Feedback regulation of steady-state epithelial turnover and organ size

Abstract

Epithelial organs undergo steady-state turnover throughout adult life, with old cells being continually replaced by the progeny of stem cell divisions. To avoid hyperplasia or atrophy, organ turnover demands strict equilibration of cell production and loss. However, the mechanistic basis of this equilibrium is unknown. Here we show that robustly precise turnover of the adult Drosophila intestine arises through a coupling mechanism in which enterocyte apoptosis breaks feedback inhibition of stem cell division. Healthy enterocytes inhibit stem cell division through E-cadherin, which prevents secretion of mitogenic epidermal growth factors (EGFs) by repressing transcription of the EGF maturation factor rhomboid. Individual apoptotic enterocytes promote divisions by loss of E-cadherin, which releases cadherin-associated β-catenin (Armadillo in Drosophila) and p120-catenin to induce rhomboid. Induction of rhomboid in the dying enterocyte triggers activation of the EGF receptor (Egfr) in stem cells within a discrete radius. When we blocked apoptosis, E-cadherin-controlled feedback suppressed divisions, and the organ retained the same number of cells. When we disrupted feedback, apoptosis and divisions were uncoupled, and the organ developed either hyperplasia or atrophy. Together, our results show that robust cellular balance hinges on the obligate coupling of divisions to apoptosis, which limits the proliferative potential of a stem cell to the precise time and place at which a replacement cell is needed. In this way, localized cell-cell communication gives rise to tissue-level homeostatic equilibrium and constant organ size.

Extended Data Fig. 1. Midgut lineage and morphology, esg^F/O labeling system, and work-flow for semi-automated cell counting

a, Lineage of the adult Drosophila midgut^,. Stem cells are, in general, the only cells capable of division. Asymmetric stem cell divisions typically produce absorptive enterocytes; less frequently, they produce secretory enteroendocrine cells. Enterocytes arise through direct maturation of transient, post-mitotic intermediates called enteroblasts. Stem and enteroblast cells express the Snail-family transcription factor escargot (esg). b, Compartments of the female adult midgut^,,. The R4ab compartment (also known as P1–2) was used for all experiments in this study. Schematic adapted from Ref. . c–e, Identification of R4ab using morphological landmarks. As defined in Ref. , R4ab is bounded by: (1) the apex of the midgut tube’s most distal 180° turn (blue arrowheads in d) and (2) the first prominent muscle constriction distal to this 180° turn (red arrowheads in d). e, The R4ab distal muscle constriction (red arrowheads) is particularly apparent in confocal optical sections. Visceral muscle stained with phalloidin. Midguts in panels c–d and e are two different samples. f, Genetic schema of the esg^F/O system. Stem and enteroblast cells are induced to express heritable GFP by temperature shift from 18°C to 29°C. The temperature shift inactivates GAL80^ts, which allows esgGAL4 to drive expression of both UAS-GFP and UAS-flp in stem and enteroblast cells. In these cells, flp recombinase renders GFP expression permanent and heritable by excising a CD2 ‘flp-out’ cassette to generate a functional actGAL4; once generated, actGAL4 drives expression of UAS-GFP (and UAS-flp) irrespective of cell type. Thus, after temperature shift, all mature cells that arise from undifferentiated cells will express GFP. g, Pipeline for semi-automated, comprehensive cell counts of 3D, reconstructed midgut regions. (1) Confocal microscope z-stacks capturing the entire depth of the organ are visualized in Fiji. (2) The R4ab region of the midgut (yellow outline) is digitally isolated and exported to Imaris. (Only the top half of the gut tube is shown.) Note that different midgut regions have different rates of turnover: R4ab undergoes complete turnover between adult days 4–8 (at 29°C). However, other regions undergo slower turnover, as shown by large unlabeled regions outside of R4ab. See Methods for further discussion. (3) To quantify total cells, nuclei (DAPI) are mapped to surface objects using Imaris. To quantify newly-added cells in the esg^F/O system, GFP⁺ cells are recognized in Imaris by co-localization of GFP and DAPI channels, and subsequently mapped to surface objects. Scale bars: 100µm.

Extended Data Fig. 2. Genetic schema of system to simultaneously manipulate enterocyte expression and trace stem cell divisions

a, Detailed explanation of the genetic system in Fig. 1f. Animals are raised at 18°C; at this temperature, GAL80^ts represses mex-driven GAL4 in enterocytes (mex^ts), and lacZ labeling of stem cells is not induced. At 4 days post-eclosion, animals are temperature-shifted to 29°C; consequent inactivation of GAL80^ts allows mexGAL4 to express genes of interest (UAS-gene X) specifically in enterocytes. After 1 day of UAS gene expression (5 days post-eclosion), animals are shifted to 38.5°C for one hour to induce ubiquitous expression of flp recombinase, which is under control of a heat-shock promoter (hs-flp). Flp catalyzes trans-recombination of the two FRTs to place the α-tubulin promoter upstream of the promoter-less nls-lacZ cassette and, consequently, turn on permanent nls-lacZ expression. After heat shock, animals are returned to 29°C to maintain UAS-transgene expression. Midguts are harvested for clonal analysis 4 days after the 38.5°C heat shock (9 days post-eclosion). See also Methods. b, Validation of genetic system using mex^ts>his2av::RFP. β-galactosidase marks a stem cell clone (outlined) in a background of His2av::RFP⁺ enterocytes. Within the 5-cell clone, only the enterocyte (yellow asterisk, polyploid) expresses his2av::RFP.

Extended Data Fig. 3. Quantifications of organ size and EGFR activation in genetically manipulated midguts

a, Lengths of the R4ab compartment. N=10–12 midguts per genotype or condition, analyzed after 4 days of UAS-transgene expression. b, dpERK⁺ cells in the R4ab compartment. N=4 midguts per genotype or condition, analyzed after 2 days of UAS-transgene expression. One of two replicate experiments was quantified. In a and b, values are means ± S.D. p values: unpaired t-test, compared to control.

Extended Data Fig. 4. Analysis of epithelial architecture, polarity, and barrier function

a–f, Apoptotic inhibition (b, e) or E-cad depletion (c, f) in enterocytes does not disrupt either epithelial architecture or apical-basal polarity. Images show vertical sections through the midgut epithelium after 4 days of either mex^ts>p35 or mex^ts>E-cadRNAi expression. Enterocytes remain as a coherent monolayer. Apical-basal polarity is intact, as revealed by immunolocalization of apical, actin-rich microvilli (a–f; SiR-Actin, red) and of two apico-lateral septate junction proteins, Coracle (a–c, green) and Discs-large (d–f, green). At the basal surface of the epithelium (white dotted lines), midgut visceral muscle cells stain brightly for actin and Discs-large. Scale bars: 25µm. g–j, Depletion of E-cad in enterocytes does not compromise the intestinal barrier. To test the intestinal barrier, animals were subjected to Smurf assays in which a blue, non-absorbable food dye is administered by feeding. The dye remains within the midgut when the barrier is intact (g, non-Smurf) but leaks into the body cavity when the barrier is compromised, such as after consumption of 1% SDS (h, Smurf). After 10 days of mex^ts>E-cadRNAi expression, midguts still retain the blue dye; no Smurf phenotypes are observed (i–j).

Extended Data Fig. 5. Depletion of E-cad has distinct cell-autonomous and tissue-level effects on cell death

In Fig. 2h, total cell counts show that midguts accumulate excess cells when E-cad is depleted from apoptosis-inhibited enterocytes but not apoptosis-competent enterocytes. To shed light on this difference, we examined whether E-cad depletion itself promotes cell death. Two approaches, mosaic knockdown and pan-enterocyte knockdown, were used to distinguish direct, cell-autonomous effects from indirect, tissue-level effects. a–c, Mosaic knockdown of E-cad does not promote cell-autonomous death. To generate a mosaic epithelium, MARCM labeling was used to induce sparse, multicellular, GFP-marked clones in a background of unmarked, genetically unperturbed cells. a–b, Dotted outlines show representative control and E-cadRNAi clones (green). Sytox (red) identifies dying cells. c, Percentage of GFP⁺ cells that are also Sytox⁺. Dying cells occur with near-equal frequency within control and E-cadRNAi clones. N=5 midguts per genotype, analyzed 9 days after clone induction; n=873 cells in control clones and 698 cells in E-cadRNAi clones. d–f, Pan-enterocyte knockdown of E-cad promotes cell death, likely through a non-autonomous effect. d–e, Representative images of mex^ts control and mex^ts>E-cadRNAi epithelia. Sytox (red) identifies dying cells. f, Quantification of Sytox⁺ cells in the R4ab compartment. The number of dying cells increases ~2.5× in E-cadRNAi midguts compared to control. N=5 midguts per genotype, analyzed after 3 days of transgene induction. In both c and f, values are means ± S.D; comparisons by unpaired t-test. Scale bars are 25 µm. g, Summary. The unaltered frequency of dying cells in E-cadRNAi mosaic clones indicates that loss of E-cad does not cause cell-autonomous death. This result suggests that elevated death in mex^ts>E-cadRNAi guts is a non-autonomous, tissue-level effect, possibly due to excess divisions (Fig 2b) and consequent crowding. These findings may explain why p35, E-cadRNAi guts accumulate excess cells whereas E-cadRNAi guts retain a normal number of cells (Fig. 2h).

Extended Data Fig. 6. Loss of enterocyte E-cad activates EGFR, but not Wg, Hpo, or Upd/JAK/STAT

a, Effect of enterocyte E-cad depletion on target mRNAs of known midgut regulatory pathways. mRNAs were measured by qPCR of mex^ts control or mex^ts>E-cadRNAi midguts. Relative to control (red line), mRNAs do not increase for: the Wg targets frizzled-3 (fz3) and senseless (sens), the Hpo/Yki targets expanded (ex) and diap1, the injury-associated cytokines upd and upd3, and the JAK/STAT target windpipe (wdp). The other JAK/STAT target, Socs36E, is elevated, likely reflecting its occasional activation in enterocytes (panel f). By comparison, the EGFR target pointed (pnt) is slightly increased, and the EGFR target cyclinE (cycE) is substantially increased. Values are means ± S.D. from 3 independent experiments. Midguts analyzed 4 days post-induction. b–d, The number of upd3-lacZ⁺ enterocytes in the R4ab compartment is unchanged by enterocyte E-cad depletion. e–g, The number of 10XSTAT-GFP⁺ diploid cells in R4ab is unchanged by enterocyte E-cad depletion. Occasional activation of 10XSTAT-GFP⁺ occurs in E-cad-depleted enterocytes (asterisk in f), consistent with elevated Socs36E (panel a). h–j, The number of cycE⁺ diploid cells in R4ab increases following enterocyte E-cad depletion. In panels d, g, and j, values are means ± S.D of 4 midguts, analyzed 2 days post-induction; comparisons by unpaired t-test. k, dpERK immunostaining is limited to stem cells (HRP⁺, Su(H)lacZ⁻; arrowheads in k' and k") and does not mark enteroblasts (HRP⁺, Su(H)lacZ⁺; asterisks in k' and k"), even in mex^ts>E-cadRNAi midguts^,. l, Expression of upd3 is not associated with physiological apoptosis. Most enterocytes that express upd3-lacZ are non-apoptotic, as assessed by staining for cleaved Caspase-3. Values are means ± S.D of 4 midguts, analyzed 6 days post-eclosion. Representative images in all panels. Scale bars: 25 µm.

Extended Data Fig. 7. Two E-cad-associated transcription factors, Armadillo and p120-catenin, activate rho following loss of E-cad in enterocytes

a, rho mRNA levels were measured by qPCR of either mex^ts control (red line) or mex^ts>E-cadRNAi midguts, the latter with additional manipulation of candidate rho regulators as indicated. Five candidates were examined: Yki, a transcriptional co-activator in the Hpo pathway; Groucho, a co-repressor known to target rho in some tissues^,; puckered (puc), an inhibitor of Basket/JNK, the latter of which can enhance EGF signaling; and Arm and p120, co-activators that are inhibited by sequestration at E-cad adherens junctions. Knockdown of either arm or p120 significantly reduces rho activation. b, Overexpression of p120, but not arm^S10, in enterocytes is sufficient to increase rho mRNAs; control (red line). In a and b, values are means ± S.D. of 3 independent experiments; midguts were analyzed 4 days post-induction. c–f, Depletion of E-cad or overexpression of p120 induces rho-lacZ in enterocytes and not in diploid cells. f, Quantification. g–l, Enterocyte arm and p120, but not yki or upd3, are necessary for activation of stem cell EGFR (dpERK immunostain) following loss of E-cad. m–o, Enterocyte rho is necessary and sufficient for activation of stem cell EGFR. p–r, Enterocyte p120, but not arm, is sufficient to activate stem cell EGFR. See also Extended Data Fig. 3b. s, Overexpression of enterocyte rho increases the number of mitotic (phospho-histone H3⁺) stem cells. In f and s, values are means ± S.D of N=4 midguts, assessed after 2 days of transgene expression. p values: unpaired t-test, comparisons to control. Representative images shown in all panels. Scale bars: 25 µm.

Extended Data Fig. 8. Loss of rho, arm, or p120 in enterocytes results in organ atrophy

a, Total cell counts. Depletion of rho, arm, or p120 in enterocytes reduces total cells compared to control. Values are means ± S.D from 1 of 3 replicate experiments. N=4 midguts per genotype, analyzed after 6 days of induction. b, Depletion of enterocyte rho, arm, or p120 reduces the length of the R4ab compartment compared to control. N=10–12 midguts per genotype, analyzed after 6 days of induction. In a and b, comparisons to control by unpaired t-test. c–d, Depletion of enterocyte rho leads to organ atrophy. Representative whole mount images. A, anterior; P posterior. Scale bar: 200µm.

Fig. 1. Enterocyte apoptosis regulates the rate of stem cell division for homeostatic mainte-nance of total cell number

a–e, Kinetics of midgut epithelial turnover: Cartoon (a), images (b–d) and quantitation (e) of esg^F/O>GFP labeling. Progenitors (a, small circles) express GFP upon induction; new, but not old, enterocytes (a, large hexagons) inherit GFP from progenitors after induction. Quantitation of total and GFP⁺ cells over time shows complete replacement of unlabeled cells by GFP⁺ cells after 4 days (e, means ± S.D.; 3 midgut R4ab compartments per timepoint). See Extended Data Fig. 1. f, Genetic schema and experimental timeline for tracing stem cell divisions (split-lacZ clones) in a background of genetically manipulated enterocytes (mex^ts). See Extended Data Fig. 2. g–i, Sizes (g, means ± S.D; p values, Mann-Whitney test) and images (h, i) of stem cell clones following enterocyte inhibition of apoptosis (mex^ts>p35). Clone sizes are reduced by apoptotic inhibition. N=4–5 midguts per genotype. j, EdU incorporation in diploid cells is reduced by apoptotic inhibition. k, Total cell counts of the R4ab compartment with control or apoptosis-inhibited (p35, diap1) enterocytes. For j, k: Means ± S.D. shown; p values from unpaired t-test. N=4 midguts per genotype. One of three replicate experiments shown in each graph. All scale bars, 25 µm.

Fig. 2. Homeostatic size control requires E-cad on enterocytes, but not on stem cells

a, E-cad::m-Tomato (red-hot LUT) is absent from cell-cell junctions (arrowheads) of dying enterocytes (Sytox⁺, asterisks). Tracheal autofluorescence appears as bright, red-yellow lines (arrows). b–g, Sizes (b) and images (c–g) of stem cell clones following enterocyte manipulation of E-cad, with or without apoptotic inhibition (p35; experimental schema in Fig. 1f). Clone sizes are reduced by enterocyte E-cad. h–j. Total cell counts (h, means ± S.D. shown; p values, unpaired t-test) and whole-organ images (i, j; A, anterior; P, posterior). Midguts become hyperplastic following enterocyte co-expression of p35 and E-cadRNAi, but not expression of either p35 or E-cadRNAi alone, or co-expression of p35 and edRNAi. N=4 midguts per genotype. See Extended Data Figs. 3a, 5. k, Sizes of stem cell clones are unchanged by stem cell/enteroblast manipulation of E-cad. Experimental schema similar to Fig. 1f, but using stem/enteroblast esg^tsGAL4. For b, k: Means ± S.D. shown; p values, Mann-Whitney test. N=4–5 midguts per genotype. One of three replicate experiments shown in each graph. Scale bars, 25 µm or as indicated.

Fig. 3. Enterocyte E-cad inhibits stem cell EGFR via a dispersed signal for homeostatic size control

a–e, Immunostain for activated, diphospho-ERK (dpERK) following enterocyte manipulation of E-cad, without or with EGFR inhibition (AG1478, egfr^tlsa). ERK activation in stem cells (Extended Data Fig. 6k) is repressed by E-cad and requires EGFR. See Extended Data Fig. 3b. f, Sizes of stem cell clones (means ± S.D; p-values, Mann-Whitney test) after induction of enterocyte p35 and E-cadRNAi, without or with AG1478. EGFR inhibition suppresses stem cell divisions. N=4–5 midguts per condition. g, Total cell counts (means ± S.D; p-values, unpaired t-test compared to control). Midgut hyperplasia (mex^ts>p35, E-cadRNAi) requires EGFR and enterocyte spi and krn. N=4 midguts per genotype or condition. See Extended Data Fig. 3a. h–k, The spatial distribution of dpERK cells around a single, E-cadRNAi enterocyte distinguishes direct and dispersed activation mechanisms (h). dpERK⁺ cells are both directly adjacent to and dispersed from GFP-marked, E-cadRNAi enterocytes (i), consistent with a dispersed mechanism. dpERK⁺ cells are infrequent near marked control enterocytes (j). Distribution of distances between dpERK⁺ cells and E-cadRNAi enterocytes (k, n=53 dpERK⁺ cells; N=4 midguts, 5 day induction). Green bar represents dpERK⁺ cells directly adjacent to enterocytes (0 µm). One of three replicate experiments shown in each graph. All scale bars, 25 µm.

Fig. 4. Enterocyte apoptosis activates stem cell division by disrupting E-cad-controlled inhibition of rhomboid

a, qPCR of whole midgut mRNAs following enterocyte-specific E-cad manipulation. E-cad represses rho but not EGFs (vn, spi, krn) or other EGF-related factors (egfr, aos, star). Red line indicates control. Means ± S.D. of 3 independent experiments. b, c, Enterocyte rho is required for ERK hyperactivation following depletion of enterocyte E-cad. See Extended Data Fig. 3b. d–f, rho-lacZ induction during physiological apoptosis. Immunostains for β-gal and activated Caspase-3 (Cas-3) mark identical enterocytes. Images show different fields in planar (d) and vertical (e) sections; dotted line in e indicates basal. Quantitation of Cas-3⁺, rho-lacZ⁺ enterocytes (f, means ± S.D). n=188 enterocytes from 3 experiments; N=3–4 midguts (6 days post-eclosion) per experiment. g–j, ERK activation is suppressed by apoptotic inhibition and depends on E-cad and rho. See Extended Data Fig. 3b. k, Total cell counts (means ± S.D; p values, unpaired t-test compared to control). Hyperplasia of mex^ts>p35, E-cadRNAi midguts requires rho, arm, and p120. Hyperplasia is induced by either rho or p120 alone. N=4 midguts per genotype. One of three replicate experiments shown. See Extended Data Fig. 3a. l, Model for steady-state equilibrium of division and death. All scale bars, 25 µm.