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

A. Top row: live image of a control RNAi animal followed by three examples of map3k1 RNAi animals with disorganized eyes (white arrows) (n=156/200 >1 ectopic eye, after 4 weeks of RNAi). Bottom row: control RNAi FISH images followed by three map3k1 RNAi examples visualizing OC cells (RNA probe pool to catalase/tyrosinase/glut3); PRNs are visualized with either an RNA probe to opsin (PRN cell bodies) or an anti-Arrestin antibody (PRN cell bodies and projections). The left and middle map3k1 RNAi FISH examples show single ectopic PRNs (opsin, left; anti-Arrestin, middle) and OC cells (catalase/tyrosinase/glut3) scattered below and around the eyes after 3 weeks of RNAi. The far FISH example shows ectopic OC cells and PRNs (anti-Arrestin) after 6 weeks of RNAi. Dorsal up. Scale bars, 100μm B. FISH showing ectopic PRNs (anti-Arrestin) and OC cells (catalase/tyrosinase/glut3) in the trunk and tail region of a map3k1 RNAi animal after 5 weeks of RNAi (n=10/10 with at least one cell in the trunk) (see panel 1A for control RNAi). Scale bar, 100μm. Magnified panels 1 and 2 scale bars, 50μm. White arrows point to all ectopic cells. C. FISH showing the tail regions of control and map3k1 RNAi animals; single OC (catalase/tyrosinase/glut3) cells are observed in the tail after one week of map3k1 RNAi (n=12/18 with at least one cell in the tail). Scale bar, 200μm. D. Top graph depicts the number of ectopic PRNs (1 week: n=15; 2 weeks: n=9; 3-4 weeks: n=22). Control shown at 3-4 weeks (n=11). Bottom graph depicts the number of ectopic OC cells (1 week: n=16; 2 weeks: n=7; 3-4 weeks: n=17) per map3k1 RNAi animal along the AP axis over time. Control shown at 3-4 weeks (n=18). map3k1 RNAi resulted in higher ectopic cell numbers along the AP axis (p<0.0001; Poisson generalized linear mixed model) compared to the control condition for both PRNs and OC cells at 3-4 weeks. E. Schematic comparing previously identified eye-patterning RNAi phenotypes (after ndk, wnt5, slit, and notum RNAi) with the map3k1 RNAi phenotype. F. FISH examples of eye-specialized neoblasts (ovo+; smedwi-1+ cells) in tails of control and map3k1 RNAi animals at 3 weeks of RNAi. Scale bar, 20μm. Right graph shows no significant difference (p=0.181; Permutation test, 10,000 permutations; two-tailed) in the frequency of eye-specialized neoblasts in the tails of control (n=10) and map3k1 RNAi (n=10) animals. All images, dorsal up. Bottom left numbers indicate the number of animals exhibiting the shown phenotype out of the total number of animals observed.

map3k1 RNAi results in posterior ectopic differentiation of some neurons and gland cells.

A. map3k1 RNAi animals exhibit posterior, ectopic brain branches that project dorsolaterally from ventral nerve cords (5 weeks of RNAi). These branches contain chat+ and pc2+ neurons. Photoreceptor axons (visualized with an anti-Arrestin antibody) extend along the ventral nerve cords and into ectopic brain branches. See also Figure 2- figure supplement 1A. Ventral up. Scale bar, 100μm. B. FISH showing gluR+ (dd_16476+) neurons in ectopic brain branches after 3 weeks of map3k1 RNAi. Scale bars, 200μm; higher magnification scale bar, 100μm. A, B. Ventral up. C. FISH images showing no change in cintillo+ neuron distribution in the head, but expansion of dd_17258+ neurons along the entire AP axis after 3 weeks of map3k1 RNAi. See also Figure 2-figure supplement 1B, C); Ventral up. Scale bars, 200μm. D. Graph showing no significant difference in the number of dd_17258+ neurons in the heads (AP_1) of map3k1 and control RNAi animals (p=0.410; Mann-Whitney test) but a significant difference in the number of ectopic cells observed along the entire AP axis (AP_2→AP_6: ***p<0.0006; Multiple Mann-Whitney tests). Counted animals underwent 3-4 weeks of RNAi; 2 replicates were used. No ectopic cintillo+ neurons were observed for both control and map3k1 RNAi animals. E. FISH images of unaffected (dd_9223) and affected (dd_7131 and dd_8476) parenchymal cell types in map3k1 RNAi animals (3 weeks of RNAi). Ventral up. Scale bar, 200μm. Right graph shows more dd_7131+ (p<0.0001; Negative binomial regression) and dd_8476+ (p<0.0001; Negative binomial regression) cells in the tails of map3k1 RNAi animals compared to control RNAi animals. Counted animals underwent 3-4 weeks of RNAi; 3 replicates each. Bottom left numbers indicate the number of animals with the result displayed in the image out of the total number of animals observed.

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

A. FISH images of control and map3k1 RNAi animals showing anterior expansion and dispersal of vitrin+ (pharynx) single cells (white arrows) and clusters of cells (pink arrows and boxes) at variable positions along the AP and ML axes at 3 weeks (n=20/20; 2 replicates) and 4 weeks (n=12/12; 1 replicate) of RNAi, between the cephalic ganglia (1), near the ventral nerve cords (2), and lateral to the pharynx (lower right panel). Scale bars, 200μm; magnified images scale bars, 20μm. Control animals, 3 weeks of RNAi. B Left panels: FISH images showing anterior clusters of mhc-1+ (pharynx muscle) cells (pink arrows) in map3k1 RNAi animals. Middle panel shows ectopic mhc-1+cells and vitrin+ cells near the brain in map3k1 RNAi animals (n=5/8; 1 replicate, white arrows). Left and middle panels, 3 weeks RNAi. Bottom right panel shows ectopic large clusters of mhc-1+ cells between the cephalic ganglia after 6 weeks of RNAi (n=6/6; 1 replicate, white arrows). Ventral up. Scale bar, 100μm. C. FISH images of map3k1 RNAi animals showing NB.22.1e+ mouth cells anterior to the normal location (pink brackets; 3 weeks RNAi) and dispersed around the pharynx (white arrows; 8 weeks RNAi). Scale bar, 100μm. D. FISH images showing clusters of dd_554+ cells (white arrows) (intermediate pharynx progenitor population (Zhu et al., 2015)) and single dd_554+ cells dispersed around and anterior to the pharynx (vitrin) in 3-week map3k1 RNAi animals. Counted animals for all panels underwent 3-4 weeks of RNAi; 3 replicates were used. Ventral up. Scale bar, 200μm. E. FISH showing examples of FoxA+; smedwi-1+ cells in the head region of both control and map3k1 RNAi animals (white boxes). 3 weeks RNAi, 2 replicates. See also Figure 3 – Figure supplement 1E. Scale bars, 200μm; magnified image scale bars, 10μm. A, B, C, D. All panels, ventral up. Numbers in each panel indicate number of animals displaying the result shown in the image out of total animals observed.

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

A. Top schematic depicts eye resection experimental design. Live images of control and map3k1 RNAi animals prior to (d0; left column) and the day after (d1; middle column) eye resection. Right column shows live and FISH images of control and map3k1 RNAi animals 10 days following eye resection. PRNs are visualized with anti-Arrestin (PRN cell bodies and projections) and an RNA probe to opsin (PRN cell bodies). Optic cup cells are visualized using a pool of catalase1/ tyrosinase/glut3 RNA probes. 3 weeks of RNAi was performed prior to resections; FISH and live images are from different animals. Dorsal, up. Scale bars, 200μm. See Figure 4 - figure supplement 1A for d0 FISH. B. Diagram of pharynx resection on the left. Live images at d1 and d10 after pharynx resection showing pharynx regeneration in the correct location for both map3k1 RNAi and control animals. Pharynx regeneration in map3k1 RNAi animals is disorganized; FISH contains RNA probes to vitrin (pharynx-specific), NB.22.1e (mouth and esophagus), and mhc-1 (pharynx muscle). See also Figure 4- figure supplement 1B for d0 FISH. Ventral up. Live images, scale bars, 200μm; FISH images, scale bars, 100μm. C. Live image of d10 head, trunk, and regenerating tail fragments. map3k1 RNAi tail fragments regenerate pharynges (white arrow) in a more anterior location compared to control animals. C, D. 3 weeks of RNAi was performed prior to resection. Scale bar, 100μm. D. FISH images showing anterior vitrin+ pharynx cells (white arrows) and NB.22.1e+ mouth cells (white brackets) in map3k1 RNAi day 10 trunk regenerates. All map3k1 RNAi trunks and tails fully regenerate eyes (anti-Arrestin). Day 10 tails regenerate pharynges in a more anterior location compared to control animals. Ventral up. Scale bar, 200μm. E. Top graph shows no difference in the AP location of original pharynges in control and map3k1 RNAi d10 trunk regenerates (p=0.1054; Welch’s t-test) but a significant difference between control original pharynges and ectopic pharyngeal cell clusters map3k1 RNAi trunks (****p<0.0001; Mann-Whitney test). Bottom graph shows an anterior shift in pharynx regeneration in map3k1 RNAi tail fragments compared to control animals (p<0.0001; Welch’s t-test) Numbers in each panel indicate number of animals displaying the result shown in the image out of total animals observed.

Positional information remains largely unaffected in map3k1 RNAi animals

A. FISH panel of position control gene expression (sFRP-1, ndl-4, ndl-5, ndl-2, ndl-3, wntP-2, axinB, sp5, ptk7, wnt11-1, and slit) shows no obvious changes to expression domains in map3k1 RNAi animals. Animals from 3 and 4 weeks of RNAi were used. Pink arrowheads mark the end of PCG expression domains. Scale bars, 200μm. Ventral up. B. FISH example showing some dispersion of notum+; chat+ cells in the brain of a map3k1 RNAi animal after 3 weeks of RNAi (sample numbers from 3 and 4 weeks of RNAi). No obvious changes in wnt1+ posterior pole organization was observed after 2 and 3 weeks of RNAi (example FISH, 2 weeks RNAi). Scale bars, 200μm. Ventral up. C. Top row panels show example FISH images of opsin+(PRNs), dd_17258+ (neuron type), and vitrin+(pharynx) cells outside of typical PCG expression domains (ndl-5, ndl-5, and wntP-2, respectively). Bottom row panels show example FISH images of opsin+, dd_17258+, and vitrin+ cells inside atypical PCG expression domains (wntP-2, wnt11-1, and ndl-5/ndl-2, respectively). Far right panels on top and bottom, ventral up. All other panels, dorsal up. Animals from 3 and 4 weeks of RNAi were used. Scale bars, 200μm. Numbers in each panel indicate number of animals displaying the result shown in the image out of total animals observed.

map3k1 RNAi eye progenitors prematurely differentiate along a normal migratory path

A. Schematic of head-shielded irradiation experimental design. Animals were fixed 12-14 days following the first map3k1 RNAi feeding (2 days after irradiation), depending on health by inspection. Right graph shows ectopic differentiation events were more likely to occur in the anterior half of the animal (surviving neoblast region; marked by a smedwi-1+ RNA probe) versus outside of (distal or proximal to) the neoblast region for both opsin+and dd_17258+ neurons (p<0.0001; Binomial exact test). All observed ectopic PRNs (opsin) and dd_17258+ neurons outside of the neoblast region were present proximal to (within 50μm) the neoblast region. Bottom FISH panels depict examples of ectopic PRN (anti-Arrestin) and dd_17258+ neuron differentiation events within the neoblast region. Scale bar, 200μm; magnified boxes 1-4 scale bars, 50μm. B. Schematic of tail-shielded irradiation experimental design. Animals were fixed 10-12 days following the first map3k1 RNAi feeding (2 days after irradiation), depending on health by inspection. Right graph shows ectopic events were more likely to occur in the tail (area of surviving neoblasts) versus outside of (distal or proximal to) the neoblast region for dd_17258+ neurons (p<0.0001; Binomial exact test). All observed ectopic PRNs dd_17258+ neurons outside of the neoblast region were present proximal to (within 50μm) the neoblast region. FISH panels depict examples (white arrows) of ectopic PRNs (opsin) and dd_17258+ neurons in the tail of a tail-shielded, irradiated map3k1 RNAi animal. Scale bars, 200μm; magnified boxes 1-5 scale bars, 50μm. C. Schematic of EdU-labeled graft transplant experimental design; bottom graph shows a significant number of total ectopic eye cells (****p<0.0001; Mann-Whitney test) and ectopic EdU-positive eye cells (**p=0.002; Mann-Whitney test) in recipient wild-type animals compared to control. Animals after 2 and 3 weeks of RNAi prior to the EdU-pulse were used for transplantation. D. FISH example of EdU-positive ectopic eye cells (white arrows) differentiated in wild-type animals (n=13/20) with EdU-positive ectopic cells; n=19/20 exhibited any ectopic eye cells outside of the transplant area). Scale bars, 200μm; zoom in scale bars, 20μm. A, B, C, D. All panels are dorsal up. Numbers in each panel indicate number of animals displaying the result shown in the image out of total animals observed.

map3k1 RNAi results in differentiation in incorrect organs and teratoma formation

A. FISH showing clusters of mhc-1+ cells within the lobes of the brain and ventral nerve cords, and NB.22.1e+cells (epidermis, mouth) present within the eye after 8 weeks of map3k1 RNAi. Brain, ventral up; eye, dorsal up. Scale bars, 100μm. B. FISH examples of vitrin+ (pharynx) cells present within eyes (anti-Arrestin) at day 10 tail regeneration, following 3 weeks of map3k1 RNAi. See also figure 7- figure supplement 1B. Dorsal up. Scale bars, 200μm; scale bars for magnified eye images, 50μm. C. Left panels: live images of small growths in animals at 12 weeks of map3k1 RNAi feedings, accompanied by DAPI images of similarly positioned growths. Right panels: live images of advanced teratomas (pink arrows) in animals at 12 weeks of map3k1 RNAi, accompanied by a FISH example showing photoreceptors (opsin and anti-Arrestin) and OC cells (tyrosinase, catalase1, glut3) scattered in and around the teratomas. Bottom left animal, 8 weeks RNAi. C, D. Scale bars, 200μm. Dorsal up. D. Left panels: FISH images showing chat+, mhc-1+, and NB.22.1e+ cells are common in teratomas, often excluding vitrin+ (pharynx) and mag-1+ (gland cells). Scale bars, 100μm; panels 1-4 scale bars, 50μm. Right panels show examples of other cell types commonly found in outgrowths: cintillo+ (neuron), dd_17258+ (neuron), dd_3534+ (neuron), estrella+ (glia), anti-Arrestin+ (photoreceptors), NB.22.1e+, lamin+ (mouth and epidermis), and colF-2+ (muscle). 6-8 weeks of RNAi. Scale bars, 100μm. Dorsal up. Numbers in each panel indicate number of animals displaying the result shown in the image out of total animals observed.

Model: Map3K1 restricts migratory progenitor differentiation until the correct target is reached

A. Schematic showing the inhibition of differentiation of a migratory progenitor cell, via map3k1, until reaching its target tissue at the target zone. B. Migratory precursors can differentiate in the incorrect PCG expression locations following map3k1 RNAi. C. Patterning abnormalities that can occur without suitable restriction of differentiation in migratory progenitors, demonstrating the patterning properties yielded by this mechanism.