Ectopic head regeneration after nervous system ablation in a sea anemone

Introduction

When you think of regeneration, you may think of an axolotl rebuilding a limb or of a flatworm regenerating an entire half of its body. But, have you ever stopped to think about how these organisms know just how much tissue they’ve lost and what structures they need to regrow? This concept, also known as positional memory, remains to be completely elucidated in many regenerative organisms and raises additional questions regarding what cells or tissues harbor these signals and, in turn, if other cells or tissues are also involved with regulating position-specific expression. 

So far, studies in planarians and acoels have characterized the role of subepidermal muscle cells as regulators of positional memory [1,2]. More specifically, these cells express position control genes that help set up axial polarity during regeneration, leading to proper regeneration of the corresponding structures. Within the cnidarians, on the other hand, Hydra has been shown to depend on its epithelial cells for positional information during regeneration [3,4]. 

In this preprint, the authors set out to characterize another popular cnidarian model–the sea starlet Nematostella vectensis–and how it regenerates in the absence of its nervous system. In doing so, they characterize a fascinating phenomenon that may point towards the nervous system as the harbor of positional memory in the starlet.

Main Findings

Inducing successful nervous system ablation using a conditional ablation tool & regeneration of the nervous system

To assess whether the nervous system plays a role in Nematostella regeneration, the authors first generated a double transgenic animal that would allow them to both label the neurons and ablate them using a nitroreductase construct. Using the elav1 promoter, they observed expression in large parts of the epidermal and gastrodermal nervous system. Following this confirmation, the authors ablated the elav1+ neurons of six-week-old juvenile polyps by exposing the animals to Nifurpirinol for 72 hours. Using confocal and electron microscopy, the authors saw an absence of elav1+ neurons and loss of neurite bundles along muscles of the body wall, evidence of successful ablation. Additionally, they observed new neurons four days after ablation, with neurons reaching pre-ablation levels around 25 days post-ablation, indicating successful regeneration of the nervous system. 

Lastly, to confirm that the knock-out was specific to elav1+ neurons, the authors crossed their double transgenic animals to a reporter line labeling a non-overlapping population of sensory cells as well as one labeling the retractor muscle. Following confirmation that these cell populations were unaffected by elav1+ nitroreductase ablation, the authors determined that they had successfully induced nervous system ablation in Nematostella.

Ablation of the nervous system induces an ectopic head phenotype in head fragments of regenerating Nematostella

Having induced ablation of the nervous system and observed successful regeneration, the authors next wanted to understand what role, if any, the nervous system plays during this process. To do so, they ablated the neurons, washed out the Nifurpirinol, and cut the polyps in half to assess their regeneration. Interestingly, they found that, while all “foot” fragments were able to regenerate their corresponding head, all of the head fragments regenerated a second head instead of a foot. The second head persisted for months after the regenerative event as well, eventually separating into individual polyps via transverse fission.

To test whether the regeneration of a second head was tied to the ablation of the nervous system, the authors re-bisected the double-headed polyps at different times following the initial cut. They found that regeneration of a second head decreased as the time between the two cuts increased, meaning that ectopic head regeneration preceded the regeneration of elav1+ neurons. In order to rule out that apoptosis induced by the nitroreductase construct was responsible for ectopic head formation, the authors used UV irradiation to induce unspecific apoptosis in juvenile polyps. Afterwards, they bisected the polyps and observed regeneration. In all cases, they did not identify any instances of ectopic head formation, indicating that apoptosis by itself is not responsible for this phenomenon.

Wnt signaling is involved with ectopic head formation in ablated animals & mimics expression dynamics seen in non-ablated foot fragments

Previous work on positional signaling in Nematostella has shown that Wnt signaling controls head formation, and that overactivation of Wnt results in ectopic head formation. To see whether Wnt was being upregulated during the regeneration of ectopic heads in their nervous-system ablated animals, the authors isolated tips of regenerating head and foot fragments from both ablated and non-ablated animals and performed qPCR to assess expression of select genes. The authors observed that Wnt4 expression in head fragments of ablated animals showed elevated expression at 0 hours post amputation (hpa) and further increased expression from 6 to 24 hpa, similar to the dynamics of non-ablated foot fragments regenerating a head. Additionally, when they assessed the expression of an aboral master gene, otxC, they saw that expression was no longer upregulated by 72 hpa in ablated head fragments. Taken together, the authors determined that non-ablated foot fragments and ablated head fragments both appear to use an overlapping set of Wnt signaling genes.

Why I Chose To Highlight This Preprint

Regeneration is a phenomenon with a lot of moving parts. An organism attempting to regenerate a limb or organ needs to undergo a wide array of events, beginning with understanding what was lost, to re-initiation of developmental programs and/or deployment of novel regenerative programs, to transduction of local and long-range signals responsible for coordinating regeneration. In order to understand the big picture of how these events are occurring and how they influence each other, it is important to characterize and understand them in isolation first. The question of positional memory is a fascinating one that is still actively under investigation in multiple regenerative organisms. This preprint–in addition to describing a rare case of “faulty” regeneration in response to the loss of the nervous system–marks an exciting step towards delineating how existing cell types and tissues can inform positional memory and patterning in a regenerative organism. 

Questions for the Authors

1. Have you attempted to inhibit Wnt signaling in the ablated head fragments to rescue the foot regeneration phenotype and prevent ectopic head formation? 
2. In the discussion, you mention the possibility that directional neural activity may be tied to Wnt signaling. Have you tried to visualize or assay whether Wnt activity is tied to neural-driven behaviors, such as peristaltic constriction?
3. Removal of interstitial stem cells in Hydra eventually leads to an absence of neurons, but does not cause any issues with polarity or positional memory during regeneration. Given that Hydra are part of the hydrozoans and Nematostella are part of the anthozoans, what may be the evolutionary implications of this divergence in mechanism of positional information?

References

[1] Raz, A.A., Srivastava, M., Salvamoser, R. et al. (2017). Acoel regeneration mechanisms indicate an ancient role for muscle in regenerative patterning. Nat Commun 8, 1260. https://doi.org/10.1038/s41467-017-01148-5

[2] Witchley, J.N., Mayer,  M., Wagner, D.E., Owen, J.H., and Reddien, P.W. (2013). Muscle cells provide instructions for planarian regeneration. Cell Rep. 4, 633–641.        10.1016/j.celrep.2013.07.022

[3] Marcum, B.A., and Campbell, R.D. (1978). Development of Hydra lacking nerve and interstitial cells. J Cell Sci 29, 17–33. 10.1242/jcs.29.1.17     

[4] Sugiyama, T., and Fujisawa, T. (1978). Genetic analysis of developmental mechanisms in Hydra. II. Isolation and characterization of an interstitial cell-deficient strain. J Cell Sci 29, 35–52. 10.1242/jcs.29.1.35  

Tags: cnidarian, nematostella, nervous system, regeneration, wnt signaling

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