IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment.

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

In situ regeneration of inner hair cells in the damaged cochlea by temporally regulated co-expression of Atoh1 and Tbx2.

Cochlear inner hair cells (IHCs) are primary sound receptors, and are therefore a target for developing treatments for hearing impairment. IHC regeneration in vivo has been widely attempted, although not yet in the IHC-damaged cochlea. Moreover, the extent to which new IHCs resemble wild-type IHCs remains unclear, as is the ability of new IHCs to improve hearing. Here, we have developed an in vivo mouse model wherein wild-type IHCs were pre-damaged and nonsensory supporting cells were transformed into IHCs by ectopically expressing Atoh1 transiently and Tbx2 permanently. Notably, the new IHCs expressed the functional marker vGlut3 and presented similar transcriptomic and electrophysiological properties to wild-type IHCs. Furthermore, the formation efficiency and maturity of new IHCs were higher than those previously reported, although marked hearing improvement was not achieved, at least partly due to defective mechanoelectrical transduction (MET) in new IHCs. Thus, we have successfully regenerated new IHCs resembling wild-type IHCs in many respects in the damaged cochlea. Our findings suggest that the defective MET is a critical barrier that prevents the restoration of hearing capacity and should thus facilitate future IHC regeneration studies.

Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1.

During development, neural progenitors are in proliferative and immature states; however, the molecular machinery that cooperatively controls both states remains elusive. Here, we report that cyclin D1 (CCND1) directly regulates both proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates S309 of ATOH1, which inhibits additional phosphorylation at S328 and consequently prevents S328 phosphorylation-dependent ATOH1 degradation. Additionally, PROX1 downregulates Ccnd1 expression by histone deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. Moreover, WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT-PROX1-CCND1-ATOH1 signaling cascade cooperatively controls proliferative and immature states of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which are suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.

Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development.

Cochlear hair cells (HCs) in the inner ear are responsible for sound detection. For HC fate specification, the master transcription factor Atoh1 is both necessary and sufficient. Atoh1 expression is dynamic and tightly regulated during development, but the cis-regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized Atoh1 enhancer, defined here as Eh1, failed to impair HC development. By using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we discovered two additional Atoh1 enhancers: Eh2 and Eh3. Notably, Eh2 deletion was sufficient for impairing HC development, and concurrent deletion of Eh1 and Eh2 or all three enhancers resulted in nearly complete absence of HCs. Lastly, we showed that Atoh1 binds to all three enhancers, consistent with its autoregulatory function. Our findings reveal that the cooperative action of three distinct enhancers underpins effective Atoh1 regulation during HC development, indicating potential therapeutic approaches for HC regeneration.

Mosaic CRISPR-stop enables rapid phenotyping of nonsense mutations in essential genes.

CRISPR-stop converts protein-coding sequences into stop codons, which, in the appropriate location, results in a null allele. CRISPR-stop induction in one-cell-stage zygotes generates Founder 0 (F0) mice that are homozygous mutants; this avoids mouse breeding and serves as a rapid screening approach for nonlethal genes. However, loss of function of 25% of mammalian genes causes early lethality. Here, we induced CRISPR-stop in one of the two blastomeres of the zygote, a method we name mosaic CRISPR-stop, to produce mosaic Atoh1 and Sox10 F0 mice; these mice not only survived longer than regular Atoh1/Sox10 knockout mice but also displayed their recognized cochlear phenotypes. Moreover, by using mosaic CRISPR-stop, we uncovered a previously unknown role of another lethal gene, Rbm24, in the survival of cochlear outer hair cells (OHCs), and we further validated the importance of Rbm24 in OHCs by using our Rbm24 conditional knockout model. Together, our results demonstrated that mosaic CRISPR-stop is reliable and rapid, and we believe this method will facilitate rapid genetic screening of developmentally lethal genes in the mouse inner ear and also in other organs.

The proper timing of Atoh1 expression is pivotal for hair cell subtype differentiation and the establishment of inner ear function.

Atoh1 overexpression is essential for hair cell (HC) regeneration in the sensory epithelium of mammalian auditory and vestibular organs. However, Atoh1 overexpression alone cannot induce fully mature and functional HCs in the mammalian inner ear. In the current study, we investigated the effect of Atoh1 constitutive overexpression in native HCs by manipulating Atoh1 expression at different developmental stages. We demonstrated that constitutive overexpression of Atoh1 in native vestibular HCs did not affect cell survival but did impair vestibular function by interfering with the subtype differentiation of HCs and hair bundle development. In contrast, Atoh1 overexpression in cochlear HCs impeded their maturation, eventually leading to gradual HC loss in the cochlea and hearing dysfunction. Our study suggests that time-restricted Atoh1 expression is essential for the differentiation and survival of HCs in the inner ear, and this is pivotal for both hearing and vestibular function re-establishment through Atoh1 overexpression-induced HC regeneration strategies.

Efferent axons in the zebrafish lateral line degenerate following sensory hair cell ablation.

The zebrafish lateral line is a frequently used model to study the mechanisms behind peripheral neuronal innervation of sensory organs and the regeneration thereof. The lateral line system consists of neuromasts, a cluster of protruding hair cells, which are innervated by sensory afferent and modulatory efferent neurons. These flow-sensing hair cells are similar to the hair cells in the mammalian ear. Though, while hair cell loss in humans is irreversible, the zebrafish neuromasts are regarded as the fastest regenerating structure in vertebrates, making them an ideal model to study regeneration. However, one component of the lateral line system, the efferent projections, has largely been omitted in regenerative studies. Here, for the first time, we bring insights into the fate of efferent axons during ablation and regeneration of the hair cells in the zebrafish lateral line. Our behavioral analysis showed functional recovery of hair cells and sensory transmission within 48 h and their regeneration were in line with previous studies. Analysis of the inhibitory efferent projections revealed that in approximately half the cases the inhibitory efferent axons degenerated, which was never observed for the sensory afferent axons. Quantification of hair cells following ablation suggests that the presence of mature hair cells in the neuromast may prevent axon degeneration. Within 120 h, degenerated efferent axons regenerated along the axonal tract of the lateral line. Reanalysis of published single cell neuromast data hinted to a role for Bdnf in the survival of efferent axons. However, sequestering Bdnf, blocking the Trk-receptors, and inhibiting the downstream ERK-signaling, did not induce axon degeneration, indicating that efferent survival is not mediated through neurotrophic factors. To further explore the relation between hair cells and efferent projections, we generated atoh1a mutants, where mature hair cells never form. In larvae lacking hair cells, inhibitory efferent projections were still present, following the tract of the sensory afferent without displaying any innervation. Our study reveal the fate of efferent innervation following hair cell ablation and provide insights into the inherent differences in regeneration between neurons in the peripheral and central nervous system.

POU4F3 pioneer activity enables ATOH1 to drive diverse mechanoreceptor differentiation through a feed-forward epigenetic mechanism.

During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type-specific enhancers remain poorly understood. Here, we show that the transcriptional "master regulator" ATOH1-which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis-is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.

Fgf, Hh, and pax2a differentially regulate expression of pax5 and pou3f3b in vestibular and auditory maculae in the zebrafish otic vesicle.

BACKGROUND: The vertebrate inner ear contains distinct sensory epithelia specialized for auditory or vestibular function. In zebrafish, the first sensory epithelia form at opposite ends of the otic vesicle and are functionally distinct: the anterior utricular macula is essential for vestibular function whereas the posterior saccular macula is critical for hearing. Mechanisms distinguishing these maculae are not clear. Here, we examined the effects of manipulating Fgf or Hh on expression of pax5 and pou3f3b, unique markers of utricular and saccular identity. We also examined the roles of pax2a and atoh1a/b, early regulators of sensory specification. RESULTS: fgf3 and fgf8a were uniquely required for pax5 and pou3f3b, respectively. Elevating Fgf or blocking Hh expanded expression of pax5 but repressed pou3f3b, while blocking Fgf had the opposite effect. Blocking sensory specification did not affect pax5 or pou3f3b, but both markers were lost in pax2a-/- mutants. Maintenance of pax2a expression requires Fgf, Hh and Pax2a itself. CONCLUSION: Specification of utricular identity requires high Fgf and is repressed by Hh, whereas saccular identity requires Hh plus low Fgf. pax2a acts downstream of Fgf and Hh to maintain both fates. Comparison with mouse suggests this may reflect a broadly conserved developmental mechanism.

Structure of Long-Range Direct and Indirect Spinocerebellar Pathways as Well as Local Spinal Circuits Mediating Proprioception.

Proprioception, the sense of limb and body position, generates a map of the body that is essential for proper motor control, yet we know little about precisely how neurons in proprioceptive pathways are wired. Defining the anatomy of secondary neurons in the spinal cord that integrate and relay proprioceptive and potentially cutaneous information from the periphery to the cerebellum is fundamental to understanding how proprioceptive circuits function. Here, we define the unique anatomic trajectories of long-range direct and indirect spinocerebellar pathways as well as local intersegmental spinal circuits using genetic tools in both male and female mice. We find that Clarke's column neurons, a major contributor to the direct spinocerebellar pathway, has mossy fiber terminals that diversify extensively in the cerebellar cortex with axons terminating bilaterally, but with no significant axon collaterals within the spinal cord, medulla, or cerebellar nuclei. By contrast, we find that two of the indirect pathways, the spino-lateral reticular nucleus and spino-olivary pathways, are in part, derived from cervical Atoh1-lineage neurons, whereas thoracolumbar Atoh1-lineage neurons project mostly locally within the spinal cord. Notably, while cervical and thoracolumbar Atoh1-lineage neurons connect locally with motor neurons, no Clarke's column to motor neuron connections were detected. Together, we define anatomic differences between long-range direct, indirect, and local proprioceptive subcircuits that likely mediate different components of proprioceptive-motor behaviors.SIGNIFICANCE STATEMENT We define the anatomy of long-range direct and indirect spinocerebellar pathways as well as local spinal proprioceptive circuits. We observe that mossy fiber axon terminals of Clarke's column neurons diversify proprioceptive information across granule cells in multiple lobules on both ipsilateral and contralateral sides, sending no significant collaterals within the spinal cord, medulla, or cerebellar nuclei. Strikingly, we find that cervical spinal cord Atoh1-lineage neurons form mainly the indirect spino-lateral reticular nucleus and spino-olivary tracts and thoracolumbar Atoh1-lineage neurons project locally within the spinal cord, whereas only a few Atoh1-lineage neurons form a direct spinocerebellar tract.

Pax2a, Sp5a and Sp5l act downstream of Fgf and Wnt to coordinate sensory-neural patterning in the inner ear.

In zebrafish, sensory epithelia and neuroblasts of the inner ear form simultaneously in abutting medial and lateral domains, respectively, in the floor of the otic vesicle. Previous studies support regulatory roles for Fgf and Wnt, but how signaling is coordinated is poorly understood. We investigated this problem using pharmacological and transgenic methods to alter Fgf or Wnt signaling from early placodal stages to evaluate later changes in growth and patterning. Blocking Fgf at any stage reduces proliferation of otic tissue and terminates both sensory and neural specification. Wnt promotes proliferation in the otic vesicle but is not required for sensory or neural development. However, sustained overactivation of Wnt laterally expands sensory epithelia and blocks neurogenesis. pax2a, sp5a and sp5l are coregulated by Fgf and Wnt and show overlapping expression in the otic placode and vesicle. Gain- and loss-of-function studies show that these genes are together required for Wnt's suppression of neurogenesis, as well as some aspects of sensory development. Thus, pax2a, sp5a and sp5l are critical for mediating Fgf and Wnt signaling to promote spatially localized sensory and neural development.

Atoh1+ secretory progenitors possess renewal capacity independent of Lgr5+ cells during colonic regeneration.

During homeostasis, the colonic epithelium is replenished every 3-5 days by rapidly cycling Lgr5+ stem cells. However, various insults can lead to depletion of Lgr5+ stem cells, and colonic epithelium can be regenerated from Lgr5-negative cells. While studies in the small intestine have addressed the lineage identity of the Lgr5-negative regenerative cell population, in the colon this question has remained unanswered. Here, we set out to identify which cell(s) contribute to colonic regeneration by performing genetic fate-mapping studies of progenitor populations in mice. First, using keratin-19 (Krt19) to mark a heterogeneous population of cells, we found that Lgr5-negative cells can regenerate colonic crypts and give rise to Lgr5+ stem cells. Notch1+ absorptive progenitor cells did not contribute to epithelial repair after injury, whereas Atoh1+ secretory progenitors did contribute to this process. Additionally, while colonic Atoh1+ cells contributed minimally to other lineages during homeostasis, they displayed plasticity and contributed to epithelial repair during injury, independent of Lgr5+ cells. Our findings suggest that promotion of secretory progenitor plasticity could enable gut healing in colitis.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

Development of the cochlea.

The cochlea, a coiled structure located in the ventral region of the inner ear, acts as the primary structure for the perception of sound. Along the length of the cochlear spiral is the organ of Corti, a highly derived and rigorously patterned sensory epithelium that acts to convert auditory stimuli into neural impulses. The development of the organ of Corti requires a series of inductive events that specify unique cellular characteristics and axial identities along its three major axes. Here, we review recent studies of the cellular and molecular processes regulating several aspects of cochlear development, such as axial patterning, cochlear outgrowth and cellular differentiation. We highlight how the precise coordination of multiple signaling pathways is required for the successful formation of a complete organ of Corti.

Fate-mapping analysis of cochlear cells expressing Atoh1 mRNA via a new Atoh13*HA-P2A-Cre knockin mouse strain.

BACKGROUND: Atoh1 is recognized to be essential for cochlear hair cell (HC) development. However, Atoh1 temporal and spatial expression patterns remain widely debated. Here, we aimed to obtain evidence to resolve the controversies regarding Atoh1 expression by generating a new knockin mouse strain: Atoh13*HA-P2A-Cre . RESULTS: Fate-mapping analysis of Atoh13*HA-P2A-Cre/+ ; Rosa26-CAG-LSL-tdTomato (Ai9)/+ mice enabled us to concurrently characterize the temporal expression of Atoh1 protein (through HA-tag immunostaining) and visualize the cells expressing Atoh1 mRNA (as tdTomato+ cells). Our findings show that whereas Atoh1 mRNA expression is rapidly turned on in early cochlear progenitors, Atoh1 protein is only detected in differentiating HCs or progenitors just committed to the HC fate. Cre activity is also stronger in Atoh13*HA-P2A-Cre/+ than in previous mouse models, because almost all cochlear HCs and nearby supporting cells here are tdTomato+. Furthermore, tdTomato, but not HA, is expressed in middle and apical spiral ganglion neurons. CONCLUSION: Collectively, our findings indicate that Atoh13*HA-P2A-Cre can serve as a powerful genetic model in the developmental biology field.

Three-dimensional live imaging of Atoh1 reveals the dynamics of hair cell induction and organization in the developing cochlea.

During cochlear development, hair cells (HCs) and supporting cells differentiate in the prosensory domain to form the organ of Corti, but how one row of inner HCs (IHCs) and three rows of outer HCs (OHCs) are organized is not well understood. Here, we investigated the process of HC induction by monitoring Atoh1 expression in cochlear explants of Atoh1-EGFP knock-in mouse embryos and showed that only the cells that express Atoh1 over a certain threshold are selected for HC fate determination. HC induction initially occurs at the medial edge of the prosensory domain to form IHCs and subsequently at the lateral edge to form OHCs, while Hedgehog signaling maintains a space between IHCs and OHCs, leading to formation of the tunnel of Corti. These results reveal dynamic Atoh1 expression in HC fate control and suggest that multi-directional signals regulate OHC induction, thereby organizing the prototype of the organ of Corti.

Relationship between ATOH1 and tumor microenvironment in colon adenocarcinoma patients with different microsatellite instability status.

BACKGROUND: Colon adenocarcinoma (COAD) is one of the major varieties of malignant tumors threatening human health today. Immune checkpoint inhibitors (ICIs) have recently begun to emerge as an effective option for the treatment of COAD patients, but not all patients can benefit from ICI treatment. Previous studies have suggested that ICIs boast significant clinical effects on patients with microsatellite instability-high (MSI-H), while conversely patients with microsatellite-stable/microsatellite instability-low (MSS/MSI-L) have shown limited response. METHODS: We used ATAC-seq, RNA-seq, and mutation data from The Cancer Genome Atlas Colon adenocarcinoma (TCGA-COAD) cohort to perform multi-omics differential analysis on COAD samples with different MSI statuses, then further screened genes by additionally combining these results with survival analysis. We analyzed the effects of the screened genes on the tumor microenvironment and immunogenicity of COAD patients, and subsequently determined their influence on the efficacy of ICIs in COAD patients using a series of predictive indexes. RESULTS: Twelve genes were screened in the TCGA-COAD cohort, and after the combined survival analysis, we identified ATOH1 as having significant effects. ATOH1 is characterized by high chromatin accessibility, high expression, and high mutation in COAD patients in the MSI-H group. COAD patients with high ATOH1 expression are associated with a better prognosis, unique immune microenvironment, and higher efficacy in ICI treatment. Enrichment analysis showed that COAD patients with high ATOH1 expression displayed significant upregulation in their humoral immunity and other related pathways. CONCLUSIONS: We speculate that ATOH1 may influence the efficacy of ICIs therapy in patients with COAD by affecting the immune microenvironment and immunogenicity of the tumor.

Simultaneous gentamicin-mediated damage and Atoh1 overexpression promotes hair cell regeneration in the neonatal mouse utricle.

Loss of hair cells from vestibular epithelium results in balance dysfunction. The current therapeutic regimen for vestibular diseases is limited. Upon injury or Atoh1 overexpression, hair cell replacement occurs rapidly in the mammalian utricle, suggesting a promising approach to induce vestibular hair cell regeneration. In this study, we applied simultaneous gentamicin-mediated hair cell ablation and Atoh1 overexpression to induce neonatal utricular hair cell formation in vitro. We confirmed that type I hair cells were the primary targets of gentamicin. Furthermore, injury and Atoh1 overexpression promoted hair cell regeneration in a timely and efficient manner through robust viral transfection. Hair cells regenerated with type II characteristics in the striola and type I/II characteristics in non-sensory regions. Rare EdU+/myosin7a+ cells in sensory regions and robust EdU+/myosin7a+ signals in ectopic regions indicate that transdifferentiation of supporting cells in situ, and mitosis and differentiation of non-sensory epithelial cells in ectopic regions, are sources of regenerative hair cells. Distinct regeneration patterns in in situ and ectopic regions suggested robust plasticity of vestibular non-sensory epithelium, generating more developed hair cell subtypes and thus providing a promising stem cell-like source of hair cells. These findings suggest that simultaneously causing injury and overexpressing Atoh1 promotes hair cell regeneration efficacy and maturity, thus expanding the understanding of ectopic plasticity in neonatal vestibular organs.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

Intestinal phenotype is maintained by Atoh1 in the cancer region of intraductal papillary mucinous neoplasm.

Intraductal papillary mucinous neoplasm (IPMN) is a precancerous lesion of pancreatic cancer. Although there are 4 types of IPMN, among which intestinal-type IPMN is likely to progress into invasive cancer known as colloid carcinoma, no information regarding the involvement of the intestinal phenotype in the carcinogenesis of IPMN exists. The present study was conducted to explore how the intestinal differentiation system is maintained during the tumor progression of intestinal-type IPMN using surgical resection specimens. Results showed that Atoh1, a critical transcriptional factor for intestinal differentiation toward the secretory lineages of intestinal epithelial cells, was expressed in an invasive-grade IPMN. To determine the function of Atoh1 in pancreatic cancer, we generated a pancreatic ductal adenocarcinoma (PDAC) cell line overexpressing Atoh1. In a xenograft model, we successfully induced an IPMN phenotype in PDAC cells via Atoh1 induction. Finally, for the first time, we discovered that GPA33 is expressed in intestinal-type IPMN, thereby suggesting a novel target for cancer therapy. In conclusion, the intestinal differentiation system might be maintained during tumor progression of intestinal-type IPMN. Further analysis of the function of Atoh1 in IPMN might be useful for understanding the molecular mechanism underlying the malignant potential during the tumor progression of IPMN.