Adult caudal fin shape is imprinted in the embryonic fin fold

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

Posted on: 28 August 2024

Preprint posted on 19 July 2024

Shhaping the caudal fin: @McmenaminLab describes how shha overexpression induces a novel fin shape in zebrafish

Selected by Isabella Cisneros

Categories: cell biology, developmental biology, molecular biology

Background

The caudal fin, commonly known as the “tail” of a fish, is an important evolutionary innovation found within the ray-finned fishes. Caudal fins can vary widely in their shape due to the swimming ecologies that influence them [1]. However, two different shape classes have emerged from this variation: the truncate shape, where central and peripheral rays (the outer ventral and dorsal rays) have the same length, and the forked fin shape, where peripheral rays are longer than central rays.

Many studies have focused on deciphering how the caudal fin is patterned. From these, we have been able to define a few key players and mechanisms. When it comes to how the caudal fin is shaped, Hox factors, ion channels and gap junctions, thyroid hormone, and osteoclast activity all play a role [2,3,4]. However, while all of these factors influence different aspects of fin ray growth–such as ray length, identity, growth, and location–the pathways that regulate the shape of the caudal fin remain unknown.

To get at this question, the authors of this preprint focused on sonic hedgehog (Shh), which is known to regulate limb growth and skeletal identity. Here, the authors show how overexpression of sonic hedgehog a (shha) in the embryonic fin fold can alter overall fin shape and positional memory, further refining our understanding of developmental patterning in the fin and its consequences for processes like regeneration.

Main Findings

Transient overexpression of shha during early embryonic development alters overall fin shape

In order to probe the role of shha during fin development, the authors used a transgenic zebrafish line (hsp70l:shha-EGFP) that allowed them to induce transient overexpression of shha using heat shock. They began their experiments by inducing a global pulse at 2 days post fertilization and observing the structure and growth of the larval fin. The authors noticed that, compared to control siblings, zebrafish treated with the shha pulse had significantly longer central rays. This elongation led the fin to adopt a truncate, or flat, shape instead of the usual forked shape. Additionally, the shha pulse also caused the development of pigmented vertical arches in the fin as opposed to the horizontal stripes that usually characterize the forked fin. To ensure that the phenotypes they observed had been mediated through the Shh signaling pathway, the authors inhibited Smoothened, a Shh effector, for a period of 24 or 48 hours following a global shha pulse. These experiments resulted in a partial rescue of the forked fin shape, indicating that disruption of Shh signaling is responsible for the truncate phenotype.

Phenotypic outcomes of shha overexpression depend on developmental stage, fin position, and dose

Next, the authors wanted to determine the developmental stage at which a shha pulse can induce a truncate fin phenotype. To do so, they heat-shocked zebrafish embryos and larvae at multiple timepoints. They found that embryos heat-shocked at 2 or 3 days post fertilization develop truncate fins, but not those at later stages. The authors also wanted to see if a more localized shha pulse could still induce a truncate caudal fin. To test this, they used local heat shock techniques to activate the transgene at two positions along the zebrafish body axis. Here, they found that only activation of Shh at the posterior end of the tail was able to induce the truncate fin phenotype. Next, the authors wanted to test if the amount of shha transgenic activation affected the extent of the truncate fin phenotype. Using GFP brightness and genomic GFP quantity, they were able to predict the severity of the phenotype in relation to the amount, indicating a dose-dependent effect.

Overexpression of shha causes differences in skeletal growth rates and regional cell proliferation in the larval fin

Having characterized the effects of the shha pulses in greater detail, the authors decided to zoom in further into the truncate fin phenotype by analyzing fin ray ossification and hypural chondrogenesis during development. Compared to controls, the shha-pulsed fish lacked a diastema and experienced delayed fin ray growth. To assess whether the truncate fin phenotype is a result of delayed growth, the authors quantified the growth rate between central and peripheral rays in the fin. They found that peripheral rays in shha-pulsed caudal fins grow at the same rate as control siblings. However, this was not the case for central rays, which grow at a 35% faster rate than controls.

To see if these changes in growth rates corresponded to changes in regional cell proliferation, the authors used a cell-proliferation reporter transgenic line to quantify growth in peripheral and central regions of the fin. Starting at a standard length (a measure of body size) of about 5.9mm, peripheral cell proliferation rates increase compared to those of center regions in control fish. In shha-pulsed fish, however, proliferation in central cell populations increased, leading to the development of a truncate shape in the fin.

Treatment with shha alters positional memory of the fin in adulthood

Because fin regeneration relies on developmental programs of fin patterning, the authors wanted to see if a shha pulse could also alter memory of fin shape during regeneration. Indeed, this was what they observed: shha-pulsed fish regenerate truncate fins, not forked fins. Additional experiments using longfin and shortfin zebrafish mutants, as well as hypothyroid zebrafish, also resulted in the regeneration of a truncate fin following an embryonic shha pulse. Taking these results together, the authors suggest that, though elements like length, patterning, and shape appear to be regulated by independent signaling pathways, these pathways can be decoupled to generate phenotypic variation in fins.

Why I Highlighted This Preprint

As someone who studies caudal fin regeneration, it is always exciting to see work focusing on developmental patterning of the fin–the basis for our work! I find it fascinating that overexpression of sonic hedgehog a can drive such a drastic phenotype. In revealing this new factor’s involvement, this study raises multiple questions related to the cellular and molecular mechanisms driving fin shape patterning during development. It also reveals just how important–and how persistent–embryonic positional memory is for regeneration during adult stages, something to keep in mind for those of us who study this phenomenon.

Questions For The Authors

What do you make of the pigmentation defect created by the shha pulse? Is it possible that shha has co-opted a pigmentation patterning role in the fin?
Do you have any plans to test sonic hedgehog b overexpression in additional heat shock experiments? Is there any possibility that shha and shhb have evolutionarily diverged in their patterning roles?

References

Giammona, F. Form and Function of the Caudal Fin Throughout the Phylogeny of Fishes, Integrative and Comparative Biology, Volume 61, Issue 2, August 2021, Pages 550–572, https://doi.org/10.1093/icb/icab127
Sakaguchi et al., Medaka unextended-fin mutants suggest a role for Hoxb8a in cell migration and osteoblast differentiation during appendage formation. Dev Biol. 2006 May 15;293(2):426–38. https://doi.org/10.1016/j.ydbio.2006.02.017
Stewart et al., longfin causes cis-ectopic expression of the kcnh2a ether-a-go-go K+ channel to autonomously prolong fin outgrowth. Dev Camb Engl. 2021 Jun 1;148(11):dev199384. https://doi.org/10.1242/dev.199384
Harper et al., Thyroid hormone regulates proximodistal patterning in fin rays. Proc Natl Acad Sci. 2023 May 23;120(21):e2219770120. https://doi.org/10.1073/pnas.2219770120
Wang et al., Genetic reprogramming of positional memory in a regenerating appendage. Curr Biol CB. 2019 Dec 16;29(24):4193–4207.e4. https://doi.org/10.1016/j.cub.2019.10.038

Tags: caudal fin, fin development, fin patterning, sonic hedgehog, zebrafish

doi: https://doi.org/10.1242/prelights.38240

Read preprint

(No Ratings Yet)

Author's response

Sarah McMenamin shared

1. What do you make of the pigmentation defect created by the shha pulse? Is it possible that shha has co-opted a pigmentation patterning role in the fin?

We actually think that the pigment phenotype is a ’side effect’ of the shifts in developmental timing caused by the shha pulse. Pigment cells end up in the wrong place at the wrong time, and go along for the ride when the notochord flexes upward to reorient the caudal fin tissues posteriorly. These ectopic melanophores set up a horizontal pattern that we believe subsequent stripes follow. (It is also possible that the shh is directly stimulating the pigment cells or directing the pattern, but I think that’s actually less likely).

2. Do you have any plans to test sonic hedgehog b overexpression in additional heat shock experiments? Is there any possibility that shha and shhb have evolutionarily diverged in their patterning roles?

We’ve shown that a pulse of hyperphysiological levels of shha can cause this truncate fin phenotype, but importantly, this doesn’t necessarily imply that shha is essential for normal patterning during these same stages. Indeed, when we transiently block Shh signaling during wild-type larva development, it doesn’t cause much of a phenotype. So to address your second question: it is entirely possible that early overexpression of shhb (or even of other pathways!) might result in similar sorts of phenotypic disruptions by altering the timing of different imprinting processes during fin fold formation.

Have your say Cancel reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Also in the cell biology category:

The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during Drosophila melanogaster oogenesis

Samantha N. Milano, Livia V. Bayer, Julie J. Ko, et al.

Selected by 04 September 2024

Jonathan Townson

Discussion

Significantly reduced, but balanced, rates of mitochondrial fission and fusion are sufficient to maintain the integrity of yeast mitochondrial DNA

Brett T. Wisniewski, Laura L. Lackner

Selected by 30 August 2024

Leeba Ann Chacko

Discussion

Triglyceride metabolism controls inflammation and APOE4-associated disease states in microglia

Roxan A. Stephenson, Kory R. Johnson, Linling Cheng, et al.

Selected by 22 August 2024

Gustavo Stelzer, Marcus Oliveira

Also in the developmental biology category:

Specialized signaling centers direct cell fate and spatial organization in a limb organoid model

Evangelia Skoufa, Jixing Zhong, Oliver Kahre, et al.

Selected by 05 September 2024

Ryan Harrison

Discussion