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

Introduction

In mammals, intestinal homeostasis is maintained by the balance between intestinal stem cell (ISC) proliferation, directed differentiation, and removal of dead cells in adults [1]. The precise mechanism by which proliferation and differentiation of stem cells is lost with age and/or oxidative stress is unknown. These effects on stem cells result in age-related diseases, such as cancer [2,3]. Therefore, it is important to understand how age-related changes in ISC proliferation and differentiation contribute to age-associated intestinal diseases, including cancer. Recently, Drosophila melanogaster was shown to be an excellent model system for the study of ISC biology and aging. It was demonstrated that proliferating progenitor cells reside within the intestinal epithelium of adult Drosophila, similar to vertebrate intestine [4,5]. Adult Drosophila midgut cells can be identified as ISCs, enteroblasts (EBs), enteroendocrine cells (EEs) and enterocytes (ECs) with specific markers. ISCs can be identified by the expression of Delta, a Notch receptor ligand [6]. Escargot (esg) is a marker for ISCs and EBs [5]. Prospero is a marker for EEs [4,5]. The Su(H)GBE-lacZ reporter construct is induced by Notch activity. Therefore, expression of lacZ is a marker for EBs, since the fates of ISC daughter cells are specified via differential Notch activity modulated by expression levels of Delta in ISCs [6]. Previously, we reported age-related increases in ISC proliferation and in the number of Delta-/esg-/Su(H)GBE-positive cells [7]. We also showed that oxidative stress can mimic age-related changes in ISCs and that a PDGF/VEGF-like growth factor, PVF2/PVR, is involved in age and oxidative stress-related changes [7]. Biteau et al. reported that this increase in the number of Delta-/esg-/Su(H)GBE-positive cells is due to the accumulation of mis-differentiated ISC daughter cells such as EC-like large esg-positive cells. In addition they showed that the age-related changes are associated with aberrant Delta/Notch and JNK signaling [8]. More recently, involvement of the insulin receptor and the JAK-STAT signaling pathways was shown in ISC proliferation in response to tissue damage and during immune response, respectively [9,10].

In mammals, it has been well demonstrated that p38 MAPK is activated in response to various physical and chemical stresses, such as oxidative stress, UV irradiation, hypoxia and ischemia [11]. Fu et al. reported increased p38 MAPK activation in ISCs and their daughter cells in the small intestine after ischemia [12]. In Drosophila, two p38 MAPK isoforms have been identified, D-p38a and D-p38b. D-p38s are activated by various stresses, including UV, lipopoly-saccharide (LPS), and osmotic stress [13,14]. It was reported that D-p38b mRNA levels were detected in the developing posterior midgut [14]. We previously observed an increase in expression of a D-p38b-lacZ reporter construct in the posterior midgut by septic injury [15]. However, the role of p38 MAPK in ISC proliferation and differentiation remains unknown.

In the current study, we tested whether D-p38b MAPK is involved in age and oxidative stress-associated modulation of ISC proliferation and differentiation in the adult Drosophila midgut. We further examined the serial relationship between D-p38b MAPK and PVR signaling in age-associated intestinal changes.

Results

Discussion

In a previous study, we reported age-related changes including an increase in stem cell proliferation and a defect in stem and progenitor cell differentiation to EC. In addition we demonstrated that a PDGF/VEGF-like growth factor, PVF2/PVR, is involved in the age and oxidative stress-associated modulation in the Drosophila midgut [7]. Recently, Biteau et al. also reported the phenomenon of aged intestinal epithelium due to aberrant JNK activity [8]. Here, we investigated whether D-p38b MAPK activity is required for age and oxidative stress-associated changes in intestinal epithelium.

We found an age-related increase of a D-p38b reporter construct in ISCs and EBs in the adult Drosophila midgut. In mammals, it was reported that activated p38 MAPK was detected in the ISCs and their daughter cells after ischemia [12]. We analyzed whether D-p38b MAPK plays a role in the age-related increase of ISC proliferation in Drosophila midgut. Flies expressing D-p38b^as in ISCs and EBs and two D-p38b mutants did not show an age-related increase of ISC proliferation. Previously, we reported that PVF2/PVR signaling is involved in an age and oxidative stress-related increase in ISC proliferation [7]. Here, we showed that PVR-induced increase in ISC proliferation was suppressed by D-p38b MAPK knockdown in ISCs and EBs. These data suggest that D-p38b MAPK acts downstream of PVR signaling in ISC proliferation. However, it should be noted that D-p38b expression in ISCs and EBs partially suppressed the PVR-induced ISC proliferation.

Recently, Biteau et al. reported that JNK activity contributes to the age-associated increase in stem cell proliferation [8]. It was reported that Wg signaling, insulin receptor signaling pathway, and the JAK-STAT pathway are involved in ISC proliferation in the Drosophila adult midgut [9,10,17]. Jiang and Edgar reported that EGFR/RAS/ERK pathway is required for proliferation of adult midgut progenitors [18]. Therefore, cross-talk between these signaling pathways and PVR-p38 MAPK signaling in the regulation of ISC proliferation would be an interesting subject to investigate.

In a previous study, we also found that the number of EBs differentiating to ECs increased with age [7]. Ohlstein and Spradling reported that ISC within the adult midgut normally produce EB via asymmetric division. Most EBs differentiate to ECs, while a few differentiate to EEs [6]. It was reported that the ratio of EE to total cells did not change in the midgut with age [8]. These data suggest that there is an age-related increase in the number of EBs giving rise to ECs. Here, we showed that loss of D-p38 signaling suppressed the age-related increase of Su(H)GBE-positive cells and increased the ratio of EE to total cells, especially in old flies. In addition, the esg-positive cells with aberrant D-p38 signaling were spherical instead of angular shaped. Recently, Maeda et al. reported that knockdown of E-cad in the gut resulted in esg-positive cells with a spherical cell shape and an increase of the number of EEs [19]. Our data suggest that D-p38b MAPK is required for ISC commitment to EB differentiation to EC.

Recently, it was reported accumulation of aberrant EC-like esg-positive cells is an indicator for misdifferentiation of ECs. It was also proposed that aberrant expression of Delta in ISCs and progenitors is associated with the accumulation of large esg-positive cells [8]. In this study, expression of D-p38b^as in ISCs and EBs suppressed the age-related and oxidative stress-induced accumulation of EC-like esg-positive cells, and partially suppressed the mis-differentiation induced by ectopic PVR. Furthermore, loss of D-p38b MAPK signaling in ISCs and EBs suppressed age and stress-induced Delta expression, and partially suppressed PVR-induced Delta expression. These data suggest a cross-talk between PVR and D-p38b MAPK signaling in the regulation of Delta expression. Furthermore, our data suggest that D-p38b MAPK acts downstream of PVR signaling, and aberrant D-p38b MAPK signaling results in mis-differentiation in the midgut.

It was reported that in wild-type gut, ECs turnover approximately within one week [5]. Interestingly, we observed that D-p38b knockdown in ECs likely results in cell turnover longer than one week. We also detected that EC size in the guts of flies expressing D-p38b^as in ISCs and EBs was larger than those of controls with age (data not shown), suggesting that D-p38b MAPK may be involved in EC death. Recent studies in mammals demonstrated that p38 MAPK is involved in intestinal epithelial cell apoptosis through activation of Bax in stress conditions [20, 21].

In light of these findings, we propose that D-p38b MAPK plays a crucial role in the balance between regeneration of intestinal epithelium and proper differentiation.

Materials and Methods

Fly stock. Fly stocks were maintained at 25 °C on standard food under an ~12 h/12 h light/dark cycle. The food consisted of 79.2% water, 1% agar, 7% cornmeal, 2% yeast, 10% sucrose, 0.3% bokinin and 0.5% propionic acid. To avoid larval overpopulation in all vials, 50-60 adult flies per vial were transferred to new food vials every 2-3 days for a period of 50-60 days or longer. We previously established the reporter transgenic flies carrying promoter region of D-p38b gene [15]. UAS-D-p38b⁺ and UAS-D-p38b^as were kindly provided by T. Adachi-Yamada [16]. Su(H)GBE-lacZ was kindly provided by Sarah Bray [22]. UAS-PVR was generously provided by Pernille Rørth [23]. y w hs-Flp[122]; act>y⁺>gal-4 UAS-GFP (AyGal4) was kindly provided by K.D. Irvine [24].esg-GAL4 was kindly provided by the Drosophila Genetic Resource Center. Oregon-R was used as wild-type. p38b^EY11174 and p38b^KG01337 were established by Bellen et al. [25] and acquired from the Bloomington Drosophila Stock Center.

The UAS-D-p38b⁺ or UAS-D-p38b^as/+;esg-GAL,UAS-GFP/+;Su(H)GBE-lacZ/+ flies were obtained from a cross of the UAS-D-p38b⁺ or UAS-D-p38b^as/ y or UAS-D-p38b^as;+;Su(H)GBE-lacZ /TM6 males to the esg-GAL4,UAS-GFP/CyO females. The esg-GAL4,UAS-GFP/+;Su(H)GBE-lacZ/+flies from a cross of the Su(H)GBE-lacZ/TM3 males to the esg-GAL4,UAS-GFP/CyO females were used as a control. The UAS-D-p38b^as/+;UAS-PVR/esg-GAL4,UAS-GFP flies were obtained from a cross of the UAS-D-p38b^as/y;UAS-PVR/UAS-PVR males to the esg-GAL4,UAS-GFP/CyO females.

All experiments were conducted and evidenced similar results in both females and males. The results described in this present study were obtained from the females.

Oligonucleotides. Oligonucleotide primers of rp49 and Delta were previously described [8]. All oligonucleotides were chemically synthesized.

Quantitative RT-PCR. Total RNA from the adult midguts was isolated with Trizol Reagent (Molecular Research Center, Cincinnati, OH, USA) in accordance with the protocols recommended by the manufacturer. In brief, adult midguts were dissected, chloroform was added (Sigma, St. Louis, MO, USA) and then the samples were repeatedly centrifuged at 19326 × g at 4 °C for 15 min. The supernatant was moved to a new microtube, isopropanol was added, and the samples were incubated at 25 °C for 15 min, centrifuged repeatedly at 19326 × g at 4 °C for 15 min, washed with 70% EtOH, and dried. cDNAs from the prepared mRNA extracts were synthesized. Denatured mRNA with M-MLV-RT buffer, 2.5 mM dNTP, oligo dT, 100 mM DTT and M-MLV-reverse transcriptase (Promega, Madison, WI, USA) was incubated at 42 °C for 1 h. The real-time RT-PCR products were analyzed using OpticMonitor3.

Immunochemistry. The intact adult guts were dissected, fixed at room temperature for 1 h in 4% formaldehyde (Sigma), washed with PBT [0.1% Triton X-100 in phosphate-buffered saline (PBS)], and incubated overnight with primary antibody at 4 °C. After washing and blocking [2% bovine serum albumin (BSA) in PBT], the samples were incubated for 1 h with secondary antibodies at 25 °C, washed in PBT, mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA), and analyzed using a Zeiss Axioskop 2plus microscope (Carl Zeiss Inc., Gottingen, Germany). For the quantitative analysis of esg -, Delta- and Su(H)GBE-positive cells, EC and EE cells, images were processed in Photoshop (Adobe Systems, San Jose, CA, USA). The numbers of esg -, Delta- and Su(H)GBE-positive cells and ECs were counted in 0.06 × 0.04 cm area and the number of EEs were counted in 0.06 × 0.04 cm area of the posterior midgut.

BrdU labeling. BrdU staining was conducted via standard methods and modified as follows [5]. Flies were cultured on standard food supplemented with BrdU (final concentration 0.2 mg/ml) for 4 days. Flies were subsequently cultured at 25 °C and transferred to new media every 2 days. The entire guts were removed and fixed in ethanol : acetic acid (3 : 1) for 2 min and DNA was denatured by incubating tissue in 2M HCl for 10 mins, then incubated with primary antibody overnight at 4 °C. Primary antibody was removed and the samples were washed in PBT. The samples were incubated for 1 h with secondary antibodies and DAPI, washed in PBT and mounted with Vectashield.

Mosaic analysis. For clonal analysis, clones were generated by Flp-out cassette [17]. In this case, mitotic clones were conditionally induced on heat-shock treatments for flipase expression, and clones were marked by GFP. Fly crosses established and cultured at 18 °C to generate adults of the genotype y w hs-Flp[122] /UAS-D-p38b⁺; act>y⁺>gal-4 UAS-GFP/+ or y w hs-Flp[122] /UAS-D-p38b^as; act>y⁺>gal-4 UAS-GFP/+ or y w hs-Flp[122] /+; act>y⁺>gal-4 UAS-GFP/+ as a control flies. Equal numbers of adult flies were then divided into experimental and control groups. Experimental animals were subjected to between one and three 37 °C heat shocks for clone induction. After heat shock, flp-out clones were grown at 18 °C to minimize background signal from the Gal/UAS system. Midguts were examined 7 days after clone induction.

Antisera. The following primary antibodies diluted in PBT were used in these experiments: rabbit anti-β-gal (Cappel, Solon, OH, USA) 1 : 500; rabbit anti-phosphohistone H3 (Upstate, Charlottesville, VA, USA) 1 : 500; mouse anti-Armadillo, mouse anti-Prospero, mouse anti-Delta and mouse anti-BrdU (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) 1 : 100; mouse anti-GFP, rabbit anti-GFP (Molecular Probes, Eugene, OR, USA) 1 : 1000. The following secondary antibodies diluted in PBT + 2% BSA were used: goat anti-rabbit FITC (Cappel) 1 : 400; goat anti-rabbit Cy3 (Jackson ImmunoResearch, West Grove, PA, USA) 1 : 400; goat anti-mouse FITC (Jackson ImmunoResearch) 1 : 400; goat anti-mouse Cy3 (Jackson Immuno-Research) 1 : 400; DAPI (Molecular Probes) 1 : 1000.

X-gal staining. The adult guts were dissected on ice and fixed for 10 min with 1% glutaraldehyde (Sigma) in 1× PBS. The samples were then washed in 1× PBS for 1 h, and stained with 0.2% X-gal (USB, Cleveland, OH, USA) in staining buffer containing 6.1 mM K₄Fe(CN)₆, 6.1 mM K₃Fe(CN)₆, 1 mM MgCl₂, 150 mM NaCl, 10 mM Na₂HPO₄ and 10 mM NaH₂PO₄ in the dark at 25 °C during overnight.

Statistical analyses. Significance testing was conducted via Student's t -test.

Supplementary Materials

Supplementary Figure 1

Effect of D-p38b overexpression in ISCs and EBs on ISC DNA synthesis. Increase of BrdU incorpora-tion by ectopic D-p38b MAPK in ISCs and EBs. One-day-old esg>+ (a) or esg>UAS-D-p38b+ (b) flies were fed on 0.2 mg/ml BrdU media for 4 days and stained with anti-BrdU. Overlay (DAPI, blue; anti-BrdU, red). Scale bar, 5 μM.

Supplementary Figure 2

D-p38b MAPK plays a role in the differentiation of ISCs and progenitor cells. (A) Graph showing an increase in the ratio of EE to total cells in the guts of D-p38b mutants. Number of the Prospero-positive cells detected per midgut of 30-day-old flies control, p38bEY11174 or p38bKG01337 flies. The number of prospero-positive cells detected per posterior midgut of 30-day-old wild-type flies was set as 1. white bar, wild-type; black bar, p38bEY11174; gray bar, p38bKG01337. P-values were calculated using Student’s t-test and compared to each control. (B) Effect of p38b mutant allele expression on EE cell production. The guts of 30-day-old flies were labeled with anti-prospero and DAPI. (a), wild-type; (b), p38bEY11174; (c), p38bKG01337. (anti-prospero, red; DAPI, blue). Scale bar, 5 μM.

Supplementary Figure 3

Effect of D-p38b MAPK overexpression in ISCs and EBs on differentiation of ISC progenitor cells. (A) Graph showing the ratio of Su(H)GBE-positive to total cells. The 5-day-old midguts of esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38b+;Su(H)GBE-lacZ flies were stained with DAPI, anti-?-gal and anti-GFP. 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 of 5-day-old flies was set as 1. White square, esg>+;Su(H)GBE-lacZ; black square, esg>UAS-D-p38b+;Su(H) GBE-lacZ. P-values were determined using Student’s t-test. (B) Graph showing the ratio of ECs to Su(H)GBE-positive cells. Midguts of five-day old esg>+;Su(H)GBE-lacZ or esg>UAS-D-p38b+;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 the posterior midgut. White square, esg>+; black square, esg>UAS-D-p38b+. The ratio of ECs to Su(H)GBE-positive cell of 5-day-old flies was set as 1. P-values were determined using Student’s t-test. (C) Effect of p38b MAPK overexpression in ISCs and EBs on the size of esg- and Su(H)GBE-positive cells. The guts of 5-day-old flies were labeled with anti-β-gal and anti-GFP. (a-c) esg>+;Su(H)GBE- lacZ, (d-f) esg>UAS-D-p38b+;Su(H)GBE-lacZ. a and d, anti-GFP; b and e, anti-β-gal; c and f, merged image. (DAPI, blue; anti-?-gal, red; anti-GFP, green). Scale bar, 5 μM.