The adult Drosophila gastric and stomach organs are maintained by a multipotent stem cell pool at the foregut/midgut junction in the cardia (proventriculus)

Abstract

Stomach cancer is the second most frequent cause of cancer-related death worldwide. Thus, it is important to elucidate the properties of gastric stem cells, including their regulation and transformation. To date, such stem cells have not been identified in Drosophila. Here, using clonal analysis and molecular marker labeling, we identify a multipotent stem-cell pool at the foregut/midgut junction in the cardia (proventriculus). We found that daughter cells migrate upward either to anterior midgut or downward to esophagus and crop. The cardia functions as a gastric valve and the anterior midgut and crop together function as a stomach in Drosophila; therefore, we named the foregut/midgut stem cells as gastric stem cells (GaSC). We further found that JAK-STAT signaling regulates GaSCs’ proliferation, Wingless signaling regulates GaSCs’ self-renewal, and hedgehog signaling regulates GaSCs’ differentiation. The differentiation pattern and genetic control of the Drosophila GaSCs suggest the possible similarity to mouse gastric stem cells. The identification of the multipotent stem cell pool in the gastric gland in Drosophila will facilitate studies of gastric stem cell regulation and transformation in mammal.

Acknowledgements

We thank G. Baeg, K. Basler, J.P. Vincent, V. Hartenstein, S. Hyashi, J. Merriam, M. Pankratz, J. Skeath, T. Xie, S. DiNardo, and the Bloomington stock center for fly stocks; J. Skeath, F. Matszaki, and B. Patterson for antibodies; Dr. Stephen Lockett and Optical Microscopy and Analysis Laboratory (OMAL) staff for help with the confocal microscopy; Dr. David King for help in identifying cell types in the cardia; and Maritta Perry Grau for editing the manuscript. This research was supported by the Intramural Research Program, National Cancer Institute of the National Institutes of Health.

Figures and Tables

Figure 1 The molecular markers expression and proliferation in cardia. (A,B) The cardia of a Stat92E-GFP fly was stained with GFP (green) and DAPI (blue). (B) The high magnification of (A). (C) The cardia of a Stat92E-GFP fly was stained with GFP (green) and Odd (red) antibodies. (D, D′) the cardia of a wg-Gal4 UAS-GFP fly was stained with GFP (green) and Ptc (red) antibodies and DAPI (blue). (D′) A high magnification picture of (D), in which a part of wg-Gal4 UAS-GFP and anti-Ptc co-localize (yellow—highlighted by blue dashed line). (E) The cardia of a Stat92E-GFP fly was analyzed after five days of BrdU pulse for cell proliferation. F/M junction cells outlined by a white dotted line; anti-GFP (green), anti-BrdU (red), and DAPI (blue). The red arrows show the cell proliferation at anterior midgut region, white arrow shows the cell proliferation at foregut region (F) The Stat92E-GFP-positive cells are proliferating at the F/M junction after five days of BrdU pulse and five days chase period. Only the Stat92E-GFP-positive F/M junction cells retain BrdU, outlined by a white dotted line; anti-GFP (green), anti-BrdU (red), and DAPI (blue). (G) The cardia of a Stat92E-GFP fly was analyzed after five days BrdU pulse and 17 days chase. All BrdU-labeled cells at the F/M junction were gone. The cardia was stained with anti-GFP (green), anti-BrdU (red), and DAPI (blue). The F/M junction cells are outlined by a white dotted line (H, H′) the wg-Gal4 UAS-GFP flies were stained with phospho-histone H3 antibody, which labels only a rare population of cell with small nuclei (highlighted in white box; anti-PH3, red; anti-GFP, green; and DAPI, blue). (I) wg-Gal4 UAS-GFP flies were stained with anti-GFP and Apoptag kit to detect dead cells. Anterior is at the top in all panels. Scale bars: 50 µm (A); 5 µm ( B, D′ H′); 10 µm (C, D, E, H, I); 20 µm (F, G).

Figure 2 F/M junction cells are multipotent stem cells. (A) MARCM control flies without heat shock (NHS, no heat shock). (B–G) MARCM clones (green) induced in adult cardia. MARCM clone imaged two days after clone induction (ACI) (B), four days (C), ten days (D–F; E, outer view; F, inner view), and 24 days (G) after clonal induction. Clones are highlighted in insets. White arrow in C points to the migration direction of GFP-marked cells from the F/M junction to foregut. Red dotted arrow in (E) points to the migration direction of GFP-marked cells from the F/M junction to foregut and crop. Yellow dotted arrow in (E) points to the migration direction of GFP-marked cells from the F/M junction to midgut. (H) The average number of GFP clones per cardia at the indicated times ACI. Anterior is at the top in all panels. Scale bars: 10 µm (A–G).

Figure 3 F/M junction cells are multipotent stem cells of the gastric and stomach organs. (A) Schematic diagram of the cell lineage marking system. After shifting the flies with genotype UAS-FLP/+; wg-Gal4 UAS-GFP/actin5C-FRT-draf-FRT-tau-lacZ; tub-Gal80^ts/+ to 29°C, the Gal4 was activated and drove GFP (green) and FLP recombinase expression. The FLP recombinase then removed the ‘FLP-out’ cassette so that the constitutive actin5C promoter drove lacZ expression (red) permanently within all subsequent daughter cells. Flies with the genotype UAS-FLP/+; wg-Gal4 UAS-GFP/actin5C-FRT-draf-FRT-tau-lacZ; tub-Gal80^ts/+ (B–G) were cultured at the permissive temperature (18° C, B) and then shifted to the restricted temperature (29°C) for 8 hours (C), 1 day (D), 2 days (E), 4 days (F), and 7 days (G). The cardia was stained with anti-GFP (green), anti-β-gal (red), and DAPI (blue). Anterior is at the top in all panels. Scale bars: 50 βm (B, G); 20 βm (C–F).

Figure 4 The GaSCs are multipotent gastric stem cells. (A and B) MARCM clones (4 days) to see the expression of Ptc in the clones. Ptc expressing cells are stem cells in (A and B). Cardia was stained with Ptc (red), GFP (green) and DAPI (blue). White dotted line in (B) shows the expression of Ptc is limited in stem cells (C) The cardia of wg-Gal4 UAS-GFP flies were stained with GFP (green) and Fu (red in C). (D) The flies with MARCM clones were dissected and stained with anti-GFP (green) and anti-Fu (red in D). (E) The Stat92E-GFP flies were stained with GFP (green) and Dve (red in E) antibodies. (F–H) The flies with MARCM clones were dissected and stained with anti-GFP or anti-Dve (red in F) or anti-Na/K ATPase α-subunit α5 (red in G) or anti-MEF2 (red in H). White line highlighted Fu-positive (D) or Dve-positive (F) cells. Black arrow in G points to Na/K ATPase α-subunit α5-positive cells. Anterior is at the top in all panels. Scale bars: 5 µm (A, B); 10 µm (C–H).

Figure 5 Asymmetric division of GaSCs. (A) Schematic diagram of the cell lineage marking system. After shifting the flies with genotype UAS-FLP/+; wg-Gal4 UAS-RFP/Act5C-FRT-y⁺-FRT-EGFP; tub-Gal80^ts/+ to 29°C, the Gal4 was activated and drove RFP (red) and FLP recombinase expression. The FLP recombinase then removed the ‘FLP-out’ cassette so that the constitutive actin5C promoter drove EGFP expression (green) permanently within all subsequent daughter cells. Flies with the genotype UAS-FLP/+; wg-Gal4 UAS-RFP/ Act5C-FRT-y⁺-FRT-EGFP; tub-Gal80^ts/+ (B–G, I, I′) were cultured at the permissive temperature (18°C, B) and then shifted to the restricted temperature (29°C, C–G, I, I′). At 18°C no EGFP/RFP expression (B). Flies shifted to 29°C for 12 hrs (C), 1 day (D, high magnification in D′), 2 days (E, E′, high magnification in E″, F, G). (G) 2 days (Z-stack merged). Division of GaSCs is highlighted in E′ (arrow) and E″ F (round dotted lines). In B–G the cardia was dissected and examined under confocal microscope without staining (live imaging). (H, H′) The flies with MARCM clones were dissected and stained with anti-GFP (green), anti-Ptc (red) and DAPI (blue). (I, and high magnification in I′) UAS-FLP/+; wg-Gal4 UAS-RFP/Act5C-FRT-y⁺-FRT-EGFP; tub-Gal80^ts/+ flies (2 days) were stained with RFP (red), GFP (green), Ptc (purple) and DAPI (blue). Ptc is expressed in GaSCs as highlighted by dotted lines in H, H′. Anterior is at the top in all panels except in I, I′, where anterior is in right. Scale bars: 10 µm (B–E); 2 µm (E′, F, G, D′ E″, I′); 5 µm (H, H′ I).

Figure 6 Wg and Hh signaling regulate GaSCs maintenance and differentiation, respectively. The cardia of a wg-Gal4 UAS-GFP fly was stained with anti-GFP (green) and DAPI (blue). White arrows point to the F/M junction. (B) Flies with the genotype Stat92E-GFP/+; Act-Gal4/+; tub-Gal80ts/UAS-wg were cultured at the restricted temperature (29°C) for 4 days. The cardia was stained with anti-GFP (green), and DAPI (blue). (C) Flies with the genotype Stat92E-GFP/+; Act-Gal4/+; tub-Gal80^ts/UAS-dnTCF were cultured at the restricted temperature (29°C) for 4 days. The cardia was stained with anti-GFP (green) and DAPI (blue). (D) Flies with the genotype Stat92E-GFP/+; Act-Gal4/+; tub-Gal80^ts/UAS-dnTCF were given food containing BrdU for 5 days, followed by a 5-day chase at the restricted temperature (29°C). The cardia was stained with anti-GFP (green), anti-BrdU (red), and DAPI (blue). (E) The cardia of a wg-Gal4 UAS-GFP flies (as control) were stained with Apoptag (red) GFP (green) for dying cells. (F) Stat92E-GFP/+; Act-Gal4/+; tub-Gal80^ts/ UAS-dnTCF flies were stained for Apoptag (red) for dying cells. (G) The cardia of an hh-Gal4 UAS-GFP fly stained with anti-GFP (green), anti-Arm (red), and DAPI (blue). White arrows point to the F/M junction. (H, I) Flies with the genotype Stat92E-GFP/+; Act-Gal4/+; tub-Gal80^ts//UAS-Ci^Cell were given food containing BrdU for 5 days, followed by a 5-day chase at the restricted temperature (29°C). The cardia was stained with anti-GFP (green), anti-BrdU (red), and DAPI (blue). Anterior is at the top in all panels. Scale bars: 10 µm (A–D, G–I); 20 µm (E, F).

Figure 7 JAK-STAT signaling regulates GaSCs proliferation. (A) The cardia of a upd-Gal4 UAS-GFP fly was stained with anti-GFP (green), anti-Arm (red), and DAPI (blue). White arrows point to the F/M junction. (B) The cardia of upd-Gal4 UAS-GFP/ STAT92E-lacZ flies were stained with anti-GFP (green), anti-β-gal (red), and DAPI (blue). White arrows point to the upd-Gal4 UAS-GFP (green) and red arrow point to the STAT92E-lacZ (red). (C) Flies with the genotype Stat92E-GFP/+; Stat92E⁰⁶³⁴⁶/Stat92E^F were given food containing BrdU for five days, followed by a five day chase at the restricted temperature (29°C). The cardia was stained with anti-GFP (green), anti-BrdU (red), and DAPI (blue). (D,E) Flies with the genotype Stat92E-GFP/+; Act-Gal4/+; tub-Gal80^ts/UAS-upd were cultured at the restricted temperature (29°C) for four days. The cardia was stained with anti-GFP (green), anti-Ptc (red in D–E), and DAPI (blue). (F,G) Diagram showing behavior and regulation of GaSCs in Drosophila. (F) The slow proliferative GaSCs first give rise to the fast proliferative progenitors in both foregut and anterior midgut. The progenitors then migrate and differentiate into the terminally differentiated cell types in both crop and midgut. (G) Schematic diagram summarizing the functions of JAK-STAT, Wg, and Hh pathways in regulating GaSCs′ behaviors in Drosophila. Anterior is at the top in all panels. Scale bars: 10 µm (A, C–F); 5 µm (B).