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).

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).

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).

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).

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).

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).

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).