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Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system

2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês

10.1016/j.jbc.2023.102928

1083-351X

Masatoshi Ito, Natsuko Fujii, Saori Kohara, Shuho Hori, Masayuki Tanaka, Christopher Wittwer, Kenta Kikuchi, Takatoshi Iijima, Yu Kakimoto, Kenichi Hirabayashi, Daisuke Kurotaki, Henning J. Jessen, Adolfo Saiardi, Eiichiro Nagata,

Endoplasmic Reticulum Stress and Disease

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography–mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7—a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system. Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography–mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7—a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system. Myo-inositol phosphates (IPs) are ubiquitously synthesized in all organisms and are involved in pleiotropic biological processes, most importantly in intracellular signaling (1Irvine R.F. Schell M.J. Back in the water: the return of the inositol phosphates.Nat. Rev. Mol. Cell Biol. 2001; 2: 327-338Crossref PubMed Scopus (547) Google Scholar). Among the IP family, inositol hexakisphosphate (IP6) is the most abundant and serves as a precursor of inositol pyrophosphates (PP-IPs) possessing diphosphate moieties at specific carbon positions (2Saiardi A. How inositol pyrophosphates control cellular phosphate homeostasis?.Adv. Biol. Regul. 2012; 52: 351-359Crossref PubMed Scopus (52) Google Scholar, 3Wilson M.S. Livermore T.M. Saiardi A. Inositol pyrophosphates: between signalling and metabolism.Biochem. J. 2013; 452: 369-379Crossref PubMed Scopus (191) Google Scholar, 4Shears S.B. Inositol pyrophosphates: why so many phosphates?.Adv. Biol. Regul. 2015; 57: 203-216Crossref PubMed Scopus (91) Google Scholar, 5Shah A. Ganguli S. Sen J. Bhandari R. Inositol pyrophosphates: energetic, omnipresent and versatile signalling molecules.J. Indian Inst. Sci. 2017; 97: 23-40Crossref PubMed Scopus (21) Google Scholar). 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Tsukamoto H. et al.Inositol hexakisphosphate kinase 2 promotes cell death in cells with cytoplasmic TDP-43 aggregation.Mol. Neurobiol. 2016; 53: 5377-5383Crossref PubMed Scopus (13) Google Scholar), and Alzheimer's disease (23Crocco P. Saiardi A. Wilson M.S. Maletta R. Bruni A.C. Passarino G. et al.Contribution of polymorphic variation of inositol hexakisphosphate kinase 3 (IP6K3) gene promoter to the susceptibility to late onset Alzheimer's disease.Biochim. Biophys. Acta. 2016; 1862: 1766-1773Crossref PubMed Scopus (17) Google Scholar). Therefore, PP-IPs are currently being considered as potential therapeutic targets for several diverse human disorders (24Shears S.B. Towards pharmacological intervention in inositol pyrophosphate signalling.Biochem. Soc. Trans. 2016; 44: 191-196Crossref PubMed Scopus (11) Google Scholar, 25Chakraborty A. The inositol pyrophosphate pathway in health and diseases.Biol. Rev. Camb. Philos. Soc. 2018; 93: 1203-1227Crossref PubMed Scopus (56) Google Scholar). However, we are unaware of any systematic studies that have directly and comprehensively analyzed PP-IP distribution in mammalian tissues, which could provide valuable insights into the effects of pharmacological interventions on the PP-IP system. Over the past decade, extensive efforts have been made to develop analytical methods for detecting PP-IPs. Traditionally, PP-IPs have been studied using radioisotopic 3H-inositol labeling coupled with anion exchange chromatography (26Azevedo C. Saiardi A. Extraction and analysis of soluble inositol polyphosphates from yeast.Nat. Protoc. 2006; 1: 2416-2422Crossref PubMed Scopus (86) Google Scholar), which allows sensitive detection of metabolically labeled PP-IPs from cultured cells. Electrophoretic separation and colorimetric visualization of PP-IPs (27Losito O. Szijgyarto Z. Resnick A.C. Saiardi A. Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis.PLoS One. 2009; 4e5580https://doi.org/10.1371/journal.pone.0005580Crossref PubMed Scopus (100) Google Scholar) have also become alternative standard methods for distinguishing PP-IPs. However, PP-IPs in mammalian tissues can neither be radioisotopically labeled nor explicitly detected using colorimetric visualization. A mass spectrometric method coupled with capillary electrophoretic separation (capillary electrophoresis–mass spectrometry) (28Qiu D. Wilson M.S. Eisenbeis V.B. Harmel R.K. Riemer E. Haas T.M. et al.Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry.Nat. Commun. 2020; 11: 6035Crossref PubMed Scopus (49) Google Scholar) was recently reported for sensitive analysis of PP-IPs in biological samples at the isomer level. However, the instrument setup involved is complex and requires skillful handling, and this method is therefore rarely available in research institutes. We recently developed an analytical method that directly detects mammalian-derived IP7 and its precursor IP6 using conventional liquid chromatography–tandem mass spectrometry (MS/MS) coupled with hydrophilic interaction liquid chromatography (HILIC) (29Ito M. Fujii N. Wittwer C. Sasaki A. Tanaka M. Bittner T. et al.Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the quantitative analysis of mammalian-derived inositol poly/pyrophosphates.J. Chromatogr. A. 2018; 1573: 87-97Crossref PubMed Scopus (19) Google Scholar), enabling the previously impossible quantitation of PP-IPs in mammalian tissues. In this study, we analyzed PP-IP and their precursor IP6 levels in mammalian organs using a refined HILIC-MS/MS protocol. We found that IP7 was present at explicit levels in the mammalian central nervous system (CNS), where IP6Ks are highly expressed. Surprisingly, we also discovered that the highest IP7 production was observed in the gastrointestinal tract (GIT), even after depletion of dietary derived IP7. Of the major GIT cells, enteric neurons selectively expressed IP7-synthesizing enzyme IP6K2, which was revealed by assessment of single-cell RNA sequencing (scRNA-seq) data sets and confirmed by immunohistochemical detection. Our HILIC-MS/MS survey using IP6K2-knockout (IP6K2−/−) mice exhibited that IP6K2-dependent enhanced IP7 metabolism exists in the gut and duodenal muscularis externa where the myenteric plexus is located. We further performed whole transcriptome analysis of IP6K2-deficient and wildtype (WT) duodenal muscularis externa to define a physiological role of IP6K2-IP7 pathway in the enteric nervous system (ENS). Before investigating PP-IP metabolism in mammalian tissues, we improved our HILIC-MS/MS analysis protocol for unequivocal detection and more precise quantitation of PP-IPs. Medronic acid was shown to improve the chromatographic detection of phosphorylated compounds (30Hsiao J.J. Potter O.G. Chu T.W. Yin H. Improved LC/MS methods for the analysis of metal-sensitive analytes using medronic acid as a mobile phase additive.Anal. Chem. 2018; 90: 9457-9464Crossref PubMed Scopus (62) Google Scholar). Consistently, a form of this solvent additive optimized for HILIC analysis (InfinityLab deactivator additive, Agilent Technologies) significantly improved the chromatographic peak shapes of IP6 and IP7 (Fig. S1A) and achieved clear detection of low abundance (10 pmol) of IP7 (Fig. S1B). We also determined the selected reaction monitoring (SRM) conditions for IP8 (Table S1) by learning its fragmentation pattern (Fig. S1D) to quantitate IP8 simultaneously with IP6 and IP7 (Fig. S1E). To benchmark this method for the detection of endogenous PP-IPs, we analyzed HCT116 cells treated with NaF, which is known to increase intracellular PP-IPs level (31Menniti F.S. Miller R.N. Putney Jr., J.W. Shears S.B. Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells.J. Biol. Chem. 1993; 268: 3850-3856Abstract Full Text PDF PubMed Google Scholar). Our HILIC-MS/MS analysis detected explicit IP7 and IP8 SRM peaks in NaF-treated cells (Fig. S1F). We also observed a dose-dependent reduction in IP7 level and the IP7/IP6 ratio in HCT116 cells treated with the IP6K inhibitor TNP (Fig. S2). Thus, our refined HILIC-MS/MS protocol achieved robust, sensitive, and reliable detection of endogenous IP6, IP7, and IP8 in biological samples. Using the newly developed HILIC-MS/MS protocol, we investigated the distribution of PP-IPs in experimental model rodents fed with a standard plant-based diet (CE-2; Clea, Japan). Fifteen organs, including the CNS and GIT, were harvested from standard diet–fed C57BL/6J male mice. Surprisingly, HILIC-MS/MS analysis showed that the GIT had the highest levels of IP6 and IP7, even after extensive rinsing of the organs with phosphate-buffered saline (PBS) to wash out the digested contents (Fig. 1, A and B andTable S2). Importantly, the IP7/IP6 ratio in the GIT was remarkably high, by far the highest in all organs examined (Fig. 1C and Table S2). A subtle IP8 SRM peak was detected in stomach and small intestine samples, wherein IP7 was abundant (Fig. 1D) but was not detected in other organs. While IP7 SRM peaks were clearly detected in CNS samples (Fig. 1E), IP7 levels in the CNS were modest compared with those in the GIT. Moreover, the IP7/IP6 ratio in the spinal cord appeared to be higher than that in the cerebrum (Fig. 1C). Several reports have shown that IPs (mainly IP6, known as phytic acid) are present in a variety of crop seeds (32Dorsch J.A. Cook A. Young K. Anderson J.M. Bauman A.T. Volkmann C.J. et al.Seed phosphorus and inositol phosphate phenotype of barley low phytic acid genotypes.Phytochemistry. 2003; 62: 691-706Crossref PubMed Scopus (160) Google Scholar, 33Liu X. Villalta P.W. Sturla S.J. Simultaneous determination of inositol and inositol phosphates in complex biological matrices: quantitative ion-exchange chromatography/tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2009; 23: 705-712Crossref PubMed Scopus (40) Google Scholar, 34Kolozsvari B. Firth S. Saiardi A. Raman spectroscopy detection of phytic acid in plant seeds reveals the absence of inorganic polyphosphate.Mol. Plant. 2015; 8: 826-828Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 35Duong Q.H. Clark K.D. Lapsley K.G. Pegg R.B. Quantification of inositol phosphates in almond meal and almond brown skins by HPLC/ESI/MS.Food Chem. 2017; 229: 84-92Crossref PubMed Scopus (17) Google Scholar); moreover, plants also generate PP-IPs, which are crucial for phosphorus-starvation responses (36Dong J. Ma G. Sui L. Wei M. Satheesh V. Zhang R. et al.Inositol pyrophosphate InsP8 acts as an intracellular phosphate signal in arabidopsis.Mol. Plant. 2019; 12: 1463-1473Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 37Ried M.K. Wild R. Zhu J. Pipercevic J. Sturm K. Broger L. et al.Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis.Nat. Commun. 2021; 12https://doi.org/10.1038/s41467-020-20681-4Crossref PubMed Scopus (73) Google Scholar, 38Riemer E. Qiu D. Laha D. Harmel R.K. Gaugler P. Gaugler V. et al.ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis.Mol. Plant. 2021; 14: 1864-1880Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Therefore, we assumed that the plant-based CE-2 diet contains IP6 and PP-IPs, and explicit chromatographic peaks of IP6, IP7, and IP8 were observed in CE-2 samples (Fig. 2A, upper panel). We next investigated their concentrations in purified diets with minimal levels of plant-derived components (Fig. 2A, middle and lower panels). The two purified diets examined (iVid-neo and 70% casein) contained low amounts of IP6 and negligible amounts of IP7 and IP8. Quantitative analysis revealed that the levels of all PP-IPs in both purified diets were less than 2% of those in CE-2 (Fig. 2B). The concentrations of IP6 and IP7 and the relative level of IP8 in CE-2 and two purified diets were summarized in Table S3. Thus, the abundances of IP6 and PP-IPs differ among different mouse diets, and standard mouse diet CE-2 substantially contains IP6 and PP-IPs, suggesting that dietary IP6 and IP7 affect the total levels of these molecules in the GIT of standard diet–fed mice. Two possibilities were considered to explain the high IP7/IP6 ratios in the stomach and duodenum (Fig. 1C): increased absorption of dietary IP7 or the presence of endogenous IP7 in these two GIT organs. To verify the former possibility (selective intestinal absorption of IP7), we analyzed the feces of mice fed on CE-2 and estimated the loss of IP6 and IP7 in the digestive system. Similar to those for CE-2 samples, IP6 and IP7 SRM peaks were clearly observed in mouse feces samples (Fig. 2C), suggesting that considerable amounts of IP6 and IP7 still remain in the feces. Quantitative analysis showed that approximately 50% of IP6 and IP7 in ingested food remained in the feces (Fig. 2D). Since the IP7/IP6 ratio remained unchanged between undigested CE-2 and feces (Fig. 2E), dietary IP6 and IP7 might not be disproportionately degraded and absorbed throughout the digestive system. Therefore, we could exclude the possibility that dietary IP7 is preferentially absorbed in the GIT, further highlighting that the high IP7/IP6 ratios of the stomach and duodenum are probably attributable to cellular IP7 metabolism. Dietary IP6 and PP-IPs present in the diet blur the direct detection of mammalian-derived IP6 and PP-IPs in the GIT of standard diet–fed mice. To more precisely validate the presence of endogenously synthesized IP6 and PP-IPs in the GIT, we prepared C57BL/6J mice maintained under two different conditions both of which remove dietary PP-IPs, namely, 2 months feeding of purified diet iVid-neo containing negligible amounts of PP-IPs (Fig. 2, A and B and Table S3) and fasting for 48 h, and compared their tissue concentrations of IP6 and PP-IPs with those from standard CE-2 diet–fed counterparts (Fig. 3A). The GIT of both purified diet–fed and fasted mice showed a reduction in IP6 and IP7 levels compared with those of standard diet–fed mice; however, the levels were still close to (in the case of IP6) or far greater than (in the case of IP7) the CNS levels (Fig. 3, B and C). IP8 was not detected in any of the tested organs of purified diet–fed and fasted mice. The SRM chromatograms of both purified diet–fed and fasted mice samples had explicit IP7 SRM peaks (Fig. 3D). Importantly, the stomach and duodenum of these mice showed prominently higher IP7/IP6 ratios than those of their standard diet–fed counterparts (Fig. 3E), implying further enhanced IP7 metabolism compensated for the overall reduced IP7 level. On the other hand, both purified diet–fed and fasted mice did not show any changes in the IP6 and IP7 levels as well as the IP7/IP6 ratio in the CNS and testis compared with those of mice fed a standard diet. However, as with standard diet–fed mice, both purified diet–fed and fasted mice showed higher IP7/IP6 ratios in the spinal cord than in the cerebrum, implying heterogeneous IP7 metabolic activity in the rostral and caudal CNS. We also investigated IP7 levels in the GIT of purified diet (70% casein)-fed Sprague–Dawley rats (Fig. S3A). Analogous to the results observed in the mouse model, both IP6 and IP7 levels in the GIT of these rats were drastically reduced compared with those in the standard diet–fed GIT and comparable with those in the CNS (Fig. S3, B and C). In addition, the IP7/IP6 ratio was higher in the stomach and duodenum of purified diet–fed rats compared with that of standard diet–fed rats (Fig. S3D), further demonstrating very active IP7 metabolism in the mammalian proximal GIT. To investigate the expression levels of the three IP6Ks in each GIT cell type, we used scRNA-seq datasets and compared the expression levels of IP6Ks among GIT cell types. Quantitative analysis using a human embryonic intestinal cell scRNA-seq dataset (39Fawkner-Corbett D. Antanaviciute A. Parikh K. Jagielowicz M. Gerós A.S. Gupta T. et al.Spatiotemporal analysis of human intestinal development at single-cell resolution.Cell. 2021; 184: 810-826.e23Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) showed that enteric neural cells expressed the highest levels of IP6K2 among different intestinal cells (Fig. 4A, upper panel). In enteric neural cells, IP6K2 was selectively expressed across enteric neuron subsets, such as motor neurons, interneurons, and neuroendocrine cells but not in glial cells (Fig. 4A, lower panel). This analysis was further supported by the IP6K quantitation using both E15.5 (Fig. S4A) and E18.5 (Fig. 4B) mouse embryonic ENS scRNA-seq datasets (40Morarach K. Mikhailova A. Knoflach V. Memic F. Kumar R. Li W. et al.Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.Nat. Neurosci. 2021; 24: 34-46Crossref PubMed Scopus (91) Google Scholar). As in humans, IP6K2 isoform expression level in mouse enteric neurons was higher than in other neural cells such as neuroblasts, progenitors, glial cells, and Schwann cells. Moreover, the transcriptional analysis–based data were verified using immunohistochemical analyses. IP6K2 colocalized with the neuronal marker HuC/D in the mouse duodenal muscle layer, suggesting IP6K2 was expressed in the myenteric plexus (Fig. 4C). Other than enteric neurons, several cell types, including secretory progenitor cells, also expressed relatively high levels of IP6K2 (Fig. S4B). In addition, mouse enteric epithelial cell scRNA-seq data (41Haber A.L. Biton M. Rogel N. Herbst R.H. Shekhar K. Smillie C. et al.A single-cell survey of the small intestinal epithelium.Nature. 2017; 551: 333-339Crossref PubMed Scopus (814) Google Scholar) showed that IP6K2 is expressed in mouse enteroendocrine cells (Fig. S4C). Expression levels of IP6K1 and IP6K3 in entire embryonic intestinal cells were low and negligible, respectively (Fig. 4, A and B). These results suggest that IP6K2 is highly expressed in mammalian enteric neurons. To estimate the importance of IP6K2 in endogenous IP7 synthesis in the mammalian organs including GIT, we employed a genetically modified mouse in which IP6K2 exon 6, encoding the kinase domain, was specifically deleted (Fig. 5A, left panel) (42Rao F. Cha J. Xu J. Hu R. Vandiver M.S. Tyagi R. et al.Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2.Mol. Cell. 2014; 54: 119-132Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). To avoid any contamination of dietary-derived IPs in our analysis, IP6K2-knockout (IP6K2−/−) or wildtype (WT) mice raised on the standard CE-2 diet were switched to a purified diet (iVid-neo) for 1 week and then fasted for 48 h before sacrifice (Fig. 5A, right panel). In WT mice, IP6K2 mRNA containing the exon 6 sequence was expressed in the proximal GIT but only marginally compared with the expression in the CNS (Fig. S5A). As expected, the IP6K2 transcript was absent in IP6K2−/− mouse organs. We confirmed the loss of IP6K2 expression in IP6K2−/− mice at the protein level using cerebrum lysate (Fig. S5B), because it has high IP6K2 protein expression and thus was useful for clearly validating the loss of IP6K2 in IP6K2−/− mice. HILIC-MS/MS analysis showed that IP6K2−/− mice had significantly lower levels of IP7 in various organs, including the stomach and duodenum, compared with those in their WT counterparts, while IP6 levels in each organ were almost the same between IP6K2−/− and WT mice (Fig. 5, B and C). As previously observed (Figs. 3E and S3D), the IP7/IP6 ratios in the stomach and duodenum of WT mice were much higher than those in the other organs examined (Fig. 5D). These two organs of IP6K2−/− mice exhibited significant reduction in the IP7/IP6 ratios compared with those of WT mice, suggesting active IP6K2 pathway works in stomach and duodenum. Consistently, the IP7 SRM peaks for the IP6K2−/− mouse stomach and duodenum were smaller compared with those of WT mice, while IP6 levels were unchanged (Fig. S5, C and D). Collectively, these data demonstrate that IP6K2 is required for enhanced IP7 metabolism in the mammalian proximal GIT. Since IP7-synthesizing kinase IP6K2 is selectively expressed in enteric neurons (Fig. 4), we next sought to investigate IP7 metabolism in the mammalian ENS. We collected the stomach and the consecutive 5-cm segments of duodenum, jejunum, and ileum from standard diet–fed (dietary PP-IPs provided) or fasted (dietary PP-IPs depleted) mice to more precisely survey the PP-IP profile in the proximal GIT. Some of these organs collected were subsequently used to isolate the muscularis externa where the myenteric plexus is located. These total GIT tissues and their muscularis externa were subjected to HILIC-MS/MS analysis to compare their IP7 metabolism (Fig. 6A). Similar to the results shown in Figure 3, 48 h fasting of mice rendered drastic reduction of IP6 and IP7 levels with concomitant increase of the IP7/IP6 ratio in total GIT tissues (Figs. 6B and S6). Although the muscularis externa contained less IP6 and IP7 than total GIT tissues, the muscle layer exhibited a higher IP7/IP6 ratio than total GIT tissues, which was less dependent on dietary conditions. The IP7/IP6 ratio of the duodenal muscularis externa was highest among the corresponding muscle layers of the neighboring GITs, implying highly active IP7 metabolism in the duodenal ENS. To verify the relationship between IP6K2-IP7 axis and the ENS, we first attempted to visualize the duodenal myenteric plexus of IP6K2−/− mice by whole mount immunostaining (Fig. S7A). We found that IP6K2 deletion largely affected neither the morphological features nor the neuronal cell density in the duodenal myenteric plexus (Fig.