Re-entry into mitosis and regeneration of intestinal stem cells through enteroblast dedifferentiation in Drosophila midguts

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.

ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to 23 generate ISCs under physiological conditions remains unknown. Here we show that infection of 24 pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the 25 mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal 26 growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in 27 EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined 28 whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell 29 marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage 30 tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs 31 after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new 32 role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during 33 regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation 34 6 arrows, K; 5.5 PH3/gut, n=12) and significant increase of ISC mitosis (PH3 + GFP ─ , Figure  with PH3 (Figure 1─figure supplement 1). 117

EGFR-Ras signaling in EBs is up-regulated upon bacterial infection. 118
Bacterial infection activates signaling pathways such as EGFR-Ras pathway through up-119 regulation of ligands (Jiang et al., 2011) to induce ISC proliferation. To assess the expression 120 level of ligands of the EGFR-Ras pathway, we performed qRT-PCR analysis and confirmed that 121 P. e infection upregulated expression of the ligand Vn and Krn (Figure 2A). To determine whether 122 EGFR-Ras signaling was up-regulated in EBs upon P. e infection, we examined the activity of 123 mitogen-activated protein kinase (MAPK), which marks the activation of EGFR-Ras signaling, by 124 using an antibody against the diphosphorylated and active form of MAPK (dpERK) (Gabay et al., 125 1997). We found that the dpErk level was high in GFP ─ ISCs ( Figure 2B

EGFR-Ras signaling in EBs is required for bacterial infection induced EB mitosis. 131
The up-regulation of EGFR-Ras signaling in EBs by P. e infection led us to hypothesize that 132 activation of EGFR-Ras signaling in EBs may induce EB mitosis. To test this, we activated EGFR-133 Ras signaling in EBs and examined the expression of PH3. The inducible Gal4/Gal80 ts system 134 with EB specific Gal4 (Su(H)-Gal4 Tub-Gal80 ts ; referred to as Su(H) ts ) was used to were raised to adults at 18℃ (Gal4 is 'off') and then shifted to 29℃ to degrade Gal80 ts (Gal4 is 149 'on') for indicated days so that Su(H)-Gal4 can drive expression of UAS-gene. Our assay with 150 expression of Ras V12 in EBs found PH3 in GFP + EBs, indicating that activation of Ras in EBs 151 induced cell-autonomous EB mitosis (GFP + PH3 + , Figure 3C, E, E', L, red arrows; 12.7 PH3/gut 152 in Ras V12 vs. 0 PH3/gut in the control, n=13). In addition, we found that activation of Ras in EBs 153 non-cell-autonomously promote ISC proliferation (PH3 + GFP ─ , Figure 3C  To confirm that these GFP + EBs did not gain ISC identity as a prerequisite to divide, we 166 performed the immunostaining with ISC marker (Delta, Dl) and found that these PH3 + ISCs 167 without GFP did have Dl expression ( Figure 3N-O', P-Q'), but these EBs marked by Su(H)>GFP 168 with PH3 did not have Dl expression ( Figure 3P, R-R', 100%, n=42), suggesting that these dividing 169 EBs do not gain ISC identity. In summary, activation of EGFR-Ras signaling in EBs induces cell-170 autonomous re-entry into mitosis in EBs and non-cell-autonomously promotes ISC proliferation. 171 To determine whether EGFR-Ras signaling in EBs is necessary for P. e infection induced re-  were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test).

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The observation that P.e infection induces EB dedifferentiation suggests that activation of 247 EGFR−Ras signaling in EBs may induce EB dedifferentiation. Indeed, after we performed the 248 lineage tracing experiments with the same Flip-out system and overexpression of UAS-Ras V12 in 249 EBs for 6d, we found LacZ + EB cells with GFP (GFP + LacZ + , Figure 4F-F''', arrows) and LacZ + 250 cells without GFP (GFP − LacZ + , Figure 4F-F''', red arrowheads). In these GFP ─ LacZ + cells, some 251 showed Dl expression (GFP ─ LacZ + Dl + ) ( Figure 4F-F''', red and blue arrowheads, and 4G), 252 indicating that activation of EGFR-Ras signaling in EBs can induce EB dedifferentiation to 253 generate ISCs. 254 To determine whether P. e infection induces EB dedifferentiation through EGFR-Ras signaling. 255 We inactivated the EGFR-Ras signaling in EBs by knocking down EGFR with the Flp-out system 256 and examined whether the new regenerated ISCs were blocked upon P.e infection. We found 257 that the total number of regenerated ISCs from EBs was reduced with EGFR knockdown in 258 response to P. e infection (total number of GFP ─ LacZ + Dl + cells in five guts, 28.5 in P. e infection 259 vs. 11.3 in EGFR-RNAi with P. e infection, Figure 4H). 260 Our next question is whether these new Dl + stem cells from EB dedifferentiation are functional. 261 To test this, differentiation of ISCs into EE cells marked by expression of Prospero (Pros) was 262 used as a readout for functional ISCs. After activating EGFR-Ras signaling in EBs with Ras V12 or 263 EGFR A887T expression for 6d or P.e infection for 36h, we found that some LacZ expressed cells 264 without GFP have gained Pros expression ( Figure 5A-D), indicating that these ISCs from EB 265 dedifferentiation are functional so that mature EE cells are generated. 266

The symmetric division of EBs produces two ISCs upon activation of EGFR-Ras signaling 267 in EBs. 268
Our results show that EBs can re-enter mitosis and ISCs are generated from EBs, and the next 269 question is whether EBs generate ISCs after mitosis or EBs gain stem cell identity before 270 mitosis. Firstly, we excluded the possibility that these dividing EBs have gained ISC property 271 marked by Dl expression (Figure 3P, R-R'). Secondly, we compared mitosis in EBs to that in 272   Previous studies found that only resident ISCs in Drosophila midguts localized at the basal side 323 of the gut epithelium undergo asymmetric cell division to produce renewed ISCs and EBs 324 (Micchelli and Perrimon, 2006;Ohlstein and Spradling, 2006). Our studies showed that EBs can 325 dedifferentiate to generate ISCs (Figure 7). There are two possibilities about the process of 326 dedifferentiation with mitosis. One could be that EBs directly revert to ISCs, like induced 327 pluripotent stem cells (Takahashi and Yamanaka, 2006), and then start mitosis. The other one 328 could be that EBs re-enter mitosis, and then dedifferentiate to produce ISCs. Our results from 329 immunostaining with Dl support the latter, and the two-color lineage tracing experiments further 330 demonstrate that two regenerated ISCs are generated from one division of EBs. This process of 331 dedifferentiation is distinct from amitosis, another dedifferentiation process in the intestine, which 332 was found in re-fed condition in starved Drosophila midguts to produce ISCs through reduction of 333 ploidy (Lucchetta and Ohlstein, 2017). Therefore, activating EGFR-Ras signaling in EBs firstly 334 enforces these cells to re-enter the mitotic cycle, and then both EGFR-Ras signaling and mitosis 335 may play a role in the fate determination towards ISCs. As dedifferentiation has emerged as a 336 conserved mechanism underlying the replenishment of stem cell pool, dedifferentiation through 337 mitosis could be a conserved mechanism from Drosophila to mammals. 338 Infection of two Gram-negative bacteria induces the same EB mitosis phenotype, indicating 339 that the immunity signaling pathways might be involved in EB mitosis and dedifferentiation. The    Quantification of PH3 + in ISCs (GFP ─ ) and EBs (GFP + ). n = 12 guts for each genotype. Three 586 independent experiments were performed, and error bars ± SEM. ***, P < 0.001 (Student's t-test). 587