A, E, H: Average RPKM values per embryo for A) embryonic gut transcripts cathepsin L1-like (CTSL-like: SMED30023322), lysosomal alpha glucosidase-like (LYAG-like: SMED30028442, SMED30008977), macrophage-expressed gene 1 like-3 (MPEG1-like-3: SMED30015696), E) gamma (g) class neoblast transcripts (gata456a, hnf4, prox-1, nkx2.2), and H) transcripts with enriched expression in the adult gut (Forsthoefel et al., 2012; Vu, 2015; Wurtzel et al., 2015) Yolk (Y), S2-S8. Early embryonic gut transcript expression was validated by WISH on staged embryo collections. H: Adult gut-enriched transcripts with enriched expression during S5 and/or S6 (top, n=146), or S7 and/or S8 (bottom, n=292). Adult gut enriched transcripts are flagged in lists of S5-S8 enriched transcripts (Figure 1 – source data 5, Figure 1 – source data 6, Figure 1 – source data 7, Figure 1 – source data 8). 74% (n=1112) of the intestinal phagocyte enriched transcripts reported in (Forsthoefel et al., 2012) were identified in the smed20140614 transcriptome; 129 (11%) of the cross-referenced transcripts were enriched during S5, S6, S7 and/or S8. 90% (n=425) of the gut-enriched transcripts reported in (Wurtzel et al., 2015) were identified in the smed20140614 transcriptome; 44% (n=186) of the cross-referenced transcripts were enriched during S5, S6, S7 and/or S8.

B-D: CTSL-like (B), LYAG-like (C) and MPEG1-like-3 (D) expression (blue) in the temporary embryonic pharynx (S2-S4), four primitive gut cells abutting the temporary embryonic pharynx (S4), and yolk-laden gut cells forming an irregular lattice beneath the embryonic wall (S5-S6). Expression of these markers was downregulated as branching morphogenesis proceeded during S7.

F-G: gata456a (F) and hnf4 (G) expression (blue) during embryogenesis, S2-S8. Staining was detected in the presumptive temporary embryonic pharynx (S2), and was later detected in scattered parenchymal cells from S5 onwards. Expression of both markers became more prominent in the developing gut over time, especially after branching morphogenesis was underway during S7-S8.

I: porcn-A expression (blue) during embryogenesis, S2-S8. Hazy, faint expression was detected in the gut during S5-S6, with increasing signal following the initiation of branching morphogenesis during S7-S8.

B-D, F-G, I: Anterior: top (S6-S8). O: oral hemisphere. A: aboral hemisphere. D: dorsal. V: ventral. Black arrowheads: temporary embryonic pharynx. Black arrows: primitive gut cells. Red arrowheads: definitive pharynx. Scale bars: 100 µm.') ,

Four yolk-laden primitive gut cells associate with the temporary embryonic pharynx (Figure 1 – figure supplement 13 B-D, black arrows), and can frequently be detected in unstained specimens. Yolk ingested through the temporary embryonic pharynx collects in the central gut cavity.

Definitive gut development may begin as early as S4, with the production of isolated phagocytic cells that gradually form a continuous, honeycomb-like lattice beneath the embryonic wall (Figure 1 – figure supplement 13 B-D). Embryonic gut markers, identified amongst the S4-S5 enriched transcripts, were expressed in gut tissue through S6 and were subsequently downregulated as branching morphogenesis proceeded in elongated S7 embryos (Figure 1 – figure supplement 13 A-D). The progenitor population(s) for embryonic gut cell type(s) are not known. These early intestinal cells may represent a transient population, turning over during S6-S7, or they may persist, changing their expression signature as morphogenesis proceeds.

Expression of gamma (g) class neoblast transcripts, including gata456a and hnf4, was detected in the temporary embryonic pharynx during S2, and in scattered cells in the embryonic wall during S5 (Figure 1 – figure supplement 13 E-G, Figure 6 B); a similar expression pattern was reported for Spol gata456a (Martín-Durán and Romero, 2011). The anarchic distribution of putative gut progenitors during embryogenesis is reminiscent of the systemic distribution of gut progenitors in the adult parenchyma, as well as the uniform incorporation of neoblast progeny into the adult gut during growth and homeostasis (Forsthoefel et al., 2011). In the adult gut, neoblast progeny incorporated into both new and preexisting gut tissue during regeneration, and morphallaxis of the gut required stem cell activity (Forsthoefel et al., 2011). Branching morphogenesis begins during S6-S7, as gut cells ingress from the anterior dorsoventral margins of the embryo, forming the secondary branches (Figure 1 – figure supplement 13 F-G, I). As in the adult, branching morphogenesis may proceed locally and may be reliant upon the incorporation of differentiating progeny, and/or remodeling of differentiated gut tissue. Separation of the two posterior gut branches also occurs during S6-S7, concomitant with the development of the definitive pharynx and a medial, muscular septum running from the pharynx pouch to the posterior pole (Figure 1 – figure supplement 13 F-G, I). Many molecular markers of adult gut tissue are expressed during S5 or later (Forsthoefel et al., 2012; Wurtzel et al., 2015), suggesting that gut maturation is a gradual process (Figure 1 – figure supplement 13 H). Newborn hatchlings are born with yolk-filled intestines, and it may take up to one week to completely digest and clear the yolk from the gut.

Forsthoefel, D.J., James, N.P., Escobar, D.J., Stary, J.M., Vieira, A.P., Waters, F.A., and Newmark, P.A. (2012). An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians. Dev Cell 23, 691-704.

Forsthoefel, D.J., Park, A.E., and Newmark, P.A. (2011). Stem cell-based growth, regeneration, and remodeling of the planarian intestine. Dev Biol 356, 445-459.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Wurtzel, O., Cote, L.E., Poirier, A., Satija, R., Regev, A., and Reddien, P.W. (2015). A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians. Dev Cell 35, 632-645.

Figure 1 – figure supplement 15: Molecular markers for the definitive epidermis

A, C, F: Average RPKM values per embryo for: A) zeta (z) class neoblast transcripts C) Category 2 and Category 3 transcripts, and F) Category 4 and 5 transcripts, Y (yolk), S2-S8 (Eisenhoffer et al., 2008; Pearson and Sánchez Alvarado, 2010; Tu et al., 2015; van Wolfswinkel et al., 2014; Wagner et al., 2012; Zhu et al., 2015). Transcripts shown had enriched expression during S5, S6, S7 and/or S8 and are flagged in excel tables for S5-S8 enriched transcripts (Figure 1 – source data 5, Figure 1 – source data 6, Figure 1 – source data 7, Figure 1 – source data 8).

B, D-E, G-I: WISH with riboprobes complementary to: B) p53, D) prog-1, E) AGAT-1, G) zpuf-6, H) vim-3, I) crocc (blue), S3-S8. Anterior: top (S6-S8). D: dorsal. V: ventral. Red arrowheads: definitive pharynx. Red arrows: ciliated protonephridial tubules. Scale bars: 100 µm.

Definitive epidermis development begins during S5, with the advent of p53 expression in piwi-1+ cells (Figure 6 A, Figure 1 – figure supplement 15 A-B, Figure 1 – source data 5). p53 and zfp-1, transcriptional regulators upregulated during S5, are required for production of epidermal progenitors in adults, and knock-down of either gene results in loss of the epidermal lineage(s) (Pearson and Sánchez Alvarado, 2010; van Wolfswinkel et al., 2014; Wagner et al., 2012). Epidermal lineage progression during embryogenesis likely utilizes the same transition states elucidated during adult homeostasis. Transcripts expressed in post-mitotic epidermal progenitors, including the Category 2 genes prog-1 (NB.21.11e), prog-2 (NB.32.1g), prog1-1, prog2-2, and prog2-3, the Category 3 genes AGAT-1, AGAT-2, AGAT-3, Cyp1A1, egr-5, SMED30000058, SMED30001567, SMED30002242, pmp-10, SMED30008120, SMED30025584, odc-1/DCOR-1, litaf, pmp-6 and the Category 4 gene zpuf-6 were among the enriched transcripts in S5 embryos (Figure 1 – source data 5) (Eisenhoffer et al., 2008; Tu et al., 2015; van Wolfswinkel et al., 2014; Zhu et al., 2015). Consistent with the RNA-sequencing expression trends, the Category 2 and 3 progenitor markers, prog-1 and AGAT-1, respectively, were first detected by WISH during S5, in scattered parenchymal cells (Figure 1 – figure supplement 15 D-E). Notably, p53, prog-1, AGAT-1 and zpuf-6 positive cells were more numerous on the presumptive dorsal side of elongating S6 embryos (Figure 1 – figure supplement 15 B,D,E,G), and cells appeared to be more densely packed along the anterior dorsoventral margin. This positional bias in epidermal progenitors was transient, with little difference in the number or density of positive cells observed during S7 or later. Markers of late epidermal progenitor population(s) present within the epidermal monolayer, such as vim-1, vim-2 and vim-3 (Tu et al., 2015; van Wolfswinkel et al., 2014), showed enriched expression during S7 and S8 (Figure 1 – source data 7, Figure 1 – source data 8). vim-3 was first detected by WISH during S6, predominantly on the dorsal side of elongating embryos (Figure 1 – figure supplement 15 H). By S7, an uninterrupted layer of vim3+ cells blanketed the embryos, with dorsoventral biases in epidermal cell density becoming less apparent as development proceeded. crocc (rootletin), a marker or terminally differentiated, ciliated epithelial cells (Scimone et al., 2011), was detected during S6 and thereafter (Figure 1 – figure supplement 15 I).

Eisenhoffer, G.T., Kang, H., and Sánchez Alvarado, A. (2008). Molecular analysis of stem cells and their descendants during cell turnover and regeneration in the planarian Schmidtea mediterranea. Cell Stem Cell 3, 327-339.

Pearson, B.J., and Sánchez Alvarado, A. (2010). A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages. Development 137, 213-221.

Tu, K.C., Cheng, L.C., Tk Vu, H., Lange, J.J., McKinney, S.A., Seidel, C.W., and Sánchez Alvarado, A. (2015). egr-5 is a post-mitotic regulator of planarian epidermal differentiation. Elife 4, e10501.

van Wolfswinkel, J.C., Wagner, D.E., and Reddien, P.W. (2014). Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment. Cell Stem Cell 15, 326-339.

Wagner, D.E., Ho, J.J., and Reddien, P.W. (2012). Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis. Cell Stem Cell 10, 299-311.

Zhu, S.J., Hallows, S.E., Currie, K.W., Xu, C., and Pearson, B.J. (2015). A mex3 homolog is required for differentiation during planarian stem cell lineage development. Elife 4, e07025.

Figure 1 – figure supplement 17: Molecular markers for the developing musculature

A, C: Expression of the muscle progenitor marker myoD and the mature muscle marker mhc-1, Stages (S), S2-S8. Anterior: top (S6-S8). O: oral hemisphere. A: aboral hemisphere. D: dorsal. V: ventral. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. Cyan arrows: cephalic ganglia. Cyan arrowheads: ventral nerve cords. Scale bars: 100 µm.

B, D: Average RPKM values per embryo for the putative muscle progenitor marker myoD (B) and transcripts enriched in adult muscle (D), Y (yolk), S2-S8. Transcripts in D showed enriched expression during S5, S6, S7 and/or S8 (n=166 transcripts, or 42% of the muscle cell enriched transcripts reported in (Wurtzel et al., 2015). Muscle-enriched transcripts are flagged in excel tables for S5-S8 enriched transcripts (Figure 1 – source data 5, Figure 1 – source data 6, Figure 1 – source data 7, Figure 1 – source data 8).

S2-S4 embryos solely contain radial muscle fibers within the temporary embryonic pharynx (Figure 1 – figure supplement 17 C). Presumptive progenitors for body wall musculature, cells that coexpress piwi-1 and myoD, emerged during S5 (Figure 1 – figure supplement 17 A-B, Figure 6 C). myoD upregulation was first noted during S4, and was significantly enriched in S5 and S6 embryos (albeit the adjusted p-values were higher than the threshold set for inclusion in the S5 and S6 enriched transcript lists) (Figure 1 – figure supplement 17 A-B, Figure 1 – source data 5, Figure 1 – source data 6). While a genetic requirement for myoD in muscle cell regeneration has not been demonstrated conclusively during adulthood, myoD expression within the adult neoblast compartment has been observed previously (Scimone et al., 2014), and myoD knock-down impaired regeneration in two independent reports (Cowles et al., 2013; Reddien et al., 2005). mhc-1+ cells were first observed in the parenchyma during S5, without noticeable bias in distribution toward either the oral or aboral hemispheres (Figure 1 – figure supplement 17 C). Expression within the temporary embryonic pharynx remained strong through S6. During S6, fusiform mhc-1+ cells became more numerous, and were more densely packed beneath the presumptive dorsal surface of elongating embryos. As elongation proceeded, mhc-1+ cells were densely packed along the dorsoventral margin, particularly in the anterior half of the embryo (Figure 1 – figure supplement 17 C). The dorsal bias in mhc-1+ cell density was transient, and was not obvious by S7. myoD+ cells were located around the developing cephalic ganglia and ventral nerve cords, in S6-S8 embryos (Figure 1 – figure supplement 17 A). Concentrations of myoD+ cells, as well as mhc-1+ muscle cells, were seen around the definitive pharynx rudiment and along the developing tail stripe, a muscular septum that bifurcates the posterior gut and connects the dorsal and ventral body wall musculature (Figure 1 – figure supplement 17 A,C). Generation of robust, increasingly dense transverse and longitudinal body wall muscle fiber networks and enteric musculature proceeded during S7-S8 (Figure 1 – figure supplement 17 C).

Cowles, M.W., Brown, D.D., Nisperos, S.V., Stanley, B.N., Pearson, B.J., and Zayas, R.M. (2013). Genome-wide analysis of the bHLH gene family in planarians identifies factors required for adult neurogenesis and neuronal regeneration. Development 140, 4691-4702.

Reddien, P.W., Bermange, A.L., Murfitt, K.J., Jennings, J.R., and Sánchez Alvarado, A. (2005). Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev Cell 8, 635-649.

Scimone, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.

Asynchronously cycling piwi-1+ cells fuel embryogenesis, giving rise to all temporary and definitive tissues. During S2, some piwi-1+ blastomeres (purple cells) exit the cell cycle and differentiate into temporary embryonic tissues (primitive ectoderm, temporary embryonic pharynx and primitive endoderm). The remaining piwi-1+ blastomeres, located in the embryonic wall (purple cells, S3-S4), continue to divide and express both early embryo enriched transcripts (turquoise arrow) and adult asexual neoblast enriched transcripts (red arrow). As organogenesis begins during S5, early embryo enriched transcripts are downregulated throughout the compartment (purple cells transition into red). Concomitantly, progenitor subpopulations required for definitive organ formation are specified via the heterogeneous expression of developmental transcription factors within the piwi-1+ population (colored cells denote different progenitor subpopulations). Adult pluripotent neoblasts, themselves a lineage, are established during S5 (red cells). Embryonic donor cells harvested during or after S6 function similarly to adult neoblasts (cNeoblast activity, gray arrow). Pluripotent and lineage-primed neoblasts established during embryogenesis are maintained throughout the lifetime of the animal, where they are required for tissue maintenance during homeostasis and formation of new tissue during regeneration.

Figure 1 – figure supplement 16: Molecular markers for the developing nervous system

A, C: Average RPKM values per embryo for validated and putative adult neural progenitor transcripts (Cowles et al., 2013; Currie and Pearson, 2013; Lapan and Reddien, 2012; März et al., 2013; Monjo and Romero, 2015; Scimone et al., 2014; Wenemoser et al., 2012) (A) and adult neural classifier transcripts identified in single cell sequencing experiments (Wurtzel et al., 2015) (C), Y (yolk), Stage (S) S2-S8. Neural transcripts that showed enriched expression during S5, S6, S7 and/or S8 are flagged in excel tables for S5-S8 enriched transcripts (Figure 1 – source data 5, Figure 1 – source data 6, Figure 1 – source data 7, Figure 1 – source data 8). 90% (n=533) of the neural enriched transcripts reported in (Wurtzel et al., 2015) were identified in the smed20140614 transcriptome; 60% (n=323) of the cross-referenced transcripts were enriched during S5, S6, S7 and/or S8.

B, D: Expression of the neural progenitor marker pax6a (B) and the neural marker synaptotagmin (syt, D) (blue), S2-S8. Anterior: top (S6-S8). O: oral hemisphere. A: aboral hemisphere. D: dorsal. V: ventral. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. Cyan arrows: cephalic ganglia. Cyan arrowheads: ventral nerve cords. Scale bars: 100 µm.

WISH developmental time course data for two markers expressed in differentiated neurons, synaptotagmin and PC-2, suggests that the nervous system is limited to neurons in the temporary embryonic pharynx of S2-S5 embryos (Figure 1 – figure supplement 16 D, Figure 1 – figure supplement 12 A). Validated and presumptive neural progenitor markers showing low to undetectable expression during early embryogenesis are upregulated as organogenesis commences during S5 (Figure 1 – figure supplement 16 A) (Cowles et al., 2013; Currie and Pearson, 2013; Lapan and Reddien, 2012; März et al., 2013; Monjo and Romero, 2015; Scimone et al., 2014; Wenemoser et al., 2012), and many transcripts exhibiting enriched expression in adult neurons show upregulated expression during and after S5 (Figure 1 – figure supplement 16 C). During S6 and S7, neural progenitors and their descendants must migrate, interact, and organize themselves into two bilaterally symmetric cephalic ganglia and the attendant ventral nerve cords, commissural and peripheral neurons (Figure 1 – figure supplement 16 D). Differentiating neurons accumulate in the presumptive anterior region of the embryo, adjacent to the D/V margin, as the cephalic ganglia form (Figure 1 – figure supplement 16 B,D, cyan arrows). Ventral nerve cord formation is evident during S6, and appears to proceed from anterior to posterior (Figure 1 – figure supplement 16 D, cyan arrowheads). Gross morphology of the nervous system is comparable to that of adult animals during S7-S8 (Figure 1 – figure supplement 16 B,D).

Currie, K.W., and Pearson, B.J. (2013). Transcription factors lhx1/5-1 and pitx are required for the maintenance and regeneration of serotonergic neurons in planarians. Development 140, 3577-3588.

Lapan, S.W., and Reddien, P.W. (2012). Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration. Cell Rep 2, 294-307.

März, M., Seebeck, F., and Bartscherer, K. (2013). A Pitx transcription factor controls the establishment and maintenance of the serotonergic lineage in planarians. Development 140, 4499-4509.

Monjo, F., and Romero, R. (2015). Embryonic development of the nervous system in the planarian Schmidtea polychroa. Dev Biol 397, 305-319.

Scimone, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.

Wenemoser, D., Lapan, S.W., Wilkinson, A.W., Bell, G.W., and Reddien, P.W. (2012). A molecular wound response program associated with regeneration initiation in planarians. Genes Dev 26, 988-1002.

Figure 1 - figure supplement 18: Molecular markers for the developing excretory system

A, C: Average RPKM per embryo for transcripts expressed in protonephridia progenitors (pou2/3, six1/2-2, sal1, eya, osr) (Scimone et al., 2011) (A) or differentiated protonephridia (Rink et al., 2011; Scimone et al., 2011; Wurtzel et al., 2015) (C). Transcripts shown were enriched during S5, S6, S7 and/or S8 and are flagged in excel tables for S5-S8 enriched transcripts (Figure 1 – source data 5, Figure 1 – source data 6, Figure 1 – source data 7, Figure 1 – source data 8).

B, D: WISH developmental time course with riboprobes complementary to the protonephridial progenitor and tubule cell marker pou2/3 (B) or the non-ciliated tubule marker CAVII-1 (D) (blue), S2-S8. Anterior: top (S6-S8). D: dorsal. L: lateral. Red arrowheads: definitive pharynx. Scale bars: 100 µm.

The transcription factors pou2/3, six1/2-2, eya, sal1, all of which were required for protonephridia specification during regeneration, as well as osr, a transcription factor expressed in protonephridial progenitors, showed enriched expression during S5 (Figure 1 – figure supplement 18 A, Figure 1 – source data 5) (Scimone et al., 2011). Scattered pou2/3+ cells were detected by WISH in S5 embryos (Figure 1 – figure supplement 18 B), and preferential accumulation of pou2/3+ cells around the presumptive D/V margin of the embryo was observed as tubules began to form during S6. Branching morphogenesis of the tubules proceeded during S6-S8, with a noted dorsolateral bias in positioning of the developing filtration units (Figure 1 – figure supplement 18 D). Transcripts with enriched expression in adult protonephridial cell type(s) were frequently expressed at low to undetectable levels prior to S5, and were upregulated during S5-S8 (Figure 1 – figure supplement 18 C) (Scimone et al., 2011; Vu, 2015; Wurtzel et al., 2015). crocc (rootletin), a marker of ciliated epithelial cells (Scimone et al., 2011), was expressed in the developing protonephridia during S6-S8 (Figure 1 – figure supplement 15 I).

Scimone, M.L., Srivastava, M., Bell, G.W., and Reddien, P.W. (2011). A regulatory program for excretory system regeneration in planarians. Development 138, 4387-4398.

Vu, H.T.-K., Jochen C Rink, Sean A McKinney, Melainia McClain, Naharajan Lakshmanaperumal, Richard Alexander, Alejandro Sánchez Alvarado (2015). Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. Elife 4, e07405.

Wurtzel, O., Cote, L.E., Poirier, A., Satija, R., Regev, A., and Reddien, P.W. (2015). A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians. Dev Cell 35, 632-645.

Figure 1 – figure supplement 19: Molecular markers for the developing eyes

A, C: Average RPKM per embryo for transcripts required for eye progenitor specification (ovo, six-1/2, eya (purple)), for photoreceptor neuron differentiation (otxA,(red)), or pigment cup differentiation (sp6-9, dlx (blue)) (A), or with enriched expression in adult eye tissue (Lapan and Reddien, 2012) (C). Yolk (Y), S2-S8. Transcripts shown were enriched during S5, S6, S7 and/or S8, and are flagged in excel tables for S5-S8 enriched transcripts (Figure 1 – source data 5, Figure 1 – source data 6, Figure 1 – source data 7, Figure 1 – source data 8).

B, D-E: WISH developmental time course with riboprobes complementary to ovo (B), opsin (D), and tyrosinase (E), S5-S8. Anterior: top (S6-S8). D: dorsal. Purple Arrowheads: developing eye tissue. Blue arrowheads: trail cells (eye progenitors). Scale bars: 100 µm.

Genes required for production of all eye progenitors (eya, six-1/2), photoreceptor neuron differentiation (otxA), or pigment cup differentiation (sp6-9, dlx) were enriched during S5-S6 (Figure 1 – figure supplement 19 A, Figure 1 – source data 5, Figure 1 – source data 6)(Lapan and Reddien, 2011, 2012). The transcription factor ovo, a master regulator of eye development in planarians, showed statistically significant upregulation during S6, albeit with adjusted p-values and fold-changes that precluded its inclusion on the S6 enriched transcript list (Lapan and Reddien, 2012). ovo expression was first detected by WISH during S5-S6, in only a few cells per embryo (Figure 1 – figure supplement 19 B). During S7, two dorsolateral trails of ovo+ progenitors appeared along the anterior head margin, and these cells and their differentiating descendants likely generate the eye primordia (Figure 1 – figure supplement 19 B, blue arrowheads). The photoreceptor neurons, marked by opsin (Figure 1 – figure supplement 19 D), and the non-neuronal pigment cups, marked by tyrosinase (Figure 1 – figure supplement 19 E), were first visible during S7, consistent with published expression patterns for Spol-opsin and Spol-tph in elongated embryos (Martín-Durán et al., 2012). Growth of the eyes continued through S8. Moreover, co-expression of ovo and transcription factors required during eye differentiation, including eya, klf, six-1/2, soxB, sp6-9, otxA, meis and FoxQ2, was reported in elongated embryos (S7-S8) (Lapan and Reddien, 2012), suggesting that eye development during embryogenesis and adulthood may rely upon common genes and genetic regulatory networks.

Lapan, S.W., and Reddien, P.W. (2011). dlx and sp6-9 Control optic cup regeneration in a prototypic eye. PLoS Genet 7, e1002226.

Lapan, S.W., and Reddien, P.W. (2012). Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration. Cell Rep 2, 294-307.

Martín-Durán, J.M., Monjo, F., and Romero, R. (2012). Morphological and molecular development of the eyes during embryogenesis of the freshwater planarian Schmidtea polychroa. Dev Genes Evol 222, 45-54.

Zygote-derived blastomeres undergo dispersed cleavage among yolk cells: they divide asynchronously and are not in direct contact with one another (Bardeen, 1902; Cardona et al., 2005; Hallez, 1887; Ijima, 1884; Le Moigne, 1963; Metschnikoff, 1883; Vara et al., 2008).

During sphere formation (Stage 2), some blastomeres differentiate into temporary embryonic cell types that provide form and function to the embryo (Cardona et al., 2005; Hallez, 1887). Undifferentiated blastomeres remain in the embryonic wall of nascent spheres (Cardona et al., 2005; Hallez, 1887).

piwi-1 is expressed in all undifferentiated blastomeres of S3 embryos.

SPIM reconstructed S3 embryo costained with piwi-1 (red) and EF1a-like-1 (green). piwi-1 is expressed in all undifferentiated blastomeres in the embryonic wall (piwi-1+, EF1a-like-1+ cells). piwi-1 is not expressed in differentiated tissues marked by EF1a-like-1 alone, including the primitive ectoderm and temporary embryonic pharynx (green). Several fluorescent beads used for 3-dimensional reconstruction are visible (red).

Mitotic activity is restricted to piwi-1+ blastomeres, which cycle asynchronously.

SPIM reconstructed S4 embryo costained with piwi-1 and LYAG-like (both in green) and H3S10p antibodies (red). LYAG-like marks the temporary embryonic pharynx and is not expressed in piwi-1+ blastomeres. Several examples of piwi-1+, H3S10p+ cells are evident.

Cell cycle activity is restricted to blastomeres in the embryonic wall of S3-S5 embryos. All blastomeres are cycling, and they cycle asynchronously.

piwi-1+ blastomeres co-express the adult asexual neoblast enriched gene piwi-2.

SPIM reconstructed S4 embryo costained with piwi-1 (red) and piwi-2 (green). piwi-1+ blastomeres co-express the nuage factor piwi-2, and virtually all piwi-2+ cells co-express piwi-1. Several fluorescent beads used for 3-dimensional reconstruction are visible (green).

piwi-1+ blastomeres co-express the adult asexual neoblast enriched gene piwi-3.

SPIM reconstructed S4 embryo costained with piwi-1 (red) and piwi-3 (green). piwi-1+ blastomeres co-express the nuage factor piwi-3, and virtually all piwi-3+ cells co-express piwi-1.

piwi-1+ blastomeres co-express the adult asexual neoblast enriched gene tud-1.

SPIM reconstructed S4 embryo costained with piwi-1 (red) and tud-1 (green). piwi-1+ blastomeres co-express the nuage factor tud-1, and virtually all tud-1+ cells co-express piwi-1.

piwi-1+ blastomeres co-express the adult asexual neoblast enriched gene bruli-1.

SPIM reconstructed S4 embryo costained with piwi-1 (red) and bruli-1 (green). piwi-1+ blastomeres co-express the stem cell maintenance gene bruli-1, and virtually all bruli-1+ cells co-express piwi-1. Several fluorescent beads used 3-dimensional reconstruction are visible (red).

Blastomeres express many adult asexual neoblast markers. Shared elements of the blastomere and neoblast expression signatures likely include genes implicated in DNA replication and repair, cell cycle control, chromatin remodeling and/or modification, genome surveillance and pluripotency, all prominent features of an evolutionarily conserved gene expression signature for metazoan primordial stem cells (Alie et al., 2015). For additional information, see Figure 4 and associated text.

Colorimetric WISH depicting expression of the early embryo enriched (EEE) transcripts tct-like, *BTF3-like*, *DDX5-like* and *eIF4a-like* (blue) in S2-S8 embryos. EEE transcript expression declines dramatically during S5 and remains low through subsequent stages. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. O: oral. V: ventral. Scale bars: 100 µm.

**Early Embryo Enriched transcripts were expressed throughout the *piwi-1+* population in S3-S4 embryos.**
Fluorescent double WISH with riboprobes against *piwi-1* (red) and the EEE transcripts *tct-like*, *BTF3-like*, *DDX5-like* and *eIF4a-like* (green) in S4 embryos. Percentage *piwi-1+* cells coexpressing the indicated EEE marker (red) and percentage EEE+ cells coexpressing *piwi-1* (green) appear in lower left corner of merged images.

Blastomeres express early embryo enriched (EEE) transcripts, providing a molecular metric to distinguish the expression profiles of blastomeres and neoblasts. EEE transcripts expressed throughout the undifferentiated piwi-1+ blastomere population in S3-S4 embryos are downregulated as definitive organogenesis begins during S5. These transcripts likely represent a key temporal shift in the expression profile of piwi-1+ cells during embryogenesis. For additional information, see Figure 5 and associated text.

The blastomere to neoblast transition occurs during S5, as EEE transcripts are downregulated. Concomitantly, regulators of lineage commitment and differentiation are upregulated heterogeneously within the blastomere population. The observed changes in gene expression within the blastomere population mirror functional distinctions observed between S4, S5 and S6 embryonic donor cells in heterochronic transplantation assays. Blastomeres, albeit similar in many respects to adult neoblasts, do not persist in an adult microenvironment. In contrast, embryos undergoing organogenesis harbor cells with neoblast activity: they engraft, proliferate, differentiate and can ultimately rescue adult hosts devoid of stem cells from lethality. For additional information, see Figures 6-9 and associated text.

Alie, A., Hayashi, T., Sugimura, I., Manuel, M., Sugano, W., Mano, A., Satoh, N., Agata, K., and Funayama, N. (2015). The ancestral gene repertoire of animal stem cells. Proc Natl Acad Sci U S A 112, E7093-7100.

Bardeen, C.R. (1902). Embryonic and regenerative development in planarians. Biol Bull 3, 262-288.

Cardona, A., Hartenstein, V., and Romero, R. (2005). The embryonic development of the triclad Schmidtea polychroa. Dev Genes Evol 215, 109-131.

Hallez, P. (1887). Embryogénie des Dendrocoeles d’eau douce, Vol 16.

Ijima, I. (1884). Untersuchungeniiber den Bau und die Entwicklungsgeschichte der stisswasser-Dendrocoelen(Tricladen). Vol Bd. 40.

Le Moigne, A. (1963). Etude du developpement embryonnaire de Polycelis nigra (Turbellarie - Triclade). Bulletin de la Societe Zoologique de France 88, 403-422.

Metschnikoff, E. (1883). Die embryologie von Planaria polychroa. Zeitschrift fur wissenschaftliche Zoologie 38, 331-354.

Vara, D.C., Leal-Zanchet, A.M., and Lizardo-Daudt, H. (2008). Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola). Braz J Biol 68, 889-895.

Forsthoefel, D.J., Park, A.E., and Newmark, P.A. (2011). Stem cell-based growth, regeneration, and remodeling of the planarian intestine. Dev Biol 356, 445-459.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Wurtzel, O., Cote, L.E., Poirier, A., Satija, R., Regev, A., and Reddien, P.W. (2015). A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians. Dev Cell 35, 632-645.

Figure 1 – figure supplement 11: Molecular markers for the primitive ectoderm

A: Average RPKM values per embryo for primitive ectoderm markers gelsolin-like (SMED30014940, blue) and spondin1-like (SMED30032088, red) during embryogenesis, Y (yolk), S2-S8.

B-C: The number of primitive ectoderm cells remained constant while embryo volume increased during S3-S4. Primitive ectoderm cell nuclei were scored in SPIM reconstructed S3 and S4 embryos. B) S3: Average number of primitive ectoderm nuclei per embryo: 21.5 +/- 2.9. S4: Average number of primitive ectoderm nuclei per embryo: 22 +/- 1.4. n=5 embryos. Unpaired t-test, two tailed p value = 0.72. C) Average S3 embryo volume: 7.4 x 10⁷ µm³. Average S4 embryo volume: 1.5 x 10⁸ µm³. Unpaired t-test, two tailed p value = 0.01. Embryo volumes were calculated by generating a masked surface in Imaris. Embryos scored, S3: n= 22. S4: n=5. Error bars: Standard deviation of the mean.

D-E: gelsolin-like (D) and spondin1-like (E) expression (blue) during embryogenesis, S2-S8. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. Yellow arrows: primitive ectoderm cells. O: oral hemisphere. A: aboral hemisphere. V: ventral. Scale bars: 100 µm.

Sphere formation begins during S2 when some of the blastomeres exit the cell cycle and commit to primitive ectoderm fate (Hallez, 1887; Ijima, 1884; Le Moigne, 1963; Metschnikoff, 1883). These cells flatten, elaborate numerous processes, and ultimately generate a single cell layer bounding the sphere. The number of primitive ectoderm cells varied, with a mean of 21.5 +/- 2.9 per S3 embryo (Figure 1 – figure supplement 11 B). This observation is consistent with primitive ectoderm cell number estimations in Polycelis nigra embryos (Le Moigne, 1963). Moreover, the primitive ectoderm cell number remained constant during S4 as the embryo ingested yolk and increased in volume (Figure 1 – figure supplement 11 B-C). Consistent with this finding, cycling cells were not observed in the primitive ectoderm (Figure 3 A-E). Pan-embryonic cell markers, including EF1a-like-1 (SMED30004295), EF1a-like-2 (SMED30012165), and eIF4a-like (SMED30003202) were expressed in primitive ectoderm cells of S2-S3 embryos, (Figure 2A-B, Figure 5 E, Figure 5 – source data 3). gelsolin-like (SMED30014940) was expressed in blastomeres during S2, and primitive ectoderm cells and the temporary embryonic pharynx in S3-S4 embryos (Figure 1 – figure supplement 11 D). gelsolin-like mRNA was detected throughout the primitive ectoderm cell layer, with concentrated perinuclear staining. spondin-1-like (SMED30032088) was frequently detected in blastomeres during S2, and was expressed in primitive ectoderm cells with star-like morphology, as well as parenchymal cells, in S3-S4 embryos (Figure 1 – figure supplement 11 E). Early embryo enriched (EEE) transcripts were not detected in the primitive ectoderm beyond S4 (Figure 5 – source data 3).

Hallez, P. (1887). Embryogénie des Dendrocoeles d’eau douce, Vol 16.

Ijima, I. (1884). Untersuchungeniiber den Bau und die Entwicklungsgeschichte der stisswasser-Dendrocoelen(Tricladen). Vol Bd. 40.

Le Moigne, A. (1963). Etude du developpement embryonnaire de Polycelis nigra (Turbellarie - Triclade). Bulletin de la Societe Zoologique de France 88, 403-422.

Metschnikoff, E. (1883). Die embryologie von Planaria polychroa. Zeitschrift fur wissenschaftliche Zoologie 38, 331-354.

Figure 1 – figure supplement 12: Molecular markers for the temporary embryonic pharynx

A-B: The temporary embryonic pharynx is innervated by PC-2+ neurons (A) and contains mhc-1+ radial muscle fibers (B). For simplicity, only S4 is shown. The image shown in B is also shown in Figure 1 – figure supplement 17 C.

C: Average RPKM values per embryo for the temporary embryonic pharynx markers venom allergen-like (VAL-like; SMED30015313), macrophage expressed gene 1 like-1 (MPEG1-like 1; SMED30000139), MPEG1-like 2 (SMED30034696) and netrin-like (SMED30023593) during embryogenesis, Y (yolk), Stage (S) S2-S8.

D-G: Expression of temporary embryonic pharynx specific markers VAL-like (D, S3-S7), netrin-like (E, S2-S7), MPEG1-like-1 (F, S3-S4) and MPEG1-like-2 (G, S3-S4).

A-B, D-G: Anterior: top (S6-S8). Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. O: oral hemisphere. A: aboral hemisphere. D: dorsal. V: ventral. Scale bars: 100 µm.

The temporary embryonic pharynx is an innervated, muscular pump that ingests yolk into the gut cavity (Figure 1 – figure supplement 12 A-B). It forms during S2 and likely functions during S3 through S5. Temporary embryonic pharynx-specific markers were identified among the S2-S4 enriched transcripts, including VAL-like (Figure 1 – figure supplement 12 C-D), netrin-like (Figure 1 – figure supplement 12 C,E), MPEG1-like-1 (Figure 1 – figure supplement 12 C,F), MPEG1-like-2 (Figure 1 – figure supplement 12 C,G). Expression of foxA1, a developmental transcription factor required for maintenance and regeneration of the pharynx during adulthood (Adler et al., 2014; Scimone et al., 2014), was detected in the epithelial cells lining the lumen of the temporary embryonic pharynx during S3-S5 (Figure 1 – figure supplement 14 A, black arrowheads). The temporary embryonic pharynx degenerates during S6, as the definitive pharynx primordium develops beneath it (Martín-Durán and Romero, 2011). Temporary embryonic pharynx markers are no longer detectable by S7 (Figure 1 – figure supplement 12 C-D).

Adler, C.E., Seidel, C.W., McKinney, S.A., and Sánchez Alvarado, A. (2014). Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. Elife 3, e02238.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Scimone, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.

Figure 1 – figure supplement 14: Molecular markers for the definitive pharynx

A: WISH developmental time course using foxA1 riboprobes (blue), S3-S8. foxA1 expression was consistently detected in the embryonic pharynx lumen during S3-S5 (black arrowheads). Anterior: top (S6-S8). Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. O: oral hemisphere. A: aboral hemisphere. D: dorsal. V: ventral. Scale bars: 100 µm.

B: Average RPKM values per embryo for the definitive pharynx markers foxA1, meis, laminin, npp-1 during embryogenesis (Adler et al., 2014; Scimone et al., 2014), Y (yolk), Stage (S) S2-S8.

foxA1, a pioneer transcription factor required for pharynx maintenance and regeneration during adulthood (Adler et al., 2014; Scimone et al., 2014), may similarly be required for construction of the definitive pharynx during embryogenesis. Development of the definitive pharynx, the single opening of the Smed digestive tract, commenced during S4-S5 with the onset of foxA1 expression in parenchymal cells, many of which were located in the oral hemisphere (Figure 1 – figure supplement 14A). The distribution of foxA1+ cells remained concentrated in and around the developing definitive pharynx rudiment during S6-S8, a pattern reminiscent of that observed in S. polychroa embryos (Martín-Durán et al., 2010), as well as in intact and regenerating Smed asexual adults (Adler et al., 2014; Scimone et al., 2014). The definitive pharynx develops beneath the degenerating temporary embryonic pharynx, and marks the ventral side of the embryos during S6 and thereafter (Martín-Durán and Romero, 2011). foxA1 upregulation during S5-S8 was statistically significant, albeit the adjusted p-values were above the thresholds set for inclusion in the enriched transcript lists presented in Figure 1 – source data 5, Figure 1 – source data 6, Figure 1 – source data 7, Figure 1 – source data 8. meis, a transcription factor coexpressed in foxA1+ neoblasts and expressed within the regenerating pharynx (Scimone et al., 2014), was among the S5 enriched transcripts (Figure 1 – source data 5); its expression trend was similar to foxA1 during embryogenesis (Figure 1 – figure supplement 14B). Two markers exhibiting pharynx-restricted expression in adults, laminin and npp-1 (Adler et al., 2014), were upregulated during S6-S8, after development of the definitive pharynx rudiment was evident (Figure 1 – figure supplement 14B).

Adler, C.E., Seidel, C.W., McKinney, S.A., and Sánchez Alvarado, A. (2014). Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. Elife 3, e02238.

Martín-Durán, J.M., Amaya, E., and Romero, R. (2010). Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa. Dev Biol 340, 145-158.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Scimone, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.

Zygote(s) and yolk cells are packaged into egg capsules laid by the parent. The single cell stage can be visualized in histological cross-sections: spherical zygotes, ~ 25-30 µm in diameter, are surrounded by a halo of yolk cells reminiscent of the corona radiata. During early cleavage divisions blastomeres cycle asynchronously, without retaining direct cell-cell contacts, within a yolk syncytium. Embryos are readily apparent in histologically stained sections but may be difficult to locate in live, unstained specimens.

Embryos undergoing the dynamic process of sphere formation are called protospheres, and appear as loosely adherent masses of shaggy yolk, roughly 200 µm in diameter, in live preparations. During sphere formation some blastomeres differentiate into temporary embryonic tissues, providing form and function to the embryo. The primitive ectoderm forms a single cell layer bounding the sphere. Other blastomeres differentiate into the temporary embryonic pharynx and associated primitive gut cells. The temporary embryonic pharynx is an innervated, muscular pump that ingests yolk into the primitive gut cavity. The primitive gut consists of phagocytic cells associated with the temporary embryonic pharynx. Some undifferentiated blastomeres remain in the embryonic wall, the parenchymal space between the primitive gut and ectoderm.

1) Pairwise comparison (S2 vs Y): adjusted p-value < 1e-5 S2 vs mixed reference comparison: adjusted p-value <1e-52) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only)3) Average scaled RPKM value ≥ 1.0 for S24) Transcript has ≥ 1 ORF5) Transcripts derived from transposase or retroviruses were omitted, as were S2 enriched transcripts also upregulated in yolk in the mixed reference comparison.

Tabs in this excel file contain 1) pairwise comparison data (if applicable), 2) mixed stage reference comparison data, 3) cluster membership, average RPKM values across embryogenesis (Y-S8), and in C4 and SX adults, as well as best BLASTx hits (E < 0.001) versus the NR, Swiss-Prot, C. elegans, D. melanogaster, D. rerio, X. tropicalis, M. musculus and H. sapiens REF-Seq databases, 4) GO analysis: Manually curated and categorized Biological Process (BP) GO IDs and 5) GO analysis: unabridged results. S2-S8: Stages 2-8. Y: Yolk. C4: C4 asexual adults. SX: Mature sexual adults

Blastomeres continue to divide asynchronously in the embryonic wall, the parenchymal space between the primitive gut and ectoderm. The temporary embryonic pharynx ingests yolk into the gut cavity of nascent spheres, which range in size from ~ 200-400 µm in diameter. Four primitive gut cells abutting the pharynx digest yolk; few, if any, differentiating gut cells are seen at other locations in the embryo.

1) Pairwise comparison (S2 vs S3): adjusted p-value < 1e-5 S3 vs mixed reference comparison: adjusted p-value <1e-52) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only)3) Average scaled RPKM value ≥ 1.0 for S34) Transcript has ≥ 1 ORF5) Transcripts derived from transposase or retroviruses were omitted, as were S3 enriched transcripts also upregulated in yolk in the mixed reference comparison.

Blastomeres continue to divide asynchronously in the embryonic wall. The temporary embryonic pharynx ingests yolk cells into the gut cavity of spherical embryos, ranging in size from ~300-500 µm in diameter. Islands of differentiating gut cells appear within and beneath the parenchyma. It is not known whether the embryonic gut created during stage 4 is a temporary or definitive tissue

1) Pairwise comparison (S3 vs S4): adjusted p-value < 1e-5 S4 vs mixed reference comparison: adjusted p-value <1e-52) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only)3) Average scaled RPKM value ≥ 1.0 for S44) Transcript has ≥ 1 ORF5) Transcripts derived from transposase or retroviruses were omitted, as were S4 enriched transcripts also upregulated in yolk in the mixed reference comparison.

Spherical embryos continue to grow via yolk ingestion, reaching maximal size by the end of Stage 5 (~400-800 µm in diameter). Blastomeres fill the parenchyma, forming a dense collection of cells blanketing the sphere. Yolk-laden gut cells generate a continuous, honeycomb-like lattice encircling the central ingested yolk mass. Many early embryo enriched transcripts expressed in S4 blastomeres are downregulated during S5. Progenitors for many of the definitive organs, including the epidermis, nervous system, muscle, gut, pharynx, protonephridia, eyes and primordial germ cells first appear during Stage 5, marking the onset of organogenesis. Pluripotent neoblasts and adult lineage progenitors arise in the blastomere population during S5.

1) Pairwise comparison (S4 vs S5): adjusted p-value < 1e-5 S5 vs mixed reference comparison: adjusted p-value <1e-52) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only)3) Average scaled RPKM value ≥ 1.0 for S54) Transcript has ≥ 1 ORF5) Transcripts derived from transposase or retroviruses were omitted.

Spherical embryos begin to flatten and elongate, with the anterior head margin becoming apparent. The posterior portion of the embryo usually appears larger and more bulbous than the head prior to formation of the tail stripe, a muscular septum connecting the dorsal and ventral surfaces that bifurcates the posterior gut. Embryos remain unpigmented and immotile during S6. Differentiation of the definitive organ systems continues. Parenchymal epidermal progenitors are abundant and are more densely packed on the dorsal side of the embryo, and are often more numerous in the anterior. Fusiform muscle cells, once uniformly distributed across the sphere, become more densely packed on the presumptive dorsal side of the elongating animal. The gut cavity, still filled with yolk, is ovoid and largely unbranched. Differentiated gut cells expressing embryonic gut markers are detected through S6; gut cells expressing definitive gut markers increase during S6. The definitive pharynx develops beneath the degenerating temporary embryonic pharynx on the ventral side of the embryo, near the presumptive posterior pole. Aggregations of differentiating neurons accumulate along the anterior head margin, along the presumptive dorsoventral boundary, and constellations of nerve cells form the ventral nerve cords, beginning from the base of the cerebral ganglia and progressively proceeding posteriorly. Although eye progenitors are present and differentiating, the eyes are generally not visible in live specimens during S6.

1) Pairwise comparison (S5 vs S6): adjusted p-value < 1e-5 S6 vs mixed reference comparison: adjusted p-value <1e-202) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only)3) Average scaled RPKM value ≥ 1.0 for S64) Transcript has ≥ 1 ORF5) Transcripts derived from transposase or retroviruses were omitted.

Elongated embryos continue to flatten, but have clearly adopted the bilaterally symmetric body plan of juvenile and adult animals. Pigmentation, if present, is sparse. Animals are motile: slow gliding locomotion is apparent, but animals cannot flip over. Mature, ciliated epidermal cells largely cover the dorsal and ventral surfaces of the embryos. A dense network of elongated, anteroposterior and circular fibers of body wall muscle is evident beneath the epidermis. Gut development continues: branching morphogenesis is evident, as is clear separation of the two posterior gut branches. piwi-1+ cells occupy the parenchymal space between the gut branches and along the tail stripe. Expression of embryonic gut markers ceases, and expression of mature gut markers becomes robust. Resorption of the temporary embryonic pharynx is complete, and definitive pharynx formation continues. The bilobed brain and ventral nerve cords are well developed, and lateral and commissural neurons continue to develop. The eyes, noted by the presence of developing pigment cups in live animals, first become visible during S7.

Criteria for flagging differentially expressed transcripts: 1) Pairwise comparison (S6 vs S7): adjusted p-value < 1e-5 S7 vs mixed reference comparison: adjusted p-value <1e-202) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only)3) Average scaled RPKM value ≥ 1.0 for S74) Transcript has ≥ 1 ORF5) Transcripts derived from transposase or retroviruses were omitted.

Elongated, bilaterally symmetric animals continue to mature and are capable of developing outside of the egg capsule in 1x Montjuic water. Gliding speed increases, and animals acquire the ability to flip over. Outgrowth of the anterior head margin becomes evident as the epidermis matures. The body wall musculature grows denser. Gut branching and morphogenesis continues, and undigested yolk remains in the gut at the time of hatching. The piwi-1+ compartment occupies the parenchymal space between the gut branches. The eyes enlarge, and both the photoreceptors and pigment cups become visible. Pigmentation, usually sparse in newborn hatchlings, progressively increases during juvenile development.

Criteria for flagging differentially expressed transcripts: 1) Pairwise comparison (S7 vs S8): adjusted p-value < 1e-5 S8 vs mixed reference comparison: adjusted p-value <1e-202) log2 ratio ≥ 2.322 (5-fold upregulation, both comparisons) or log2 ratio ≤ -2.322 (5 fold downregulation, pairwise comparison only)3) Average scaled RPKM value ≥ 1.0 for S84) Transcript has ≥ 1 ORF5) Transcripts derived from transposase or retroviruses were omitted.

Forsthoefel, D.J., James, N.P., Escobar, D.J., Stary, J.M., Vieira, A.P., Waters, F.A., and Newmark, P.A. (2012). An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians. Dev Cell 23, 691-704.

Forsthoefel, D.J., Park, A.E., and Newmark, P.A. (2011). Stem cell-based growth, regeneration, and remodeling of the planarian intestine. Dev Biol 356, 445-459.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Wurtzel, O., Cote, L.E., Poirier, A., Satija, R., Regev, A., and Reddien, P.W. (2015). A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians. Dev Cell 35, 632-645.

Eisenhoffer, G.T., Kang, H., and Sánchez Alvarado, A. (2008). Molecular analysis of stem cells and their descendants during cell turnover and regeneration in the planarian Schmidtea mediterranea. Cell Stem Cell 3, 327-339.

Pearson, B.J., and Sánchez Alvarado, A. (2010). A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages. Development 137, 213-221.

van Wolfswinkel, J.C., Wagner, D.E., and Reddien, P.W. (2014). Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment. Cell Stem Cell 15, 326-339.

Wagner, D.E., Ho, J.J., and Reddien, P.W. (2012). Genetic regulators of a pluripotent adult stem cell system in planarians identified by RNAi and clonal analysis. Cell Stem Cell 10, 299-311.

Zhu, S.J., Hallows, S.E., Currie, K.W., Xu, C., and Pearson, B.J. (2015). A mex3 homolog is required for differentiation during planarian stem cell lineage development. Elife 4, e07025.

Cowles, M.W., Brown, D.D., Nisperos, S.V., Stanley, B.N., Pearson, B.J., and Zayas, R.M. (2013). Genome-wide analysis of the bHLH gene family in planarians identifies factors required for adult neurogenesis and neuronal regeneration. Development 140, 4691-4702.

Reddien, P.W., Bermange, A.L., Murfitt, K.J., Jennings, J.R., and Sánchez Alvarado, A. (2005). Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev Cell 8, 635-649.

Cowles, M.W., Brown, D.D., Nisperos, S.V., Stanley, B.N., Pearson, B.J., and Zayas, R.M. (2013). Genome-wide analysis of the bHLH gene family in planarians identifies factors required for adult neurogenesis and neuronal regeneration. Development 140, 4691-4702.

Currie, K.W., and Pearson, B.J. (2013). Transcription factors lhx1/5-1 and pitx are required for the maintenance and regeneration of serotonergic neurons in planarians. Development 140, 3577-3588.

Lapan, S.W., and Reddien, P.W. (2012). Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration. Cell Rep 2, 294-307.

März, M., Seebeck, F., and Bartscherer, K. (2013). A Pitx transcription factor controls the establishment and maintenance of the serotonergic lineage in planarians. Development 140, 4499-4509.

Monjo, F., and Romero, R. (2015). Embryonic development of the nervous system in the planarian Schmidtea polychroa. Dev Biol 397, 305-319.

Scimone, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.

Wenemoser, D., Lapan, S.W., Wilkinson, A.W., Bell, G.W., and Reddien, P.W. (2012). A molecular wound response program associated with regeneration initiation in planarians. Genes Dev 26, 988-1002.

Scimone, M.L., Srivastava, M., Bell, G.W., and Reddien, P.W. (2011). A regulatory program for excretory system regeneration in planarians. Development 138, 4387-4398.

Vu, H.T.-K., Jochen C Rink, Sean A McKinney, Melainia McClain, Naharajan Lakshmanaperumal, Richard Alexander, Alejandro Sánchez Alvarado (2015). Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. Elife 4, e07405.

Wurtzel, O., Cote, L.E., Poirier, A., Satija, R., Regev, A., and Reddien, P.W. (2015). A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians. Dev Cell 35, 632-645.

Lapan, S.W., and Reddien, P.W. (2011). dlx and sp6-9 Control optic cup regeneration in a prototypic eye. PLoS Genet 7, e1002226.

Lapan, S.W., and Reddien, P.W. (2012). Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration. Cell Rep 2, 294-307.

Martín-Durán, J.M., Monjo, F., and Romero, R. (2012). Morphological and molecular development of the eyes during embryogenesis of the freshwater planarian Schmidtea polychroa. Dev Genes Evol 222, 45-54.

Zygote-derived blastomeres undergo dispersed cleavage among yolk cells: they divide asynchronously and are not in direct contact with one another (Bardeen, 1902; Cardona et al., 2005; Hallez, 1887; Ijima, 1884; Le Moigne, 1963; Metschnikoff, 1883; Vara et al., 2008).

During sphere formation (Stage 2), some blastomeres differentiate into temporary embryonic cell types that provide form and function to the embryo (Cardona et al., 2005; Hallez, 1887). Undifferentiated blastomeres remain in the embryonic wall of nascent spheres (Cardona et al., 2005; Hallez, 1887).

Cell cycle activity is restricted to blastomeres in the embryonic wall of S3-S5 embryos. All blastomeres are cycling, and they cycle asynchronously.

Alie, A., Hayashi, T., Sugimura, I., Manuel, M., Sugano, W., Mano, A., Satoh, N., Agata, K., and Funayama, N. (2015). The ancestral gene repertoire of animal stem cells. Proc Natl Acad Sci U S A 112, E7093-7100.

Bardeen, C.R. (1902). Embryonic and regenerative development in planarians. Biol Bull 3, 262-288.

Cardona, A., Hartenstein, V., and Romero, R. (2005). The embryonic development of the triclad Schmidtea polychroa. Dev Genes Evol 215, 109-131.

Hallez, P. (1887). Embryogénie des Dendrocoeles d’eau douce, Vol 16.

Ijima, I. (1884). Untersuchungeniiber den Bau und die Entwicklungsgeschichte der stisswasser-Dendrocoelen(Tricladen). Vol Bd. 40.

Le Moigne, A. (1963). Etude du developpement embryonnaire de Polycelis nigra (Turbellarie - Triclade). Bulletin de la Societe Zoologique de France 88, 403-422.

Metschnikoff, E. (1883). Die embryologie von Planaria polychroa. Zeitschrift fur wissenschaftliche Zoologie 38, 331-354.

Vara, D.C., Leal-Zanchet, A.M., and Lizardo-Daudt, H. (2008). Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola). Braz J Biol 68, 889-895.

Forsthoefel, D.J., James, N.P., Escobar, D.J., Stary, J.M., Vieira, A.P., Waters, F.A., and Newmark, P.A. (2012). An RNAi screen reveals intestinal regulators of branching morphogenesis, differentiation, and stem cell proliferation in planarians. Dev Cell 23, 691-704.

Forsthoefel, D.J., Park, A.E., and Newmark, P.A. (2011). Stem cell-based growth, regeneration, and remodeling of the planarian intestine. Dev Biol 356, 445-459.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Wurtzel, O., Cote, L.E., Poirier, A., Satija, R., Regev, A., and Reddien, P.W. (2015). A Generic and Cell-Type-Specific Wound Response Precedes Regeneration in Planarians. Dev Cell 35, 632-645.

Hallez, P. (1887). Embryogénie des Dendrocoeles d’eau douce, Vol 16.

Ijima, I. (1884). Untersuchungeniiber den Bau und die Entwicklungsgeschichte der stisswasser-Dendrocoelen(Tricladen). Vol Bd. 40.

Le Moigne, A. (1963). Etude du developpement embryonnaire de Polycelis nigra (Turbellarie - Triclade). Bulletin de la Societe Zoologique de France 88, 403-422.

Metschnikoff, E. (1883). Die embryologie von Planaria polychroa. Zeitschrift fur wissenschaftliche Zoologie 38, 331-354.

Adler, C.E., Seidel, C.W., McKinney, S.A., and Sánchez Alvarado, A. (2014). Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. Elife 3, e02238.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Scimone, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.

Adler, C.E., Seidel, C.W., McKinney, S.A., and Sánchez Alvarado, A. (2014). Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. Elife 3, e02238.

Martín-Durán, J.M., Amaya, E., and Romero, R. (2010). Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa. Dev Biol 340, 145-158.

Martín-Durán, J.M., and Romero, R. (2011). Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa. Dev Biol 352, 164-176.

Scimone, M.L., Kravarik, K.M., Lapan, S.W., and Reddien, P.W. (2014). Neoblast specialization in regeneration of the planarian Schmidtea mediterranea. Stem Cell Reports 3, 339-352.