Fig. 4: Elucidation of downstream effectors mediating Hox gene regulation of asexual reproduction.

a Diagram depicting a two-part strategy to elucidate downstream effectors of Hox genes that regulate fission: (I) use RNAseq analysis to identify putative effector genes from the differentially expressed genes in Hox RNAi-treated animals, then, (II) use RNAi to determine the effects of putative effector genes on fission. b Heatmap of 1724 DEGs from RNAseq that are significantly regulated following RNAi of Hox genes. c Heatmap of the top 24/423 genes from RNAi screen of putative downstream fission effectors (see Supplementary Fig. 8) 16–19 Bacterial RNAi feedings. Heatmap depicts normalized fission number on Day 12 across six RNAi-treated worms. Effector genes are color coded with respect to their putative upstream Hox gene: hox1 (red), lox5b (orange), hox3 (green), post2b (purple). Source data are provided as a Source Data file. d Representative images of post2b effector RNAi-treated animals 15 days post fission induction (n = 6 animals, 17 RNAi feedings, two independent repeats). Scale, 1 mm. e Double fluorescent whole-mount in situ staining of post2b effectors rcn-1 and syt1, ifb, or lmnAC (n = 6–7 animals, two independent repeats). Whole animal image (top) and zoom in of posterior lateral section (bottom). Scale, 500 and 100 μm, respectively. f Expression of post2b effector genes detected by whole-mount in situ hybridization in control, hox3a + hox3b, and post2b RNAi-treated animals (n = 4–13 animals, 1 independent repeat). Scale, 500 μm. g Plot of the number of fission planes/animal length following post2b effector RNAi treatment (n = 4, 10, 11, 10, 10, 7, 4, and 17 animals for Ctl, rcn-1, plg, syt1, ifb, lmnAC, post2a, and post2b, respectively; Ctl vs. rcn-1, plg, syt1, ifb, lmnAC, post2a, and post2b p-values = 0.9581, 0.6815, 0.1552, 0.1634, 0.5967, 0.4605, and 0.0001, respectively; replicates pooled from 1 to 4 independent repeats, 21 bacterial RNAi feedings). p-Values were calculated by Welch’s two-tailed t-test. Source data are provided as a Source Data file. h Plot of the total number of fission attempts for RNAi-treated animals (n = 14, 5, 5, and 6 animals for Ctl, syt1, post2a, and all other RNAi samples, respectively; Ctl vs. rcn-1, plg, syt1, ifb, lmnAC, post2a, and post2b p-values = 0.0145, <0.0001, <0.0001, 0.0006, 0.0157, and 0.0218, respectively; 21 bacterial RNAi feedings). Box and whisker plot depicts individual data points, median (center), 25th/75th percentile (bounds of box), and minima/maxima (whiskers). p-values were calculated by Welch’s two-tailed t-test. Source data are provided as a Source Data file. i Scanning electron micrographs of the lateral edge of the tail containing the marginal adhesive gland in control and post2b RNAi-treated animals (n = 3 animals; representative images; 30 bacterial RNAi feedings; two independent repeats). j Transverse scanning electron micrographs of the marginal adhesive gland in control and post2b RNAi-treated animals (n = 3 animals; representative images; 30 bacterial RNAi feedings; single experiment). Annotations demarcate the dorsal (D), ventral (V), lateral edge (LE), epithelium (E), basal lamina (BL), parenchyma (P), viscid cell extensions crossing basement lamina (arrowheads), clustered (V’) or individual (V) viscid cell glands in the epidermis, and adhesive papillae (A). k Diagram summarizing the roles of the Hox genes as regulators of the adult tissue patterning and behavior required for asexual reproduction (top). Zoom-in panel describing requirement of post2b in the initiation of fission behavior via effector gene regulation in the adhesive cells and anchor cells of the planarian marginal adhesive glands (bottom).