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GFAP-directed Inactivation of Men1 Exploits Glial Cell Plasticity in Favor of Neuroendocrine Reprogramming

Open AccessPublished:July 11, 2022DOI:https://doi.org/10.1016/j.jcmgh.2022.06.009

      Background & Aims

      Efforts to characterize the signaling mechanisms that underlie gastroenteropancreatic neoplasms (GEP-NENs) are precluded by a lack of comprehensive models that recapitulate pathogenesis. Investigation into a potential cell-of-origin for gastrin-secreting NENs revealed a non-cell autonomous role for loss of menin in neuroendocrine cell specification, resulting in an induction of gastrin in enteric glia. Here, we investigated the hypothesis that cell autonomous Men1 inactivation in glial fibrillary acidic protein (GFAP)-expressing cells induced neuroendocrine differentiation and tumorigenesis.

      Methods

      Transgenic GFAPΔMen1 mice were generated by conditional GFAP-directed Men1 deletion in GFAP-expressing cells. Cre specificity was confirmed using a tdTomato reporter. GFAPΔMen1 mice were evaluated for GEP-NEN development and neuroendocrine cell hyperplasia. Small interfering RNA-mediated Men1 silencing in a rat enteric glial cell line was performed in parallel.

      Results

      GFAPΔMen1 mice developed pancreatic NENs, in addition to pituitary prolactinomas that phenocopied the human MEN1 syndrome. GFAPΔMen1 mice exhibited gastric neuroendocrine hyperplasia that coincided with a significant loss of GFAP expression. Men1 deletion induced loss of glial-restricted progenitor lineage markers and an increase in neuroendocrine genes, suggesting a reprogramming of GFAP+ cells. Deleting Kif3a, a mediator of Hedgehog signaling, in GFAP-expressing cells attenuated neuroendocrine hyperplasia by restricting the neuroendocrine cell fate. Similar results in the pancreas were observed when Sox10 was used to delete Men1.

      Conclusions

      GFAP-directed Men1 inactivation exploits glial cell plasticity in favor of neuroendocrine differentiation.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      ACTH (adrenocorticotropic hormone), BG (Brunner’s glands), BSA (bovine serum albumin), CHGA (chromogranin A), DEG (differently expressed genes), EGC (enteric glial cells), ELISA (enzyme-linked immunosorbent assay), FBS (fetal bovine serum), GCG (glucagon), GEP-NENs (gastroenteropancreatic neuroendocrine neoplasms), GFAP (glial fibrillary acidic protein), GH (growth hormone), GI (gastrointestinal), GRP (gastrin-releasing peptide), HPF (high power field), HRP (horseradish peroxidase), INS1 (insulin), IRX2 (iroquois homeobox protein 2), MEN1 (multiple endocrine neoplasia 1), NET (neuroendocrine tumors), NF-H (neurofilament-H), OCT (optimal cutting temperature), PBS (phosphate buffered saline), pitNET (pituitary neuroendocrine tumor), PNEC (pancreatic neuroendocrine carcinoma), PNEN (pancreatic neuroendocrine neoplasm), PNET (pancreatic neuroendocrine tumor), qPCR (quantitative polymerase chain reaction), RT (room temperature), SHH (sonic hedgehog), siRNA (small interfering RNA), SOX10 (SRY-box transcription factor 10), SST (somatostatin), SYP (synaptophysin), VIP (vasoactive intestinal peptide), WT (wild-type)
      Loss of the tumor suppressor protein menin in glial fibrillary acidic protein-expressing cells stimulates gastric neuroendocrine hyperplasia and neuroendocrine tumors in the pituitary and pancreas. Deletion of menin favors glial cell reprogramming toward a neuroendocrine phenotype with tumorigenic potential.
      Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) comprise a heterogeneous group of malignancies showing an increase in incidence and prevalence across the United States.
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      Deletion of Men1 and somatostatin induces hypergastrinemia and gastric carcinoids.
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      Deletion of Men1 and somatostatin induces hypergastrinemia and gastric carcinoids.
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      Gastrin induces nuclear export and proteasome degradation of menin in enteric glial cells.
      Taken together, these observations suggest that hyperplastic G-cells might emerge from reprogrammed neural crest-derived cells in addition to endoderm-derived enteroendocrine cells. Indeed, prior studies have shown that multipotent glial cells marked by GFAP or SRY-box transcription factor 10 (SOX10) can generate neuroendocrine cells during normal development
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      Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells.
      Therefore, we tested the hypothesis that cell autonomous deletion of Men1 in GFAP-expressing cells promotes neuroendocrine cell development. Here, we show that conditional deletion of Men1 in GFAP-expressing glial cells results in NET development in the pituitary and pancreas, in addition to neuroendocrine hyperplasia in the stomach. These events coincided with loss of the GFAP-restricted lineage and emergence of an endocrine phenotype with tissue-specific hormone expression, suggesting a direct role for glial cell reprogramming upon removal of menin. Directing Men1 deletion to SOX10-expressing cells recapitulated pancreatic NET development and gastric neuroendocrine hyperplasia, further supporting the premise that a neural crest-derived cell might be a cell-of-origin for NETs.

      Results

      GFAPΔMen1 Mice Develop Pancreatic Neuroendocrine Neoplasms (PNENs)

      To determine whether loss of menin in GFAP-expressing cells was sufficient to drive the development of GEP-NENs, we conditionally deleted Men1 under the control of 2.2 kb of the human GFAP promoter (GFAPΔMen1).
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      After 15 to 24 months of age, 11 of 22 GFAPΔMen1 mice developed macroscopic tumors throughout the pancreas (Table 1). Histological analysis of the pancreas identified significant islet hyperplasia and features characteristic of neuroendocrine neoplasms (Figure 1, A). Pancreatic lesions varied from poorly differentiated with a solid sheet-like morphology to highly differentiated with a rosette pattern (Figure 1, B–D). Consistent with their unique histology, highly differentiated neoplasms exhibited cytoplasmic or absent menin expression (Figure 1, E) and strong immunoreactivity for the neuroendocrine markers synaptophysin (SYP) and chromogranin A (CHGA) (Figure 1, E–F). In contrast, poorly differentiated tumors were negative for CHGA and exhibited low expression of SYP (Figure 1, G). To assess whether these tumors were indeed of pancreatic islet origin, we examined the expression of iroquois homeobox protein 2 (IRX-2), a tissue-restricted transcription factor required for alpha cell lineage specification.
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      we stained GFAPΔMen1 neoplasms for Ki-67 and classified the neoplasms as pancreatic neuroendocrine tumors (PNETs) (<2% Ki-67+ cells per high power field, HPF) (Figure 1, H–I) or pancreatic neuroendocrine carcinomas (PNECs) (>20% Ki-67+ cells per HPF) (Figure 1, J). Classification of the Ki-67HIGH/CHGANEG/SYPLOW neoplasms as PNECs is consistent with human NECs exhibiting reduced expression of the classical neuroendocrine biomarkers.
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      Further characterization of highly differentiated PNETs revealed the presence of both hyperplastic INS1 (insulin)-expressing beta-cells and glucagon (GCG)-expressing alpha-cells (Figure 1, K–M). Consistent with positive staining for neuroendocrine markers, PNETs exhibited a 10-fold increase in Chga transcript and a trending but nonsignificant increase in Syp mRNA expression compared with normal pancreas (Figure 1, N).
      Table 1Summary of Phenotypes Observed in GFAP Cre; Men1FL/FL Mice Aged 13 to 24 Months
      Men1Pancreatic hyperplasia/tumor, n/N; average agePituitary adenoma, n/NAntral hyperplasia/tumor, n/NBG/duodenal polyp, n/NSI lipoma, n/N
      WT0/30; 17.5 mo0/300/300/300/30
      FL/+1/9; 17.9 mo1/91/91/90/9
      FL/FL11/36; 17.9 mo18/3911/362/362/36
      11/22; 15 mo and older
      • 7/13 female
      • 4/9 male
      • 17/30 female

      • 1/9 male
      • 8/27 female

      • 3/9 male
      • 2/27 female• 2/27 female
      0/14; Under 14 mo
      BG, Brunner’s glands; SI, sacroiliac; WT, wild-type.
      Figure thumbnail gr1
      Figure 1GFAP-directed inactivation of Men1 promotes pancreatic islet hyperplasia and the development of pancreatic neuroendocrine neoplasms. (A) Hematoxylin and eosin (H&E) stains of well differentiated and poorly differentiated tumors in GFAPΔMen1 mice compared with WT and heterozygous groups. (B–C) H&E stains of well differentiated GFAPΔMen1 PNETs compared with a poorly differentiated PNEC (D). (E) Immunofluorescent staining of a PNET for CHGA and menin. Insets: representative images of a PNET showing absent or cytoplasmic menin in the tumor (white). Immunofluorescent staining of a PNET (F) and PNEC (G) for SYPand the alpha cell-specification factor IRX-2. Insets: IRX-2 is expressed in the nucleus of tumor cells (white). H–I) Ki-67 and smooth muscle actin (SMA) staining of PNETs compared with a PNEC (J). Proliferative index (PI) is indicated by the percentage of Ki67-positive tumor cells in each HPF. K) H&E stain of a GFAPΔMen1 PNET stained for GCG (L) and IS1 (M). (N) Quantitation of Chga and Syp mRNA in WT pancreas and GFAPΔMen1 PNETs. N = 3–5 mice per group;∗∗P < .01. Data are represented as mean ± standard deviation. (O) Number of mice presenting with pancreatic neuroendocrine neoplasms as stratified by sex. (P) Number of PNETs per mouse in males vs females. Levels of GCG (Q) IS1 (R) and glucose (S) in sera of WT and GFAPΔMen1 mice, with symbols indicating male or female mice with and without tumors. N = 11–12 mice per group; ∗P < .05; ∗∗P < .01. Data are represented as mean ± standard deviation.
      PNENs were predominantly comprised of PNETs (approximately 83% of all PNEN-bearing mice), whereas PNECs were only observed in 2 mice (comprising 17% of all PNEN-bearing mice, one of which was heterozygous for Men1 deletion). Notably, there was not a mixture of NETs and NECs in the same mice. As gender-related differences have been previously reported in patients with MEN1 syndrome,
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      we further stratified the occurrence of GFAPΔMen1 PNECs and PNETs by sex (Table 1; Figure 1, O). Both cases of PNECs occurred in female mice. The incidence of PNETs was slightly higher in female mice compared with male mice, occurring in 7 of 13 females vs 4 of 9 males over the age of 15 months (53% vs 44%, respectively). Interestingly, female GFAPΔMen1 mice tended to present with multiple lesions compared with males that presented with a solitary tumor (Figure 1, P). Of the 7 female tumor-bearing mice, 6 (86%) had 2 or more PNETs compared with only 1 of 4 (25%) males. As anticipated, GFAPΔMen1 mice exhibited significantly elevated levels of circulating GCG and INS1 hormones, with tumor-bearing mice generally exhibiting the highest hormone expression irrespective of sex (Figure 1, Q–R). Consistent with the opposing effects of GCG and INS1 on glucose regulation, serum glucose levels were unchanged in GFAPΔMen1 mice regardless of sex and tumor status (Figure 1, S).

      Development of PNETs in GFAPΔMen1 Mice Coincides With Loss of GFAP Expression

      Due to the low overall incidence of PNECs, we focused our subsequent investigation on PNETs only. To determine the effect of Men1 loss on GFAP expression in PNETs, we stained normal pancreas and PNETs for GFAP. Consistent with previous reports, GFAP expression was strongly localized to the periphery of SYP+ and hormone-expressing islets in both wild-type and GFAPΔMen1 mice;
      • Regoli M.
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      Glial fibrillary acidic protein (GFAP)-like immunoreactivity in rat endocrine pancreas.
      however, GFAP expression was uniquely lost in cells encircling the PNETs (Figure 2, A–B). Consistent with this loss, the expression of glial lineage transcripts Gfap and S100b were significantly lower in PNETs compared with the pancreas of wild-type (WT) mice, whereas Men1 mRNA exhibited a trending decrease (Figure 2, C). The specificity of GFAP marking the glial lineage was confirmed by showing that GFAP did not colocalize with phospo-Neurofilament-H (NF-H)-expressing nerve fibers in the pancreas and in PNETs (Figure 2, A–B). To further evaluate the specificity of GFAP Cre expression, we generated GFAP Cre-loxP-tdTomato mice and confirmed that fluorescent tdTomato protein expression associated with GFAP and the glial lineage markers S100B and p75NTR in frozen mouse pancreas sections, as previously reported
      • Wang Z.
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      • Sundaresan S.
      • Ding L.
      • Hayes M.
      • Sahoo N.
      • Xu H.
      • Merchant J.L.
      Isolation of enteric glial cells from the submucosa and lamina propria of the adult mouse.
      (Figure 2, D–E). As expected, GFAP-tdTomato expression did not co-localize with NF-H+ nerve fibers, further confirming the specificity of GFAP marking the glial lineage. In summary, development of well-differentiated PNETs in this model coincided with loss of GFAP expression from the periphery of normal pancreatic islets.
      Figure thumbnail gr2
      Figure 2GFAPΔMen1 PNETs exhibit loss of GFAP expression and maintain stemness in 3D culture. (A) Immunofluorescent staining of a WT mouse pancreatic islet for the glial-restricted progenitor lineage marker GFAP, the nerve fiber marker NF-H, SYP, the beta-cell hormone INS1, the alpha-cell markers GCG and IRX-2, and menin. (B) Representative images of a normal islet and GFAPΔMen1 PNET stained for GFAP in green and the nerve fiber marker NF-H in red, indicating the absence of GFAP expression in and surrounding the PNET. (C) Quantitation of Gfap, S100b) and Men1 mRNA in WT pancreas and GFAPΔMen1 PNETs. N = 4–5 mice per group; ∗P < .05; ∗∗P < .01. Data are represented as mean ± standard deviation. (D) Schematic of GFAPΔMen1-tdTomato construct for endogenous labeling of GFAP. (E) Immunofluorescent images of frozen pancreas sections from WT GFAP-tdTomato mice co-stained for the glial-restricted progenitor lineage markers GFAP, S100B, and P75NTR, in addition to NF-H as a negative marker for the glial lineage. Immunofluorescent images were merged on a transmitted light micrograph to distinguish islets from acinar cells. (F) Top to bottom: Macroscopic image of a GFAPΔMen1 pancreas, with an arrow indicating the presence of a PNET and phase contrast image of a corresponding PNET organoid. PNET organoids were stained for CHGA, SYP, GCG, INS1, and Ki-67 and imaged by confocal microscopy. Images are representative of PNET organoids derived from 4 tumor-bearing mice (n = 4).
      We next evaluated whether GFAPΔMen1 PNETs carried the potential to propagate in 3-D organoid culture as a measure of stemness. Organoid lines were established from PNETs from 4 mice and cultured for 3 weeks prior to staining or cryopreservation (Figure 2, F). Similar to parent tumors, the organoids strongly expressed SYP, CHGA, GCG, and INS1, but only a few cells exhibited Ki-67 positivity (Figure 2, F). Therefore, PNET-derived organoids recapitulated the biological features of their parent tumors and exhibited stem-like features in the absence of Ki-67 expression.

      GFAPΔMen1 Mice Develop PitNETs That Phenocopy Human MEN1 Prolactinomas

      Nine of 30 female GFAPΔMen1 mice presented with clinical signs of hydrocephalus starting at 15 months of age, including presentation of a domed head appearance, loss of muscle coordination, seizures, and repetitive motor movements. Following the onset of hydrocephalus, mice were necropsied, and brains were analyzed for potential effects of GFAP-directed Men1 inactivation. The overarching rationale for these studies was emphasized by the fact that GFAP is the main intermediate filament protein in brain astrocytes and other cells of the glial-restricted lineage.
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      GFAP in health and disease.
      We discovered that GFAPΔMen1 mice developed hemorrhagic pituitary adenomas (Figure 3, A–B). Subsequent immunostaining of GFAPΔMen1 adenomas demonstrated diffuse immunoreactivity for SYN and CHGA as expected (Figure 3, C). Strong staining for prolactin showed these tumors to be prolactinomas (Figure 3, D). As prolactin is produced by lactotropes of the PIT-1+ lineage, we stained pituitary NETs for the cell-patterning transcription factor PIT-1. As expected, tumors exhibited strong expression of PIT-1, thus confirming that both pancreatic and pituitary NETs arise in the context of endocrine lineage-specific transcription factor expression (Figure 3, E). Additionally, Ki-67 inversely correlated with the expression of neuroendocrine markers, with stronger neuroendocrine status associated with lower Ki-67 staining (Figure 3, E). Because cells of the Pit-1+ lineage also comprise growth hormone (GH)-expressing somatotrophs but not adrenocorticotropic hormone (ACTH)-expressing corticotropes,
      • Zhang S.
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      we stained pituitary tumors for GH and ACTH expression. Tumors exhibited patchy but generally robust expression of GH, whereas the expression of ACTH was restricted to only a few cells (Figure 3, F). These observations were consistent with the fact that pituitary tumors arising in the human MEN1 syndrome are most commonly comprised of prolactinomas and pluri-hormone-secreting adenomas (ie, secreting prolactin and a second hormone), with growth hormone-producing tumors being the next most common tumor type.
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      Pituitary disease in MEN type 1 (MEN1): data from the France-Belgium MEN1 multicenter study.
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      Pituitary tumors in patients with MEN1 syndrome.
      Figure thumbnail gr3
      Figure 3GFAPΔMen1 mice develop pitNETs that phenocopy human MEN1 prolactinomas. (A) Hematoxylin and eosin (H&E) image of a sagittal section of a GFAPΔMen1 pitNET labeled with adjacent brain structures (C.b., Cerebellum; Hyp., hypothalamus; M.b., midbrain). Inset shows a representative macroscopic image of a pitNET with an arrow indicating to the tumor. (B) Higher magnification image of a pitNET showing classical “salt and pepper” nuclei characteristic of well-differentiated NETs. Immunofluorescent staining of a GFAPΔMen1 pitNET for SYN and CHGA (C), prolactin (D), and (E) Ki-67 and the pituitary specification factor PIT-1. Green arrow indicates a single Ki-67+ cell in the tumor. (F) Representative immunofluorescent image of a GFAPΔMen1 pitNET stained for the anterior pituitary hormones ACTH and GH, showing increased expression of the latter. As pitNETs were highly vascularized, immunofluorescent images were merged on a transmitted light micrograph to distinguish tumor cells from red blood cells (Cap., Capillary). (G) Number of mice presenting with pitNETs as stratified by sex. (H) Serum prolactin levels in male and female mice with or without pitNETs. N = 12–15 mice per group; ∗P < .05 by the unpaired Student t test. Data are represented as mean ± standard deviation. (I) Quantitation of neuroendocrine-related transcripts in WT pituitary and GFAPΔMen1 pitNETs. N = 4–5 mice per group, with the exception of the WT pituitary group, which represents 4 samples of 3 pooled pituitaries, for a total of 12 tissues in this group; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (J) PitNETs show absent expression of menin and GFAP compared with the adjacent hypothalamus (Hyp). Left panel: a capillary (Cap.), characterized by auto fluorescent red blood cells, separates the 2 compartments. Middle panel: Robust GFAP staining (red) in a GFAPΔMen1 hypothalamus with few cells co-expressing menin (green). Right panel: Tumor is negative for menin and GFAP. (K) Quantitation of Gfap and Men1 mRNA in WT pituitary and GFAPΔMen1 pitNETs. Data are represented as mean ± standard deviation. (L) Representative images of tumor neurospheres generated from 3 tumor-bearing mice (n = 3). Phase contrast images show growth at days (d) 2, 10, and 21. Tumor neurospheres were stained for prolactin (PRL) and the neural stem cell markers nestin and SOX2. (M) Specificity of select antibodies was tested by preadsorption. Immunofluorescent images of a pitNET stained with non-adsorbed and pre-adsorbed GH and SOX2 antibodies.
      Similar to previous reports of gender differences in human MEN1 patients, the emergence of pituitary tumors in mice was strikingly sex-dependent.
      • Vergès B.
      • Boureille F.
      • Goudet P.
      • Murat A.
      • Beckers A.
      • Sassolas G.
      • Cougard P.
      • Chambe B.
      • Montvernay C.
      • Calender A.
      Pituitary disease in MEN type 1 (MEN1): data from the France-Belgium MEN1 multicenter study.
      ,
      • Goudet P.
      • Bonithon-Kopp C.
      • Murat A.
      • Ruszniewski P.
      • Niccoli P.
      • Ménégaux F.
      • Chabrier G.
      • Borson-Chazot F.
      • Tabarin A.
      • Bouchard P.
      • Cadiot G.
      • Beckers A.
      • Guilhem I.
      • Chabre O.
      • Caron P.
      • Du Boullay H.
      • Verges B.
      • Cardot-Bauters C.
      Gender-related differences in MEN1 lesion occurrence and diagnosis: a cohort study of 734 cases from the Groupe d’etude des Tumeurs Endocrines.
      ,
      • Cohen-Cohen S.
      • Brown D.A.
      • Himes B.T.
      • Wheeler L.P.
      • Ruff M.W.
      • Major B.T.
      • Singh Ospina N.M.
      • Atkinson J.L.D.
      • Meyer F.B.
      • Bancos I.
      • Young W.F.
      • Van Gompel J.J.
      Pituitary adenomas in the setting of multiple endocrine neoplasia type 1: a single-institution experience.
      Female GFAPΔMen1 mice (17 of 30; 57%) developed macroscopic pituitary adenomas as early as 15 months of age, whereas only 1 of 9 males (11%) developed a pituitary tumor (Table 1; Figure 3, G). Consistent with tumors staining strongly for prolactin, GFAPΔMen1 mice exhibited significantly higher serum prolactin levels compared with age-matched littermate controls (Figure 3, H). Neuroendocrine transcripts Chgb, Syp, and Enolase 2 (Eno2) were significantly elevated in GFAPΔMen1 pitNETs compared with WT pituitary. Moreover, mRNA levels of prolactin and vasoactive intestinal peptide (Vip) hormones were 15-fold higher in pitNETs compared with normal pituitary (Figure 3, I). Further evaluation of GFAP and menin expression in GFAPΔMen1 prolactinomas showed absent GFAP staining and low expression of menin within the tumor compared with the adjacent hypothalamus (Figure 3, J). Similar to the previous observations in PNETs, Gfap and Men1 mRNA expression was significantly reduced in prolactinomas compared with WT pituitary (Figure 3, K).
      Because GFAP marks nestin+ subventricular neural stem cells
      • van den Berge S.A.
      • Middeldorp J.
      • Zhang C.E.
      • Curtis M.A.
      • Leonard B.W.
      • Mastroeni D.
      • Voorn P.
      • van de Berg W.D.
      • Huitinga I.
      • Hol E.M.
      Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP-delta.
      • Liu Y.
      • Namba T.
      • Liu J.
      • Suzuki R.
      • Shioda S.
      • Seki T.
      Glial fibrillary acidic protein-expressing neural progenitors give rise to immature neurons via early intermediate progenitors expressing both glial fibrillary acidic protein and neuronal markers in the adult hippocampus.
      • Raponi E.
      • Agenes F.
      • Delphin C.
      • Assard N.
      • Baudier J.
      • Legraverend C.
      • Deloulme J.C.
      S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage.
      and interacts with menin,
      • Lopez-Egido J.
      • Cunningham J.
      • Berg M.
      • Oberg K.
      • Bongcam-Rudloff E.
      • Gobl A.
      Menin’s interaction with glial fibrillary acidic protein and vimentin suggests a role for the intermediate filament network in regulating menin activity.
      ,
      • Poisson A.
      • Zablewska B.
      • Gaudray P.
      Menin interacting proteins as clues toward the understanding of multiple endocrine neoplasia type 1.
      we determined whether GFAP-negative GFAPΔMen1 pitNETs share features with neural stem cells by generating tumor neurosphere lines from multiple GFAPΔMen1 pituitary prolactinomas. Subsequent immunostaining of GFAPΔMen1 tumor neurospheres revealed that tumor cells co-expressed prolactin and the neural stem cell markers SOX2 and nestin, suggesting that the hormone-expressing tumor cells arose from a neural stem cell lineage (Figure 3, L). Positive SOX2+ expression in tumor neurospheres was consistent with strong SOX2-immunoreactivity in parent pitNETs (Figure 3, M). Thus, like PNETs that carried the potential to propagate as organoids, pitNETs also exhibited features of stemness that allowed them to be subcultured as 3-D neurospheres.

      Hyperplastic Reprogramming of the GI Epithelium in GFAPΔMen1 Mice

      To determine whether loss of menin in enteric GFAP-expressing cells supports the development of NENs in the luminal GI tract, we evaluated the stomach and proximal small intestine of GFAPΔMen1 mice for changes in enteroendocrine cell numbers and composition. Although no gastrinomas or small intestinal NENs were observed at 14 to 24 months of age, 11/36 GFAPΔMen1 mice (30%) showed hyperplastic changes in the proximal and distal stomach (Table 1; Figure 4, A). We further observed the development of small intestinal lipomas and duodenal polyps emerging from the BGs at a low incidence (2/36 mice; 5%). In contrast to the previous observations of sex differences in the occurrence of NETs, the incidence of gastric hyperplasia was similar between male and female mice (33% and 30%, respectively) (Figure 4, A).
      Figure thumbnail gr4
      Figure 4GFAP-directed deletion of Men1 stimulates hyperplastic reprogramming of the gastro-intestinal epithelium and promotes neuroendocrine cell hyperplasia. (A) Number of mice presenting with gastric hyperplasia and antral tumors, as stratified by sex. (B) Representative hematoxylin and eosin (H&E) and immunofluorescent images of WT and GFAPΔMen1 corpus stained for CHGA, SYN, and gastrin. Bottom H&E indicates parietal cell atrophy consistent with metaplasia. (C) Representative H&E and immunofluorescent images of WT and GFAPΔMen1 gastric antrum showing increased numbers of CHGA- (green), SYN- (red), and gastrin- (white) positive cells. Colored arrows indicate the degree of co-localization amongst the 3 markers: yellow indicates cells where all 3 markers are expressed, whereas magenta indicates cells expressing gastrin and SYN only. (D) Expression of Chga, Syp, and Gast mRNA in WT and GFAPΔMen1 corpus (CP), gastric antrum (AT), and duodenal mucosa (DUO). N = 6 mice per group; ∗P < .05; ∗∗P < .01. (E) Average number of CHGA-, SYN-, and gastrin-positive cells per crypt counted across three 400× fields in the mid-antrum. N = 7 mice per group. ∗P < .05 by 2-way analysis of variance. (F) GFAPΔMen1 mice exhibit elevated serum gastrin levels compared with WT littermates. N = 12–15 mice per group; ∗P < .05 by unpaired t test. (G) Evaluation of gastric acidity in GFAPΔMen1 mice and controls. N = 8–9 mice per group. ∗P < .05 by unpaired t- test. The expression of non-enteroendocrine cell transcripts was evaluated in the corpus (H) and gastric antrum (I) of WT and GFAPΔMen1 mice. N = 6–8 mice per group; ∗P < .05; ∗∗P < .01 by 2-way analysis of variance. All data are represented as mean ± standard deviation.
      Compared with WT mice, GFAPΔMen1 mice exhibited histological evidence of hyperplasia and metaplasia marked by parietal cell atrophy (Figure 4, B–C). These events coincided with increased expression of CHGA and SYP in the corpus, and CHGA, SYP, and gastrin in the gastric antrum. Notably, CHGA and SYP did not co-localize in all cells of the proximal and distal stomach, suggesting the presence of divergent endocrine cell populations. Whereas all gastrin+ cells appeared to express SYP, not all G cells expressed CHGA (Figure 4, C). Increased expression of Chga, Syp, and Gast mRNA was confirmed by quantitative polymerase chain reaction (qPCR), and this was consistent with increased numbers of CHGA+, SYP+, and gastrin+ cells in the gastric antra of GFAPΔMen1 mice (Figure 4, D–E). No differences in endocrine mRNA levels were observed in the proximal duodenum, indicating that hyperplastic reprogramming of endocrine cells was limited to the stomach. Consistent with increased G cell numbers and tissue gastrin expression, GFAPΔMen1 mice exhibited a small but significant increase in serum gastrin levels compared with littermate controls (Figure 4, F). Previous studies in transgenic mice identified hypergastrinemia-induced changes to the gastric epithelium, including parietal cell atrophy.
      • Xiao C.
      • Ogle S.A.
      • Schumacher M.A.
      • Orr-Asman M.A.
      • Miller M.L.
      • Lertkowit N.
      • Varro A.
      • Hollande F.
      • Zavros Y.
      Loss of parietal cell expression of Sonic hedgehog induces hypergastrinemia and hyperproliferation of surface mucous cells.
      ,
      • Shirai N.
      • Choudhary S.
      • Houle C.
      Gastric neuroendocrine tumors with parietal cell atrophy in a long-term carcinogenicity study in rats.
      Here, we also observed decreased gastric acid levels in the setting of hypergastrinemia in GFAPΔMen1 mice, consistent with the presence of metaplasia (Figure 4, G). qPCR analysis of other gastric cell types further confirmed decreased expression of the parietal cell-specific marker Atp4b and significant upregulation of Gif, a marker of zymogenic chief cells (Figure 4, H–I). Therefore, conditional deletion of menin in enteric GFAP-expressing cells stimulated neuroendocrine hyperplasia that is specific to the corpus and gastric antrum. However, unlike the pancreatic islets and pituitary, glial-directed Men1 deletion was insufficient to convert the hyperplastic endocrine cells into NETs.

      Loss of Menin Reduces GFAP Expression in GFAPΔMen1 Mice and In Vitro

      Because NET development in the pituitary and pancreas coincided with loss of GFAP expression, we examined the impact of Men1 deletion on the fate of GFAP-expressing cells in vivo. We performed fluorescent imaging of both whole tissue mounts and frozen sections of GFAP Cre-tdTomato mice to confirm the specificity of GFAP Cre expression throughout the stomach (Figure 5, A–B). Consistent with previous characterization of this model,
      • Wang Z.
      • Ocadiz-Ruiz R.
      • Sundaresan S.
      • Ding L.
      • Hayes M.
      • Sahoo N.
      • Xu H.
      • Merchant J.L.
      Isolation of enteric glial cells from the submucosa and lamina propria of the adult mouse.
      fluorescent tdTomato protein expression was strongly localized to the myenteric plexus in whole tissue mounts (Figure 5, A). Further, tdTomato expression co-localized at the cellular level with GFAP protein and the glial lineage markers S100B and p75NTR, but not with NF-H+ nerve fibers, as expected (Figure 5, B). Surprisingly, ex vivo fluorescent imaging of tissues from GFAPΔMen1 mice revealed a ∼4-fold reduction in the tdTomato signal across the upper GI tract compared with wild-type mice (Figure 5, C–D). Imaging of frozen tissue sections confirmed near-loss of tdTomato fluorescence in the mucosa and lamina propria, whereas some signal was still retained in the submucosal and myenteric plexi (Figure 5, E). Therefore, deletion of Men1 in GFAP+ cells extinguished GFAP expression.
      Figure thumbnail gr5
      Figure 5Conditional deletion of menin in GFAP+ cells stimulates reprogramming from a glial-restricted progenitor lineage. (A) Whole tissue mounts of proximal duodenum from Cre-negative and tdTomato-expressing mice showing tdTomato fluorescence localized to the myenteric plexus (MP). (B) Immunofluorescent images of frozen stomach sections from WT GFAP-tdTomato mice co-stained for the glial-restricted progenitor lineage markers GFAP, S100B, and p75NTR, showing strong localization to the same cell types. Negative localization with the nerve fiber marker NF-H serves as a control. Immunofluorescent images were merged on a transmitted light micrograph to distinguish submucosal layers. Widefield images (C) and quantitation (D) of tdTomato signal in the stomach and duodenum of WT and GFAPΔMen1 mice expressing tdTomato reporter. N = 4–5 mice per group; ∗∗∗P < .001; ∗∗∗∗P < .0001. (E) Representative images of cryosections of corpus (CP), gastric antrum (AT), and proximal duodenum (DUO) from WT and GFAPΔMen1 mice expressing tdTomato. (F) Co-immunoprecipitation (Co-IP) of menin from rat EGC lysate followed by Western blot for GFAP. Input is 5% of lysate used for IP. (G) Quantitation of band density in 3 Co-IP experiments comparing expression of GFAP with IP with IgG isotype control in cell extracts. (H) Expression of Men1 and the glial transcripts Gfap and S100b following siRNA-mediated Men1 silencing in cultured rat EGCs. N = 3 independent experiments; ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001. (I) Western blot of menin and GFAP in whole cell lysates following 72-hour Men1 silencing in rat EGCs with (J) quantitation of band density normalized to loading control. N = 3 experiments; ∗∗P < .01; ∗∗∗P < .001 by unpaired t test. All data are represented as mean ± standard deviation.
      A prior study showed that GFAP and menin proteins interact in astrocytes, suggesting that the 2 proteins might be interdependent.
      • Lopez-Egido J.
      • Cunningham J.
      • Berg M.
      • Oberg K.
      • Bongcam-Rudloff E.
      • Gobl A.
      Menin’s interaction with glial fibrillary acidic protein and vimentin suggests a role for the intermediate filament network in regulating menin activity.
      To test whether this interaction also occurs in GFAP-expressing glial cells, we performed co-immunoprecipitation using cytoplasmic extracts from a rat enteric glial cell line (EGC) and confirmed interaction between GFAP isoforms and menin (Figure 4, F–G). Studies were performed in EGCs as they are known to express GFAP and menin, and these cells also exhibit robust growth in culture compared with primary glial cells. To test whether loss of menin in a GFAP-expressing cell type modulates the expression of GFAP, we used small interfering RNA (siRNA) to knockdown Men1 expression in GFAP+ EGCs. Consistent with the in vivo observations, Men1 silencing decreased the expression of GFAP and S100B at the transcript and protein levels (Figure 5, H–J). Taken together, these results indicated a role for menin in regulating glial cell identity by modulating GFAP expression.

      Transcriptome-wide Analysis of Gastric Neuroendocrine Hyperplasia and Pituitary NETs Supports Glial-to-neuroendocrine Reprogramming

      Because GFAP-specific deletion of menin was sufficient to drive NET formation in the pituitary and pancreas but not in the stomach or intestine, we used RNA-Seq to identify the molecular pathways that might contribute to the transition from neuroendocrine hyperplasia to NET development. GFAPΔMen1 pitNETs, gastric antrum, and their respective WT counterparts were submitted for bulk RNA sequencing. Unsurprisingly, the number of significant differentially expressed genes (DEGs) was 10 times higher when comparing normal pituitary with pitNETs vs the antral tissues of both genotypes (Figure 6, A–B). As expected, a number of genes related to neuroendocrine differentiation were upregulated in both datasets (eg, Grp, Vip, Syp, and Ncam1/2 in antra and Prl, Chgb, Eno2, Cckbr, Vip, and Ascl2 in pitNETs) (Figure 6, C–D). Genes associated with the neuroglial-restricted lineage were significantly upregulated in GFAPΔMen1 tissues compared with WT controls (Figure 6, E). Furthermore, most upregulated antral genes were correlated with neural differentiation and acquisition of a neuronal cell fate, including Ngfr, Elav3/4, Nrsn1/2, Phox2b, Nos1, Tubb3, Uchl1, Ret, and Hoxb (Figure 6, E). PitNETs also exhibited increased expression of neural-specific transcripts (eg, Elav3/4, Nrsn1/2, and Tubb3) that coincided with significant downregulation of glial lineage-associated genes, including Gfap and others (Figure 6, F). Consistent with their ability to propagate in 3-D culture, pitNETs exhibited increased expression of neural crest cell transcripts related to stemness, including Vim, Fabp7, and Sox11 (Figure 6, F).
      Figure thumbnail gr6
      Figure 6Transcriptome-wide analysis of gastric neuroendocrine hyperplasia and NENs identifies a role for glial-to-neural reprogramming in epithelial differentiation. (A) Volcano plot of significant DEGs in gastric antra of wild type and GFAPΔMen1 mice. (B) Volcano plot of significant DEGs in pooled WT pituitaries and pitNETs of GFAPΔMen1 mice. Heat maps of significant DEGs mapped to neuroendocrine differentiation in GFAPΔMen1 gastric antra (C) and pitNETs (D). Heatmap of significant DEGs mapped to the neuroglial lineage in GFAPΔMen1 gastric antra (E) and pitNETs (F) compared with littermate WT controls. Arrow indicates GFAP as among the genes most significantly downregulated in tumors. (G) Immunofluorescent staining for GRP and SYP in WT and GFAPΔMen1 gastric antra, shown as green and red) respectively. Insets: Co-localization of GRP and SYP in the gastric mucosa (yellow). (H) Immunofluorescent staining for GRP and the nerve fiber marker NF-H, indicating localization of NF-H to the myenteric plexus, with no overlap among GRP+ cells of the mucosa. (I) Staining of a WT mouse gastric antrum with non-adsorbed and pre-adsorbed GRP antibody to confirm antibody specificity. (J) Quantitation of Syp and Grp mRNA following 48-hour siRNA-mediated silencing of Men1 in rat EGCs. N = 3 independent experiments; ∗P < .05 and ∗∗P < .01 by unpaired Student t test. Data are represented as mean ± standard deviation.
      Enteric neurons express gastrin-releasing peptide (GRP),
      • Costa M.
      • Furness J.B.
      • Yanaihara N.
      • Yanaihara C.
      • Moody T.W.
      Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine.
      which was identified in the RNA-seq dataset as the most significantly upregulated transcript in GFAPΔMen1 gastric antra. Increased GRP provides a direct mechanism for increased antral gastrin levels in these mice. Hence, we evaluated whether GFAP-directed deletion of Men1 stimulates GRP expression leading to G cell expansion by staining the gastric antra of GFAPΔMen1 mice for GRP. As anticipated, GFAPΔMen1 mice exhibited increased antral expression of GRP, and its expression co-localized with SYP in the epithelial mucosa but did not overlap with NF-H+ nerve fibers (Figure 6, G–H). The specificity of GRP antibody binding was further validated by preadsorption with recombinant GRP protein (Figure 6, I). Lastly, we assessed whether loss of menin in GFAP+ EGCs directly stimulates the expression of Grp and Syp to further explain a mechanism for the induction of Grp and other neuroendocrine transcripts in GFAPΔMen1 mice. Indeed, Men1 silencing in rat EGCs induced Grp and Syp mRNA expression (Figure 6, J). Taken together, these results demonstrate that Men1 deletion suppresses the mature glial lineage in favor of a neuroendocrine phenotype, perhaps due to loss of GFAP.

      Transcriptome Analysis Identifies Molecular Targets in Neuroendocrine Differentiation Leading to Gastric Neuroendocrine Hyperplasia and NET Development in GFAPΔMen1 Mice

      To further home in on the molecular changes leading to gastric neuroendocrine hyperplasia and NET development, we identified significant DEGs associated with growth and developmental pathways. Consistent with the role of menin in regulating proliferation and endocrine cell development, GFAPΔMen1 pitNETs exhibited increased expression of genes involved in cell cycle inhibition, senescence, and differentiation (eg, Cdkn1a, Cdkn2a, and Cend1), as well as tissue development (e.g. Shh, Nog, Bmp2/7, Bdnf, and Ezh2) (Figure 7, A–B). Increased expression of genes related to NET differentiation and cellular senescence identified by RNA-seq were further validated by qPCR of pituitary and pancreatic NETs and age-matched WT tissues (Figure 7, C–D). Datasets comparing GFAPΔMen1 gastric antra and pitNETs with their respective WT tissues showed an enrichment of genes involved in neuroactive ligand receptor interaction and synaptic signaling pathways known to be upregulated in NET development (Figure 7, E–F). Interestingly, GFAPΔMen1 pitNETs uniquely showed perturbations in the Hedgehog signaling pathway, which was consistent with increased expression of Shh mRNA and ciliary motor proteins known to facilitate transduction of Hedgehog signaling (Figure 7, B, C, and E). In contrast, increased expression of neuroendocrine and neural-lineage genes in GFAPΔMen1 antral extracts coincided with downregulation of cytokeratins and immune-related transcripts marking epithelial and immune cells, respectively (Figure 7, H–I). Collectively, transcriptome-wide sequencing identified the involvement of dysregulated cell cycle and developmental pathways prefacing NET development and suggests a potential role for Hedgehog signaling in mediating the transition from neuroendocrine hyperplasia to tumorigenesis.
      Figure thumbnail gr7
      Figure 7Transcriptome analysis identifies potential pathways in neuroendocrine differentiation leading to gastric neuroendocrine hyperplasia and NET development in GFAPΔMen1 mice. Heat maps of significant DEGs mapped to cell cycle regulation (A) and development (B) in GFAPΔMen1 pitNETs compared with WT pituitaries. (C) Validation of select DEGs in pitNETs vs WT pituitary by qPCR. N = 4–5 mice per group. WT pituitary group represents 4 samples of 3 pooled pituitaries, for a total of 12 tissues in this group. ∗P < .05; ∗∗∗P < .001 by 2-way analysis of variance. (D) Cross-validation of select pitNET-enriched transcripts in GFAPΔMen1 PNETs and WT pancreas. Data are represented as mean ± standard deviatin. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) Ontology Pathway analysis of wild type pituitary and GFAPΔMen1 pitNETs showing the number of genes mapped to enriched pathways and their level of statistical significance. (F) KEGG Ontology Pathway analysis of gastric antra from WT and GFAPΔMen1 mice showing the number of genes mapped to enriched pathways and their level of statistical significance. (G) Heat map showing enrichment of kinesin motor protein transcripts in GFAPΔMen1 pitNETs. Heat maps depicting downregulation of cytokeratins (H) and immune-related transcripts (I) in GFAPΔMen1 antral extracts.

      Impairment of Sonic Hedgehog Signaling (SHH) in Enteric GFAP+ Cells Attenuated Neuroendocrine Cell Hyperplasia in GFAPΔMen1 Mice

      Hedgehog signaling mediated by primary cilia is known to promote premature differentiation of neural progenitor cells toward the neuronal lineage.
      • Dutton R.
      • Yamada T.
      • Turnley A.
      • Bartlett P.F.
      • Murphy M.
      Sonic hedgehog promotes neuronal differentiation of murine spinal cord precursors and collaborates with neurotrophin 3 to induce Islet-1.
      As GFAPΔMen1 pitNETs exhibited significantly elevated SHH transcript levels, we investigated whether loss of KIF3A, a ciliary motor protein required for SHH signal transduction, blocks GFAPΔMen1 glia from developing a neuroendocrine phenotype. To further stimulate gastric neuroendocrine hyperplasia, GFAPΔMen1 mice were crossed onto a somatostatin null (Sst–/–) background. Somatostatin (SST) suppresses hormone secretion and gene expression, including gastrin, thus removal of Sst-mediated feedback inhibition increases gastrin levels.
      • Sundaresan S.
      • Meininger C.A.
      • Kang A.J.
      • Photenhauer A.L.
      • Hayes M.M.
      • Sahoo N.
      • Grembecka J.
      • Cierpicki T.
      • Ding L.
      • Giordano T.J.
      • Else T.
      • Madrigal D.J.
      • Low M.J.
      • Campbell F.
      • Baker A.M.
      • Xu H.
      • Wright N.A.
      • Merchant J.L.
      Gastrin induces nuclear export and proteasome degradation of menin in enteric glial cells.
      The effect of Kif3a deletion on neuroendocrine hyperplasia was accomplished by breeding GFAPΔMen1;Sst–/– mice onto a Kif3aFL/FL genetic background (GFAPΔMen1;ΔKif3a). We next investigated whether Kif3a deletion could reverse the gastric hyperplasia observed in GFAPΔMen1 mice. Compared with littermate controls, GFAPΔMen1;ΔKif3a mice more frequently presented with severe dermatitis and polycystic livers, and thus were euthanized and necropsied at 12 months of age. GFAPΔMen1;ΔKif3a mice exhibited reduced hyperplasia in the corpus and gastric antrum compared with age-matched GFAPΔMen1 mice (Figure 8, A). To determine the effect of Kif3a deletion on enteroendocrine cell composition, we analyzed the expression of CHGA and gastrin in the stomach after sequential deletion of Sst, Men1, and Kif3a genes. Whereas GFAPΔMen1 mice exhibited hyperplasia of CHGA+ and gastrin+ cells, additive deletion of KIF3A appeared to reverse these events (Figure 8, A). These observations were supported by associated fluctuations in Chga and Gast transcript levels. Whereas deleting Men1 significantly increased the expression of Chga and Gast mRNA in the gastric antra, loss of Kif3a in the presence of Sst and Men1 deletions inhibited this induction (Figure 8, B–C). Similarly, analysis of gastric antral extracts from these mice showed reduced gastrin peptide levels in GFAPΔMen1;ΔKif3a mice compared with those carrying the Men1 deletion alone (Figure 8, D). Thus, blockade of SHH-mediated signaling in enteric GFAP+ cells attenuated neuroendocrine cell hyperplasia in the stomach.
      Figure thumbnail gr8
      Figure 8Blockade of Shh signaling in GFAP+ cells attenuates gastric neuroendocrine differentiation in GFAPΔMen1 mice. (A) Representative hematoxylin and eosin (H&E) and immunofluorescent images of corpus and gastric antrum of GFAPΔMen1;Sst-/-, GFAPΔMen1;ΔKif3a;Sst-/- and littermate control mice showing reduced numbers of CHGA- and gastrin-positive cells with additive Kif3a deletion. CHGA (B) and gastrin (C) mRNA expression is reduced in the gastric antra following Kif3a deletion. (D) Expression of antral gastrin peptide among the different genotypes as evaluated by enzyme immunoassay. N = 7–8 mice per group; ∗∗P < .01; ∗∗∗P < .001. All data are represented as mean ± standard deviation.

      SHH Signaling Through Primary Cilia Modulates GFAP Expression

      Because disruption of SHH signaling on GFAP+/menin-deficient cells resulted in reduced neuroendocrine cell hyperplasia, we further investigated a potential mechanism to bridge these events. We first confirmed that deletion of Kif3a resulted in the impairment of primary cilia on GFAP+ cells. Shortened primary cilia were shown by immunostaining for acetylated tubulin-positive hair-like projections on GFAP+ cells of the gastric antrum. Compared with WT mice, GFAPΔMen1;ΔKif3a mice exhibited significantly shortened primary cilia on GFAP+ cells residing in the gastric mucosa (Figure 9, A–B). Hedgehog signaling is mediated through activation of the GLI1/2/3 family of transcriptional effectors.
      • Villavicencio E.H.
      • Walterhouse D.O.
      • Iannaccone P.M.
      The sonic hedgehog-patched-gli pathway in human development and disease.
      Thus, we assessed whether deleting Kif3a affects the expression of GLI2, a known suppressor of gastrin gene expression in antral G cells.
      • Ding L.
      • Sontz E.A.
      • Saqui-Salces M.
      • Merchant J.L.
      Interleukin-1β suppresses gastrin via primary cilia and induces antral hyperplasia.
      Whereas GFAP-directed Men1 deletion increased antral GLI2 expression, loss of Kif3a reduced the expression of GLI2 protein without significantly influencing transcript levels (Figure 9, C–D). We next assessed whether GFAPΔMen1;ΔKif3a mice showed reduced GFAP-tdTomato fluorescence compared with GFAPΔMen1 mice with intact primary cilia. As expected, loss of GFAP-tdTomato fluorescence was reversed in the GFAPΔMen1;ΔKif3a mice (Figure 9, E–F). Further, Kif3a deletion was associated with a trending but nonsignificant increase in antral Gfap mRNA expression (Figure 9, G), suggesting that modulation of GFAP by hedgehog signaling is most apparent at the protein level.
      Figure thumbnail gr9
      Figure 9Loss of the ciliary motor protein Kif3a in GFAP-expressing cells impairs SHH signaling and GFAP expression. (A) Immunofluorescent staining for acetylated-tubulin (Ac-TUB) and GFAP in the gastric antra of WT and GFAPΔMen1;ΔKif3a;Sst-/- mice. Arrows indicate primary cilia marked by acetylated tubulin. (B) Quantitation of primary cilia length in GFAP-expressing cells of the gastric antrum. N = 34 cells counted across 10 random 1000× magnification images per group. ∗∗∗∗P < .0001 by the unpaired Student t test. (C) Western blot of GLI2 protein in gastric antral lysates of Sst-/- (Con), GFAPΔMen1;Sst-/-, and GFAPΔMen1;ΔKif3a;Sst-/- mice. N = 3 mice. (D) Gli2 mRNA in gastric antra and duodenal mucosa of respective genotypes. N = 4–7 mice per group. (E) Representative images of cryosections of corpus (CP), gastric antrum (AT), and proximal duodenum (DUO) from WT, GFAPΔMen1;Sst-/- and GFAPΔMen1;ΔKif3a;Sst-/- mice expressing tdTomato. (F) Quantitation of relative tdTomato fluorescence intensity following whole tissue ex vivo imaging. ∗P < .05; ∗∗P < .01 by 2-way analysis of variance. (G) Quantitation of GFAP mRNA in the gastric antrum and duodenum of different groups. N = 4–7 mice per group. All data are represented as mean ± standard deviation.

      SOX10-driven Deletion of Men1 Recapitulates Hyperplastic Reprogramming of the Gastric Epithelium and Leads to Accelerated Development of PNETs

      GFAP is expressed at low levels in nestin+ neural stem cells and turns on more strongly in terminally differentiated cells along the glial lineage in addition to other cell types, including stellate cells.
      • Imura T.
      • Kornblum H.I.
      • Sofroniew M.V.
      The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP.
      Therefore, we postulated that targeting Men1 deletion by a more specific driver of glial cell specification might potentiate NET development. The nuclear transcription factor Sry-related HMG Box gene 10 (Sox10) is expressed by neural crest cells and its expression is maintained in glial and melanocyte cell lineages, but not in other neural crest cell derivatives.
      • Britsch S.
      • Goerich D.E.
      • Riethmacher D.
      • Peirano R.I.
      • Rossner M.
      • Nave K.A.
      • Birchmeier C.
      • Wegner M.
      The transcription factor Sox10 is a key regulator of peripheral glial development.
      Thus, we conditionally deleted Men1 in Sox10-expressing cells using the Sox10 Cre system expressed on the same Sst-/- background described previously. Unlike GFAPΔMen1 mice that showed no apparent adverse health effects, Sox10ΔMen1 mice initially presented with reduced body weight and increased morbidity by 10 weeks of age; however, no significant fluctuations in gastric pH were observed (Figure 10, A–C). These events were later circumvented by delaying the age of weaning by 1 week and providing mice with moistened chow. Following these interventions, Sox10ΔMen1 mice showed similar survival to littermate controls with no signs of adverse health or weight loss.
      Figure thumbnail gr10
      Figure 10Sox10-driven deletion of Men1 recapitulates hyperplastic reprogramming of the gastric epithelium and leads to accelerated development of PNETs. (A) Survival curve for Sox10ΔMen1 mice prior to delaying the age of weaning from 3 weeks of age to 4 weeks. Body weight (B) and gastric pH (C) of 10-week-old Sox10ΔMen1 mice prior to adjusting the age of weaning from 3 weeks to 4 weeks of age. Blue triangles = male mice; Red circles = female mice. (D) Number of Sox10ΔMen1 mice presenting with PNETs by 11 months of age, as stratified by sex. Levels of serum GCG (E) glucose (F) and prolactin (G) in Sox10ΔMen1 mice compared with littermate controls, with symbols indicating male and female mice with and without the tissue-involved tumors. N = 6–7 mice per group; ∗∗P < .01 by the unpaired Student t test. (H) Representative hematoxylin and eosin and immunofluorescent images of a well-differentiated Sox10ΔMen1 PNET stained for neuroendocrine and hormone markers. Hormone-expressing tumor cells are negative for expression of the nerve fiber marker NF-H. (I) Macroscopic images of tissues from a 21-month-old Sox10ΔMen1 mouse presenting with corpus hyperplasia, multiple antral adenocarcinomas, and a PNET. (J) Hematoxylin and eosin and immunofluorescent staining of the previous stomach tissues indicating the presence of a gastric NET and neuroendocrine cell hyperplasia. Colored arrows indicate the degree of co-localization of CHGA (green), SYN (red), and gastrin (white), with several gastrin+ cells expressing Syp but not Chga. (K) Serum gastrin peptide levels in 6–11-month-old Sox10ΔMen1 mice as evaluated by enzyme immunoassay. N = 9–12 mice per group; ∗P < .01 by the unpaired Student t test. All data are represented as mean ± standard deviation.
      We next assessed whether surviving Sox10ΔMen1 mice aged 10 to 12 months developed endocrine hyperplasia akin to GFAPΔMen1 mice. Four of 7 Sox10ΔMen1 mice (57%) presented with PNETs, with the incidence being slightly higher in females compared with male mice when age-matched (60% vs 50%) (Table 2; Figure 10, D). Like GFAPΔMen1 mice, Sox10ΔMen1 mice also exhibited significantly elevated serum GCG levels compared with Sst-/- littermate controls (Figure 10, E). Intriguingly, serum INS1 levels were too low for detection using the same high range standard curve for INS1 determination in GFAPΔMen1 sera (1 ng/mL as the limit of detection). Surprisingly, glucose levels also trended lower in Sox10ΔMen1 mice (Figure 10, F). Although no pituitary tumors were observed in this strain at 10 to 12 months of age, Sox10ΔMen1 mice exhibited a trending but nonsignificant increase in circulating prolactin levels (Figure 10, G). Subsequent histological analysis of PNETs revealed rosette-like features typical of well-differentiated NETs (Figure 10, H). PNETs exhibited strong immunoreactivity for SYP; however, CHGA expression was restricted to a discreet cell population. Robust SYP expression overlapped with GCG, whereas CHGA+ cells appeared to overlap with INS1-expressing cells within tumors (Figure 9, E). Moreover, SYP+ tumor cells did not overlap with NF-H+ nerve fibers, indicating that tumor cells neither arise from, nor associate with nerve fibers (Figure 9, E).
      Table 2Summary of Phenotypes Observed in Sox10 Cre; Men1FL/FL Mice Aged 10 to 21 Months
      Men1Pancreatic hyperplasia/tumor, n/N; average agePituitary adenoma, n/NAntral hyperplasia/tumor, n/N
      WT0/6; 12.6 mo0/60/6
      FL/+1/1; 21 mo

      • 1/1 male
      0/11/1 (gastric carcinoid and adenocarcinoma)
      FL/FL4/7; 11 mo0/71/7
      • 3/5 female

      • 1/2 male
      • 1/5 female
      WT, Wild-type.
      An additional 21-month-old male mouse heterozygous for Men1 deletion (Men1FL/+) presented with PNETs, severe corpus hyperplasia, and multiple macroscopic antral tumors (Table 2; Figure 9, F). Subsequent staining for endocrine markers identified the presence of a CHGA+/SYP+ gastric NET in the fundic submucosa (Figure 9, G). Moreover, the gastric antrum was characterized by hyperplasia of CHGA+, SYP+, and gastrin+ cells, consistent with increased serum gastrin levels in these mice (Figure 9, G–H). Similar to GFAPΔMen1 mice, all G cells expressed SYP but not CHGA. Thus, cell autonomous inactivation of Men1 using Sox10 Cre as a more selective driver of the glial lineage accelerated the development of PNETs and recapitulated the hyperplastic gastric phenotype observed in GFAPΔMen1mice.

      Discussion

      Progress in understanding GEP-NET pathogenesis is impeded by a lack of comprehensive in vivo models, in part due to tissue heterogeneity from which the neoplasms arise, and the paucity of driver mutations identified that precede malignancy.
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      Neuroendocrine cell hyperplasia and NET development coincided with an unexpected loss of GFAP expression and induction of neuronal and neuroendocrine-related genes in the stomach, pancreas, and pituitary. In contrast to pituitary and pancreatic NETs, which exhibited global reduction in GFAP protein and mRNA expression, the gastric antra of GFAPΔMen1 mice showed reduced GFAP-reporter expression but did not exhibit significant loss of GFAP at the transcript level. This observation may be explained in part by the fact that menin interacts and colocalizes with GFAP.
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      • Wells J.M.
      Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells.
      Subsequent analysis of ENCCs identified elevated expression of the SHH-induced posteriorizing factors BMP4 and BMP7, and that inhibition of these factors in ENCCs attenuates BG formation in organoid co-cultures.
      • Eicher A.K.
      • Kechele D.O.
      • Sundaram N.
      • Berns H.M.
      • Poling H.M.
      • Haines L.E.
      • Sanchez J.G.
      • Kishimoto K.
      • Krishnamurthy M.
      • Han L.
      • Zorn A.M.
      • Helmrath M.A.
      • Wells J.M.
      Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells.
      Consistent with our observations, ENCCs and their progeny carry the potential to direct epithelial differentiation in favor of an endocrine phenotype, and these events are strongly influenced by SHH-mediated signaling. Importantly, our observations prompt further investigation into both glial cell autonomous and non-cell autonomous mechanisms presaging these outcomes.
      In summary, we report the development of 2 glial-directed mouse models of human MEN1 syndrome with the aim of defining the contribution of neural crest cell reprogramming in neuroendocrine cell differentiation and tumorigenesis. Because it remains unknown at what point during the cell’s lifespan it transitions to a hormone-producing endocrine cell upon loss of menin, we used a constitutive Cre as reported here. Future work requires lineage tracing coincident with inducible deletion of Men1 using glial-restricted promoters to directly track GFAP+ and SOX10+ cells in NET development. By addressing a previously uncharacterized compartment of GEP-NETs, this study carries the potential to identify a unique cell-of-origin for these neoplasms. The importance of these discoveries is underscored by the fact that GEP-NETs comprise remarkably diverse neoplasms that vary in location, mutational profile, and response to therapy. Such heterogeneity may in part be explained by divergent cellular origins for NETs as a function of different tissue sites. Previous work centered on mapping their unique transcriptional signatures suggests the potential for neuroglial reprogramming in the development of human duodenal gastrinomas. Defining the cells-of-origin and the events preceding neoplastic transformation will be critical to informing molecular signaling pathways that can then be targeted therapeutically.

      Materials and Methods

      Animal Studies

      All animal studies received approval by the University of Arizona Institutional Animal Care and Use Committee and conform to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Mice were housed in individually ventilated caging, with food and water ad lib. All mice were fasted overnight with water ad lib prior to blood collection. Mouse strains, Research Resource Identifiers, and vendors are listed in Table 3. GFAP-Cre transgenic mice on a C57BL/6J genetic background were purchased from Jackson Laboratories and bred onto a Men1FL/FL background to conditionally delete Men1 in GFAP-expressing cells (named GFAPΔMen1). A subset of wild type and GFAPΔMen1 mice were bred onto a somatostatin-null background (Sst-/-). GFAPΔMen1;Sst-/- mice were further bred onto a Kif3aFL/FL background to conditionally delete Kif3a under the control of the Gfap promoter (named GFAPΔMen1;ΔKif3a;Sst-/-). WT, GFAPΔMen1, GFAPΔMen1;Sst-/-, and GFAPΔMen1;ΔKif3a;Sst-/- mice were also bred to mice carrying a lox-Stop-lox-tdTomato sequence to selectively express the tdTomato fluorescent reporter in GFAP+ cells. GFAPΔMen1 strains were necropsied at 8 to 9 months of age and between 13 and 24 months of age. All histological characterization and downstream comparisons were made using littermate controls that genotyped negative for Cre recombinase expression and included mice with and without the relevant floxed alleles. Sox10-Cre transgenic mice were bred onto the Men1FL/FL and Sst-/- background to generate Sox10ΔMen1 mice. Sox10ΔMen1 mice were necropsied at 10 to 12 months of age, except for one Men1FL/+ male mouse taken at 21 months old. Histological characterization and serum analyses were compared with Cre-negative littermate controls.
      Table 3List of Mouse Strains and Vendors
      Mouse strain nameStrain #RRID #Vendor/provider
      FVB-Tg(GFAP-cre)25Mes/J004600IMSR_JAX:004600Jackson Laboratories
      129S(FVB)-Men1tm1.2Ctre/J005109IMSR_JAX:005109Jackson Laboratories
      B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J007914IMSR_JAX:007914Jackson Laboratories
      B6;CBA-Tg(Sox10-cre)1Wdr/J025807IMSR_JAX:025807Jackson Laboratories
      Sst-/-Strain provided by Dr. Malcolm Low and described in PMID: 11413165 (not available in Jackson Laboratories to the authors’ knowledge).
      RRID, Research Resource Identification.

      Cell and Tissue Culture

      PNET organoids

      Organoids were generated from four 17 to 18 month old GFAPΔMen1 mice presenting with pancreatic neuroendocrine tumors. Tumors were carefully removed from adjacent pancreas tissue and washed in ice-old Dulbecco’s phosphate buffered saline (PBS) (minus Ca2+/Mg2+) containing RNase inhibitor. Tumors were minced using a razor blade, then collected in 10 mL of collagenase solution consisting of 1.5 mg/ml Type VIII collagenase, 40 μg/ml DNase I, 10 mM HEPES, and 5% fetal bovine serum (FBS) dissolved in prewarmed Dulbecco’s PBS (minus Ca2+/Mg2+). Tissue was digested at 200 rpm for 40 minutes at 37 °C. The suspension was shaken 5 to 7 times prior to filtering through a 100-μm cell strainer and centrifuging at 400 × g for 5 minutes at 4 °C. The pellet was gently washed once with prewarmed media and re-pelleted. The pellet was resuspended in ice-cold Matrigel (Corning, Corning, NY), and 25 μL was pipetted into each well of a prewarmed 24-well plate. The plate was inverted and incubated at 37 °C for 15 minutes before supplementing with complete PNET organoid growth media consisting of a 1:1 solution of L-WRN conditioned media and Basal Growth Media (advanced DMEM/F12, 1× B-27 supplement, 1× N-2 supplement, 10 mM HEPES, 2 mM GlutaMAX, 1× penicillin-streptomycin, 1 mM N-acetylcysteine). Growth media was supplemented with ascorbic acid (284 μM, Sigma), INS1 (20 μg/mL, Tocris), hydrocortisone (0.25 μg/mL, Sigma), retinoic acid (100 nM, Sigma), FGF-10 (100 ng/mL, Biolegend), Y-27632 (10 μM, Tocris), and A8301 (500 nM, Tocris).

      PitNET neurospheres

      Primary tumor neurospheres were generated from three 17- to 18-month-old female GFAPΔMen1 mice presenting with pituitary adenomas. A 4 × 2 mm piece of tissue was cut from the tumor and minced into 1-mm pieces on ice. The tissue was transferred to a 10 mL solution of Dispase/DNase solution warmed to 37 °C (0.1% dispase, 0.01% DNase, 10 mM HEPES, DPBS without calcium and magnesium) and incubated in a shaking water bath (60 rpm) at 37 °C for 80 minutes. Halfway through the incubation, the tissue was shaken manually 3 times to facilitate dissociation. The tissue was triturated 10 times with a 10 mL serological pipette, then filtered through a 40-μm cell strainer before centrifuging at 123 × g for 10 minutes at 4 °C. The cell pellet was resuspended in Neurosphere Media consisting of DMEM/F12 (ThermoFisher, Waltham, MA), 1× GlutaMAX (Invitrogen, Carlsbad, CA), 1× N2 (Gibco, Waltham, MA), 1× B-27 (Gibco), 100 U penicillin-streptomycin (Invitrogen), 10 mM HEPES (Invitrogen), 30 ng/mL mouse recombinant FGF-10 (Biolegend, San Diego, CA), and 30 ng/mL recombinant human EGF (R&D Systems, Minneapolis, MN). Cells were plated in a non-tissue culture treated 6-well plate and incubated overnight at 37 °C. Media was changed every 2 to 3 days by collecting neurospheres, centrifuging, and reseeding the pellet in fresh medium. Tumor neurosphere generation was repeated with 3 prolactinoma-bearing mice.
      A rat enteric glial cell line was purchased from ATCC (#CRL-2690) and grown in DMEM supplemented with 10% FBS and 100 U penicillin-streptomycin (EGC/PK060399egfr, ATCC, Manassas, VA). Upon reaching 30% to 50% confluency, glial cells were switched to 5% FBS-containing media and used for siRNA experiments. Both the non-targeting siRNA and Men1 siRNA consisted of a SMARTpool of 4 ON-TARGETplus siRNA constructs (#L-090784-02-0005 for Men1 pool and #D-001810-10-05 for non-targeting pool, Dharmacon Horizon Discovery, Lafayette, CO). Cells were transfected with 25 nM siRNA using Lipofectamine 3000 without p3000 reagent according to manufacturer instructions (ThermoFisher). Following 48 to 72 hours post-transfection, cells were harvested for downstream RNA or protein extraction.

      Immunohistochemistry and Immunofluorescent Staining

      Mouse tissues were fixed overnight at 4 °C in 4% paraformaldehyde. Tissues were paraffin-embedded, cut into 5-μm sections, and placed on frosted glass slides. Slides were deparaffinized in 3 xylene washes, then rehydrated in 100%, 90%, and 70% ethanol washes. Slides were washed in TBS with 0.05% Tween-20 (TBST) before proceeding with antigen retrieval. Slides were boiled in 1× sodium citrate buffer pH 6.0 for 30 minutes and allowed to cool to room temperature (RT) for 20 minutes prior to washing with TBST. Slides were incubated for 1 hour at RT in blocking buffer consisting of 10% donkey serum, 1% bovine serum albumin (BSA), 0.1% Triton X-100 in TBST. In some instances, tissues were permeabilized with 0.5% Triton X-100 for 5 minutes at RT prior to blocking. Tissues were incubated in primary antibody overnight at 4 °C. Primary antibodies were prepared in blocking solution according to the dilutions listed in Table 4. Following overnight incubation, slides were washes in TBST and incubated in Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Eugene, OR; Invitrogen) diluted 1:500 in 1% BSA and TBST for 1 hour at RT in the dark. Slides were washed in TBS before mounting with #1.5 coverslips using Prolong Gold anti-fade mounting medium with DAPI (Life Technologies, Rockville, MD). Immunofluorescence was visualized at 200×, 400×, and 1000× magnification using the Olympus BX53F epifluorescence microscope (Center Valley, PA).
      Table 4List of Primary Antibodies and Dilutions
      Primary antibodiesVendorCatalog #RRID #Dilution/application
      Menin (Rabbit pAb)Bethyl LaboratoriesA300-105AAB_21433061:2,000 (IHC/IF TSA) 1:10,000 (WB)
      Chromogranin AAbcam15160AB_3017041:100 (IHC/IF)
      SynaptophysinCell Signaling Technologies9020AB_26310951:200 (IHC/IF)
      Irx-2Abcam1967551:100 (IHC/IF)
      Ki-67Cell Signaling Technologies9449AB_27977031:400 (IHC/IF)
      ⍺-smooth muscle actinAbcam5694AB_2223021:200 (IHC/IF)
      GlucagonCell Signaling Technologies2760AB_6598311:100 (IHC/IF)
      InsulinCell Signaling Technologies3014AB_21265031:1000 (IHC/IF)
      GFAP (Rabbit pAb)Dako Agilent TechnologiesZ0334AB_100133821:400 (IHC/IF)
      1:2000 (WB)
      GFAP (Rat mAb)Invitrogen13-0300AB_25329944 μg/mg lysate (IP)
      GFAP (Chicken pAb)InvitrogenPA1-10004AB_10746201:1000 (IHC/IF)
      Neurofilament-HNovus BiologicalsNBP1-05209AB_15563281:100 (IHC/IF)
      S100BAbcamab52642AB_8824261:200 (IHC/IF)
      p75NTRAbcamab52987AB_8816821:1000 (IHC/IF)
      ACTHNovus BiologicalsNBP2-32911AB_10699551:100 (IHC/IF)
      Growth hormoneR&D SystemsAF1067AB_3545731:100 (IHC/IF)
      NestinNovus BiologicalsNB100-1604AB_22826421:500 (IHC/IF)
      1:100 (ICC/IF)
      Sox2Novus BiologicalsNB110-37235AB_7920701:200 (ICC/IF)
      Pit-1Novus BiologicalsNBP1-92273AB_110303101:500 (IHC/IF)
      ProlactinAbcam183967AB_28148391:200 (IHC/ICC/IF)
      Gastrin (Rabbit mAb)Dako Agilent TechnologiesA0568AB_27572701:1000 (IHC/IF)
      Gastrin (Goat pAb)Santa Cruz TechnologiesSc-7783AB_21082611:1000 (IHC/IF)
      Acetylated-tubulinSigmaMABT868AB_28191781:200 (IHC/IF)
      Gastrin-releasing peptideInvitrogenPA5-115272AB_28999081:200 (IHC/IF)
      GAPDHCell Signaling Technologies5174SAB_106220251:2000 (WB)
      Gli2Santa Cruz Technologies271786AB_107081241:1000 (WB)
      ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; RRID, Research Resource Identification; TSA, tyramide signal amplification; WB, Western blot.
      For immunofluorescent staining of PNET organoids and PitNET neurospheres, organoids and neurospheres were collected and centrifuged. The pellet was resuspended in 3.7% paraformaldehyde and transferred to a 24-well 0.17-mm glass-bottom plate. Organoids and neurospheres were fixed for 20 minutes at RT, then washed with 1× TBST. Samples were permeabilized with 0.5% triton X-100 for 10 minutes at RT, washed, then blocked in 10% donkey serum, 1% BSA, and 0.2% triton in TBST for 1 hour at RT. Primary antibodies were diluted in 5% donkey serum 1% BSA, 0.1% triton in TBST and added to wells for overnight incubation at 4 °C. Samples were washed thoroughly with TBST and incubated for 1 hour at RT in Alexa Fluor-conjugated secondary antibodies diluted 1:500 in 1% BSA and TBST. Organoids and neurospheres were washed with TBS, then stored in TBS containing Prolong gold antifade mounting medium with DAPI. Samples were imaged at 600× magnification using a Nikon Ti2-E inverted microscope with a Crest X-Light V2 L-FOV Spinning Disk Confocal and a Photometrics Prime 95B-25MM Back-illuminated sCMOS Camera (Nikon, Tokyo, Japan).
      Mouse tissues with endogenous tdTomato reporter expression were fixed in 4% paraformaldehyde for 1 hour on ice before dehydrating overnight at 4 °C in a solution of 30% sucrose-PBS. Tissues were then embedded in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and frozen on dry ice. OCT blocks were cryosectioned to a 10-μm thickness and melted onto frosted glass slides prior to mounting with Prolong Gold anti-fade mounting medium containing DAPI. TdTomato signal was visualized at 200× (whole mount) and 1000× magnification (co-stained images) using the Olympus BX53F epifluorescence microscope. Images drawing comparisons between groups were acquired with identical laser acquisition settings (Center Valley, PA).

      Antibody Pre-adsorption

      Select antibodies were tested for specificity by pre-adsorption with recombinant proteins. Briefly, human recombinant protein was mixed with respective antibody at a 5-fold excess by weight. Antibody-antigen solution was diluted to a working concentration in 10% donkey serum, 1% BSA, 0.1% triton in TBST and incubated overnight at 4 °C on a rotator. The antibody-antigen solution was then used for immunohistochemistry/immunofluorescence staining as previously described and compared with staining with antibody only. Images were acquired using identical acquisition settings for comparison of fluorescence signal.

      qPCR

      RNA was extracted from frozen mouse tissues using the Purelink RNA Extraction Kit (Invitrogen). Tissues were homogenized with a rotor-stator homogenizer in RNA lysis buffer containing 1% β-mercaptoethanol, and RNA was isolated following manufacturer’s instructions. Extracted RNA was treated with ezDNase to remove any residual genomic DNA and then prepared for cDNA synthesis using Superscript VILO IV Master Mix according to manufacturer’s instructions (Invitrogen). Real-time qPCR was performed using PowerUp SYBR Green Master Mix (Invitrogen) with 20 ng cDNA added to each reaction. Predesigned forward and reverse primer sets were purchased from Integrated DNA Technologies (IDT, Coralville, IA) and used at a final 500 nM concentration. qPCR was performed using the QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA) with the following cycling conditions: 2 minutes at 50 °C, 2 minutes at 95 °C, denaturing step for 1 second at 95 °C, extension and annealing for 1 minute at 60 °C, followed by a dissociation melt curve stage to confirm primer specificity. All forward and reverse primers were purchased as validated predesigned PrimeTime qPCR Assay primer sets used for SYBR Green dye (Integrated DNA Technologies, Coralville, IA). qPCR data was expressed as fold-change using the established 2-ddCt method and log-transformed to fit a normal distribution prior to performing statistical analysis.
      • Pfaffl M.W.
      A new mathematical model for relative quantification in real-time RT-PCR.
      ,
      • Arocho A.
      • Chen B.
      • Ladanyi M.
      • Pan Q.
      Validation of the 2-DeltaDeltaCt calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts.

      Ex Vivo Fluorescence Imaging

      At 8 to 9 months of age, GFAP Cre-tdTomato mice were euthanized following overnight fasting, and the stomach and proximal duodenum were collected for ex vivo imaging prior to fixation for OCT embedding. The stomach and proximal duodenum were opened along the greater curvature, and contents were flushed with cold PBS. Both tissues were placed in cold PBS with mucosa facing up and imaged in a dark room using a wide field fluorescence imaging system. Illumination was provided by a 300W xenon arc lamp source (LS-OF30, Sutter instruments, Novato CA). Illumination light was filtered with a 554 nm excitation filter with 23 nm bandwidth (FF01-554/23-25, Semrock Inc., Rochester NY) and delivered using a fiber bundle (LLG, Sutter Instruments). Images were collected with an 0.5” CCD Monochrome Camera (EO-1312M, Edmund Optics, Barrington NJ) with a 35 mm fixed focal length lens (#59-872, Edmund Optics) and a 594 nm long-pass filter (BLP01-594R-25, Semrock) mounted on a rigid arm above the sample plane. Images were collected with an exposure time of 60 ms and saved in 16 bit .tif format. Each day, a flat-field image was collected by imaging a white diffuse reflectance target (#58-609, Edmund Optics) to correct for spatial non-uniformity using unfiltered illumination. Additionally, a power measurement was collected for the filtered illumination light at the sample plane using a power meter (S120C, Thorlabs, Newton NJ) to adjust for day-to-day variations of light source intensity. The collected images were first normalized through division by the flat-field image and the light source power measured on the day of sample acquisition. Regions of interest were then manually drawn around the forestomach, corpus, antrum and duodenum using ImageJ. Fluorescence intensity was calculated in the region of interest and averaged over the number of valid pixels. Any pixels with 5% of saturation (eg, digital value of 65535) were discarded from the analysis.

      RNA Sequencing

      RNA was extracted from 3 GFAPΔMen1 PNETs, pitNETs, and the pyloric antrum using the Purelink RNA Extraction kit as previously described (Invitrogen). Pancreas, pituitary, and antra of age-matched WT mice were used as controls. Due to its small size, 3 normal pituitaries were pooled for each sample, and a total of 3 samples were submitted for sequencing (ie, 9 WT pituitaries total). RNA was processed on the bioanalyzer for quality control prior to proceeding with mRNA library construction and bulk RNA sequencing. Due to degradation of pancreas tissues by pancreatic enzymes, only antral extracts and pituitary samples were usable for downstream library construction and sequencing. Samples were sequenced by Novogene using Ilumina sequencing platforms and the resulting calculated fragments per kilobase of transcript sequence per millions base pairs sequenced values were analyzed for differential gene expression, principal component analysis, and Gene Ontology enrichment analysis. Differential expression between WT and GFAPΔMen1 groups was determined using the DESeq2 R package (1.20.0) using a negative binomial distribution model. P values were adjusted for multiple testing using the Benjamini and Hochberg’s method for controlling false discovery rate and genes with adjusted P value < .05 were considered significantly differentially expressed. The statistical enrichment of DEGs in Kyoto Encyclopedia of Genes and Genomes pathways was tested using the clusterProfiler R package with correction for gene length bias. Gene Ontology terms with a corrected P value < .05 were considered significantly enriched. Heatmaps of significant DEGs assigned to specific signatures of interest were generated using Python 3 using the open-source packages Seaborn and Pandas. For each gene, the Z-score was calculated and plotted as a function of color, with the color white being fixed to a Z-score of 0.

      Western Blot Analysis

      Seventy-two hours following siRNA-mediated Men1 silencing, rat enteric glial cells were harvested by washing twice in cold DPBS and lysing in cold RIPA buffer containing phosphatase and protease inhibitors. Cells were collected by scraping, then homogenized by passing through a 20G needle syringe 5 times. The resulting lysate was centrifuged at 15,000 × g for 20 minutes at 4 °C. The supernatant was collected and used for Western blot analysis as follows. Lysates were prepared in reducing conditions in 1× SDS buffer with 5% β-mercaptoethanol and denatured by boiling at 95 °C for 5 minutes. Fifteen μg of protein was loaded into a NuPage 4% to 12% Bis-Tris Mini Protein Gel and allowed to migrate for 1 hour at 140 V in 1× MOPS Gel Electrophoresis Buffer. Proteins were transferred onto a pre-wetted polyvinylidene fluoride membrane using the iBlot2 transfer system (P0 setting, 7 minutes), then blocked in 5% BSA TBST for 1 hour at RT. Membranes were incubated overnight at 4 °C in primary antibody diluted in blocking buffer at the dilutions listed in Table 4. Membranes were washed in TBST, then incubated for 1 hour at RT in a corresponding host IgG horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:3000 in blocking buffer. Membranes were washed again in TBST and incubated in Pierce ECL reagent (ThermoFisher) for 1 minute prior to developing on film in a dark room.
      For analysis of tissue extracts, gastric antra of 8- to 9-month-old mice were homogenized in ice-cold RIPA buffer using a rotor-stator homogenizer. Samples were centrifuged at 15,000 × g for 15 minutes, and the supernatant (20 μg) was evaluated for Gli2 protein expression (Table 5).
      Table 5List of RT-qPCR Primers From Integrated DNA Technologies
      GenePrimer sequence 1 (5' to 3')Primer sequence 2 (5' to 3')IDT PrimeTime ID
      ChgaCCAAGGTGATGATGAAGTGCGTTGGTGTCGCAGGATAGAGAMm.PT.58.41352258
      SypCAGACAGGAAACACATGCAAGTCTCCTTGAACACGAACCATAGMm.PT.58.29275406
      GfapGCATCTCCACAGTCTTTACCAAACCGCATCACCATTCCTGMm.PT.58.31297710
      GfapCATCTCCACCGTCTTTACCACAACCGCATCACCATTCCTGRn.PT.58.36145160
      S100bTCACTTTGTCCACCACTTCCGCCCTCATTGATGTCTTCCATRn.PT.58.36785742
      Men1CCCAAGCATGATCTTCAGACACCCACATCCAGTCCCTCTTMm.PT.58.30625021
      Men1CATCTCTGAGACCTAGAGCCTCACATCCAGTCTCTCTTCAGCRn.PT.58.9524749
      ChgbCCTTTCTCTTCACCTTTGACCTCCTATCCAAGTCCAGTGTTCCMm.PT.58.11605373
      Eno2ACATCCATACCGATCACCATCCAAGGACAAGTATGGCAAGGAMm.PT.58.31414881
      PrlATATCAGCAACAGGAGGAGTGGTTCTCTCAGGCCATCTTGGMm.PT.58.9092137
      VipGAGTATCAGGAATGCCAGGAAGAACTTCAGCACCCTAGACAGMm.PT.58.32346790
      GastCCAAAGTCCATCCATCCGTAGTCCTCAACACTTCATAGCAGACMm.PT.58.9850738
      GrpCCTTGTCGTTGTCCCTTCAGCCTCTCAGTCTCCAGCCTMm.PT.58.41177438
      GrpCCTTGTCATGGTCCCTTCAGATGGCAGCTACTTCAGTGATGRn.PT.58.45266321
      Gli2ATCATGCGTTGTAGGTCGAGCATCTCCATGACTACCTCAACCMm.PT.58.11001473
      Cdkn2aCTCTGCTCTTGGGATTGGCGTGCGATATTTGCGTTCCGMm.PT.58.43961185
      Cdkn2bGTGCACAGGTCTGGTAAGGAGATCCCAACGCCCTGAAMm.PT.58.7138437
      Cend1CTCACCTCTGGGCATTATATGGGAAACTCCTGAGCACTCCTCMm.PT.58.8515727
      CckbrCAGAAAGATCACCGCATAAAGGTCCCTCCTCAACAGCAGTAGMm.PT.58.45922724
      ShhCGTAAGTCCTTCACCAGCTTGGAATCCAAAGCTCACATCCACMm.PT.58.14105875
      Atp4bCCTCTTTCCCCATACACGTCATCTATGTGCTGATGCAGACCMm.PT.58.5892771
      GifCTTGGTAGGTTGCTCAGGTGAGGCTATTGTGGAGAAGATCAGMm.PT.58.13185585
      Lgr5GTCAAAGCATTTCCAGCAAGACTCCAACCTCAGCGTCTTCMm.PT.58.12492947
      Muc5acCTGGTTGAGTGGTTGTGTGTCCCATGTGTATTCCTCTCCCAMm.PT.58.42279692
      Muc6GCAGTTGGAGACACAAAGGTACATGACATCCACTCTCACACCMm.PT.58.33323120
      Hprt1AACAAAGTCTGGCCTGTATCCCCCCAAAATGGTTAAGGTTGCMm.PT.39a.22214828
      Hprt1GCTTTTCCACTTTCGCTGATGGGTGAAAAGGACCTCTCGAAGRn.PT.39a.22214832
      RT-qPCR, Real-time quantitative polymerase chain reaction.

      Immunoprecipitation Assay

      Immunoprecipitation of menin was performed in rat enteric glial cells. Prior to lysis, cells were collected in ice-cold DPBS, centrifuged, and washed once with cold DPBS. The cell pellet was resuspended in cytoplasmic lysis buffer to generate a cytoplasmic extract. Buffer was supplemented with 1× HALT protease and phosphatase inhibitor. Cytoplasmic extracts were pipetted up and down 20×, incubated on ice for 10 minutes, then vortexed for 15 seconds prior to centrifuging at 15,000 × g for 5 minutes at 4 °C. The supernatant was saved as the cytoplasmic fraction. Protein concentration in freshly prepared lysates was evaluated by bicinchoninic assay. To form an antigen-antibody complex, 1 mg lysate was incubated with either mouse anti-menin monoclonal antibody (Bethyl Laboratories, #A500-003A, 3μg per mg lysate) or mouse IgG1 Isotype control (Cell Signaling Technologies, 3μg per mg lysate) overnight at 4 °C. Fifty μL of prewashed Protein A/G agarose beads (Pierce, #20423) was added to each antigen-antibody complex and incubated for 2 hours at 4 °C on a rotator. Bead-complexes were pelleted at 3000 × g for 2 minutes and washed 6 times with lysis buffer. Bound proteins were eluted by mixing with 2× SDS sample buffer. Samples were incubated at 50 °C for 10 minutes, vortexed briefly, then centrifuged to remove beads. Dithiothreitol was added to the protein suspension at a final concentration of 10 mM prior to boiling at 95 °C for 5 minutes. Samples were then evaluated by Western blot as previously described. Extracts were probed using rabbit anti-GFAP antibody (Dako Agilent Technologies, #Z033429, 1:3000). Immunoprecipitation was repeated 3 times in independent experiments and quantified via densitometry analysis using Image J Fiji software.

      Hormone Immunoassays

      GCG hormone was evaluated in mouse serum using the Mouse Glucagon enzyme-linked immunosorbent assay (ELISA) Kit according to manufacturer’s instructions (Crystal Chem, #81518). Serum was diluted 1:10 (10 μL serum per well) and incubated with anti-mouse GCG and HRP-secondary antibody overnight at 4 °C. Glucagon concentration was determined by HRP enzymatic activity, and the absorbance at 450 nm was averaged across duplicate wells. INS1 hormone was measured in mouse serum using the Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem, #90080). Five μL serum (1:20) was evaluated in duplicate and incubated with anti-mouse INS1 antibody overnight at 4 °C. INS1 concentration was interpolated from a high range standard curve of 1–64 ng/mL). Serum glucose was measured using a Mouse Glucose Assay Kit (Crystal Chem, #81692). Two μL serum (1:50) was incubated with an oxidizing enzyme solution for 10 minutes at 37 °C to yield hydrogen peroxide and generate a red dye. Absorbance at 505 nm was measured and averaged across technical duplicates. Glucose concentration was interpolated from a standard curve ranging from 10.5 to 675 mg/dL. Serum prolactin levels were measuring using the Mouse Serum Prolactin ELISA Kit (Invitrogen, #EMPRL). Serum was diluted 1:20 (5 μL serum per well) and incubated with anti-mouse prolactin antibody overnight at 4 °C. HRP-secondary enzymatic activity was evaluated by measuring absorbance at 450 nm, and optical density values were averaged prior to interpolating concentrations from a standard curve of 27.43 to 20,000 pg/mL.
      Gastrin peptide was evaluated in mouse serum using the Gastrin Enzyme Immunoassay according to manufacturer’s instructions (Sigma, #RAB0200). Serum was diluted 1:4 (25 μL serum per well) and incubated with biotinylated gastrin peptide anti-gastrin-1 antibody overnight at 4 °C. Gastrin concentration was determined by HRP enzymatic activity, and the average absorbance at 450 nm was calculated across duplicate wells. Gastrin concentration was interpolated from a standard curve of 0.1 to 1000 pg/mL gastrin-1 peptide that was plotted using a 4-parameter logistic regression. For tissue, gastrin peptide was extracted from the antra of 8- to 9-month-old mice by boiling tissues in 200 μL of nuclease-free water and vigorously vortexing. The resulting solution was centrifuged to pellet debris, and 50 μL of the supernatant was evaluated in duplicate.

      Gastric Acid Determination

      Following overnight fasting and necropsy, whole stomachs were removed, opened, and flushed with 5 mL of 0.9% saline solution. The stomach acid-solution was centrifuged at 2000 × g for 5 minutes, and the supernatant was collected for pH determination using a pH meter (Orion Star A111). The hydrogen ion concentration was measured by base titration with 0.005 N sodium hydroxide. Acid levels were calculated following normalization to mouse body weight.

      Statistical Analysis

      Assays comparing one parameter (eg, gastrin hormone) across 2 genotypes were evaluated for statistical significance by applying the unpaired Student t test. All qPCR data expressed as fold-change (2-ddCt) were log-transformed to fit a normal distribution prior to performing statistical analysis. Comparisons consisting of 3 or more genotypes or groups were evaluated for significance using 1-way analysis of variance, with Tukey post-test. Group comparisons of 2 or more parameters (eg, multiple genes) across genotypes and tissues were evaluated for significance using 2-way analysis of variance with Sidak post-test. Significance was determined as ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 using Graphpad Prism (v9) software.

      CRediT Authorship Contributions

      Suzann Duan, PhD (Conceptualization: Supporting; Data curation: Lead; Formal analysis: Lead; Methodology: Lead; Validation: Lead; Visualization: Lead; Writing – original draft: Lead; Writing – review & editing: Supporting)
      Travis W. Sawyer, PhD (Data curation: Supporting; Formal analysis: Supporting; Software: Supporting)
      Ricky A. Sontz, MS, BS (Methodology: Supporting; Validation: Supporting)
      Bradley A. Wieland, BS (Data curation: Supporting)
      Andres F. Diaz, MS, BS (Data curation: Supporting)
      Juanita L. Merchant, MD, PhD (Conceptualization: Lead; Funding acquisition: Lead; Supervision: Lead; Writing – original draft: Supporting; Writing – review & editing: Lead)

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