Axis reset is rate limiting for onset of whole-body regenerative abilities during planarian development

Background

The capacity to undergo whole-body regeneration, or the ability to regenerate any lost body part, varies widely across the metazoans. While some organisms are only able to regenerate certain organs or tissues, such as fish and amphibians, others can regenerate entire bodies from just a few small fragments, such as planarians [1-3]. However, how these abilities are first established during development–and how this establishment compares to other species–has not been fully characterized.

While previous work has focused on comparisons of regenerative ability between developmental and adult stages of multiple organisms, the molecular mechanisms underlying this process remain unclear [4-6]. To better characterize the mechanisms that underlie the acquisition of regenerative ability during development, the authors decided to study a close relative of the landmark regeneration model Schmidtea mediterranea (Smed) known as Schmidtea polychroa(Spol), which is capable of producing directly developing embryos. In this study, the authors identify the onset of regenerative ability in developing Spol and the factors that influence this onset, particularly those that play a role in limiting it.

Main Findings

Spol embryos acquire regenerative abilities throughout development and vary in their ability to regenerate anterior vs. posterior structures

The authors began their study by assessing the regenerative capabilities of Spol adults. They found that, regardless of the position or plane of the cut, all fragments successfully regenerated, indicating that Spol possesses the same robust regenerative abilities as Smed. Having characterized adult regeneration, the authors next set out to assess regeneration during different stages of development in Spol. They began by performing amputation assays during multiple embryonic stages (S7, S7.5, and S8) as well as juvenile stages (J0, J1, and J2), cutting immediately anterior to the developing pharynx. Anterior and posterior fragments were left to regenerate and were assessed at 14 days post cut (dpc) for successful regeneration of different structures and swimming behavior. They found that anterior fragments at all stages were able to successfully regenerate, exhibiting normal locomotion and negative phototaxis. The posterior fragments, however, showed more variation in their regenerative abilities. Fragments cut at S7 did not regenerate anterior tissues, S7.5 and S8 fragments had an intermediate regenerative phenotype reminiscent of Smed RNAi animals, and fragments cut at J1 and J2 were able to successfully regenerate all anterior structures and organs.

Tissue composition, axial position, and developmental stage are important for the acquisition of head regeneration competency in posterior fragments

Seeing these differences between anterior and posterior fragment regeneration, the authors wanted to understand what factors may be underlying these regenerative variabilities. After ruling out fragment size, they decided to assess whether the position of the cut plays a role in head regeneration competence. The authors performed three different axial cuts–one posterior to the eyes (P1), one between the eyes and pharynx (P2), and one immediately anterior to the pharynx (P3)–for S7, S7.5, S8, and J2 stage animals. When they assessed the fragments at 14 dpc, they saw that anterior fragments were still capable of successful regeneration regardless of stage. However, depending on the cut, posterior fragments had different regenerative outcomes: P1 posterior fragments regenerated at all stages, P2 posterior fragments had lower frequency of regeneration at S7 and S7.5 stages, and P3 fragments failed to regenerate at S7 and only showed limited regeneration abilities at stages S7.5 and S8. However, animals that underwent sagittal cuts regenerated completely at all stages, indicating that anterior patterning cues are needed for the onset of head regeneration capabilities.

The authors were curious to see if these posterior fragments could acquire regenerative capabilities following maturation. To test this, they cut S7 stage animals at the P3 position and subjected them to a second cut at the J2 stage. Unfortunately, this re-amputation did not result in expanded regenerative capabilities, as the S7 fragments did not reliably regenerate their heads successfully. Interestingly, the authors noticed that S7 fragments that were re-amputated through the pharynx underwent whole-body regeneration at high frequencies. To test if pharynx presence influences head regenerative competence, the authors removed the pharynx via a dorsal incision and repeated the experiment. They observed that, while the pharynx does regenerate, neither its removal nor the dorsal incision alone resulted in improved head regenerative capabilities. Together, these findings led the authors to conclude that acquisition of head regenerative competence depends on tissue composition, axial position, and developmental stage of the posterior fragment.

Piwi-1+ cells are required for successful regeneration and axis reset acts as a rate-limiting factor for the establishment of whole-body regenerative competence

Having characterized the acquisition of regenerative abilities in developing Spol, the authors next wanted to explore the role of piwi-1+ cells in regeneration. These cells, known as neoblasts, have previously been characterized as adult pluripotent stem cells and are necessary for regeneration in Smed [7]. After they successfully developed a gamma irradiation protocol for the elimination of these cells, they decided to perform amputation assays on treated S7 embryos to see whether piwi-1+ cells are required for regeneration. Treated fragments were not able to be scored on regenerative outcomes due to early death following irradiation, indicating a complete regenerative block. For the authors, this suggested that piwi-1+ cells are necessary for regeneration. Interestingly, however, they observed that piwi-1+ cells were present in S7 posterior fragments of unirradiated Spol. Additionally, they detected mitotic cells in S7 and J2 fragments at 48 hours post cut. These results led the authors to conclude that while piwi-1+ cells are needed for successful regeneration, they are not sufficient for the induction of head regeneration competency.

To conclude their study, the authors wanted to understand if posterior fragments were successfully resetting their anteroposterior (AP) axis during regeneration. They assessed expression of anterior pole markers such as notum, follistatin, and foxD in the blastema post-cut in S7 and J2 posterior fragments. Here, they observed that while J2 posterior fragments expressed these markers in the blastema within the first 3 days dpc, S7 posterior fragments did not, even up to 14 dpc. They also assayed these stages for expression of sFRP-1, a head margin marker. Again, the S7 posterior fragments did not express this marker, meaning that they did not adopt anterior identity. To see if Wntinhibition–a necessary precursor to head regeneration in Smed–could elicit head regeneration in Spol, the authors performed RNAi knockdown of β-catenin-1 in S7 posterior fragments. They found that 97% of these fragments were able to successfully regenerate their heads, including the relevant organs, and expressed markers of anterior identity. Taking these results together, the authors concluded that the ability to reset the main body axis after amputation is a rate-limiting factor for the successful establishment of whole-body regeneration.

Why I highlight this preprint

I was very excited to see this preprint when it came out–in part because of its fascinating findings, but also because a previous labmate of mine, Clare Booth, spearheaded the work. This study marks an important step towards understanding an aspect of regeneration that is commonly overlooked: its developmental onset. While it is important to understand the extent and limitations of processes like whole-body regeneration, it is also crucial to consider how establishment of regenerative abilities can affect competency throughout different life stages. Though there is still much to learn when it comes to regeneration, this preprint represents a pioneering step towards understanding this phenomenon in its entirety.

Questions for the authors

1. In the discussion, you mention a diversity of regeneration phenotypes exhibited by planarians. With that in mind, what do you think is the functional relevance of partitioning regenerative ability along the AP axis during development as opposed to other forms of regeneration?
2. Do you have plans to expand upon this study using other model systems? What do you think the results of such a comparison can tell us about regeneration as a whole?

References

1. Gemberling, M., Bailey, T.J., Hyde, D.R., and Poss, K.D. (2013). The zebrafish as a model for complex tissue regeneration. Trends Genet. 29, 611–620. doi:10.1016/j.tig.2013.07.003
2. Bölük, A., Yavuz, M., Demircan, T. (2022). Axolotl: A resourceful vertebrate model for regeneration and beyond. Developmental Dynamics, 251, 1914-1933. https://doi.org/10.1002/dvdy.520
3. Ivankovic, M., Haneckova, R., Thommen, A., Grohme, M.A., Vila-Farré, M., Werner, S., and Rink, J.C. (2019). Model systems for regeneration: planarians. Development, 146. doi:10.1242/dev.167684
4. Henry, L.-A., & Hart, M. (2005). Regeneration from injury and resource allocation in sponges and corals – a review. Int. Rev. Hydrobiol. 90, 125–158. doi:10.1002/iroh.200410759
5. Vickery, M.C., Vickery, M.S., Amsler, C.D., and McClintock, J.B. (2001). Regeneration in echinoderm larvae. Microsc. Res. Tech. 55, 464–473. doi:10.1002/jemt.1191
6. Boyd, A.A., & Seaver, E.C. (2023). Investigating the developmental onset of regenerative potential in the annelid Capitella teleta. Invertebr. Biol. doi:10.1111/ivb.12411
7. Reddien, P.W. (2018). The cellular and molecular basis for planarian regeneration. Cell, 175, 327-345. 10.1016/j.cell.2018.09.021

Tags: development, neoblasts, planarian, regeneration

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