The activity of Drosophila MAPK was assayed by anti-dpERK staining. A, A’. MAPK activity in the mock-infected control midgut. B, B’. MAPK activity after infecting with Pe for 1 day. ISCs and EBs were marked by esgGal4-driven GFP expression and indicated by arrowheads and arrows respectively (A, B). C, C’. MAPK activity after infecting with Pe for 2 days. Differentiating ECs (pre-ECs, medium nucleus) and newly formed mature ECs (large nucleus) were indicated by arrowheads and arrows respectively. D, D’. MAPK activation induced by ectopic expression of sSpi (MyoIA^ts>sSpi). E, E’. Cell autonomous MAPK activation induced by activated Ras (esg^tsF/O>Ras^V12).

A-D. MARCM analysis of ISC clones. Wildtype (A) and mutant ISC clones (B-D) were induced using the MARCM system, and examined 8 days later. The number of cells in each clone were indicated. E. Quantification of ISC clone sizes. The number of clones counted for each genotype were indicated inside each bar. F. Quantification of progenitor cells in the posterior midguts of GFP and EGFR knockdown. Progenitor cells (esg⁺) were indicated by diamonds, EBs (esg+, Su(H)⁺) were indicated by squares and presumed ISCs (esg⁺, Su(H)⁻) were indicated by triangles. Filled symbols, esg^ts>GFP; Open symbols, esg^ts>EGFR RNAi. G-J. Midgut epithelium turnover assay. EGFR suppression inhibited midgut turnover (H, esg^tsF/O>Egfr RNAi). Furthermore, GFP⁺ progenitor cells were depleted after long-term EGFR knockdown (I). In control midgut, GFP were present in both progenitors and large polyploid cells (likely ECs) after 2 weeks (J, esg^tsF/O>GFP). K. Quantification of compensatory ISC proliferation induced by Pe infection. EGFR signaling was suppressed in the progenitor cells by esg^tsF/O-driven Egfr or Raf RNAi. L-O. Midgut turnover in mock (L, M) or Pe-infected (N, O) animals. Midgut turnover was assayed using the esg^tsF/O system. P, Q. Quantification of compensatory ISC proliferation in spi, vn and Krn mutants. We used viable Krn null mutant (Krn^27-7-B), lethal spi null mutant (spi^A14, in a heterozygous background), spi RNAi knockdown in progenitors (esg^ts>spi IR) or ECs (MyoIA^ts>spi IR), or vn RNAi knockdown in VMs (24B^ts>vn IR). IR, inverted repeats.

A. ISC proliferation induced by EGFR and Jak/Stat signaling. With the exception of co-expressing sKrn and Upd in the ECs (MyoIA^ts>Upd + sKrn), all the other ectopic expression experiments were performed using the esg^tsF/O driver. Midgut mitotic indices (PH3⁺) were quantified after activating the transgenes for 2 days. B-J. ISC clonal assay. GFP-marked ISC clones were induced using the MARCM system and analyzed 4 or 8 days later. The sizes of the ISC clones were indicated. Vn-induced ISC proliferation is dependent on Jak/Stat signaling (B-D). Activated Ras (Ras^V12)-induced ISC proliferation is independent of Jak/Stat signaling (F-G). Some EB clones overexpressing Ras^V12 underwent extra round of endoreplication (E). Upd-induced ISC proliferation is dependent on EGFR signaling (H-J). K. Quantification of ISC clone sizes. The sizes of ISC clones were measured 4 or 8 days after clone induction (ACI) using the MARCM system. L. RT-qPCR analysis of the induction of Jak/Stat and EGFR signalings by Pe infection in the absence of either pathway (esg^tsF/O>Stat or Egfr RNAi).

A. RT-qPCR quantification of Drosophila EGFR ligands (vn, spi and Krn) and MKP3 (MAP kinase phosphatase-3) mRNA expression in the regenerating midgut. The midgut was induced to regenerate by activating the JNK pathway in the ECs (MyoIA^ts>Hep^Act, 24hrs or puc RNAi, 72hrs), or inducing EC apoptosis (MyoIA^ts>Rpr, 24hrs), or Pe infection (48hrs). STDEV and P-value (t-test) were shown. B-E. Expression of vn-lacZ reporter in control (B) or regenerating posterior midguts (C-E). 2 of the 4 rows of circular visceral muscle cells (VM) were shown. F, G. vn fluorescent in situ hybridization. The strongest vn signals were in the nucleus (arrows) of VMs (asterisks), most likely the loci of Vn transcription. H, I. Krn fluorescent in situ hybridization. The strongest Krn signals were in the nucleus of ECs (arrows). In mock-infected control midguts, vn and Krn were expressed at low levels in the VM and ECs respectively (F, H).

A. Ectopic ISC proliferation induced by Vn. Vn was induced in the midgut using the inducible VM-specific driver, 24B^ts. B. ISC proliferation induced by activated EGFR signaling. Transgenes were induced in the midgut for 2 days using the esg^tsF/O or MyoIA^ts system. Midguts were scored for PH3⁺ mitotic figures in both A and B. C-E. Adult midgut growth measured using the esg^tsF/O system. Both sSpi (D) and λTOP (E) promoted significant new midgut cell formation. F-F”. Ras^V12 also promoted the formation of new mature midgut cells. Most of the newly formed large polyploid midgut cells (GFP⁺, arrows) were positive for mature EC marker, PDM-1.

A. Activating EGFR signaling induced Jak/Stat signaling in the midgut. The expression levels of Drosophila cytokines (upds) and downstream target gene, Socs36E, in the midgut were analyzed by RT-qPCR. B. Induction of vn expression in the midgut by Jak/Stat signaling as quantified by RT-qPCR. Jak/Stat signaling was activated in the VM by ectopic expression of Upd in the ECs (MyoIA^ts>Upd) or Hop directly in the VM (24B^ts>Hop). C, D. Induction of the upd-lacZ reporter in the midgut epithelium by activated Ras (esg^tsF/O>Ras^V12, D). E, F. Induction of the Upd3.1-lacZ reporter in ECs by activated Ras (MyoIA^ts>Ras^V12, F). G, H. Induction of the vn-lacZ reporter in the VM by ectopic expression of Upd (MyoIA^ts>Upd, H).

Stressed or dying ECs induce the expression of fly cytokines (such as Upd3 and Upd2) and EGFs (such as Krn and Vn) in the midgut, which activate the Jak/Stat and EGFR pathways in the midgut progenitor cells. While EGFR signaling functions mainly to promote ISC proliferation, Jak/Stat signaling functions to promote both ISC proliferation and EB differentiation.