Endocrine Journal 2004, 51 (3), 255–264

REVIEW

Neurogenin 3 is a Key Factor for Differentiation of the Endocrine

HIROTAKA WATADA

Department of Medicine, Metabolism and Endocrinology, School of Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

Key words: NeuroD, Hes1, Pax4, Nkx2.2, Insulin

Introduction how these factors function. Accumulation of such knowledge has revealed that transcription factors THE pancreas is composed of exocrine and endocrine orchestrate the intricate pathways of cellular growth, compartments. The endocrine compartment consists death, and differentiation by direct regulation of of the islets of Langerhans, which contain clusters expression. Amongst the transcription factors in this comprising four types of cells: -producing well-organized cascade, neurogenin 3 (Ngn3) plays a  cells, somatostatin-producing  cells, pancreatic key role in determining the fate of cells in the endo- polypeptide-producing PP cells, and insulin-producing crine pancreas. This article discusses the role of Ngn3  cells. Because an inadequate mass of functioning during pancreatic development, the regulation of Ngn3 pancreatic  cells is a feature of both type 1 and type 2 expression and how Ngn3 activates the expression of ,  cell replacement therapy is considered to downstream . be a potential curative treatment. If we can understand how  cells develop normally, it may become possible to use that knowledge to reprogram various cell types Brief outline of development of to differentiate into new  cells. Thus, studies of the endocrine pancreas pancreatic development may contribute to overcoming diabetes. In mice, the pancreas initially arises as two buds One of the main roles of  cells is secretion of from the dorsal and ventral sides of the gut at the junc- insulin in response to an increase of the blood glu- tion of the foregut and the midgut at the embryonic age cose level. To accomplish this, pancreatic  cells 9.5 days (e9.5). At this stage of development, cells express numerous genes that are essential for glucose- appear as the first differentiated cells of the pancreas responsive insulin secretion, such as glucokinase, and -cells appear after then. As the gut rotates during Kir6.2, and SUR1, as well as insulin. To allow the development, the ventral bud becomes fused to the expression of such strictly selected multiple sets of dorsal bud, giving rise to the single organ seen in genes, various differentiation steps are required during adults. Endocrine cells continue to differentiate from pancreatic development. As is the case for other types cells into duct-like structures throughout pancreatic of cells, recent studies have identified several tran- development. At about e14, the termini of the duct-like scription factors that control the activation and repres- structures begin to form acini and differentiate into the sion of a large number of genes during pancreatic exocrine cells. At the same time, there is a dramatic development and cell biology studies have revealed rise in the insulin level and the number of  cells and for the first time, well-differentiated  cells with Correspondence to: Dr. Hirotaka WATADA, Department of characteristic insulin-containing granules are detected. Medicine, Metabolism and Endocrinology, School of Medicine, These changes of  cells have been termed the “sec- Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, ondary transition” [1, 2]. The first  cells also appear Tokyo 113-8421, Japan around this time, while the PP cells appear shortly 256 WATADA before birth. The ultimate result of embryogenesis is pancreatic development is evident, its expression is not that the pancreas becomes divided into two major restricted to pancreatic buds in the early stage of dif- types of cells, which are endocrine cells and exocrine ferentiation [5, 6]. On the other hand, Ptf1a is exclu- cells. The endocrine cells are further segregated into sively expressed by the pancreatic buds. Using Ptf1a-  and PP cells, which form circumscribed clus- Cre knock-in mice, Kawaguchi et al. found that most ters within the islets of Langerhans in the mature pan- pancreatic cells are derived from Ptf1a-expressing creas. cells [7]. Thus, these cell tracing studies have revealed During these morphological changes, scattered cells that mature pancreatic cells are derived from cells in in the pancreatic buds begin to differentiate into endo- the pancreatic buds that express Pdx1 and Ptf1a. crine cells. Recent cell tracing analyses using Cre-Lox These cells initially differentiate to Ngn3-expressing method system have helped to identify the cell line- common endocrine precursor cells, and then differen- ages involved in development of pancreas. The general tiate into mature endocrine cells. Accordingly,  cells strategy of these studies is that transgenic mice carry- are derived from these common precursor cells and not ing Cre-recombinase driven by the of the tar- from glucagon-positive cells (Fig. 1) [3, 8]. get gene are crossed with mice in which the reporter gene is designed to only be expressed in cells express- ing Cre recombinase. By this method, cells that ex- Importance of Ngn3 during differentiation of press Cre recombinase even temporarily are labeled the endocrine pancreas irreversibly, and the fate of a cell that expresses the target gene can thus be investigated. Using glucagon Ngn3 is a containing a basic- promoter-Cre mice, Herrera demonstrated that insulin- helix-loop-helix domain (bHLH) as its DNA-binding positive cells are not derived from glucagon-positive domain. The involvement of bHLH in pancre- cells, because there were no cells that co-expressed in- atic cell function was initially demonstrated by analy- sulin and the reporter gene [3]. Using Pdx-1-Cre mice, sis of the insulin promoter [9, 10]. Adjacent to the and Ngn3-Cre mice, Gu et al. found that all pancreatic Pdx1-binding site, conserved CANNTG sequences are cells are derived from Pdx-1-expressing cells and all found in the insulin promoters of various organisms pancreatic endocrine cells are derived from Ngn3- [11]. This sequence is called the E box and is critical expressing cells [4]. While the importance of in for basal insulin promoter activity. In general, cell-

Fig. 1. Process of endocrine differentiation in the developing pancreas. The relationship between the state of differentiation and the expression of key transcription factors for each step is shown. Ngn3 AND ISLET DEVELOPMENT 257 specific bHLH (class B) form dimers with of endocrine cells is due to misspecification and Ngn3 ubiquitously expressed bHLH transcription factors is essential for differentiation toward endocrine cells. such as E47 and E12 (class A), bind to the E box se- Further, these mice do not express several transcrip- quence and are involved in cell-specific gene expres- tion factors that function in the later stage of endocrine sion. In mature  cells, NeuroD1 seems to be the most pancreatic development, including NeuroD1, Pax4 [22], abundantly expressed class B transcription factor and and Pax6 [23, 24]. These transcription factors seem to one of the main regulators of insulin transcription [12]. be either direct or indirect downstream targets of Ngn3. In the pancreas, NeuroD1 is expressed by all endocrine According to these observations, cells expressing Ngn3 cells, but only during the postmitotic stage of develop- in the developing pancreas are considered to be endo- ment [13]. Targeted disruption of NeuroD1 causes a crine precursor cells that start to differentiate toward decrease of endocrine cell numbers, but this occurs    or PP cells soon after the disappearance of secondary to an increase of apoptosis and, not due to Ngn3. This concept is also supported by cell tracing blocking the differentiation of endocrine cells [14]. experiments [4]. Beyond insulin gene expression, NeuroD1 also seems Ngn3 is not just an essential factor for the differen- to regulate the expression of genes involved in the pro- tiation of endocrine cells. Overexpression of Ngn3 in cess of differentiation after determination of cell fate multipotent pancreatic bud cells with the ability to to endocrine pancreas. differentiate into any kind of cell in the pancreas NeuroD1 is also expressed in the developmental provokes differentiation to glucagon-producing cells neuron and plays an important role in [20, 25]. This action is shown to be shared by [15, 16]. In myogenesis and neurogenesis, different NeuroD1 and a similar result was obtained by transient bHLH proteins act to control the sequential stages in expression of Ngn3 in chick endoderm [26]. Thus, the the development of a given cell lineage as well as to Ngn3-NeuroD1 axis seems to be sufficient for control- specify different sublineages. In various organisms, ling the differentiation of endocrine cells. Drosophila atonal homologue, a neurogenin subfamily The importance of Ngn3 in the control of endocrine that is distinguishable from the NeuroD subfamily, is cell differentiation can also be seen in the stomach and expressed by proneuronal cells [17]. The neurogenin intestine, which are also derived from the endoderm. subfamily determines the fate of neurons and induces All kinds of gastrointestinal epithelial cells, including the expression of NeuroD1, which then regulates enteroendocrine cells, are derived from stem cells re- terminal differentiation of neurons [18]. Similar to siding in the base of the crypts. While migrating from neurogenesis, the neurogenin subfamily controls endo- crypts to the tips of the villi, the stem cells differentiate crine cell differentiation during the development of the into each kind of gastrointestinal epithelial cell. Intes- pancreas. tinal enteroendocrine cells are known to comprise at Ngn3 was discovered by the degenerate RT-PCR least 15 different types of cells. According to studies method using RNA from -derived Monc-1 of mice lacking the Ngn3 gene, enteroendocrine cells cells. Unlike Neurogenin 1 and 2 that belong to the are derived from Ngn3-expressing progenitor cells. same family, Ngn3 is expressed by the developing Intestinal enteroendocrine cells cannot differentiate pancreas in addition to neural cells [19]. In mice, without the expression of Ngn3. In contrast, lack of Ngn3 expression is initiated at the time of pancreatic Ngn3 does not impair the differentiation of gastric budding, when glucagon cells first differentiate. Rela- enteroendocrine cells [27, 28]. These results suggest tively low levels of Ngn3 expression are then observed that the importance of Ngn3 for control of endocrine until e13.5, after which the major peak of Ngn3 gene cell differentiation depends upon its position in the expression occurs concomitantly with the secondary hierarchy of transcription factors in each tissue. transition [20]. The importance of Ngn3 in pancreatic development has been shown by studies of mice lacking the Ngn3 Regulation of Ngn3 gene expression gene. Expression of Ngn3 is not observed in cells ex- pressing pancreatic hormones, and these mice fail to Ngn3 promotes switching from pancreatic precursor generate any pancreatic endocrine cells without show- cells to pancreatic proendocrine cells. How are cells ing an increase in apoptotic events [21]. Thus, the lack scattered in the pancreatic bud selected to become 258 WATADA endocrine cells? In neurogenesis, the expression of ing the development of the pancreas. Mice deficient proneural bHLH genes is regulated by hairy and for Delta-like gene 1 (Dll1) or the intracellular media- of split (HES)-type proteins [29–31], which tor RBP-Jk show accelerated differentiation of pan- are defined as anti-neural bHLH genes. Studies using creatic endocrine cells [25]. On the other hand, mice HES1-deficient mice have revealed that HES1 has a with overexpresson of Notch1 ICD show inhibited role in pancreatic development. These mice show differentiation of both endocrine and exocrine cells severe pancreatic hypoplasia caused by accelerated [36, 37]. These results suggest that the Dll1-Notch1- differentiation of glucagon-producing cells [32]. The Hes1 signal transduction pathway is also essential for pancreatic phenotype of mice with HES1 deficiency is maintenance of the undifferentiated state during pan- similar to that caused by overexpression of Ngn3 in creatic development, and that cells eluding these sig- the pancreatic buds. These results suggest that HES1 nals begin to express Ngn3 and then become endocrine inhibits the expression of Ngn3, and thus maintains cells. cells in an undifferentiated state in the pancreas. Anal- In addition to signals from adjacent cells, signals ysis of the Ngn3 gene promoter also supports this from neighboring tissues are also important in each observation. This promoter contains at least three HES1- process of pancreatic development. Their role during binding sites adjacent to the TATA box sequence, as early differentiation, the so-called “initiation process”, confirmed by electophoretic mobility shift assay, and was extensively studied and reviewed by Kim and expression of HES1 strongly inhibits Ngn3 gene pro- Hebrok [38]. In addition, after the formation of the moter activity in a manner that depends on the sequence pancreatic buds, the mesenchymal tissue surrounding containing the HES1-binding sites [33]. Beyond its the central epithelial cells has an inductive effect on role in the pancreas, the importance of the HES1-Ngn3 the development of the exocrine pancreas and a repres- axis has been demonstrated for the determination of sive effect on the development of the endocrine pan- cell fate in the biliary tree, which arises from foregut creas [39]. Follistatin antagonizes the actions of near the site where the ventral pancreatic bud forms. members of the TGF- family and is produced by the In Hes1-deficient mice, biliary epithelial cells express mesenchyme, after which it mimics the effects of the ectopic Ngn3 and differentiate into pancreatic endo- mesenchyme on differentiation of cells in the pancreas crine and exocrine cells. These results suggest that ex- [40, 41]. These results suggest that members of the pression of Hes1 plays a key role in targeting cells TGF- family are needed for proper differentiation of towards the biliary epithelium, probably by repressing pancreatic endocrine cells. One of the candidate mole- the expression of Ngn3 [34]. cules is activin. For transmembrane signaling, activin During neurogenesis, a cell-cell signaling pathway requires two types of receptors containing the / called “lateral inhibition” [35] controls the state of dif- threonine kinase domain, which are activin ferentiation. Cells that start to differentiate inhibit their type I (ActRI) and type II (ActRII) [42]. Activin di- neighbors from entering the neural differentiation rectly binds to ActRII and this complex then associates pathway so that only a subset of the cells that are ini- with ActRI, resulting in hyperphosphorylation of ActRI tially competent to form neurons are allowed to pro- by the kinase activity of ActRII [43]. While disruption ceed down the default pathway to neural differentiation. of the ActRIIB gene affects the AP patterning of The key molecules in this process are a cell surface foregut-derived organs, compound heterozygous mice receptor, Notch, and its two membrane bound ligands, (ActRIIA+/–IIB+/–) develop the hypoplastic pancreatic Delta and Serrate. The ligands Delta and Serrate bind islets without showing axial defects [44]. Pancreatic to Notch and thus induce a signaling pathway. The in- expression of a dominant-negative type II activin tracellular domain of Notch (Notch ICD) interacts with receptor in transgenic mice also results in islet hypo- RBP-J, a DNA-binding protein without a transcrip- plasia [45, 46]. These results suggest that activin sig- tional activation domain, and then translocates to the naling might be involved in pancreatic endocrine cell nucleus. Notch ICD seems to have a transactivation differentiation. domain, and so the Notch ICD/RBP-Jk complex binds AR42J cells were originally derived from a chemi- and activates the Hes1 promoter. Cells that accept the cally induced pancreatic tumor and possess both Notch signal remain in an undifferentiated state. A exocrine and neuroendocrine properties. Upon treat- similar mechanism directs the endocrine cell fate dur- ment with activin A, AR42J cells stop growing and Ngn3 AND ISLET DEVELOPMENT 259 extend neurites. In the presence of activin A plus vated kinase 1- mitogen-activated protein kinase betacellulin or hepatocyte growth factor (HGF), these kinase 3 (MKK3)- p38 mitogen-activated protein cells differentiate into insulin-expressing cells [47, 48]. kinase (p38MAPK) pathway regulates the function of Thus, AR42J cells provide a model for studying the a critical region of the Ngn3 promoter, probably by molecular mechanisms of cell differentiation in re- influencing the binding of transcription factors between sponse to activin. In this cell line, activin A and HGF –402 bp and –326 bp [50]. can provoke expression of Ngn3 and activate its pro- Signals from neighboring cells and tissues are not moter [49]. A 5’ deletion study of the Ngn3 promoter sufficient to induce the expression of Ngn3 in proendo- identified the region between –402 bp and –326 bp of crine cells, because a longer promoter sequence than Ngn3 as the activin A- and HGF-responsive DNA the region containing Notch and the ActR-responsive sequence. This region seems to function as a repressor elements seems to be needed to direct its expression by in AR42J cells, while activin A and HGF antagonize proendocrine cells in the pancreas [33]. Endodermal such repression. transcriptional activators of the HNF6 [51], HNF1, and Signaling via TGF- superfamily receptors leads FoxA [33] families all bind to this Ngn3 promoter and to phosphorylation of receptor-activated Smads (R- may play an important role in the expression of Ngn3 Smads), such as Smad2 or Smad3, which bind to the (Fig. 2). At least in the case of HNF6, its importance related factor Smad4 and translocate into the nucleus. in regulating Ngn3 gene has been demonstrated by a Once inside the nucleus, this Smad complex associates study using HNF6 gene-disrupted mice, which showed with other transcription factors to activate the tran- that expression of Ngn3 is remarkably impaired in scription of target genes [43]. On the other hand, these mice and only a few mature endocrine cells are activin-responsive activation of Ngn3 is not mediated observed [51]. by a Smad-related pathway. Instead, the TGF- acti-

Fig. 2. Proposed model of differentiation to proendocrine cells. Ngn3 is regulated by signals from neighboring cells (epithelial-epithelial cell interactions) and mesenchymal cells (epithelial- mesenchymal cell interactions). Intrinsic endodermal transcription factors are also essential. 260 WATADA

Regulation of gene expression by Ngn3  cell differentiation [22]. During development of the pancreas, the peak of Pax4 expression is observed Ngn3 is a transcription factor, and to better under- around the secondary transition stage and it shows stand its function, it is essential to identify the factors partial coexpression with Ngn3 [53]. However, its ex- that it regulates directly. NeuroD1 was one of the can- pression is not observed in mature  cells. Reporter didate factors, because it is known that neurogenin gene analysis in mice and cell lines has revealed that family regulates the proteins of the NeuroD1 family the region around –1900 bp from the transcription during neurogenesis. In fact, injection of Ngn3 mRNA initiation site of the Pax4 gene is important for cell- into Xenopus embryos induces ectopic expression of specific expression. The HNF4-binding site, HNF1- NeuroD1. The promoter fragments that recapture the binding site, and E box are located within this region expression of native NeuroD1 contains 17 E boxes. and play an important role in the expression of Pax4 Among them, the sequences of two of the E boxes are [54]. Among them, introduction of a mutation within critical for expression of NeuroD1, since Ngn3 can the HNF1-binding site or E box abolishes its enhancer bind to and activate the NeuroD1 promoter through the activity almost completely. HNF1 and Ngn3 can bind proximal E boxes [52]. to each site, physically interact and synergistically Because Ngn3 is only expressed transiently by activate the promoter. These data suggest that Ngn3 proendocrine cells during differentiation of the pan- cooperates with HNF1, recruit a transcriptional co- creas, factors that co-express ngn3 in the pancreas like activator, and activate Pax4 expression [55]. NeuroD1 seem to be the direct downstream target of At e15.5, a burst of cell growth is observed, and the Ngn3. One such factor may be the paired homeodomain majority of Ngn3-expressing cells also express Nkx2.2. transcription factor Pax4. Disruption of the Pax4 gene On the other hand, Isl1, Brn4,and PDX1 are never impairs  and  cell differentiation, but does not affect coexpressed with Ngn3 [20]. These results suggest

Fig. 3. Potential direct downstream targets of Ngn3. Ngn3 can activate expression of NeuroD1. For the regulation of Nkx2.2 and Pax4 gene expression, Ngn3, physically interacts with endodermal transcription factors and synergistically activates the promoter of each target gene. Interestingly, Ngn3 represses its own promoter although it does not have a strong repression domain. Ngn3 AND ISLET DEVELOPMENT 261 that Nkx2.2 is one of the possible direct downstream Application of Ngn3 to  cell regeneration targets of Ngn3. Unlike Ngn3 and Pax4, the expres- sion of Nkx2.2 is also observed in mature islets. Dis- After the formation of mature islets, there is con- ruption of the Nkx2.2 gene impairs the differentiation tinuous renewal and loss of islet cells throughout life, of  and  cells [56] and analysis of this gene has re- and regeneration is involved in the renewal process. vealed that Nkx2.2 possesses at least three alternative Although the molecular mechanisms that control islet first exons (exons 1a, 1b, and 1c). Sequencing of the regeneration have yet to be clarified, the process seems 5’ flanking regions of these exons has suggested that to resemble the developmental process. Several stud- each fragment functions as a promoter. Studies of ies have indicated that cells residing in islets and duct transgenic mice with each of the three promoters cells can differentiate into endocrine cells [59, 60]. driving the bacterial LacZ gene have revealed that the Based on cell tracing data, the majority of the progeni- 1b promoter (the sequence upstream from exon 1b) tors of duct and exocrine/endocrine cells separate be- specifically restricts expression to islet precursors. On fore e12.5 [4]. Overexpression of Ngn3 in human duct the other hand, the 1a promoter (the sequence upstream cells provokes differentiation to  cells, a capability from exon 1a) predominantly directs expression in that is shared by NeuroD1. Accordingly the Ngn3- mature islet cells. The transition from proendocrine NeuroD axis might serve as a master switch that drives cells to differentiated endocrine cells should involve transdifferentiation to an endocrine phenotype when progressive changes in gene expression program. Us- misexpressed by adult duct cells [61]. It was shown ing different sequences within the Nkx2.2 gene, cells previously that overexpression of Ngn3 in immature in different stages of endocrine differentiation can ex- cells like pancreatic buds induces differentiation into  press Nkx2.2. Expression of the 1a promoter depends cells [25] [20]. These differences might be derived on the Foxa-binding site and E box, which are located from variations in the ability to become  cells. The proximal to the transcription initiation site. Foxa2 and state of maturity of various cells might be one of the Ngn3 or NeuroD1 can bind to each site to, interact and determinants of their fates as  or  cells. Recently, synergistically activate the promoter [57]. These re- Kojima et al. showed that overexpression of NeuroD1 sults suggest that Ngn3 or NeuroD1 cooperates with in the liver leads to ectopic formation of islets in this Foxa2, in order to recruit a transcriptional co-activator organ and normalized hyperglycemia induced by and activate the expression of Nkx2.2. Considering streptozotocin. Although the lineage of the cells dif- the timing of expression of the Ngn3-NeuroD1 axis, ferentiating into endocrine cells was unclear, this re- Ngn3 may initiate the enhanced expression of Nkx2.2. sult indicates the usefulness of NeuroD1 for promoting After the disappearance of Ngn3, NeuroD1 may act to regeneration of  cells from progenitor cells residing maintain the expression of Nkx2.2. in adult organs [62]. The other candidate as a downstream factor directly influenced by Ngn3 is Ngn3 itself. Ngn3 is expressed by proendocrine cells, but its expression is rapidly Future prospects turned off. Unlike the Pax4 and Nkx2.2 promoter, the Ngn3 promoter is repressed by Ngn3 itself. This re- Genetic and cell biology studies have revealed that pressor activity is also mediated by the E box sequence Ngn3 is a key factor in the determination of endocrine located proximal to the transcription initiation site. differentiation. However, we still lack information This repressor effect seems to be mediated at least about the major targets of Ngn3. Disappearance of partly by direct competitive binding of another strong Ngn3 expression and detection of neuroD1 expression transcription factor, because Ngn3 itself does not show are coincident with exiting of cells from the cell cycle, transcription repressor activity [58]. suggesting that one of the main downstream targets of Thus, Ngn3 transactivates islet transcription factors Ngn3 and/or NeuroD1 is a factor that controls cell such as Pax4 and Nkx2.2 in cooperation with other en- cycling [13]. Further, Ngn3-positive endocrine pre- dodermal transcription factors like HNF1 and Foxa2, cursor cells can differentiate into the four types of cells while repressing its own promoter. that exist in pancreatic islets. Although several factors are essential for such differentiation, identifying the mechanisms that determine the fate of cells after Ngn3 262 WATADA expression is important and will assist in developing Acknowledgements an efficient method for generation of pancreatic  cells from non- cells. The author would like to thank Dr. Yoshio Fujitani (Vanderbilt University Medical Center) for his valuable suggestions to this manuscript, and Dr. Mike German for his great mentorship.

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