https://orcid.org/0009-0000-7791-8272
This is an uncorrected proof.
Abstract
Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like wnt11-6/wntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Consistent with these results, eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. map3k1(RNAi) ectopic eyes emerged in the regions normally occupied by migratory eye progenitors, and these animals produced a net excess of differentiated eye cells. Furthermore, the formation of ectopic eyes after map3k1 inhibition coincided with an increase to numbers of differentiated eye cells, a decrease in numbers of ovo+ eye progenitors, and also was preceded by eye progenitors prematurely expressing opsin/tyosinase markers of eye cell terminal differentiation. Therefore, map3k1 negatively regulates the process of terminal differentiation within the eye lineage. Similar ectopic eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations.
Author summary
During adult regeneration, progenitors must migrate and differentiate at the proper locations in order to successfully restore lost or damaged organs and tissues, yet the mechanisms underlying these abilities are not fully understood. The planarian eye is a model to study this problem, because this organ is regenerated using migratory progenitors that travel long distances through the body in an undifferentiated state prior to terminal differentiation upon their arrival at target destinations. We determined that a pathway involving the MAP kinase kinase kinase map3k1 holds planarian eye progenitors in an undifferentiated state during their transit. Inhibition of map3k1 caused a dramatic body transformation in which migratory progenitors differentiate inappropriately early, and in the wrong locations, into mature eyes. By analyzing this phenotype and measuring the change to eye progenitor abundance after map3k1 inhibition, we found that map3k1 prevents ectopic differentiation of eye cells rather than mediating body-wide patterning through the Wnt pathway. Our study argues that whole-body regeneration mechanisms involve separate steps to control patterning and progenitor differentiation.
Figures
Citation: Lo KC, Petersen CP (2025) map3k1 suppresses terminal differentiation of migratory eye progenitors in planarian regeneration. PLoS Genet 21(3): e1011457. https://doi.org/10.1371/journal.pgen.1011457
Editor: Tin Tin Su,, University of Colorado, UNITED STATES OF AMERICA
Received: October 9, 2024; Accepted: February 19, 2025; Published: March 17, 2025
Copyright: © 2025 Lo, Petersen. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by National Institutes of Health grant NIGMS R35GM149280 (to C.P.P.), and NSF-GRFP to K.C.L (DGE-2234667). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The process of regeneration restores damaged tissue in a spatially coordinated manner that produces new parts in the correct locations and proportions. This process requires precise control of programs which detect injury, re-establish global body axis, activate stem cell proliferation and differentiation, target progenitor to the proper areas, and ultimately assemble cells into functional tissues and organs. While the pathways controlling progenitor specification for regeneration have become increasingly resolved, still little is known about the processes enabling the targeting of these cells to appropriate locations. The planarian Schmidtea mediterranea can replace nearly any adult tissue after injury and are a model for understanding whole-body regeneration mechanisms [1]. This ability requires the neoblast population of pluripotent stem cells, which constitute the organism’s only adult somatic proliferative cell type [2,3]. Elimination of neoblasts by gamma-irradiation prevents regeneration, while transplantation of single neoblasts into irradiated animals rescues this regeneration defect [2]. After injury, neoblasts proliferate and specify into specialized progenitors in order to differentiate into the different cell types required for building new tissue. The progenitors building regionalized tissues such as eyes and pharynx are specified in broader domains along the body axes and are believed to subsequently migrate to specific target positions in order to build new organs through regeneration or to homeostatically maintain existing organs through gradual cell replacement [4]. However, the mechanisms ensuring that stem cells terminally differentiate only at the correct locations are not well understood.
The planarian eye is a paradigm for understanding the mechanisms of organ regeneration because of its simple structure, easy observation in live animals, and its ability to be manipulated through gene perturbation or surgical resection. Planarians have two bilateral eyes residing in the anterior part of body, containing populations of opsin+ rhabdomeric photoreceptor neurons (PRNs), as well as tyrosinase+ optic cup cells (also known as pigment cups cells, PCCs). PRNs produce ARRESTIN+ axons which project ipsilaterally and contralaterally to enervate to the ventral brain. Axon guidance involves interactions with a specific set of notum+ and frizzled5/8-4+ muscle cells which act as guidepost cells to provide cues for the proper formation of the neuronal circuit and visual axon bundles [5]. The lateral portion of each eye is composed of PCCs believed to use melanin to allow light shadowing of PRN inputs enabling appropriate left/right discrimination for negative phototaxis [6–8]. New eyes can regenerate after surgical eye ablation, damage to the eye regions, or after decapitation during head regeneration. Eye regeneration initially involves specification of naïve neoblasts into eye progenitors through expression of transcription factors ovo, eyes absent (eya), and sine oculis-1/2 (six-1/2). In uninjured animals, ovo+ eye progenitors are dispersed throughout the anterior half of the body, and in eye regeneration, these cells coalesce into “trails” located posterior and lateral to the newly forming eyes. BrdU pulse-chase experiments suggest these trails of eye progenitors migrate and are the source of new eye cells during regeneration [7]. Additionally, eye transplantation and resection experiments found that eyes and/or their surrounding tissue form self-organizing centers which have the ability to attract migratory eye progenitors to sustain themselves homeostatically [9]. During specification, eye progenitors are partitioned into distinct eye cell subpopulations: otxA+/ovo+ progenitors which differentiate into opsin+ PRNs and sp6-9+/dlx+/ovo+ progenitors that generate tyrosinase+ PCCs [6,7]. The expression of these fate-specifying transcription factors is also retained after eye progenitors terminally differentiate into mature eye cells. Proper formation of each subclass of PRN cells also involves additional eye transcription factors soxB, meis, klf, and foxQ2, as their inhibition by RNA interference (RNAi) resulted in aberrant optic cup phenotypes such as small eyes and elongated optic cup morphology, without decreasing eye progenitor numbers [6]. Eye differentiation was also reduced following inhibition of egfr-4 [10] and bcat-4 [11], and formation of PRNs increased after inhibition of the NuRD complex component p66 [12]. Thus, a complex network of factors regulates eye specification and regeneration, but the signals controlling the propensity for migratory cells to mature remain poorly understood.
In addition to controlled eye cell specification, body-wide patterning factors ensure that eyes form in the correct position and proportion. Position control genes (PCGs) are signaling factors expressed in the body-wall muscle which establish global body axis and determine regional tissue identities [13,14]. The anterior-posterior (AP) axis in planarians is broadly defined by a gradient of β-catenin dependent Wnt signaling, with wnt1 expression defining the posterior and Wnt inhibitors such as notum expressed in the anterior [15–22]. Inhibition of notum in uninjured animals or in regenerating head fragments causes an anterior shift of regional head identity resulting in formation of a set of eyes anterior to the original pre-existing eyes [23]. Inhibition of other head patterning factors such as wnt11-6 (also known as wntA), a Wnt signaling factor which regulates brain size via a negative feedback loop with notum [24], and nou darake (ndk), a fibroblast growth factor receptor-like (FGFRL) gene which restricts brain tissues to the head [25,26], can also cause a set of ectopic eyes to form posterior to pre-existing eyes. Inhibition of src-1, a non-receptor tyrosine kinase, caused phenotypes resembling a combination of the Wnt/FGFRL pathways to result in posterior expansion of head, formation of posterior ectopic eyes, as well as ectopic trunk formation [27]. Inhibition of slit and wnt5, regulators of the medial-lateral (ML) axis, cause medial or more lateral ectopic eyes to form, respectively [28]. Modification of Wnt/FGFRL signaling and Wnt5/Slit signaling also causes AP and ML changes to brain structure, respectively, suggesting these patterning signals more broadly control head regional identity that also affects the eyes [24–29]. Intriguingly, although regeneration of new eyes occurs at precise locations defined by PCG genes, eye homeostasis can occur in broader domains. Eyes transplanted into the anterior but not posterior regions of the body are homeostatically maintained by migratory eye progenitors [9], and inhibition of patterning factors can shift the site of new eye regeneration without altering eye homeostasis [23,27]. However, it is not yet clear what signals may coordinate eye progenitors to their target destinations and enable their proper differentiation specifically at the location of existing eyes.
Here we identify a pathway involving mitogen-activated protein kinase kinase kinase 1 (map3k1) that specifically regulates the site of terminal differentiation of migratory eye progenitors. map3k1 (also known as MEKK1) is a mitogen-associated protein kinase kinase kinase known to transduce extracellular signals into a diverse set of intracellular responses, including cell survival, cell migration, and in apoptosis [30]. MAP3K1 factors contain a C-terminal kinase domain characteristic of MAP kinases, as well as a RING domain with E3 ubiquitin ligase activity [31,32], a unique combination among MAP3Ks. Our research identifies a novel role for planarian map3k1 in suppressing terminal differentiation and preserving the eye progenitor state until they reach their target destination.
Results
map3k1 RNAi causes formation of ectopic eyes
We conducted a small-scale RNAi screen of signaling molecules whose expression had been described previously as activated by injury. We identified a single planarian homolog of map3k1 (dd_Smed_v6_5198_0_1) which encodes a protein with predicted kinase and RING finger domains typical of MAP3K1 family members (S1A Fig). Prior studies in Schmidtea mediterranea had identified the map3k1 transcript as rapidly induced within the first 30 minutes after injury [33,34] but the role of this gene in regeneration was unclear. In order to investigate the function of this gene, we inhibited map3k1 by RNAi using 6 dsRNA doses administered over 2 weeks, then challenged the worms to regenerate. map3k1(RNAi) animals succeeded at formation of blastemas, but newly formed heads produced ectopic eyes in positions that appeared in lateral-posterior locations within the blastema (Fig 1A). The ectopic eye phenotype had the greatest penetrance in head fragments (15/15 animals, 100% penetrance), compared to regenerating trunks (13/15 animals, 87%) and regenerating tail fragments (9/15, 60%). We used FISH to confirm that ectopic eyes in map3k1(RNAi) animals contain both opsin+ PRNs and tyrosinase+ PCCs (Fig 1B), and anti-ARRESTIN antibody staining shows that map3k1(RNAi) ectopic eyes can also make axon projections to the brain (Fig 1C). These results suggest that map3k1(RNAi) ectopic eyes possess the morphology of normal eyes.
(A-B) Animals were fed control or map3k1 dsRNA 6 times over 2 weeks, then amputated into head, trunk and tail fragments and allowed to regenerate for 14 days or left uninjured for an equal amount of time, followed by (A) live imaging or (B) FISH to detect opsin and tyrosinase markers of eye cells. In each type of regenerating fragment, map3k1 RNAi caused formation of ectopic eyes (red arrowheads) that contained opsin+ photoreceptor neurons and tyrosinase+ pigment cup cells. (C) Homeostatic map3k1(RNAi) and control animals were stained with anti-ARRESTIN antibody to mark photoreceptor neuron axons. map3k1 inhibition caused formation of ectopic ARRESTIN+ axons projecting toward the brain (white arrowheads). Scale bars 50μm. (D) Uninjured animals were fed with dsRNA twice per week for the times indicated and live imaged in a timeseries to visualize the progression of the map3k1(RNAi) phenotype. map3k1(RNAi) caused a progressive formation of additional eyes mainly located within the head region. Scorings indicate the number of animals which appeared normal (controls) or because of map3k1 RNAi had any ectopic eyes (A, B, D) or ectopic axons (C).
Given map3k1’s demonstrated transcriptional activation in the early wound response, we wanted to determine whether its role in eye patterning required its injury-induced gene expression. To test this possibility, we inhibited map3k1 homeostatically and examined the impact on eye formation (Fig 1A). Long-term inhibition of map3k1 in the absence of injury resulted in highly penetrant formation of ectopic eyes (15/15, 100%), indicating map3k1 regulates an eye formation process common to both regeneration and homeostasis. The map3k1(RNAi) phenotype appeared to progress over time, with ectopic eyes appearing by 2 weeks of RNAi, and additional eyes appearing over time through 3-8 weeks of treatment (Figs 1D and S1B). map3k1(RNAi) animals survived through at least 8 weeks of RNAi, suggesting that map3k1 likely does not regulate viability. Together, map3k1 inhibition led to formation of additional eyes at particular locations, with relatively normal composition and in an injury-independent manner.
map3k1 specifically regulates eye formation, not overall head patterning
In order to understand map3k1’s role in the process of eye formation and regeneration, we compared the map3k1(RNAi) phenotype to inhibition of ndk, known to cause posterior duplication of eyes and posterior expansion of the brain. We first stained map3k1(RNAi) animals for cintillo+ cells, a population of antero-lateral neurons involved in mechano- or chemo-sensation and whose numbers scale proportionally to body size [35]. While ndk(RNAi) animals expanded cintillo+ cell numbers posteriorly as expected, map3k1(RNAi) animals had no change in AP position and average number of cintillo+ cells as normalized to body area (Fig 2A). Similarly, ndk RNAi caused posterior expansion of brain branches marked by GluR+, while map3k1 inhibition did not appear to cause posterior brain expansion (Fig 2B). Additionally, map3k1 RNAi did not cause any identifiable increases in the brain size as assessed by the head domain of ChAT+ cholinergic neurons (S2 Fig). These results suggest map3k1 is unlikely to regulate eye production because of a role in global head patterning. To further examine this hypothesis, we tested markers of body regional identity to determine whether map3k1 might have other roles in AP body axis formation. map3k1 RNAi did not affect AP expression domains for a majority of regional markers, including anterior/head markers foxD, notum, and sfrp-1, head/trunk markers ndk and ndl-3, as well as tail markers wnt1, fzd4, and wntP-2 (Fig 2C). ndl-5, marking the head region, was the only exception, because map3k1(RNAi) animals had a slightly expanded ndl-5 domain compared to control animals. However, ndl-5 is unlikely to exert a strong role on eye patterning by itself, because prior work found that ndl-5 RNAi did not cause patterning phenotypes [25]. Together, we conclude map3k1 likely does not control global AP axis identity or head regional identity, and instead acts at a novel control point that primarily regulates eye formation at a regulatory step distinct from previously identified eye regulatory factors.
Homeostatic animals fixed after 3 or 4 weeks of RNAi were stained with markers to detect the effects of map3k1 on brain and body patterning. (A) ndk RNAi caused an expected increase in the domain size (left images) and number (right quantifications) of brain-associated cintillo cells, while map3k1 RNAi had no effect. Right, box plots overlayed with jittered scatterplots showing individual datapoints displaying the per-animal number of cintillo+ cells normalized to body area in millimeters2 (mm2). *, p<0.05; n.s. represents p>0.05 as calculated by 2-tailed unpaired t-test; sample sizes for each condition are n≥6. Scale bars, 100μm. (B) Likewise, map3k1 RNAi did not change the AP distribution of brain branches marked by GluR, while ndk RNAi resulted in ectopic brain branches forming throughout the body. Right, box plots showing the distance from the anterior tip of the animal to the most posterior GluR expression in the brain, relative to body length, for each of the corresponding RNAi conditions. ******, p<1E-7, n.s. represents p>0.05 as calculated by 2-tailed unpaired t-test; sample sizes for each condition are n≥7. Scale bars, 300μm. (C-D) Control and map3k1(RNAi) animals stained for position control gene (PCG) markers of (C) anterior and posterior body patterning. Expression domains were either assessed after 14 days of regeneration following 2 weeks of dsRNA feeding and amputation of tails (wnt1), or assessed in uninjured animals treated with dsRNA for 3 weeks prior to fixation (all other probes). Right, plots showing quantification of expression domain sizes normalized to body length in Fiji/ImageJ. The sizes of foxD, notum, sfrp1, ndk, and ndl5 expression domains were measured starting from the head tip to the posterior-most boundary of expression. ndl3 occupies a position that does not reach the tip of the head, so the AP extent of this domain was measured instead. wnt1, fzd4, and wntp-2 expression domain sizes were measured from the anterior-most expression to the tip of the tail. Each condition had a sample size of at least 5 animals. **, p<0.01; n.s., p>0.05 as calculated by 2-tailed unpaired t-test. Scale bars, 300μm. map3k1 RNAi did not cause a measurable change to the expression domains for the majority of genes tested, and caused a small but statistically significant increase in the expression domain of ndl5 expression. Scorings indicate the number of animals with characteristics similar to the images shown.
map3k1 likely functions independently from Wnt signaling in eye regeneration
Our analysis of the map3k1 RNAi phenotype suggested this gene likely controls a distinct step in eye formation compared to patterning signals, and we used double-RNAi analysis to further examine this possibility. In uninjured animals and regenerating head fragments, RNAi of the Wnt antagonist notum causes an anterior shift in head patterning to produce a second set of anteriorly positioned eyes [23]. We reasoned that if map3k1 functioned obligately downstream of notum, then dual inhibition of both notum and map3k1 might tend to produce only the ectopic posterior eyes phenotype from map3k1 RNAi. To test this, we inhibited notum and map3k1 together under homeostatic conditions, and then compared this outcome to single-gene inhibitions spiked with similar amounts of competing control dsRNA (Fig 3). Under these conditions, notum(RNAi) animals all formed anterior ectopic eyes (13/13 animals, 100%) and map3k1(RNAi) animals formed ectopic posterior ectopic eyes (14/14 animals, 100%). By contrast, the majority of notum;map3k1(RNAi) animals displayed a synthetic phenotype in which both ectopic anterior and posterior eyes formed (14/15 animals, 93%). These outcomes argue that map3k1 likely controls eyes formation at a step independent from notum/Wnt signaling and consequently head patterning.
Double RNAi experiments were conducted to test potential interactions between notum and map3k1 genes whose individual inhibition causes spatially distinct ectopic eye phenotypes. Animals were fed dsRNA every 2-3 days for 4 weeks before live scoring (top panels) and fixation to detect opsin+ eye cells (bottom panels). As expected, notum(RNAi) animals formed anterior ectopic eyes (13/13), while map3k1(RNAi) animals formed posterior ectopic eyes (14/14). However, nearly all map3k1;notum(RNAi) animals formed a synthetic phenotype in which both anterior and posterior ectopic eyes formed (14/15). Therefore, it is likely that map3k1 and Wnt pathways regulate distinct processes in eye formation. Yellow arrows are used to highlight anterior ectopic eyes while green arrows are used to highlight posterior ectopic eyes. Single-RNAi conditions involved combining control dsRNA with an equal amount of experimental dsRNA so that all treatments received the same total amount of dsRNA. Scale bars, 100μm.
In normal uninjured animals, eye removal by resection results in regeneration of a new eye at precisely the original location. By contrast, removal of eyes in animals inhibited for patterning factors wnt11-6/wntA, fzd5/8-4, src-1 or notum, eyes only regenerate at the location of the newly formed ectopic eyes, and not at the location of pre-existing eyes, despite being able to be maintained for long periods of time homeostatically [23,27]. In principle, map3k1 could control patterning within the head region but in a manner specific to eye placement and not brain patterning. To test this possible model, we measured the outcomes of original and ectopic eye removal in map3k1(RNAi) animals. In homeostatic map3k1(RNAi) animals, removal of the original eyes resulted in eye regeneration at that location in 100% of animals (S3 Fig). Similarly, removal of ectopic eyes from these animals resulted in regeneration in these same locations in 50% of animals. Therefore, in these experiments, map3k1 inhibition did not shift the target location for eye regeneration away from the original eyes. Instead, eye regeneration became permissible in an expanded domain. These outcomes further support a model in which map3k1 likely acts independently of Wnt/notum signaling and suggests a role distinct from known patterning factors.
map3k1 is expressed broadly throughout the body, including in eye cells
We then investigated the expression of map3k1 transcripts in the body, with particular interest with whether this factor is expressed in differentiated eye cells, undifferentiated eye progenitors, or other cell types. By analysis of published single-cell RNAseq data of intact animals, map3k1 expression was broad and present in many cell types, including muscle, gut, neurons, and protonephridia (S4A Fig, top panels) [36]. Similarly, we re-analyzed a single-cell RNAseq dataset of neoblasts FACS-sorted from 72-hour head blastemas to determine map3k1 expression in these cells. At this early stage of regeneration, eye progenitors are strongly marked by expression of six-1/2 [37], which operates in conjunction with ovo to specify these cells from naïve neoblasts [6]. In this dataset, map3k1 expression was detected weakly across all neoblasts, including within the eye progenitor clusters (S4A Fig, bottom panels). FISH using a map3k1 riboprobe displayed broad staining across most regions of the animals, putatively in locations around the brain, flame cells, and also near the eyes, based on anatomical location (S4B Fig). We then used double-FISH to co-detect map3k1 with either opsin or ovo to verify whether map3k1 is expressed in differentiated eye cells or eye progenitors, respectively. map3k1 FISH signal was weak and broad and in most cells took on a sparse punctate appearance, and was detected throughout the opsin+ cells in all animals (4/4, 100%) (S4C Fig). We also found similarly weak map3k1 expression within a subset of ovo+ progenitors (S4D-E Fig, yellow arrowheads) (22/36 ovo+ cells were positive for map3k1 FISH signal), while map3k1 could not be detected in other ovo+ cells (14/36 ovo+ cells were negative for map3k1 FISH signal). We also noticed some nearby ovo- cells of unknown cell type that had higher levels of map3k1, which could either represent cells of an unknown function in eye regeneration or could represent cells unrelated to the eye system (S4D Fig, green arrows). These results are consistent with map3k1 acting either within mature eye cells or migratory eye progenitors, or could indirectly regulate eye formation through action in an alternative cell type.
map3k1 RNAi increases numbers of differentiated eye cells
Given that map3k1 has broad expression that included eyes and eye progenitors, but functions independently of Wnt signaling and head patterning, we considered whether map3k1 might instead regulate the process of eye cell differentiation. If map3k1 only regulated the locations but not extent of eye cell differentiation, we predicted the total numbers of mature eye cells in each animal would be unaltered after map3k1 RNAi, similar to prior results that determined notum(RNAi) animals with more eyes do not contain more differentiated eye cells [23]. By contrast, map3k1 RNAi might instead produce more eyes through excess or premature differentiation, resulting in a greater number of differentiated eye cells in each animal. To investigate map3k1’s influence on eye cell abundance, we quantified the total number of opsin+ differentiated eye cells in control versus map3k1(RNAi) homeostatic animals. To make these measurements, we sampled regularly-spaced confocal slices from z-stacks of the animal eyes, detected number of eye cells per slice using 2D segmentation of nuclei followed by measuring overlap with opsin+ FISH signal after applying thresholding, and summed opsin+ nuclei across slices sampled for each animal (see methods, Fig 4A). This analysis revealed that map3k1(RNAi) animals contained significantly more differentiated opsin+ cells compared to control animals (Fig 4B and 4C) and were also distributed in a broader spatial range compared to control animals. The detection of an increase in opsin+ cell numbers after map3k1 RNAi was also robust to variation of the exact threshold and inter-slice distance chosen (see Methods). To examine the possibility of any systematic differences in segmentation quality across the two treatments, we calculated a Jaccard Similarity Index (JSI, also known as the “Intersection over Union” metric) on randomly selected animals and z-slices, then compared these metrics between control and map3k1(RNAi) conditions. We found that the mean JSIs for control and map3k1(RNAi) samples were 0.55 and 0.49, respectively, and did not differ from each other as measured by a 2-tailed unpaired t-test (p=0.23, S2 Table). Therefore, the differences in estimated cell numbers were not likely due to any systematic differences in the segmentation and counting efficiency across the treatments. We conclude that map3k1 RNAi causes an increase in total opsin+ eye cell numbers per animal, in addition to causing the formation of ectopic eyes. Together, these observations suggest that map3k1 normally limits both the location and extent of eye cell differentiation.
Uninjured control and map3k1(RNAi) animals fixed after 3 weeks of RNAi and stained with opsin riboprobe to quantify numbers of eye cells in each condition. Eye cells are present in close association with each other, necessitating an image analysis workflow for their quantification. Z-stacks capturing all eye cells were obtained through confocal imaging, then slices 5-microns apart were selected to represent each stack for 2D segmentation using Stardist, followed by assignment of nuclei as opsin+ using a global threshold and summing number of positive cells across the selected stacks for each animal. (A) Example of an image slice after nuclei segmentation and assignment of opsin+ nuclei (red overlay, right) of how opsin+ cells were counted for one z-stack. (B) Scaled images of example z-stacks from control and map3k1(RNAi) animals showing that map3k1 inhibition resulted in an expansion of eye regions. (C) Total number of opsin+ cells counted in control versus map3k1(RNAi) animals. map3k1 inhibition caused an increase to the number of measured eye cells. Plots show data points overlaid with boxplots. **p=0.01 by 2-tailed unpaired t-test. n=8 animals. Scale bars, 25μm.
map3k1(RNAi) ectopic eyes form in regions normally occupied by eye progenitors
We hypothesized that map3k1 might control the terminal differentiation of eye progenitors and reasoned that the locations of ectopic eye formation might provide additional information useful for distinguishing this role from the patterning systems regulating eye positioning. We noted that map3k1(RNAi) ectopic eyes seemed to emerge in a particular region of the body laterally and posteriorly to the normal eye location. By contrast, extra eyes forming after inhibition of wnt11-6/wntA, ndk, or notum often appear symmetrically and in specific locations [23,26]. To examine these distributions, we systematically quantified eye position in images of live animals following homeostatic RNAi of map3k1, wnt11-6/wntA, notum, and ndk. Because planarians lack a fixed size and readily grow or de-grow while maintaining proportionality, we normalized the AP and ML position of each ectopic eye with respect to the original eye locations in each animal (see Methods). This analysis confirmed that map3k1(RNAi) ectopic eyes generally form across a broad region located laterally and posterior to the original eyes, whereas the majority of wnt11-6(RNAi) and ndk(RNAi) ectopic eyes form in a more concentrated region located directly posterior to the original eyes (Fig 5A-C). This analysis provides further confirmation that map3k1 likely controls a distinct step in eye formation compared to wnt11-6/wntA and ndk.
Homeostasis animals were treated with the indicated dsRNA for 3 weeks (control RNAi, wnt11-6 RNAi) or 6 weeks (control RNAi, map3k1 RNAi, ndk RNAi) followed by live imaging to detect the location of eyes or fixing and staining to detect the location of migratory ovo+ cells (eye progenitors) in unfed uninjured animals. (A) Planarians lack a fixed size, so in order to make comparisons across treatments, locations of ectopic eyes and eye progenitors in each image were defined and then registered and normalized to the location of the original eyes in order to create a common coordinate system (see Methods). These data were plotted along normalized AP (y.coord) and ML (x.coord) axes with units equal to one-half of the distance between the normal eyes of control animals or between the original eyes in ndk(RNAi), wnt11-6(RNAi), and map3k1(RNAi) conditions. Eye position measurements and assignment to a coordinate system are shown for a representative map3k1(RNAi) animal. (B) Scatterplot of control eyes (black dots, from 5 animals) versus map3k1(RNAi) ectopic eyes (light blue dots, from 8 animals) shows that map3k1 inhibition caused formation of ectopic eyes in a distribution located laterally and posteriorly compared to control eye locations. However, rare map3k1(RNAi) ectopic eyes were also identified slightly anterior to the original eyes (2 blue dots with y.coord > 0). (C, top) Plots of ectopic eye locations in ndk(RNAi) (green dots) or wnt11-6(RNAi) animals (purple dots) with respect to control eyes (black dots). (C, bottom) Graphs comparing locations of ectopic eyes in map3k1(RNAi) (blue dots) versus either ndk(RNAi) animals (green dots) or wnt11-6(RNAi) animals (purple dots). Both ndk RNAi and wnt11-6 RNAi caused a tighter distribution of ectopic eyes that were located more directly posterior to the original eyes compared to the broader distribution of map3k1(RNAi) ectopic eyes located more laterally. (D, left) Locations of migratory ovo+ eye progenitors from control uninjured animals (pink dots) were compared to ovo+ mature eyes (black). (D, right) Locations of ovo+ eye progenitors (pink dots) were compared to locations of ectopic eyes from map3k1(RNAi) animals (blue dots). map3k1 inhibition caused formation of ectopic eyes in a set of locations overlapping with the location of the eye progenitors from control animals.
The position of map3k1(RNAi) ectopic eyes were instead reminiscent of the distribution of ovo+ eye progenitor “trails” leading to the eye that are most evident 5-8 days after decapitation, during the late stages of head regeneration [6]. We hypothesized that if map3k1 limits the terminal differentiation of migratory progenitors without influencing patterning, then ectopic eyes in map3k1(RNAi) animals might be most likely to form within regions that ordinarily contain ovo+ cells. To make a systematic comparison, we employed a similar positional mapping strategy used above, to measure the location of ovo+ cells in homeostatic control animals, then plotted this distribution in comparison to the position of map3k1(RNAi) eyes (Fig 5D). map3k1(RNAi) ectopic eyes were located within a subset of the region occupied by ovo+ cells in control animals (Fig 5D). In the process of undertaking this analysis, we noticed a few very rare occurrences in which ectopic eyes from map3k1(RNAi) animals formed anterior to the original eyes (2/8 animals had 1 anterior eye each in this experiment, Fig 5B). These observations, while encompassing only a small fraction of eyes surveyed, provide additional evidence that map3k1 likely does not exclusively control the process of anterior versus posterior eye placement. Similarly, the majority of ovo+ cells were posterior to the existing eyes, as reported previously, but rare ovo+ cells could also be detected anterior to the eyes (Fig 5D). This observation is consistent with a model in which ovo+ eye progenitor specification normally takes place over a broad anterior region, including to some extent anterior to the eyes, but receive signals which hone progenitors to migrate and incorporate into eyes for cell replacement and growth [6,9]. Furthermore, the distribution of map3k1(RNAi) ectopic eyes is consistent with a model in which map3k1 normally maintains ovo+ migratory progenitors in an undifferentiated state prior to arrival at the precise location of terminal differentiation.
map3k1 suppresses differentiation of migratory ovo + eye progenitors
Based on these findings, we hypothesized map3k1 might regulate terminal differentiation rather than the initial specification of ovo+ cells. To test this possibility, we examined the number of ovo+ cells across different stages of head regeneration, beginning with ovo+ cell specification from neoblasts within 2-3 days of injury to their subsequent localization into posterolateral trails that support the differentiation of new eye cells within the blastema. In map3k1(RNAi) trunk fragments, a single set of seemingly normal eyes formed by 3-7 days after amputation, followed by formation of ectopic eyes after 2 weeks of regeneration (Fig 6A). We used a FISH experimental strategy to quantify numbers of eye progenitors during this process. Double-FISH simultaneously detecting ovo along with a mixture of opsin and tyrosinase riboprobes allowed distinguishing progenitors from mature eye cells. We then compared the number of ovo+ progenitor cells in control versus map3k1(RNAi) trunk fragments across a time series of regeneration (Fig 6B). The number of undifferentiated ovo+ eye progenitors were unchanged between map3k1(RNAi) and control animals at days 0, 2, 5, and 8 post-amputation. However, map3k1 RNAi caused a decrease to the number of ovo+ eye progenitors at 14 days post-amputation, at a time coinciding with the emergence of ectopic eyes in these animals. Based on this result and our analysis showing that map3k1(RNAi) homeostatic animals have greater numbers of differentiated eye cells (Fig 4), we also expected to observe an increase in the number of differentiated eye cells coinciding with the decrease in number of eye progenitors during head regeneration. To investigate this possibility, we counted the number of opsin/tyrosinase+ differentiated cells present in mature eyes using a similar strategy as described in Fig 4, by confocal imaging, 2D segmentation of nuclei, and summation of nuclei determined to be opsin/tyrosinase+ across the stack (see methods). The total number of opsin/tyrosinase+ differentiated eye cells was similar in control and map3k1(RNAi) animals at 8 days post-amputation (Fig 6C), a time point at which there were no differences in the numbers of ovo+ eye progenitors and these animals had not yet formed ectopic eyes. However, by 14 days, map3k1(RNAi) animals had greater total numbers of differentiated eye cells per animal than controls at the same time they had a decrease to numbers of eye progenitors. Therefore, map3k1 inhibition decreases the number of eye progenitors while also increasing the number of terminally differentiated eye cells, suggesting that map3k1 regulates the terminal differentiation of eye progenitors, rather than their specification from neoblasts. Consistent with this model, in map3k1(RNAi) animals, we also found that a small subset of migratory ovo+ cells (i.e., ovo+ cells located in trails outside of mature eye structures) prematurely expressed opsin/tyrosinase markers of terminal eye cell differentiation (Fig 6A, yellow arrows). Quantifying numbers of these isolated ovo+opsin/tyrosinase+ cells outside of mature eyes, we found map3k1(RNAi) animals indeed had significantly more of these cells than control animals at both 8 and 14 days post-amputation (Fig 6D). These observations directly support a model in which map3k1 inhibition causes eye progenitors to prematurely express markers of terminal differentiation. Taken together, these results indicate that map3k1 likely does not function in the initial specification of naïve neoblasts into ovo+ migratory eye progenitors, but instead negatively regulates the process of eye progenitor cell terminal differentiation.
(A) Animals were treated with control and map3k1 dsRNA for 2 or 3 weeks prior to amputation of heads, and the resulting regenerating trunk fragments were fixed in a time series followed by staining with an ovo riboprobe to detect migratory eye progenitors (red) and simultaneously with mixture of opsin and tyrosinase riboprobes (green) to detect mature eye cells. Yellow coloring indicates overlap of green and red signals, with mature eyes expressing both ovo and opsin/tyrosinase. Data for 0dpa and 2dpa were aggregated from two experiments. Data for 5dpa, 8dpa, and 14dpa were aggregated from two different experiments. In map3k1(RNAi) animals, a subpopulation of migratory ovo+ cells located outside of mature eye structures expressed high levels of opsin/tyrosinase+ (examples indicated by yellow arrows, insets show magnifications of representative cells), while ovo+ progenitors in control animals had minimal opsin/tyrosinase+ expression (insets) (qualifications in panel D). (B) Boxplots showing the numbers of ovo+ migratory progenitors for each timepoint and condition quantified by manual scoring of maximum projection images. The number of undifferentiated ovo+ eye progenitors did not change significantly in map3k1(RNAi) worms at 0, 2, 5, and 8dpa. At 14dpa, a time when ectopic eyes began to emerge, map3k1(RNAi) worms showed a decrease in ovo+ eye progenitors. (C) Boxplots showing the total number of opsin/tyrosinase+ differentiated eye cells in animals at the indicated conditions and timepoints. The total number of opsin/tyrosinase+ differentiated eye cells in each animal were counted as in Fig 4 by confocal imaging of whole eyes, 2D segmentation of nuclei using Stardist on z-slices selected every 5 microns, assigning nuclei as opsin/tyrosinase+ using a global threshold, and summing the total number positive cells across all z-stacks for each animal. Numbers of opsin/tyrosinase+ differentiated eye cells were the same between control and map3k1(RNAi) animals at 8 dpa but their numbers increased in map3k1(RNAi) animals at 14 dpa. (D) Boxplots quantifying numbers of ovo+ cells away from mature eyes that prematurely expressed opsin/tyrosinase (denoted by yellow arrows in (A) in animals of each condition at 8 dpa and 14 dpa, as manually scored from maximum projection confocal images. map3k1 RNAi significantly elevated the number of migratory cells expressing opsin/tyrosinase markers of terminal eye cell differentiation at both 8dpa and 14dpa. Plots show data points from individual animals overlaid with boxplots. *p<0.05, **p<0.01, ***p<0.001, n.s. represents p>0.05 by a 2-tailed unpaired t-test. Scale bars, 100μm.
map3k1 likely signals through map2k-4 and map2k-7 to suppress eye formation
We used the molecular identity of map3k1 to inform targeted secondary RNAi screens in order to determine a candidate pathway of action for this gene in eye formation. In a typical MAP Kinase pathway, MAP3Ks signal to MAP2Ks, which then activate MAPK in order to regulate downstream responses. To identify the map3k1 pathway relevant for eye formation in planaria, we first identified all 8 MAP2K genes then screened these individually by RNAi to determine whether any would phenocopy the ectopic eye effect. However, knockdown of planarian MAP2K genes individually did not produce any eye phenotypes (Fig 7A), suggesting a potential redundancy in their uses. We then inhibited combinations of genes and found that map2k4;map2k7(RNAi) animals formed ectopic eyes in regenerating head fragments (4/10, 40%) similar in appearance, though at a lower penetrance, to the effects of map3k1 RNAi (Fig 7B). MAP2K4 and MAP2K7 are known to transduce mammalian MAP3K1 signals, and frequently do so by regulating p38 and/or JNK downstream MAPKs [38]. We therefore inhibited the two planarian p38 genes in combination, as well as jnk, by administering 6 dsRNA feedings over 2 weeks followed by 14 days of regeneration. Under these conditions, inhibition of jnk or both homologs of p38 caused ectopic eye phenotypes similar to map3k1 inhibition, though again at a low penetrance (Fig 7C). The reduced penetrance from inhibitions of downstream MAP2K and MAPK factors, compared to map3k1 inhibition, could indicate further uncharacterized redundancy, or perhaps inefficient protein knockdown from RNAi. These factors likely also have independent uses and inputs in planarians, as previously argued from other studies using alternate dsRNA dosing schedules and methods to perturb JNK and p38 signaling [39–42]. However, our results argue that map3k1 likely signals via MAP2K4/MAP2K7 and JNK/p38 in order to suppress differentiation of planarian eye cells.
Members of MAP2K and MAPK gene families were inhibited in order to identify signals downstream of map3k1 regulating eye formation. Animals were treated with indicated dsRNAs for 2 weeks (A-B) or 3 weeks (C) prior to amputation of tails and allowed to regenerate for 7 days (B) or 14 days (A, C) followed by live imaging. (A) While map3k1(RNAi) animals developed ectopic eyes as expected (red arrows), single gene inhibitions of map2k genes did not cause formation of ectopic eyes. (B) map2k4;map2k7(RNAi) animals were observed to develop posterior ectopic eyes (red arrow) through live imaging. Double FISH detecting opsin+ photoreceptor neurons and tyrosinase+ pigment cup cells showed that the ectopic eyes contained both opsin+ and tyrosinase+ cells (white arrows). (C) Inhibition of both homologs of the planarian p38, or jnk, caused ectopic eyes to form in regenerating head fragments. Scorings indicate the number of animals displaying the phenotype depicted in each panel.
Discussion
Our analysis identifies a critical and novel role for map3k1 in controlling the site of eye progenitor differentiation during eye regeneration in planarians (Fig 8). Several lines of evidence suggest map3k1 controls a distinct step in eye regeneration compared to other known patterning factors. map3k1 silencing caused eye-specific effects rather than also affecting brain:body patterning, map3k1 interacted synthetically with notum, the locations of ectopic eyes in map3k1(RNAi) differed from those in wnt11-6(RNAi) and ndk(RNAi) animals, and map3k1 RNAi did not cause the target location for eye regeneration to shift away from the location of the original eyes. Additionally, we found that map3k1 inhibition increases numbers of terminally differentiated eye cells, decreases eye progenitor numbers in regeneration, and causes eye progenitors to prematurely express markers of terminal differentiation. These results together suggest that map3k1 signaling regulates a novel control point involved in preventing terminal differentiation during the migration of eye progenitors to their target destinations. In our experiments, we observed expression of map3k1 at very low levels in a subset of eye progenitors, within differentiated eye cells, and also broadly in most tissues of the animal. Therefore, map3k1 could either operate directly within eye progenitors or control eye terminal differentiation indirectly from a different cell type.
Model of the role of map3k1 in eye regulation. Head patterning factors such as notum and wnt11-6/wntA regulate a target zone for eye placement in regeneration, while map3k1 suppresses terminal differentiation of migratory eye progenitors by activating Map2k4/Map2k7-JNK/p38 downstream signals. map3k1 and downstream factors could operate in migratory progenitors or some other cell type(s) to control eye cell terminal differentiation.
Map3k1 proteins in other organisms are known to be involved in control of multiple cellular outputs, including promoting cell survival and enabling migration, but a detailed knowledge of their roles in specific developmental pathways is still not fully understood. Mammalian Map3k1 was originally identified as a spontaneous mutant in 1966 called lidgap-Gates (lgGa) which displayed an eyes-open-at-birth (EOB) phenotype caused by a failure or delay in epithelial migration of the eyelid epidermis. lgGa was subsequently mapped and cloned as a deletion of Map3k1 [43,44]. Targeted mutations found that mice lacking the full-length map3k1 gene (Map3k1-/-) and also mice lacking the map3k1 kinase domain (Map3k1ΔKD/ΔKD) both display an EOB phenotype due to improper cell-cell interactions and failed migration of eyelid epithelial cells [32,45,46]. Eyelid closure involves a collective migration of epidermal cells and inputs from many pathways, including EGF, FGF, Wnt, TGF-beta, and JNK signaling [46–50]. However, due to the complexity of signals involved in this process, the precise signals impinging on Map3k1 are not yet fully understood. Map3k1 genes are not present in Drosophila melanogaster or Caenorhabditis elegans genomes, suggesting planarians could be a useful system for understanding the roles this pathway plays in differentiation.
Several possible developmental mechanisms could explain how map3k1 controls planarian eye cell differentiation. Prior work found that in the planarian Dugesia japonica, map3k1 (Djmekk1) was required for correct positioning of trunk tissues during regeneration [51]. In principle, these roles could be related to the control of terminal differentiation our study identifies, for example, if distinct RNAi dosing strategies reveal a broader role of map3k1 in migratory progenitor maintenance relevant to tissues beyond the eyes. It is therefore likely that the Schmidtea mediterranea map3k1 participates in signaling beyond eye formation due to the fact this gene is broadly expressed. Therefore, identifying tissue-specific upstream and downstream factors acting through map3k1 will be important for resolving the mechanisms by which this factor controls eye regeneration. Previous research determined that mammalian Map3k factors can be activated through a wide variety of signals, including EGF, FGF, or stresses such as cold shock, microtubule disruption, or LPA treatment, and pathways downstream of map3k1 can include stress-responsive MAP kinase pathways including MAP2K4 and MAP2K7 signaling to p38 or JNK [45,46,52,53]. While we not able to identify signals upstream of map3k1 in this study, we found that inhibition of map2k4 and map2k7, jnk, or p38 could cause a low-penetrance phenocopying of the map3k1 RNAi ectopic eye phenotype. Prior analysis of JNK or p38 signaling in planarians has also revealed their participation in several aspects of regenerative growth, including cell-cycle control, differentiation, and regulation of apoptosis [40–42,54,55]. However, our analysis suggests it is unlikely that map3k1 acts only to control the viability of differentiated eye cells or the proliferation of eye cells progenitors, because disrupting such processes would be predicted to change the size of mature eyes rather than generating ectopic eyes. Similarly, map3k1 likely does not promote the survival of migratory progenitors, because these cells did not increase in number following map3k1 inhibition. map3k1 could in principle promote cell death of any rare progenitors which attempt to terminally differentiate in incorrect locations. However, we suggest that the observation that eye progenitors begin to express markers of eye cell terminal differentiation after map3k1 RNAi gives stronger support for a model in which map3k1 specifically limits the eye cell terminal differentiation process. Together, our analysis reveals the potential existence of a Map3k1-Map2k4/Map2k7-JNK/p38 signaling pathway in planarians with a prominent role in controlling eye cell terminal differentiation.
Some aspects of the map3k1(RNAi) phenotype suggest complexity in the control of terminal differentiation used in regeneration. For example, in our experiments, knockdown of map3k1 did not result in complete elimination of all migratory eye progenitors through their terminal differentiation, because we were able to observe that ovo+ undifferentiated cells were still present in map3k1(RNAi) animals, though at reduced numbers during the process of ectopic eye formation. One possibility is that the map3k1 only acts directly or indirectly on a subset of eye progenitors ordinarily more prone to terminal differentiation. On the other hand, map3k1(RNAi) ectopic eyes did not simultaneously form in random positions within the head. Instead, the ectopic eye phenotype emerged progressively, with affected animals typically going through an intermediate with only 1 set of ectopic eyes followed by the emergence of another set at a more posterior location (Figs 1D and S1B). This observation implies that the effects of map3k1 inhibition display an A-P bias in which eye progenitors from successively more posterior regions terminally differentiate during the progression of the phenotype, yet without any substantial changes to position control gene expression domains in muscle. map3k1(RNAi) trunk fragments produced ectopic eyes only subsequent (by day 14) to the initial regeneration of eyes at normal locations (by day 8), consistent with the gene having some involvement in spatial regulation, perhaps dependent on existing eyes. map3k1 might specifically suppresses a signal produced from existing eyes that ordinarily induces nearby progenitors to terminally differentiate. Alternatively, map3k1 could provide a constitutive dampening of the terminal differentiation while progenitors are in transit such that progenitors traveling for longer distances are more prone to map3k1 inhibition. Planarian eyes appear to be capable of drawing in nearby progenitors to ensure their homeostatic maintenance over long periods of time, through an unknown mechanism, even if such eyes are experimentally induced to take on incorrect animal locations using transplantation or patterning gene RNAi [9,23]. Our results suggest an unexpected complexity to the process of terminal differentiation used in regeneration.
Whole-body regeneration likely involves a substantial component of progenitor sorting and migration, but these steps are still poorly understood. In planarians, progenitors for regionalized tissues like eyes and pharynx are specified in broad domains [6,56] and lack consistent spatial segregation from each other [37]. Therefore, controlling differentiation of migratory progenitors to appropriate locations is likely critical for enabling appropriate regeneration. While integrins and snail-family transcription factors are important for neoblast migration and appropriate targeting of differentiating cells [57–59], and neoblast migration can be coupled to DNA damage control [60], the signals regulating migratory progenitor behavior and targeting are not known. Body-wide patterning signals play an important role in determining the target locations of eye regeneration, yet map3k1 likely controls a distinct process that prevents migratory progenitors from differentiating too early prior to arriving at their destination. We suggest that regeneration involves the integration of positional information to define progenitor domains, signaling that targets progenitors to particular locations, and systems that can sustain the undifferentiated state during migration. Our study identifies map3k1 as a critical regulator of terminal differentiation affecting the eye progenitors used for organ regeneration.
Methods and Materials
Ethics Statement
Procedures with Schmidtea mediterranea planarians (invertebrates) were conducted according to safety and ethics procedures in line with the Northwestern Office for Research Safety, with authorization from the Institutional Biosafety Committee (IBC), approved 12/7/21. As invertebrate animal subjects, planarians are not subject to IACUC or IRB review.
Experimental model.
Asexual Schmidtea mediterranea (CIW4 strain) were cultured in 1x Montjuic salts at 18-20°C. Animals were fed pureed calf liver once a week and cleaned at least once every week for maintenance. Animals were starved for at least 7 days before experiments.
RNA interference (RNAi).
RNAi was performed by feeding dsRNA to animals every 2-3 days for the designated length of the experiment. Double stranded RNA (dsRNA) were produced by in vitro synthesis reactions (NxGen, Lucigen) using primers listed in S1 Table. Control dsRNA was produced from Caenorhabditis elegans unc-22, a gene sequence not found in the planarian genome. Liver mixtures contained 20% dsRNA, 5% food coloring dye, and 75% pureed calf liver. For homeostatic RNAi treatments, animals were fixed 4 days after the last feeding. For RNAi treatments of regenerating worms, animals were amputated 1 day after the final feeding and fixed at the indicated times. Unless otherwise noted in the text, RNAi dose schedules were performed by 6 dsRNA feedings over 2 weeks. For double-RNAi experiments, single-gene RNAi comparisons to double-RNAi conditions involved mixing equal amounts of competing control dsRNA (targeting C. elegans unc-22) along with the experimental dsRNA to ensure the same amount of overall dsRNA was present across conditions.
Fluorescence in situ hybridization (FISH) and immunostaining.
FISH protocol used were performed as described in previous work [61]. Briefly, animals were fixed in 7.5% N-Acetyl-cysteine in PBS (w/v) followed by 4% formaldehyde in PBSTx (1X PBS+0.3% Triton-X100, v/v), washed in PBSTx and stored in methanol prior to bleaching for 2-4 hours in a solution of 5% deionized formamide (v/v) and 0.36% hydrogen peroxide (v/v) in 1XSSC. Digoxigenin- or fluorescein-labeled riboprobes were synthesized as described in previous work [61] and detected with anti-digoxigenin-POD or anti-fluorescein-POD antibodies (1:2000, Roche/Sigma-Aldrich) blocked with 10% heat-inactivated Horse serum and 10% Western Blotting Blocking Reagent (Roche). Tyramide amplification was performed by depositing 0.2% rhodamine-tyramide or fluorescein-tyramide and 0.1% 4-iodophenylboronic acid (in dimethylformamide) in TSA buffer (2M NaCl, 0.1M Boric acid, pH 8.5). For double FISH, the enzymatic activity of tyramide reactions were stopped in 100mM sodium azide in TNTx. Nuclei were stained using 1:1000 Hoechst 33342 (Invitrogen) in TNTx. For immunostainings, animals were fixed in 4% formaldehyde as previously described [61], blocked with 5% heat-inactivated Horse Serum in PBSTx, incubated in goat anti-mouse HRP (1:300) to detect labeling with mouse anti-ARRESTIN (1:10,000), before tyramide amplification. Unless otherwise indicated, riboprobes were generated by PCR using primers listed in S1 Table. Other riboprobes were as previously described: GluR/gpas [26,35], ChAT (cholinergic neurons expressing choline acetyltransferase) [62], cintillo [63], ovo, opsin, tyrosinase [6,7], FoxD [64], and ndl5 [25].
Image Acquisition.
Live animals were imaged on either a LeicaMZ125 dissecting microscope with a LeciaDFC295 camera or a LeicaS6D dissecting microscope with a FlexacamC3 camera. Stained animals were imaged with a Stellaris 5 laser-scanning confocal microscope. Adjustments to brightness and contrast were made using FIJI/ImageJ or Adobe Photoshop.
Quantification and Statistical Analysis.
Stained animals were imaged using a Leica Stellaris 5 laser-scanning confocal microscope. For analysis of PCG expression domains in Fig 2, maximum projection images were analyzed in ImageJ/Fiji to measure body length, body area, and manual scoring of the extent of PCG expression. For body length, animals were measured from the head tip to the end of the tail. PCG domain measurements were conducted done by measuring the most anterior to most posterior expression for each gene. cintillo+ cells in Fig 2 were counted manually and normalized to the area of the animal as determined by Hoechst staining. To quantify undifferentiated eye progenitors in Fig 6A, ovo+ cells located outside of eyes and also lacking opsin/tyrosinase expression were manually counted from maximum projections of animal head regions imaged using a 20x objective on a Leica Stellaris 5 laser-scanning confocal microscope. Isolated ovo+ opsin/tyrosinase+ cells in Fig 6D were manually counted using the same technique. Quantification of map3k1-ovo+ and map3k1+ovo+ cells in S4D Fig was conducted through manual scoring of confocal z-stacks acquired using a 40x glycerol-immersion objective. Sample sizes for each experiment are indicated in the legends and/or through plotting individual datapoints for each experiment. Statistical tests are indicated in each figure legend and were conducted in Microsoft Excel, boxplots were generated using BoxPlotR, stacked bargraphs were generated in Microsoft Excel or Datawrapper, and 2D dot plots were generated in R. Single-cell RNAseq plots were generated from previously reported datasets. Expression of map3k1 was visualized in tSNE plots at digiworm.wi.mit.edu based on intact whole-animal cell atlas data [36]. Expression of map3k1 in neoblasts and eye progenitors was visualized through 10x Genomics single-cell RNAseq data obtained from a the previously reported project PRJNA1067154 datasets consisting of ~30,000 G2/S/M and G1 neoblasts FACS-sorted from 72-hour anterior-facing blastemas, mapped to the Dresden ddv6 transcriptome [65] and clustered using cellranger-9.0.0 implemented in the 10x Genomics cloud analysis platform, followed by UMAP visualization in Loupe v7 using default settings (10 principal components, UMAP minimum distance 0.1 and UMAP number of neighbors 15). Eye progenitor cluster was identified by expression of six-1/2 (dd15436) as described [37]. Raw data used for plotting the figures of the study is tabulated in S3 Table.
Counting differentiated opsin + or tyrosinase + cells within planarian eyes.
For each animal, confocal z-stacks capturing all fluorescently labeled eye cells were obtained on a Leica Stellaris 5 laser-scanning confocal microscope using a 40x glycerol objective and 0.3-micron slice size. For each sample, individual left and right sides of the head were imaged using equivalent laser and gain settings, and z-stack size chosen to capture the entire depth of all eye cells. Eye cell numbers were quantified using Stardist to segment nuclei (probThresh=0.60, nmsThresh=0.5) and counting nuclei ROIs whose median opsin-channel fluorescence exceeded an empirically defined threshold of detection obtained from Otsu thresholding slices that contained target cells, measured in ImageJ. Eye cell counts from individual slices taken every 5-microns were summed across a z-stack for each sample. Total number of detected cells were compared across control and map3k1(RNAi) treatments using an unpaired 2-tailed t-test. For Fig 4, the analysis was performed across a range of threshold settings (+/- 10% pixel intensity of threshold) and slice widths (from 2.5 to 10 microns), and similar differences to eye cell numbers were measured in each case across the treatment types. A Jaccard similarity index/coefficient (JSI, Intersection over Union) was calculated by randomly selecting 6 annotated z-slices collected from control and map3k1(RNAi) worms which were used in the calculating the estimated number of opsin+ cells, and manually categorizing cells as true positives, false negatives, and false positives (S2 Table). The average JSI was calculated for control and map3k1 RNAi samples separately, and an unpaired 2-tailed t-test was performed to determine whether automated cell segmentation and counting efficiency varied between treatment types.
Mapping relative positions of eyes in fixed and live animals.
In Fig 5, images of live or stained animals were overlaid on a grid, which normalized to the inter-eye distance such that the left and right pigment cup cells were positioned at (-1,0) and (1,0) respectively. Locations of each eye locations were manually scored in a consistent way across all samples, as the pixels associated with the A/P and M/L midpoint of the pigmented regions associated with each optic cup. For analysis of ectopic eye phenotypes, the pigment cup of each ectopic eye was marked, and the distance from the original eyes was measured using WebPlotDigitizer (https://apps.automeris.io/wpd/), and then normalized in units equal to one half the inter-eye distance. A similar procedure was used to map the relative positions of ovo+ progenitor cells in images of fixed and stained animals. The relative coordinates of each eye or ovo+ eye progenitor were extracted and plotted using ggplot2 in R.
Supporting information
S1 Fig.
(A) Domain structure of dd_Smed_v6_5198_0_1 (map3k1) containing a RING (E-value=0.0255) domain, a serine/threonine kinase domain (E-value=1.16e-69) characteristic of MAPKs (smart.embl-heidelberg.de). (B) Stacked bar graph quantifying the number of ectopic eyes in control (n=46) versus map3k1(RNAi) (n=48) animals over 8 weeks of RNAi showing that map3k1 inhibition caused ectopic eyes to continue forming over time.
https://doi.org/10.1371/journal.pgen.1011457.s001
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S2 Fig. Homeostatic animals fixed after 4 weeks of RNAi were stained with ChAT to detect cholinergic neurons.
map3k1 RNAi did not increase ChAT+ neuron staining, compared to the ectopic ChAT+ brain branches that formed after ndk RNAi. Scorings indicate how many animals had a ChAT expression pattern that appeared normal (controls and map3k1 RNAi) or had ectopic ChAT+ cells extending laterally from the ventral nerve cords (ndk RNAi). Scale bars, 300μm.
https://doi.org/10.1371/journal.pgen.1011457.s002
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S3 Fig. To test whether map3k1 could control eye placement and/or a subset of head patterning, animals were fed with either control or map3k1 dsRNA for 4 weeks before undergoing eye resections at different positions indicated by the cartoons.
Individual live animals were imaged before, immediately after (post-resection), and 14 days post-surgical eye resection (14dpR) to track whether eye regeneration subsequently occurred. map3k1(RNAi) animals regenerated their original eyes at a high frequency (15/16). Ectopic eyes from these animals were also capable of regeneration, though at lower frequencies. Removal of either the anterior-most ectopic eyes (6/14 eyes regenerated, “1st ectopic”) or the posterior-most ectopic eyes (4/7 eyes regenerated, “last ectopic”) could result in regeneration from the original eye. Sample size, n≥7 animals in each condition. Scorings indicate the number of animals that regenerated an eye in the positions shown.
https://doi.org/10.1371/journal.pgen.1011457.s003
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S4 Fig.
(A) Single-cell RNA sequencing expression profiles of map3k1 expression (A, top panels) in intact animals from Fincher et al. 2018 (36) plotted at digiworm.wi.mit.edu (tSNE plots), and (bottom panels) show map3k1 expression in neoblasts harvested from day 3 anterior-facing blastemas in the process of head and eye regeneration from King et al. 2024 (37) plotted with 10x genomics Loupe (UMAP plots with zoomed insets indicated with blue boxes). map3k1 expression was broad in most tissues in homeostatic animals from the Fincher et al. 2018 (36) cell atlas, including in muscle, neural, and gut clusters. (A, bottom panels) mapk31 positive cells were also present in most clusters of neoblasts isolated from anterior-facing blastemas at 72 hours in the King et al. 2024 (37) early blastema cell atlas, including within rare cells (right panels) located within a cluster of six1/2+ eye progenitors (left panels) identified by that study as produced in early regeneration (37). (B) Maximum projection images of map3k1 expression in homeostatic worms as detected by FISH show map3k1 is expressed broadly throughout the body. Right panel, image showing map3k1 expression in the head and low levels of map3k1 expression in the eyes (arrows). Sample size, n=14 animals. Scale bar, 100μm. (C) Maximum projection images of map3k1 and opsin expression in the eye show some expression of map3k1 in opsin expressing cells (4/4 animals). Scale bar, 50μm. (D-E) Double-FISH detecting ovo and map3k1 expression in homeostatic animals. Panels show either the eye region (D) or higher-magnification view of individual cells (E). Some ovo+ cells expressed low levels of map3k1 (yellow arrowheads) while other ovo+ cells did not have any detectable map3k1 expression (white arrowheads). map3k1 is also broadly expressed, so other unknown map3k1+ovo- cells were identifiable (green arrowhead). Scale bar, 50μm. Panels in (E) show 40X confocal images of ovo and map3k1 expression in homeostatic animals detected through double-FISH, focusing on ovo+ cells containing low map3k1 expression (top panels, 22/36 cells counted over 5 intact animals) and ovo+ cells containing no map3k1 expression (bottom panels, 14/36 cells over 5 animals). Together, map3k1 could act either within a subpopulation of migratory eye progenitors, or alternatively act within some other cell type, to impact eye differentiation.
https://doi.org/10.1371/journal.pgen.1011457.s004
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S2 Table. Jaccard index calculation comparing automated versus manual cell counting.
https://doi.org/10.1371/journal.pgen.1011457.s006
(XLSX)
S3 Table. Raw data used for plotting graphs.
Each tab is labeled with the figure subpanel relevant for the data therein.
https://doi.org/10.1371/journal.pgen.1011457.s007
(XLSX)
Acknowledgments
We thank members of the Petersen lab for critical comments and thoughtful discussion. We thank R. Zayas for the kind gift of the planarian anti-ARRESTIN antibody.
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