Primary Cilia are Required for Cell-Type Determination and Angiogenesis in Pituitary Development.

The functional maturation of the pituitary gland requires adequate cell differentiation and vascular network formation. Although spatiotemporal signaling and transcription factors are known to govern pituitary development, the involvement of primary cilia, nonmoving hair-like organelles, remains unclear. In this study, we uncovered the contribution of primary cilia to cell-type determination and vascular network formation during pituitary development. Homozygous knockout mice lacking a ciliary kinase, Dyrk2-/-, exhibit abnormalities in ciliary structure and pituitary hypoplasia, accompanied by varying degrees of failure in differentiation among all types of hormone-producing cells in the anterior lobe. Aberrations in cell differentiation in Dyrk2-/- mice arise from a decrease in the expression of crucial transcription factors, Lhx4, Lhx3, and Prop1, resulting from the inactivity of Hedgehog (Hh) signaling during the early stages of development. Furthermore, the loss of Dyrk2 results in vascular system abnormalities during the middle to late stages of development. Mechanistically, transcriptome analyses revealed the downregulation of vitronectin-integrin αvß3-VEGFR2 signaling, essential for orchestrating vascular development. Collectively, our findings demonstrate that primary cilia play a pivotal role as critical regulators of cell survival, cell determination, and angiogenesis during pituitary gland development through the activation of Hh signaling. These findings expand our understanding of the potential link between pituitary dysfunction in human disorders and ciliopathies.

Expression of Retinaldehyde Dehydrogenases in the Pituitary Glands of Fetus and Adult Mice.

Retinoic acid (RA) plays a critical role in cell growth and tissue development. RA is synthesized from retinoids through oxidation processes by the retinaldehyde dehydrogenase (Raldh) family. However, the expression of Raldhs during pituitary development and the identification of Raldh-expressing cells in the adult pituitary have not been fully elucidated. Here, we performed in situ hybridization to localize the three Raldh isoforms (Raldh1-3) in fetal and adult mouse pituitary glands. The results showed that Raldh2 expression was observed in Rathke's pouch from embryonic day 13.5 (E13.5), and this expression was sustained in the anterior lobe of the pituitary primordium from E15.5 to E17.5. In contrast, Raldh1 and Raldh3 were rarely detectable. Real-time PCR analysis revealed that Raldh2 was the predominant isoform expressed in the adult pituitary, although Raldh1 was also expressed to a lesser extent. In the adult pituitary, Raldh1-expressing cells were primarily observed in the posterior lobe. Raldh2-expressing cells were found in the marginal cell layer and parenchyma of the anterior lobe and were immunopositive for aldolase C (folliculostellate cells), but not for anterior pituitary hormones. These results suggest that RA is an important regulatory factor in the functions of the pituitary throughout its development in mice.

Positive regulation of Hedgehog signaling via phosphorylation of GLI2/GLI3 by DYRK2 kinase.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

Paracrine and endocrine pathways of natriuretic peptides assessed by ligand-receptor mapping in the Japanese eel brain.

The natriuretic peptide (NP) family consists of cardiac NPs (ANP, BNP, and VNP) and brain NPs (CNPs) in teleosts. In addition to CNP1-4, a paralogue of CNP4 (named CNP4b) was recently discovered in basal teleosts including Japanese eel. Mammals have lost most Cnps during the evolution, but teleost cnps were conserved and diversified, suggesting that CNPs are important hormones for maintaining brain functions in teleost. The present study evaluated the potency of each Japanese eel CNP to their NP receptors (NPR-A, NPR-B, NPR-C, and NPR-D) overexpressed in CHO cells. A comprehensive brain map of cnps- and nprs-expressing neurons in Japanese eel was constructed by integrating the localization results obtained by in situ hybridization. The result showed that CHO cells expressing NPR-A and NPR-B induced strong cGMP productions after stimulation by cardiac and brain NPs, respectively. Regarding brain distribution of cnps, cnp1 is engaged in the ventral telencephalic area and periventricular area including the parvocellular preoptic nucleus (Pp), anterior/posterior tuberal nuclei, and periventricular gray zone of the optic tectum. cnp3 is found in the habenular nucleus and prolactin cells in the pituitary. cnp4 is expressed in the ventral telencephalic area, while cnp4b is expressed in the motoneurons in the medullary area. Such CNP isoform-specific localizations suggest that function of each CNP has diverged in the eel brain. Furthermore, the Pp lacking the blood-brain barrier expressed both npra and nprb, suggesting that endocrine and paracrine NPs interplay for regulating the Pp functions in Japanese eels.

Fluctuation of CD9/SOX2-positive cell populations during the turnover of GH- and TSH-producing cells in the adult anterior pituitary gland.

The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke's cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.

Subcellular localization of Na+/K+-ATPase isoforms resolved by in situ hybridization chain reaction in the gill of chum salmon at freshwater and seawater.

The Na+/K+-ATPase (NKA) α1-isoforms were examined by in situ hybridization chain reaction (ISHCR) using short hairpin DNAs, and we showed triple staining of NKA α1a, α1b, and α1c transcripts in the gill of chum salmon acclimated to freshwater (FW) and seawater (SW). The NKA α1-isoforms have closely resembled nucleotide sequences, which could not be differentiated by conventional in situ hybridization. The ISHCR uses a split probe strategy to allow specific hybridization using regular oligo DNA, resulting in high specificity at low cost. The results showed that NKA α1c was expressed ubiquitously in gill tissue and no salinity effects were observed. FW lamellar ionocytes (type-I ionocytes) expressed cytoplasmic NKA α1a and nuclear NKA α1b transcripts. However, both transcripts of NKA α1a and α1b were present in the cytoplasm of immature type-I ionocytes. The developing type-I ionocytes increased the cytoplasmic volume and migrated to the distal region of the lamellae. SW filament ionocytes (type-II ionocytes) expressed cytoplasmic NKA α1b transcripts as the major isoform. Results from morphometric analysis and nonmetric multidimensional scaling indicated that a large portion of FW ionocytes was NKA α1b-rich, suggesting that isoform identity alone cannot mark the ionocyte types. Both immature or residual type-II ionocytes and type-I ionocytes were found on the FW and SW gills, suggesting that the chum salmon retains the potential to switch the ionocyte population to fit the ion-transporting demands, which contributes to their salinity tolerance and osmoregulatory plasticity.

Behavioural osmoregulation during land invasion in fish: Prandial drinking and wetting of the dry skin.

Osmoregulatory behaviours should have evolutionarily modified for terrestrialisation of vertebrates. In mammals, sensations of buccal food and drying have immediate effects on postprandial thirst to prevent future systemic dehydration, and is thereby considered to be 'anticipatory thirst'. However, it remains unclear whether such an anticipatory response has been acquired in the non-tetrapod lineage. Using the mudskipper goby (Periophthalmus modestus) as a semi-terrestrial ray-finned fish, we herein investigated postprandial drinking and other unique features like full-body 'rolling' over on the back although these behaviours had not been considered to have osmoregulatory functions. In our observations on tidal flats, mudskippers migrated into water areas within a minute after terrestrial eating, and exhibited rolling behaviour with accompanying pectoral-fin movements. In aquarium experiments, frequency of migration into a water area for drinking increased within a few minutes after eating onset, without systemic dehydration. During their low humidity exposure, frequency of the rolling behaviour and pectoral-fin movements increased by more than five times to moisten the skin before systemic dehydration. These findings suggest anticipatory responses which arise from oral/gastrointestinal and cutaneous sensation in the goby. These sensation and motivation seem to have evolved in distantly related species in order to solve osmoregulatory challenges during terrestrialisation.

Differentiation of stem progenitor CD9/SOX2-positive cells is promoted with increased prolactin-producing and endothelial cells in the pituitary.

Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.

The multiciliated cells in Rathke's cleft express CYP26A1 and respond to retinoic acid in the pituitary.

The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary.

Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP.

The diversified natriuretic peptide (NP) family, consisting of four CNPs (CNP1-4), ANP, BNP, and VNP, has been identified in the eel. Here, we successfully cloned additional cnp genes from the brain of eel (a basal teleost) and zebrafish (a later branching teleost). The genes were identified as paralogues of cnp4 generated by the third round of whole genome duplication (3R) in the teleost lineage, thereby being named eel cnp4b and zebrafish cnp4-like, respectively. To examine the histological patterns of their expressions, we employed a newly developed in situ hybridization (ISH) chain reaction using short hairpin DNAs, in addition to conventional ISH. Eel cnp4b was expressed in the medulla oblongata, while mRNAs of eel cnp4a (former cnp4) were localized in the preoptic area. In the zebrafish brain, cnp4-like mRNA was undetectable, while the known cnp4 was expressed in both the preoptic area and medulla oblongata. Together with the different mRNA distribution of cnp4a and cnp4b in eel peripheral tissues determined by RT-PCR and ISH, it is suggested that subfunctionalization by duplicated cnp4s in ancestral teleosts has been retained only in basal teleosts. Intriguingly, cnp4b-expressing neurons in the glossopharyngeal-vagal motor complex of the medulla oblongata were co-localized with choline acetyltransferase, suggesting an involvement of Cnp4b in swallowing and respiration functions that are modulated by the vagus. Since teleost Cnp4 is an ortholog of mammalian CNP, the identified localization of teleost Cnp4 will contribute to future studies aimed at deciphering the physiological functions of CNP.

Molecular mechanisms underlying guanylin-induced transcellular Cl- secretion into the intestinal lumen of seawater-acclimated eels.

Guanylin (GN) stimulates Cl- secretion into the intestinal lumen of seawater-acclimated eels, but the molecular mechanisms of transepithelial Cl- transport are still unknown. In Ussing chamber experiments, we confirmed that mucosal application of eel GN reversed intestinal serosa-negative potential difference, indicating Cl- secretion. Serosal application of DNDS or mucosal application of DPC inhibited the GN effect, but serosal application of bumetanide had no effect. Removal of HCO3- from the serosal fluid also inhibited the GN effect. In intestinal sac experiments, mucosal GN stimulated luminal secretion of both Cl- and Na+, which was blocked by serosal DNDS. These results suggest that Cl- is taken up at the serosal side by DNDS-sensitive anion exchanger (AE) coupled with Na+-HCO3- cotransporter (NBC) but not by Na+-K+-2Cl- cotransporter 1 (NKCC1), and Cl- is secreted by unknown DPC-sensitive Cl- channel (ClC) at the mucosal side. The transcriptomic analysis combined with qPCR showed low expression of NKCC1 gene and no upregulation of the gene after seawater transfer, while high expression of ClC2 gene and upregulation after seawater transfer. In addition, SO42- transporters (apical Slc26a3/6 and basolateral Slc26a1) are also candidates for transcellular Cl- secretion in exchange of luminal SO42. Na+ secretion could occur through a paracellular route, as Na+-leaky claudin15 was highly expressed and upregulated after seawater transfer. High local Na+ concentration in the lateral interspace produced by Na+/K+-ATPase (NKA) coupled with K+ channels (Kir5.1b) seems to facilitate the paracellular transport. In situ hybridization confirmed the expression of the candidate genes in the epithelial enterocytes. Together with our previous results, we suggest that GN stimulates basolateral NBCela/AE2 and apical ClC2 to increase transcellular Cl- secretion in seawater eel intestine, which differs from the involvement of apical CFTR and basolateral NKCC1 as suggested in mammals and other teleosts.

CD9-positive cells in the intermediate lobe of the pituitary gland are important supplier for prolactin-producing cells in the anterior lobe.

A supply of hormone-producing cells from stem/progenitor cells is critical to sustain the endocrine activity of the pituitary gland. In the adenohypophysis composing the anterior and intermediate lobe (AL and IL, respectively), stem/progenitor cells expressing sex-determining region Y-box 2 (SOX2) and S100ß are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). Our previous studies using mice and rats indicated that the tetraspanin superfamily CD9 and CD81 are expressed in S100ß/SOX2-positive cells of primary and secondary niches (named CD9/CD81/S100ß/SOX2-positive cell), and the cells located in the AL-side niches exhibit plasticity and multipotency. However, it is unclear whether CD9/CD81/S100ß/SOX2-positive cells in the IL-side primary niche are stem/progenitor cells for the AL or IL. Here, we successfully isolated pure CD9/CD81/S100ß/SOX2-positive cells from the IL-side primary niche. They had a higher level of S100ß and SOX2 mRNA and a greater pituisphere forming capacity than those of CD9/CD81/S100ß/SOX2-positive cells isolated from the AL. They also had capacity to differentiate into all types of adenohypophyseal hormone-producing cells, concomitantly with the loss of CD9 expression. Loss of CD9 and CD81 function in CD9/CD81/S100ß/SOX2-positive cells by siRNA treatment impaired prolactin cell differentiation. Consistently, in the pituitary gland of CD9/CD81 double knockout mice, dysgenesis of the MCL and a lower population of prolactin cells were observed. These results suggest that the CD9/CD81/S100ß/SOX2-positive cells in the MCL of the IL-side are potential suppliers of adult core stem cells in the AL.

Cluster of differentiation (CD) 9-positive mouse pituitary cells are adult stem/progenitor cells.

SOX2-positive cells are stem/progenitor cells that supply hormone-producing cells; they are found in the anterior lobe of the rodent pituitary gland. However, they are likely composed of several subpopulations. In rats, a SOX2-positive cell populations can be distinguished by the presence of S100ß. We identified the novel markers cluster of differentiation (CD) CD9 and CD81, members of the tetraspanin superfamily, for the identification of S100ß/SOX2-positive cells. Recently, CD9/CD81 double-knockout mice were generated. Although they grew normally until 3 weeks after birth, they exhibited atrophy of the pituitary gland. These findings suggested that CD9/CD81/S100ß/SOX2-positive cells in the mouse pituitary are adult stem/progenitor cells. To substantiate this hypothesis, we examined CD9 and CD81 expression in the adult and developing anterior lobe. Immunohistochemistry showed that CD9/CD81-positive cells began appearing from postnatal day 0 and settled in the stem cell niches (marginal cell layer and parenchyma) of the adult anterior lobe while expressing S100ß. We next isolated CD9 -positive cells from the adult anterior lobe, using the anti-CD9 antibody for cell characterisation. The cells in culture formed free-floating three-dimensional clusters (pituispheres); moreover, induction into all types of hormone-producing cells was successful. Furthermore, reduction of CD9 and CD81 mRNAs by siRNAs inhibited cell proliferation. These findings indicate that CD9/CD81/S100ß/SOX2-positive cells may play a role as adult stem/progenitor cells in SOX2-positive subpopulations, thus supplying hormone-producing cells in the postnatal anterior lobe. Furthermore, CD9 and CD81 are implicated in cell proliferation. The current findings provide novel insights into adult pituitary stem/progenitor cells.

Expression and functions of cluster of differentiation 9 and 81 in rat mammary epithelial cells.

Cluster of differentiation (CD) 9 and CD81 are closely-related members of the tetraspanin family that consist of four-transmembrane domain proteins. Cd9 and Cd81 are highly expressed in breast cancer cells; however, their expression in healthy mammary glands is unclear. In this study, we performed quantitative real-time PCR to analyze the expression levels of Cd9 and Cd81. Histological techniques were employed to identify Cd9- and Cd81-expressing cells in rat mammary glands during pregnancy and lactation. It was observed that Cd9 and Cd81 were expressed in the mammary glands, and their expression levels correlated with mammary gland development. To identify cells expressing Cd9 and Cd81 in the mammary glands, we performed double immunohistochemical staining for CD9 and CD81, prolactin receptor long form, estrogen receptor alpha, or Ki67. The results showed that CD9 and CD81 were co-expressed in proliferating mammary epithelial cells. Next, we attempted to isolate CD9-positive epithelial cells from the mammary gland using pluriBead cell-separation technology based on antibody-mediated binding of cells to beads of different sizes, followed by isolation using sieves with different mesh sizes. We successfully isolated CD9-positive epithelial cells with 96.8% purity. In addition, we observed that small-interfering RNAs against Cd9 and Cd81 inhibited estrogen-induced proliferation of CD9-positive mammary epithelial cells. Our current findings may provide novel insights into the proliferation of mammary epithelial cells during pregnancy and lactation as well as in pathological processes associated with breast cancer.

Aldolase C is a novel molecular marker for folliculo-stellate cells in rodent pituitary.

The anterior pituitary gland is composed of five types of hormone-producing cells and folliculo-stellate cells. Folliculo-stellate cells do not produce anterior pituitary hormones but they are thought to play important roles as stem cells, phagocytes, or supporting cells of hormone-producing cells in the anterior pituitary. S100ß protein has been used as a folliculo-stellate cell marker in some animals, including rats. However, since no reliable molecular marker for folliculo-stellate cells has been reported in mice, genetic approaches for the investigation of folliculo-stellate cells in mice are not yet available. Aldolase C/Zebrin II is a brain-type isozyme and is a fructose-1,6-bisphosphate aldolase. In the present study, we first used immunohistochemistry to verify that aldolase C was produced in the anterior pituitary of rats. Moreover, using transgenic rats expressing green fluorescent protein under the control of the S100ß gene promoter, we identified aldolase C-immunoreactive signals in folliculo-stellate cells and marginal cells located in the parenchyma of the anterior pituitary and around Rathke's cleft, respectively. We also identified aldolase C-expressing cells in the mouse pituitary using immunohistochemistry and in situ hybridization. Aldolase C was not produced in any pituitary hormone-producing cells, while aldolase C-immunopositive signal co-localized with E-cadherin- and SOX2-positive cells. Using post-embedding immunoelectron microscopy, aldolase C-immunoreactive products were observed in the cytoplasm of marginal cells and folliculo-stellate cells of the mouse pituitary. Taken together, aldolase C is a common folliculo-stellate cell marker in the anterior pituitary gland of rodents.

Seawater transfer down-regulates C-type natriuretic peptide-3 expression in prolactin-producing cells of Japanese eel: Negative correlation with plasma chloride concentration.

In euryhaline fishes, atrial and B-type natriuretic peptides are important hormones in hypo-osmoregulation, whereas osmoregulatory functions of C-type natriuretic peptides (CNPs) remain to be investigated. Although four CNP isoforms (CNP1-4) are mainly expressed in the brain, multiorgan expression of CNP3 was found in euryhaline Japanese eel, Anguilla japonica. Here we identified the CNP3-expressing cells and examined their response to osmotic stress in eel. CNP3 was expressed in several endocrine cells: prolactin-producing cells (pituitary), glucagon-producing cells (pancreas), and cardiomyocytes (heart). Pituitary CNP3 expression was the highest among organs and was decreased following seawater transfer, followed by a decrease in the freshwater-adaptating (hyper-osmoregulatory) hormone prolactin. We also showed the negative correlation between CNP3/prolactin expression in the pituitary and plasma Cl- concentration, but not for plasma Na+ concentration. These results suggest that CNP3 in the pituitary (and pancreas) plays a critical role in freshwater adaptation of euryhaline eel together with prolactin.

CX3CL1/CX3CR1-signalling in the CD9/S100ß/SOX2-positive adult pituitary stem/progenitor cells modulates differentiation into endothelial cells.

Approximately 8% of CD9-, S100ß- and SOX2-triple positive (CD9/S100ß/SOX2-positive) stem/progenitor cells in the anterior lobe of the rat pituitary gland have previously been shown to differentiate into endothelial cells in vitro, suggesting that they play a role in vascularisation as tissue-resident vascular precursor cells. In the present study, we focused on chemokine ligands to further characterise the CD9/S100ß/SOX2-positive cells and found that they distinctively express CX3C chemokine ligand 1 (Cx3cl1). Immunohistochemical analysis of the anterior lobe showed that CX3CL1-positive cells comprised 7.8% in CD9-positive cells. By cultivation of the CD9-positive cells on laminin-coated plates, we observed that the expression levels of Cx3cl1 decreased, while those of Sox18, an endothelial cell-progenitor marker, and Cx3cr1, a CX3CL1 receptor, increased. Furthermore, in a rat model of prolactinoma, the most common pituitary tumour, which is accompanied by frequent neo-vasculogenesis in the anterior lobe, we have confirmed a decrease in Cx3cl1 expression and an increase in Cx3cr1 expression, as well as a prominent increase in Sox18 expression. These findings suggest that CX3CL1/CX3CR1 signalling in CD9/S100ß/SOX2-positive cells plays an important role in resupplying endothelial cells for vascular remodelling in the anterior lobe.

Histological differentiation of mucus cell subtypes suggests functional compartmentation in the eel esophagus.

We investigated the morphological and histological changes in eel esophagus during the course of freshwater (FW) to seawater (SW) transfer and identified multiple types of mucus cells from tissues that were fixed using Carnoy's solution to retain the mucus structure. The FW esophageal epithelium is stratified and composed of superficial cells, mucus cells, club cells (exocrine cells with a large vacuole), and basal cells. Two types of periodic acid-Schiff (PAS)-positive mucus cells were identified, and they can be further distinguished by the periodic acid-thionin Schiff/KOH/PAS (PAT) method, indicating that C7/9- and C8-sialic acids were produced. Isolectin B4-positive mucus cells were found among the C8-sialic acid-producing cells and located at the tips of the villi at mid-posterior regions of the FW esophagus. The two different muci were immiscible and may form separate layers to protect the tissues from the high osmolality of imbibed SW during early SW acclimation. The densities of club cells and isolectin B4-positive cells decreased after SW acclimation, and cuboidal/columnar epithelial cells subsequently developed for active Na+ and Cl- absorption. Cuboidal/columnar epithelial cells proliferated in scattered array rather than at the bases of the villi, thereby retaining the characteristic of the stratified epithelium. Prominent leukocyte invasion was found at the base of the stratified epithelium at early SW transfer, indicating that the immune system was also activated in response to antigen exposure from imbibed SW. The mucus composition in FW is more complicated than that in SW, fueling further studies for their functions to form unstirred layers as osmoregulatory barriers.

Identification of TGFß-induced proteins in non-endocrine mouse pituitary cell line TtT/GF by SILAC-assisted quantitative mass spectrometry.

TtT/GF is a mouse cell line derived from a thyrotropic pituitary tumor and has been used as a model of folliculostellate cells. Our previous microarray data indicate that TtT/GF possesses some properties of endothelial cells, pericytes and stem/progenitor cells, along with folliculostellate cells, suggesting its plasticity. We also found that transforming growth factor beta (TGFß) alters cell motility, increases pericyte marker transcripts and attenuates endothelial cell and stem/progenitor cell markers in TtT/GF cells. The present study explores the wide-range effect of TGFß on TtT/GF cells at the protein level and characterizes TGFß-induced proteins and their partnerships using stable isotope labeling of amino acids in cell culture (SILAC)-assisted quantitative mass spectrometry. Comparison between quantified proteins from TGFß-treated cells and those from SB431542 (a selective TGFß receptor I inhibitor)-treated cells revealed 51 upregulated and 112 downregulated proteins (|log2| > 0.6). Gene ontology and STRING analyses revealed that these are related to the actin cytoskeleton, cell adhesion, extracellular matrix and DNA replication. Consistently, TGFß-treated cells showed a distinct actin filament pattern and reduced proliferation compared to vehicle-treated cells; SB431542 blocked the effect of TGFß. Upregulation of many pericyte markers (CSPG4, NES, ACTA, TAGLN, COL1A1, THBS1, TIMP3 and FLNA) supports our previous hypothesis that TGFß reinforces pericyte properties. We also found downregulation of CTSB, EZR and LGALS3, which are induced in several pituitary adenomas. These data provide valuable information about pericyte differentiation as well as the pathological processes in pituitary adenomas.

Immunohistochemical Study of the Laminin α5 Chain and Its Specific Receptor, Basal Cell Adhesion Molecule (BCAM), in both Fetal and Adult Rat Pituitary Glands.

Laminin, a major basement membrane protein, comprises three subunit chains: α, ß, and γ chains. Among these chains, only the laminin α chain is capable of signaling via laminin receptors. Although laminin isoforms containing the α5 chain were reported to be the first laminin produced during rat anterior pituitary gland development, the functions of these isoforms are unknown. We used immunohistochemical techniques to localize the laminin α5 chain and its specific receptor, basal cell adhesion molecule (BCAM), in fetal and adult pituitary gland. Laminin α5 chain immunoreactivity was observed in the basement membrane of the primordial adenohypophysis at embryonic days 12.5 to 19.5. Double immunostaining showed that BCAM was present and co-localized with the laminin α5 chain in the tissue. Quantitative analysis showed that the laminin α5 chain and BCAM were expressed in the anterior pituitary gland during postnatal development and in adulthood (postnatal day 60). In the adult gland, co-localization of the laminin α5 chain and BCAM was observed, and BCAM was detected in both the folliculo-stellate cells and endothelial cells. These results suggest that laminin α5 chain signaling via BCAM occurs in both the fetal adenohypophysis and adult anterior pituitary gland.