Bunched and Madm Function Downstream of Tuberous Sclerosis Complex to Regulate the Growth of Intestinal Stem Cells in Drosophila

Abstract

The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. We show here that the leucine zipper protein Bunched and the adaptor protein Madm are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, our data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells.

Keywords: Bunched; Drosophila; Intestine; Madm; Stem cells; TSC-22; Tuberous sclerosis complex.

Fig. 1

Bun and Madm are required in ISCs for proliferation. a-c Confocal images of midguts from adult flies that had the esg^ts > driving the UAS-GFP as control, or the UAS-bun^RNAi or UAS-Madm^RNAi as indicated. Flies were kept at room temperature after eclosion for 5–7 days and then shifted to 29 °C for 4 days to induce the dsRNA expression. The arrows in A indicate 2 examples of normal GFP+ precursor cells, and the arrowheads in B and C indicate examples of defective GFP+ cells after RNAi. DAPI staining for DNA is blue. The scale bar is 20 μm and all images were processed in the same way. d-e Quantification of GFP+ and Pros + cells were carried out by counting the positive cells in 40X objective confocal images of midguts from each genotype. The area per view was approximately 0.08 mm². 7 to 12 images of each genotype were counted and the average was plotted. The error bar represents standard error of the means. All P values are from Student’s t test, and ** is P < 0.01. f-j The flies were aged and temperature shifted the same way as described above. In F-H, representative confocal images are shown for GFP and phospho-histone 3 (p-H3) staining. In I-J, the flies were split into parallel vials with sucrose solution or with sucrose + 5%DSS. Fly guts were dissected and stained for the p-H3 mitotic marker and positive staining was counted in the whole gut. More than 10 guts from each experimental condition were counted and the average was plotted. * is P < 0.05. k-n MARCM clonal analyses in midgut ISCs of bun and Madm mutants. Eclosed flies were aged at room temperature for 5–7 days and MARCM clones were generated after 37 °C heat shock of the flies to induce FLP expression and mitotic recombination. The flies were returned to 18 °C (bun mutants) or room temperature (Madm mutants) for 7 days and midguts were dissected for analysis. The FRT40A wild type chromosome (control) and the FRT40A-bun ^ΔGE1292 (precise excision) chromosome were used as controls for bun mutants. The FRT82B wild type chromosome was used as the control for Madm mutants. N is the number of clones counted for each genotype. The clone size distribution and average clone size were plotted. NS is non-significant with P > 0.05. o-r Confocal images containing MARCM clones in midguts of control and mutant flies. The UAS-GFP used in the FRT40A experiment was cytoplasmic and in the FRT82B experiment was nuclear, all labeling the MARCM clones. The arrows in all these panels indicate examples of Delta (Dl) + cells. The arrowheads indicate examples of mutant cells. s Mitotic cell counts in adult midguts using the ISC-specific Delta(Dl) ^ts driver. The control flies were Dl^ts > GFP and two different bun ^RNAi and Madm ^RNAi lines were examined. Cells positively stained for p-H3 were counted for whole guts and the average was plotted

Fig. 2

Bun and Madm function independently of the major niche signaling pathways. a-b Transgenic constructs as indicated were crossed together with the esg^ts > driver. The flies were aged and shifted to 29 °C as described in Fig. 1. Staining and quantification of p-H3 positive cells were performed as described above. Transgenes located on X (bun ^RNAi2) or 2^nd (bun ^RNAi1, Madm ^RNAi1 and Madm ^RNAi2) chromosome were used depending on the chromosomal location of the other transgenes used for the experiments. c-e MARCM clonal analysis of bun mutant and gain of function Ras^v12. The control was FRT40A-bun ^ΔGE1292 precise excision crossed with UAS-Ras^v12. The mutant was FRT40A-bun ^200B crossed with UAS-Ras^v12. MARCM clones were induced as described in Fig. 1. The arrows in C indicate Delta + ISCs. The arrowheads in D indicate bun mutant cells. The GFP+ cell numbers in control and mutant bun clones were plotted as average in E. f Experimental conditions were the same as described in panels A and B, except that the UAS-hpo ^RNAi or UAS-msn ^RNAi was used to increase mitotic cell count in midguts

Fig. 3

Bun localizes in cytoplasm and acts synergistically with Madm to promote ISC proliferation. a-d Confocal images of midguts from flies that expressed the transgenic UAS-BunA or UAS-Madm or the combination driven by the esg^ts>. The shift to 29 °C was carried out for 5 days. All the fly strains also contain the UAS-GFP, represented by the green fluorescent signal of the images. DAPI staining for DNA is blue. e-h The same fly strains were used as in panel A-D. Panel E were from flies incubated at 29 °C for 5 days, and panel F-H were from flies incubated at 29 °C for 10 days. The p-H3+ staining was counted through out the midgut, and the GFP+ and Pros + cells were counted after taking microscopic images under the 40X objective. The average was plotted and more than 10 guts or images were counted for each sample. i-l Confocal images of midguts from flies that expressed the transgenic UAS-BunA tagged with SBP at N- or C-terminus. The antibody for immunofluorescent staining was directed against SBP. The expression was driven by esg^ts > and the flies were shifted to 29 °C for 24 h. The SBP-Delta (Dl) transgenic line expressed a fusion of SBP with an approximately 85 kb genomic Delta fragment containing the Delta promoter. m-n Quantification of p-H3+ staining and GFP+ cells after expressing the various combinations of transgenic UAS constructs as indicated. The BunA-nls construct contained one copy of SV40 nls and no SBP tag. The SBP-BunA-NLS construct contained multiple copies of sequences as SBP-NLS-BunA-2XNLS-SBP

Fig. 4

Bun acts downstream of Tuberous Sclerosis Complex to regulate 4EBP and ISC growth. a-d Confocal images of midguts from adults flies with the esg^ts > RNAi constructs as indicated. All crosses also contained the UAS-GFP. Pupae were reared at 29 °C from 2 days before adult fly eclosion and continued for 4 more days after eclosion; this condition gives the best cell growth after TSC2 RNAi. Midguts were dissected and used for anti-phospho-4EBP (p-4EBP) and anti-Delta (Dl) staining. e-g Confocal images showing p-4EBP staining of midguts from flies containing the UAS-GFP control, UAS-Rheb overexpression construct, or together with the bun ^RNAi as indicated. h-i The circumference of GFP+ cells in the confocal images was measured and the average was plotted. More than 30 cells were measured for each genotype. For the same cells, the p-4EBP staining signal was also quantified as fluorescent intensity in each GFP+ cell. The signal was averaged and plotted as shown. j-m Confocal images showing p-4EBP staining of midguts from flies overexpressing BunA, Madm or the combination. n A model of Bun and Madm function in the TOR pathway regulating 4EBP phosphorylation and ISC growth, which is required for ISC division. Bun and Madm should function downstream of Tuberous Sclerosis Complex 2 (TSC2) and Rheb but the molecular mechanism remains to be determined