Nondestructive and noncontact evaluation of cellulose nanofiber-reinforced composites using terahertz time-domain spectroscopy.

Cellulose nanofibers (CNFs) can be obtained from natural sources, such as plants and wood fibers. Thermoplastic resin composites reinforced with CNFs exhibit various features, including superior mechanical strength. As the mechanical properties of composites reinforced with CNFs are affected by the amount of fiber addition, it is important to determine the concentration of the CNF filler in the matrix after injection or extrusion molding. We confirmed that there was good linearity between the CNF concentration and terahertz absorption. We could distinguish the difference in the 1%-point-CNF concentration with terahertz time-domain spectroscopy. Furthermore, we estimated the mechanical properties of the CNF nanocomposites using terahertz information.

Dynamic Changes in the Localization of Neuronatin-Positive Cells during Neurogenesis in the Embryonic Rat Brain.

Neuronatin (NNAT) was first identified as a gene selectively and abundantly expressed in the cytoplasm of the newborn mouse brain, and involved in neonatal neurogenesis. However, the particular roles of NNAT in the developing prenatal brain have not been identified, especially in mid to late stages. In this study, we performed immunohistochemical analyses of NNAT and SOX2 proteins, a nuclear transcription factor and neural stem/progenitor marker, in the rat brain on embryonic days 13.5, E16.5, and E20.5. NNAT signals were broadly observed across the developing brain on E13.5 and gradually more localized in later stages, eventually concentrated in the alar and basal parts of the terminal hypothalamus, the alar plate of prosomere 2 of the thalamus, and the choroid plexus in the lateral and fourth ventricles on E20.5. In particular, the mammillary body in the basal part of the terminal hypothalamus, a region with a high number of SOX2-positive cells, evidenced intense NNAT signals on E20.5. The intracellular localization of NNAT showed diverse profiles, suggesting that NNAT was involved in various cellular functions, such as cell differentiation and functional maintenance, during prenatal neurogenesis in the rat brain. Thus, the present observations suggested diverse and active roles of the NNAT protein in neurogenesis. Determining the function of this molecule may assist in the elucidation of the mechanisms involved in brain development.

Characteristic Localization of Neuronatin in Rat Testis, Hair Follicle, Tongue, and Pancreas.

Neuronatin (Nnat) is expressed in the pituitary, pancreas, and other tissues; however, the function of NNAT is still unclear. Recent studies have demonstrated that NNAT is localized in the sex-determining region Y-box 2-positive stem/progenitor cells in the developing rat pituitary primordium and is downregulated during differentiation into mature hormone-producing cells. Moreover, NNAT is widely localized in subcellular organelles, excluding the Golgi. Here, we further evaluated NNAT-positive cells and intracellular localization in embryonic and postnatal rat tissues such as the pancreas, tongue, whisker hair follicle, and testis. Immunohistochemistry revealed that NNAT was localized in undifferentiated cells (i.e., epithelial basal cells and basement cells in the papillae of the tongue and round and elongated spermatids of the testis) as well as in differentiated cells (insulin-positive cells and exocrine cells of the pancreas, taste receptor cells of the fungiform papilla, the inner root sheath of whisker hair follicles, and spermatozoa). In addition, NNAT exhibited novel intracellular localization in acrosomes in the spermatozoa. Because the endoplasmic reticulum (ER) is excluded from spermatozoa and sarco/ER Ca2+-ATPase isoform 2 (SERCA2) is absent from the inner root sheath, these findings suggested that NNAT localization in the ER and its interaction with SERCA2 are cell- or tissue-specific properties.

Correction to: SOX10-positive cells emerge in the rat pituitary gland during late embryogenesis and start to express S100ß.

The published online version contains mistake in Table 1, Table 2, and some data in Materials and Methods.

SOX10-positive cells emerge in the rat pituitary gland during late embryogenesis and start to express S100ß.

In the pituitary gland, S100ß-positive cells localize in the neurohypophysis and adenohypophysis but the lineage of the two groups remains obscure. S100ß is often observed in many neural crest-derived cell types. Therefore, in this study, we investigate the origin of pituitary S100ß-positive cells by immunohistochemistry for SOX10, a potent neural crest cell marker, using S100ß-green fluorescence protein-transgenic rats. On embryonic day 21.5, a SOX10-positive cell population, which was also positive for the stem/progenitor cell marker SOX2, emerged in the pituitary stalk and posterior lobe and subsequently expanded to create a rostral-caudal gradient on postnatal day 3 (P3). Thereafter, SOX10-positive cells appeared in the intermediate lobe by P15, localizing to the boundary facing the posterior lobe, the gap between the lobule structures and the marginal cell layer, a pituitary stem/progenitor cell niche. Subsequently, there was an increase in SOX10/S100ß double-positive cells; some of these cells in the gap between the lobule structures showed extended cytoplasm containing F-actin, indicating a feature of migration activity. The proportion of SOX10-positive cells in the postnatal anterior lobe was lower than 0.025% but about half of them co-localized with the pituitary-specific progenitor cell marker PROP1. Collectively, the present study identified that one of the lineages of S100ß-positive cells is a SOX10-positive one and that SOX10-positive cells express pituitary stem/progenitor cell marker genes.

Correction to: Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

The published online version contains mistakes in Table 1, Table 2 and Fig. 2. See below for the corrected Tables and Figure.

Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

Clump formation in mouse pituitary-derived non-endocrine cell line Tpit/F1 promotes differentiation into growth-hormone-producing cells.

The adenohypophysis comprises six types of endocrine cells, including PIT1-lineage cells such as growth hormone (GH)-producing cells and heterogeneous non-endocrine cells, such as pituitary stem/progenitor cells as a source of endocrine cells. We determine the expression of characteristic stem cell marker genes, including sex-determining region Y-box 2 (Sox2), in mouse pituitary-derived non-endocrine cell lines Tpit/E, Tpit/F1 and TtT/GF. We observed high expression of fibroblast growth factor (FGF) receptors in Tpit/F1 cells, which we characterised by cultivation in medium containing a basic FGF and B27 supplement as used for neural stem-cell differentiation. A 4-day cultivation of Tpit/F1 produced floating embryonic stem-cell-like clumps accompanied by a three-fold increase in Sox2 expression. Passages in these clumps maintained the proliferative activity and Sox2 expression levels. After 10 days of cultivation, Tpit/F1 cell clumps were immuno-positive for SOX2 and Ki67 (proliferation marker) and loosely attached to the well bottom. An additional 10 days of cultivation induced the emergence of GH-positive/pituitary-specific transcription factor (PIT1)-negative cells showing migration from the clumps. Pit1 overexpression in attached cells could not induce GH production. Finally, we confirmed the presence of PIT1-negative GH-producing cells (3.2-7.7 % of all GH-positive cells) in rat pituitary. Thus, we demonstrate that Tpit/F1 has the plasticity to differentiate into one type of hormone-producing cell.

Gene tracing analysis reveals the contribution of neural crest-derived cells in pituitary development.

The anterior pituitary originates from the adenohypophyseal placode. Both the preplacode region and neural crest (NC) derive from subdivision of the neural border region, and further individualization of the placode domain is established by a reciprocal interaction between placodal precursors and NC cells (NCCs). It has long been known that NCCs are present in the adenohypophysis as interstitial cells. A recent report demonstrated that NCCs also contribute to the formation of pericytes in the developing pituitary. Here, we attempt to further clarify the role of NCCs in pituitary development using P0-Cre/EGFP reporter mice. Spatiotemporal analyses revealed that GFP-positive NCCs invaded the adenohypophysis in a stepwise manner. The first wave was detected on mouse embryonic day 9.5 (E9.5), when the pituitary primordium begins to be formed by adenohypophyseal placode cells; the second wave occurred on E14.5, when vasculogenesis proceeds from Atwell's recess. Finally, fate tracing of NCCs demonstrated that NC-derived cells in the adenohypophysis terminally differentiate into all hormone-producing cell lineages as well as pericytes. Our data suggest that NCCs contribute to pituitary organogenesis and vasculogenesis in conjunction with placode-derived pituitary stem/progenitor cells.

Involvement of DNA methylation in regulating rat Prop1 gene expression during pituitary organogenesis.

PROP1 is a pituitary specific transcription factor that plays a crucial role in pituitary organogenesis. The Prop1 shows varied expression patterns that promptly emerge and then fade during the early embryonic period. However, the regulatory mechanisms governing Prop1 expression remain unclear. Here, we investigated whether Prop1 was under epigenetic regulation by DNA methylation. Bisulfite sequencing was performed on DNA obtained from the pituitary glands and livers of rats on embryonic days (E) 13.5 and E14.5, and postnatal days (P) 4 and P30. The methylation of CpG sites in seven regions from 3-kb upstream of the Prop1 transcription start site through to its second intron were examined. Certain differences in CpG-methylation levels were observed in Region-1 (-2772 b to -2355 b), Region-4 (-198 b to +286 b), Region-5 (+671 b to +990 b), and Region-6 (+1113 b to +1273 b) based on comparisons between pituitary and liver DNA on E13.5. DNA methylation in pituitary glands on E14.5, P4, and P30 was generally similar to that observed in in the pituitary gland on E13.5, whereas the anterior and intermediate lobes of the pituitary gland on P4 and P30 showed only small differences. These results indicate that Prop1 is under regulation by CpG methylation during the early period of pituitary primordium development around E13.5.

Identification of THY1 as a novel thyrotrope marker and THY1 antibody-mediated thyrotrope isolation in the rat anterior pituitary gland.

Contact-dependent (juxtacrine) signaling is important for local cell-to-cell interaction and has received attention in recent years regarding its role in pituitary function, differentiation, and development. This study investigated one of the juxtacrine-related molecules, thymocyte differentiation antigen 1 (THY1), in the anterior lobe of the rat pituitary gland. Western blot analysis revealed expression of the THY1 protein in the adult rat anterior lobe. We also found that the THY1 ligand, integrin-ß2 (ITGB2), is also expressed in the pituitary gland. In situ hybridization and immunohistochemical analyses showed that both THY1 mRNA and protein were present in almost, if not all, thyroid-stimulating hormone (TSH)-immunopositive cells (thyrotropes) and that ITGB2 was co-expressed in these cells. As THY1 appeared to represent a novel marker for thyrotropes, we then attempted to isolate these cells from various anterior lobe cells by the use of a THY1 antibody and a pluriBead-cascade cell isolation system. This technology allowed the isolation of thyrotropes with 83% purity at about 17-fold enrichment. Furthermore, the isolated THY1-immunopositive cells had higher Tsh mRNA levels compared with THY1-immunonegative cells and released TSH in response to thyrotropin-releasing hormone. These findings indicated that THY1 represents a potent thyrotrope marker and that the thyrotrope isolation method using the THY1 antibody may serve as a powerful tool to analyze their function including juxtacrine regulation through THY1/ITGB2 interaction.

S100ß-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland.

The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these S100ß-positive cells, which are not classic endocrine cells but instead comprise a heterogeneous cell population with plural roles, especially as stem/progenitor cells. To better understand the roles of these S100ß-positive cells, we performed immunohistochemical analysis using several markers in S100ß/GFP-TG rats, which express GFP in S100ß-expressing cells under control of the S100ß promoter. GFP-positive cells were present as mesenchymal cells surrounding the developing pituitary gland and at Atwell's recess but were not present in the anterior lobe on embryonic day 15.5. These cells were negative for SOX2, a pituitary stem/progenitor marker, and PRRX1, a mesenchyme and pituitary stem/progenitor marker. However, three days later, GFP-positive and PRRX1-positive (but SOX2-negative) cells were observed in the parenchyma of the anterior lobe. Furthermore, some GFP-positive cells were positive for vimentin, p75, isolectin B4, DESMIN, and Ki67. These data suggest that S100ß-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.

Isolation of adult pituitary stem/progenitor cell clusters located in the parenchyma of the rat anterior lobe.

Recent studies have demonstrated that Sox2-expressing stem/progenitor cells play roles in the pituitary cell turnover. Two types of niches have been proposed for stem/progenitor cells, the marginal cell layer (MCL) and the dense cell clusters in the parenchyma. Among them, the appearance of the parenchymal-niche only after birth indicates that this niche is involved in the cell turnover required for the postnatal pituitary. However, little is known about the roles of the parenchymal-niche and its regulation. The present study aimed to isolate pituitary stem/progenitor cells from the parenchymal-niche in the adult rat pituitary. Cell dispersion by stepwise treatment with proteases allowed the isolation of dense cell clusters. Immunocytochemistry demonstrated that clusters are universally composed of SOX2-positive cells, and most of them are positive for PROP1. Taken together with the anatomical analysis, we concluded that the isolated clusters are the parenchymal stem/progenitor cell (PS)-clusters, not the MCL-one. PS-clusters cultivated by serum-free overlay 3-dimensional culture maintained their stemness, and treatment with bFGF and EGF induced cyst-formation. Moreover, PS-clusters demonstrated some differentiation capacity with GSK3ß-inhibitor treatment. Collectively, the present study demonstrates a simple method for isolating stem/progenitor cells from the parenchymal-niche, and provides tools to analyze the factors for regulating the pituitary niches.

Transcription of follicle-stimulating hormone subunit genes is modulated by porcine LIM homeobox transcription factors, LHX2 and LHX3.

The LIM-homeobox transcription factors LHX2 and LHX3s (LHX3a and LHX3b) are thought to be involved in regulating the pituitary glycoprotein hormone subunit genes Cga and Fshß. These two factors show considerable differences in their amino acid sequences for DNA binding and protein-protein interactions and in their vital function in pituitary development. Hence, we compared the DNA binding properties and transcriptional activities of Cga and Fshß between LHX2 and LHX3s. A gel mobility shift assay for approximately 1.1 kb upstream of Cga and 2.0 kb upstream of Fshß varied in binding profiles between LHX2 and LHX3s. DNase I footprinting revealed DNA binding sites in 8 regions of the Cga promoter for LHX2 and LHX3s with small differences in the binding range and strength. In the Fshß promoter, 14 binding sites were identified for LHX2 and LHX3, respectively. There were alternative binding sites to either gene in addition to similar differences observed in the Cga promoter. The transcriptional activities of LHX2 and LHX3s according to a reporter assay showed cell-type dependent activity with repression in the pituitary gonadotrope lineage LßT2 cells and stimulation in Chinese hamster ovary lineage CHO cells. Reactivity of LHX2 and LHX3s was observed in all regions, and differences were observed in the 5'-upstream region of Fshß. However, immunohistochemistry showed that LHX2 resides in a small number of gonadotropes in contrast to LHX3. Thus, LHX3 mainly controls Cga and Fshß expression.

Search for regulatory factors of the pituitary-specific transcription factor PROP1 gene.

Pituitary-specific transcription factor PROP1, a factor important for pituitary organogenesis, appears on rat embryonic day 11.5 (E11.5) in SOX2-expressing stem/progenitor cells and always coexists with SOX2 throughout life. PROP1-positive cells at one point occupy all cells in Rathke's pouch, followed by a rapid decrease in their number. Their regulatory factors, except for RBP-J, have not yet been clarified. This study aimed to use the 3 kb upstream region and 1st intron of mouse prop1 to pinpoint a group of factors selected on the basis of expression in the early pituitary gland for expression of Prop1. Reporter assays for SOX2 and RBP-J showed that the stem/progenitor marker SOX2 has cell type-dependent inhibitory and activating functions through the proximal and distal upstream regions of Prop1, respectively, while RBP-J had small regulatory activity in some cell lines. Reporter assays for another 39 factors using the 3 kb upstream regions in CHO cells ultimately revealed that 8 factors, MSX2, PAX6, PIT1, PITX1, PITX2, RPF1, SOX8 and SOX11, but not RBP-J, regulate Prop1 expression. Furthermore, a synergy effect with SOX2 was observed for an additional 10 factors, FOXJ1, HES1, HEY1, HEY2, KLF6, MSX1, RUNX1, TEAD2, YBX2 and ZFP36Ll, which did not show substantial independent action. Thus, we demonstrated 19 candidates, including SOX2, to be regulatory factors of Prop1 expression.

Expression studies of neuronatin in prenatal and postnatal rat pituitary.

The pituitary gland, an indispensable endocrine organ that synthesizes and secretes pituitary hormones, develops with the support of many factors. Among them, neuronatin (NNAT), which was discovered in the neonatal mouse brain as a factor involved in neural development, has subsequently been revealed to be coded by an abundantly expressing gene in the pituitary gland but its role remains elusive. We analyze the expression profile of Nnat and the localization of its product during rat pituitary development. The level of Nnat expression was high during the embryonic period but remarkably decreased after birth. Immunohistochemistry demonstrated that NNAT appeared in the SOX2-positive stem/progenitor cells in the developing pituitary primordium on rat embryonic day 11.5 (E11.5) and later in the majority of SOX2/PROP1 double-positive cells on E13.5. Thereafter, during pituitary embryonic development, Nnat expression was observed in some stem/progenitor cells, proliferating cells and terminally differentiating cells. In postnatal pituitaries, NNAT-positive cells decreased in number, with most coexpressing Sox2 or Pit1, suggesting a similar role for NNAT to that during the embryonic period. NNAT was widely localized in mitochondria, peroxisomes and lysosomes, in addition to the endoplasmic reticulum but not in the Golgi. The present study thus demonstrated the variability in expression of NNAT-positive cells in rat embryonic and postnatal pituitaries and the intracellular localization of NNAT. Further investigations to obtain functional evidence for NNAT are a prerequisite.

GFP-expressing S100ß-positive cells of the rat anterior pituitary differentiate into hormone-producing cells.

Some non-endocrine cells in the pituitary anterior lobe are responsible for providing stem/progenitor cells to maintain hormone-producing cells. In particular, cells expressing S100ß protein, a calcium-binding protein, have been hypothesized to be a pituitary cell resource. Accumulating data have revealed that S100ß-positive cells comprise heterogeneous populations and some of them certainly show stem/progenitor characteristics in vivo. Hence, we examine whether S100ß-positive cells have the capacity to differentiate into endocrine cells, by means of in vivo and in vitro experiments on transgenic rats expressing enhanced green fluorescent protein (EGFP) under the control of the S100ß promoter. Immunohistochemistry of the pituitary confirmed that some S100ß-positive cells expressed SOX2 (SRY [sex-determining region Y]-box 2) and had proliferative activity. Dispersed anterior lobe cells were observed by time-lapse microscopy, followed by immunostaining for hormone and pituitary-transcription-factor1 (PIT1). First, the dispersed anterior lobe cells were immunostained by an antibody against SOX2. S100ß-protein co-localizes with SOX2 (about 89 %). Although 44 of 134 S100ß-positive cells traced were proliferative but negative to any hormones, 14 cells were positive for one of the pituitary hormones and/or PIT1, confirming the presence of all types of hormone-producing cells. Notably, GFP-fluorescence appeared in two hormone-positive cells during culture. On the other hand, we observed hormone-producing cells that were not positive for S100ß at the end of the time-lapse study, despite being initially positive. These findings suggest that S100ß-positive cells cultured from the anterior lobe are capable of developing into hormone-producing cells, although this happens relatively infrequently.

Isolation of dendritic-cell-like S100ß-positive cells in rat anterior pituitary gland.

S100ß-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100ß-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100ß-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100ß protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100ß-positive cells by means of their property of adhering to laminin.

Three-dimensional studies of Prop1-expressing cells in the rat pituitary just before birth.

Recently, we demonstrated that differentiation was underway as early as embryonic day (E) 13.5 in the lateral region of the rat pituitary primordium. In this study, we analyze the heterogeneous property of cells in the pituitary at E21.5 (just before birth) leading to its biological function with the differentiation and expansion of tissue. The three-dimensional structure of the pituitary at E21.5 was built up from measurements taken from many DAPI-stained sections and cell populations positive to the stem/progenitor marker SOX2, pituitary-specific transcription factor PROP1 and paired-related homeodomain transcription factor PRX. At E21.5, the pituitary, composed of anterior and intermediate lobes, showed a flattened chestnut shape with dimensions of about 500 µm (dorsoventral axis) by 2500 µm (left-right axis) by 850 µm (rostrocaudal axis) and consisted in approximately 113,500, 16,000 and 14,800 cells in the anterior, intermediate and posterior lobes, respectively. Five cell types were observed expressing Sox2, Prop1 and Prx; these were heterogeneously distributed in the mediolateral and dorsoventral axes. In the anterior lobe, the marginal cell layer (MCL) was mostly occupied by stem/progenitor cells positive for SOX2, with the co-expression of Prop1 and/or Prx, whereas more SOX2-single-positive cells than those for PROP1 and PRX were scattered in the parenchyma. PRX-positive cells of mesenchymal origin invaded the parenchyma, together with PECAM- and NESTIN-positive cells, indicating the advance of vasculogenesis. Thus, marked developmental progress occurs regarding the transition of stem/progenitor cells in the MCL and regarding vasculogenesis in the parenchyma during the prenatal pituitary growth wave.

Coxsackievirus and adenovirus receptor-positive cells compose the putative stem/progenitor cell niches in the marginal cell layer and parenchyma of the rat anterior pituitary.

The pituitary gland is a slow generative tissue but actively responds to demands by changing homeostasis. The marginal cell layer (MCL) facing the residual lumen has long been indicated as a stem/progenitor cell niche of the pituitary. On the other hand, the coxsackievirus and adenovirus receptor (CAR), which localizes at the tight-junction of the polarized epithelium, is known to participate in the development, differentiation and regeneration of specified tissues. The present study attempts to characterize the cells lining the MCL during pituitary development by immunohistochemistry of CAR. Consequently, we found that CAR localizes in an apical surface of the single cell layer facing the oral cavity in the invaginating oral epithelium on rat embryonic day (E) 11.5. On E13.5, when this single layer constructs the MCL in the pituitary primordium Rathke's pouch, CAR-positive cells occupied the MCL and this localization pattern of CAR was persistently maintained throughout life. Moreover, clusters of CAR-positive cells were also found in the parenchyma. CAR-positive cells were positive for stem/progenitor cell markers sex-determining region Y-box 2 (SOX2) and epithelial calcium-dependent adhesion (E-cadherin). However, prior to the postnatal growth wave, cells positive for CAR in the basolateral surface constructed multiple cell layers beneath the MCL and cell-type transition to a putative migratory cell phenotype by fading of SOX2 and E-cadherin occurred, suggesting the composition of new putative niches in the parenchyma. These data, together with our previous reports, suggest that CAR-positive cells are pituitary stem/progenitor cells and compose putative stem/progenitor cell niches in the MCL and parenchyma.