Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine

The initiator caspase Dronc is the only CARD-domain containing caspase in Drosophila and is essential for apoptosis. Here, we report that homozygous dronc mutant adult animals are short-lived due to the presence of a poorly developed, defective and leaky intestine. Interestingly, this mutant phenotype can be significantly rescued by enteroblast-specific expression of dronc+ in dronc mutant animals, suggesting that proper Dronc function specifically in enteroblasts, one of four cell types in the intestine, is critical for normal development of the intestine. Furthermore, enteroblast-specific knockdown of dronc in adult intestines triggers hyperplasia and differentiation defects. These enteroblast-specific functions of Dronc do not require the apoptotic pathway and thus occur in a non-apoptotic manner. In summary, we demonstrate that an apoptotic initiator caspase has a very critical non-apoptotic function for normal development and for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.

www.nature.com/scientificreports/ epithelial cells and make up the majority of the cells in the midgut. More recently, it has also been suggested that EEs can be directly generated by asymmetric ISC division without going through an EB intermediate [23][24][25] . The cell lineage in the midgut is under strict control to ensure proper function and homeostasis of the midgut (reviewed in 22,26 ). ECs and EEs are regularly turned over and need to be replaced by new cells due to ISC mitosis. There is feedback from dying EC cells to control ISC activity 27 . Imbalances of this control can lead to malfunction of the intestine, dysplasia, premature aging and death of the animal. Because mammalian intestines are also subject to a similar cell lineage 28 , a clear understanding of the mechanisms involved in the control of this cell lineage may help to understand disease and identify potential targets for the cure of the disease.
Here, we show that the initiator caspase Dronc has essential functions for development and homeostasis of the Drosophila adult midgut. Interestingly, this function is primarily required in EBs and appears to be non-apoptotic in nature. Loss of dronc in EBs causes hyperplasia due to increased proliferation. Furthermore, there are significant differentiation defects. Specifically, loss of dronc results in an increased number of EBs and accumulation of cells that have features of both EBs and ECs. There is also a significant increase in the number of EEs. These data demonstrate that an apoptotic initiator caspase has a very critical function for the control of the cell lineage in the adult midgut and therefore for proper physiology and homeostasis.  I24 /dronc I29 intestines. The density of nuclei in 2500 μm 2 fields was analysed by unpaired t test, two tailored and plotted ± SEM. ****p < 0.0001. n = 6 (wt), 5 (dronc I24 /dronc I29 ). (d,e) Wild-type (Canton S) and dronc I24 /dronc I29 mutant animals, subjected to a SMURF assay.

Results
Homozygous dronc mutants die prematurely due to fragile and leaky intestines. Homozygous dronc mutants are pupal lethal. However, at a very low frequency (less than 1% of the expected offspring), adult homozygous dronc I24 /dronc I29 mutant flies can be recovered. These dronc alleles carry premature stop codons at codons 28 and 53 and encode strong, if not null, alleles of dronc 11 . With the exception of down-curved and opaque wings 11 , these mutant flies do not have any obvious phenotypic abnormalities. Nevertheless, they are very short-lived and die within 3 to 4 days after eclosion suggesting that they may have some internal defects.
To identify the possible cause of this premature death, we examined the internal organs of these mutants. When dissecting the intestines of dronc I24 /dronc I29 flies, we noticed that they are very fragile. By phallodin labelings, these intestines displayed structural irregularities (Fig. 1a,b). Furthermore, labelings with the nuclear dye DAPI show that the mutant intestines have a higher density of cells (Fig. 1a' ,b'; quantified in c).
To examine if these structural defects cause a dysfunction of the intestine, we performed SMURF assays with homozygous dronc I24 /dronc I29 flies. In a SMURF assay, a blue dye is mixed into the food and fed to the flies 29,30 . Flies with an intact intestine keep the blue food in the intestine which can be easily seen through the abdominal cuticle (Fig. 1d). However, in flies with an intestinal barrier dysfunction, the blue dye penetrates into every tissue of the fly, generating a SMURF phenotype 30 . We examined five homozygous dronc I24 /dronc I29 mutants in the SMURF assay and all of them displayed the SMURF phenotype (Fig. 1e). Furthermore, while wild-type flies start feeding almost immediately, we noticed that dronc I24 /dronc I29 mutant flies do not feed for the first 24 to 48 h after eclosion. These data suggest that dronc mutant intestines have structural defects and a defective barrier function causing a leaky gut.

EB-specific expression of dronc rescues semi-lethality and restores gut function of dronc mutants.
To determine if the defective intestine causes the premature organismal lethality of homozygous dronc I24 /dronc I29 mutants, we asked if cell-type specific expression of UAS-dronc wt can rescue the strong semilethality and short lifespan as well as the defective and leaky gut phenotype of homozygous dronc I24 /dronc I29 animals. As a control, we expressed the catalytic UAS-dronc C318A mutant. Interestingly, expression of dronc wt in EBs using the EB-specific driver Su(H)GBE-Gal4 (from now on Su(H)) gave the best rescue of the lethality ( Fig. 2a; genotype 3). About 75% of the expected Su(H) > dronc wt ; dronc I24 /dronc I29 progeny was recovered as adults. With the esg-Gal4 driver, which is expressed in ISCs and to some extent in EBs, a weaker rescue was recorded ( Fig. 2a; genotype 2). The weakest rescue was scored when dronc wt was expressed in mature ECs using NP1-Gal4 ( Fig. 2a; genotype 4). Expression of the catalytic mutant dronc C318A using all three Gal4 drivers was not able to rescue the lethality of dronc I24 /dronc I29 animals ( Fig. 2a; genotypes 5-7). These rescue crosses suggest that for development of the intestine, Dronc function is most critical in EBs and requires its catalytic activity.
However, these data have the caveat that the Gal4 drivers used are also expressed in other tissues during development. We can therefore not conclude, that the exclusive expression of dronc in EBs is sufficient for normal development and survival. Nevertheless, EB-specific expression of dronc does not rescue all known phenotypes of dronc mutants. The wings of Su(H) > dronc wt ; dronc I24 /dronc I29 flies still have the reported dronc mutant phenotype (down-curved, opaque wings) (Fig. 2b,c). Furthermore, we observed one to two additional scutellar bristles (macrochaetae) with 100% penetrance (Fig. 2d,e) which had been reported for dark and cytochrome c-d mutants 31,32 and represents a typical phenotype when cell death is blocked. We also recovered Su(H) > dronc wt ; dronc I24 /dronc I29 flies with a split abdomen phenotype at reduced penetrance ( Fig. 2f,g). The split abdomen phenotype is likely the result of reduced cell death of larval cells in the abdomen during pupal development 33 . Therefore, despite the caveat that Su(H)-Gal4 expression is not restricted to EBs in the midgut, Su(H)-driven expression of dronc cannot rescue all phenotypes of dronc I24 /dronc I29 mutants, demonstrating that expression of dronc in select groups of cells is sufficient for development and survival of the animal.
To directly examine the effect of EB-specific expression of dronc on the physiology of the intestine, we performed SMURF assays with Su(H) > dronc wt ; dronc I24 /dronc I29 rescued animals. Of 26 rescued flies which were tested in this assay, only one animal developed a SMURF phenotype. The intestines of the other 25 flies stayed intact and were also able to clear the blue food within 24 h after they were removed from the blue food (Fig. 2h,i). In contrast, 6 out of 7 tested esg > dronc wt ; dronc I24 /dronc I29 flies (Fig. 2a, genotype 2) developed a SMURF phenotype.
Given the critical role of Dronc in the adult intestine, in particular in EBs, we examined if Dronc is expressed in these cells using a Dronc-specific antibody. To identify EBs, we expressed GFP using the EB-specific driver Su(H)-Gal4. This analysis reveals that Dronc is expressed in EBs (Fig. 2j, yellow arrowheads) as well as in other cell types as demonstrated recently 34 . A specificity control of the anti-Dronc antibody is shown in Supplementary  Fig. S1. Taken together, these results indicate that Dronc function in the intestine, in particular in EBs, is essential for intestinal integrity and organismal survival of the animal.

Down-regulation of dronc in EB cells causes hyperplasia.
The above data indicate that dronc has a very important function in EB cells for proper formation of the intestine during development. Additionally, we examined if dronc also has an important role for homeostasis of the adult midgut. For that purpose, we downregulated dronc in EB cells by RNAi, using Su(H)-Gal4 ts . The ts in this annotation indicates the presence of Gal80 ts which allows down-regulation of dronc using this Gal4 driver by temperature shift to 29 °C after the animals have fully developed and eclosed. dronc C318A carries a mutation of the catalytic Cys318 residue to an Ala and represents a catalytic mutant. dronc I24 / dronc I29 (genotype 1) is strongly pupal semi-lethal and less than 1% of the expected progeny can be recovered as adults. The obtained progeny in the rescue crosses is plotted as the percentage of the expected Mendelian progeny. Genotypes of expected rescued progeny are indicated on the right.   www.nature.com/scientificreports/ intestines were analysed 5-6 days later. Preferentially, region R4ab of the posterior midgut was examined in these assays 35,36 . There is a significant increase in the total number of cells in the midgut (as revealed by DAPI labelings (Fig. 3a-c'' , quantified in d)), and GFP-positive EB cells were overabundant compared to controls (Fig. 3a'-c' , quantified in e). Consistent results were obtained with 2 independent dronc RNAi lines (Fig. 3b-e). In addition to the EB overabundance of Su(H) ts > dronc RNAi midguts, EBs (marked by GFP) also appear to change shape and are much larger than normal (Fig. 3a'-c'). This observation is also statistically significant (Fig. 3f).
Given that dronc has an important function in apoptosis 11,37-39 , we considered the possibility that this EBspecific dronc phenotype is caused by loss of apoptosis. However, we did not observe a similar EB-overabundance phenotype in response to EB-specific (using Su(H)-Gal4 ts ) dark RNAi (Fig. 3g; see additional example and quantification in Supplementary Fig. S2), which encodes the adaptor protein for incorporation of Dronc into the apoptosome during apoptosis 3,4,6 . EB-specific RNAi targeting drICE, the most important effector caspase in Drosophila 40,41 , also did not phenocopy the dronc RNAi phenotype (Fig. 3h; Supplementary Fig. S2). Finally, EB-specific expression of the effector caspase inhibitor p35 in otherwise wild-type midguts using Su(H)-Gal4 ts did not replicate the dronc RNAi phenotype (Fig. 3i; Supplementary Fig. S2). EB-specific dark RNAi , drICE RNAi and p35 expression also did not phenocopy other aspects of the dronc RNAi phenotype such as the increase in overall cell number and in EB cell size (Supplementary Fig. S2). Therefore, we can rule out that the Su(H) ts > dronc RNAi phenotypes in adult midguts are caused entirely by loss of apoptosis. The functionality of the dark RNAi , drICE R-NAi and p35 transgenic lines was validated by the ability of these lines to suppress the GMR-reaper eye ablation phenotype, a commonly used apoptosis model 42 (Supplementary Fig. S2).
It was previously shown that an accumulation of EBs can cause increased ISC proliferation 43,44 . Consistently, based on PH3-labeling experiments, we found that cell proliferation is increased in Su(H) ts > dronc RNAi midguts (Fig. 3j,k). This was confirmed with two dronc RNAi lines (Fig. 3m). In contrast, EB-specific dark RNAi , drICE RNAi and p35 expression did not phenocopy this phenotype ( Supplementary Fig. S2) suggesting that dronc controls cell proliferation in a non-apoptotic manner. Combined, these observations suggest that EB-specific dronc RNAi triggers a hyperplastic phenotype in the intestine. Consistently also, mosaic analysis of adult intestines with dronc I24 and dronc I29 showed that dronc mutants form larger clones than control clones (Supplemental Fig. S3).

Down-regulation of dronc in EB cells causes differentiation defects.
Given that ECs are much larger in size than EBs and that EBs in Su(H) > dronc RNAi midguts are significantly enlarged (Fig. 3f), we considered that GFP-positive EBs in dronc RNAi midguts also display features of EC cell fate. To examine this possibility, we labelled these midguts with Pdm-1 antibody, a marker for EC fate 45,46 . Consistently, in Su(H) ts > dronc RNAi ,GFP midguts we observed multiple examples where Su(H) ts -driven GFP overlaps with Pdm-1 labeling suggesting that these cells have properties of both EBs and ECs (Fig. 4a-c). While in control midguts, expression of Pdm-1 in EBs was also observed at a low frequency (~ 2% of total EBs), this number was significantly higher in Su(H) ts > dronc RNAi midguts (Fig. 4g). These data imply that in normal midguts, Dronc is required for the appropriate differentiation of EBs into ECs.
We also examined enteroendocrine (EE) cells in Su(H) ts > dronc RNAi midguts using the EE marker Prospero (Pros) and observed an increased number of Pros-positive cells (Fig. 4d'-f '; quantified in 4 h), indicating that the number of EEs is elevated. This EE overabundance phenotype was not observed in response to EB-specific dark RNAi , drICE RNAi and p35 expression in adult midguts ( Supplementary Fig. S2) suggesting that this dronc RNAidependent phenotype is independent on its role in apoptosis. These observations further indicate that Dronc function in EBs is required for proper differentiation of intestinal cell types.

Discussion
In the absence of dronc during development, the adult intestine has structural defects, is fragile and leaky, limiting the lifespan of homozygous adult escapers to only 3-4 days. Interestingly, expression of dronc specifically in EBs can significantly rescue these phenotypes suggesting that dronc has a very important function in EBs for development of the intestine. Nevertheless, because Su(H)-Gal4 is also expressed in cells outside the intestine, we cannot exclude the possibility that other developmental defects contribute to the lethality of dronc mutant animals. Because the catalytic mutant dronc C318A cannot rescue these phenotypes, Dronc likely requires its catalytic activity for proper function in the midgut. The observation that expression of dronc in ECs using NP1-Gal4 cannot significantly rescue the lethality of dronc mutants (Fig. 2a) does not mean that Dronc does not have a function in ECs. It only means that NP1-Gal4 is not expressed in those cells (such as EBs) where dronc has an essential function for survival.
EB-specific knockdown of dronc in adult intestines causes differentiation defects and hyperplasia due to increased proliferation. Therefore, Dronc function is critical for proper control of the cell lineage in the midgut and loss of dronc disrupts this homeostasis. The observation that loss of other genes in the apoptosis pathway (dark, drICE) or overexpression of the effector caspase inhibitor p35 do not replicate the EB-specific dronc phenotypes suggests that Dronc mediates this role in a non-apoptotic manner. Similar results were recently reported by Arthurton et al. 47 . This adds control of proper proliferation and differentiation of the adult midgut to the growing list of non-apoptotic functions of Dronc.
There are several interesting aspects of the EB-specific dronc phenotypes in the midgut. First, the observed hyperplasia is puzzling. In the Drosophila intestine, only ISCs are mitotically active, EBs are not 19,20 . In fact, EBs are the daughter cells of the asymmetric division of ISCs. The increased mitotic activity of ISCs in response to EB-specific dronc knockdown suggests that Dronc is involved in a feedback mechanism between EBs and ISCs. Elucidating the molecular mechanism of this feedback mechanism and the role Dronc plays in this will be an exciting avenue for future research. www.nature.com/scientificreports/ Second, the increased number of EBs and accumulation of EBs expressing the EC marker Pdm-1 suggests that Dronc is involved in the differentiation process from EBs to ECs. The function of Dronc in this process can be explained in one of two opposite ways. Dronc may be required for an important step in the differentiation process from EBs to ECs. In the absence of dronc, while EBs are able to increase in size and induce expression of the EC marker Pdm-1, they cannot complete the differentiation program into ECs and get stuck along the way. This would result in an increased number of EBs in the intestine. However, the opposite explanation, that Dronc inhibits the differentiation of EBs into ECs under normal conditions, is formally also possible. In that case, the differentiation into ECs occurs so fast, that the EB-specific expression of GFP (which is Su(H)-Gal4 dependent) is not turned off early enough to avoid overlap of EB-and EC-specific markers. Future experiments will clarify by which mechanism Dronc controls the differentiation of EBs to ECs.
Third, the increase of EEs in response to EB-specific knockdown of dronc suggests that Dronc negatively controls the formation and differentiation of EEs. Because it is not clear whether EEs are also differentiating from EBs, as originally suggested [19][20][21] , or if they are direct descendants of ISCs 23-25 , it is not clear whether the role of Dronc in this process is autonomous or non-cell autonomous. Because we do not observe an overlap of EB-specific GFP expression (driven by Su(H)-Gal4) with EE-specific Pros labelling ( Fig. 4d-f), suggest a noncell autonomous control of EE fate by Dronc, but other explanations may be possible, too. In any case, what these data show is that under normal conditions, Dronc controls the number of EEs in an EB-specific manner.
Another important question for the future is how Dronc mediates the homeostatic effect in the adult intestine. There are several possibilities. It was shown that loss of the transcription factor escargot (esg) has similar phenotypes compared to dronc: increased differentiation into ECs and EEs 48,49 . Esg suppresses the expression of the differentiation-promoting factor Pdm-1 in progenitor cells 48 . Thus, Dronc may be involved in Esg-mediated control of Pdm-1 expression. Dronc may also be involved in the control of some of the signalling pathways that operate in the differentiation process, such as Notch (N) signalling. Given that N signalling is also controlled by esg 48,49 , it is possible that Dronc participates in this complex signalling network. However, because many signalling pathways are involved in the control of the cell lineage in the intestine (reviewed in 22,26 ), Dronc may also control any of these pathways. Given that the only known biochemical function of Dronc is proteolytic activity, it will be a challenge in the future to identify the cleavage substrate(s) of Dronc in this process.
Finally, how is Dronc activated in this non-apoptotic context? During apoptosis, activation of Dronc occurs by incorporation into the Dark apoptosome [7][8][9] . However, in EBs of the intestine, this occurs in an apoptosomeindependent manner because Dark is not involved (Fig. 3g; Supplementary Fig. S2). Dronc may be incorporated into a different protein complex for activation. For example, mammalian initiator caspases can be recruited into different complexes such as the apoptosome and the inflammosome 50 . A different complex may provide different properties to Dronc such that it does not cleave its apoptotic substrates and can act in a non-apoptotic manner. Alternatively, it is possible that a different protease may cleave and activate Dronc.
Impaired caspase function may also provide a contributing factor for the development of colon cancer in human patients. For example, Caspase-9, the Dronc ortholog in humans, is epigenetically silenced in almost 50% of colon cancer cases 51,52 . Other caspases, such as Caspase-7 are down-regulated in up to 85% of colon cancer cases 51,52 . While this silencing is likely a means for evading apoptosis, it could also trigger additional effects such as increased proliferation and differentiation defects, thus further supporting tumorigenesis. Therefore, revealing the mechanism by which Dronc expression in EBs maintains tissue homeostasis may also have important implications for understanding of the tumor-promoting effect of loss of caspase-9 in humans. Given that the function of Dronc for maintaining tissue homeostasis in the intestine is non-apoptotic, characterizing the role of Dronc in EBs does provide a convenient opportunity to study this function in the absence of its apoptotic function which may not be as simple for Caspase-9 in humans.

Materials and methods
Drosophila husbandry. All crosses were performed on standard cornmeal-molasses medium (60 g/L cornmeal, 60 ml/L molasses, 23.5 g/L bakers yeast, 6.5 g/L agar, 4 ml/L acid mix and 0.13% Tegosept). Crosses not involving conditional expression of transgenes were incubated at room temperature. Crosses involving conditional expression of transgenes including RNAi were incubated at 18 °C until adult offspring eclosed. Adults were kept at 18 °C for 5 days before they were incubated at 29 °C for another 5-6 days prior to dissection. Flies were provided fresh, yeasted food every day. Only female midguts were dissected and analysed. SMURF assays were performed as described 29   To induce dronc mosaics in the intestine (Supplemental Fig. S3), the MARCM 58 (mosaic analysis with a cell repressible marker) technique was used which positively labels mutant clones with GFP. dronc I24 and dronc I29 were analysed which both carry FRT80 for mitotic recombination. 6 days old female flies of the correct genotype were heat shocked for 60 min at 37 °C in a water bath. Mosaic flies were kept at 25 °C for another 7 days before dissection.
Dissection and immunolabeling of adult guts. Intact female midguts were dissected using standard protocols 59  www.nature.com/scientificreports/ Prospero (Pros, 1:20; DSHB; Prospero (MR1A) was deposited to the DSHB by C.Q. Doe); Pdm-1 61 (1:1,000; a kind gift of Yu Cai). DAPI was used to counterstain nuclei. Phalloidin labeling was used to assess the physical properties of the guts. Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. If not noted otherwise, region R4ab 35,36 in the posterior midgut was imaged and analysed. Images were obtained with a Zeiss LSM 700 confocal microscope, analysed with Zen 2012 imaging software (Carl Zeiss) and processed with Adobe Photoshop CS6.

Counts of DAPI-, Pdm-1-, Pros-and PH3-positive cells. DAPI-, Pdm-1-and Pros-positive cells were
counted manually by detecting signal-positive cells as spots in region R4ab and normalized to areas of 2,500 µm 2 . PH3 counts were performed across the entire intestine. At least three independent experiments for every genotype were performed. Analysis and graph generation was done using GraphPad Prism 8.30. The statistical method used is indicated in the legends to the respective panels.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).