map3k1 RNAi results in ectopic eyes and ectopic isolated eye cells.

A. Live images (upper panels) showing posterior eyes in map3k1 RNAi animals. Upper panels, 6 feedings of RNAi over 36 days. FISH (bottom panels): single optic cup and photoreceptor cells are present ectopically with RNA probes for opsin (photoreceptors), a catalase/tyrosinase/glut3 pool (optic cup), and an anti-Arrestin antibody (labeling photoreceptors and their projections). B. Examples showing a diversity of ectopic eyes and eye cells in map3k1 RNAi animals. A, B. Dorsal up; Scale bar, 200μm. C. Schematic comparing previously identified eye-patterning phenotypes with the map3k1 RNAi phenotype. D. FISH of single cells differentiated proximal to the pharynx and in the tail of map3k1 RNAi animals. Dorsal up; scale bar, 100μm. E. FISH examples of ovo+; smedwi-1+ cells in the tail. F. Left graph depicts the number of ectopic optic cup differentiation events per animal along the AP axis over 10, 16, and 26 days. Right graph depicts the number of ectopic photoreceptor neurons at 26 days of map3k1 RNAi; See also Figure 1- figure supplement 1.

map3k1 RNAi results in differentiated eye cells throughout the AP axis, and no overt change in eye progenitor distribution.

A. Domain structure of human and Schmidtea mediterranea MAP3K1. % identity/ similarity is displayed above each domain. B. FISH of map3k1 RNAi animals at 10 days, 16 days, and 6 weeks of RNAi shows optic cup (catalase1, tyrosinase, glut3) in the posterior at early timepoints. C. FISH with an RNA probe to the map3k1 gene in a wild-type animal. Scale bar, 200μm for full body images; Scale bar, 100μm for closeup of the brain. D. FISH showing double-positive ovo+ ; smedwi-1+ cells near the posterior half of the pharynx. Scale bars, 50μm. E. quantification of smedwi-1 high, smedwi-1 low, and smedwi-1 negative ovo+ cells in the tails of map3k1 RNAi and control animals. No significant change is observed between the number of smedwi-1+ ; ovo+ cells in the tail between map3k1 RNAi and control animals.

map3k1 RNAi results in ectopic neuron classes and gland cells along the AP axis.

A. map3k1 RNAi animals exhibit posterior ectopic brain branches with chat+ neurons and gluR+ (dd_16476) neurons. Photoreceptor axons extend along ventral nerve cords and into ectopic brain branches. Ventral up; Scale bars, 200μm; Closeup images, scale bar, 100μm. B. FISH images showing no change in cintillo+ neuron distribution, but expansion of dd_17258+ cells down the entire AP axis. ventral up; scale bars, 200μm; See also Figure 2- figure supplement 1. C. Graph showing distribution of dd_17258+ cells along the AP axis in control and map3k1 RNAi animals. D. Quantification of cells in the tail for dd_7131 and dd_8476. More cells are observed in the tails of map3k1 RNAi animals compared to control animals; Student t-test using Graphpad Prism software ***p<.0001; **p<.001 . E. FISH examples of unaffected (dd_9223) and affected (dd_7131 and dd_8476) parenchymal cell types in map3k1 RNAi animals. ventral up; scale bar, 200μm. Numbers of animals are represented on the bottom left of each panel.

map3k1 RNAi results ectopic brain branches posterior to the brain and posterior expansion of some neural cell types.

A. Close-up FISH of ventral nerve cord (VNC) and branches in a map3k1 RNAi animal. Photoreceptor projections (anti-Arrestin) are travel along the VNC and into ectopic branches labeled by the pan-neural markers chat and pc2. Scale bar, 100μm. B. FISH of dd_17258+ cells and optic cup cells (catalase1, tyrosinase, glut3), together with antibody labeling of photoreceptors (anti-Arrestin), extending down the AP axis. All scale bars, 200μm, unless otherwise specified in the lower left. C. FISH showing expansion of dd_17258 cells down the entire AP axis in a map3k1 RNAi animal. Scale bar, 200μm. D. FISH showing an unchanged neural cell type (gad) in map3k1 RNAi and control animals. Scale bar, 200μm.

map3k1 RNAi results in pharynx cell types in ectopic anterior locations.

A. FISH images showing anterior expansion and disorganization of vitrin+ (pharynx-specific marker) cells along the AP-axis. Ventral up; scale bars, 100μm. B. FISH showing anterior expansion of NB.22.1e (mouth and esophagus) cells along the AP axis. map3k1 RNAi animals exhibit a low frequency of NB.22.1e cells in the tail, posterior to the normal mouth location. C. FISH shows anterior expansion of mhc1+ cells in map3k1 RNAi animals compared to control animals. ventral up, scale bar, 100μm. D. FISH shows anterior expansion of dd_554 cells (pharynx progenitors) in map3k1 RNAi animals compared to control animals. Ventral, up. Scale bar, 100μm. E. FISH shows FoxA+; smedwi-1+ cells close to the brain lobes and eyes. Scale bar, 50μm.

map3k1 RNAi animals display tissue-specific regeneration at target zones.

A. Top, schematic depicts experimental design. Bottom panels show live image examples of control and map3k1 RNAi animal eye resections at d0, d1, and d10 post eye resection. Scale bar, 200μm. B. Diagram, pharynx resection. Control and map3k1 RNAi animals at d1 and d10 post resection; accompanied by FISH of d10 pharynx regeneration using probes to vitrin (pharynx-specific), NB.22.1e (mouth and esophagus), and mhc1 (pharynx muscle). Scale bar, 200μm; See also Figure 4- figure supplement 1. C. d10 regeneration in head, trunk, and tail fragments. An anterior-shifted pharynx regenerated in a map3k1 RNAi tail fragment. Scale bar, 200μm. D. FISH images of d10 trunk and tail regenerates showing regeneration of photoreceptors (anti-Arrestin), pharynx (vitrin), and mouth and esophagus (NB.22.1e). An anterior shift in vitrin+ cells in d10 tail regenerate. Scale bar, 200μm. E. AP distributions of original trunk pharynges in control and map3k1 RNAi animals and ectopic differentiation events in map3k1 RNAi trunk regenerates **** p <.0001 Mann-Whitney-U test. F. AP distributions of regenerated pharynges in tail fragments shows an anterior shift in pharynx regeneration in map3k1 RNAi animals compared to control animals. *** p <.0001 Mann-Whitney-U test.

map3k1 RNAi animals can undergo tissue-specific and whole-body regeneration, with some errors in pharynx organization.

A. FISH following pharynx resection at day zero (d0) with the markers vitrin (pharynx-specific), NB.22.1e (mouth and esophagus), and mhc1 (pharynx muscle). Scale bars, 200μm. B. Example of map3k1 RNAi d10 pharynx resection. FISH shows dorsal (left) and ventral (right) views of the same animal, showing disorganized pharynx regeneration in map3k1 RNAi animals. C. FISH of d10 head regeneration showing disorganized regeneration of the pharynx using vitrin and NB.22.1e RNA probes. Scale bars, 200μm. n=10/10. D. FISH showing dispersed notum+ cells in a d7 tail regenerate compared to control animals. Scale bar, 200μm

Positional information remains largely unaffected in map3k1 RNAi animals

A. FISH panel of position control genes (sFRP-1, ndl2, ndl5, ndl3, wntP-2, axinB, and slit) shows no obvious changes to positional information. Sample numbers are indicated in the lower left of each panel. Scale bar, 200μm. B. FISH showing some dispersion of notum+ cells in the heads of map3k1 RNAi animals compared to control animals. Right panels, no obvious changes in posterior pole organization. Sample numbers are indicated in the lower left of each panel, scale bars, 200μm.

map3k1 is expressed in neoblasts and post-mitotic progenitors.

A. map3k1 is expressed broadly across neoblast clusters. Plot points are ordered. B. map3k1 is expressed broadly across post-mitotic progenitors; plot points are ordered. C. map3k1 is expressed in some six-1/2+ neoblasts, which contain eye neoblasts; plot points are ordered. D. map3k1 is expressed in some G0 post-mitotic eye progenitors; plot points are ordered. E. map3k1 is expressed within the predicted progenitor cluster (Neural 1) of the affected cell types: dd_17258 and dd_16476; plot points are ordered. F. dd_17258 mature marker expression within its predicted progenitor cluster (Neural 1); plot points are ordered. G. dd_16476 mature marker expression within its predicted progenitor cluster (Neural 1); plot points are ordered. H. map3k1 expression in neural, parenchymal, pharyngeal, and six1/2+ neoblast clusters. I. map3k1 expression in neural, parenchymal, and pharyngeal post-mitotic progenitor clusters.

map3k1 RNAi eye progenitors prematurely differentiate along their typical migratory path.

A. Schematic of head-shielded irradiation experimental design. B. FISH showing instances of ectopic photoreceptor (anti-Arrestin), optic cup (catalase, tyrosinase, glut3), and dd_17258 cells born in the upper half of the animal (n=3/3 with good shielding and cells present correspond to area of leftover neoblasts). C. Schematic of EdU-labeled transplant experimental design. D. FISH and antibody staining showing examples of EdU+ eye cells from map3k1 RNAi animals ectopically differentiated in wild-type animals (n=7/18 with EdU+ ectopic cells; n=16/18 exhibiting any ectopic eye cells outside the transplant area). Scale bar, 50μm.

map3k1 RNAi animals display to improper progenitor targeting and teratoma formation.

A. FISH showing EdU+ photoreceptors (anti-Arrestin) in the eye of the recipient animal. These cells differentiated from map3k1 RNAi progenitors born during the EdU pulse of the donor animal, following wild-type migratory cues out of the graft and into the recipient eye. Scale bar, 200μm. B. FISH image of EdU-ectopic eye cells in recipient wild-type animals after EdU plug transplantation. Scale bar, 200μm. C. Live images of early growths that will eventually become teratomas in map3k1 RNAi animals. Scale bar, 100μm.

map3k1 RNAi results in differentiation in incorrect organs and teratoma formation.

A. FISH of long term (>6 weeks) map3k1 RNAi animal showing clusters of mhc1 cells that appear to have formed small structures within the lobes of the brain and ventral cords. Right panels depict NB.22.1e cells incorporated into the eye (anti-Arrestin). Number of animals analyzed is indicated in the lower left of each panel; Left panel scale bar, 200μm; all other panels scale bars, 50μm. B. FISH showing vitrin+ cells incorporated into the eyes of a d10 tail regenerate. n=9/24 animals contained at least one vitrin+ cell in one eye; left panels scale bar, 200μm. All other scale bars, 50μm. C. Left panel contains live images of early developing growths in map3k1 RNAi animals, accompanied by a DAPI image of an outgrowth in between the brain lobes. Scale bars 200μm; The right panel contains live images and FISH examples of late-stage teratomas from map3k1 RNAi animals between 12 and 16 feedings, accompanied by FISH examples showing eyes dispersed around the teratomas. Scale bars, 200μm. D. Left panels are FISH images showing an exclusion of vitrin+ cells from lateral outgrowths but a common presence of NB.22.1e+ cells in outgrowths. mag1, a parenchymal marker with transcripts abundant in cells in the neck region is also mostly excluded from outgrowths. Right panel shows examples of cell types commonly found in outgrowths: cintillo (neuron), dd_17258 (neuron), dd_3534 (neuron), estrella (glia), anti-Arrestin (photoreceptor projections), NB.22.1e, lamin (mouth, epidermis), and col2 (muscle). Scale bar 100μm.

Model: Map3K1 restricts migratory progenitor differentiation until they reach their target.

A. Schematic showing the inhibition of differentiation of a specific progenitor until reaching its target tissue at the target zone. B. Defects observed with map3k1 RNAi, highlighting significance of restricting differentiation until suitable contextual cues are reached.