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Conversion of Gastrointestinal Somatostatin-Expressing D Cells Into Insulin-Producing Beta-Like Cells Upon Pax4 Misexpression

Type 1 diabetes results from the autoimmune-mediated loss of insulin-producing beta-cells. Accordingly, important research efforts aim at regenerating these lost beta-cells by converting pre-existing endogenous cells. Following up on previous results demonstrating the conversion of pancreatic somatostatin delta-cells into beta-like cells upon Pax4 misexpression and acknowledging that somatostatin-expressing cells are highly represented in the gastrointestinal tract, one could wonder whether this Pax4-mediated conversion could also occur in the GI tract. We made use of transgenic mice misexpressing Pax4 in somatostatin cells (SSTCrePOE) to evaluate a putative Pax4-mediated D-to-beta-like cell conversion. Additionally, we implemented an ex vivo approach based on mice-derived gut organoids to assess the functionality of these neo-generated beta-like cells. Our results outlined the presence of insulin+ cells expressing several beta-cell markers in gastrointestinal tissues of SSTCrePOE animals. Further, using lineage tracing, we established that these cells arose from D cells. Lastly, functional tests on mice-derived gut organoids established the ability of neo-generated beta-like cells to release insulin upon stimulation. From this study, we conclude that the misexpression of Pax4 in D cells appears sufficient to convert these into functional beta-like cells, thus opening new research avenues in the context of diabetes research.

Interestingly, most of these approaches rely on the modulation of developmental genes, a majority encoding for transcription factors. Among these, Pax4 plays a crucial role in the differential endocrine cell allocation during pancreas morphogenesis (34)(35)(36). Accordingly, mice depleted of Pax4 are hyperglycaemic and die shortly postpartum due to a lack of insulin-producing beta-and somatostatin-secreting delta-cells (37)(38)(39). Conversely, the number of glucagon-expressing alphacells is proportionally increased in Pax4 mutant mice. These results indicate that, during development, Pax4 promotes the allocation to the beta-and delta-cell lineages at expense of the alpha-(but also epsilon-) cell fate(s) (40).
Importantly, mice misexpressing Pax4 in alpha-cells were found able to recover from several cycles of chemically-induced diabetes as a result of beta-like cell neogenesis (20,21,41,42). Specifically, it was demonstrated that the ectopic expression of Pax4 in pancreatic glucagon-producing alpha-cell was sufficient to induce their neogenesis and their subsequent conversion into insulin-producing beta-like cells (20,22). Furthermore, it was recently shown that in the pancreas of mice ectopically expressing Pax4 in somatostatin cells, larger and increased numbers of islets were observed when compared with matched controls, such difference progressively increasing with age. This increment in islets size was correlated with an increase in the number of insulin + cells (41). The results obtained demonstrated that somatostatin cells were continuously regenerated and converted into insulin-expressing cells. Further analysis confirmed that these beta-like cells were fully functional and could partially reverse chemical-induced diabetes. Altogether, these results indicated that the neo-generated insulin-expressing cells were phenotypically and functionally true beta-cells. It is worth noting that the vast majority of circulating somatostatin originates from the D cells located along the GI tract (43,44), accounting for 65% of the total amount of the hormone, whereas the central nervous system is responsible for producing around 25% of it and the pancreas releases approximately 5% of the total somatostatin (44,45). D cells are found scattered into the gastrointestinal mucosa, being highly represented in the duodenum and in the stomach (43,44,(46)(47)(48). In fact, D cells, which produce somatostatin and are scattered along the gut epithelium, produce approximately 65% of the whole-body circulating hormone. One could therefore wonder whether the Pax4-mediated conversion of somatostatinproducing cells into insulin-expressing cells could also occur in the gastrointestinal tract, an information of great importance for type 1 diabetes research.

Mouse Strain: SSTCrePOE
A bi-transgenic mouse line, the SSTCrePOE mouse line (41), was generated by crossing two single transgenic mouse lines: SST-Cre (49) and Pax4-OE (20) which were created using classical pronuclear injection. This transgenic mouse model allows the ectopic and constitutive expression of Pax4 in somatostatinproducing cells.

Animal Procedures and Ethics Statement
Experiments were performed on transgenic and control mice, control mice corresponding to SST-Cre-negative and Pax4-OE littermates. Transgenic and control animals were found to be viable, healthy, and fertile, with no premature death observed. All animals were housed on a standard 12:12-h light/dark cycle, with standard diet food and water ad libitum. Animal protocols were reviewed and approved by an institutional ethics committee (Ciepal-Azur) at the University of Nice, and all colonies were maintained following European animal research guidelines. This project received approval from our local ethics committee.

b-Galactosidase-Based Lineage Tracing Experiments
Tissues were isolated and fixed for 30 min at 4°C in a solution containing 1% formaldehyde, 0.2% glutaraldehyde, and 0.02% NP-40 and washed three times in cold PBS. The tissues were dehydrated in PBS-25% sucrose overnight at 4°C. Tissues were washed 2h in Jung freezing medium (Leica Biosystems) and embedded in freezing medium on dry ice. 10mm sections were applied to glass slides, dried, and washed three times in PBS. After 45min blocking in PBS-FCS 5%, sections were incubated with a rabbit anti-bgalactosidase primary antibody (1/8,000; MP-Cappel).

RNA Analyses
Mice were euthanized by cervical dislocation and a piece of tissue was isolated and placed in cold RNA later solution (Thermo Fisher Scientific) for 48 h at 4°C. RNA was isolated (RNAeasy; QIAGEN), and cDNA synthesis (SuperScript choice system; Invitrogen) was performed according to the manufacturer's instructions.

Gene Expression Analyses
Quantitative RT-PCR was performed using validated primers (QIAGEN) and the QuantiTect SYBR Green RT-PCR kit (QIAGEN) following the manufacturer's instructions. PCR reactions and detection were performed on a Mastercycler ep realplex cycler using GAPDH as an internal control for normalization purposes.

Glucose Tolerance Tests and Blood Glucose Level Measurements
For challenge purposes, animals were fasted for 6-8h and injected intraperitoneally with 2g/kg body weight glucose. Blood glucose levels were measured at the indicated time points post injection with a ONE TOUCH Vita glucometer (Life Scan, Inc.).

Organoid Generation
Organoids were generated following the protocols described in literature (50,51). Briefly, animals were sacrificed by cervical dislocation or by decapitation and duodenum was harvested and the villi were scraped and removed. The duodenum was incubated in 2mM EDTA-DPBS for 1h at 4°C. The supernatant was removed, and 10ml of 10% FBS-DPBS were added and the samples were vigorously shaken. The supernatant was collected and passed through a 70mm strainer. The strainer flow-through was centrifuged at 300g for 5min so the crypts were spun down. The number of crypts was calculated using a Neubauer chamber and the crypts were cultured at a range of 5000 crypts per well. To proceed with plating the crypts, the sample was spun down at 200g for 2min, the supernatant was aspirated, and the pellet was resuspended in 50% Matrigel-Growth medium. 50ml of the solution was plated and placed into the incubator at 37°C and 5% of CO 2 for 2min in order to allow the Matrigel to solidify and after, 500ml of growth medium was added into each well. The organoids were fed every 3 days and split in a ratio 1:6 every 10 days.

Organoid Immunofluorescence
Organoids were assayed by immunofluorescence following a previously protocol (51). Briefly, medium was aspirated from the wells. PFA 4%, was added into the wells and incubated for 20min at RT. Once the incubation finished, the fixative was removed and DPBS was added. All the well content was transferred in a 15ml falcon tube and centrifuged for 5min at 800rpm. The supernatant was discarded and a permeabilization buffer, which is a solution of 0.2% Triton in DPBS, was poured into the organoids pellet and incubated for 30min at 4°C on a rocking plate. Once the incubation was completed, samples were centrifuged 5min at 800rpm and the supernatant was discarded. A blocking step was performed by adding during 30min at RT and on a rocking plate 10% FBS-DPBS. Then, the normal immunofluorescence steps were performed (primary antibody ON at 4°C and secondary antibody for 45min at RT).

Glucose-Stimulated Insulin Secretion (GSIS)
The medium was aspirated and 1ml of fresh prepared 2.8mM Glucose-Krebs Ringer Buffer was added. The organoids were incubated for 30min at 37°C. The medium was aspirated and 500ml of new 2.8mM Glucose-Krebs Ringer Buffer added. The samples underwent another incubation of 30min at 37°C. The medium was collected and snap-freeze to determine the basal insulin concentration. Afterwards, 500ml of 16.8mM Glucose-Krebs Ringer Buffer was added into the wells and samples were incubated. After a 30-minute incubation, the medium was collected and snap freeze in liquid nitrogen. This medium will serve to assess insulin release upon glucose stimulation. Finally, RIPA buffer was added into each well pipetted back and forth and well content was collected into Eppendorf's. The tubes were centrifuged for 20min at 4°C at 10000rpm and supernatant was collected and frozen to perform a BCA assay. The samples were analysed by using the Mouse Ultrasensitive Elisa kit (Mercodia).

Data Analyses
All values are depicted as mean ± SEM of data from at least three animals. Data were analysed using GraphPrism 7 software. Normality was tested using a D'Agostino-Pearson omnibus normality test, and appropriate statistical tests were performed. Results are considered significant if P < 0.001 (***), P < 0.01 (**), and P < 0.05 (*).

Pax4 Misexpression in D Cells Results in Their Conversion Into Insulin-Expressing Cells
To investigate the consequences of Pax4 misexpression in gastrointestinal D cells, we made use of the SSTCrePOE transgenic mouse model allowing the ectopic and constitutive expression of Pax4 in somatostatin-producing cells ( Figure 1A). Such double transgenic animals harboured a first transgene (SST-Cre) composed of the Somatostatin (Sst) promoter driving the expression of the Cre recombinase and a second (Pax4-OE) encompassing the ubiquitously expressed pCAG promoter upstream of a LoxP-GFP-STOP-LoxP cassette followed by a Pax4-IRES-b-Galactosidase cassette (20,41). Thus, SSTCrePOE animals both allowed Pax4 misexpression in somatostatin + cells and the tracing of their lineage. Control mice were SST-Cre-negative Pax4-OE littermates. Importantly, a significant increase in Pax4 expression (up to 599%) was detected both in the duodenum and the stomach of SSTCrePOE animals when compared to age matched controls ( Figures 1B, C). Unfortunately, due to the lack of working anti-Pax4 antibodies, the detection of Pax4 at the protein level could not be performed. However, the exclusive misexpression of Pax4 in somatostatin + cells had been previously validated in SSTCrePOE pancreata (41). D cells are generally found scattered along the gut epithelium throughout life. Accordingly, using different approaches, we confirmed the presence of D cells both in the stomach and the duodenum. Specifically, somatostatin + cells were detected by immunofluorescence in control duodenum and in the stomach at all ages tested, that are 15, 90, 150, 240 and 360 days of age (Figures 2A, B). Importantly, a strong reduction in the content in somatostatin + cells was evidenced in SSTCrePOE animals at all ages tested, although few cells could still be detected ( Figures 2C, D). Due to the difficulties to quantify scattered somatostatin-expressing cells in organs as large as the duodenum and the stomach, we resorted to qRT-PCR analyses. These confirmed our observations with a massive decrease in somatostatin transcript contents in SSTCrePOE transgenic samples (up to 97% depending on the (st) age analysed) when compared to control littermates (Figures 2E, F). In line with this strong reduction in somatostatin expression, somatostatin circulating levels were also found reduced by 35% when compared to controls (Supplementary Figure 1A), while no significant difference was observed in circulating levels of IL-1beta and TNF-alpha proinflammatory cytokines (Supplementary Figures 1B, C), suggesting that the gastrointestinal barrier function of SSTCrePOE animals remained unaltered.
Aiming to determine whether the reduction in D cell numbers observed in transgenic animals could be explained by the acquisition of an alternative identity, as previously reported in the pancreas (41), further analyses were undertaken with a focus on insulin. It is important to note that insulin + cells are normally never detected in the gastrointestinal tract of wild-type mice ( Figures 3A, B). However, and most surprisingly, insulin + cells were clearly observed in SSTCrePOE transgenic duodenum and stomach ( Figures 3C, D). Accordingly, gene expression studies confirmed a dramatic increase in insulin transcript contents (up to 495%) at all (st)ages tested both in the duodenum and stomach of SSTCrePOE mice when compared to control samples ( Figures 3E, F).
Additional analyses were carried out to pinpoint the exact origin of gastrointestinal insulin-expressing cells. We first evaluated the main cell subtypes from the gastrointestinal mucosa ( Supplementary Figures 2A-E) focusing on epithelial, Goblet, Paneth, Stem and Enteroendocrine cells. The results obtained showed that the insulin + cells found in SSTCrePOE did express chromogranin A (ChgA), a marker of EECs, while being negative for Muc2, Lyz and Dcamkl1 markers of goblet, Paneth, and stem cell respectively. As gastrointestinal insulin-expressing cells were positive-labelled with ChgA (Supplementary Figure 2E Figures 4A, B). Indeed, 65% of insulin + cells appeared to be bihormonal (Sst + /Ins + ), while 24,5% of these were exclusively expressing this hormone, supporting the notion of a progressive/gradual conversion of somatostatin + cells into insulin + cells ( Figure 4E). Importantly, somatostatin-expressing cells are mostly found located closer to the crypts whereas insulin-labelled cells are detected closer to the tip of the villi ( Figure 4G), thus supporting the hypothesis that somatostatin + cells are converted into insulin + cells as they progress from the crypts to the top of the villi. Further investigations were then undertaken to determine the exact origin of gastrointestinal insulin-expressing cells. Towards this goal, we took advantage of the b-galactosidase reporter gene included in SSTCrePOE animals to trace the lineage of somatostatin + cells ( Figure 4F). Interestingly, the co-detection of both insulin and bgalactosidase ( Figures 4C, D) demonstrated the conversion of D cells (permanently labelled with b-galactosidase upon somatostatin expression) into insulin + cells. All insulin-expressing cells were thus found labelled with b-galactosidase. Together, these results suggest  that the misexpression of the Pax4 gene in D cells is sufficient to promote their reprogramming into insulin-producing cells.

Gastrointestinal Insulin + Cells Display a Beta-Like Cell Phenotype
Aiming to assess the identity of SSTCrePOE gastrointestinal insulin-expressing cells, a thorough characterisation was performed. Insulin + cells from both SSTCrePOE duodenum and stomach were thus found to express bona fide beta-cell markers, such as C-peptide, Kir6.2 and NeuroD1 ( Figures 5A-F). Moreover, gastrointestinal insulin + cells were also positive for PC1/3, an enzyme not exclusively expressed in beta-cells but required for the maturation of insulin ( Figures 5G, H). In addition to this immunohistochemical characterisation, a gene expression profile was established in gastrointestinal tissues by means of qRT-PCR. The expression levels of MafA, Kcnj11 (encoding for Kir6.2), Nkx6.1, Ucn3, Pcsk2 (encoding for PC2) and FoxA2 were all found increased in both the duodenum (up to 550%) and the stomach (up to 480%) of SSTCrePOE mice when compared to the control littermates ( Figure 6). Taken together, these results suggest that, overall, gastrointestinal insulinexpressing cells display a beta-like cell identity.
To determine whether somatostatin-expressing cells were fully losing their characteristics upon conversion into beta-like cells, the expression of the D cell-specific marker, Hhex, was examined ( Figures 7A, B). Interestingly, the results obtained in SSTCrePOE animals outlined three subpopulations: (1) insulin + cells retaining the expression of Hhex in their nuclei which could suggest a residual activity of the transcription factor ( Figure 7B),   depleted of Hhex signal ( Figure 7B). Such variability of Hhex expression could, yet again, be explained by the apparent progressive/gradual conversion process with the detection of cells at different stages, some of these thus displaying a mixed identity. Intriguingly, upon focusing on Mnx1 (Figures 7C, D), which repression induces the inverse conversion (beta-to-delta) (52), a significantly increase in the expression of this gene was noted when comparing transgenic versus control gastrointestinal tissues, suggesting a putative role of Mnx1 in the reprogramming of D cells into beta-like cells.
Taking into consideration our previous results, the functionality of gastrointestinal insulin + cells was next explored. The results obtained with regards to controls animals outlined an improved glucose tolerance of SSTCrePOE mice with a lower glycemia peak and a faster return to euglycemia (Supplementary Figure 3). However, it is important to mention that one cannot directly correlate this improvement in glucose handling to the sole release of gastrointestinal insulin, as pancreatic delta-cells are also converted into insulin + cells (41) and could therefore also contribute to this ameliorated glucose tolerance.

Generation of an Ex Vivo Model
The aforementioned limitations of our in vivo model prompted us to turn to an ex vivo approach based on gut organoids. Gut organoids were thus generated from control and SSTCrePOE transgenic mice and thorough analyses were performed at day 90. As seen in vivo at the protein level, we confirmed that somatostatin-expressing cells were found in all gut organoids analysed ( Figures 8A, C). Importantly, insulin + cells were exclusively detected in organoids derived from SSTCrePOE transgenic mice (Figures 8B, D). Somatostatin transcript levels were found 38% decreased in gut organoids derived from SSTCrePOE mice as compared to controls ( Figure 8E). Importantly, insulin expression levels appeared 256% increased (and up to 805% augmented in some organoids) when comparing control and transgenic organoids ( Figure 8F), again mimicking the results obtained in vivo. Another validation of such an ex vivo model came from the detection of insulinlabelled cells co-stained with somatostatin, again replicating in vivo observations where 65% of cells were found to express both hormones ( Figure 9A). Furthermore, and most importantly, lineage-tracing experiments proved that cells positive for insulin were also labelled with b-galactosidase ( Figure 9B). In a continued effort to determine whether gut organoids could faithfully mimic the in vivo mouse model, we assessed gene expression levels of key beta-cell markers ( Figures 9C-H). A clear increase in the expression levels of MafA, Kcnj11 (encoding for Kir6.2), Nkx6.1, Ucn3, Pcsk2 (encoding for PC2) and FoxA2 was outlined (up to 275%), thus confirming in vivo data. Altogether, being the results obtained using gut organoids in agreement with the in vivo data, we could validate the use of this ex vivo approach as a model to explore the functional outcome of gastrointestinal beta-like cells.

Gastrointestinal Beta-Like Cells Can Release Insulin Upon Stimulation
Once the gut organoids showed their ability to replicate in vivo data, we wondered whether the neo-generated beta-like cells were functional. Therefore, glucose-stimulated insulin secretion (GSIS) analyses were performed. The medium from control and transgenic organoids was collected and the amount of insulin contained in the medium was analysed by ELISA ( Figure 10). Interestingly, transgenic organoids released more insulin in the medium at basal glucose stimulation (2.8mM) when compared to control organoids (depleted of beta-cells). Equally important was the finding that the same organoids, when stimulated with a high glucose concentration (16.8mM), were able to secrete significantly higher amounts of insulin than with basal stimulation, suggestive of the ability of gastrointestinal beta-like cells to sense glucose and respond by secreting insulin in a regulated manner. As a last proof, organoids were challenged with KCl, which induces membrane depolarisation and, subsequently, an exocytosis of the secretory vesicles containing insulin. Yet again, a significantly higher insulin content was detected in the medium of SSTCrePOE organoids when compared to controls.
Together, these results clearly demonstrate that SSTCrePOEderived organoids can release insulin upon glucose stimulation and that such secretion is regulated depending on the glucose concentration.

DISCUSSION
In the present study, we demonstrate that the misexpression of Pax4 in gastrointestinal D cells triggers their conversion into insulin-expressing cells. These cells display a beta-like cell phenotype and can sense and respond to glucose by releasing insulin as demonstrated in an ex vivo environment. Our work thus demonstrates that Pax4 gene modulation could represent an interesting path for reprogramming intestinal cells into beta-like cells, a result of great potential for diabetes research. Somatostatin originating from the gastrointestinal tract has been reported to play an inhibitory role on other hormones secretion as well as to control gut motility and absorption rate (47,(53)(54)(55)(56)(57). Somatostatin-producing D cells are found scattered throughout the gastrointestinal mucosa, gastric D cells representing the major source of this hormone (44,48,58). Accordingly, we confirmed the greatest concentration in D cells in the stomach and, to a lesser extent, in the duodenum. Interestingly, the misexpression of Pax4 was found sufficient to induce a dramatic decrease in D cells and consequently in somatostatin expression, in both tissues. However, few D cells could still be detected while circulating somatostatin levels were only reduced by 35%, indicating that the remaining D cells were able to compensate for their lost counterparts. This could be easily explained by the continued regeneration of the gastrointestinal tissue, and thus of D cells.
Taking in consideration previous results demonstrating that, in the pancreas, the misexpression of Pax4 in somatostatinexpressing delta-cells was associated with their conversion into beta-like cells (41), we wondered whether such conversion was similarly occurring in the gastrointestinal tissues. At the protein level multiple insulin + cells were detected in stomach and duodenum of SSTCrePOE, both tissues being normally devoid of insulin-producing cells. Supporting this observation, insulin expression levels were found to be increased by up to 495% depending on the (st)age analysed in both duodenum and stomach transgenic samples. Lineage tracing experiments confirmed that all insulin-expressing cells were labelled with bgalactosidase (more than 100 cells examined), demonstrating that the sole misexpression of Pax4 can turn D cells into insulinproducing cells. This was further confirmed by the detection of cells co-expressing both insulin and somatostatin. Of note, such detection of cells displaying a mixed identity suggest an initial slow and gradual process of conversion eventually resulting in single-hormone insulin-expressing cells (Figure 11).
Aiming to determine whether these insulin-expressing cells displayed a true beta-cell phenotype, we investigated the expression of bona fide beta-cell markers (59). At both the transcript and protein levels, the data obtained was of interest as we could prove a parallel increase (up to 480%) between the FoxA2 (H) (6 < n < 17). All beta-cell markers were found considerably increased at the expression level when comparing intestinal organoids derived from SSTCrePOE mice versus controls. *P < 0.05, **P < 0.01.

FIGURE 10 | Regulated insulin secretion in SSTCrePOE gut organoids.
Quantification of insulin amounts released from control (red) and SSTCrePOE (blue) gut organoids upon stimulation (7 < n < 15) with 2.8mM glucose, 16.8mM glucose and 70mM KCl. At higher concentration of glucose (16.8mM), transgenic intestinal organoids release significant higher amounts of insulin when compared to control and to SSTCrePOE organoids stimulated with lower concentration of glucose (2.8mM), suggesting that insulin from intestinal beta-like cells can be secreted in a regulated manner. The KCl stimulation acts as a positive control of the test, as KCl induces the exocytosis of the insulin vesicles. ¶ P < 0.05, *P < 0.05, **P < 0.01. insulin + cell counts and the levels of expression of key genes involved in defining the beta-cell identity (MafA, Kcnj11, Nkx6.1, Ucn3, Pcsk2, FoxA2 and Mnx1), these being normally not expressed in the gastrointestinal tract of wild-type animals. Moreover, at the protein level, the observation of insulin + cells co-labelled with C-peptide was also equally interesting, as it suggested that insulin went through all the classical maturation steps prior to initiating C-peptide expression. We also investigated the expression of PC1/3, an enzyme involved in the maturation of insulin itself: PC1/3 was thus clearly detected in insulin + cells, a result demonstrating the normal posttranslational processing of insulin. Similarly, the co-labelling of insulin + cells with Kir6.2, which is the major subunit of the K ATP channel involved in insulin secretion, indicated that the insulin + cells could potentially release the hormone.
Interestingly and as previously discussed, such Pax4mediated D-to-beta-like conversion appeared to be progressive, as outlined by the detection of cells displaying a mixed identity ( Figure 11). This is further supported when focusing on the D cell marker, Hhex, a key transcription factor for their specification (60). As expected, Hhex was detected (1) in the nuclei of D cells, but also (2) in the nucleus or cytoplasm of a subpopulation of insulin + cells, suggestive of D-to-beta-like transition phase. A last subpopulation (3) of insulin + cells was outlined, these being depleted of Hhex, highlighting the completion of the identity shift.
One may therefore envision two hypotheses concerning the mechanisms underlying the Pax4-mediated D-to-beta-like cell conversion. In the first, Pax4 would directly inhibit Hhex expression. Accordingly, Pax4 binding sites have been detected within the Hhex locus at the position chr10:94451176-94451184 (61,62)), which could explain the reduction of Hhex expression and the loss of the D cell identity. The other alternative would be a putative role of Pax4 in modulating Mnx1 expression. In previous reports, the repression of Mnx1 was indeed shown to trigger pancreatic beta-to-delta-cell conversion (52). Accordingly, pancreata from animals lacking Pax4 were found depleted of Mnx1 expression, suggesting that Mnx1 might act downstream of Pax4 in b-cell determination (39). Hence, the increased Mnx1 expression observed in SSTCrePOE gastrointestinal samples could underly the reverse conversion, that is from somatostatin-producing cells to insulin-expressing cells. Interestingly, according to the JASPAR database, Mnx1 is predicted to regulate Hhex, which could suggest a putative indirect role of Pax4 in Hhex inhibition. Obviously, additional studdies and alternative analyses focusing on D cell markers would be necessary to confirm these results but, for the latter, none of those tried did work in our hands when assayed by immunohistochemistry.
Focusing further on the mechanisms underlying the D-to-betalike conversion observed in SSTCrePOE animals, one may wonder whether this process involved a dedifferentiation step prior to a redifferentiation event. To address this question, we assayed the expression of the endocrine precursor cell marker Ngn3 (data not shown). No change in Ngn3 expression or Ngn3-content was noted. One may thus conclude that the Pax4-mediated D-to-betalike conversion most likely occurs through direct conversion (or via a limited de-differentiation/re-differentiation). Nevertheless, additional work would be required to gain further insight into the molecular mechanisms involved.
Considering the fact that the delta-to-beta-like cell conversion previously observed in SSTCrePOE pancreas prevented us from assessing the metabolic changes exclusively provoked by the gastrointestinal D-to-beta-like cell conversion, an ex vivo approach was preferred. We thus developed gut-derived mouse organoids to evaluate the functionality of the neo-generated gastrointestinal insulin + cells. Importantly, intestinal beta-like cells appeared able to release insulin in a glucose dependent FIGURE 11 | Model proposed for the Pax4-mediated conversion of D cells into beta-like cells. Representation of the gastrointestinal structure. In the crypts, tissue stem cells (orange) continuously (re)generate the intestinal epithelium, some of which eventually differentiating into D cells (red). In our model, D cells are genetically modified to ectopically express Pax4. This genetic alteration triggers the conversion of D cells into beta-like cells (blue). We hypothesised that the Pax4-mediated conversion occurs through the inactivation of Hhex, directly repressed by Pax4 and/or indirect modulated through Mnx1. Interestingly, this conversion appears to be progressive, as supported by the observation of cells displaying a mixed identity. Hhex is expressed exclusively in the nucleus of D cells. However, Hhex was found in the nucleus and translocated in the cytoplasm of insulin + cells, while the expression of Mnx1 was increasing. At the last step of this conversion, insulin + cells only express beta-cell markers. Nevertheless, one cannot exclude the possibility that some D cells are directly converted into beta-like cell without passing through a transitory bi-hormonal phase. Equally important is the observation that D cells lost to conversion are continuously replaced and yet again turned into betalike cells. manner. Hence, from this work combined to thorough qPCR analyses focusing on beta-cell markers, we can conclude that the insulin + cells detected in the gastrointestinal tract of SSTCrePOE transgenic mice display most beta-cell features and are functional. The gastrointestinal tract harbours a great potential for T1D research due to its continuously regenerating nature. This feature is of great interest in the context of type 1 diabetes as reprogramming/ engineering the gastrointestinal epithelium to generate insulin + cells could allow for their continued replenishment despite a putative autoimmune attack. Here, we reported the conversion of D cells into functional beta-like cells upon the misexpression of Pax4. It is worth considering that the neo-generated gastrointestinal insulinproducing cells are (1) directly exposed to the ingested food, (2) continuously replaced, and (3) scattered in an immune-tolerogenic environment, which altogether renders the aforementioned conversion of great interest in the context of type 1 diabetes research. However, prior to considering such translational approach, some limitations need to be addressed. Indeed, (1) determining whether human D cells harbour an equivalent Pax4mediated plasticity when compared to their mouse counterparts would be required, despite reports suggesting comparable transcriptomes and peptidomes (63). Similarly, (2) understanding whether the neo-generated gastrointestinal insulin-expressing cells would escape the autoimmune attack and/or promote systemic immune tolerance would need to be investigated. Altogether, this work suggests that identifying Pax4 targets, and putative druggable targets, would be of great interest to gain further insight into the molecular mechanisms involved in this Pax4-mediated conversion, but also for the development of therapies aiming at regenerating the endogenous beta-cell mass.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Ciepal-Azur.

AUTHOR CONTRIBUTIONS
AG-U, ND, and PC conceived, designed the experiments, and discussed the results. AG-U performed most of the experiments, acquired the data and analysed/interpreted the results. CA, MF, SB, TN, and SS supported designing and performing some experiments. JA contributed to data acquisition. AG-U drafted and wrote the manuscript. AG-U, CA, MF, JA, SB, TN, SS, ND, and PC revised the manuscript and approved the final version.
PC is the guarantor of this work. All authors contributed to the article and approved the submitted version.