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The role of p38b MAPK in age-related modulation of intestinal stem cell proliferation and differentiation in Drosophila.

Park JS, Kim YS, Yoo MA - Aging (Albany NY) (2009)

Bottom Line: In addition, this pathway is involved in age and oxidative stress-associated mis-differentiation of enterocytes and upregulation of Delta, a Notch receptor ligand.Furthermore, we also show that D-p38b acts downstream of PVF2/PVR signaling in these age-related changes.Taken together, our findings suggest that p38 MAPK plays a crucial role in the balance between ISC proliferation and proper differentiation in the adult Drosophila midgut.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Pusan National University, Busan 609-735, Korea.

ABSTRACT
It is important to understand how age-related changes in intestinal stem cells (ISCs) may contribute to age-associated intestinal diseases, including cancer. Drosophila midgut is an excellent model system for the study of ISC proliferation and differentiation. Recently, age-related changes in the Drosophila midgut have been shown to include an increase in ISC proliferation and accumulation of mis-differentiated ISC daughter cells. Here, we show that the p38b MAPK pathway contributes to the age-related changes in ISC and progenitor cells in Drosophila. D-p38b MAPK is required for an age-related increase of ISC proliferation. In addition, this pathway is involved in age and oxidative stress-associated mis-differentiation of enterocytes and upregulation of Delta, a Notch receptor ligand. Furthermore, we also show that D-p38b acts downstream of PVF2/PVR signaling in these age-related changes. Taken together, our findings suggest that p38 MAPK plays a crucial role in the balance between ISC proliferation and proper differentiation in the adult Drosophila midgut.

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D-p38b MAPK plays a role in age-related defects in the differentiation of ISCs and progenitor cells. (A) Graph showing the ratio of                                                Su(H)GBE-positive to total cells. Effects of D-p38b activity on age-related                                                changes in the number of Su(H)GBE-positive cells in the posterior midgut.                                                Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ                                                flies were stained with DAPI, anti-β-gal and anti-GFP. The numbers of                                                each cell type were counted in a 0.06 x 0.04 cm area of the                                                posterior midgut. The ratio of Su(H)GBE-positive to total cells counted in                                                the posterior midgut of 5-day-old flies was set as 1. White square,                                                5-day-old flies; black square, 30-day-old flies. P-values were determined                                                using Student's t-test. (B) Graph showing the ratio of EE to total                                                cells. Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ                                                flies were stained with DAPI, anti-Prospero and anti-GFP. Numbers of each                                                cell type were counted in a 0.06 x 0.04 cm area of posterior midgut.                                                The ratio of EE to total cells counted in the posterior midgut of 5-day-old                                                flies was set as 1. White square, 5-day-old flies; black square, 30-day-old                                                flies. P-values were determined using Student's t-test. (C) Effects                                                of D-p38bas expression on ISC and EB cell morphology of                                                esg-positive cells and differentiation of EEs. Midguts of esg>+                                                (a-b) or esg>UAS-D-p38bas (c-d) flies were stained with                                                anti-Prospero (red), anti-GFP (green) and DAPI (blue). Enlarged images,                                                panels b and d. Scale bar, 5 μM. Arrow heads indicate esg-positive                                                cells. Asterisks indicate EEs.                                                (D) Graph showing the ratio of                                                EC to Su(H)GBE-positive cells. Midguts of esg>+; Su(H)GBE-lacZ or                                                esg>UAS-D-p38bas; Su(H)GBE-lacZ flies were stained                                                with DAPI, anti-β-gal and anti-GFP. Numbers of each cell type were                                                counted in a 0.06 x 0.04 cm area of posterior midgut. The ratio of                                                EC to Su(H)GBE-positive cells counted in the posterior midgut of 5-day-old                                                flies was set as 1. White square, 5-day-old flies; black square, 30-day-old                                                flies. P-values were determined using Student's t-test. (E) Effect                                                of D-p38bas expression in ISCs and EBs on age-related                                                accumulation of EC-like large esg- and Su(H)GBE-positive cells. The guts of                                                5- and 30-day-old flies were labeled with anti-β-gal and anti-GFP.                                                (a-f) esg>+;Su(H)GBE-lacZ, (g-l) esg>UAS-D-p38bas;Su(H)GBE-lacZ,                                                (m-r) esg>UAS-D-p38b+; Su(H)GBE-lacZ. (a, d, g, j, m,                                                and p - green) anti-GFP; (b, e, h, k, n, and q - red) anti-β-gal; (c,                                                f, I, l, o, and r) merged image. (DAPI, blue). Arrow indicates EC-like                                                large esg-GAL4. Asterisk indicates large Su(H)GBE-positive cell. Scale bar,                                                5 μM. (F)                                                Effect of D-p38b activity on the expression levels of Delta mRNA ISCs and                                                EBs in adult guts. Delta mRNA was measured by quantitative RT-PCR in cDNA                                                prepared from dissected guts from 5- and 30-day-old esg>+, esg>UAS-D-p38bas,                                                wild-type, p38bEY11174, or p38bKG01337 flies.                                                Expression was normalized to the expression of rp49. Expression level of                                                Delta mRNA in the midgut of 5-day-old flies was set as 1. White bar,                                                5-day-old flies; black bar, 30-day-old flies. P-values were determined                                                using Student's t-test. (G) Effect of D-p38b+ on the                                                expression of Delta in ISCs and EBs in adult gut. The level of Delta mRNA                                                was measured by real-time RT-PCR in cDNA prepared from dissected gut from                                                5-day-old esg>+ or esg>UAS-D-p38b+ flies.                                                Expression was normalized to the expression of rp49. Expression level of                                                Delta mRNA in midgut of 5-day-old flies was set as 1. White bar, esg>+;                                                black bar, esg>UAS-D-p38b+. P-value was determined                                                using Student's t-test.
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Figure 4: D-p38b MAPK plays a role in age-related defects in the differentiation of ISCs and progenitor cells. (A) Graph showing the ratio of Su(H)GBE-positive to total cells. Effects of D-p38b activity on age-related changes in the number of Su(H)GBE-positive cells in the posterior midgut. Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ flies were stained with DAPI, anti-β-gal and anti-GFP. The numbers of each cell type were counted in a 0.06 x 0.04 cm area of the posterior midgut. The ratio of Su(H)GBE-positive to total cells counted in the posterior midgut of 5-day-old flies was set as 1. White square, 5-day-old flies; black square, 30-day-old flies. P-values were determined using Student's t-test. (B) Graph showing the ratio of EE to total cells. Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ flies were stained with DAPI, anti-Prospero and anti-GFP. Numbers of each cell type were counted in a 0.06 x 0.04 cm area of posterior midgut. The ratio of EE to total cells counted in the posterior midgut of 5-day-old flies was set as 1. White square, 5-day-old flies; black square, 30-day-old flies. P-values were determined using Student's t-test. (C) Effects of D-p38bas expression on ISC and EB cell morphology of esg-positive cells and differentiation of EEs. Midguts of esg>+ (a-b) or esg>UAS-D-p38bas (c-d) flies were stained with anti-Prospero (red), anti-GFP (green) and DAPI (blue). Enlarged images, panels b and d. Scale bar, 5 μM. Arrow heads indicate esg-positive cells. Asterisks indicate EEs. (D) Graph showing the ratio of EC to Su(H)GBE-positive cells. Midguts of esg>+; Su(H)GBE-lacZ or esg>UAS-D-p38bas; Su(H)GBE-lacZ flies were stained with DAPI, anti-β-gal and anti-GFP. Numbers of each cell type were counted in a 0.06 x 0.04 cm area of posterior midgut. The ratio of EC to Su(H)GBE-positive cells counted in the posterior midgut of 5-day-old flies was set as 1. White square, 5-day-old flies; black square, 30-day-old flies. P-values were determined using Student's t-test. (E) Effect of D-p38bas expression in ISCs and EBs on age-related accumulation of EC-like large esg- and Su(H)GBE-positive cells. The guts of 5- and 30-day-old flies were labeled with anti-β-gal and anti-GFP. (a-f) esg>+;Su(H)GBE-lacZ, (g-l) esg>UAS-D-p38bas;Su(H)GBE-lacZ, (m-r) esg>UAS-D-p38b+; Su(H)GBE-lacZ. (a, d, g, j, m, and p - green) anti-GFP; (b, e, h, k, n, and q - red) anti-β-gal; (c, f, I, l, o, and r) merged image. (DAPI, blue). Arrow indicates EC-like large esg-GAL4. Asterisk indicates large Su(H)GBE-positive cell. Scale bar, 5 μM. (F) Effect of D-p38b activity on the expression levels of Delta mRNA ISCs and EBs in adult guts. Delta mRNA was measured by quantitative RT-PCR in cDNA prepared from dissected guts from 5- and 30-day-old esg>+, esg>UAS-D-p38bas, wild-type, p38bEY11174, or p38bKG01337 flies. Expression was normalized to the expression of rp49. Expression level of Delta mRNA in the midgut of 5-day-old flies was set as 1. White bar, 5-day-old flies; black bar, 30-day-old flies. P-values were determined using Student's t-test. (G) Effect of D-p38b+ on the expression of Delta in ISCs and EBs in adult gut. The level of Delta mRNA was measured by real-time RT-PCR in cDNA prepared from dissected gut from 5-day-old esg>+ or esg>UAS-D-p38b+ flies. Expression was normalized to the expression of rp49. Expression level of Delta mRNA in midgut of 5-day-old flies was set as 1. White bar, esg>+; black bar, esg>UAS-D-p38b+. P-value was determined using Student's t-test.

Mentions: To assess whether D-p38b MAPK is required for age-related changes in ISC and progenitor cell dif-ferentiation, we analyzed the ratio of Su(H)GBE-positive to total cells to determine the frequency of EBs differentiation to ECs. It was reported that high Su(H)GBE-positive EBs become ECs [6]. Consistent with our previous study, the number of Su(H)GBE-positive cells in the guts of control esg>Su(H)GBE-lacZ flies increased with age (Figure 4A) [7]. In contrast, the number of Su(H)GBE-positive cells in the guts of 30-day-old esg>UAS-D-p38bas;Su(H)GBE-lacZ flies was 0.5-fold less than that of 5-day-old flies (Figure 4A). Overexpression of D-p38b+ in ISCs and EBs resulted in an increased number of Su(H)GBE-positive cells compared to control flies at 5-days-old (Supplementary Figure 3A). We also examined the ratio of Prospero-positive cells, to determine the frequency of EBs differentiation to EEs. It was reported that the ratio of EEs to total cells does not change [8]. Interestingly, in guts expressing D-p38bas in ISCs and EBs, the ratio of EE to total cells increased with age. A 1.48-fold increase was observed in 30-day-old compared to 5-day-old esg>UAS-D-p38bas;Su(H)GBE-lacZ flies, while of the ratio of EE to total cells did not change in control flies (Figure 4B). Esg-positive cells had a spherical cell shape in the guts of esg>UAS-D-p38bas, distinguishing them morphologically from their angularly shaped counterparts in the guts of esg>+ (Figure 4C). We also detected that the ratio of EE to total cells in the 30-day-old posterior midgut of two D-p38b mutant flies was 2.2-fold and 2.1-fold higher than that in the 30-day-old gut of control flies (Supplementary Figure 2A and B). These results indicate that D-p38b MAPK is required for the age-related increase in ISC differentiation to ECs in the adult posterior midgut.


The role of p38b MAPK in age-related modulation of intestinal stem cell proliferation and differentiation in Drosophila.

Park JS, Kim YS, Yoo MA - Aging (Albany NY) (2009)

D-p38b MAPK plays a role in age-related defects in the differentiation of ISCs and progenitor cells. (A) Graph showing the ratio of                                                Su(H)GBE-positive to total cells. Effects of D-p38b activity on age-related                                                changes in the number of Su(H)GBE-positive cells in the posterior midgut.                                                Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ                                                flies were stained with DAPI, anti-β-gal and anti-GFP. The numbers of                                                each cell type were counted in a 0.06 x 0.04 cm area of the                                                posterior midgut. The ratio of Su(H)GBE-positive to total cells counted in                                                the posterior midgut of 5-day-old flies was set as 1. White square,                                                5-day-old flies; black square, 30-day-old flies. P-values were determined                                                using Student's t-test. (B) Graph showing the ratio of EE to total                                                cells. Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ                                                flies were stained with DAPI, anti-Prospero and anti-GFP. Numbers of each                                                cell type were counted in a 0.06 x 0.04 cm area of posterior midgut.                                                The ratio of EE to total cells counted in the posterior midgut of 5-day-old                                                flies was set as 1. White square, 5-day-old flies; black square, 30-day-old                                                flies. P-values were determined using Student's t-test. (C) Effects                                                of D-p38bas expression on ISC and EB cell morphology of                                                esg-positive cells and differentiation of EEs. Midguts of esg>+                                                (a-b) or esg>UAS-D-p38bas (c-d) flies were stained with                                                anti-Prospero (red), anti-GFP (green) and DAPI (blue). Enlarged images,                                                panels b and d. Scale bar, 5 μM. Arrow heads indicate esg-positive                                                cells. Asterisks indicate EEs.                                                (D) Graph showing the ratio of                                                EC to Su(H)GBE-positive cells. Midguts of esg>+; Su(H)GBE-lacZ or                                                esg>UAS-D-p38bas; Su(H)GBE-lacZ flies were stained                                                with DAPI, anti-β-gal and anti-GFP. Numbers of each cell type were                                                counted in a 0.06 x 0.04 cm area of posterior midgut. The ratio of                                                EC to Su(H)GBE-positive cells counted in the posterior midgut of 5-day-old                                                flies was set as 1. White square, 5-day-old flies; black square, 30-day-old                                                flies. P-values were determined using Student's t-test. (E) Effect                                                of D-p38bas expression in ISCs and EBs on age-related                                                accumulation of EC-like large esg- and Su(H)GBE-positive cells. The guts of                                                5- and 30-day-old flies were labeled with anti-β-gal and anti-GFP.                                                (a-f) esg>+;Su(H)GBE-lacZ, (g-l) esg>UAS-D-p38bas;Su(H)GBE-lacZ,                                                (m-r) esg>UAS-D-p38b+; Su(H)GBE-lacZ. (a, d, g, j, m,                                                and p - green) anti-GFP; (b, e, h, k, n, and q - red) anti-β-gal; (c,                                                f, I, l, o, and r) merged image. (DAPI, blue). Arrow indicates EC-like                                                large esg-GAL4. Asterisk indicates large Su(H)GBE-positive cell. Scale bar,                                                5 μM. (F)                                                Effect of D-p38b activity on the expression levels of Delta mRNA ISCs and                                                EBs in adult guts. Delta mRNA was measured by quantitative RT-PCR in cDNA                                                prepared from dissected guts from 5- and 30-day-old esg>+, esg>UAS-D-p38bas,                                                wild-type, p38bEY11174, or p38bKG01337 flies.                                                Expression was normalized to the expression of rp49. Expression level of                                                Delta mRNA in the midgut of 5-day-old flies was set as 1. White bar,                                                5-day-old flies; black bar, 30-day-old flies. P-values were determined                                                using Student's t-test. (G) Effect of D-p38b+ on the                                                expression of Delta in ISCs and EBs in adult gut. The level of Delta mRNA                                                was measured by real-time RT-PCR in cDNA prepared from dissected gut from                                                5-day-old esg>+ or esg>UAS-D-p38b+ flies.                                                Expression was normalized to the expression of rp49. Expression level of                                                Delta mRNA in midgut of 5-day-old flies was set as 1. White bar, esg>+;                                                black bar, esg>UAS-D-p38b+. P-value was determined                                                using Student's t-test.
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Figure 4: D-p38b MAPK plays a role in age-related defects in the differentiation of ISCs and progenitor cells. (A) Graph showing the ratio of Su(H)GBE-positive to total cells. Effects of D-p38b activity on age-related changes in the number of Su(H)GBE-positive cells in the posterior midgut. Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ flies were stained with DAPI, anti-β-gal and anti-GFP. The numbers of each cell type were counted in a 0.06 x 0.04 cm area of the posterior midgut. The ratio of Su(H)GBE-positive to total cells counted in the posterior midgut of 5-day-old flies was set as 1. White square, 5-day-old flies; black square, 30-day-old flies. P-values were determined using Student's t-test. (B) Graph showing the ratio of EE to total cells. Midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38bas;Su(H)GBE-lacZ flies were stained with DAPI, anti-Prospero and anti-GFP. Numbers of each cell type were counted in a 0.06 x 0.04 cm area of posterior midgut. The ratio of EE to total cells counted in the posterior midgut of 5-day-old flies was set as 1. White square, 5-day-old flies; black square, 30-day-old flies. P-values were determined using Student's t-test. (C) Effects of D-p38bas expression on ISC and EB cell morphology of esg-positive cells and differentiation of EEs. Midguts of esg>+ (a-b) or esg>UAS-D-p38bas (c-d) flies were stained with anti-Prospero (red), anti-GFP (green) and DAPI (blue). Enlarged images, panels b and d. Scale bar, 5 μM. Arrow heads indicate esg-positive cells. Asterisks indicate EEs. (D) Graph showing the ratio of EC to Su(H)GBE-positive cells. Midguts of esg>+; Su(H)GBE-lacZ or esg>UAS-D-p38bas; Su(H)GBE-lacZ flies were stained with DAPI, anti-β-gal and anti-GFP. Numbers of each cell type were counted in a 0.06 x 0.04 cm area of posterior midgut. The ratio of EC to Su(H)GBE-positive cells counted in the posterior midgut of 5-day-old flies was set as 1. White square, 5-day-old flies; black square, 30-day-old flies. P-values were determined using Student's t-test. (E) Effect of D-p38bas expression in ISCs and EBs on age-related accumulation of EC-like large esg- and Su(H)GBE-positive cells. The guts of 5- and 30-day-old flies were labeled with anti-β-gal and anti-GFP. (a-f) esg>+;Su(H)GBE-lacZ, (g-l) esg>UAS-D-p38bas;Su(H)GBE-lacZ, (m-r) esg>UAS-D-p38b+; Su(H)GBE-lacZ. (a, d, g, j, m, and p - green) anti-GFP; (b, e, h, k, n, and q - red) anti-β-gal; (c, f, I, l, o, and r) merged image. (DAPI, blue). Arrow indicates EC-like large esg-GAL4. Asterisk indicates large Su(H)GBE-positive cell. Scale bar, 5 μM. (F) Effect of D-p38b activity on the expression levels of Delta mRNA ISCs and EBs in adult guts. Delta mRNA was measured by quantitative RT-PCR in cDNA prepared from dissected guts from 5- and 30-day-old esg>+, esg>UAS-D-p38bas, wild-type, p38bEY11174, or p38bKG01337 flies. Expression was normalized to the expression of rp49. Expression level of Delta mRNA in the midgut of 5-day-old flies was set as 1. White bar, 5-day-old flies; black bar, 30-day-old flies. P-values were determined using Student's t-test. (G) Effect of D-p38b+ on the expression of Delta in ISCs and EBs in adult gut. The level of Delta mRNA was measured by real-time RT-PCR in cDNA prepared from dissected gut from 5-day-old esg>+ or esg>UAS-D-p38b+ flies. Expression was normalized to the expression of rp49. Expression level of Delta mRNA in midgut of 5-day-old flies was set as 1. White bar, esg>+; black bar, esg>UAS-D-p38b+. P-value was determined using Student's t-test.
Mentions: To assess whether D-p38b MAPK is required for age-related changes in ISC and progenitor cell dif-ferentiation, we analyzed the ratio of Su(H)GBE-positive to total cells to determine the frequency of EBs differentiation to ECs. It was reported that high Su(H)GBE-positive EBs become ECs [6]. Consistent with our previous study, the number of Su(H)GBE-positive cells in the guts of control esg>Su(H)GBE-lacZ flies increased with age (Figure 4A) [7]. In contrast, the number of Su(H)GBE-positive cells in the guts of 30-day-old esg>UAS-D-p38bas;Su(H)GBE-lacZ flies was 0.5-fold less than that of 5-day-old flies (Figure 4A). Overexpression of D-p38b+ in ISCs and EBs resulted in an increased number of Su(H)GBE-positive cells compared to control flies at 5-days-old (Supplementary Figure 3A). We also examined the ratio of Prospero-positive cells, to determine the frequency of EBs differentiation to EEs. It was reported that the ratio of EEs to total cells does not change [8]. Interestingly, in guts expressing D-p38bas in ISCs and EBs, the ratio of EE to total cells increased with age. A 1.48-fold increase was observed in 30-day-old compared to 5-day-old esg>UAS-D-p38bas;Su(H)GBE-lacZ flies, while of the ratio of EE to total cells did not change in control flies (Figure 4B). Esg-positive cells had a spherical cell shape in the guts of esg>UAS-D-p38bas, distinguishing them morphologically from their angularly shaped counterparts in the guts of esg>+ (Figure 4C). We also detected that the ratio of EE to total cells in the 30-day-old posterior midgut of two D-p38b mutant flies was 2.2-fold and 2.1-fold higher than that in the 30-day-old gut of control flies (Supplementary Figure 2A and B). These results indicate that D-p38b MAPK is required for the age-related increase in ISC differentiation to ECs in the adult posterior midgut.

Bottom Line: In addition, this pathway is involved in age and oxidative stress-associated mis-differentiation of enterocytes and upregulation of Delta, a Notch receptor ligand.Furthermore, we also show that D-p38b acts downstream of PVF2/PVR signaling in these age-related changes.Taken together, our findings suggest that p38 MAPK plays a crucial role in the balance between ISC proliferation and proper differentiation in the adult Drosophila midgut.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Pusan National University, Busan 609-735, Korea.

ABSTRACT
It is important to understand how age-related changes in intestinal stem cells (ISCs) may contribute to age-associated intestinal diseases, including cancer. Drosophila midgut is an excellent model system for the study of ISC proliferation and differentiation. Recently, age-related changes in the Drosophila midgut have been shown to include an increase in ISC proliferation and accumulation of mis-differentiated ISC daughter cells. Here, we show that the p38b MAPK pathway contributes to the age-related changes in ISC and progenitor cells in Drosophila. D-p38b MAPK is required for an age-related increase of ISC proliferation. In addition, this pathway is involved in age and oxidative stress-associated mis-differentiation of enterocytes and upregulation of Delta, a Notch receptor ligand. Furthermore, we also show that D-p38b acts downstream of PVF2/PVR signaling in these age-related changes. Taken together, our findings suggest that p38 MAPK plays a crucial role in the balance between ISC proliferation and proper differentiation in the adult Drosophila midgut.

Show MeSH
Related in: MedlinePlus