(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 one like-3 (MPEG1-like-3: SMED30015696), (E) gamma (γ) class neoblast transcripts (gata456a, hnf4, prox-1, nkx2.2), and (H) transcripts with enriched expression in the adult gut (Forsthoefel et al., 2012; Vu et al., 2015; Wurtzel et al., 2015). S2–S8, Stage 2–8; Y, yolk. 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 Figure 1—source data 5–8. 74% (n = 1,112) 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). A, aboral hemisphere; D, dorsal; O, oral hemisphere; V, ventral. Black arrowheads: temporary embryonic pharynx. Black arrows: primitive gut cells. Red arrowheads: definitive pharynx. Scale bars: 100 µm.