Abstract
A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the etiology of chronic disorders such as inflammatory bowel diseases and cancer1. We used the Drosophila midgut2 to investigate this and discovered that during regeneration a subpopulation of cholinergic3 neurons triggers Ca2+ currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. We found that down-regulation of the conserved cholinergic enzyme Acetylcholinesterase4 in the gut epithelium enables acetylcholine from specific TNF/Egr5-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca2+ that spreads in the epithelium through Inx2/Inx7 gap junctions6, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki/Yap activation7, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel diseases8. Altogether, the conserved cholinergic pathway facilitates epithelial currents that heal the intestinal epithelium. Our findings demonstrate nerve- and bioelectric9-dependent intestinal regeneration and advance our current understanding of how a tissue returns to homeostasis after injury.
The cholinergic pathway is an ancient conserved pathway used by peripheral neurons to communicate with internal organs3. The two cholinergic receptors, nicotinic and muscarinic, and enzymes modulating acetylcholine (ACh) metabolism, e.g. Acetylcholinesterase (AChE/Ace), are highly conserved and expressed in non-neuronal tissues3. Cholinergic receptors regulate ion transport in the intestinal epithelium which is vital for water and nutrient absorption10. Recently, attention has been given to the anti-inflammatory properties of the cholinergic pathway, with reduced ACh responsiveness associated with intestinal diseases11.
The Drosophila midgut, equivalent to mammalian small intestine, has been used to identify conserved molecular pathways that trigger inflammation and regeneration in the injured epithelium1,12. The midgut epithelium is single-layered and comprised of enterocytes (ECs), large polyploid epithelial cells specialized in absorption, secretory enteroendocrine cells (EEs) and progenitor cells (PCs)2,13,14. The visceral muscle and trachea surround the midgut epithelium, whereas anterior and posterior midgut regions are innervated by enteric neurons2. When the epithelium is injured, intestinal stem cells (ISCs) divide rapidly, giving rise to daughter cells (EBs) that differentiate into ECs and EEs2. Depending on the injury or infection, a multifaceted interplay of conserved inflammatory and regenerative pathways (e.g., EGFR, JAK-STAT, Wnt, BMP, Yki/Yap) activate ISC proliferation so that a sufficient PC (ISC/EB) pool replenishes the epithelium15. Despite the in-depth understanding of how repair is triggered, it is unclear how these pathways are dampened once the epithelium transitions to homeostasis. Following specific damage, the BMP pathway has been reported to have dual roles first promoting proliferation and later ISC-quiescence16,17.
Here, we provide evidence for the fundamental role of an epithelial bioelectric mechanism controlled by cholinergic neurons that occurs as the midgut transitions from colitis-like injury to homeostasis – a phase we refer to as recovery. We show that during recovery, ECs become more sensitive to ACh by downregulating Ace and upregulating nAChR subunit β3 (nAChRβ3). Also, specific TNF/Egr-regulated cholinergic neurons, that we refer to as ARCENs, strengthen the axonal properties of their enteric innervations. We demonstrate that transition to homeostasis relies on the healing functions of nAChR-mediated Ca2+ currents among ECs, that spread through Inx2/Inx7 gap junctions and are triggered by local ARCEN-EC cholinergic signaling.
ECs are sensitive to ACh during recovery
To study the Drosophila intestinal epithelium while it transitions to homeostasis after injury, we damaged the gut with dextran sodium sulfate (DSS), then returned flies to standard food. DSS induces colitis in mammals8 and has been used in Drosophila to identify conserved proliferative pathways15. We fed flies DSS for 4 days (injury) followed by two or four days of standard food (recovery, Fig.1a). Gut damage elevated the expression of effector Drosophila caspase 1 (Dcp1), indicative of cell death (Fig.1b), and of conserved inflammatory cytokines such as IL6-like unpaired 3 (upd3)18 and TNF homolog eiger (egr)5 (Ext. Data Fig.1a), resembling DSS-induced colitis8. Once flies were transferred to standard food, the epithelium required 4 days to return to homeostasis as determined by the levels of i) inflammatory cytokines (Ext. Data Fig.1a), ii) cell death (Fig.1b), iii) ISC proliferation (with mitotic marker anti-phospho-Histone3 (pH3), Fig. 1c), and iv) expression of PC marker escargot (esg; Ext. Data Fig.1b) and two markers of mature cells, pdm1, marker of ECs, and prospero (pros), marker of EEs (Ext. Data Fig.1b).
To search for recovery-specific differentially expressed genes, we performed snRNAseq on the 2nd day of recovery (Ext. Data Fig.1c). We identified 14 clusters from 8073 nuclei recovered, which we assigned to different epithelial and progenitor cell populations, as well as to cardia and LFC/Cu/Fe gut cells (Ext. Data Fig.1c-e). We analyzed differential gene expression between homeostasis and recovery and observed that Ace, is highly enriched in ECs and significantly downregulated during recovery (Ext. Data Fig.1f, Ext. Data Fig.2a-b, S. Table 1-2). Ace hydrolyzes ACh to choline and acetate and thus defines a cell’s sensitivity to ACh.
We observed ~75% reduction of Ace during recovery (Fig.1d). Previous intestinal RNA-seq profiling after bacterial infections also detected Ace downregulation (Flygut-EPFL data)19, which we confirmed (Ext. Data Fig.2c). Next, we challenged the gut with injury-inducing chemical Bleomycin which triggers different pathways as compared with DSS20. This led to significant Ace decrease (Ext. Data Fig.2c), indicating that Ace downregulation occurs consistently after different types of intestinal epithelial damage.
To test the role of Ace during regeneration, we used CRISPR/Cas9 activation (Ext. Data Fig.2d). We found that 4 days of Ace overactivation in ECs (using the Gal4 myo1A-driver together with the repressor Tubulin-Gal80TS, referred as myo1ATS) led to excessive ISC proliferation during recovery (Fig.1e), while same activation during homeostasis or injury did not affect proliferation (Fig.1e). Similarly, consecutive DSS challenges while Ace is conditionally overexpressed in ECs using the myo1ATS or mexTS driver (another EC driver) led to recovery-specific ISC over-proliferation (Fig.1f, Ext. Data Fig.2e). We next tested if Ace perturbations in visceral muscle or immune cells regulate ISC proliferation, which they did not (Ext. Data Fig.2f). These findings reveal that overexpressing Ace in ECs during recovery prevents ISCs from becoming quiescent, causing an excessive regenerative response. We next tested whether the role of Ace during recovery is consistent after different types of epithelial damage. Overexpressing Ace in ECs after Ecc15 infection or Bleomycin-injury consistently caused over-proliferation (Ext. Data Fig.2g-h).
ACh has been proposed to modulate ion transport in the intestinal epithelium in a Ca2+-dependent manner10. Thus, to test for epithelial changes in ACh sensitivity during homeostasis and recovery, we visualized Ca2+ by conditionally expressing the Ca2+ indicator GCAMP7c21 in ECs. Using ex vivo live imaging, we found that Ca2+ levels in ECs are significantly higher during recovery following ACh administration than during homeostasis and that this is attenuated upon overexpression of Ace (Fig.1g, Ext. Data Fig.2i and Videos S1-S3). These results indicate that during recovery ECs become more sensitive to ACh by decreasing Ace and this change is required for transition to homeostasis after injury.
nAChRβ3 is required in ECs for recovery
Cholinergic receptors are G-protein-coupled muscarinic receptors (mAchR) or ligand-gated ion channel nicotinic receptors (nAChR) made of five homomeric or heteromeric subunits (α1, α2, α3, α4, α5, α6, α7, β1, β2 or β3). To identify which cholinergic receptor becomes activated during recovery, we screened all nAChR subunits and different mAchR subtypes by knocking down their expression in ECs. Conditional RNAi expression targeting nAChRβ3 in ECs caused over-proliferation during recovery (Fig.2a-b, Ext. Data Fig.3a-c) and after Ecc15 infection (Ext. Data Fig.3d). Furthermore, nAChRβ3 knockdown in ECs combined with repeated DSS-injury (Recovery 2x) led to hyperplasia (Fig.2b-c). Conditional knockdown of nAChRβ3 in ECs significantly reduced the Ca2+ response after ACh administration during recovery (Ext. Data Fig.3e, Ext. Data Fig.3j). Together, these data suggest that reducing nAChRβ3 in ECs leads to phenotypes resembling Ace upregulation. This effect is specific to ECs, as conditionally knocking down nAChRβ3 in PCs, EBs alone, EEs, visceral muscle or hemocytes had no effect on proliferation (Ext. Data Fig.3f).
The profiling depth of our snRNAseq was not sufficient to conclude if nAChRβ3 expression is altered between homeostasis and recovery, despite being solely found in ECs (Ext. Data Fig.3g). To visualize the expression of nAChRβ3, we inserted a Flag tag within nAChRβ3 (nAChRβ3-flag) (Fig.2d, Ext. Data Fig.3h). Endogenous nAChRβ3 was significantly enriched in ECs by day 2 of recovery (Fig.2d), whereas a decrease in nAChRβ3 levels coincided with return to homeostasis (Ext. Data Fig.3i). nAChRβ3 was localized to the basal side of ECs and some ECs had more nAChRβ3 clustered on their basal side (Fig.2d-e).
Next, we administered the cholinergic agonist nicotine, which activates nAChRs and cannot be hydrolyzed by Ace. Nicotine administration significantly increased Ca2+ in ECs during recovery compared to homeostasis, reminiscent of ACh-sensitivity, and this was diminished when nAChRβ3 was knocked down (Fig.2f-g, Ext. Data Fig.3k and Videos S4-S6). We conclude that nAChRβ3 in ECs is essential for gut recovery and recovery-specific enrichment of nAChRβ3 provides an additional level of regulation that likely renders ECs more responsive to ACh while the epithelium transitions to homeostasis.
nAChRβ3-mediated Ca2+ promotes recovery
ISC proliferation can be triggered by the release of cytokines which vary depending on the stimulus15. To identify pathways responsible for unrestrained proliferation during recovery after nAChRβ3 knockdown in ECs, we tested the expression of known cytokines (Fig.3a, Ext. Data Fig.4a-b). We detected unpaired 2 (upd2) and upd3 JAK-STAT ligands, together with EGF-like ligand vein (vn) and egr, to be significantly upregulated (Fig.3a, Ext. Data Fig.4a-b). Egr is associated with cell death22, whereas Upd2, Upd3 and Vn are up-regulated on activation of the Hippo pathway effector Yorkie (Yki/YAP) in damaged ECs23,24. Supporting this, knocking down nAChRβ3 in ECs during recovery significantly increased cell death (Ext. Data Fig.4c) as well as expression of Yki targets Diap1 and Ex (Ext. Data Fig.4d-e). Also, knockdown of nAChRβ3 during recovery significantly reduced the transcript and protein levels of pdm1 (Fig. 3b, Ext. Data Fig.4f-g), while pros remained unchanged (Ext. Data Fig.4h). These data suggest that disruption of nAChRs in ECs during recovery impairs ECs, leading to cell death, Yki activation and subsequent production of inflammatory signals that induce unwarranted ISC proliferation.
Cholinergic receptors regulate ion transport in the mammalian epithelium10,25. We asked whether nAChRs have similar functions in ECs using dyes that detect Na+ (SodiumGreen) or Cl− (MQAE), and the Ca2+ transcriptional reporter NFAT-CalexA26. Reduction of nAChRβ3 during recovery caused significant ion imbalance in the epithelium, with reduced Cl− and Na+ levels (Ext. Data Fig.4i). We observed that Ca2+ is significantly upregulated the first day of recovery before returning to levels resembling homeostasis (Fig.3c). This endogenous Ca2+ increase disappears when nAChRβ3 is knocked down in ECs (Fig.3d). Also, Ca2+ increase occurs only in ECs during recovery, as ISCs that use Ca2+ for proliferation27,28 show Ca2+ decline during recovery (Ext. Data Fig.4j). To examine the importance of nAChR-mediated Ca2+ during recovery, we genetically compensated for Ca2+ in nAChRβ3-deficient ECs. Conditional overexpression of Ca2+ channel Orai combined with knockdown of nAChRβ3 in ECs was sufficient to restore i) ISC proliferation (Fig.3e, Ext. Data Fig.5a), ii) the number of pdm1+ ECs (Fig.3f, Ext. Data Fig.5b), and iii) Cl− (Ext. Data Fig.5c) to levels identical to controls. Next, we over-expressed the vertebrate Ca2+ buffer protein parvalbumin (PV) in ECs to reduce the amount of intracellular Ca2+ for four days during recovery (Ext. Data Fig.5d). This led to over-proliferation during recovery while having no effect during homeostasis (Ext. Data Fig.5d), supporting the importance of Ca2+ in ECs during recovery.
To further study the effect of nAChRβ3 during gut regeneration, we generated flies that overexpress nAChRβ3 in ECs using the Gal4 or LexA system (UAS- nAChRβ3 and LexAop-nAChRβ3, Ext. Data Fig.5e-f). Also, we generated an EC-LexA driver (mex-LexA::GAD) together with Tubulin-Gal80TS, referred as mexLexATS (Ext. Data Fig.5f). Conditionally overexpressing nAChRβ3 in ECs during homeostasis doubles the amount of Ca2+ in ECs after nicotine administration (Ext. Data Fig.5g). Also, nAChRβ3 overexpression in ECs significantly expedited recovery, with ISC proliferation and pdm1 expression reaching levels indistinguishable from unchallenged guts at two days, half the expected time (Fig.3g-h). nAChRβ3 overexpression in ECs during homeostasis and injury did not change ISC proliferation (Fig.3g). Additionally, overexpressing nAChRβ3 in ECs during recovery significantly reduced inflammatory cytokine levels and cell death (Fig.3i, Ext. Data Fig.5h-j).
Together, our data show that high intracellular Ca2+ in ECs triggered by nAChRs control intestinal epithelium recovery by promoting EC maturation and ion balance. Disruption of nAChR-mediated Ca2+ causes EC deficiency, ion imbalance, elevated cell death and Yki activation, followed by over-inflammation and over-proliferation. In contrast, increasing Ca2+ in ECs via overexpressing nAChRβ3 expedites return to homeostasis by advancing EC maturation and decreasing cell death, subsequently reducing inflammation and proliferation.
Neuro-EC interactions promote recovery
ACh is released by neuronal and non-neuronal cells that express ChAT (Choline acetyltransferase), the enzyme that catalyzes ACh synthesis3. To identify the source of ACh responsible for nAChR-mediated recovery, we first tested midgut cells (PCs, ECs, EEs), the visceral muscle and immune cells (hemocytes). ISC proliferation during recovery remained unaffected when ChAT was conditionally knocked down in these cells (Ext. Data Fig.6a). Similarly, EE-less guts29 do not over-proliferate during recovery (Ext. Data Fig.6b). Altogether, these data point to a neuronal source of ACh. The importance of neurons during regeneration has been reported in different tissues30. For the gut, studies have highlighted the anti-inflammatory properties of mammalian enteric neurons31,32, while limited associations have been made between neurons and ISC proliferation in Drosophila33,34.
Since ACh is a short-distance neurotransmitter/local neurohormone, the most likely source during recovery is the enteric nervous system. Drosophila enteric neurons innervate the midgut in anterior (R1, R2) and posterior regions (R4, R5), even though their cell bodies reside in the brain, hypocerebral ganglion (HCG), or the adult ventral nerve cord (VNC)2. Also, enteric innervations reach the intestinal epithelium34. To identify a driver for cholinergic enteric neurons, we screened a set of neuronal drivers (FlyLight)35. We identified a Gal4 driver, R49E06-Gal4, that is expressed in ~43 neurons in the abdominal ganglion of the VNC (Ext. Data Fig.6c-g), has no expression in the gut, and very limited expression in the brain (Ext. Data Fig.6c&FlyLight). We tested the cholinergic nature of R49E06-neurons using the cholinergic Gal80 repressor (ChAT-Gal80), which inhibited GFP expression in most VNC R49E06-neurons (Ext. Data Fig.6d). We found that ~35 of the ~43 neurons in the abdominal ganglia are ChAT+/cholinergic (Ext. Data Fig.6e-f) and in their majority do not express Prospero (Pros, Ext. Data Fig.6g). We observed that some R49E06-neurons innervate the midgut and are ChAT+ (Ext. Data Fig.6h), suggesting that a subpopulation of R49E06-neurons are cholinergic enteric neurons. To confirm that R49E06-innervations can release ACh to the gut, we used an antibody against the synaptic vesicle membrane protein Synaptotagmin1 (Syt1) which is essential for neurotransmitter release. We observed that R49E06-innervations run between the muscle and intestinal epithelium while carrying Syt1+ swellings (Ext. Data Fig.6i). We refer to Syt1+ swellings as presynaptic boutons because they resemble en passant varicosities described in the autonomous nervous system and are located at sites where a neurotransmitter diffuses to receptors located in nearby (innervated) cells. Together these data indicate that R49E06-neurons are in their majority cholinergic and include a subpopulation of cholinergic enteric neurons that innervate the intestinal epithelium and muscle.
To further characterize R49E06-enteric innervations, we sectioned flies with a vibratome so that innervations and gut remained intact (Fig.4a). R49E06-neurons innervate the midgut at distinct locations in R2, R4 and R5 (Fig.4a). To distinguish between the axonal and dendritic domains, we expressed the synaptic vesicle marker Syt1HA and the dendritic indicator DenMark for two days during recovery. We observed that Syt1HA accumulated at the terminal of R49E06-innervations in the vicinity of the gut (Fig.4a, Ext. Data Fig.6j), whereas DenMark accumulated upstream (Ext. Data Fig.6k). We observed that Syt1HA-innervations are close to ECs (Fig.4b, Ext. Data Fig.6l and Video S7), suggesting that R49E06 neurons innervate ECs and could be the source of ACh.
To test whether R49E06 neurons regulate gut regeneration, we conditionally knocked down ChAT in these neurons. This caused: i) over-proliferation during recovery and after repetitive DSS-injury (Fig.4c), (ii) recovery-specific reduction of pdm1 (Fig. 4d), iii) no significant change of pros (Ext. Data Fig.7a), and iv) elevated expression of inflammatory cytokines during recovery (Ext. Data Fig.7b). Also, co-expression of the VNC repressor Tsh-Gal8036, while reducing ChAT in R49E06-neurons, prevented ISC over-proliferation during recovery (Ext. Data Fig.7c). Further, ChAT decrease in R49E06 neurons led to ISC over-proliferation after Ecc15 infection (Ext. Data Fig.7d). Thus, reduction of ACh synthesis from R49E06-neurons in VNC leads to lasting unresolved injury, resembling nAChRβ3-deficiency in ECs. These data indicate that intestinal repair is under neuronal control, leading us to name these neurons Anti-inflammatory Recovery-regulating Cholinergic Enteric Neurons (ARCENs).
To test whether ARCENs are required for nAChR-mediated recovery, we blocked neurotransmitter release with the UAS-shibireTS transgene while simultaneously overexpressing nAChRβ3 (Fig.4e). We found that 24hr expression of shibireTS in ARCENs was sufficient to prevent nAChRβ3 overexpression in ECs from rapidly decreasing proliferation (Fig.4e). To verify that ARCENs release ACh to the intestinal epithelium, we overexpressed the ion channel TrpA1 and thermo-genetically depolarized ARCENs (Ext. Data Fig.7e). TrpA1-mediated induction of ARCENs the first 6 hours of recovery significantly reduced ISC proliferation, whereas additionally expressing the cholinergic repressor ChAT-Gal80 restored proliferation to levels identical to controls (Ext. Data Fig.7e). Also, TrpA1-induction of ARCENs significantly reduced the expression of gut inflammatory cytokines (Ext. Data Fig.7f). Moreover, we used the QF system to generate R49E06-QF (ARCEN-QF) driver (Ext. Data Fig.7g). We used ARCEN-QF together with light-gated cation channel CsChrimson to depolarize ARCENs while conditionally expressing in ECs the Ca2+ transcriptional reporter NFAT-CaLexA (Fig.4f). Optogenetic activation of ARCENs the first 6 hours of recovery was sufficient to significantly increase endogenous Ca2+ and levels of pdm1+ ECs (Fig.4f). Together, these data support that ARCENs provide ACh to ECs to promote transition to homeostasis after injury by activating nAChR-mediated Ca2+ influx, increasing mature ECs, reducing proliferation, and decreasing inflammation in the intestinal epithelium. However, we cannot completely rule out that the subpopulation of ARCENs without enteric innervations may have mediator roles that promote recovery by activating non-ARCEN cholinergic enteric innervations. Our findings demonstrate nerve-dependent intestinal regeneration, placing the intestinal epithelium among the tissues whose ability to regenerate depends on neurons.
ARCENs depend on TNF signaling
The cholinergic anti-inflammatory reflex has been proposed to sense inflammatory signals like TNF and reduce them by triggering ACh release across neuro-immune interactions11,37. As Drosophila peripheral neurons have been reported to respond to Egr through wengen (wgn)38, one of the two known Egr receptors (wgn and grindelwald, grnd)39,40, we tested whether the protective role of ARCENs is linked to Egr. Reduction of wgn but not grnd in ARCENs led to significant ISC over-proliferation specifically during recovery, which was rescued when Tsh-Gal80 was co-expressed (Fig.4g, Ext. Data Fig.7h).
We searched for the source of TNF signaling by conditionally knocking down egr in gut and immune cells (Ext. Data Fig.7i). Reduction of egr in PCs and ECs did not impact ISC proliferation during recovery and knocking down egr in hemocytes caused under-proliferation (Ext. Data 7i). However, decreasing egr in all three populations concurrently caused significant over-proliferation during recovery (Ext. Data Fig.7i-j). This suggests that ARCENs likely sense Egr from multiple sources. Also, knocking down egr in all three cell populations during injury caused ISC under-proliferation (Ext. Data Fig.7j), consistent with the proposed proliferative role of Egr41.
Further, we tested whether secreted Egr reaches ARCENs. We expressed in the transmembrane of ARCENs an extracellular nanobody-based GFP trap (morphotrap)42 for two days during recovery while endogenous Egr was fused to GFP (Egr-GFP). We observed GFP accumulation in ARCEN-projections expressing the morphotrap (Ext. Data Fig.7k), indicating that secreted Egr reaches ARCENs. GFP accumulation was only observed in ARCEN-projections near the posterior midgut and not near the VNC (Ext. Data Fig.7k). Since near the posterior midgut ARCEN-innervations are enriched with dendritic sites (Ext. Data Fig.6k), this raises the possibility that wgn receptors are present in this region and ARCENs likely sense Egr through their enteric projections during recovery. As TNF has been proposed to promote synaptic plasticity and strengthening43 and axonal strengthening is associated with elevated firing44, we examined whether existing synaptic boutons of ARCEN-innervations undergo Egr-dependent changes (Fig.4h). ARCEN-innervations undergo significant increase in density and volume of synaptic boutons during recovery compared to homeostasis (Fig. 4h). This increase is diminished upon wgn reduction (Fig.4h), indicating that ARCEN-innervations respond to Egr by strengthening their axonal properties, likely to boost ACh release. Altogether, our data support that the function of ARCENs during gut regeneration is linked to TNF signaling, reminiscent of the cholinergic anti-inflammatory reflex37.
Ca2+ spreads via Inx2/Inx7 gap junctions
We observed that opto-activation of ARCENs led to broad Ca2+ increase across ECs in the posterior gut (Fig.4f), despite the limited innervations, suggesting that Ca2+ likely propagates between innervated and non-innervated ECs. To test this, we performed ex vivo Ca2+ live imaging utilizing split-Gal4 drivers45 that when combined are specific to a small population of ECs between R4 and R5 (R4c, Ext. Data Fig.8a), which we refer as spECs. We transiently increased Ca2+ in spECs using the light-gated CsChrimson channel, while recording Ca2+ changes in neighboring ECs (Fig. 5a, Ext. Data Fig.8b). Activation of CsChrimson in spECs increased Ca2+ in distant ECs as far as 180μm (Fig.5a, Ext. Data Fig.8b, Video S8) and the gap junction blocker Heptanol, significantly reduced Ca2+ propagation (Ext. Data Fig.8b). These data suggest that when Ca2+ is elevated in a subpopulation of ECs, gap junctions spread Ca2+ in more ECs. The endogenous flow of ions among cells (bioelectric signaling) has been proposed to regulate regeneration9,46,47. Therefore, we tested if generating Ca2+ currents via CsChrimson-activation in spECs could impact gut regeneration (Fig.5b). We found that 7hr opto-activation of spECs led to significantly faster decrease in proliferation the first day of recovery (Fig.5b), reminiscent of ARCEN activation (Ext. Data Fig.7e).
To investigate further how bioelectric signaling regulates gut recovery we analyzed in our single nuclei data the expression of innexins, the components of gap junctions in invertebrates6,48. Innexin2 (Inx2) and Innexin7 (Inx7) are similarly enriched in ECs whereas their expression in PC and EE clusters is lower (Ext. Data Fig.8c-e). We confirmed expression in ECs using an antibody for Inx2 (Ext. Data Fig.8f). Inx7 reduction in ECs disrupted Inx2 gap junction formation (Ext. Data 8g-h), suggesting that Inx2 and Inx7 form heteromeric gap junctions. Moreover, knocking down Inx2 or Inx7 in ECs led to recovery-specific over-proliferation (Fig.5c-d), whereas no significant changes occurred during homeostasis or injury. Further, knocking down Inx2 while opto-activating spECs prevented rapid decrease in ISC proliferation during recovery (Ext. Data Fig.8i). Also, knocking down Inx2 or Inx7 in ECs during recovery caused significantly reduced and irregular Ca2+ distribution together with significantly decreased pdm1+ levels (Fig.5e).
Gap junctions are activated by membrane potential changes, including increases in intracellular Ca2+6. This suggests that nAChR-induced Ca2+ during recovery could prompt gap junction activation in ECs. To test this, we overexpressed nAChRβ3 for two days while knocking down Inx2 in ECs during recovery (Fig.5f, Ext. Data Fig.8j). Knocking down Inx2 was sufficient to attenuate rapid decrease in ISC proliferation and block fast increase of pdm1+ ECs, thereby blocking the expedited recovery triggered by nAChRβ3 overexpression (Fig.5f, Ext. Data Fig.8j). Finally, we tested whether Inx2/Inx7 gap junctions facilitate Ca2+ responses in ECs during the nicotinic sensitivity assay. Conditionally reducing Inx2 expression resulted in significantly dampened nicotine-triggered Ca2+ increase during recovery (Fig.5g, Ext. Data Fig.8k and Videos S9-S10). We observed that Ca2+ distribution among ECs was not only reduced but also uneven (Fig.5g, Ext. Data Fig.8k and Videos S9-S10). This suggests that during recovery gap junctions facilitate nAChR-induced Ca2+ to spread evenly among ECs. Altogether, our data support that Inx2/Inx7 gap junctions are required for nAChR-mediated Ca2+ to spread in ECs during recovery and that disruption of this bioelectric signaling prevents transition to homeostasis.
Discussion
We address a fundamental question, how does transition to homeostasis occur after injury? We found that the cholinergic pathway directs the gut to return to homeostasis by coordinating neuro-epithelial interactions and bioelectric signaling (Ext. Data Fig.9). Our findings reveal that nAChR-mediated high Ca2+ in ECs is essential for recovery (Fig.3, Ext. Data Fig.5). It is reported that Ca2+ increase after nAChR activation is augmented autonomously by opening of voltage gated Ca2+ channels and release of Ca2+ from intracellular stores49. This could occur in nAChR-activated ECs during recovery likely to assist in sufficiently elevating Ca2+. We discovered that elevated Ca2+ from few ECs spreads via gap junctions to more ECs (Fig.5, Ext. Data Fig.8), which is consistent with a recent report of Ca2+ waves in R3 of the midgut50. The use of endogenous ion currents that electrically couple multiple cells so that they behave as one unit, has been linked to growth and tissue-patterning during development and regeneration9,47. Our study supports that bioelectric mechanisms regulate midgut regeneration to ensure that important physiological functions like ion transport are evenly restored across the epithelium.
Despite the increasing knowledge of protective anti-inflammatory roles for peripheral neurons, many aspects remain unclear11. We discovered that cholinergic signaling from ARCENs is required for the transition of the intestinal epithelium from injury to homeostasis (Fig.4, Ext. Data Fig.7). During recovery ARCEN-innervations undergo Egr-dependent synaptic strengthening (Fig.4h) and ECs change the levels of cholinergic components (Fig.1d, 2d-e). This coordinated plasticity, in neuro-epithelial cholinergic interactions likely occurs to control the initiation, strength, and duration of nAChR-mediated Ca2+ currents across the epithelium and precisely promote recovery. In addition, the cholinergic anti-inflammatory reflex has been proposed to be a cholinergic neuro-immune response that senses and counteracts inflammatory signals11,37. We propose that a similar mechanism exists in Drosophila and is regulated by ARCENs which reside in the posterior VNC, potentially the fly equivalent of vagus nerve. ARCENs sense Egr likely through their projections (Ext. Data Fig.6k, Ext. Data Fig.7k) and counteract gut inflammation and proliferation via neuro-EC cholinergic signaling (Fig.4, Ext. Data Fig.7). Altogether, our study broadens our current understanding of how regeneration ends by revealing how cooperation between peripheral neurons and epithelial bioelectric signaling directs a tissue towards homeostasis. This work may help identify the etiology of chronic intestinal diseases and provides a link between neurological disorders and intestinal pathologies.
Methods
Flies were crossed and raised between 19-23°C in standard fly food. All adult flies were tested 3-5 days after hatching. All experiments were done in female flies. Detailed description of experimental methods is found on Supplementary Information (SI).
Extended Data
Supplementary Material
Acknowledgments:
We thank Stephanie Mohr and Justin Blau for comments on the manuscript. Confocal imaging was conducted at MicRoN Facility at Harvard Medical School, and we thank Paula Montero Llopis for advice. We thank the Cepko lab at Harvard Medical School for sharing their vibratome. We also thank Mike Levin for discussions and Hugo Bellen, Guy Tanentzapf, Kate O'Connor-Giles, Xiaohang Yang, Chris Potter, Todd R. Laverty, Gerry Rubin, Janelia FlyLight, DSHB, DRSC/TRiP, VDRC and the Bloomington Stock Center for fly lines, antibodies, and reagents. We thank Frederik Wirtz-Peitz, Sudhir Gopal Tattikota, Rich Binari and Haofan Li for help in this project and Patrick Jouandin, Pedro Saavedra, Liz Lane, David Doupé, Justin Bosch, Ben Ewen-Campen, Lucy Liu, Charles Xu, Misty Rose Riddle, Tyler Huycke for advice and reagents. We thank Christians Villalta and Bestgene for fly injections, Hunter Elliot and Marcelo Cicconet at the Image and Data Analysis Facility (IDAC) and Simon Norrelykke at the Image Analysis Collaboratory (IAC) at Harvard Medical School for advice on Imaris, Shahar Alon for advice on expansion microscopy, and the Biopolymers Facility and Computing facilities and PCMM Flow Cytometry Facility at Harvard Medical School. All illustrations were created with BioRender.com.
Funding:
During this study AP was a Good Ventures fellow of the Life Science Research Foundation and next was supported by the Center for the Study of Inflammatory Bowel Disease (DK043351). NP is an investigator of the Howard Hughes Medical Institute. YH, YL and AC were supported by P41GM132087 and BBSRC-NSF/BIO (DBI-2035515). YL was supported by the Finnish Cultural Foundation.
Footnotes
All authors declare that they have no competing interests.
Supplementary Information and Source Data Files are available for this paper.
Data availability:
Raw data from main and Extended Data Figures are available in the Source Data files provided with this work. Reagents are available upon request. The snRNA-seq datasets generated in this work are publicly available in the Gene Expression Omnibus (GEO) databases under GSE218641 accession code.
GSE218641: [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218641], snRNA-seq dataset of gut from Ore R females flies during Homeostasis and Recovery
Single-nuclei profiling data from this study can be found at https://www.flyrnai.org/tools/rna_seq_base/web/showProject/39/plot_coord=1/sample_id=all , to allow users to query the expression of any gene of interest.
All other data are available in Figures, Ext. Data Figures and SI files (SI, SI figure, S. videos, S. Tables)
Code availability:
This study does not use any custom codes for analysis. snRNA-seq dataset were analyzed using standard Seurat pipeline.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Raw data from main and Extended Data Figures are available in the Source Data files provided with this work. Reagents are available upon request. The snRNA-seq datasets generated in this work are publicly available in the Gene Expression Omnibus (GEO) databases under GSE218641 accession code.
GSE218641: [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218641], snRNA-seq dataset of gut from Ore R females flies during Homeostasis and Recovery
Single-nuclei profiling data from this study can be found at https://www.flyrnai.org/tools/rna_seq_base/web/showProject/39/plot_coord=1/sample_id=all , to allow users to query the expression of any gene of interest.
All other data are available in Figures, Ext. Data Figures and SI files (SI, SI figure, S. videos, S. Tables)
This study does not use any custom codes for analysis. snRNA-seq dataset were analyzed using standard Seurat pipeline.