Warts and Yorkie mediate intestinal regeneration by influencing stem cell proliferation

Abstract

Homeostasis in the Drosophila midgut is maintained by stem cells [1, 2]. The intestinal epithelium contains two types of differentiated cells that are lost and replenished: enteroendocrine (EE) cells and enterocytes (ECs). Intestinal stem cells (ISCs) are the only cells in the adult midgut that proliferate [3, 4], and ISC divisions give rise to an ISC and an enteroblast (EB), which differentiates into an EC or an EE cell [3-5]. If the midgut epithelium is damaged, then ISC proliferation increases [6-12]. Damaged ECs express secreted ligands (Unpaired proteins) that activate Jak-Stat signaling in ISCs and EBs to promote their proliferation and differentiation [7, 9, 13, 14]. We show that the Hippo pathway components Warts and Yorkie mediate a transition from low- to high-level ISC proliferation to facilitate regeneration. The Hippo pathway regulates growth in diverse organisms and has been linked to cancer [15, 16]. Yorkie is activated in ECs in response to tissue damage or activation of the damage-sensing Jnk pathway. Activation of Yorkie promotes expression of unpaired genes and triggers a nonautonomous increase in ISC proliferation. Our observations uncover a role for Hippo pathway components in regulating stem cell proliferation and intestinal regeneration.

Fig. 1. Yki and Wts acts in ECs to influence ISC proliferation

A) Schematic of cell types in the intestine. ISCs divide (red arrows) asymmetrically to an EB and an ISC. EBs then differentiate (black arrows) into ECs or EEs. Cell-type specific markers are indicated in green. B–P) Show portions of the adult posterior midgut, stained for DNA (blue, using DAPI), esg (green, using esg-lacZ, or esg-Gal4 and UAS-GFP), Arm (red), Actin (green, using phalloidin), pH3 (green), EdU (red), or Dl (red/white), as indicated. Panels marked prime show individual channels of the stain to the left. B) Control (without Gal4) Tub-Gal80^ts UAS-wts-RNAi after temperature shift induction for 7 days. C) Tub-Gal80^ts Tub-Gal4 UAS-dcr2 UAS-wts-RNAi after 7 days induction. D) esg-Gal4 tub-Gal80^ts UAS-yki^S168A:V5 UAS-GFP, 10 days after induction. E) esg-Gal4 tub-Gal80^ts UAS-GFP control, 10 days after induction. F) MyoIa-Gal4 tub-Gal80^ts esg-lacZ control, 2 days after induction. G) MyoIa-Gal4 tub-Gal80^ts control, 2 days after induction; image shows an optical cross-section through the center of the intestine. H) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5, 2 days after induction; image shows an optical cross-section through the center of the intestine. I) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5 esg-lacZ, 2 days after induction. J) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5, 5 days after induction. K, L) MyoIa-Gal4 tub-Gal80^ts UAS-GFP wts RNAi UAS-dcr2, 6 days after induction. K shows a projection through several horizontal sections. L shows a vertical section, yellow arrows point to Dl-expressing cells basal to MyoIa-Gal4 expression. M) MyoIa-Gal4 tub-Gal80^ts control, 11 days after induction, stained and labeled for EdU. N) MyoIa-Gal4UAS-dcr2 tub-Gal80^ts control, 6 days after induction, stained and labeled for EdU and pH3. O) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5, 3 days after induction. P) MyoIa-Gal4UAS-dcr2 wtsRNAi tub-Gal80^ts, 6 days after induction, stained and labeled for EdU and pH3. White arrows point to some examples of pH3-stained mitotic cells. Q) Quantitation of pH3-stained mitotic cells in posterior midguts of the indicated genotypes, error bars indicate sem. See also Supplementary Figure S1.

Fig. 2. Non-autonomous influence of Yki on ISC proliferation

Portions of the adult posterior midgut, stained for DNA (blue, using DAPI), esg (green, using esg-lacZ), pH3 (magenta), EdU (red), or Dl (red/white), as indicated. Panels marked prime show separate channels of the stain to the left. A) Tub-Gal4 MARCM control clones, 7 days after induction. B) Tub-Gal4 UAS-yki^S168A:V5 MARCM clones, 7 days after induction. C) Tub-Gal4 UAS-yki^S168A:V5 Flp-out clones, 2 days after induction, cf. Fig 1F. D) wts^X1 mutant clones (marked by expression of GFP, green), 8 days after clone induction, stained for DNA, Dl and pH3. D' shows Dl expression relative to clones, D" shows pH3 staining relative to the clones. Regions devoid of clones (yellow asterisk) do not have elevated Dl or pH3 staining. E) Tub-Gal4 MARCM control clones, 10 days after induction, stained and labeled for EdU. Clones (marked by GFP, green) are small, and EdU labeling (red) is rare. Arrow points to an example of an EdU labeled nucleus. F) Tub-Gal4 UAS-yki^S168A:V5 MARCM clones, 10 days after induction, stained and labeled for EdU. Clones (green) are larger. Regions devoid of clones (yellow asterisk) do not have elevated EdU labeling. See also Figure S2.

Fig. 3. Yki regulates the Jak-Stat pathway

Portions of the adult posterior midgut, stained for DNA (blue, DAPI), upd-lacZ (red) Stat-DGFP (green), Dl (red), pH3 (green) or Arm (red), as indicated. Experimental and control images were stained in parallel and captured with identical confocal settings. A) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5 upd-lacZ, 2 days after induction. B) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5 upd-lacZ control (maintained at 18°C). C) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5 10X-Stat-DGFP 1 day after induction. D) MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5 10X-Stat-DGFP control (maintained at 18°C). E–G) examples of E) wts^X1, F) wts^X1 stat92E⁰⁶⁴³⁶, and G) control MARCM clones (marked by expression of GFP, green), 8 days after clone induction, stained for Dl. H) Quantitation of average clones size for clones of the indicated genotypes (wt= wild type control), error bars show sem. I–K) Examples of I) wts RNAi, J) hop RNAi, K) hop; wts RNAi, 7 days after induction by temperature shift, stained and labeled for Dl and pH3. L) Fold change in RNA levels of upd genes, by quantitative RT-PCR, between MyoIa-Gal4 tub-Gal80^ts UAS-yki^S168A:V5 and MyoIa-Gal4 tub-Gal80^ts (control), 1 day after temperature shift induction, normalized to RpL32, error bars indicate standard deviation. See also Figure S3.

Fig. 4. Yki activity is regulated by damage response pathways

Portions of the adult posterior midgut, stained for DNA (blue, DAPI), Yki (green), Dl (red/white), pH3 (red), ex-lacZ (magenta), or Arm (red), as indicated. A) Bleomycin treatment for 2 days increases Dl-expressing cells. B) MyoIa-Gal4 tub-Gal80^ts UAS-RNAi-yki UAS-dcr2 with induction of yki RNAi for 7 days and bleomycin treatment for 2 days. C) Quantitation of pH3 mitoses per posterior midgut in animals treated and stained as in D, E. Error bars indicate sem. D) Bleomycin treatment for 2 days increases pH3-staining mitoses cells. E) MyoIa-Gal4 tub-Gal80^ts UAS-RNAi-yki UAS-dcr2 with induction of yki RNAi for 10 days and bleomycin treatment for 2 days, stained for pH3. F) ex-lacZ flies without bleomycin treatment. G) ex-lacZ flies with 2 days bleomycin treatment, stained and imaged under the same conditions as F; quantitation of staining intensity identified a 2.4 fold increase in ex-lacZ relative to controls. H) Bleomycin treatment for 2 days results in partial translocation of Yki into the nucleus. I) MyoIa-Gal4 tub-Gal80^ts control flies without Bleomycin treatment, Yki is cytoplasmic. J) Flip-out tub-Gal4 UAS-hep^CA clones; flies were kept at 18C for 5 days after clone induction and then shifted to 29C 24 h before dissection. Dashed line outlines a clone. K) MyoIa-Gal4 tub-Gal80^ts UAS-hep^CA, 1 day after induction. See also Figure S4.