e15734 Background: Microsatellite stable colorectal cancer (CRC) is historically poorly responsive to immunotherapy, due in part to low neoantigen burden, an immunosuppressive tumor microenvironment, and poor T-cell infiltration. Dipeptidase-1 (DPEP1) is a GPI-anchored protein involved in glutathione and leukotriene metabolism and was identified as a part of a 4-gene immune cell exclusion signature associated with worse overall and progression free survival. Here we investigated the incidence and distribution of DPEP1 isoforms in data from The Cancer Genome Atlas (TCGA) and the in vitro properties of both isoforms. Methods: To determine the prevalence of DPEP1 Isoform B in human CRCs, we aligned TCGA Colon Adenocarcinoma (COAD) and Rectal Adenocarcinoma (READ) sample transcripts to the RefSeq (NCBI) genome database which has annotation of both DPEP1 isoforms. Murine CRC MC38 cells overexpressing human DPEP1 isoform A or isoform B were generated via lentiviral infection. The resulting cells underwent RNA sequencing followed by genome ontology and KEGG pathway over-representation analysis using the WebGestalt R package with FDR q-values ≤ 0.05. These DPEP1-expressing cells were injected into the tail veins of host mice to determine the effect of each isoform on metastasis, differences in means determined via Welch’s T-test. Results: Bulk RNA sequencing of 640 samples from 615 patients revealed DPEP1 A was found in 61% of CRC samples, while DPEP1 B was found in 91% of CRC samples, making DPEP1 B the predominant isoform. Furthermore, samples expressing high levels of DPEP1 B as compared to low or no expression, were associated with left-sided primary tumor location, MSS status, younger age at diagnosis, and no prior history of colon polyps. Transcriptional profiling revealed three distinct gene expression clusters that were correlated with DPEP1 isoform expression. Cluster one was associated with low to no DPEP1 A but high DPEP1 B. This cluster was enriched for non-coding RNAs and alternative spicing genes including RUNX1T1 and the small nuclear spliceosome RNAs. Cluster two was associated with high levels of both DPEP1 A and B expression. This was enriched for multiple G2/M cell cycle and Myc-associated genes. RNA sequencing revealed an upregulation in Myc-targets, EMT, and TNFα pathways in MC38 cells expressing isoform B. This expression of isoform B led to an increased metastatic burden in a mouse model compared to isoform A (p < 0.05). Conclusions: This marks the discovery of a novel isoform of DPEP1 that is upregulated in colorectal cancer patients. These data support the continued exploration of DPEP1 as a predictive biomarker for response to immune checkpoint inhibitors in MSS CRC as well as an emerging therapeutic target given its association with Myc and increased metastatic burden in preclinical models.

Background and Aims EGFR signaling maintains intestinal homeostasis by modulating proliferation and differentiation within the stem cell compartment with excess EGFR signaling predisposing to neoplasia. EGFR protein levels are central to EGFR function. These levels are negatively regulated by LRIG1, which accelerates receptor internalization and degradation, supporting a tumor suppressor role for LRIG1. LRIG3 is a less studied family member that has been reported, in a context-dependent manner, to both cooperate with and oppose the effects of LRIG1 on EGFR. Methods To examine the function of LRIG3 relative to LRIG1 on EGFR levels and downstream signaling in vivo, we generated an Lrig3 floxed allele and intercrossed these mice with Lrig1CreERT2 mice that are Lrig1 null in the homozygous state. This allowed us to assess the effects of Lrig3 loss in Lrig1-null intestinal cells. We also tested how LRIG3 depletion in Lrig1-expressing cells affects colorectal tumors in Lrig1CreERT2/+; Apcfl/+ mice. Results Unlike the heightened EGFR and Wnt activity caused by Lrig1 loss, loss of Lrig3 in Lrig1Null cells markedly reduced activity in both pathways. In this setting, a widening of the intestinal crypt base was observed with the appearance of intermediate-lineage secretory cells at the expense of stem cells. In marked contrast to multiple distal colonic tumors in Lrig1CreERT2/+; Apcfl/+ mice, deletion of a single Lrig3 allele in Lrig1 haploinsufficient mice decreased EGFR signaling and markedly reduced colonic tumor formation. Conclusions These findings indicate that LRIG3 and LRIG1 oppose one another to balance EGFR signaling, maintain intestinal homeostasis and minimize the likelihood colonic neoplasia.

Immune checkpoint blockade (ICB) is an effective treatment for microsatellite instability-high (MSI-H) colorectal cancers (CRCs) that are highly infiltrated by CD8+ T cells. Microsatellite stable (MSS) CRCs are unresponsive to ICB, at least in part, due to the paucity of intratumoral CD8+ T cells. We recently identified Discoidin Domain Receptor 1 (DDR1) as one of four genes associated with CD8+ T-cell exclusion in MSS CRC. There are conflicting reports about the presence and role of the cleaved ectodomain (cECD) of DDR1 in mouse models of breast and pancreatic cancer. To explore the role of the DDR1 cECD in human CRC, we developed Collagen Alignment and Spatial Transcriptomics Analysis (CASTA), which revealed that genes involved in fibroblast contractility were associated with both DDR1 tumor expression and collagen alignment as determined by label-free second harmonic generation (2HG) imaging of the tumor collagen. Using 3D collagen co-cultures of CRC spheroids and fibroblasts, we show that DDR1 promotes collagen alignment and CD8+ T-cell exclusion. We found large amounts of DDR1 cECD in MSS CRC supermeres, 25-35 nm secreted amembranous nanoparticles. Supermeres containing DDR1 cECD were sufficient to induce contraction of human colonic fibroblasts. We propose a model in which supermeres containing DDR1 cECD promote the contraction of stromal fibroblasts in MSS CRC, leading to collagen alignment and CD8+ T-cell exclusion. These results support DDR1 cECD as an attractive therapeutic target in MSS CRC.

Abstract Protein isoforms play significant roles in cancer progression as they can impact localization, binding partners, and functions, especially when it comes to pro-tumorigenic properties. One protein in particular, dipeptidase-1 (DPEP1), was identified 25 years ago as a cell-surface protein upregulated in adenomas and colorectal cancers (CRCs) in comparison to normal colonic tissue. We have found that a diffuse staining pattern of DPEP1, not a cell-surface localization, leads to worse overall and progression-free survival for CRC patients. Spurred by the inconsistency of an intracellular DPEP1 staining pattern for a cell surface protein, we discovered two isoforms of DPEP1 that differ in their C-terminal sequence. This difference reflects an absence of a glycosylphosphatidylinositol anchorage signal sequence in a novel, undescribed isoform of DPEP1, that we have termed DPEP1 Isoform B. We found expression of DPEP1 Isoform B in CRC patient tissue samples which, upon immunohistochemical staining, revealed an intracellular localization of DPEP1 in cells. DPEP1 Isoform B expression transforms a non-tumorigenic cell line to form large, invasive tumors in nude mice and upregulates pro-tumorigenic gene programs including Myc targets, epithelial-to-mesenchymal transition, and angiogenesis as identified by RNA-seq. Surprisingly, in a CRC cell line, we find that DPEP1 Isoform B binds to Myc, increasing its half-life 2-fold. Utilizing TCGA data sets, we deployed a strategy to examine long transcripts and found that DPEP1 Isoform B was present in 91% of CRC patients, with 28% of patients having high expression of DPEP1 Isoform B. To contrast, DPEP1 Isoform A was present in 68% of CRC patients, having high expression in 26% of patients. Overall, our work demonstrates the discovery of a novel isoform of DPEP1, which given its high incidence in CRC, warrants further investigation as a predicative biomarker as well as a novel therapeutic target. Citation Format: Elizabeth Grace Fisher, Sarah E. Glass, Chelsie K. Sievers, Zheng Cao, Matthew E. Bechard, Samuel T. Ellis, Radhika Aramnadla, Ping Zhao, Ryan T. Smith, Yu Wang, James N. Higginbotham, Frank Revetta, M. Kay Washington, Martha J. Shrubsole, Qi Liu, Ken S. Lau, Bruce Aronow, Robert J. Coffey. Uncovering a highly transforming, Myc-stabilizing isoform of dipeptidase-1 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4760.

The mammalian intestine regenerates rapidly after damage, yet the clonal dynamics and species-specific regulation of different populations remain poorly understood. Here we used synthetic or naturally occurring DNA alterations to reconstruct clonal histories of the mouse and human intestinal epithelium at single-cell resolution. In mice, we uncovered the clonal architecture of different cell types and their roles in regeneration, supporting a hierarchical regenerative response model. We identified a rare embryonic precursor population that persisted in the adult and was crucial for regeneration after irradiation. This population was marked by Tob2, which is required for nuclear transport of Ascl2. A parallel clonal analysis of 65 human colonic biopsies revealed secretory lineage bias and an age-associated decline in clonal diversity in the distal colon. Unlike highly proliferative murine Lgr5+ stem cells, human LGR5+ cells were found largely quiescent, revealing species-specific difference in clonal potency, and suggesting a distinct regulation of intestinal stemness.

Dipeptidase-1 (DPEP1) is highly upregulated in colorectal cancer (CRC), with its enzymatic function linked to invasion and metastasis. More recently, DPEP1 was found to serve as a receptor for neutrophils when expressed by activated endothelial cells. It is unknown whether neutrophils bind to DPEP1-expressing CRC cells and whether this impacts features of CRC. Neutrophils have been shown to be tumor promoting in cancers including CRC, where they act to exclude CD8+ T cells. Herein, we show that neutrophils bind DPEP1-expressing CRC cells. In addition, DPEP1 is preferentially expressed in microsatellite-stable (MSS) CRCs, in which there are a paucity of CD8+ T cells, whereas DPEP1 is negatively correlated with microsatellite-unstable (MSI-H) CRCs, which are T cell rich and are more responsive to immunotherapy. Remarkably, carcinogen-treated Dpep1-null mice develop multiple, large, plaque-like, locally invasive adenocarcinomas and squamous cell cancers in the distal colon. These adenocarcinomas exhibit a marked reduction in neutrophils and an influx CD8+ T cells, along with reduced expression of mismatch repair proteins, consistent with features of MSI-H CRC. These results establish DPEP1’s importance in maintaining MSS CRC and its ability to shape the tumor microenvironment.

Both Ménétrier's disease (MD) and juvenile polyposis syndrome (JPS) are rare premalignant conditions that can lead to gastric cancer. MD is an acquired disease without known causative mutations that is characterized by increased expression of an EGF receptor (EGFR) ligand, transforming growth factor-alpha (TGFα), in the stomach. JPS is inherited in an autosomal dominant pattern and is caused by BMPR1A or SMAD4 mutations. Although there are distinct clinico-pathological features that differ between the two diseases, they also share similar features that often lead to misdiagnosis. To identify diagnostic markers for MD and to better understand the pathogenesis of the disease, we performed transcriptomic profiling of stomach tissues from normal (NL), MD, and JPS patients. Comparative analysis between MD and JPS revealed both common and differential gene signatures. Common gene signatures included estrogen receptor signaling, integrin signaling, mTOR signaling, and others, which may be responsible for histopathological similarities. Among differential gene signatures, we found that Hedgehog (Hh) signaling is upregulated in MD and confirmed that protein expression of Hh signaling downstream targets, GLI1 (glioma-associated oncogene homolog 1) and HHIP (Hedgehog-interacting protein), is higher in MD than in JPS, particularly in foveolar cells by immunohistochemistry. We also demonstrated that treatment with an Hh pathway inhibitor partially rescued the histopathological phenotypes in an MD mouse model. This study provides valuable insights into the potential mechanisms underlying the similar clinico-pathological features observed in MD and JPS. We also identified GLI1 and HHIP as diagnostic markers that can help to distinguish MD from JPS. Furthermore, Hh signaling was shown to play an important role in the pathogenesis of MD and may serve as a potential therapeutic target. © 2025 The Pathological Society of Great Britain and Ireland.

Abstract DPEP1 is a well-established GPI-linked dipeptidase whose expression is highly upregulated in colorectal cancer (CRC). Its enzymatic function includes extracellular processing of glutathione and leukotrienes, which has been linked to invasion and metastasis in CRC. More recently, it was found to serve as a receptor for neutrophils when expressed by activated endothelial cells. It is unknown whether neutrophils bind to DPEP1-expressing CRC cells and whether this impacts features of CRC. Neutrophils have been shown to accumulate and exclude cytotoxic CD8+ T cells in CRC with DPEP1 being identified as a key gene in immune exclusion that has not been functionally evaluated. Herein, we show that neutrophils bind CRC cells that express DPEP1 and that DPEP1 is preferentially expressed in microsatellite stable CRCs, in which there are a paucity of CD8+ T cells. In contrast, DPEP1 is negatively correlated with microsatellite unstable CRCs, which are T cell-rich and have a much higher propensity to respond to immunotherapy. Notably, carcinogen-treated DPEP1 null mice develop multiple, large, plaque-like, locally invasive adeno-squamous carcinomas with markedly reduced DNA repair protein expression and neutrophil presence but exhibited an influx of CD8+ T cell within the tumor. These results establish DPEP1’s importance in maintaining MSS CRC and its ability to shape the tumor microenvironment. Citation Format: Sarah Glass, Matthew Bechard, Zheng Cao, Radhika Aramnadla, Ping Zhao, Samuel Ellis, Elizabeth Fisher, Frank Revetta, M. Kay Washington, Gregory Ayers, Cody Heiser, Annika Windon, Nicholas Markham, Martha Shrubsole, Ken Lau, Robert J Coffey. DPEP1 maintains microsatellite stability in colorectal cancer at the tumor microenvironment interface [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A003.

The cellular census of the colonic crypt is tightly regulated, yet the molecular mechanisms that regulate this census are not fully understood. Lrig3, a transmembrane protein, is expressed in colonic crypt epithelial cells, including the stem, progenitor, and differentiated cell types. Mice missing Lrig3 have a disruption in their cellular census: using a novel Lrig3 -/- mouse we demonstrate that Lrig3 -/- mice have more cells per crypt, a greater mucosal area, and longer colons compared to wildtype mice, suggesting the expression of Lrig3 is required for both the total number of epithelial cells in the mouse colon, as well as colon length. In addition, we show Lrig3 -/- mice have significantly more stem, progenitor, and deep crypt secretory cells, yet harbor a normal complement of enteroendocrine, Tuft, and absorptive cells. Lrig3 -/- mice also have a concomitant decrease in phosphorylated Extracellular signal-related kinases, indicating the loss of Lrig3 leads to an expansion of the colonic stem cell compartment, in an Erk-dependent manner. Our study describes the expression of Lrig3 within the colon, defines perturbations in mice lacking Lrig3 , and supports a role for Lrig3 in the establishment of both colonic crypt structure and cellular census, defined as the epithelial cell type and number in colon crypts. Graphical Abstract

Bert Vogelstein and co-workers identified DPEP1 in 2001 as one of the top six upregulated genes encoding a membrane or secreted protein in colorectal adenomas and cancers (CRCs). DPEP1 is a GPI-linked, zinc-dependent dipeptidase whose enzymatic activity includes processing extracellular glutathione and converting LTD4 to LTE4. Recently, it was reported to act nonenzymatically as a receptor for neutrophils on liver and lung endothelial cells and to influence monocyte migration. DPEP1 has been reported to have roles in cancer proliferation, invasion and metastasis. We recently showed that DPEP1 is the most abundant protein in small extracellular vesicles (sEVs) released from CRC cell lines by mass spectrometry and is present in a subset of EGFR+/CD81+ exosomes along with other clinically relevant biomarkers by fluorescence-activated vesicle sorting (FAVS) (Q Zhang et al., Nature Cell Biology 23:1240-1254, 2021). We also found that DPEP1/CEACAM5 double-positive exosomes are increased in the plasma from three CRC patients compared to normal controls. Immunohistochemical staining for DPEP1 in clinically well-annotated adenoma and CRC tissue microarrays has revealed staining in 27% of adenomas and 71% of CRCs, whereas no staining was observed in normal colonic tissue. Moreover, diffuse cytoplasmic staining in CRCs was associated with a worse progression-free and overall survival. We have now generated adenoma organoids, some of which express endogenous DPEP1 that localizes to the apical plasma membrane in 3D organoids and when cultured as polarized monolayers on Transwell filters. We plan to examine the bi-directional effects of cellular DPEP1 and DPEP1-containing EVs and freshly isolated neutrophils and/or their EVs in a co-culture system that is part of our recently funded Translational and Biological Science in Early Lesions (TBEL) NCI U54 grant. Citation Format: Matthew E. Bechard, Sarah E. Glass, Robert J. Coffey. Dipeptidase-1 is a highly upregulated protein in CRC exosomes: Potential clinical relevance [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B030.

Metastatic outgrowth is supported by metabolic adaptations that may differ from the primary tumor of origin. However, it is unknown if such adaptations are therapeutically actionable. Here we report a novel aminopyridine compound that targets a unique Phosphogluconate Dehydrogenase (PGD)-dependent metabolic adaptation in distant metastases from pancreatic cancer patients. Compared to structurally similar analogs, 6-aminopicolamine (6AP) potently and selectively reversed PGD-dependent metastatic properties, including intrinsic tumorigenic capacity, excess glucose consumption, and global histone hyperacetylation. 6AP acted as a water-soluble prodrug that was converted into intracellular bioactive metabolites that inhibited PGD in vitro, and 6AP monotherapy demonstrated anti-metastatic efficacy with minimal toxicity in vivo. Collectively, these studies identify 6AP and possibly other 6-aminopyridines as well-tolerated prodrugs with selectivity for metastatic pancreatic cancers. If unique metabolic adaptations are a common feature of metastatic or otherwise aggressive human malignancies, then such dependencies could provide a largely untapped pool of druggable targets for patients with advanced cancers.

Our organoid generation technique has allowed for the development of downstream organoid applications. Here, we detail an accessible, straightforward protocol for immunofluorescent staining and imaging of thyroid cancer organoids, particularly those with tumor de-differentiation. Immunofluorescence is a powerful tool to help understand the localization of cell types within organoids and determine the interactions between those cells. As organoids have been shown to recapitulate patient tumor morphology, immunofluorescent staining and imaging of organoids allows for enhanced understanding of near in vivo structures. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020).

Although metastasis is the most common cause of cancer deaths, metastasis-intrinsic dependencies remain largely uncharacterized. We previously reported that metastatic pancreatic cancers were dependent on the glucose-metabolizing enzyme phosphogluconate dehydrogenase (PGD). Surprisingly, PGD catalysis was constitutively elevated without activating mutations, suggesting a non-genetic basis for enhanced activity. Here we report a metabolic adaptation that stably activates PGD to reprogram metastatic chromatin. High PGD catalysis prevents transcriptional up-regulation of thioredoxin-interacting protein (TXNIP), a gene that negatively regulates glucose import. This allows glucose consumption rates to rise in support of PGD, while simultaneously facilitating epigenetic reprogramming through a glucose-fueled histone hyperacetylation pathway. Restoring TXNIP normalizes glucose consumption, lowers PGD catalysis, reverses hyperacetylation, represses malignant transcripts, and impairs metastatic tumorigenesis. We propose that PGD-driven suppression of TXNIP allows pancreatic cancers to avidly consume glucose. This renders PGD constitutively activated and enables metaboloepigenetic selection of additional traits that increase fitness along glucose-replete metastatic routes.

Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (H4MPT) as a one-carbon carrier instead of tetrahydrofolate. In H4MPT biosynthesis, β-ribofuranosylaminobenzene 5'-phosphate (RFAP) synthase catalyzes the production of RFAP, CO2, and pyrophosphate from p-aminobenzoic acid (pABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from Methanothermobacter thermautotrophicus was produced in Escherichia coli, and site-directed mutagenesis was used to alter arginine 26 (R26) and aspartic acid 19 (D19), located in a conserved sequence of amino acids resembling the pABA binding site of dihydropteroate synthase. Replacement of R26 with lysine increased the KM for pABA by an order of magnitude relative to wild-type enzyme without substantially altering the KM for PRPP. Although replacement of D19 with alanine produced inactive enzyme, asparagine substitution allowed retention of some activity, and the K M for pABA increased about threefold relative to wild-type enzyme. A molecular model developed by threading RFAP synthase onto the crystal structure of homoserine kinase places R26 in the proposed active site. In the static model, D19 is located close to the active site, yet appears too far away to influence ligand binding directly. This may be indicative of the protein conformational change predicted previously in the Bi-Ter kinetic mechanism and/or formation of the active site at the interface of two subunits. Due to the vital role of RFAP synthase in H4MPT biosynthesis, insights into the mode of substrate binding and mechanism could be beneficial for developing RFAP synthase inhibitors designed to reduce the production of methane as a greenhouse gas.

During mouse pancreas organogenesis, endocrine cells are born from progenitors residing in an epithelial plexus niche. After a period in a lineage-primed Neurog3(LO) state, progenitors become endocrine committed via upregulation of Neurog3 We find that the Neurog3(LO) to Neurog3(HI) transition is associated with distinct stages of an epithelial egression process: narrowing the apical surface of the cell, basalward cell movement and eventual cell-rear detachment from the apical lumen surface to allow clustering as nascent islets under the basement membrane. Apical narrowing, basalward movement and Neurog3 transcriptional upregulation still occur without Neurog3 protein, suggesting that morphogenetic cues deployed within the plexus initiate endocrine commitment upstream or independently of Neurog3. Neurog3 is required for cell-rear detachment and complete endocrine-cell birth. The ROCK-nmMyoII pathway coordinates epithelial-cell morphogenesis and the progression through Neurog3-expressing states. NmMyoII is necessary for apical narrowing, basalward cell displacement and Neurog3 upregulation, but all three are limited by ROCK activity. We propose that ROCK-nmMyoII activity, Neurog3 gene-dose and Notch signaling integrate endocrine fate allocation with epithelial plexus growth and morphogenesis, representing a feedback control circuit that coordinates morphogenesis with lineage diversification in the endocrine-birth niche.

Pancreatic ductal adenocarcinoma (PDAC) adopts several unique metabolic strategies to support primary tumor growth. Whether additional metabolic strategies are adopted to support metastatic tumorigenesis is less clear. This could be particularly relevant for distant metastasis, which often follows a rapidly progressive clinical course. Here we report that PDAC distant metastases evolve a unique series of metabolic reactions to maintain activation of the anabolic glucose enzyme phosphogluconate dehydrogenase (PGD). PGD catalytic activity was recurrently elevated across distant metastases, and modulating PGD activity levels dictated tumorigenic capacity. Metabolomics data raised the possibility that distant metastases evolved a core pentose conversion pathway (PCP) that converted glucose-derived metabolites into PGD substrate, thereby hyperactivating the enzyme. Consistent with this, each individual metabolite in the PCP stimulated PGD catalysis in distant metastases, and knockdown of each individual PCP enzyme selectively impaired tumorigenesis. We propose that the PCP manufactures PGD substrate outside of the rate-limiting oxidative pentose phosphate pathway (oxPPP). This enables PGD-dependent tumorigenesis by providing adequate substrate to fuel high catalytic activity, and raises the possibility that PDAC distant metastases adopt their own unique metabolic strategies to support tumor growth.

Neurogenin 3 (Neurog3) is a pro-endocrine transcription factor required for endocrine-lineage specification during mouse pancreas development. It was a long-standing notion that Neurog3 activation in Sox9C epithelial cells, a subset of early multipotent progenitors that produce ductal and endocrine lineages, triggers a rapid progression to a post-mitotic, Neurog3 endocrine-committed precursor state. We challenged this model by demonstrating that a mitotic progenitor state, in which Neurog3 is transcriptionally active but at a low level (Neurog3), pre-empts endocrine-commitment. We postulated a new model in which this Neurog3 progenitor state is a mitotic, metastable condition in which low Neurog3 levels regulate decisions regarding progenitor maintenance and endocrine-lineage specification. Our work showed that Neurog3 progenitors resemble other progenitor populations, such as intestinal and haematopoietic progenitors, in their ability to undergo multiple rounds of division that seem to be symmetric and produce either more Neurog3 progenitors, or two endocrine-committed daughters. This modification of the endocrine-lineage specification model focuses attention on the Neurog3 progenitor state as being the stage wherein the critical decision is made as to which endocrine cell fate will be established within committed cells emerging from Neurog3 progenitors. Below, we discuss potential connections between the cell cycle and Neurog3 protein levels in Neurog3 progenitors, and the implications of bringing this model together with a systems-level understanding of the 3-dimensional and dynamic nature of an epithelial plexus state that represents the endocrine-birth niche. Understanding how cycling Neurog3 progenitors integrate their behavior within the surrounding dynamics of a developing epithelial endocrine birth niche will be important. For example, what determines progenitor-maintaining or symmetric endocrinebirth divisions, and the end-fate endocrine potential of Neurog3 progenitors, could inform on how to generate multilineage islets rather than simply populations of b cells. Gain and loss of function studies show the power of Neurog3 in instructing endocrine differentiation, but other studies had already begun to suggest correlations between a more finely adjusted Neurog3 expression level and the potential of the Neurog3 population to produce endocrine cells (Fig. 1). Wang et al. (2010) reported that a strong Neurog3 hypomorphic condition significantly decreased the degree of endocrine biasing, diverting many cells to non-endocrine acinar or ductal fates (Fig. 1). Our analysis of the same hypomorphic condition showed a doubling of the mitotic index of Neurog3 progenitors, but not of the surrounding nonendocrine-biased cells, and expansion of the Neurog3 pool at the expense of endocrine-committed cells. In addition to low-level Neurog3 expression within the endocrine-biased mitotic population, a number of Neurog3 downstream targets are also expressed at relatively low levels, suggesting that endocrine-biased cells undertake a concerted effort to build the framework of a gene regulatory network that primes intraepithelial Neurog3 progenitors toward the endocrine lineage, before their committment. Such a concept has been referred to as lineage-priming and is a familiar concept regarding haematopoietic and intestinal stem cells. We propose that the Neurog3 state represents a mitotic metastable progenitor state in which low Neurog3 levels, under normal conditions, begin to drive cells toward the endocrine lineage (Fig. 1). However, small reductions of the Neurog3 level within Neurog3 progenitors allow movement toward other lineages (Fig. 1). The notion that Neurog3 can maintain a metastable progenitor state at low levels, but at higher levels trigger cell-cycle exit and endocrine-lineage commitment is a concept not without precedent. In neural progenitors, the Neurog3 sister protein Neurog2 is actively phosphorylated by Cdks during S-G2-M phases, resulting in a low-level unstable form that activates progenitor-associated target genes. During G1, however, the increased activity of Cdk-inhibitors results in accumulation of underphosphorylated Neurog2 which is stabilized and targets different suites of genes that promote neural differentiation. Similarly, in the pancreas, we found that Neurog3 protein levels are low-to-undetectable in cycling Neurog3 progenitors and transition to an endocrine-committed Neurog3 state »2–3 hours after the symmetric mitotic division, during G1. Previous work using Xenopus egg extract showed stabilization of Neurog3 in the presence of the Cdk-inhibitor p27, and that high Neurog3 levels promote expression of the

During pancreas organogenesis, Neurog3HI endocrine-committing cells are generated from a population of Sox9+ mitotic progenitors with only a low level of Neurog3 transcriptional activity (Neurog3TA.LO ). Low-level Neurog3 protein, in Neurog3TA.LO cells, is required to maintain their mitotic endocrine-lineage-primed status. Herein, we describe a Neurog3-driven FUCCI cell-cycle reporter (Neurog3P2A.FUCCI ) derived from a Neurog3 BAC transgenic reporter that functions as a loxed cassette acceptor (LCA). In cycling Sox9+ Neurog3TA.LO progenitors, the majority of cells in S-G2 -M phases have undetectable levels of Neurog3 with increased expression of endocrine progenitor markers, while those in G1 have low Neurog3 levels with increased expression of endocrine differentiation markers. These findings support a model in which variations in Neurog3 protein levels are coordinated with cell-cycle phase progression in Neurog3TA.LO progenitors with entrance into G1 triggering a concerted effort, beyond increasing Neurog3 levels, to maintain an endocrine-lineage-primed state by initiating expression of the downstream endocrine differentiation program prior to endocrine-commitment.