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Strategy for identifying genes required for neoblast-dependent formation of the anterior pole. (A) in situ hybridizations to detect expression of notum after amputation in untreated animals or animals exposed to gamma irradiation (6000 Rads) 48 hours prior to surgery. Late (72 hours) expression of notum at the anterior pole is irradiation sensitive (arrow), but not early (18 hours) expression of notum in disperse cells near the wound site. Cartoon shows surgery (red lines) and enlarged region (green box). Bars, 300 microns. (B) Cartoon showing strategy to identify genes expressed differentially within neoblasts in head and tail regeneration. Animals were amputated transversely at either planes (i) or (ii), and X1 neoblasts purified by FACS from macerated tissue fragments from regions near injury sites engaged in either head or tail regeneration in a time series. Expression profiling was performed to identify genes whose expression significantly changed at 1, 2, or 3 days after amputation as compared to neoblasts isolated from regionally matched non-regenerating tissue. (C) Heat map showing only genes upregulated as detected by microarray (yellow, log2 fold-change (FC), chosen with false-discovery rate <10%) in head and/or tail regeneration. (D) qPCR experiment showing expression of 21/24 candidate genes upregulated in neoblasts due to regeneration whose expression behavior was confirmed upregulated. (C–D) Heat maps show microarray and qPCR data for genes ordered using hierarchical clustering with both microarray and qPCR expression values as inputs and broadly categorize into those upregulated primarily in anterior or posterior regeneration, or both (yellow, log2 fold-change versus 0 hour timepoint for each anterior or posterior series). We further analyzed a subset of genes that reproducibly increased in expression specific to anterior regeneration.
Source publication
Mechanisms that enable injury responses to prompt regenerative outgrowth are not well understood. Planarians can regenerate essentially any tissue removed by wounding, even after decapitation, due to robust regulation of adult pluripotent stem cells of the neoblast population. Formation of pole signaling centers involving Wnt inhibitors or Wnt liga...
Citations
... However, amputation of pak1(RNAi) animals failed to form a detectable blastema even after 5 dpa (SI Appendix, Fig. S3C). Failure to form blastema can be a result of reduced stem cell proliferation, failed neoblast migration to the site of injury, disrupted neoblast differentiation, or failed patterning (49)(50)(51)(52). We measured neoblast proliferation in pak1(RNAi) animals using anti-phospho-Histone H3(Ser10) (H3P) antibody, which marks cells in the G2/M phase of cell cycle (53,54). ...
... Besides shaping body axes, Smed-pak1 is required for blastema formation. It is unclear whether this phenotype in pak1(RNAi) animals is due to anomalies along the body axes, considering that failure to form anterior or posterior poles is known to result in small blastemas (50)(51)(52). However, delay in blastema formation could also be due to impaired wound healing, as a similar process called "dorsal closure" during Drosophila embryogenesis requires Pak1 (88,89). ...
Successful regeneration of missing tissues requires seamless integration of positional information along the body axes. Planarians, which regenerate from almost any injury, use conserved, developmentally important signaling pathways to pattern the body axes. However, the molecular mechanisms which facilitate cross talk between these signaling pathways to integrate positional information remain poorly understood. Here, we report a p21-activated kinase ( smed-pak1 ) which functionally integrates the anterior–posterior (AP) and the medio-lateral (ML) axes. pak1 inhibits WNT/β-catenin signaling along the AP axis and, functions synergistically with the β-catenin-independent WNT signaling of the ML axis. Furthermore, this functional integration is dependent on warts and merlin —the components of the Hippo/Yorkie (YKI) pathway. Hippo/YKI pathway is a critical regulator of body size in flies and mice, but our data suggest the pathway regulates body axes patterning in planarians. Our study provides a signaling network integrating positional information which can mediate coordinated growth and patterning during planarian regeneration.
... In any case a combination of intrinsic and extrinsic signals and stimuli emanating from differentiated tissues and organs play a role in regulating neoblast biology [6][7][8][9][10] and need to be tightly coordinated and controlled for a successful regeneration. [32,33], the musculature [34,35], the excretory system [36], as well as for establishing axial polarity [37][38][39][40][41][42][43][44]. ...
... Open Biol. 13: 230327 as foxD, zic, follistatin or notum (electronic supplementary material, file S1) [39,[81][82][83][84]. To clarify whether the expression of islet2 in the anterior pole (figure 1) could also be associated with the establishment of polarity, we analysed the late and stem-cell-dependent expression of genes associated with anterior (notum) [81] and posterior (wnt1) [85] identity in islet2(RNAi) treated animals. ...
Adult planarians can regenerate the gut, eyes and even a functional brain. Proper identity and patterning of the newly formed structures require signals that guide and commit their adult stem cells. During embryogenesis, LIM-homeodomain (LIM-HD) transcription factors act in a combinatorial ‘LIM code’ to control cell fate determination and differentiation. However, our understanding about the role these genes play during regeneration and homeostasis is limited. Here, we report the full repertoire of LIM-HD genes in Schmidtea mediterranea. We found that lim homeobox (lhx) genes appear expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian, with some of them being co-expressed in the same cell types. We have identified that Smed-islet1, -lhx1/5-1, -lhx2/9-3, -lhx6/8, -lmx1a/b-2 and -lmx1a/b-3 are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while Smed-lhx1/5.2 and -lhx2/9.2 are required for the proper expression of intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in controlling axonal pathfinding (lhx6/8), axial patterning (islet1, lhx1/5-1, lmx1a/b-3), head/body proportions (islet2) and stem cell proliferation (lhx3/4, lhx2/9-3, lmx1a/b-2, lmx1a/b-3). Altogether, our results suggest that planarians might present a combinatorial LIM code that controls axial patterning and axonal growing and specifies distinct neuronal and intestinal cell identities.
... Specific muscle cells express positional control genes (PCGs) to maintain and regenerate patterning of new tissues and organs, and their disruption can cause the abolishment of regeneration similar to what we observed in brg1 or smarcc2 RNAi animals [49]. Our ATACseq datasets (derived from intact worms, but dissociated during processing for flow cytometry) indicated that PCGs, particularly anterior-pole genes such as prep, and zic1 [78,79], were less accessible in neoblasts upon disruption of the BAF complex (Fig. 5A). Genes involved in body wall and non-body wall muscle cell generation, myoD and foxF-1 [75,84], were less accessible with disruption of the BAF complex (Fig. 5A). ...
... Furthermore, stem cell RNAseq data demonstrated that PCGs and musclerelated genes, including prep, zic-1, and foxF-1, were downregulated in both brg1(RNAi) and smarcc2(RNAi) neoblasts (Fig. 5B,C) [82]. To determine whether downregulation of genes critical to the anterior pole in intact planarian stem cells actually resulted in less anterior pole cells during regeneration, we assayed anterior pole markers notum, sfrp1, zic1, prep, and the posterior marker wnt1 at 3 and 10 days of regeneration in each RNAi condition [79,85,86]. Although notum was not downregulated in our brg1 or smarcc2 neoblast datasets, its expression would distinguish whether the pole can regenerate or not. ...
Background
The flatworm planarian, Schmidtea mediterranea, has a large population of adult stem cells (ASCs) that replace any cell type during tissue turnover or regeneration. How planarian ASCs (called neoblasts) manage self-renewal with the ability to produce daughter cells of different cell lineages (multipotency) is not well understood. Chromatin remodeling complexes ultimately control access to DNA regions of chromosomes and together with specific transcription factors determine whether a gene is transcribed in a given cell type. Previous work in planarians determined that RNAi of core components of the BAF chromatin remodeling complex, brg1 and smarcc2, caused increased ASCs and failed regeneration, but how these cellular defects arise at the level of gene regulation in neoblasts is unknown.
Results
Here, we perform ATAC and RNA sequencing on purified neoblasts, deficient for the BAF complex subunits brg-1 and smarcc2. The data demonstrate that the BAF complex promotes chromatin accessibility and facilitates transcription at target loci, as in other systems. Interestingly, we find that the BAF complex enables access to genes known to be required for the generation of mesoderm- and ectoderm-derived lineages, including muscle, parenchymal cathepsin, neural, and epithelial lineages. BAF complex knockdowns result in disrupted differentiation into these cell lineages and functional consequences on planarian regeneration and tissue turnover. Notably, we did not detect a role for the BAF complex in neoblasts making endodermal lineages.
Conclusions
Our study provides functional insights into how the BAF complex contributes to cell fate decisions in planarian ASCs in vivo.
... Transcription factors (TFs) play key roles in multiple aspects of animal development. In planarians, many conserved TFs have been identified (Suzuki-Horiuchi et al., 2021) and characterized to be required for the regeneration of multiple cells, tissues and organs including the intestine (Flores et al., 2016;Forsthoefel et al., 2012;Forsthoefel et al., 2020;González-Sastre et al., 2017), the eyes (Lapan and Reddien, 2011;Lapan and Reddien, 2012;Mannini et al., 2004;Pineda et al., 2000), the central nervous system (Brown et al., 2018;Coronel-Córdoba et al., 2022;Cowles et al., 2013;Cowles et al., 2014;Currie and Pearson, 2013;Fraguas et al., 2014;März et al., 2013;Roberts-Galbraith et al., 2016;Ross et al., 2018), the epidermis (Dubey et al., 2022;Tu et al., 2015), the pharynx (Adler et al., 2014), the pigment cells (He et al., 2017;Wang et al., 2016), the musculature (Scimone et al., 2017;Scimone et al., 2018), the excretory system (Scimone et al., 2011), as well as for the establishment of axial polarity (Blassberg et al., 2013;Chen et al., 2013;Felix and Aboobaker, 2010;Hayashi et al., 2011;Pascual-Carreras et al., 2023;Scimone et al., 2014;Tian et al., 2021;Vásquez-Doorman and Petersen, 2014). both vertebrate and invertebrate organisms, the expression of different combinations of lim homeobox genes (lhx) genes are considered to form a "LIM code" that specifies neural types within a tissue or organ and guide the establishment of topographically arranged connections (reviewed in Bachy et al., 2002;Gill, 2003;Hobert and Westphal, 2000;Shirasaki and Pfaff, 2002). ...
Adult planarians can regenerate the gut, eyes, and even a functional brain in just a few days after injury. Proper regeneration of these complex structures requires that signals guide and restrict the commitment of their adult stem cells and ensure the identity and patterning of the newly formed structures. During embryogenesis of both vertebrates and invertebrates, LIM Homeodomain (LIM-HD) transcription factors act in a combinatorial 'LIM code' that controls crucial aspects of cell fate determination and cell differentiation, including specification of neuronal cell type identity and axonal guidance. So far, however, our understanding about the role these genes may play during regeneration is limited. Here, we report the identification and functional characterization of the full repertoire of LIM-HD genes in Schmidtea mediterranea. We found that these lim homeobox genes (lhx) appear mainly expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian. By functional RNAi based analysis we have identified that several Smed-lhx genes (islet1, lhx1/5-1, lhx2/9-3, lhx6/8, lmx1a/b-2 and lmx1a/b-3) are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while others (Smed-lhx1/5.2 and Smed-lhx2/9.2) are required for the proper expression of diverse intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in the control of axonal pathfinding (lhx6/8), axial patterning (islet1 and lmx1a/b-3), head/body proportions (islet2) and stem cell proliferation (lhx3/4, lhx2/9-3, lmx1a/b-2 and lmx1a/b-3) in planarians. Altogether, our results suggest that planarian LIM-HD could provide a combinatorial LIM code to control axial patterning, axonal growing as well as to specify distinct neuronal and intestinal cell identities during regeneration.
... Importantly, during the first hours of regeneration, both notum and wnt1 are expressed in differentiated cells of any wound, and it is not until 36-48 h of regeneration that they are restricted to their respective tip 22,24,31 , forming the anterior and the posterior organizing regions. It is known that this late localized expression of notum and wnt1 depends on the proliferation of stem cells, and that it requires the expression of specific transcription factors and kinases, such as foxD, zicA, prep, or pbx for anterior tips [32][33][34][35][36] and islet, pitx, mob, striatin, and teashirt (tsh) for posterior tips [37][38][39][40][41] . However, the triggering of the early expression of notum and wnt1, which does not depend on stem cell proliferation, is not understood. ...
... Genes required for the late expression of notum and wnt1, localized in the midline, have been identified. foxD and zicA (RNAi) animals do not show the late expression of notum and do not regenerate a proper head [32][33][34] ; islet and pitx (RNAi) animals do not show the late expression of wnt1 and are tailless 37,38,46 . Little was known about the regulation of the expression of notum and wnt1 in disperse cells of early wounds. ...
For successful regeneration, the identity of the missing tissue must be specified according to the pre-existing tissue. Planarians are ideal for the study of the mechanisms underlying this process; the same field of cells can regrow a head or a tail according to the missing body part. After amputation, the differential activation of the Wnt/β-catenin signal specifies anterior versus posterior identity. Initially, both wnt1 and notum (Wnt inhibitor) are expressed in all wounds, but 48 hours later they are restricted to posterior or anterior facing wounds, respectively, by an unknown mechanism. Here we show that 12 hours after amputation, the chromatin accessibility of cells in the wound region changes according to the polarity of the pre-existing tissue in a Wnt/β-catenin-dependent manner. Genomic analyses suggest that homeobox transcription factors and chromatin-remodeling proteins are direct Wnt/β-catenin targets, which trigger the expression of posterior effectors. Finally, we identify FoxG as a wnt1 up-stream regulator, probably via binding to its first intron enhancer region. Any planarian fragment regenerates the missing head and tail in the proper end. Early activation of the Wnt/β-catenin signaling pathway changes the chromatin accessibility of the cells of the posterior-facing wound to regenerate a tail.
... The online version of this article includes the following figure supplement(s) for figure 4: linked to tbx genes (tbx-20 142 motifs, tbx-2/3c 34 motifs, tbx-2/3a 11 motifs) ( Figure 6A, B and D, Supplementary file 2 and). tbx-20 had a clear intronic enhancer-like region and a distal enhancer-like region, both containing motifs for TFs implicated as positional control genes (PCGs) (hox1, hox4a, prep, smad1, smad9, zic-1, sp5) (Molina et al., 2007, Reddien et al., 2007Felix and Aboobaker, 2010;Vásquez-Doorman and Petersen, 2014;Tewari et al., 2019), TFs involved in neoblast migration (snail-1 and snail-2) (Abnave et al., 2017), and various other TFs ( Figure 6D). ...
... (D) The neural FSTF soxB1-1 has a distal enhancer-like region marked in red (width 1000 bp) upstream of the gene body. Prep, zic-1, isl-1, and foxD1 are PCGs that are expressed in the anterior pole of both intact and regenerating planarians (Felix and Aboobaker, 2010;Vásquez-Doorman and Petersen, 2014;Vogg et al., 2014). Interestingly, we found that these TFs are bound to several motifs in enhancer-like regions of X1 cells (Supplementary file 1 enhancers, prep 132 motifs, zic-1/2 330 motifs, isl-1 42 motifs, foxD1/2/3 42 motifs) and a few motifs were found to bind to the enhancer-like regions linked to prep, zic-1, isl-1, and foxD1 ( Figure 7C, Supplementary file 2, Supplementary file 5, and Supplementary file 6). ...
Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to predict enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify regions with histone modifications consistent with enhancer identity and activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for predicted transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Foot-printing analyses provided further evidence that these potential TF binding sites are potentially occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of transcription factors in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. We found that our predicted GRNs were independently supported by existing TF RNAi/RNA-seq data sets, providing further evidence that our work predicts active enhancers regulating adult stem cells and regenerative mechanisms.
... Clusters of specialized muscle cells, with organizer-like activity influencing blastema pattern, found at the head and tail tips, are called anterior and posterior poles, respectively. Poles are specified from neoblasts and pole progenitors coalesce during regeneration at the DVB and at the pre-existing midline of the amputated fragment [20][21][22][23] . bmp4 RNAi tail fragments frequently failed to regenerate an anterior pole ( Supplementary Fig. 1c). ...
Regeneration often involves the formation of a blastema, an outgrowth or regenerative bud formed at the plane of injury where missing tissues are produced. The mechanisms that trigger blastema formation are therefore fundamental for regeneration. Here, we identify a gene, which we named equinox, that is expressed within hours of injury in the planarian wound epidermis. equinox encodes a predicted secreted protein that is conserved in many animal phyla. Following equinox inhibition, amputated planarians fail to maintain wound-induced gene expression and to subsequently undergo blastema outgrowth. Associated with these defects is an inability to reestablish lost positional information needed for missing tissue specification. Our findings link the planarian wound epidermis, through equinox, to regeneration of positional information and blastema formation, indicating a broad regulatory role of the wound epidermis in diverse regenerative contexts. Many regenerative animals form an outgrowth at wound sites called a blastema. Here the authors identify equinox, which is expressed in the planarian wound epidermis and essential to initiate positional information regeneration and blastema formation.
... Following the discovery of epidermal-committed ζ-neoblast and gut-committed γ-neoblasts (van Wolfswinkel et al. 2014), intense research was focused on identifying FSTFs that were also expressed in smedwi-1 + cells and, thus, probably involved in neoblast commitment versus a specific lineage. In this way, putative neoblast precursors for cells of the eye, protonephridia, nervous system, pharynx, anterior pole, and gut were identified (Scimone et al. 2011(Scimone et al. , 2014a(Scimone et al. , 2014bLapan and Reddien 2012;Currie and Pearson 2013;Cowles et al. 2013;Adler et al. 2014;Vásquez-Doorman and Petersen 2014;Flores et al. 2016). Recently, Plass et al. (2018) performed highly parallel droplet-based single-cell transcriptomics and by applying a partition-based graph abstraction algorithm, combined with independent computational and experimental approaches, derived a consolidated lineage tree that includes all identified cell types rooted to a single stem cell cluster. ...
The regeneration process assumes the presence in the body of
cells capable of self-renewal and subsequent differentiation into specialized cells. Whether these cells are stem cells or are present in circulating fluids or tissues as a pool of reserve progenitor cells, or whether they appear following dedifferentiation/transdifferentiation of specialized cells of individual tissues, are the main questions that scientists are focusing on. Understanding the origin and pathways of differentiation in coelomic fluid cells and coelomocytes of the starfish
Asterias rubens was the aim of this research. The coelomic epithelium is considered as a possible source of coelomocytes. Further effective studies of coelomocyte replenishment are difficult due to the lack of protein markers characterizing various cell morphotypes. Additional difficulties lie in the heterogeneity of analyzed cell populations. In the present study, we separated cells of the coelomic fluid and the coelomic epithelium, and a subpopulation of the coelomic epithelium enriched with poorly differentiated cells, which are proposed precursors of some types of coelomocytes, in a Percoll density gradient. Characterization of the cell morphology of different fractions and their behavior in vitro (functional characteristics) revealed
an enrichment of the gradient fractions in two of eight types of coelomocytes and three of eight morphotypes of cells of the coelomic epithelium.
... An activin-dependent process restricts the initial 6-18 hours of notum 111 expression to anterior-facing wounds, resulting in a low Wnt environment that leads to head 112 regeneration (Cloutier et al., 2021). At later times in regeneration (by 24-72hours) and throughout 113 homeostasis, stem cell-dependent processes (Hayashi et al., 2011;Currie and Pearson, 2013;Marz 114 et al., 2013;Scimone et al., 2014;Vasquez-Doorman and Petersen, 2014;Vogg et al., 2014;115 Tejada- Romero et al., 2015;Schad and Petersen, 2020) generate cells expressing wnt1 and notum 116 in muscle cells at the posterior and anterior midline termini respectively (termed poles) where they 117 may function to control region-specific patterning or act at the tip of a hierarchy of AP regulatory 118 factors (Adell et al., 2009;Petersen and Reddien, 2009;Gurley et al., 2010;Stuckemann et al., 119 2017; Schad and Petersen, 2020). 120 Other Wnt-dependent pathways may function downstream or in parallel to pole identity 121 and tissue polarization. ...
Tissue identity determination is critical for regeneration, and the planarian anteroposterior (AP) axis uses positional control genes expressed from bodywall muscle to determine body regionalization. Canonical Wnt signaling establishes anterior versus posterior pole identities through notum and wnt1 signaling, and two Wnt/FGFRL signaling pathways control head and trunk domains, but their downstream signaling mechanisms are not fully understood. Here we identify a planarian Src homolog that restricts head and trunk identities to anterior positions. src-1(RNAi) animals formed enlarged brains and ectopic eyes and also duplicated trunk tissue, similar to a combination of Wnt/FGFRL RNAi phenotypes. src-1 was required for establishing territories of positional control gene expression, indicating it acts at an upstream step in patterning the AP axis. Double RNAi experiments and eye regeneration assays suggest src-1 can act in parallel to at least some Wnt and FGFRL factors. Co-inhibition of src-1 with other posterior-promoting factors led to dramatic patterning changes and a reprogramming of Wnt/FGFRLs into controlling new positional outputs. These results identify src-1 as a factor that promotes robustness of the AP positional system that instructs appropriate regeneration.
Highlights
Src-1 suppresses head and trunk identity
Src-1 can regulate positional control gene domains
Src-1 likely acts independently of notum /Wnt and FGFRL signals
Src-1 inhibition broadly sensitizes animals to AP pattern disruption
... After this wound-induced phase of notum expression, other anterior PCGs are expressed in muscle and progenitors for the anterior pole are specified. The anterior pole is a signaling center [23][24][25][26] comprised of muscle cells generated from neoblasts during regeneration [23][24][25], and the anterior pole also expresses notum [12]. Thus the two phases of notum expression in head regeneration involve distinct cells; i.e., the wound-induced notum + cells do not directly form the anterior pole [12,23,25]. ...
... After this wound-induced phase of notum expression, other anterior PCGs are expressed in muscle and progenitors for the anterior pole are specified. The anterior pole is a signaling center [23][24][25][26] comprised of muscle cells generated from neoblasts during regeneration [23][24][25], and the anterior pole also expresses notum [12]. Thus the two phases of notum expression in head regeneration involve distinct cells; i.e., the wound-induced notum + cells do not directly form the anterior pole [12,23,25]. ...
... The anterior pole is a signaling center [23][24][25][26] comprised of muscle cells generated from neoblasts during regeneration [23][24][25], and the anterior pole also expresses notum [12]. Thus the two phases of notum expression in head regeneration involve distinct cells; i.e., the wound-induced notum + cells do not directly form the anterior pole [12,23,25]. The asymmetric wound induction of notum is the earliest known difference in gene expression that exists between wounds that go on to make a head instead of a tail [12,19]. ...
Planarians are flatworms and can perform whole-body regeneration. This ability involves a mechanism to distinguish between anterior-facing wounds that require head regeneration and posterior-facing wounds that require tail regeneration. How this head-tail regeneration polarity decision is made is studied to identify principles underlying tissue-identity specification in regeneration. We report that inhibition of activin-2 , which encodes an Activin-like signaling ligand, resulted in the regeneration of ectopic posterior-facing heads following amputation. During tissue turnover in uninjured planarians, positional information is constitutively expressed in muscle to maintain proper patterning. Positional information includes Wnts expressed in the posterior and Wnt antagonists expressed in the anterior. Upon amputation, several wound-induced genes promote re-establishment of positional information. The head-versus-tail regeneration decision involves preferential wound induction of the Wnt antagonist notum at anterior-facing over posterior-facing wounds. Asymmetric activation of notum represents the earliest known molecular distinction between head and tail regeneration, yet how it occurs is unknown. activin-2 RNAi animals displayed symmetric wound-induced activation of notum at anterior- and posterior-facing wounds, providing a molecular explanation for their ectopic posterior-head phenotype. activin-2 RNAi animals also displayed anterior-posterior (AP) axis splitting, with two heads appearing in anterior blastemas, and various combinations of heads and tails appearing in posterior blastemas. This was associated with ectopic nucleation of anterior poles, which are head-tip muscle cells that facilitate AP and medial-lateral (ML) pattern, at posterior-facing wounds. These findings reveal a role for Activin signaling in determining the outcome of AP-axis-patterning events that are specific to regeneration.
