A genetic and microscopy toolkit for manipulating and monitoring regeneration in Macrostomum lignano

R Nelson Hall; Hongquan Li; Chew Chai; Sidney Vermeulen; Robin R Bigasin; Eun Sun Song; Souradeep R Sarkar; Jesse Gibson; Manu Prakash; Andrew Z Fire; Bo Wang

doi:10.1016/j.celrep.2024.114892

. Author manuscript; available in PMC: 2025 Sep 16.

Published in final edited form as: Cell Rep. 2024 Oct 19;43(11):114892. doi: 10.1016/j.celrep.2024.114892

A genetic and microscopy toolkit for manipulating and monitoring regeneration in Macrostomum lignano

R Nelson Hall ^1,^*, Hongquan Li ², Chew Chai ¹, Sidney Vermeulen ³, Robin R Bigasin ¹, Eun Sun Song ⁴, Souradeep R Sarkar ¹, Jesse Gibson ¹, Manu Prakash ¹, Andrew Z Fire ^3,⁵, Bo Wang ^1,^6,^*

PMCID: PMC12435804 NIHMSID: NIHMS2038918 PMID: 39427313

SUMMARY

Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of tissue regeneration in the flatworm Macrostomum lignano, including a high-throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate genetic cellular ablation and reveal the limited regenerative capacity of neurons and their essential role during wound healing, contrasting muscle cells’ rapid regeneration after ablation. Finally, we build an open-source tracking microscope to continuously image freely moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection is highly robust and proceeds independently of regeneration.

In brief

Hall et al. present an advanced toolkit for manipulating and monitoring regeneration in the flatworm Macrostomum lignano, enabling tissue-specific labeling, ablation, and long-term tracking. These methods reveal critical insights into neural repair and regeneration, opening avenues for regeneration research and the functional characterization of marine meiofauna.

Graphical Abstract

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INTRODUCTION

In response to injury, regenerative animals initiate a cascade of dynamic processes, including signaling, apoptosis, proliferation, differentiation, and patterning, to restore lost tissues.^1–3 While most studies infer dynamics based on snapshots in time, live imaging can allow for continuous observation of these dynamic processes.^4–6 However, imaging the entire course of regeneration has been challenging for three major reasons. First, very few systems offer both optical transparency and minimal autofluorescence required for fluorescence imaging. Second, except for a few genetically tractable systems,^7–10 most regenerative organisms lack tissue-specific transgenic labeling necessary for imaging cells in vivo. Finally, restraining or paralyzing animals for prolonged imaging over days, the timescale relevant for observing regeneration, can negatively affect the organism’s physiology and change the course of regeneration. In this study, we address these challenges by combining the favorable physiological and genetic features of the flatworm Macrostomum lignano with advanced microscopy techniques to enable sensitive and long-term imaging of regeneration and illustrate the mechanistic insights that may be gained through such studies.

The interstitial, marine flatworm M. lignano, studied for diverse biological processes¹¹ including sexual selection,^12–14 bioadhesion,¹⁵ genome evolution,^16–18 and host-microbiome interactions,¹⁹ is capable of regenerating all tissues posterior to the pharynx but not anterior structures, presenting a unique opportunity to compare the molecular and cellular basis of regenerative and non-regenerative outcomes.^20,21 In addition, M. lignano is conducive to live imaging thanks to its small body size, optical transparency, minimal autofluorescence, and robust physiology.

In contrast to other commonly studied flatworm models such as planarians, which have limited transgenic tools,²² M. lignano has the ability to integrate exogenous DNAs into its genome, with transgenesis^23–25 further facilitated by the animal’s short egg-to-egg generation time (3 weeks at 20°C) and abundant, injectable zygotes.²⁶ However, the repertoire of transgenic tools in M. lignano is still limited, and microscopy tools needed for imaging organisms in their native physiological state throughout regeneration have been lacking. Here, we integrate three key technologies to facilitate the use of M. lignano as a model system for regenerative biology research.

First, we established a modular library of genetic parts to enable the rapid assembly of complex expression cassettes. This high-throughput cloning is coupled with a refined injection procedure allowing a single person to comfortably inject ~200 fertilized eggs within a 2-h session, routinely producing 1–2 germline-transmitting transformants per session. Using this pipeline, we have generated various tissue-specific reporter lines to reveal key steps during the regeneration of complex organs.

Second, we demonstrate targeted cell ablation by expressing in specific tissues nitroreductase (NTR2.0), a genetically encoded, inducible toxin engineered to minimize bystander effects of toxins released by dying cells into the local environment.²⁷ To circumvent ablation-induced autofluorescence that obscures ablation outcomes, we built a multi-modal epifluorescence/luminescence microscope to detect remaining cells and observed a lack of neural regeneration following ablation. Furthermore, neural-ablated animals failed to heal their wounds, revealing the critical role played by the nervous system in the organism’s regenerative ability.

Third, we designed and built an epifluorescence tracking microscope to image free-moving animals for up to a week, allowing us to track posterior regeneration and identify milestones in neural regeneration with high temporal and spatial resolution. A particularly striking observation is that the rate of nerve cord extension depends on the initial length of the remaining nerve cord after amputation.

Finally, combining all these techniques, we investigated the function of β-catenin, a central regulator of the Wnt signaling pathway, in M. lignano regeneration. We found that β-catenin RNAi inhibited body extension and regeneration of posterior structures after injury, without significantly affecting stem cell proliferation. Nevertheless, nerve cord reconnection proceeded normally, suggesting that it is separated from other regenerative processes. Altogether, the toolkit we created helps to establish M. lignano as a powerful platform for live imaging studies to reveal dynamic cellular processes during tissue regeneration.

RESULTS

A modular cloning protocol enables rapid generation of tissue-specific reporter lines

To streamline the cloning process of complex transgene cassettes and facilitate resource sharing, we built upon the 3G Assembly pipeline²⁸ and implemented a single-day protocol to generate multi-transcriptional unit (TU) plasmids (Figure 1A). This method breaks down transgenes into “parts”—promoters, genes, and terminators—and hierarchically assembles them through simple reactions while minimizing cloning scar sites. We introduced a backbone into which all parts can be cloned, thereby reducing the reliance on PCR for generating part fragments. This allows for long-term storage of parts as glycerol stocks and simplifies part sharing and reuse. We have built a parts library (Table S1, and Data S1) and expect to continue expanding it.

We created a series of transgenic lines carrying increasingly complex expression cassettes. First, we generated lines ubiquitously expressing green nanolantern (GeNL), a fusion of mNeonGreen and nanoluciferase (Nluc),²⁹ under the control of either the enolase (Figure 1B) or eukaryotic elongation factor 1α (Eef1α) promoter. Notably, the pEnolase:GeNL:tEnolase line exhibited more uniform expression, especially in the epidermis (Figures 1B, 1C, S1A, and S1B), allowing us to visualize various anatomical features including epidermal cilia, seminal vesicles, stylet, testes, muscles, and intestine (Figure 1C and Video S1).

Next, we created a line co-expressing GeNL and NTR2.0 in neurons, driven by the promoter of a prohormone convertase 2 (PC2) homolog (Figure 1D), which is broadly accessible across all neural cell types in M. lignano³⁰ (Figure S1C). Consistently, the pPC2:GeNL-P2A-NTR2.0:tPC2 line showed strong transgene expression throughout the nervous system. NTR was incorporated for targeted chemical ablation of the labeled cells, which is discussed below.

Finally, we produced lines with transgenes expressed in multiple distinct tissues. We used the APOB and MHY6 promoters to co-express GeNL and NTR2.0 in intestinal and muscle cells, respectively²³ (Figures S1C, S2A, and S2B), with an additional expression cassette, pEef1α:mScarlet:tEef1α, as a co-transfection marker. This marker was crucial in determining the locations of GeNL⁺ cells in various organs as described below. Overall, these results demonstrate that our approach can quickly generate lines carrying complex transgenes.

Tissue-specific labeling resolves detailed anatomy

These tissue-specific reporter lines allowed us to characterize the fine anatomy of various tissues in live animals using confocal microscopy. In the PC2 reporter line, the cephalic ganglia was clearly labeled above the photoreceptors, projecting a battery of ciliated sensory neurons toward the anterior (Figure 2A). The pharynx was encircled by a highly enervated ring (Figure 2A and Video S1). The two photoreceptors were connected by visual axons (Figure 2B). Along the body, a pair of major ventral nerve cords (VNCs) and several minor nerve cords were situated medially, which often projected horizontally to connect in between (Figure 2C). The tail was highly innervated, with VNCs looping around the tail base. Along the exterior of the tail was an array of neurons, possibly integrating sensory information to regulate the adhesive organs (Figure 2D), as they both connected to the VNC and extended to the edge of the tail (Figure 2E). Finally, we discovered unique arrangements of neurons around the stylet opening (Figure 2F) and the antrum (Figure 2G), the opening from which eggs are deposited. Intriguingly, similar groupings of neurons around the vulva regulate copulatory behaviors and egg laying in C. elegans.³¹ Notably, many of these neural structures were not visible in previous studies of the nervous system using immunofluorescence.^21,32–34

Figure 2. — (A) Confocal MIP of the nervous system in the anterior. Arrowheads (from top to bottom): anterior sensory projections, neuropil, and nerve ring around the pharynx.

(B) A close-up of the two photoreceptor cells and the visual axons.

(C) The major ventral nerve cord (VNC, left) and minor nerve cords (middle and right).

(D) An overview of the nervous system in the tail showing VNCs looping around the tail base.

(E) Neurons posterior to the VNC loop each sending a projection into the VNC and another outward.

(F) A circular arrangement of neurons around the opening of the stylet.

(G) Dense nerve fibers around the opening of the antrum.

(H) Tiled gut cells with highly vacuolated cytoplasm.

(I) Large pAPOB::GeNL⁺ cells (green) enclosing other cells (magenta) present in the anterior of the animal. Arrowheads: enclosed cells.

(J) Ovaries (magenta) are wrapped by pAPOB::GeNL⁺ cell bodies (green) on the exterior, which extend cytoplasmic processes around individual oocytes. (J′)A magnified view.

(K) A pAPOB::GeNL⁺ cell (green) sits on the exterior of the testes (magenta). (K′) A magnified view of this cell, which extends a long process down the length of the testes.

(L) Crosshatched circular and diagonal muscles (green) with intestinal cells beneath (magenta).

(M) An overview of the muscular structure in the tail showing the male copulatory apparatus (green). Arrowhead: the stylet opening.

(N) The male copulatory apparatus, with the false seminal vesicle (fsv), seminal vesicle (sv), and stylet (st) wrapped by muscles (green). Abundant prostate gland cells (magenta) surround the seminal vesicles and stylet.

(O) Individual muscle cell bodies hang like beads from circular muscle fibers (green) around the gut (magenta). Arrowheads: gut muscle fibers.

(P) Body-wall muscles and gut muscles (green) sandwich the ovaries (magenta). Dashed line: gut boundary. Arrows trace the two layers of muscles.

(Q) The antrum is surrounded by concentric muscle rings intersected by a second set of perpendicular radial muscle cells (green). Arrows trace the radial and perpendicular muscle fibers.

(R) Schematics showing the regions imaged in (A)–(Q) (dashed boxes).

Scale bars: 50 μm (A, D), 20 μm (B, C, G, H, L–O, Q), and 10 μm (E, F, I–K, P). All animals were imaged live and anesthetized with 7.14% MgCl₂. See also Figures S1 and S2; Videos S1 and S2.

In the APOB line, the gut architecture was revealed as a dense tiling of highly vacuolated cells. While both APOB and Eef1α promoters drove expression in the gut, the ratio of their expression varied between cells (Figure 2H), consistent with our single-cell data³⁰ suggesting that eef1α is highly expressed in one of the subpopulations of intestinal cells (Figure S1C). In the head, large pAPOB::GeNL⁺ cells extended numerous cytoplasmic processes, often encasing neighboring cells (Figure 2I). Surprisingly, pAPOB::GeNL⁺ cells were present within the ovaries, with their cell bodies located at the periphery and cytoplasmic processes surrounding oocytes (Figures 2J and 2J′). We also observed pAPOB::GeNL⁺ cells straddling the exterior of the testes (Figures 2K and 2K′). These observations are consistent with the single-cell analysis that revealed a population of apob⁺ cathepsin⁺ cells³⁰ (Figure S1C), suggesting that these previously undescribed extraintestinal cells could play phagocytic roles patrolling through various organs.

The MHY6 line showed distinct layers of circular and diagonal body-wall muscles beneath the epidermis (Figures 2L and 2M).³⁵ Additionally, the seminal vesicle, false seminal vesicle, and stylet were all wrapped in muscular rings, which likely control sperm expulsion and stylet movement during copulation (Figure 2N). Nearby, pEef1α:mScarlet expression uncovered clusters of presumptive prostate gland cells,³³ extending into the stylet (Figures 2N and S2C). Within the trunk, gut muscles pressed against highly vacuolated gut cells (Figure 2O), with testes and ovaries sandwiched between gut and body-wall muscles (Figure 2P and Video S2). Finally, the antrum was surrounded by concentric muscle rings (Figure 2Q), aligning with the neural ring (Figure 2G), suggesting that the muscles are innervated to regulate egg laying. Together, these observations demonstrate that combining tissue-specific and ubiquitous transgenes can resolve anatomical details with high resolution, establishing many anatomical landmarks that should be instrumental in evaluating the regenerative process.

Time-course imaging using tissue-specific reporters reveals stages of posterior regeneration

To investigate how tissue structures regenerate, we amputated each transgenic line (PC2, APOB, and MYH6) and performed live imaging at regular time points over a week. Using the PC2 line, we observed that severed VNCs produced fan-like axonal projections toward wounds at 6 h post amputation (hpa) (Figure 3A). Occasionally, VNCs could connect with minor nerve cords forming loops (Figure 3B). By 48 hpa, VNCs fully reconnected, although no neurons posterior to the nerve loop were yet observed (Figure 3C). By 4 days post amputation (dpa), we observed an increasingly elaborate array of neurons in this region (Figure 3D), consistent with prior observations that the duo-adhesive system regenerates by 3 dpa.²⁰ By 7 dpa, the tail plate regained its normal club-shaped appearance (Figure 3E). These results suggest a potential role for axonal guidance cues during wound healing as existing nerve cords seek out connections, followed by neurogenesis in the regenerating tail plate.

Turning to the APOB line, at 6 hpa, the epidermis at wounds appeared distorted and stretched, resembling a drawstring being pulled tight (Figure 3F). The gut sat directly beneath the epidermis with no tissue in between (Figure 3G). By 24 hpa, the blastema had grown between the gut and epidermis, often with a subset of cells encircled by pAPOB::GeNL⁺ cells (Figures 3H and 3H′). This pattern was similarly observed for the pAPOB::GeNL⁺ cells within the head during homeostasis, hinting at a possible role of presumptive phagocytes in removing extra cells.

Finally, we analyzed the musculature during regeneration using the MYH6 line. Consistent with prior observations,³⁶ circular muscle fibers formed wavy concentric rings around the wound at 6 hpa (Figure 3I) and enclosed it by 24 hpa (Figures 3J and 3J′). We often observed muscle fibers with termini forming multiple filamentous projections in both pre-existing and regenerating tissues (Figure 3K), suggesting that the muscle network was in the process of reforming during regeneration.

The MYH6 line also revealed key steps during organogenesis of the male copulatory apparatus. Starting at 3 dpa, a ring of pMYH6:GeNL⁺ cells appeared in tail blastema with a small rosette of pEef1α:mScarlet⁺ cells at the center (Figure 3L). Next, this ring developed into three connecting rings that later became seminal vesicles and the stylet sheath, inside of which a small stylet primordium began to form (Figure 3M). The rings continued to grow, as did the stylet. By this point, many prostate gland cells were present around the edges of the rings and extended long processes down into the stylet, sometimes traveling many cell bodies in distance (Figures 3N and S2D). Finally, the rings became fully formed chambers sheathed in muscle fibers, and the stylet adopted a sharp bend at its base, which was heavily invaded by processes of the surrounding prostate glands (Figure 3O). These results highlight the complex dynamic processes within regenerating tissues, providing multiple examples of intricate cellular processes with continuous coordination between cells and across tissues.

Luminescence imaging tracks neural ablation outcomes

Cell-type-specific expression not only facilitates the observation of tissue dynamics during regeneration but also enables targeted ablation using conditional, genetically encoded toxins such as NTR2.0,²⁷ which can help elucidate the function of specific cell populations. For 7 days, we treated each of our strains with 0.5 or 5 mM metronidazole (MTZ), a prodrug reduced by NTR to induce cytotoxicity. Compared to vehicle controls, the PC2 strain showed a gradual loss of muscle tone and paralysis (Video S3 and Figure S3A), the APOB animals lost their gut (Figure S3B), and the MYH6 line, though still capable of moving, contracted into spherical shapes (Video S3), likely due to the disruption of circular muscle fibers responsible for body elongation. However, the drastically increased autofluorescence after treatment obscured the extent of cell ablation (Figure 4A).

To overcome this challenge, we utilized the open-source Squid platform³⁷ to develop an upgraded version of our previous luminescence microscope²² by incorporating full motorization, dual-color fluorescence imaging, and a Python-based graphical user interface (Figures S3C and S4A). To make luminescence imaging more broadly accessible, we used a cooled CMOS camera for luminescence detection, which boasts high peak quantum efficiency (91%), extremely low dark current, and a cost reduced by more than an order of magnitude compared to traditional electron multiplying CCD cameras. Comparing fluorescence and luminescence images of the same animal, we confirmed that luminescence was consistent with fluorescence but had reduced background, permitting the visualization of even fine neural processes (Figure S3D).

Luminescence imaging revealed a progressive reduction of neural abundance in the PC2 animals kept in 5 mM MTZ (Figure 4B). By 11 days post ablation (dpab), only a few neurons remained, yet our microscope was able to detect their faint luminescent signal. Some neurons, particularly those in the lateral ganglia, resisted complete ablation.

The ability to unambiguously validate ablation results opens the possibility to explore the regenerative capacity of the nervous system post-ablation. For this, we removed the animals from MTZ at 6 dpab, followed by 7 days of recovery in artificial seawater (ASW), but we found no substantial changes to the de-generated nervous system (Figure 4C). This indicates a lack of neural regeneration after extensive ablation.

Next, we explored whether the nervous system is necessary for regeneration and whether injury can activate neural regeneration after ablation. We amputated animals at 7 dpab and monitored their regeneration without MTZ. Both MTZ-treated non-transgenic and DMSO (vehicle)-treated PC2 controls fully regenerated by 7 dpa (Figure S3E). In contrast, the posterior wound remained open for PC2 animals subjected to neural ablation with MTZ (Figure 4D), eventually leading to lysis.

Finally, we wondered whether impaired healing was unique to neural ablation. We treated the MYH6 strain with MTZ and observed extensive loss and disruption of the circular and diagonal muscles by 7 dpab. After amputation, unlike in neural ablation, the animals healed their wounds, regenerated, and restored much of their muscle network once removed from the drug (Figure 4E). These results highlight the differential recovery capacity of tissues after ablation and ruled out the potential off-target toxicity or stress caused by MTZ treatment as factors preventing wound healing or regeneration.

Tracking microscopy enables continuous imaging of posterior neural regeneration in free-moving animals

Live imaging over long periods of time often requires restraining the organism, which may interfere with regeneration. Using the Squid platform,³⁷ we built an epifluorescence microscope capable of tracking a freely moving animal while continuously capturing high-resolution images for over a week.

The animal is imaged in bright field with low-intensity infrared (IR) light, which does not elicit photophobic responses in M. lignano or heat up the water. The images are acquired at a frequency of ~6 Hz and segmented in real time to determine the animal’s centroid and adjust the stage’s position to keep the animal in the center of the field of view (FOV). In parallel, the microscope acquires fluorescence images through a separate optical path in up to four possible channels (Figures 5A and S4B). To avoid the loss of tracking due to vertical movements of the animal, we engineered a flat imaging chamber that confines the animal in the z axis (Figure 5B) without affecting its normal behavior and long-term survival (Figure 5C).

Figure 5. — (A) (Left) Diagram of the tracking microscope. (Right) Overview of the tracking routine involving IR imaging, segmentation, and stage repositioning.

(B) Exploded diagram of the long-term imaging chamber.

(C) Survival curve of ten animals placed in individual chambers and maintained at room temperature in darkness. All animals were phenotypically normal and active up to 9 days, when half of the animals began reducing their movement and forming mucus cysts. By week 3, only one animal had lysed.

(D) Representative images of neural repair and regeneration from a head fragment taken from a continuous week-long tracking fluorescence microscopy session. Images are from the region highlighted in the cartoon (dashed box). Scale bar: 100 μm.

(E) Cartoon showing the different stages of neural repair and regeneration following a horizontal cut.

(F) Representative fluorescence images of neural repair and regeneration from an oblique cut. Arrowheads: termini of the ventral nerve cords. Scale bar: 100 μm.

(G) Cartoon showing the course of neural repair and regeneration after an oblique cut. The oblique cut introduces an asymmetry evident in the uneven extension of the VNCs and offsets the location of regenerated tail plate. In the first 30 hpa, the left nerve cord extends a longer distance than the right, eventually meeting slightly off-center by 40 hpa. By 65 hpa, the tail plate begins to form adjacent to the point of nerve cord reconnection, consistent with the anatomical posterior of the animal, and the tail continues to re-center by 80 hpa.

See also Figure S5 and Video S4.

We horizontally bisected the PC2 strain and imaged the head fragments. The severed VNCs gradually extended toward each other and finally reconnected at ~30 hpa. Thereafter, the posterior tissue began to grow outward, forming a new tail plate between 40 and 50 hpa. Additional neurons started to emerge posterior to the nerve cords around 55 hpa, eventually arranging into their normal configuration around the tail plate by ~100 hpa (Figures 5D and 5E; Video S4).

We wondered whether VNCs extend at an intrinsic rate independent of injury type. To investigate, we amputated animals at a 45° angle and monitored regeneration. The shorter VNC quickly extended toward the posterior, while the longer one grew laterally only a slight distance (Figure 5F). This asymmetry resulted in the VNCs meeting far from the initial midpoint of the two termini. By ~65 hpa, neurons posterior to the nerve cord loop began forming at the anatomical midline adjacent to the VNC closure site (Figure 5G and Video S4), suggesting that VNC reconnection and posterior regeneration may be distinct processes not only separated in time but also in the patterning cues to which they respond. To further explore this, we amputated an animal at an extreme angle, causing the remaining posterior tissue to wrap around the wound site. This led to VNCs reconnecting on the far side of the animal. Despite this large-scale tissue movement, the tail plate again regenerated at the anatomical midline, far from the site of VNC reconnection (Figures S5A and S5B), further supporting the idea that VNC closure and posterior regeneration are controlled by separate cues throughout wound healing and regeneration.

Continuous live imaging allows quantification of regeneration progress across scales

With continuous tracking and live imaging, we can simultaneously quantify the regenerative process across tissue, organismal, and behavioral scales in a single experiment. For example, at the tissue level, we annotated the nerve cords and measured the distance between their termini or their length following a horizontal or 45° oblique cut, respectively. In the case of a symmetric amputation, the nerve cord termini joined at a linear rate (Figure S5C). Strikingly, after an oblique cut, the shorter nerve cord extended linearly at a rate twice higher than the longer VNC (~10 μm/h vs. ~5 μm/h) (Figure 6A), indicating an adaptive mechanism by which the extension rate can be adjusted.

Figure 6. — (A) (Left) Schematic of oblique amputation. Nerve cords are manually annotated from the lateral ganglia (large upper dots) to the termini of the nerve cords (large lower dots). Scale bar: 50 μm. (Middle) The length of each nerve cord is plotted over time, showing linear extension. (Bottom) The Euclidian distance between the nerve cord termini also decreases linearly. Dashed lines: time points corresponding to the fluorescence images shown above.

(B) Example showing body extension during regeneration (top left). Scale bar: 50 μm. Quantification of body length in three animals. The length of the animal is normalized to the starting length, and the data are fit to a curve $y = y_{0} - y_{1} e^{- k x}$ , where $y$ is the length of the animal, $y_{0}$ is the final length, $y_{0} - y_{1}$ is the starting length of the animal, $k$ is the time constant, and $x$ is time. Dots: data points. Lines: best fit.

(C) Activity score of a tracked animal over ~140 h. The highlighted regions correspond to a period of quiescence (left) in which the animal remains stationary, and a period of high activity (right) in which the animal explores much of its enclosure (dashed circle). Trajectories of the animal’s position over time in each time period are shown above.

(D) Example fluorescence images of tracked animals during the quiescent and active periods. Each image is spaced ~20–25 min apart. During times of high activity, the animal is often stretched, whereas the animal occasionally bends during the quiescent period. Scale bar: 100 μm.

(E) Normalized average activity within 10-h windows measured from the activity score in (C). Dotted line: low average activity during the first 30 hpa, followed by an increase in average activity.

See also Figure S5.

At the organismal level, the animals rescaled their bodies as regeneration progressed. Unlike linear VNC extension, the animal length grew following a saturation curve, with the rate of regeneration proportional to the amount of tissue to be regenerated. Surprisingly, the time constant of this saturation appeared consistent across animals at 0.027 ± 0.004 h⁻¹ (Figure 6B), suggesting a potentially characteristic timescale for Macrostomum posterior regeneration.

At the behavioral level, the microscope’s tracking feature allowed us to quantify animal movement using the stage’s position and the animal’s location within the FOV. We calculated a behavioral activity score^38,39 with high scores corresponding to animals actively exploring their environment, punctuated by body extension and scrunching (Figures 6C and 6D). After amputation, we observed a decrease in average activity during the first 20 h followed by a 50-h bout of hyperactivity, eventually returning to the baseline level (Figure 6E). Correlating the behavioral output with the processes in tissue regeneration (Figure 5D), our data suggested that the animal’s activity was suppressed during wound healing. Together, these multi-scale data can help us to understand how cellular and tissue-level processes translate into functional outcomes at both the organismal and behavioral levels.

Multi-modal imaging characterizes posterior regeneration defects induced by β-catenin RNAi

We next investigated whether VNC reconnection depends on posterior regeneration—a critical question, as in the planarian flatworm, Schmidtea mediterranea, the regeneration of guidepost cells is essential for guiding the reconnection of visual axons.⁴⁰ Given that β-catenin is a conserved wound-response gene responsible for resetting the primary body axis in several regenerative animals,^41,42 we developed a hybridization chain reaction (HCR) protocol to detect both β-catenin and its Wnt ligand, wnt-1, in M. lignano (Figures S6A and S6B). In intact animals, β-catenin expression was diffuse but concentrated around the gonads and in cells surrounding the pharynx, while wnt-1 was expressed in a band of cells in the posterior, adjacent to the adhesive glands stained by peanut agglutinin (PNA)⁴³ (Figure 7A). Following amputation, β-catenin expression was broadly induced in both anterior and posterior fragments, with strongest expression at wound sites at 6 hpa. By 24 hpa, β-catenin expression became localized to wound sites, whereas wnt-1 expression was activated specifically in the posterior blastema (Figure 7B). Knockdown of β-catenin using RNAi,⁴⁴ targeting all three homologs with double-stranded RNA (dsRNA) against a common sequence (Figure S6A), blocked the regeneration of posterior structures (Figures 7C and 7D) but did not abolish wnt-1 expression during homeostasis (Figure S6C). This deficiency was not due to reduced stem cell proliferation, as staining for mitotic cells using histone H3 phosphorylation (H3P) showed no statistically significant change after β-catenin RNAi (Figures S6D and S6E).

Figure 7. — (A) Homeostatic expression of *β-catenin* (cyan) and *wnt-1* (red) in intact animal. (i) An overview of the whole animal. (ii) A magnified view showing adhesive glands (yellow) in relation to *wnt-1* expression (red). (iii) The same view as (ii) showing just *wnt-1* expression. Dashed line: outline of the tail plate. (iv) *wnt-1*⁺ cells and PNA-stained adhesive glands are adjacent to each other in the posterior. Scale bars: 50 μm (i), 20 μm (ii and iii), and 10 μm (iv).

(B) *β-catenin* (cyan) and *wnt-1* (red) expression after amputation. By 0.5 hpa, neither *β-catenin* nor *wnt-1* expression is activated at wounds. By 6 hpa, *β-catenin* is upregulated around the gut in both anterior and posterior fragments and highly concentrated at wounds. Finally, at 24 hpa, *β-catenin* expression is largely restricted to the wounds in both anterior and posterior fragments, with *wnt-1* expression beginning to appear in the new posterior pole of the anterior fragment. Arrowheads: wound sites. Box and inset: *β-catenin* and *wnt-1* expression localized to the posterior pole at 24 hpa. Scale bars: 50 μm.

(C) Cartoon of the amputation made (far left). The posteriors of control (left, n = 4/4) showing a regenerated array of adhesive glands and *wnt-1*⁺ cells. In contrast, *β-catenin* RNAi animals show none (middle, n = 16/22) or few (right, n = 6/22) *wnt-1*⁺ cells and no adhesive glands at 7 dpa. Scale bars: 20 μm.

(D and E) Luminescence imaging of control-treated (left) and *β-catenin* RNAi-treated (right) PC2 animals. At 24 hpa (D), animals in both groups heal the wound and the VNCs reconnect. At 7 dpa (E), control animals regenerate a full tail with neurons innervating the tail plate (arrow), while *β-catenin* RNAi animals show no tail regeneration or tail-plate neurons radiating from the ventral nerve cord. Scale bars: 100 μm.

(F) Normalized body length of control (blue) or *β-catenin* RNAi animals (red) showing no growth after *β-catenin* knockdown. Dots: individual data points. Lines: best fit.

(G) Activity score of control animals (top) compared to *β-catenin* RNAi animals (bottom) showing the lack of recovery after *β-catenin* knockdown.

Animals in (A)–(C) were fixed and mounted for imaging, while animals in (D) and (E) were imaged live and anesthetized with 7.14% MgCl₂ supplemented with FFz for luminescence imaging. See also Figures S6 and S7.

Remarkably, despite the impaired regeneration in β-catenin knockdowns, VNCs still extended and reconnected normally by 24 hpa, as observed in the PC2 reporter strain through luminescence imaging (Figure 7D). Yet regeneration ceased beyond that point (Figure 7E). Tracking microscopy revealed no significant changes in the animal length over time, indicating a complete lack of regenerative growth (Figure 7F). Furthermore, tracking data revealed a lack of recovery in the behavioral activity of the β-catenin knockdown animals compared to controls in the process of regeneration (Figure 7G). These findings suggest that nerve cord reconnection is part of the wound healing process, which can occur independently of regeneration. This is further supported by the ability of nerve cords to reconnect in the anterior of the non-regenerative tail fragments (Figure S7A). Together, these results provide insights into the dynamics of Wnt/β-catenin activity during wound healing and regeneration in M. lignano and demonstrate the utility of our toolbox in dissecting complex regenerative phenotypes caused by genetic manipulations.

DISCUSSION

Regeneration is a complex, dynamic, organism-wide process involving many different cell types.^1,3,45 Here, by deploying several key techniques—transgenesis, ablation, and continuous live imaging—we pave the way for tagging and ablating various cell types to systematically delineate their roles during regeneration and to explore the crosstalk between these cell types. For example, our neural ablation experiments highlighted the critical role of the nervous system in facilitating wound healing. The tools are now available to identify which neuronal populations are essential in this process and whether neurons communicate directly with epidermal cells, as recently noted in the fly gut after injury,⁴⁶ or indirectly via other cell types, such as muscles or phagocyte-like cells we observed within the blastema.

Our tools also open avenues for studying axonal repair. Following an oblique cut, VNCs extended at drastically different rates, suggesting that nerve cord repair may be adaptive. We hypothesize that the extension rate is set soon after amputation and is inversely related to the remaining length of the nerve cord, since the rates of extension remained constant even as the animal continued to grow. Given that VNCs reconnect in both the anterior and posterior wounds (Figure S7A), we propose that midline genes, known to be crucial for axonal guidance in various systems,⁴⁷ might play a role in instructing nerve cord extension, potentially acting in conjunction with other morphogenic cues⁴⁸ or genes that may regulate mediolateral patterning, such as the non-canonical Wnt signaling or planar cell polarity pathway.^49,50 By integrating RNAi and tracking microscopy to study the neural reporter strain, M. lignano can become a powerful system to dissect the molecular logic underlying axonal guidance in the context of regeneration.

Finally, our characterization of the β-catenin RNAi phenotype in M. lignano unveiled significant functional variations in the Wnt signaling pathway during regeneration across regenerative animals. Unlike the planarian S. mediterranea, which regenerates a head in place of a tail after β-catenin RNAi,^41,51 M. lignano simply failed to regenerate anything. Consequently, while β-catenin RNAi can often rescue deficient anterior regeneration in other planarian species,^52,53 it failed to induce anterior regeneration in M. lignano tail fragments (Figure S7B). Another key difference lies in the regulation of β-catenin and wnt-1 activation following injury. In the planarian, injury-induced wnt-1 expression is β-catenin independent and precedes the transcriptional activation of β-catenin at both anterior and posterior wounds.^41,54 In contrast, in M. lignano, β-catenin was induced earlier than wnt-1, and β-catenin RNAi largely abolished wnt-1 activation at the posterior wounds after amputation (Figure 7C) but not during homeostasis (Figure S6C). These expression kinetics also differ from Hydra, where β-catenin and Wnt ligands are upregulated simultaneously early in regeneration.⁴² Furthermore, unlike in Hydra and the acoel Hofstenia, where stem cell proliferation depends on Wnt signaling,⁵⁵ M. lignano showed no significant differences in proliferation between β-catenin RNAi and control animals. Overall, these differences highlight the importance of studying M. lignano as a critical model for understanding the functions and evolutionary modifications of Wnt signaling in regulating regeneration.

Limitations of the study

While our protocol has made injection easier, rates of germline transmission remain low (~0.5%–2%). In an effort to enhance transformation efficiency, we experimented with injecting recombinant Tol2 transposase mixed with transgenes flanked by Tol2 inverted repeats. To date, this approach has not yielded higher efficiencies compared to the current method,²⁵ as the strains shown here are likely also the result of random integration.^23–25 However, our modular cloning toolkit should facilitate comparisons and screening of alternative integration methods.

Regarding the microscopy techniques, using the same objective for both tracking and imaging in the tracking microscope introduces a trade-off between spatial resolution and tracking consistency, as higher magnification reduces the FOV, thereby increasing the likelihood of losing track of the animal. Moreover, the current setup relies on wide-field epifluorescence for imaging, which has the limitations of reduced signal-to-noise ratio due to out-of-focus light and disruption in tracking cellular structures when they move out of focus due to the animal’s rotation or deformation. These issues may be addressed in future iterations by using separate objectives for tracking and imaging and incorporating optical sectioning and/or multi-focal imaging technologies.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed toward and will be fulfilled by the lead contact, Bo Wang (wangbo@stanford.edu).

Materials availability

Plasmids used in this study are available upon request, and all plasmid maps and sequences are available in Data S1 and Table S1. Fluorescent/luminescent strains generated are available upon request.

Data and code availability

Imaging and tracking data reported in this paper will be shared by the lead contact upon request.
Software and graphical user interface for operating (https://github.com/hongquanli/octopi-research) Squid microscopes and for tracking (https://github.com/prakashlab/squid-tracking) can be found on GitHub.
Transcript sequences for wnt-1, β-catenin, and genes utilized for generating transgenes are publicly available (https://sc455.macgenome.org/) with contig numbers listed in Table S1 and uploaded to NCBI GenBank under the accession numbers GenBank: PQ329335–PQ329342.

STAR★METHODS

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animal care and maintenance

M. lignano strain NL12¹¹ and derived strains were maintained at 20°C and 60% humidity with a 14/10 day/night cycle in ASW with a salinity of 32 parts per thousand (ppt), measured by a manual refractometer. The diatom Nitzschia c.f. curvilineata was seeded in 150 mm glass dishes and grown in F/2 media. When confluent and ready for feeding to animals, the F/2 media was poured off and replaced with fresh ASW into which animals were transferred. Feeding was performed once a week. To maintain cultures suitable for egg preparation, age synchronized cultures were collected by removing adults from a plate after feeding and allowing the eggs to hatch. Juveniles from multiple plates were pooled and used to seed a fresh plate of age synchronized animals.

Transgenic lines were established by raising injected animals to sexual maturity and screening populations for fluorescent, transgenic progeny. Fluorescent juveniles were collected and sub-cultured. Animals were continuously screened for transgene expression when transferring to fresh plates of diatoms to increase the transgene allele frequency within the population. This is essential because karyotype polymorphism frequently occurs in M. lignano.¹⁷ Transgenic animals used in experiments were screened for strong expression of the transgene prior to collection, with no observed variation in expression patterns between individuals.

METHOD DETAILS

Cloning

All primers and oligos used are listed in Table S1, and all parts and constructs are included in Data S1. Parts (promoters, genes, terminators) were assembled by PCR amplification of desired sequences with overhangs including Gibson assembly homology arms and BsaI restriction sites. These sequences are listed in Table S1.

GeNL²⁹ and NTR2.0²⁷ were codon optimized using https://www.macgenome.org/codons/ without introns and synthesized by Twist Bioscience. GeNL-P2A-NTR2.0 was assembled by Gibson assembly. Promoters from enolase and pc2 were amplified from genomic DNA. mScarlet as well as promoters from eef1α, apob, and myh6 were amplified from plasmids described previously.²³

pEmpty was linearized with EcoRV-HF, run on a 1% agarose gel, and extracted using the Zymo Gel Extraction kit. Gibson assembly was performed by combining a part amplicon and linearized pEmpty in a 2:1 mass ratio in 2× NEB Gibson Assembly Master Mix and incubating at 50°C for 2 h before transformation. Clones were sequenced using M13 Forward (5′-GTAAAACGACGGCCAGT-3′) or M13 Reverse (5′-CAGGAAACAGCTATGAC-3′) primers.

Unique sequence (UNS) oligos were annealed by combining sense and antisense oligos at 5 μM in duplex buffer, incubated at 94°C for 2 min, and cooled slowly at a rate of 1°C/min. The duplexed oligos where then diluted to 50 nM in H₂O.

Parts were assembled into TUs via Golden Gate assembly. 0.5 μL of each part (promoter, gene, and terminator, 30 nM), 0.5 μL of 5′ and 3′ UNS oligos each (50 nM) corresponding to their position in the final plasmid, 0.5 μL of 10× T4 DNA Ligase Buffer, 0.25 μL of T4 DNA Ligase, and 0.25 μL of BsaI-HFv2, were mixed to a final volume of 10 μL in nuclease free water. The samples were cycled with the following program: [37°C for 3 min, 16°C for 4 min] × 12, 50°C for 5 min, hold at 4°C.

For example, a single TU vector would require only UNS1A and UNS10D oligos in the Golden Gate reaction and would be amplified using UNS1F and UNS10R primers. Alternatively, dual TU vectors would require the first TU to use UNS1A and UNS3D oligos and the second to use UNS3A and UNS10D oligos and would be amplified by UNS1F/UNS3R and UNS3F/UNS10R primers, respectively.

Assembled TUs were amplified by PCR from the prior Golden Gate reaction products. 2 μL of Golden Gate reaction were added to 25 μL of 2× Phusion Polymerase Master Mix, mixed with 18 μL of H₂O, and 3 μL of 10 μM forward and reverse primers corresponding to the terminal UNS sequences used for that TU. The samples were cycled with the following program: 98°C for 30 s, [98°C for 30 s, 60°C for 30 s, 72°C for 30 s per kb] × 35–40, 72°C for 5 min, hold at 4°C. The resulting product was run on a 1% agarose gel and the band corresponding to the TU was extracted using the Zymo Gel Extraction kit and eluted in 11 μL of H₂O.

pDest and pTol2Dest were constructed by performing primer extension PCR and Gibson assembly to insert an EcoRV-HF restriction site between 5′ UNS1A and 3′ UNS10D sequences. pDest was constructed by amplification of pDEST_R4-R3 with primers BW-NH-710 and BW-NH-711 while pTol2Dest was constructed by amplification of pT2/HE (a gift from Perry Hackett) with BW-NH-792 and BW-NH-793. The backbone amplicons were then self-ligated using Gibson assembly. Unlike pDest, pTol2Dest contains terminal repeats for integration via Tol2 transposase.

TUs were assembled into the destination vectors, pDest or pTol2Dest, via Gibson assembly. Destination vectors were linearized using EcoRV-HF, run on a 1% agarose gel, and extracted using the Zymo Gel Extraction kit. Gibson assembly was performed combining TUs and linearized destination vector in a 2:1 mass ratio in 2× NEB Gibson Assembly Master Mix. Samples were incubated at 50°C for 2 h before transformation.

Egg preparation

To prepare fertilized eggs for microinjection, ~200–300 gravid animals (evident by a large, well-developed oocyte present in their posterior) from age-synchronized populations at ~4–7 days post feeding were collected into two separate 60 mm dishes filled with ASW. Every hour, freshly laid eggs were transferred using an eyelash pick to the upside down lid of a 100 mm polystyrene Petri dish, which was cooled to 4°C to halt the development at 1-cell stage. Once ~100–150 eggs were lined up on the lid, the eggs were washed in 1.5 mM DTT in ASW for 5–10 min, swirling the plate occasionally. The eggs were then washed three times with ASW before microinjection.

Microinjection

Eggs were injected on a Zeiss Primovert inverted stereomicroscope equipped with a Sutter XenoWorks electronic micromanipulator, controller, and pressure source. A manual 3-axis stage was used to manipulate the holding pipette. Needles were pulled from borosilicate glass capillaries on a Sutter P97 needle puller using a 3-step pulling protocol (1. Heat = 754, Pull = 90, Velocity = 8, Time = 250; 2. Same as 1. 3. Heat = 754, Pull = 85, Velocity = 8, Time = 250) with a box filament heater. A 20-degree bend at the tip of the needle was introduced using a Narishige MicroForge. Holding pipettes were pulled from borosilicate glass capillaries (Heat = 738, Pull = None, Velocity = 150, Time = None) and cut, flame polished, and bent to a 30-degree angle.

Injection mix was prepared by adding 1 μL of recombinant Tol2 (rTol2) transposase (optional) and 0.5 μL of Fast Green FCF followed by plasmid DNA and rTol2 storage buffer (10 mM HEPES, 300 mM KCl, pH 6.9) to a final plasmid concentration of 50 ng/μL. Injection in the absence of rTol2, however, yielded similar transformation efficiencies. The solution was mixed and centrifuged for 1 min to pellet any small particles that may clog the needle. Injection mix was then loaded by back filling. Injections were performed using an initial pressure of 1,000 hPa and back-pressure of +100 hPa, adjusted depending on the flow of the needle. Without applying negative pressure, the holding pipette was placed behind the egg as a backstop. PiezoXpert was used to assist in penetrating the zygote’s membrane with settings “Int = 86, Speed = 10, Pulse”. Injection mix was ejected until a bolus of fluid became visible within the zygote. Typical rates of germline transmission are around 0.5–2%.

Amputations

Animals were anesthetized in 7.14% 6H₂O×MgCl₂ for 5 min and then amputated using a stainless-steel scalpel. Fragments were transferred to ASW to recover. For live imaging, fragments were transferred to chambers with fresh ASW once they became mobile. We note that MgCl₂ treatment may reduce the contraction of circular muscles after injury, which could result in the leakage of intestinal tissues.

Luminescence/fluorescence microscope

A thermoelectrically cooled camera using the Sony IMX571 sensor (ToupTek Cat# ITR3CMOS26000KMA) was chosen for its high quantum efficiency (91%), low read noise (<1.5e-at 12 dB gain), and low dark current. To increase photon capture efficiency, a 10 MP, f = 50 mm imaging lens was used as a tube lens to provide demagnification to enable the use of higher NA objectives. The light tight enclosure was constructed from opaque black acrylic, laser cut, and assembled into a box with electrical tape to prevent light bleeding through the seams (Figure S4A). Existing and new Squid components were designed for optomechanical integration, with CAD files available at https://squid-imaging.org.

Tracking microscope

Components were constructed from modules described in ref. 37 and https://squid-imaging.org. Animals were illuminated with an IR LED light source at 850 nm. IR images for tracking were acquired at ~6 Hz using a monochrome camera (Daheng Imaging, Cat# MER-1220–32U3M) at 10× magnification (BoliOptics, Cat# 03033331). Images were binarized by user-input threshold in the GUI. The binarized images were eroded and dilated to remove noise and fill gaps. OpenCV was used to calculate the centroid of the largest contiguous region. Centroid tracking was performed using a nearest neighbor approach in which the animal’s centroid in the subsequent frame was determined within a search radius centered on the animal’s previous centroid position. The displacement of the centroid was converted into x-y stage movements using a proportional integral derivative (PID) controller, implemented on an Arduino Due microcontroller. The stage position was then adjusted by a stepper motor with optical encoder to maintain the centroid in the center of the FOV. Code for tracking was adapted from https://github.com/prakashlab/squid-tracking.

While tracking, fluorescence images were acquired every 50 s. Fiber-coupled 405/488/561/638 nm lasers were despeckled through a Molex despeckler and used as excitation. A quad-bandpass dichroic filter set (405/488/561/640) was used to split the emission light (Figure S4B). Additional bandpass filters can be installed to reduce background noise from animal and algal autofluorescence. The configuration used in this study achieves a resolution of 1.22 μm at 500 nm with a pixel size of 0.44 μm. The microscope is compatible with infinity corrected objectives for standard diffraction limited wide field imaging. The image acquisition software can be found at https://github.com/hongquanli/octopi-research.

Live luminescence and fluorescence confocal imaging

Animals were first anesthetized in 7.14% 6H₂O×MgCl₂ and 2% methylcellulose in deionized water and then transferred to a coverslip slide in a 20 μL droplet. For luminescence imaging, 0.5 μL of 8.7 mM Fluorofurimazine (FFz) in PBS was added to the droplet and mixed thoroughly. The corners of a coverslip were then scraped across clay to make four small clay ‘feet’ and gently placed over the droplet. Using forceps, corners of the coverslip were pressed down to firmly restrict the animal in place. If the animal continued to move, a paper towel was used to wick away excess water from the underside of the slide to decrease the distance between the coverslip and the slide. The slide was then imaged on the luminescence microscope using an Olympus 20× objective (NA = 0.75) with an exposure time varying between 10 and 60 s. For confocal microscopy, slides were prepared as above, skipping the FFz step. Imaging was performed on a Zeiss LSM800 AxioObserver using a 40× water-immersion objective (NA = 1.1). After short imaging sessions (~10–20 min), animals may be recovered by adding a droplet of ASW to the corner of the coverslip and gently lifting it with a razor blade.

Preparation of long-term imaging chambers

Imaging chambers were produced by gluing a 3 mm imaging spacer to a glass slide and sticking 1–2 stacked circular cryolabels in the center of the spacer. Approximately 2 mL of 2% low melting-point agarose in ASW was pipetted into the imaging spacer until it completely fills the chamber. A second slide was pressed flat over the agarose carefully not to trap any bubbles. The agarose block was solidified in the fridge. A scalpel was used to separate the agarose block from the sides of the imaging spacer, and the block was placed upside down on a slide. A single animal, starved for 2 d to reduce gut autofluorescence, was placed in 1–2% methylcellulose in ASW and then pipetted onto the depression in the agarose made by the cryolabels. A second slide with a 3 mm imaging spacer (without cryolabels) was then slowly lowered upside down onto the agarose block without generating any bubbles or removing the animal. Once placed, the whole assembly was flipped back right side up to remove the top glass slide. Finally, vacuum grease was applied to the top of the imaging spacer and a coverslip was placed over the top to seal the contents within the imaging spacer.

Chemical ablation

MTZ was dissolved in DMSO to a stock concentration of 1 M. Animals starved for 2 d and placed in 3 mL of ASW containing either 0.5 mM (gut ablation) or 5 mM MTZ (neural and muscle ablation). The ASW-MTZ mixture was pipetted up and down until there were no visible crystals of MTZ remaining. Controls contained an equivalent percentage of DMSO. ASW and MTZ was replaced every other day for the duration of ablation.

RNAi

Primers (Table S1) were used to amplify a ~500 bp region of β-catenin and TA-cloned into pJC53.2.⁵⁶ Linear templates flanked by T7 promoters were generated using PCR with EX primers (Table S1). In vitro RNA synthesis was performed using T7 polymerase, RNA was precipitated using ammonium acetate (NH₄Ac, 10 M) and ethanol, denatured, then re-annealed. Control RNAi was derived from the ccdB insert of the unmodified pJC53.2 plasmid.

RNAi was performed by soaking. Animals were starved overnight to eliminate diatoms from their gut. In a 24-well plate, ~15–20 animals were placed in 1 mL of ASW containing 2 μg of dsRNA. The ASW and dsRNA mixture was replaced every other day for 3 weeks, with a single day of feeding once per week, until amputation.

Fixation

Animals were starved overnight in fresh ASW. Before fixation, animals were washed twice with ASW for 5 min each. ASW was then replaced with 2 mL of 7.14% 6H₂O3MgCl₂ for 5 min to relax the animals. 500 μL of the MgCl₂ solution was removed and 500 μL of 16% paraformaldehyde (PFA) was added to a final concentration of 4% PFA. After 15 min of fixation, 100 μL of 10% NP-40 was added. After 45 min of fixation, the fixative was replaced with PBS containing 0.1% Tween 20 (PBSTw). Animals were then washed twice with PBSTw for 5 min each, then dehydrated by incubating for 5 min in increasing concentrations of methanol (25%, 50%, 75%, 100%), and stored at −20°C.

H3P immunostaining

Fixed animals were rehydrated by incubating in 1:1 volume ratio of PBS containing 0.1% Triton X (PBSTx) to methanol for 10 min. Animals were washed 4 times with PBSTx for 5 min each. Animals were blocked in 10% horse serum in PBSTx for 2 h. Blocking solution was replaced with 1:400 anti-phospho-Histone H3 (Ser10) diluted in pre-cooled blocking solution and incubated overnight at 4°C. Animals were washed 5 times with PBSTx for 20 min each. Animals were incubated in 1:1000 secondary antibody (anti-rabbit IgG, AF488) and 1:1000 DAPI (10 mg/mL) in blocking solution for 2 h. Animals were washed twice with PBSTx for 10 min each and then mounted on coverslips in 80% glycerol. Unless otherwise specified, all incubations were performed at room temperature (RT) with gentle shaking.

Hybridization chain reaction

Fixed animals in methanol were rehydrated by 5 min incubations in increasing concentrations of PBSTw (25%, 50%, 75%, 100%). Animals were incubated in a 1:1 volume ratio of PBSTw to probe hybridization buffer (Molecular Instruments) for 10 min at RT. The solution was then replaced with pre-hybridization solution (Molecular Instruments) for 1 h at 37°C. The pre-hybridization solution was then replaced with probe solution (4 pmol of probe mixture for every 500 μL of probe hybridization buffer). Probes targeting wnt-1 and β-catenin were designed using https://github.com/rwnull/insitu_probe_generator and ordered from IDT. Probe sequences are included in Table S1.

Samples were incubated overnight (~20 h) at 37°C. Probe solution was removed, and the samples were washed 4 times with 500 μL of probe wash buffer (Molecular Instruments), pre-heated to 37°C, for 20 min each at 37°C, and then washed twice for 5 min each with 5× SSC supplemented with 0.1% Tween 20 (5× SSCT). Pre-amplification was performed by incubating samples with 500 μL amplification buffer (Molecular Instruments) for 30 min at RT. 30 pmol of hairpin H1 and 30 pmol of hairpin H2 in 10 μL of 3 μM stock solutions were snap cooled (heated to 95°C for 90 s and cooled to RT in the dark for 30 min) separately for every 500 μL of amplification buffer. Snap cooled hairpins H1 and H2 (10 μL each) were added to a tube of 500 μL amplification buffer. Amplification buffer was removed from the samples and replaced with the hairpin-containing amplification buffer and incubated overnight (~12–16 h) in the dark at RT. On the following day, excess hairpins were removed by washing the samples with 500 μL of 5× SSCT at RT (2× for 5 min, 2× for 30 min, and finally once for 5 min). The samples were transferred to PBSTw by incubating in increasing concentrations of PBSTw in 53 SSCT (25%, 50%, 75%, 100%) for 5 min each. For visualizing the adhesive organ, samples were incubated in 1:200 FITC-PNA and 1:5000 DAPI (10 mg/mL) for 30 min. Samples were washed 2× with PBSTw for 5 min each and mounted in VectaShield Antifade mounting solution and stored at 4°C.

QUANTIFICATION AND STATISTICAL ANALYSIS

Image processing

Confocal images were processed in the Zeiss Zen 2 software. Confocal stacks were aligned using the z stack alignment tool. Dualcolor images were spectrally de-mixed. Epifluorescence and luminescence images were processed using ImageJ v1.53k. All images presented were confirmed in more than three animals, with no observed variations between individuals.

Tracking microscope image processing

Raw images were processed using Python 3.8, Scikit-Image and OpenCV2. Images were first binarized by Gaussian thresholding. An ellipse was fitted to the segmented shape and the image was rotated to align the major axis of the ellipse vertically. The image was then rotated another 180° if the animal was detected with the anterior facing downward. Images were cropped based on a common bounding box which encompassed the animal across all frames of the video. Nerve cords were manually annotated using a custom GUI for loading images and selecting anatomical landmarks. The body length of the animal was determined by the longest projection among the radially projecting rays every 5° from the centroid of the animal. Activity scores were calculated by denoising the instantaneous velocity, using a Morlet wavelet transformation and summing scales 1–31.³⁸ The activity scores are baseline subtracted.

Supplementary Material

NIHMS2038918-supplement-1.pdf^{(3.1MB, pdf)}

NIHMS2038918-supplement-2.xlsx^{(41.9KB, xlsx)}

NIHMS2038918-supplement-3.xlsx^{(16.5KB, xlsx)}

Download video file^{(33MB, mp4)}

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NIHMS2038918-supplement-8.zip^{(67.5KB, zip)}

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples

Nitzschia c.f. curvilineata	Eugene Berezikov	N/A

Chemicals, peptides, and recombinant proteins

Artificial Sea Water salts	Bulk Reef Supply	Cat# 208269
F/2 Media	Bigelow	Cat# MKf250L
EcoRV-HF	NEB	Cat# R3195S
10× T4 DNA Ligase Buffer	NEB	Cat# B0202S
T4 DNA Ligase	NEB	Cat# M0202L
BsaI-HFv2	NEB	Cat# R3733S
23 Phusion Polymerase Master Mix	Thermo Fisher	Cat# F531L
IDT duplex buffer	IDT	Cat# 11–05-01–12
Dithiothreitol (DTT)	Thermo Fisher	Cat# R0861
6H₂O×MgCl2	Fisher Scientific	Cat# BP214–500
Methylcellulose	Sigma-Aldrich	Cat# M0512–100G
Fluorofurimazine	Promega	Cat# N4100
Low melting point agarose	GoldBio	Cat# A-204–100
Metronidazole	Sigma-Aldrich	Cat# M1547–5G
16% paraformaldehyde	Fisher Scientific	Cat# 50–980-487
FITC-PNA	Vector Laboratories	Cat# FL-1071–10
DAPI	Sigma-Aldrich	Cat# D9542–1MG
VectaShield Antifade Media	Fisher Scientific	Cat# NC9532821
Recombinant Tol2	Creative BioMart	Cat# Tol2 transposase-12O
Fast Green FCF	Abcam	Cat# ab146267
Dimethyl sulfoxide (DMSO)	Fisher Scientific	Cat# D1391
Ammonium acetate	Sigma-Aldrich	Cat# A7330–100G
10×Phosphate buffered saline (PBS)	Fisher Scientific	Cat# AM9625
Tween 20	Sigma-Aldrich	Cat# 11332465001
Nonidet P 40 substitute (NP-40)	Sigma-Aldrich	Cat# 74385
Sodium citrate	Thermo Fisher	Cat# S279–500
Anti-phospho-Histone H3 (Ser10)	Millipore Sigma	Cat# 04–817; RRID:AB_1163440
Anti-rabbit IgG, AF488	Millipore Sigma	Cat# A-11008; RRID:AB_143165

Critical commercial assays

Zymo Gel Extraction Kit	Zymo	Cat# D4008
2× Gibson Assembly Master Mix	NEB	Cat# E2611S
Molecular Instruments HCR RNA-FISH Kit	Molecular Instruments	N/A

Experimental models: Organisms/strains

Macrostomum lignano	Eugene Berezikov	NL12
Macrostomum lignano (SU2)	This paper	pEnolase:GeNL:tEnolase
Macrostomum lignano (SU5)	This paper	pPC2:GeNL-NTR2.0:tPC2
Macrostomum lignano (SU6)	This paper	pAPOB::GeNL-NTR2.0:tSV40 pEef1α:mScarlet:tEef1α
Macrostomum lignano (SU7)	This paper	pMYH6:GeNL-NTR2.0:tSV40 pEef1α:mScarlet:tEef1α

Oligonucleotides

Primers used in this study	This paper	See Table S1

Recombinant DNA

Plasmids used in this study	This paper	See Table S1, Data S1
pT2/HE	Perry Hackett	Addgene #26557
pDEST_R4_R3	Thermo Fisher	N/A
pDONOR221	Thermo Fisher	Cat# 12536017
pJC53.2	Collins et al.⁵⁶	Addgene #26536

Software and algorithms

Fiji	Schindelin et al.⁵⁷	http://fiji.sc/
Scikit-image	Van Der Walt et al.⁵⁸	https://scikit-image.org/
OpenCV2	N/A	https://opencv.org/
Zen 2	Zeiss	N/A

Other

Tracking microscope	This paper	See Table S2
Luminescence microscope	This paper	See Table S2
18 × 18 mm coverslips	Fisher Scientific	Cat# 12–541AP
60 × 24 mm coverslips	Fisher Scientific	Cat# 12–545-MP
Zeiss Primovert	Zeiss	Cat# 491206–0006-000
Sutter XenoWorks Microinjector	Sutter	Cat# BRE
Sutter 3-axis micromanipulator	Sutter	Cat# MPC-200
Sutter 3-axis micromanipulator controller	Sutter	Cat# ROE-200
3-axis manual stage	Newport	Cat# 9067-XYZ-R-V-M
Borosilicate glass capillary (with filament)	MPI	Cat# 1B100F-3
Borosilicate glass capillary (thin walled)	MPI	Cat# TW100–3
Sutter P97 needle puller	Sutter	Cat# P-97
Narishige MicroForge	Narishige	Cat# MF-900
Eppendorf PiezoXpert	Eppendorf	Cat# 5194000024
Refractometer	Bulk Reef Supply	Cat# 205171B
Zeiss LSM800	Zeiss	N/A
3 mm imaging spacers	SunJin Lab	Cat# S013
Cryolabels	Fisher Scientific	Cat# 03–391-102

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Highlights.

A toolbox enables live imaging and tissue ablation in the flatworm Macrostomum lignano
Neurons are essential for wound healing and regeneration but do not regenerate after ablation
Severed nerve cords extend at a linear rate inversely proportional to their remaining length
Nerve cord reconnection is separate from Wnt/β-catenin-dependent posterior regeneration

ACKNOWLEDGMENTS

We thank E. Berezikov and J. Wudarski for sharing the M. lignano NL12 strain, plasmids, and the experimental protocols for animal husbandry and E. Davies along with members of the Berezikov and Davies labs for stimulating discussions. This work is supported by a Stanford Bio-X Interdisciplinary Initiative seed grant (IIP11-40), an EDGE grant from NSF (IOS-1923534), and NIH grants 1R35GM138061 to B.W. and R35GM130366 to A.Z.F.

Footnotes

DECLARATION OF INTERESTS

H.L. and M.P. are co-founders of Cephla, commercializing the Squid platform.

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114892.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS2038918-supplement-1.pdf^{(3.1MB, pdf)}

NIHMS2038918-supplement-2.xlsx^{(41.9KB, xlsx)}

NIHMS2038918-supplement-3.xlsx^{(16.5KB, xlsx)}

Download video file^{(33MB, mp4)}

Download video file^{(7.3MB, mp4)}

Download video file^{(74.4MB, mp4)}

Download video file^{(124.6MB, mp4)}

NIHMS2038918-supplement-8.zip^{(67.5KB, zip)}

Data Availability Statement

Imaging and tracking data reported in this paper will be shared by the lead contact upon request.
Software and graphical user interface for operating (https://github.com/hongquanli/octopi-research) Squid microscopes and for tracking (https://github.com/prakashlab/squid-tracking) can be found on GitHub.
Transcript sequences for wnt-1, β-catenin, and genes utilized for generating transgenes are publicly available (https://sc455.macgenome.org/) with contig numbers listed in Table S1 and uploaded to NCBI GenBank under the accession numbers GenBank: PQ329335–PQ329342.

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[R8] 8.Ricci L, and Srivastava M. (2021). Transgenesis in the acoel worm Hofstenia miamia. Dev. Cell 56, 3160–3170.e4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] 9.Weissbourd B, Momose T, Nair A, Kennedy A, Hunt B, and Anderson DJ (2021). A genetically tractable jellyfish model for systems and evolutionary neuroscience. Cell 184, 5854–5868.e20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Paix A, Basu S, Steenbergen P, Singh R, Prevedel R, and Ikmi A. (2023). Endogenous tagging of multiple cellular components in the sea anemone Nematostella vectensis. Proc. Natl. Acad. Sci. USA 120, e2215958120. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] 11.Wudarski J, Egger B, Ramm SA, Schärer L, Ladurner P, Zadesenets KS, Rubtsov NB, Mouton S, and Berezikov E. (2020). The free-living flatworm Macrostomum lignano. EvoDevo 11, 5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Brand JN, Wiberg RAW, Pjeta R, Bertemes P, Beisel C, Ladurner P, and Schärer L. (2020). RNA-Seq of three free-living flatworm species suggests rapid evolution of reproduction-related genes. BMC Genom. 21, 462. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Brand JN, Viktorin G, Wiberg RAW, Beisel C, and Schärer L. (2022). Large-scale phylogenomics of the genus Macrostomum (Platyhelminthes) reveals cryptic diversity and novel sexual traits. Mol. Phylogenet. Evol 166, 107296. [DOI] [PubMed] [Google Scholar]

[R14] 14.Marie-Orleach L, Vellnow N, and Schärer L. (2021). The repeatable opportunity for selection differs between pre- and postcopulatory fitness components. Evol. Lett 5, 101–114. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Lengerer B, Pjeta R, Wunderer J, Rodrigues M, Arbore R, Schärer L, Berezikov E, Hess MW, Pfaller K, Egger B, et al. (2014). Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein. Front. Zool 11, 12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Wasik K, Gurtowski J, Zhou X, Ramos OM, Delás MJ, Battistoni G, El Demerdash O, Falciatori I, Vizoso DB, Smith AD, et al. (2015). Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano. Proc. Natl. Acad. Sci. USA 112, 12462–12467. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Zadesenets KS, Schärer L, and Rubtsov NB (2017). New insights into the karyotype evolution of the free-living flatworm Macrostomum lignano (Platyhelminthes, Turbellaria). Sci. Rep 7, 6066. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] 18.Wiberg RAW, Brand JN, Viktorin G, Mitchell JO, Beisel C, and Schärer L. (2023). Genome assemblies of the simultaneously hermaphroditic flatworms Macrostomum cliftonense and Macrostomum hystrix. G3 Genes, Genomes, Genet. 13, jkad149. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Ma Y, He J, Sieber M, von Frieling J, Bruchhaus I, Baines JF, Bickmeyer U, and Roeder T. (2023). The microbiome of the marine flatworm Macrostomum lignano provides fitness advantages and exhibits circadian rhythmicity. Commun. Biol 6, 289. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Egger B, Ladurner P, Nimeth K, Gschwentner R, and Rieger R. (2006). The regeneration capacity of the flatworm Macrostomum lignano - On repeated regeneration, rejuvenation, and the minimal size needed for regeneration. Dev. Gene. Evol 216, 565–577. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] 21.Egger B, Gschwentner R, Hess MW, Nimeth KT, Adamski Z, Willems M, Rieger R, and Salvenmoser W. (2009). The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano. BMC Dev. Biol 9, 41. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Hall RN, Weill U, Drees L, Leal-Ortiz S, Li H, Khariton M, Chai C, Xue Y, Rosental B, Quake SR, et al. (2022). Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection. Cell Rep. Methods 2, 100298. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Wudarski J, Simanov D, Ustyantsev K, de Mulder K, Grelling M, Grudniewska M, Beltman F, Glazenburg L, Demircan T, Wunderer J, et al. (2017). Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano. Nat. Commun 8, 2120. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Ustyantsev K, Wudarski J, Sukhikh I, Reinoite F, Mouton S, and Berezikov E. (2021). Proof of principle for piggybac-mediated transgenesis in the flatworm Macrostomum lignano. Genetics 218, iyab076. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Wudarski J, Ustyantsev K, Reinoite F, and Berezikov E. (2022). Random integration transgenesis in a free-living regenerative flatworm Macrostomum lignano. Methods Mol. Biol 2450, 493–508. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Mouton S, Wudarski J, Grudniewska M, and Berezikov E. (2018). The regenerative flatworm Macrostomum lignano, a model organism with high experimental potential. Int. J. Dev. Biol 62, 551–558. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Sharrock AV, Mulligan TS, Hall KR, Williams EM, White DT, Zhang L, Emmerich K, Matthews F, Nimmagadda S, Washington S, et al. (2022). NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation. Nat. Methods 19, 205–215. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Halleran AD, Swaminathan A, and Murray RM (2018). Single day construction of multigene circuits with 3G assembly. ACS Synth. Biol 7, 1477–1480. [DOI] [PubMed] [Google Scholar]

[R29] 29.Suzuki K, Kimura T, Shinoda H, Bai G, Daniels MJ, Arai Y, Nakano M, and Nagai T. (2016). Five colour variants of bright luminescent protein for real-time multicolour bioimaging. Nat. Commun 7, 13718. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Chai C, Gibson J, Li P, Pampari A, Patel A, Kundaje A, and Wang B. (2024). Flexible use of conserved motif vocabularies constrains genome access in cell type evolution. Preprint at bioRxiv. 10.1101/2024.09.03.611027. [DOI]

[R31] 31.Emmons SW (2018). Neural circuits of sexual behavior in Caenorhabditis elegans. Annu. Rev. Neurosci 41, 349–369. [DOI] [PubMed] [Google Scholar]

[R32] 32.Ladurner P, Mair GR, Reiter D, Salvenmoser W, and Rieger RM (1997). Serotonergic nervous system of two Macrostomid species: recent or ancient divergence? Invertebr. Biol 116, 178. [Google Scholar]

[R33] 33.Ladurner P, Pfister D, Seifarth C, Schärer L, Mahlknecht M, Salvenmoser W, Gerth R, Marx F, and Rieger R. (2005). Production and characterisation of cell- and tissue-specific monoclonal antibodies for the flatworm Macrostomum sp. Histochem. Cell Biol 123, 89–104. [DOI] [PubMed] [Google Scholar]

[R34] 34.Morris J, Cardona A, De Miguel-Bonet MDM, and Hartenstein V. (2007). Neurobiology of the basal platyhelminth Macrostomum lignano: Map and digital 3D model of the juvenile brain neuropile. Dev. Gene. Evol 217, 569–584. [DOI] [PubMed] [Google Scholar]

[R35] 35.Rieger RM, Salvenmoser W, Legniti A, and Tyler S. (1994). Phalloidinrhodamine preparations of Macrostomum hystricinum marinum (Plathelminthes): morphology and postembryonic development of the musculature. Zoomorphology 114, 133–147. [Google Scholar]

[R36] 36.Salvenmoser W, Riedl D, and Ladurner P,RP (2001). Early steps in the regeneration of the musculature in Macrostomum sp. (Macrostomorpha). Belg. J. Zool 131, 105–109. [Google Scholar]

[R37] 37.Li H, Krishnamurthy D, Li E, Vyas P, Akireddy N, Chai C, and Prakash M. (2020). Squid: Simplifying quantitative imaging platform development and deployment. Preprint at bioRxiv. 10.1101/2020.12.28.424613. [DOI]

[R38] 38.Bray SR, Wyss LS, Chai C, Lozada ME, and Wang B. (2024). Adaptive robustness through incoherent signaling mechanisms in a regenerative brain. Cell Rep. 43, 114580. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] 39.Chen K, Wang B, Guan J, and Granick S. (2013). Diagnosing heterogeneous dynamics in single molecule/particle trajectories with multiscale wavelets. ACS Nano 7, 8634–8644. [DOI] [PubMed] [Google Scholar]

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PERMALINK

A genetic and microscopy toolkit for manipulating and monitoring regeneration in Macrostomum lignano

R Nelson Hall

Hongquan Li

Chew Chai

Sidney Vermeulen

Robin R Bigasin

Eun Sun Song

Souradeep R Sarkar

Jesse Gibson

Manu Prakash

Andrew Z Fire

Bo Wang

SUMMARY

In brief

Graphical Abstract

INTRODUCTION

RESULTS

A modular cloning protocol enables rapid generation of tissue-specific reporter lines

Figure 1. A modular cloning protocol enables rapid generation of tissue-specific reporter lines.

Tissue-specific labeling resolves detailed anatomy

Figure 2. Tissue-specific labeling resolves detailed anatomy.

Time-course imaging using tissue-specific reporters reveals stages of posterior regeneration

Figure 3. Time-course imaging using tissue-specific reporters reveals stages of posterior regeneration.

Luminescence imaging tracks neural ablation outcomes

Figure 4. Luminescence imaging tracks neural ablation outcomes.

Tracking microscopy enables continuous imaging of posterior neural regeneration in free-moving animals

Figure 5. Tracking microscopy enables continuous imaging of posterior neural regeneration in free-moving animals.

Continuous live imaging allows quantification of regeneration progress across scales

Figure 6. Continuous live imaging allows quantification of regeneration progress across scales.

Multi-modal imaging characterizes posterior regeneration defects induced by β-catenin RNAi

Figure 7. Multi-modal imaging characterizes posterior regeneration defects in β-catenin RNAi animals.

DISCUSSION

Limitations of the study

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

STAR★METHODS

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animal care and maintenance

METHOD DETAILS

Cloning

Egg preparation

Microinjection

Amputations

Luminescence/fluorescence microscope

Tracking microscope

Live luminescence and fluorescence confocal imaging

Preparation of long-term imaging chambers

Chemical ablation

RNAi

Fixation

H3P immunostaining

Hybridization chain reaction

QUANTIFICATION AND STATISTICAL ANALYSIS

Image processing

Tracking microscope image processing

Supplementary Material

Highlights.

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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