decrease (Fig. 1 G–K and SI Appendix, Fig. S2 E and K), resulting in distal segments with lengths significantly different than those in WT siblings (Fig. 1 F and J). In addition to the “flattened” slope of segment length in fins of HypoTH, we note that there is increased variability in segment length (Fig. 1G and SI Appendix, Fig. S2E), which suggests a role for TH in stabilizing placement of the segment joints. Notably, not every distal feature is dependent on TH. Leucophores still form at the peripheral, distal edges of HypoTH fins (SI Appendix, Fig. S13 C and D) and segment width tapering occurs regardless of TH titer (SI Appendix, Fig. S2 F and L). Despite enriched TH activity (SI Appendix, Figs. S13 and S14), the peripheral-most rays were never observed to produce bifurcations, even under HyperTH conditions or when Thrab was absent. Peripheral rays maintain unique expression of several genes (47), highlighting the uniqueness of these rays in the fin and potentially suggesting additional roles for TH in ray identity beyond proximodistal patterning. In the absence of TH, some distal features are eventually produced—the longest rays of HypoTH fish produce bifurcations and moderately shortened distal segments—but these processes are deployed in a delayed fashion and the ultimate fin structure is consequently highly proximalized. In the other extreme, there appears to be a limit to how distalized a fin can become by modulating TH levels: HyperTH conditions shift the proximodistal axis only slightly (Fig. 1 H–K). Indeed, HyperTH conditions did not change the total number of bifurcations in the fin (Fig. 1H), presumably because the maximum number of fin rays bifurcate even in a WT background. Although the extent of HyperTH-induced distalization is limited, it appears to be dose sensitive, as individuals heterozygous for the HyperTH mutation opallus typically showed intermediate phenotypes for aspects of proximodistal patterning (SI Appendix, Fig. S2 G–J). In all, it is likely that other TH-independent pathways buffer the proximodistal morphology of the rays, limiting the severity of the phenotypes in Hypo- and HyperTH contexts. The phenotype of a HypoTH fin can be rescued both during development (SI Appendix, Fig. S7) and regeneration (Fig. 4) by treatment with exogenous TH. Moreover, rays regenerating in a HypoTH context remain competent to produce distal features into late stages of outgrowth (Fig. 4). Treatment with TH at early stages of development (SI Appendix, Fig. S7) or during early periods of regeneration (SI Appendix, Fig. S16) produces no effects on the fin-patterning phenotype. Together, these findings suggest that the hormone acts on tissues acutely during outgrowth. During regeneration, TH is active in the epidermis and promotes high Shh pathway activity. Shh-active basal epidermal cells are required for branching morphogenesis (17, 18), and these cells still exhibit distal displacement in HypoTH fins as in WT (Fig. 5 D–G), suggesting that these components of bifurcation machinery are not dependent on TH. Nonetheless, the hormone appears to be required for the two Shh domains to separate (Fig. 5 A–C). Recent evidence suggests that bone resorption activity counteracts mineralization activity to produce branches (20). Our RNAseq data did not suggest that TH affects expression levels of genes associated with the overall abundance of osteoblasts or osteoclasts in intact, uninjured fins (SI Appendix, Fig. S9 I and J). However, these data from intact tissue may differ from expression during regeneration; osteoclast/osteoblast ratios may be altered after injury. Moreover, it is possible that TH might regulate relative activities of these cell populations along the proximodistal axis to regulate where bifurcations form along the growing axis. Indeed, preosteoblasts appear to show TH activity during regeneration (Fig. 3J), which may alter their relative activity. Fins developed under HypoTH conditions showed proximalized gene expression profiles (Fig. 1 L and M), suggesting that proximodistal identity is legitimately shifted in a TH-disrupted context. Our data do not identify a single “smoking gun” pathway or cell type through which TH likely acts to impart distalization. Instead, the hormone appears to progressively orchestrate numerous suites of genes along the axis, including pathways regulating skeletal morphogenesis, extracellular organization, as well as gas transport and adhesion (SI Appendix, Fig. S9C). We note, however, that the shifts in gene expression could be either causes or effects of the phenotypic distalization observed, and that expression patterns during outgrowth may differ from these profiles in the intact organ. The repressive activities of unliganded Thrab and the permissive context of TH appear to work in conjunction to progressively induce increasingly distal features during fin outgrowth. Unliganded TH receptors serve to repress developmental processes in other contexts, inhibiting amphibian metamorphosis (48) and gating morphogenesis of zebrafish skin and pigment (27, 49). Removing the repressive unliganded receptor rescues patterning in a HypoTH background (Fig. 3). HypoTH thrab mutant fin rays are still somewhat proximalized relative to those of WT and euthyroid mutant siblings, suggesting that TH-related mechanisms independent of Thrab also help to promote distalization. Furthermore, the euthyroid thrab mutants show patterning comparable to that of WT (despite showing less TH activity, see SI Appendix, Fig. S13 G–I), suggesting that Thrab is more active as an inhibitor than as an enhancer of distal morphology. Unlike a limb, the fin grows indeterminately from the distalmost edge throughout adult life (50). TH is active at this growing edge, even in an uninjured adult fin (SI Appendix, Fig. S13C), potentially reflecting the metabolic demands of continuous isometric growth. During regeneration, functional TH activity at the leading edge remains relatively constant as outgrowth progresses (Fig. 3K), suggesting that the hormone does not function in the “source-sink” manner of certain limb identity pathways. Indeed, distalization appears to be a progressive process along the continuously growing axis of the fin. The proximodistal patterning of the rays is actively orchestrated by TH during outgrowth. During regeneration, it is the continuous presence of TH—rather than memory of previous pattern—that dictates ray morphology along the axis. In parallel, active bioelectric signaling during outgrowth—rather than positional memory of size—determines fin length (51). We propose a model in which TH relieves the repression of Thrab to deploy bifurcation morphogenesis in the correct proximodistal contexts by coordinating suites of pathways that result in progressive distalization during outgrowth (Fig. 6E). In all, modulating the TH pathway changes the ultimate proximodistality of the appendicular skeleton. Thus, the TH axis and its downstream targets are attractive candidates for fin ray diversification and changes to axis identity in other contexts. Our study demonstrates that ray patterning changes may be largely decoupled from size, providing insight into the regulatory logic underlying fin morphogenesis during development and regeneration.

HypoTH and their WT controls were both Tg(tg:nVenus-v2a-nfnB) (24). HyperTH zebrafish were opallusb1071, which constitutively overproduce TH (24). The other transgenic strains used were: Tg(6xTRE-bglob1:eGFP) (34); Tg(−2.7shha:GFP) (38); TgBAC(ptch2:Kaede) (39); and Tg(runx2:mCherry) (Shannon Fisher Lab, Boston University). The other mutant strains used were: duoxsa9892 (29); longfindt2/kcnh2a (10, 52, 53); shortfindj7e2/cnx43 (9); and thrabvp31rc1, thraavp33rc1, and thrbvp34rc1 (27). Other species used were pink pearl danios (Danio albolineatus) transgenic for tg:nVenus-v2a-nfnB (24) and commercially obtained Japanese medaka (Oryzias latipes).

Anesthetized (using MS-222, ~0.02% in system water) or cleared and stained (54) samples were imaged on an Olympus SZX16 stereoscope using an Olympus DP74 camera, or on an Olympus IX83 inverted microscope using a Hamamatsu ORCA Flash 4.0 camera. Images were processed in CellSense (Olympus) or on FIJI and adjusted for contrast, brightness, and color balance; corresponding adjustments were made for images of both control and experimental fish. For paired images shown in figures, adult fish were size-matched as closely as possible and always within 2 mm SL (55).

Analyses were performed in RStudio (build 372). Morphological data were typically analyzed with either Welch two-sample t test or ANOVA followed by Tukey’s honest significant differences (using a 95% family-wise confidence level). In graphs showing a single comparison, significance is indicated as follows: *P < 0.05, **P < 0.001, ***P < 0.0001. In graphs showing multiple comparisons, significance is indicated on graphs by letter groups, with statistically indistinguishable groups sharing the same letter (P value threshold < 0.05). Additional methodology is explained in SI Appendix; full statistical results are shown in Dataset S1.

Sibling WT and HypoTH TgBAC(ptch2:Kaede) individuals were imaged at 5 dpa. Kaede was photoconverted by ~90s DAPI exposure at 4× magnification. Images were acquired pre- and post-DAPI exposure to confirm complete photoconversion. Fins were reimaged 24 h later (at 6 dpa) to assess new Kaede expression as in ref. 17.

All regeneration experiments were performed on adult zebrafish 18 to 23 mm SL. Caudal fins were amputated from anesthetized fish under a stereoscope at the 4th to 5th ray segment using a razor blade.

For in situ hybridization (following ref. 56), fin tissues were collected at different stages during regeneration, fixed in 4% paraformaldehyde in PBS, then stored in methanol. Probes for thrab were prepared using the primers F: agtgcacgtcctaaagagcaaac and R: acgtcacacgtcctgatcctc. Riboprobes were fractionated to an average size of ~600 nt. To ensure probe penetrance (57), a subset of specimens were embedded and cryosectioned, and in situ hybridization was subsequently performed on sectioned tissues.

Fin tissues were collected during regeneration, fixed in 4% paraformaldehyde in PBS, embedded in agarose/sucrose solution, reembedded in Tissue Plus Optimal Cutting Temperature (OCT) compound (Fisher), and frozen. Blocks were cryosectioned into 16 μM slices, dried overnight on slides, and then immunostained with anti-GFP (Sigma Aldrich) and anti-mCherry (Novus Biologicals) followed by secondary antibodies (Jackson ImmunoResearch).

A size for a region of interest was determined for each experiment, such that the smallest domain in the image series would be captured by the region. Using ImageJ, the region of interest was placed over the terminal portion of the ray to capture the mean fluorescence intensity.

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