Differential use of the Nkx2.2 NK2 domain in developing pancreatic islets and neurons.

The homeodomain transcription factor (TF) Nkx2.2 governs crucial cell fate decisions in several developing organs, including the central nervous system (CNS), pancreas, and intestine. How Nkx2.2 regulates unique targets in these different systems to impact their individual transcriptional programs remains unclear. In this issue of Genes & Development Abarinov and colleagues (pp. 490-504) generated and analyzed mice in which the Nkx2.2 SD is mutated and found that the SD is required for normal pancreatic islet differentiation but dispensable for most aspects of neuronal differentiation.

Major ß cell-specific functions of NKX2.2 are mediated via the NK2-specific domain.

The consolidation of unambiguous cell fate commitment relies on the ability of transcription factors (TFs) to exert tissue-specific regulation of complex genetic networks. However, the mechanisms by which TFs establish such precise control over gene expression have remained elusive-especially in instances in which a single TF operates in two or more discrete cellular systems. In this study, we demonstrate that ß cell-specific functions of NKX2.2 are driven by the highly conserved NK2-specific domain (SD). Mutation of the endogenous NKX2.2 SD prevents the developmental progression of ß cell precursors into mature, insulin-expressing ß cells, resulting in overt neonatal diabetes. Within the adult ß cell, the SD stimulates ß cell performance through the activation and repression of a subset of NKX2.2-regulated transcripts critical for ß cell function. These irregularities in ß cell gene expression may be mediated via SD-contingent interactions with components of chromatin remodelers and the nuclear pore complex. However, in stark contrast to these pancreatic phenotypes, the SD is entirely dispensable for the development of NKX2.2-dependent cell types within the CNS. Together, these results reveal a previously undetermined mechanism through which NKX2.2 directs disparate transcriptional programs in the pancreas versus neuroepithelium.

Gata2, Nkx2-2 and Skor2 form a transcription factor network regulating development of a midbrain GABAergic neuron subtype with characteristics of REM-sleep regulatory neurons.

The midbrain reticular formation (MRF) is a mosaic of diverse GABAergic and glutamatergic neurons that have been associated with a variety of functions, including sleep regulation. However, the molecular characteristics and development of MRF neurons are poorly understood. As the transcription factor, Gata2 is required for the development of all GABAergic neurons derived from the embryonic mouse midbrain, we hypothesized that the genes expressed downstream of Gata2 could contribute to the diversification of GABAergic neuron subtypes in this brain region. Here, we show that Gata2 is required for the expression of several GABAergic lineage-specific transcription factors, including Nkx2-2 and Skor2, which are co-expressed in a restricted group of post-mitotic GABAergic precursors in the MRF. Both Gata2 and Nkx2-2 function is required for Skor2 expression in GABAergic precursors. In the adult mouse and rat midbrain, Nkx2-2-and Skor2-expressing GABAergic neurons locate at the boundary of the ventrolateral periaqueductal gray and the MRF, an area containing REM-off neurons regulating REM sleep. In addition to the characteristic localization, Skor2+ cells increase their activity upon REM-sleep inhibition, send projections to the dorsolateral pons, a region associated with sleep control, and are responsive to orexins, consistent with the known properties of midbrain REM-off neurons.

NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing.

BACKGROUND: Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissolution might alter gene expressions. In this work, we report the development of a single-nucleus RNA sequencing (snRNA-seq) approach with targeted islet cell enrichment for endocrine-population focused transcriptomic profiling using frozen archival pancreatic tissues without islet isolation. RESULTS: We cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed. CONCLUSIONS: Our work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.

Differential regulation of Shh-Gli1 cell signalling pathway on homeodomain transcription factors Nkx2.2 and Pax6 during the medulloblastoma genesis.

BACKGROUND: Medulloblastoma is a pediatric malignant brain tumor associated with an aberrantly activated Shh pathway. The Shh pathway acts via downstream effector molecules, including Pax6 and Nkx2.2. Transcription factor Nkx2.2 plays crucial roles during early embryonic patterning and development. In this study, we aimed to determine the role of transcription factor Nkx2.2 in medulloblastoma development. METHODS AND RESULTS: Here, whole transcriptome levels and suppressive effect of transcription factor Nkx2.2 on Pax6 were assessed using one normal human brain and three surgically removed medulloblastoma samples. Additionally, protein levels of Shh, Gli1, Pax6, and Nkx2.2 and co-expression patterns of Pax6 and Nkx2.2 were assessed in 14 medulloblastoma samples. Quantitative reverse transcription-polymerase chain reaction revealed the suppressive effect of Nkx2.2 on Pax6. D283 cells were treated with the Shh pathway activator, SAG, and Gli1 inhibitor, GANT61, which revealed Pax6-Nkx2.2 regulation. Increased cell proliferation was observed in D283 cells transfected with Nkx2.2 small interfering RNA. Moreover, mRNA expression levels of Shh, Pax6, Nkx2.2, and Gli1 were assessed in Daoy cells transfected with Gli1 and Nkx2.2 small interfering RNAs using quantitative reverse transcription-polymerase chain reaction. Pax6 levels were increased in Nkx2.2 siRNA-transfected cells. CONCLUSIONS: Aberrantly activated Shh pathway leads to the ectopic expression of Pax6 in granular cells, inducing medulloblastoma development. Moreover, Nkx2.2 transcription factor acts as a suppressor of Pax6 during medulloblastoma development and maintenance. Overall, this study provides novel insights for the development of effective therapeutic strategies and suggests potential targets for medulloblastoma.

Homeodomain Transcription Factors Nkx2.2 and Pax6 as Novel Biomarkers for Meningioma Tumor Treatment.

Meningioma is a common brain tumour which has neither a specific detection nor treatment method. The Sonic hedgehog (Shh) cell signaling pathway is a crucial regulatory pathway of mammalian organogenesis and tumorigenesis including meningioma. Shh cell signalling pathway cascade function by main transcription factor Gli1 and which further regulates in its downstream to Pax6 and Nkx2.2. This current study is aimed to explore the regulation of the Sonic hedgehog-Gli1 cell signaling pathway and its potential downstream targets in meningioma samples. A total of 24 surgically resected meningioma samples were used in this current study.Cytological changes were assessed using electron microscopic techniques as well as hematoxylin & eosin and DAPI staining. The expression pattern of Gli1, Nkx2.2 and Pax6 transcription factors were determined by using immunohistochemistry. The mRNA expression was assessed using RT-qPCR assays. Later, the whole transcriptome analysis of samples was performed with the amploseq technique. Results were compared with those obtained in normal human brain tissue (or normal meninges). Compared to the normal human brain tissue, meningioma samples showed crowded nuclei with morphological changes. Transcription factor Nkx2.2 expressed highly in all samples (24/24, 100%). Twenty-one of the 24 meningiomas (88%) showed high Gli1 and Pax6 expression. Whole transcriptome analysis of two meningioma samples also exhibited a very high increase in Gli1 expression signal in meningioma samples as compare to normal control. Hence, we may conclude that the Shh-Gli1 pathway is aberrantly activated in meningioma cells and is canonically upregulating the expression of transcription factors Pax6 and Nkx2.2. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01085-1.

MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors.

The development of the nervous system requires precise regulation. Any disturbance in the regulation process can lead to neurological developmental diseases, such as autism and schizophrenia. Histone variants are important components of epigenetic regulation. The function and mechanisms of the macroH2A (mH2A) histone variant during brain development are unknown. Here, we show that deletion of the mH2A isoform mH2A1.2 interferes with neural stem cell differentiation in mice. Deletion of mH2A1.2 affects neurodevelopment, enhances neural progenitor cell (NPC) proliferation, and reduces NPC differentiation in the developing mouse brain. mH2A1.2-deficient mice exhibit autism-like behaviors, such as deficits in social behavior and exploratory abilities. We identify NKX2.2 as an important downstream effector gene and show that NKX2.2 expression is reduced after mH2A1.2 deletion and that overexpression of NKX2.2 rescues neuronal abnormalities caused by mH2A1.2 loss. Our study reveals that mH2A1.2 reduces the proliferation of neural progenitors and enhances neuronal differentiation during embryonic neurogenesis and that these effects are at least in part mediated by NKX2.2. These findings provide a basis for studying the relationship between mH2A1.2 and neurological disorders.

Nkx2-2 expressing taste cells in endoderm-derived taste papillae are committed to the type III lineage.

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields.

CSN6 aggravates Ang II-induced cardiomyocyte hypertrophy via inhibiting SIRT2.

The constitutive photomorphogenic 9 (COP9) signalosome complex subunit 6 (COPS6/CSN6) is crucial for structural integrity of the COP9 signalosome complex. CSN6 participates in various aspects of cancer progression, but its role in hypertrophic cardiomyopathy is not clear. Here, we found that the expression of CSN6 was increased in Angiotensin II (Ang II)-induced hypertrophic mice hearts and neonatal rat cardiomyocytes (NRCMs). Inhibition of CSN6 decreased the cardiomyocyte size and fetal genes' expression in Ang II-induced hypertrophic NRCMs, while overexpression of CSN6 aggravated Ang II-induced myocardial hypertrophy. Moreover, we demonstrated that the pro-hypertrophic function of CSN6 was mediated by SIRT2, which acts as a cardioprotective factor in pathological cardiac hypertrophy. CSN6 inhibited the expression of SIRT2, and re-expression of SIRT2 attenuated the myocardial hypertrophy caused by CSN6 overexpression. Further investigation discovered that CSN6 suppressed the expression of SIRT2 via up-regulating Nkx2.2, a transcription suppressor of SIRT2. Mechanistically, CSN6 blocked the ubiquitin proteasome system-mediated degradation of Nkx2.2 protein by interacting with it and inhibiting its ubiquitination directly in cardiomyocytes. Finally, our data showed that CSN6 was partially dependent on the stabilization of Nkx2.2 protein to inhibit SIRT2 and promote myocardial hypertrophy. Overall, our study identified CSN6 as a pro-hypertrophic deubiquitinase, and CSN6 inhibition may be a potential treatment strategy for heart failure.

The novel enterochromaffin marker Lmx1a regulates serotonin biosynthesis in enteroendocrine cell lineages downstream of Nkx2.2.

Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.

Cadmium-induced neurodegeneration and activation of noncanonical sonic hedgehog pathway in rat cerebellum.

BACKGROUND: Cadmium is a nonessential toxic heavy metal, which enters the body easily and damages the cellular system. The sonic hedgehog (Shh) signaling pathway is one of the key regulatory pathways, which define neural growth and development. OBJECTIVES: This study aimed to explore how cadmium exposure affects neural activities, Shh signaling cascade, and its downstream target genes. METHODS: Total 18 male Wistar rats were randomly divided into two groups, control and test groups. Test rats were administered with 3 mg cadmium/kg body weight, while the control rats were treated with vehicle continuously for 28 days. Thereafter, rats were killed and the isolated brain samples were examined using oxidative stress assessment, histological and immunohistological behavioral assessment, polymerase chain reaction (PCR), and the comet assay. RESULTS: A disturbed oxidative balance, DNA damage, and an upregulated Shh signaling pathway were observed in cadmium-treated samples. Loss of structural integrity in cerebellum and loss of motor activity were observed in cadmium-treated rats.

Development and Characteristics of Pancreatic Epsilon Cells.

Pancreatic endocrine cells expressing the ghrelin gene and producing the ghrelin hormone were first identified in 2002. These cells, named ε cells, were recognized as the fifth type of endocrine cells. Differentiation of ε cells is induced by various transcription factors, including Nk2 homeobox 2, paired box proteins Pax-4 and Pax6, and the aristaless-related homeobox. Ghrelin is generally considered to be a "hunger hormone" that stimulates the appetite and is produced mainly by the stomach. Although the population of ε cells is small in adults, they play important roles in regulating other endocrine cells, especially ß cells, by releasing ghrelin. However, the roles of ghrelin in ß cells are complex. Ghrelin contributes to increased blood glucose levels by suppressing insulin release from ß cells and is also involved in the growth and proliferation of ß cells and the prevention of ß cell apoptosis. Despite increasing evidence and clarification of the mechanisms of ε cells over the last 20 years, many questions remain to be answered. In this review, we present the current evidence for the participation of ε cells in differentiation and clarify their characteristics by focusing on the roles of ghrelin.

PAX7 immunohistochemical evaluation of Ewing sarcoma and other small round cell tumours.

AIMS: Ewing sarcoma is a small round cell tumour that affects bone and soft tissues. Although the detection of the specific fusion gene is a robust method of its diagnosis, immunohistochemistry may serve as a practical surrogate. Recent tissue microarray studies suggested that PAX7 is a novel marker, because it was expressed consistently in Ewing sarcoma, in addition to rhabdomyosarcoma and synovial sarcoma. Here, we evaluated the utility of PAX7 immunohistochemistry in whole-tissue sections of an expanded array of round cell malignancies with adequate molecular characterisation. METHODS AND RESULTS: We stained 30 molecularly confirmed Ewing sarcomas, one EWSR1-NFATC2 sarcoma and 141 non-Ewing round cell tumours by a monoclonal antibody against PAX7. Staining was considered positive if at least 5% of tumour cells were stained. PAX7 was expressed in 27 of 30 Ewing sarcomas (90%), mainly in a diffuse and strong manner. Although NKX2-2 showed similar sensitivity, PAX7 showed more extensive and strong reactivity. One EWSR1-NFATC2 sarcoma co-expressed PAX7 and NKX2-2. PAX7 was also expressed in 24 of 141 non-Ewing tumours, including alveolar rhabdomyosarcomas (seven of 10), poorly differentiated synovial sarcomas (seven of 10), BCOR-CCNB3 sarcomas (eight of 10), small-cell osteosarcoma (one of five) and desmoplastic small round cell tumour (one of 10), one-third of which showed diffuse strong reactivity. CONCLUSIONS: Although we confirmed that PAX7 is a sensitive marker for Ewing sarcoma, anti-PAX7 antibody also stained several Ewing sarcoma mimics, whose spectrum was distinct from NKX2-2-positive non-Ewing entities. Further studies are required to determine how PAX7 could be integrated into practice to classify small round cell tumours efficiently.

Integrator of Stress Responses Calmodulin Binding Transcription Activator 1 (Camta1) Regulates miR-212/miR-132 Expression and Insulin Secretion.

Altered microRNA profiles have been demonstrated in experimental models of type 2 diabetes, including in islets of the diabetic Goto-Kakizaki (GK) rat. Our bioinformatic analysis of conserved sequences in promoters of microRNAs, previously observed to be up-regulated in GK rat islets, revealed putative CGCG-core motifs on the promoter of the miR-212/miR-132 cluster, overexpression of which has been shown to increase insulin secretion. These motifs are possible targets of calmodulin binding transcription activators Camta1 and Camta2 that have been recognized as integrators of stress responses. We also identified putative NKE elements, possible targets of NK2 homeobox proteins like the essential islet transcription factor Nkx2-2. As Camtas can function as co-activators with NK2 proteins in other tissues, we explored the role of Camta1, Camta2, and Nkx2-2 in the regulation of the miR-212/miR-132 cluster and insulin secretion. We demonstrate that exposure of control Wistar or GK rat islets to 16.7 mm glucose increases miR-212/miR-132 expression but significantly less so in the GK rat. In addition, Camta1, Camta2, and Nkx2-2 were down-regulated in GK rat islets, and knockdown of Camta1 reduced miR-212/miR-132 promoter activity and miR-212/miR-132 expression, even under cAMP elevation. Knockdown of Camta1 decreased insulin secretion in INS-1 832/13 cells and Wistar rat islets but increased insulin content. Furthermore, knockdown of Camta1 reduced K(+)-induced insulin secretion and voltage-dependent Ca(2+) currents. We also demonstrate Camta1 and Nkx2-2 protein interaction. These results indicate that Camta1 is required not only for expression of the miR-212/miR-132 cluster but at multiple levels for regulating beta cell insulin content and secretion.

Nuclear import of Nkx2-2 is mediated by multiple pathways.

Nkx2-2 homeoprotein is essential for the development of the central nervous system and pancreas. Although the nuclear localization signals of Nkx2-2 have been identified, the responsible transport receptor is still unknown. Here, we demonstrate that imp α1 not only interacts with Nkx2-2 but also transports it into the nucleus in vitro by acting together with imp ß1. However, the nuclear import of Nkx2-2 in cells was not inhibited in response to knockdown expression of endogenous imp ß1 or over-expression of Bimax2. Furthermore, imp ß1 and imp 13, but not imp 4, directly interact with Nkx2-2 and are capable of transporting Nkx2-2 in an in vitro import assay. By GST pull-down assay, we demonstrate that mutation of NLS1 or NLS2 has no effect on interaction with imp α1 or imp 13, but significantly reduced binding to imp ß1. Thus, the nuclear import of Nkx2-2 is mediated not only by the classical import pathway but also directly by imp ß1 or imp 13.

Sp8 plays a supplementary role to Pax6 in establishing the pMN/p3 domain boundary in the spinal cord.

Progenitor cells are segregated into multiple domains along the dorsoventral axis of the vertebrate neural tube, and each progenitor domain generates particular types of neurons. Selective cross-repressive interactions between pairs of class I and class II transcription factors play important roles in patterning neural progenitors into domains with clear boundaries. Here, we provide evidence that the zinc-finger protein Sp8 plays a supplementary role to Pax6 in establishing the pMN/p3 domain boundary through mutually repressive interactions with the class II protein Nkx2-2. The ventral limit of Sp8 expression is complementary to the dorsal limit of Nkx2-2 expression at the pMN/p3 boundary. Sp8 and Nkx2-2 exert cross-repressive interactions, and changing the expression of Sp8 and Nkx2-2 is coupled with pMN and p3 progenitor fate conversion. Sp8 exerts its neural patterning activities by acting as a transcriptional activator. The expression of a repressive form of Sp8 results in the selective inhibition of motor neuron generation and the ectopic induction of Nkx2-2 expression. Sp8 expression is positively regulated by, but not completely dependent on, Pax6. Furthermore, whereas loss of Pax6 function alone results in disruption of the pMN/p3 domain boundary only in the rostral levels of the spinal cord, loss of both Sp8 and Pax6 functions results in disruption of the pMN/p3 domain boundary along the whole rostrocaudal axis of the spinal cord. We conclude that Sp8 plays a supplementary role to Pax6 in specifying the pMN over p3 progenitor fate through cross-repressive interactions with Nkx2-2.

Generation of a Nkx2.2(Cre) knock-in mouse line: Analysis of cell lineages in the central nervous system.

A Nkx2.2(cre) knock-in mutant mouse line was generated that on the appropriate reporter strain enables cell fate analysis of the Nkx2.2 cell lineage in the central nervous system and elsewhere. We here demonstrate that Nkx2.2 lineage-marked cells reside in the ventral p3 region along the entire length of the CNS and also in pancreas of mouse embryos. Nkx2.2(+) progenitor cells develop into V3 interneurons in spinal cord and generate the branchio-visceral motor nuclei of cranial nerves in hindbrain. Nkx2.2(+) cells in hindbrain also form serotonergic neurons and oligodendrocytes during later developmental stages. In mouse mutants lacking Nkx2.2 protein the neuronal progenitor cells in spinal cord are transformed to the distinct fate of somatic motor neurons including their axonal projections that exit the CNS ventrally and no longer cross the midline at the commissure. These data identify Nkx2.2 as key regulator to determine neuronal subtypes in the p3 domain of the central nervous system.

Arx together with FoxA2, regulates Shh floor plate expression.

Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.

Nkx2.2 is expressed in a subset of enteroendocrine cells with expanded lineage potential.

There are two major stem cell populations in the intestinal crypt region that express either Bmi1 or Lgr5; however, it has been shown that other populations in the crypt can regain stemness. In this study, we demonstrate that the transcription factor NK2 homeobox 2 (Nkx2.2) is expressed in enteroendocrine cells located in the villus and crypt of the intestinal epithelium and is coexpressed with the stem cell markers Bmi1 and Lgr5 in a subset of crypt cells. To determine whether Nkx2.2-expressing enteroendocrine cells display cellular plasticity and stem cell potential, we performed genetic lineage tracing of the Nkx2.2-expressing population using Nkx2.2(Cre/+);R26RTomato mice. These studies demonstrated that Nkx2.2+ cells are able to give rise to all intestinal epithelial cell types in basal conditions. The proliferative capacity of Nkx2.2-expressing cells was also demonstrated in vitro using crypt organoid cultures. Injuring the intestine with irradiation, systemic inflammation, and colitis did not enhance the lineage potential of Nkx2.2-expressing cells. These findings demonstrate that a rare mature enteroendocrine cell subpopulation that is demarcated by Nkx2.2 expression display stem cell properties during normal intestinal epithelial homeostasis, but is not easily activated upon injury.

Ceramide galactosyltransferase expression is regulated positively by Nkx2.2 and negatively by OLIG2.

Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.