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[Preprint]. 2024 Jul 19:2024.07.16.603744.
doi: 10.1101/2024.07.16.603744.

Adult caudal fin shape is imprinted in the embryonic fin fold

Eric Surette  1 , Joan Donahue  1 , Stephanie Robinson  1 , Deirdre McKenna  1 , Crisvely Soto Martinez  1 , Brendan Fitzgerald  1 , Rolf O Karlstrom  2 , Nicolas Cumplido  1 , Sarah K McMenamin  1
Affiliations

Adult caudal fin shape is imprinted in the embryonic fin fold

Eric Surette et al. bioRxiv. .

Abstract

Appendage shape is formed during development (and re-formed during regeneration) according to spatial and temporal cues that orchestrate local cellular morphogenesis. The caudal fin is the primary appendage used for propulsion in most fish species, and exhibits a range of distinct morphologies adapted for different swimming strategies, however the molecular mechanisms responsible for generating these diverse shapes remain mostly unknown. In zebrafish, caudal fins display a forked shape, with longer supportive bony rays at the periphery and shortest rays at the center. Here, we show that a premature, transient pulse of sonic hedgehog a (shha) overexpression during late embryonic development results in excess proliferation and growth of the central rays, causing the adult caudal fin to grow into a triangular, truncate shape. Both global and regional ectopic shha overexpression are sufficient to alter fin shape, and forked shape may be rescued by subsequent treatment with an antagonist of the canonical Shh pathway. The induced truncate fins show a decreased fin ray number and fail to form the hypural diastema that normally separates the dorsal and ventral fin lobes. While forked fins regenerate their original forked morphology, truncate fins regenerate truncate, suggesting that positional memory of the fin rays can be permanently altered by a transient treatment during embryogenesis. Ray finned fish have evolved a wide spectrum of caudal fin morphologies, ranging from truncate to forked, and the current work offers insights into the developmental mechanisms that may underlie this shape diversity.

Keywords: Caudal fin; positional information; proliferation; shape; sonic hedgehog.

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Figures

Fig. 1:
Fig. 1:. Pulse of premature shh during early fin fold development disrupts adult caudal fin shape.
(A-B) Caudal fins of control zebrafish and (C-D) transgenic zebrafish subjected to transient shh overexpression 2 dpf (shh pulse). (B and D) are cleared and stained caudal fins from juvenile zebrafish. Dashed outlines indicate the overall shape of the fins. Arrow indicates the location of the hypural diastema separating the dorsal from ventral lobes in B; asterisk indicates the absence of the diastema in (D). (E) The inheritance of the hsp70l:shha-eGFP transgene and the activation of the promoter by heat shock are both necessary in order to induce the truncate fin phenotype. Significance determined by ANOVA followed by Tukey’s post hoc test. An embryonic shh pulse (F) increases the number and variance of principal fin rays and (G) causes a loss of the hypural diastema. Significance determined using Welch’s two-sample T-tests. (H-J) Treatments with the Smoothened inhibitor BMS-833923 after shh pulse can partially rescue both (I) forked fin shape and (J) horizontal stripes of pigmentation. Significance determined by ANOVA followed by Tukey’s post hoc test. Scale bars, 1 mm.
Fig. 2:
Fig. 2:. Transient embryonic shh pulse disrupts caudal fin development in a local and dose-dependent manner.
(A) shh pulse results in truncate fin development when zebrafish are heat shock induced on 2 or 3 dpf. Significance determined by ANOVA followed by Tukey’s post hoc test. (B) Overexpression of shh during hours following heat shock. Significance determined using Welch’s two-sample T-tests, and the correlation between readout and time following heat shock determined by linear-mixed effects model. (C-E) Locally induced shha pulse is sufficient to induce truncate phenotype. (C-C’) Embryo subjected to local posterior heat shock at 2 dpf did not show GFP fluorescence and grew into an adult with a forked fin. (D-D’) Local posterior heat shock induced GFP in transgenic embryo (brackets), which grew into an adult with a truncate fin.Scale bars, 500µm. (E) Local posterior heat shocks in transgenic embryos are capable of inducing truncate fin shape. Inducing local shh pulse in the anterior of the embryo produces no change in fin shape. Significance determined by ANOVA followed by Tukey’s post hoc test. (F) Fish sorted by relative brightness of GFP expression 1 day after whole-body HS (4 dpf) show different caudal fin shapes as adults. Shown below the graph are representative images of individuals in each brightness category. Significance determined by ANOVA followed by Tukey’s post hoc test. Scale bar, 1 mm. (G) Quantified copy number of GFP transgene amplified from genomic DNA correlates with caudal fin shape. Significance between mean Rq and caudal fin shape is determined by linear-mixed effects model.
Fig. 3:
Fig. 3:. Divergent fin shape is accompanied by disruptions in skeletal growth and presaged by differences in regional cell proliferation.
Development of the caudal skeleton in (A) control and (B) shh pulsed larvae. sp7 reporter-expressing osteoblasts shown in yellow; sox10 reporter-expressing chondrocytes shown in magenta. Arrow indicates the location of the hypural diastema separating the dorsal from ventral lobes in A; asterisk indicates the absence of the diastema in B in both the ossified rays and the cartilaginous hypural complex (*). Bar, 500 uM. A’ and B’ show higher magnification images of boxed areas. (C) In these early stages of fin development (~6.0 SL), the length of the longest rays is less in shh-pulsed larvae than in control siblings. Significance determined using Welch’s two-sample T-tests. (D) control (E) and shh pulsed sibling caudal fin growth from 14 to 36 dpf. Dashed lines indicate the distal edge and overall shape of the fins. Scale bars, 500 µm. (F) The emergence of WT forked fin shape (gray lines) is the result of a lower growth rate in central rays (solid lines) relative to peripheral rays (dashed lines). Following embryonic shh pulse (green lines), central rays exhibit increased growth rates throughout development while peripheral rays retain a WT growth trajectory. (G-H) Dual Fucci reporter showing non-proliferating cells in red and cells in G2, S or M phase in cyan in the dorsal lobe of caudal fin folds of (G) control siblings and (H) larvae that experienced shh pulse. Bar, 200µm. (I) In control developing forked fins, proliferation is relatively lower in central regions, while shh pulse causes increased proliferation in central regions of the developing truncate fin Significance is determined by ANOVA followed by Tukey’s post hoc test. (J) Across the entire organ, proliferation becomes more uniform following shh pulse (closer to 1.0) compared to WT. Significance determined by Welch two-sample T-test. Difference between central / peripheral proliferation comparing WT to shh pulse is still significant when outlier in shh pulse group is removed.
Fig. 4:
Fig. 4:. Altered memory of the adult caudal fin.
(A-B) Control forked caudal fins (A) restore a forked shape 30 days after amputation. (B) Truncate fins restore a truncate shape after amputation. Bar, 1 mm. (C) Quantification showing the fin shape of each individual before and after regeneration. Significance between factors determined via a linear mixed-effects model.
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