Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

9 figures and 14 videos

Figures

Figure 1 with 19 supplements
A molecular staging series for Smed embryogenesis informed by single embryo RNA-Seq.

(A) Top: Cartoon depicting the reproductive system of a sexually mature Smed hermaphrodite. Ventral view. G, gonopore; O, ovary; OD, oviduct; P, penis papilla; SD, sperm duct . Oocytes are fertilized internally and zygote(s) are packaged with yolk produced by vitellogenic gland cells into a developing egg capsule in the genital atrium (purple). Capsules are laid through the gonopore. Bottom: Brightfield image of a live Smed hermaphrodite. Anterior: left. Dorsal view. White asterisk: pharynx. (B) Developmental timeline and staging designations for Smed embryogenesis at 20°C. Timeline: days (d) post egg capsule deposition. Gray bars and letters C–I indicate time windows, and corresponding panels (C–I), for RNA-Seq samples. Double-headed arrows: time windows for stages (S) S1–S8. (CI) Brightfield images of live embryos harvested for RNA-Seq (top), hematoxylin- and eosin-stained sections (middle), and heat maps for enriched transcripts (bottom). Scale bars: 100 µm. Yellow arrowheads: temporary embryonic pharynx. Black arrowheads: definitive pharynx. Heat maps depict cohorts of enriched transcripts at indicated stages. (J) Principal component analysis demonstrates clustering of replicates and separation of developmental time points in expression space. (K) Correlation matrix for single embryo sequencing replicates. Total transcripts with a row sum >1 CPM: 31,248. (CK) Y,yolk. 2, S2. 3, S3. 4, S4. 5, S5. 6, S6. 7, S7. 8, S8.

https://doi.org/10.7554/eLife.21052.003
Figure 1—source data 1

Molecular staging resource for Smed embryogenesis.

Tab 1 (staging overview): an overview of the molecular staging resource materials for S1–S8: developmental time interval (days post-egg capsule deposition, 20°C); names of single embryo RNA-Seq replicates; references to representative images (live brightfield and histological cross-sections); number of enriched transcripts; references to Figure 1 supplement files containing mean centered expression and raw RPKM profiles across embryogenesis for enriched transcripts; references to Figure 1 source data files (excel spreadsheets) containing enriched transcripts, organized by cluster membership, with RPKM profiles across development, BLASTx-based annotations, GO analysis, and short written descriptions of each stage (S2–S8). Additional tabs are included for: (1) pairwise comparison overview; (2) mixed stage reference overview; (3) lists of enriched transcripts for S2–-S8, compiled from both the pairwise and mixed stage reference analysis; (4) GO triage criteria; (5) categories and lists of biological process (BP) GO IDs, manually curated from the statistically significant hits for S2–S8-enriched transcripts; (6) summary table containing the number and percentage of enriched transcripts (S2–S8) assigned to BP GO ID categories.

https://doi.org/10.7554/eLife.21052.004
Figure 1—source data 2

Stage-2-enriched transcripts from pairwise and/or mixed stage reference comparisons.

Criteria for inclusion are indicated in Figure 1—source data 1, as well as the legends for Figure 1—figure supplements 23. Tabs in this excel file contain: (1) pairwise comparison data (if applicable), (2) mixed stage reference comparison data, (3) cluster membership (see Figure 1C), 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 RefSeq databases, (4) GO analysis: manually curated and categorized biological process (BP) GO IDs and (5) GO analysis: unabridged results. See also Figure 1—figure supplement 4.

https://doi.org/10.7554/eLife.21052.005
Figure 1—source data 3

Stage-3-enriched transcripts from pairwise and/or mixed stage reference comparisons.

Criteria for inclusion are indicated in Figure 1—source data 1, as well as the legends for Figure 1—figure supplements 23. Tabs in this excel file contain: (1) pairwise comparison data (if applicable), (2) mixed stage reference comparison data, (3) cluster membership (see Figure 1D), 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 RefSeq databases, (4) GO analysis: manually curated and categorized biological process (BP) GO IDs, and (5) GO analysis: unabridged results. See also Figure 1—figure supplement 5.

https://doi.org/10.7554/eLife.21052.006
Figure 1—source data 4

Stage-4-enriched transcripts from pairwise and/or mixed stage reference comparisons.

Criteria for inclusion are indicated in Figure 1—source data 1, as well as the legends for Figure 1—figure supplements 23. Tabs in this excel file contain; (1) pairwise comparison data (if applicable), (2) mixed stage reference comparison data, (3) cluster membership (see Figure 1E), 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 RefSeq databases, (4) GO analysis: manually curated and categorized biological process (BP) GO IDs, and (5) GO analysis: unabridged results. See also Figure 1—figure supplement 6.

https://doi.org/10.7554/eLife.21052.007
Figure 1—source data 5

Stage-5-enriched transcripts from pairwise and/or mixed stage reference comparisons.

Criteria for inclusion are indicated in Figure 1—source data 1, as well as the legends for Figure 1—figure supplements 23. Tabs in this excel file contain; (1) pairwise comparison data (if applicable), (2) mixed stage reference comparison data, (3) cluster membership (see Figure 1F), 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 RefSeq databases, (4) GO analysis: manually curated and categorized biological process (BP) GO IDs, and (5) GO analysis: unabridged results. See also Figure 1—figure supplement 7.

https://doi.org/10.7554/eLife.21052.008
Figure 1—source data 6

Stage-6-enriched transcripts from pairwise and/or mixed stage reference comparisons.

Criteria for inclusion are indicated in Figure 1—source data 1, as well as the legends for Figure 1—figure supplements 23. Tabs in this excel file contain; (1) pairwise comparison data (if applicable), (2) mixed stage reference comparison data, (3) cluster membership (see Figure 1G), 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 RefSeq databases, (4) GO analysis: manually curated and categorized biological process (BP) GO IDs, and (5) GO analysis: unabridged results. See also Figure 1—figure supplement 8.

https://doi.org/10.7554/eLife.21052.009
Figure 1—source data 7

Stage-7-enriched transcripts from pairwise and/or mixed stage reference comparisons.

Criteria for inclusion are indicated in Figure 1—source data 1, as well as the legends for Figure 1—figure supplements 23. Tabs in this excel file contain; (1) pairwise comparison data (if applicable), (2) mixed stage reference comparison data, (3) cluster membership (see Figure 1H), 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 RefSeq databases, (4) GO analysis: manually curated and categorized biological process (BP) GO IDs, and (5) GO analysis: unabridged results. See also Figure 1—figure supplement 9.

https://doi.org/10.7554/eLife.21052.010
Figure 1—source data 8

Stage-8-enriched transcripts from pairwise and/or mixed stage reference comparisons.

Criteria for inclusion are indicated in Figure 1—source data 1, as well as the legends for Figure 1—figure supplements 23. Tabs in this excel file contain; (1) pairwise comparison data (if applicable), (2) mixed stage reference comparison data, (3) cluster membership (see Figure 1I), 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, SwisSchmidteas-Prot, C. elegans, D. melanogaster, D. rerio, X. tropicalis, M. musculus and H. sapiens RSeq databases, (4) GO analysis: manually curated and categorized biological process (BP) GO IDs, and (5) GO analysis: unabridged results. See also Figure 1—figure supplement 10.

https://doi.org/10.7554/eLife.21052.011
Figure 1—source data 9

Molecular fate mapping resource.

Short summaries describing molecular markers for temporary embryonic tissues and definitive organ systems. Written descriptions accompany Figure 1—figure supplement 11 (primitive ectoderm), Figure 1—figure supplement 12 (temporary embryonic pharynx), Figure 1—figure supplement 13 (gut), Figure 1—figure supplement 14 (definitive pharynx), Figure 1—figure supplement 15 (definitive epidermis), Figure 1—figure supplement 16 (nervous system), Figure 1—figure supplement 17 (muscle), Figure 1—figure supplement 18 (protonephridia) and Figure 1—figure supplement 19 (eyes).

https://doi.org/10.7554/eLife.21052.012
Figure 1—figure supplement 1
Histological cross-sections of Stage one embryos.

(AD) Four independent examples of the single cell stage (S1), either metaphase II arrested oocytes or zygotes, in paraffin embedded, hematoxylin- and eosin-stained sectioned egg capsules fixed at 1–2 days post egg capsule deposition. The entire capsule is shown in (D). Black arrowheads: metaphase II arrested oocytes or zygotes, surrounded by a corona of yolk cells. Scale bars: 25 µm (AC); 100 µm (D).

https://doi.org/10.7554/eLife.21052.013
Figure 1—figure supplement 2
MA plots for pairwise comparisons.

(A) S2 vs Y. (B) S2 vs S3. (C) S3 vs S4. (D) S4 vs S5. (E) S5 vs S6. (F) S6 vs S7. (G) S7 vs S8. Upregulated transcripts: red. Downregulated transcripts: green. Criteria for flagging differentially expressed transcripts: adjusted p-value<1e-5, log2 ratio ≥2.322 (five-fold upregulation) or log2 ratio ≤−2.322 (five-fold downregulation), average scaled RPKM value ≥1.0 at indicated time point, transcript has ≥1 ORF. Transcripts derived from transposase or retroviruses were omitted. S2–S8, Stages 2–8. Y, yolk.

https://doi.org/10.7554/eLife.21052.014
Figure 1—figure supplement 3
MA plots for enriched transcripts identified in mixed-stage reference comparisons.

(A) S2, (B) S3, (C) S4, (D) S5, (E) S6, (F) S7 and (G) S8-enriched transcripts (red) identified using the following criteria: S2–S5 enriched transcripts: p adj <1e-5; S6–S8-enriched transcripts: p adj <1e-20. All stages: log2 ratio ≥2.322 (five-fold upregulation), average scaled RPKM value ≥1.0 at indicated time point, transcript has ≥1 ORF. Transcripts derived from transposase or retroviruses were omitted, as were S2, S3 and S4 transcripts also upregulated in yolk. S2–S8: Stages 2–8.

https://doi.org/10.7554/eLife.21052.015
Figure 1—figure supplement 4
Mean centered expression and average RPKM profiles for S2-enriched transcripts.

Mean centered expression and average RPKM profiles for S2-enriched transcripts, organized by cluster membership presented in Figure 1C and Figure 1—source data 2. S2–S8 Stages 2–8. Y, yolk.

https://doi.org/10.7554/eLife.21052.016
Figure 1—figure supplement 5
Mean centered expression and average RPKM profiles for S3-enriched transcripts.

Mean centered expression and average RPKM profiles for S3-enriched transcripts, organized by cluster membership presented in Figure 1D and Figure 1—source data 3. S2–S8, Stages 2–8; Y, yolk.

https://doi.org/10.7554/eLife.21052.017
Figure 1—figure supplement 6
Mean centered expression and average RPKM profiles for S4-enriched transcripts.

Mean centered expression and average RPKM profiles for S4-enriched transcripts, organized by cluster membership presented in Figure 1E and Figure 1—source data 4. S2–S8, Stages 2–8; Y, yolk.

https://doi.org/10.7554/eLife.21052.018
Figure 1—figure supplement 7
Mean centered expression and average RPKM profiles for S5-enriched transcripts.

Mean centered expression and average RPKM profiles for S5-enriched transcripts, organized by cluster membership presented in Figure 1F and Figure 1—source data 5. S2–S8, Stages 2–8; Y, yolk.

https://doi.org/10.7554/eLife.21052.019
Figure 1—figure supplement 8
Mean centered expression and average RPKM profiles for S6-enriched transcripts.

Mean centered expression and average RPKM profiles for S6-enriched transcripts, organized by cluster membership presented in Figure 1G and Figure 1—source data 6. S2–S8, Stages 2–8; Y, yolk.

https://doi.org/10.7554/eLife.21052.020
Figure 1—figure supplement 9
Mean centered expression and average RPKM profiles for S7-enriched transcripts.

Mean centered expression and average RPKM profiles for S7-enriched transcripts, organized by cluster membership presented in Figure 1H and Figure 1—source data 7. S2–S8: Stages 2–8; Y, yolk.

https://doi.org/10.7554/eLife.21052.021
Figure 1—figure supplement 10
Mean centered expression and average RPKM profiles for S8-enriched transcripts.

Mean centered expression and average RPKM profiles for S8-enriched transcripts, organized by cluster membership presented in Figure 1I and Figure 1—source data 8. S2–S8, Stages 2–8; Y, yolk.

https://doi.org/10.7554/eLife.21052.022
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. S2–S8, Stages 2–8; Y, yolk. (BC) 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) During S3, the average number of primitive ectoderm nuclei per embryo was 21.5 ± 2.9. During S4:, the average number of primitive ectoderm nuclei per embryo was 22 ± 1.4. n = 5 embryos. Unpaired t-test, two tailed p value = 0.72. (C) Average S3 embryo volume was 7.4 × 107 µm3. Average S4 embryo volume was 1.5 × 108 µm3. Unpaired t-test, two tailed p value = 0.01. Embryo volumes were calculated by generating a masked surface in Imaris. Twenty-two S3 embryos and five S4 embryos were scored. Error bars represent the standard deviation of the mean. (DE) 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. A, aboral hemisphere; O, oral hemisphere; V, ventral. Scale bars: 100 µm.

https://doi.org/10.7554/eLife.21052.023
Figure 1—figure supplement 12
Molecular markers for the temporary embryonic pharynx.

(AB) 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 17C. (C) Average RPKM values per embryo for the temporary embryonic pharynx markers venom allergen-like (VAL-like; SMED30015313), macrophage expressed gene one like-1 (MPEG1-like 1; SMED30000139), MPEG1-like 2 (SMED30034696) and netrin-like (SMED30023593) during embryogenesis. S2–S8, Stage 2–8; Y, yolk. (DG) 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). (AB, DG) Anterior: top (S6–S8). Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. A, aboral hemisphere; D, dorsal; O, oral hemisphere; V, ventral. Scale bars: 100 µm.

https://doi.org/10.7554/eLife.21052.024
Figure 1—figure supplement 13
Molecular markers for the developing gut.

(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 58. 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. (BD) 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. (FG) 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. (BD, FG, 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.

https://doi.org/10.7554/eLife.21052.025
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. A, aboral hemisphere; D, dorsal; O, oral hemisphere; V, ventral. Scale bars: 100 µm. (B) Average RPKM values per embryo for the definitive pharynx markers foxA1, meis, laminin and npp-1 during embryogenesis (Adler et al., 2014; Scimone et al., 2014). S2-S8, Stage 2–8; Y, yolk.

https://doi.org/10.7554/eLife.21052.026
Figure 1—figure supplement 15
Molecular markers for the definitive epidermis.

(A, C, F) Average RPKM values per embryo for: (A) zeta (ζ) class neoblast transcripts (C) Category 2 and Category 3transcripts, and (F) Category 4 and 5 transcripts (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). S2–S8, Stage 2–8; Y, yolk. Transcripts shown had enriched expression during S5, S6, S7 and/or S8 and are flagged in Figure 1—source data 58. (B, DE, GI) WISH with riboprobes complementary to: (B) p53, (D) prog-1, (E) AGAT-1, (G) zpuf-6, (H) vim-3 and (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.

https://doi.org/10.7554/eLife.21052.027
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), Neural transcripts that showed enriched expression during S5, S6, S7 and/or S8 are flagged in Figure 1—source data 58. 90% (n = 533) of the neural-enriched transcripts reported by 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). A, aboral hemisphere; D, dorsal; O, oral hemisphere; V, ventral. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. Cyan arrows: cephalic ganglia. Cyan arrowheads: ventral nerve cords. Scale bars: 100 µm.

https://doi.org/10.7554/eLife.21052.028
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, S2–S8. Anterior: top (S6–S8). A, aboral hemisphere; D, dorsal; O, oral hemisphere; 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). S2–S8, Stages 2–8; Y, yolk. 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 Figure 1—source data 58.

https://doi.org/10.7554/eLife.21052.029
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 Figure 1—source data 58. (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.

https://doi.org/10.7554/eLife.21052.030
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 and eya [purple]), photoreceptor neuron differentiation (otxA [(red]), or pigment cup differentiation (sp6-9 and dlx [(blue]) (A), or with enriched expression in adult eye tissue (Lapan and Reddien, 2012) (C). S2–S8, Stages 2–8; Y, yolk. Transcripts shown were enriched during S5, S6, S7 and/or S8, and are flagged in Figure 1—source data 58. (B, DE) 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.

https://doi.org/10.7554/eLife.21052.031
Blastomere anarchy drives Smed embryogenesis.

(AB) Architectural features of S2 and S3 embryos. (A) Expression of the pan embryonic cell marker EF1a-like-1 (blue) in S2 (left) and S3 (right) embryos. Cyan arrowheads: primitive ectoderm cells. Cyan arrows: undifferentiated blastomeres. Red arrowhead: temporary embryonic pharynx. Red arrows: primitive gut cells. (B) S3 embryo stained with EF1a-like-1 riboprobes (red) and sytox green nuclear counterstain (green). Cyan arrowheads: primitive ectoderm cells. Cyan arrows: undifferentiated blastomeres. Yellow arrowhead: temporary embryonic pharynx. (C) Confocal Z-slice of an ovary from a sexually mature Smed hermaphrodite stained with piwi-1 riboprobes (green) and DAPI (blue). Yellow arrows: oocytes. (D) Dispersed cleavage. S2 embryo stained with piwi-1 riboprobes (red, blastomeres) and antibodies raised against the mitotic epitope H3S10p (green). Nuclei stained with DAPI (blue). Yellow arrow: dividing blastomere. (E) piwi-1 is expressed in undifferentiated blastomeres of S3 embryos. S3 embryo costained with riboprobes complementary to piwi-1 (red) and EF1a-like-1 (green). 100% piwi-1+ blastomeres coexpressed EF1a-like-1. n = 159 cells scored, n = 5 S3 embryos. Cyan arrows: undifferentiated blastomeres. Yellow arrowhead: temporary embryonic pharynx. Red arrows: fiduciary beads used for SPIM reconstruction. (F) piwi-1+ cells are located in the embryonic wall. Paraffin-embedded cross-section of a S3 sphere stained with piwi-1 riboprobes (blue) and eosin (pink). Cyan arrows: piwi-1+ cells. GC: yolk-filled gut cavity. Inset: magnified view of a piwi-1+ cell. Scale: 25 µm. (G) Left: Average RPKM per embryo for piwi-1 (S2–S8). Right: WISH developmental time course with piwi-1 riboprobes (blue) (S2–S8). O, oral hemisphere; V, ventral. (AG) Scale: 100 µm. Left: Observed distribution of piwi-1+ cells in S3–S4 embryos (blue bars) relative to the oral-aboral axis (0–3.14 radians). Maximum likelihood analysis best described distribution by the function ((1-exp(-θ/θ’))*sin(θ), blue line). The optimal calculated θ’ was 0.45 ± 0.045 radians, based on simulations with comparably sized data sets, and was several orders of magnitude more likely to explain the observed distribution than the theoretical normal distribution, sin(θ), (θ’ = 0), red line. S3: n = 32 embryos, n = 1,746 piwi-1+ cells scored. S4: n = 8 embryos, 2,665 piwi-1+ cells scored. Right: observed piwi-1+ cell distributions for individual S3 (top) and S4 (bottom) embryos. (CG) piwi-1+ cells are detected throughout embryogenesis. (H) piwi-1+ cell positions are not stereotyped in S3–S4 embryos.

https://doi.org/10.7554/eLife.21052.032
Cell cycle activity is restricted to the piwi-1+ compartment.

(AB) Left: Colorimetric WISH depicting expression of PCNA (A) or RRM2-2 (B) during stages S2–S8. Right: Average RPKM values per embryo for PCNA (A) or RRM2-2 (B) in Y (yolk) and S2–S8. V, ventral. Scale: 100 µm. (CD) S3 (top), S4 (middle) and S5 (bottom) embryos costained with piwi-1 (red) and PCNA (green [C]) or RRM2-2 (green [D]) riboprobes. The percentage of piwi-1+ cells coexpressing the indicated cell cycle marker (red) and the percentage of PCNA+ or RRM2-2+ cells coexpressing piwi-1 (green) appear in the lower left corner of merged images. Scale bars: 100 µm. (C) S3: n = 273 cells, n = 6 embryos. S4: n = 1,267 cells, n = 4 embryos. S5: n = 734 cells, n = 3 embryos. (D) S3: n = 130 cells, n = 4 embryos. S4: n = 1,295 cells, n = 5 embryos. S5: n = 350 cells, n = 3 embryos. (E) Mitotic activity is restricted to the piwi-1+ cell compartment in S3–S5 embryos. Left: S4 embryo costained with piwi-1 and the embryonic pharynx marker LYAG-like (both in green) and antibodies against the mitotic epitope H3S10p (red). White arrows: dividing blastomeres. White arrowhead: temporary embryonic pharynx. Scale bar: 100 µm. Right: Bar graph depicting the percentage of mitotic cells scored that expressed piwi-1 in S3–S5 embryos. (F) The mitotic index for the piwi-1+ cell compartment did not vary significantly during S3–S5. Average percentage of piwi-1+ cells in mitosis during S3–S5. Error bars represent the standard deviation of the mean. Observed distribution of mitotic (piwi-1+, H3S10p+) cells in S3-–S4 embryos (blue bars) along the oral-aboral axis (0–3.14 radians). Using the function derived with maximum likelihood estimation for the piwi-1+ cell distribution, (1-exp(-θ/θ’))*sin(θ) (blue line), and simulations using equivalent sample sizes, the optimal θ’ was calculated to be 0.58 ± 0.33, and was 50-fold more likely to explain the observed trend than a simple normal distribution, sin(θ), where θ’=0 (red line). S3: n = 82 mitotic cells, n = 18 embryos. S4: n = 110 mitotic cells, n = 8 embryos. (G) Mitotic cell positions are not stereotyped in early embryos.

https://doi.org/10.7554/eLife.21052.035
Figure 4 with 1 supplement
Many adult neoblast markers are similarly expressed throughout the piwi-1+ compartment during embryogenesis.

(A) Many transcripts with adult asexual neoblast-enriched expression are expressed throughout embryogenesis. Hierarchical clustering of 242 adult asexual neoblast-enriched transcripts during embryonic development using normalized mixed stage reference comparison data. Left: Heat map. Colored bars (left) denote clusters. Right: Normalized average RPKM values per embryo, plotted as a function of developmental time, for Clusters 1–8. Y, yolk; S2–S8, Stages 2–8. (BE) Colorimetric WISH depicting expression of piwi-2 (B), piwi-3 (C), tud-1 (D) and bruli-1 (E) during embryogenesis (blue) (S2–S8). V, ventral. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. Scale bars: 100 µm. (FI) Many markers of the adult asexual neoblast compartment are also expressed in piwi-1+ blastomeres. Fluorescent WISH on S4 embryos with riboprobes against piwi-1 (red) and piwi-2 (F), piwi-3 (G), tud-1 (H) or bruli-1 (I) (green). Percentage of piwi-1+ cells coexpressing the indicated marker (red) and the percentage of the indicated adult asexual neoblast marker coexpressing piwi-1 (green) appears in the lower left corner of merged images. Scale bars: 100 µm. (F) n = 435 cells, n = 9 S3–S4 embryos. (G) n = 535 cells, n = 5 S3–S4 embryos. (H) n = 1,867 cells, n = 8 S3–S5 embryos. (I) n = 1,353 cells, n = 3 S4 embryos.

https://doi.org/10.7554/eLife.21052.039
Figure 4—source data 1

Hierarchical clustering analysis for 242 adult asexual neoblast-enriched transcripts.

Clustering was performed using normalized expression data from the mixed stage reference comparison. Normalized expression profiles, cluster membership, average RPKM values across embryogenesis (Y–S8) and for 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 RefSeq databases are provided.

https://doi.org/10.7554/eLife.21052.040
Figure 4—figure supplement 1
Adult asexual neoblast-enriched markers are coexpressed in piwi-1+ cells during embryogenesis.

(A,C) Colorimetric WISH depicting expression of SoxP-1 (A) and SoxP-2 (C) during embryogenesis (S2–S8). V, ventral. Scale bars: 100 µm. (B) Average RPKM per embryo for SoxP-1 (blue) and SoxP-2 (red). Y, yolk; S2–S8, Stages S2–S8. (DG) Fluorescent WISH costaining on S3–S5 embryos using riboprobes against piwi-1 (red) and piwi-2 (D), piwi-3 (E), tud-1 (F), or Y12 antibodies (S4–S5 only)(G) (green). Scale bars: 100 µm. (DE) S3 and S5 costained samples. S4 samples and quantification are depicted in the Figure 4F–I legend. (G) 98% piwi-1+ cells costained with Y12 antibodies. 96% Y12-positive cells costained with piwi-1, n = 913 cells, n = 7 S4–S5 embryos.

https://doi.org/10.7554/eLife.21052.041
Figure 5 with 1 supplement
Early-embryo-enriched transcripts are downregulated as organogenesis begins.

(A) Hierarchical clustering of S2–S4-enriched transcripts (n = 1,756) using scaled RPKM data. Left: Heat map. Y, yolk. Colored bars (left) denote Clusters 5, 6 and 8 containing early-embryo-enriched (EEE) transcripts. Cluster f5 sequences (blue, n = 413) were expressed at roughly equivalent levels during S2 and S3, with 66% (n = 275) transcripts showing five-fold or greater declines in average expression values between S3 and S5. Cluster 6 sequences (red, n = 523) exhibited maximal expression during S2, and average expression levels declined more than five-fold between S2 and S4 for 81% (n = 426) of these transcripts. Cluster 8 sequences (green, n = 112) showed peak expression during S4, with 52% (n = 60) of the transcripts showing five-fold or greater declines in average expression values by S5. Right: Normalized expression trends for EEE transcripts in Clusters 5 (blue), 6 (red) and 8 (green) plotted as a function of developmental time. Median 50% of transcripts based on expression maxima are plotted. (BE) Colorimetric WISH depicting expression of the EEE transcripts tct-like (B), BTF3-like (C), DDX5-like (D) and eIF4a-like (E) (blue) in S2–S8 embryos and C4 asexual adults. Black arrowheads: temporary embryonic pharynx. Red arrowheads: definitive pharynx. O, oral; V, ventral. Scale bars: 100 µm. (FI) EEE transcripts were expressed throughout the piwi-1+ compartment in S3–S4 embryos. Fluorescent double WISH with riboprobes against piwi-1 (red) and the EEE transcripts tct-like (F), BTF3-like (G), DDX5-like (H) and eIF4a-like (I) (green) in S4 embryos. Percentage piwi-1+ cells coexpressing the indicated EEE marker (red) and percentage EEE+ cells coexpressing piwi-1 (green) appear in the lower left corner of merged images. (F) n = 895 piwi-1+ cells, n = 905 tct-like+ cells, n = 7 S3–S4 embryos. (G) n = 692 piwi-1+ cells, n = 728 BTF3+ cells, n = 6 S3–S4 embryos. (H) n = 676 piwi-1+ cells, n = 681 DDX5-like+ cells, n = 5 S3–S4 embryos. (I) n = 312 piwi1+ cells, n = 332 eIF4a+ cells, n = 4 S3–S4 embryos.

https://doi.org/10.7554/eLife.21052.047
Figure 5—source data 1

Hierarchical clustering of S2–S4-enriched transcripts across embryogenesis.

S2–S4 transcripts (n = 1,756) were clustered using scaled RPKM values across embryogenesis (Y, S2–S8). Cluster membership and average RPKM values (Y–S8, C4 and SX) are provided. Early-embryo-enriched (EEE) transcripts (n = 1,048), which were downregulated by S5 and were lowly expressed through S8, comprise Clusters 5, 6 and 8. Separate tabs for EEE transcript Clusters 5, 6 and 8 include annotation based on best BLASTx hits (E < 0.001) versus the NR, Swiss-Prot, C. elegans, D. melanogaster, D. rerio, X. tropicalis, M. musculus and H. sapiens RefSeq databases.

https://doi.org/10.7554/eLife.21052.048
Figure 5—source data 2

Validation of transcript expression trends using the Nanostring nCounter platform.

Normalized Nanostring data tab: Normalized expression counts for positive ERCC spike-in controls (POS A–F), negative controls (NEG A–H), housekeeping genes, the blastomere and mitotic cell markers H2B and piwi-1, the differentiating progenitor marker prog-1, and 107 early-embryo-enriched (EEE) transcripts. Clusters tab: hierarchical clustering results for 107 EEE transcripts using normalized count data. Raw data tab: raw expression count data for positive ERCC spike-in controls (POS A–F), negative controls (NEG A–H), housekeeping genes, the blastomere and mitotic cell markers H2B and piwi-1, the differentiating progenitor marker prog-1, and 107 EEE transcripts. Nanostring probe sequences tab: target sequence region for capture and reporter probes.

https://doi.org/10.7554/eLife.21052.049
Figure 5—source data 3

EEE transcript expression patterns detected by colorimetric WISH.

Colorimetric whole mount in situ hybridization screen results detail expression patterns across embryos and C4 intact adults for select EEE transcripts. Numbers in parentheses indicate the number of embryos scored with expression in a given tissue (numerator) versus the total number of embryos scored (denominator). pT4P-EEE transcript plasmid insert and cloning primer sequences are also provided.

https://doi.org/10.7554/eLife.21052.050
Figure 5—figure supplement 1
Validation of early-embryo-enriched transcript expression trends using the Nanostring nCounter platform.

(A) Count sums for positive controls(left), housekeeping genes (middle) and experimental probes (right) for four biological replicates per sample: yolk (Y), S2–S8 embryos, C4 asexual adults (C4) and virgin sexually mature adults (SX). Note the low read counts for the experimental S2 samples. (B) Log-scale plot of count sums for positive controls (POSITIVE A–F) and negative controls (NEGATIVE A–H) across all 40 samples assayed. Positive control probes detect exogenous ERCC control sequences added to the reactions at known concentrations. Negative control probes are not homologous to any known Smed sequence and measure background signal. (C) Raw counts for the housekeeping genes ACT-B, RPL-27, G6PD, LUC7L3, clathrin-1, hgp-1, rpl13a, ubiquilin-1 and zfp207-1 across all 40 experimental samples. ACT-B showed significant variation in expression across samples. (D) Normalization factors calculated by taking the geometric mean of the positive control sequences POSITIVE A–F (left) or the suite of housekeeping genes (right) across sample replicates. (E) Normalized expression trends for piwi-1, H2B, and prog-1 during embryogenesis and adulthood (Y, S2–S8, SX, C4) mirror those observed in the single embryo RNA-Seq time course. (F) Heat map (left) generated by hierarchical clustering using normalized count data for 108 EEE transcripts across developmental time (Y, S2–S8, SX, C4). Line graphs display expression trends for Clusters 1 (blue), 2 (red), and 4 (green), which corroborate early-embryo-enriched expression observed in the RNA-Seq time course. Transcripts in Clusters 5–8 (n = 9) exhibited low expression throughout the time course and/or exhibited expression trends different from those observed using RNA-Seq.

https://doi.org/10.7554/eLife.21052.051
Figure 6 with 1 supplement
Adult lineages arise within the piwi-1+ blastomere population as organogenesis begins.

(AD) Developmental transcription factors implicated in tissue specific differentiation programs are expressed in subpopulations of piwi-1+ cells during S5. Fluorescent WISH with piwi-1 (red) and p53 (A), gata456a (B), myoD (C) and pax6a (D) (green) riboprobes on S5 embryos. Embryos in (B-D) were costained with VAL-like, a temporary embryonic pharynx specific marker (also in red). Right: Venn diagrams depict percentages of cells that were single or double positive for piwi-1 and the indicated TFs. Scale bars: 100 µm. (EG) Hierarchical clustering of zeta (ζ, E), gamma (γ, F) and sigma (σ, G) neoblast subclass-enriched transcripts during embryogenesis (Y and S2–S8), and in asexual (C4) and virgin sexual (SX) adults.

https://doi.org/10.7554/eLife.21052.052
Figure 6—source data 1

Behavior of ζ, γ and σ adult asexual neoblast subclass-enriched transcripts during embryogenesis.

Hierarchical clustering of Zeta (ζ) , Gamma (γ) and Sigma (σ) subclass transcripts was performed using normalized RPKM data from the single embryo RNA-Seq developmental time course. Average RPKM values for Y, S2–S8 embryos and for C4 and SX adults are included. Transcripts with enriched at one or more embryonic stages are flagged.

https://doi.org/10.7554/eLife.21052.053
Figure 6—figure supplement 1
PIWI-1 protein may perdure in cells committed to differentiation.

(A) Fluorescent WISH costaining on S5 embryos using riboprobes against piwi-1 (red) and antibodies against PIWI-1 protein (green). Cyan arrows: single positive cells for which PIWI-1 protein, but not piwi-1 mRNA, was detected. Scale bar: 100 µm. 99 ± 0.4% piwi-1+ cells were double positive for PIWI-1 protein, n = 4,152 piwi-1+ cells scored. 96 ± 3.1% PIWI-1 positive cells were double positive for piwi-1 mRNA, n = 4,296 PIWI-1+ cells scored. n = 4 S5 embryos.

https://doi.org/10.7554/eLife.21052.054
Embryonic cells acquire the ability to engraft, persist and proliferate in an adult microenvironment as organogenesis proceeds.

(A) Schematic depicting the workflow for heterochronic transplantation experiments. S4, S5, S6, S7 or S8 embryonic cell suspensions were injected into the tails of lethally irradiated sexual adult hosts at 3 days post-irradiation (dpi). Cohorts of transplanted animals were fixed at 1 hr and 5 days post-transplantation (1 hpt and 5 dpt, respectively) for staining with piwi-1 riboprobes and H3S10p antibodies. Lethally irradiated, uninjected host controls were fixed and stained at 5 dpt. (B) Percentage of transplanted animals fixed at 1 hpt (blue bars) or 5 dpt (red bars) containing one or more donor-derived piwi-1+ cell(s). X-axis: stage (S) of donor cells. (C) Number of donor-derived piwi-1+ cell(s) per transplant at 1 hpt and 5 dpt. Each point represents one transplanted animal. Mean ± standard deviation (black bars) are shown. Statistical tests were performed using a generalized linear model, assuming that the counts followed a Poisson distribution. S4 transplants contained significantly fewer piwi-1+ cells at 1 hpt than S5, S6, S7 or S8 transplants (Tukey post-hoc comparisons, S4 vs S5, S4 vs S6 and S4 vs S7, S4 vs S8: p<0.001). Group differences in the number of piwi-1+ cells at 1 hpt for S5 and S6 transplants were not statistically significant (p=0.21). Significantly fewer S4 and S5 donor-derived piwi-1+ cells persisted at 5 dpt than were observed for later stages (Tukey post-hoc comparisons: S4 vs S5, S4 vs S6, S4 vs S7 and S4 vs S8: p<0.001. S5 vs S6, S5 vs S7, S5 vs S8: p<0.001). (D) Percentage of transplants with mitotic piwi-1+ cell(s) at 5 dpt (green bars). X-axis: Donor cell stage. (E) Mitotic index for donor-derived piwi-1+ cells at 5 dpt. Stage-specific differences were not observed for S4–S8 embryonic cell populations using a generalized linear model, assuming counts followed a Poisson distribution and the number of piwi-1+ cells as a covariate. (BE) Numbers of transplants scored: S4: n = 36 (1 hpt), n = 43 (5 dpt), four independent experiments. S5: n = 15 (1 hpt), n = 16 (5 dpt), two independent experiments. S6: n = 31 (1 hpt), n = 29 (5 dpt), four independent experiments. S7: n = 31 (1 hpt), n = 30 (5 dpt), four independent experiments. S8: n = 19 (1 hpt), n = 20 (5 dpt), three independent experiments. (F) Confocal maximal projections of S4, S5, S6, S7 and S8 embryonic cell transplants fixed at 1 hpt and 5 dpt. Animals were stained with piwi-1 riboprobes (green), antibodies against the mitotic marker H3S10p (red, 5 dpt only) and DAPI nuclear counterstain (blue). S6, S7 and S8 insets: mitotic piwi-1+ cells. Red arrows indicate mitotic cells magnified in insets. Yellow arrows: mitotic piwi-1+ cells. Scale bar (inset): 20 µm. Scale bar (panel): 100 µm. (BC) S4–S8 embryonic piwi-1+ cells were reliably introduced into hosts. S6–S8 embryonic piwi-1+ cells persisted in an adult microenvironment. (D-E) S6–S8 embryonic piwi-1+ cells proliferated in an adult microenvironment.

https://doi.org/10.7554/eLife.21052.059
Figure 8 with 1 supplement
Embryos undergoing organogenesis contain cNeoblasts.

(A) Schematic for heterochronic transplantation experiments. S5, S6, S7 or S8 embryonic cell suspensions were injected into the tail parenchyma of lethally irradiated sexual adult hosts at 1 day post irradiation (dpi). Cohorts of transplanted animals and uninjected host controls were fixed at 5 days post-transplantation (dpt) for staining with piwi-1 riboprobes and H3S10p antibodies. The remaining animals were monitored for 70 dpt for survival and rescue. (B) Percentage of transplants with persistent, donor-derived piwi-1+ cell(s) (blue) or donor-derived mitotic (piwi-1+, H3S10p+) cell(s) (red) at 5 dpt. X-axis: Embryonic donor cell stage. (C) Number of piwi-1+ cells per transplanted host at 5 dpt for S5–S8 embryonic cell transplants. Each point represents one transplanted animal. Means ± standard deviation (SD) are shown (black bars). Statistically significant differences in the number of persistent piwi-1+ cells per transplant at 5 dpt were observed using a generalized linear model, assuming that count data followed a Poisson distribution. S5 transplants contained fewer persistent piwi-1+ cells than S6 or S7 transplants (Tukey post-hoc comparisons, S5 vs S6: p<0.0001, S5 vs S7: p<0.0001, S5 vs S8: p<0.0001). (D) Mitotic index for donor-derived piwi-1+ cells at 5 dpt for S5–S8 embryonic cell transplants. Each point represents one transplanted animal. Means ± standard deviation (SD) are shown (black bars). Statistically significant differences in the piwi-1+ cell mitotic index were observed using a generalized linear model with piwi-1+ cell counts as a covariate, assuming that count data followed a Poisson distribution. S5 transplants contained significantly fewer cycling cells than S6, S7 or S8 transplants (Tukey post-hoc comparisons, S5 vs S6: p<0.01, S5 vs S7: p<0.01, S5 vs S8: p<0.001). (E) Confocal maximal projections for S5, S6, S7 and S8 embryonic cell transplants fixed at 5 dpt and stained with piwi-1 riboprobes (green), H3S10p antibodies (red) and DAPI (blue). S6, S7 and S8 insets show mitotic piwi-1+ cells. Red arrows indicate mitotic cells magnified in the insets. Yellow arrows: mitotic piwi-1+ cells. Scale bar (inset): 20 µm. Scale bar (panel): 100 µm. (BE) Numbers of transplants scored in four independent experiments: S5 n = 22; S6 n = 24; S7 n = 21; S8 n = 27 in (C), n = 21 in (D). (F) Survival curves for S5, S6, S7 and S8 embryonic cell transplants and uninjected 6,000-Rad-irradiated host controls as a function of time (days) post-transplant. (G) Live images of regenerating S6, S7 and S8 rescue animals. Left: Tail fragment after self-amputation of head and trunk tissue. Middle: Tail fragment with unpigmented anterior blastema (yellow arrowheads). Right: Animal with new head tissue and developing eyes (yellow arrows) and a regenerated pharynx (yellow asterisk). Animals from different experiments are shown in the S7 panels; the same animals are shown in the S6 and S8 panels. Dorsal view. Anterior: top. Scale: 100 µm. (FG) Numbers of transplants scored in four independent experiments: host controls n = 89; S5 n = 105; S6 n = 90; S7 n = 92; S8 n = 85. Rescue animals were obtained in two experiments for S6 and S7 transplants, and four experiments for S8 transplants. (BE) S6, S7 and S8 embryonic donor cells persist and divide in the adult parenchyma. (FG) S6, S7 and S8 embryonic cells can rescue lethally irradiated adult hosts.

https://doi.org/10.7554/eLife.21052.060
Figure 8—figure supplement 1
Progression of irradiation-induced phenotypes, rescue or death for heterochronic transplantation assays.

(AE) Stacked bar graphs depicting the percentage of S5 (A), S6 (B), S7 (C) and S8 (D) transplanted animals or uninjected, 6,000-Rad-irradiated hosts (E) that displayed no visible phenotype (blue), head regression (red), head and tail regression (yellow), head regression and pharynx lesions (green), head regression and ventral curling (purple), death (gray) or rescue (the development of an anterior blastema and ensuing regeneration of the host animal) (orange). Bulk cell transplantation was performed as described in Figure 8A and the Materials and methods. Transplanted animals and uninjected controls were monitored for 70 days post-irradiation (dpi). S5 transplants: n = 105 animals. S6 transplants: n = 90 animals. S7 transplants: n = 92 animals. S8 transplants: n = 85 animals. Uninjected sexual adult hosts: n = 89 animals. Results were tallied from four independent experiments for each embryonic stage assayed.

https://doi.org/10.7554/eLife.21052.061
Ontogeny of the adult neoblast compartment.

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 EEE transcripts (turquoise arrow) and adult asexual neoblast enriched transcripts (red arrow). As organogenesis begins during S5, EEE 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. Neoblasts are required for tissue maintenance during homeostasis and the formation of new tissue during regeneration.

https://doi.org/10.7554/eLife.21052.062

Videos

Video 1
S3 embryo architecture.

SPIM reconstructed S3 embryo costained with EF1a-like-1 (red) and sytox green nuclear counterstain. EF1a-like-1 is a pan-embryonic cell marker that stains primitive ectoderm cells, the temporary embryonic pharynx and undifferentiated blastomeres in the embryonic wall. EF1a-like-1 staining is absent from yolk cells in the embryonic wall and gut cavity.

https://doi.org/10.7554/eLife.21052.033
Video 2
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 three-dimensional reconstruction are visible (red).

https://doi.org/10.7554/eLife.21052.034
Video 3
Cell cycle activity is restricted to piwi-1 blastomeres, and all blastomeres are cycling.

SPIM reconstructed S4 embryo costained with piwi-1 (red) and PCNA (green). PCNA expression is restricted to piwi-1+ blastomeres, and all piwi-1+ cells co-express PCNA.

https://doi.org/10.7554/eLife.21052.036
Video 4
Cell cycle activity is restricted to piwi-1 blastomeres, and all blastomeres are cycling.

SPIM reconstructed S4 embryo costained with piwi-1 (red) and RRM2-2 (green). RRM2-2 expression is restricted to piwi-1+ blastomeres, and all piwi-1+ cells co-express RRM2-2. Several fluorescent beads used for three-dimensional reconstruction are visible (red).

https://doi.org/10.7554/eLife.21052.037
Video 5
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.

https://doi.org/10.7554/eLife.21052.038
Video 6
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 three-dimensional reconstruction are visible (green).

https://doi.org/10.7554/eLife.21052.042
Video 7
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.

https://doi.org/10.7554/eLife.21052.043
Video 8
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.

https://doi.org/10.7554/eLife.21052.044
Video 9
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 for three-dimensional reconstruction are visible (red).

https://doi.org/10.7554/eLife.21052.045
Video 10
piwi-1+ blastomeres possess chromatoid bodies.

SPIM reconstructed S4 embryo costained with piwi-1 (red) and Y12 antibodies (green). Y12 antibody staining was restricted to, and present throughout, the piwi-1+ blastomere population.

https://doi.org/10.7554/eLife.21052.046
Video 11
Definitive epidermal progenitors arise in the piwi-1+ blastomere population during S5.

SPIM -reconstructed S5 embryo costained with piwi-1 (red) and p53 (green). Definitive epidermal progenitors, coexpressing piwi-1 and p53, are dispersed in the embryonic wall. As definitive epidermal progenitors differentiate, they are predicted to downregulate piwi-1 and to retain expression of p53.

https://doi.org/10.7554/eLife.21052.055
Video 12
Definitive gut progenitors arise in the piwi-1+ blastomere population during S5.

SPIM reconstructed S5 embryo costained with piwi-1 and VAL-like (both in red) and gata456a (green). Definitive gut progenitors, coexpressing piwi-1 and gata456a, are dispersed in the embryonic wall. As definitive gut progenitors differentiate, they are predicted to downregulate piwi-1 and to retain expression of gata456a. VAL-like is expressed the temporary embryonic pharynx and is not detected in piwi-1+ blastomeres.

https://doi.org/10.7554/eLife.21052.056
Video 13
Muscle progenitors arise in the piwi-1+ blastomere population during S5.

SPIM reconstructed S5 embryo costained with piwi-1 and VAL-like (both in red) and myoD (green). Muscle progenitors, coexpressing piwi-1 and myoD, are dispersed in the embryonic wall. As muscle progenitors differentiate, they are predicted to downregulate piwi-1 and to retain expression of myoD. VAL-like is expressed the temporary embryonic pharynx and is not detected in piwi-1+ blastomeres.

https://doi.org/10.7554/eLife.21052.057
Video 14
Neural progenitors arise in the piwi-1+ blastomere population during S5.

SPIM reconstructed S5 embryo costained with piwi-1 and VAL-like (both in red) and pax6a (green). Neural progenitors, coexpressing piwi-1 and pax6a, are dispersed in the embryonic wall. As neural progenitors differentiate, they are predicted to downregulate piwi-1 and to retain expression of pax6a. VAL-like is expressed the temporary embryonic pharynx and is not detected in piwi-1+ blastomeres.

https://doi.org/10.7554/eLife.21052.058

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Erin L Davies
  2. Kai Lei
  3. Christopher W Seidel
  4. Amanda E Kroesen
  5. Sean A McKinney
  6. Longhua Guo
  7. Sofia MC Robb
  8. Eric J Ross
  9. Kirsten Gotting
  10. Alejandro Sánchez Alvarado
(2017)
Embryonic origin of adult stem cells required for tissue homeostasis and regeneration
eLife 6:e21052.
https://doi.org/10.7554/eLife.21052