Saishu Yoshida

PROP1 coexists with SOX2 and induces PIT1-commitment cells

Prophet of PIT1 (PROP1) is a pituitary-specific factor and responsive gene for the combined pituitary hormone deficiency in Ames dwarf mice and human patients. Our immunohistochemical studies demonstrated that PROP1 is consistently expressed in SOX2-expressing stem/progenitor cells in the rat pituitary from embryonic (E) to postnatal periods. At E13.5, all the cells in Rathkes pouch, the primordium of the pituitary, express PROP1. Afterward, PROP1-positive cells localize along the marginal cell layer, a putative stem cell niche in the pituitary, and stratify in the parenchyma of the anterior pituitary. In the embryonic period, PROP1 coexists transiently with PIT1, which is the anterior pituitary-specific factor and is a target of PROP1, but not any hormones. Thus, the present results imply a regulatory role of PROP1 not only in pituitary organogenesis but also in conversion of PIT1-lineage cells.

Significant quantitative and qualitative transition in pituitary stem / progenitor cells occurs during the postnatal development of the rat anterior pituitary.

We reported recently that a pituitary‐specific transcription factor PROP1 is present in SOX2‐positive cells and disappears at the early stage of the transition from progenitor cell to committed cell during the embryonic development of the rat pituitary. In the present study, we examined the localisation and identification of SOX2‐positive and PROP1/SOX2‐positive cells in the neonatal and postnatal rat pituitaries by immunohistochemistry. Quantitative analysis of immunoreactive cells demonstrated that SOX2‐positive pituitary stem/progenitor cells are not only predominantly localised in the marginal cell layer, but also are scattered in the parenchyma of the adult anterior lobe. In the marginal cell layer, the number of PROP1/SOX2‐positive cells significantly decreased after postnatal day 15, indicating that a significant quantitative transition is triggered in the marginal cell layer during the first postnatal growth wave of the anterior pituitary. By contrast, other phenotypes of SOX2‐positive stem/progenitor cells that express S100β appeared in the postnatal anterior pituitary. These data suggested that quantitative and qualitative transition occurs by acquisition of a novel mechanism in terminal differentiation in the postnatal development of the anterior pituitary.

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.

Paired-Related Homeodomain Proteins Prx1 and Prx2 are Expressed in Embryonic Pituitary Stem/Progenitor Cells and May Be Involved in the Early Stage of Pituitary Differentiation

We recently cloned a paired‐related homeodomain protein Prx2 as a novel factor in the pituitary. In the present study, we investigated the ontogenic profiles of Prx2 and another cognate Prx1 in the rat embryonic pituitary. Quantitative real‐time polymerase chain reaction showed low expression of Prx2 and a marked increase of Prx1 on rat embryonic day (E)20.5. Immunohistochemical analyses using an antibody that recognises both proteins, with the aim of investigating their roles in pituitary organogenesis, demonstrated that PRXs first appear in the Rathke’s pouch on E13.5 in the pituitary stem/progenitor cells expressing Prop1 and Sox2. After E16.5, the number of Prx‐expressing cells was increased in both anterior and intermediate lobes. SOX2+ stem/progenitor cells in the intermediate lobe started to produce PRXs, and PRX+/SOX2+/PROP1+‐cells were present on the anterior side of the marginal cell layer and were scattered in the parenchyma of the anterior lobe. On the other hand, PRX+‐cells negative for PROP1 and SOX2 were located in the anterior lobe. Analysis of the relationship with pituitary endocrine cells revealed that a part of PRX+/PROP1−/SOX2−‐cells in the anterior lobe co‐expressed all types of hormones. The proportion of co‐localisation of PRXs and hormones was highest on the day each hormone first appeared. These data indicate that PRXs are produced in the pituitary progenitor cells and may play roles in the process of terminal differentiation during early pituitary organogenesis. An in vitro small interfering RNA‐knockdown experiment in the pituitary‐derived cell line, TtT/GF, revealed that PRX1 and PRX2 play roles in proliferation by different mechanisms because knockdown of Prx2, but not Prx1, induced the p21 expression. Furthermore, immunohistochemical analysis demonstrated that 76% of PRXs+ cells were positive for a cell proliferation marker Ki67 in the E18.5 pituitary. This is the first report of the involvement of PRX1 and PRX2 in organogenesis of tissue originating from the ectoderm other than the mesoderm.

PRRX1 and PRRX2 distinctively participate in pituitary organogenesis and a cell-supply system

Paired-related homeobox transcription factors, PRRX1 and PRRX2, are known to be important factors for craniofacial and limb morphogenesis. We recently cloned Prrx2 from the porcine adult pituitary cDNA library and found that only PRRX1 is present in the rat embryonic pituitary. In this study, we re-investigated the temporospatial expression and localization of PRRX1 and PRRX2 in the rat pituitary throughout life. The persistent expression of Prrx1 was ascertained after the middle stage of embryonic development, whereas significant expression of Prrx2 was found only in the postnatal pituitary. Immunohistochemistry confirmed that PRRX1-positive cells appeared inside the pituitary on embryonic day 16.5 in the marginal cell layer (MCL), a pituitary stem/progenitor cell niche, and the expanding parenchyma of the anterior pituitary. In contrast, PRRX2-positive cells first appeared in the anterior lobe and intermediate lobe sides of the MCL around postnatal day 30 when the postnatal pituitary growth wave had almost terminated. Immunostaining for PRRX1 with a stem/progenitor cell marker SOX2, a pituitary progenitor marker PROP1, or pituitary hormones revealed that PRRX1 localized in cells in the transition process from the multipotent progenitor stage to the early stage of terminal differentiation throughout life. PRRX2 emerged in cells positive for SOX2 but negative for PROP1 in the anterior and intermediate lobe sides of the postnatal MCL. Thus, PRRX1 and PRRX2 might participate distinctly in pituitary organogenesis and the postnatal cell-supply system.

Rapid transition of NESTIN-expressing dividing cells from PROP1-positive to PIT1-positive advances prenatal pituitary development.

We recently reported that the quantitative and qualitative transition of stem/progenitor cells occurs by the acquisition of a novel mechanism in the terminal differentiation during postnatal development of the anterior pituitary. We hypothesised that this novel mechanism is an alteration of a cell supply system accompanying proliferation of the progenitor cells. In the present study, we examined the proliferation activities of progenitor cells by indication of the expression of Nestin, a marker of rapidly dividing progenitor cells, aiming to verify our hypothesis and to resolve another outstanding issue regarding whether the Nestin gene is expressed in the pituitary. We found that NESTIN‐positive dividing cells certainly exist in the pituitary through all stages of development. Almost all of the PROP1‐positive progenitor cells express Nestin in early embryonic pituitary development. Thereafter, Nestin‐expressing dividing cells involved in the cell supply system transfer from PROP1‐positive progenitor cells to committed progenitor cells, such as PIT1‐positive cells, on neonatal pituitary development. Furthermore, our data, together with the findings of previous studies on cell lineage tracing analyses using Nestin‐Cre mice derived by the central nervous system (CNS)‐specific Nestin promoter, suggest that at least two regulation systems for Nestin‐expression exist in the pituitary, with the majority of these not being CNS‐specific.

Temporospatial gene expression of Prx1 and Prx2 is involved in morphogenesis of cranial placode-derived tissues through epithelio-mesenchymal interaction during rat embryogenesis

Paired-related homeobox transcription factors, PRX1 and PRX2, are verified to play essential roles in limb, heart and craniofacial development by analyses of knockout animals. Their gene expression in the embryonic primordia derived from the mesoderm and neural crest is confirmed by in situ hybridization. Nevertheless, a detailed localization of PRX1 and PRX2 was not carried out because of a lack of specific antibodies for each factor. We have previously confirmed the presence of PRX proteins in rat embryonic pituitary by using an antibody that recognizes both PRX1 and PRX2. However, the pituitary originates in the cranial placodes, not the mesoderm or neural crest. In this study, we analyze the temporospatial distribution of PRX1 and PRX2 with novel antibodies specific for each factor, together with a stem/progenitor marker SOX2 (sex-determining region Y-box 2) in the primordia formed by epithelio-mesenchymal interaction. We observe immunoreactive signals of both PRX proteins in rat embryo, showing a similar pattern to that obtained by in situ hybridization. In early embryogenesis, PRX proteins are not co-localized with SOX2 but PRX2 and/or PRX1-positive cells are present in the border or periphery of SOX2-positive primordia originating in the cranial placode. During advanced embryogenesis, either PRX2-positive cells become condensed in the border of SOX2-positive cells or PRX1 and/or PRX2 become co-localized with SOX2. Our results suggest that PRX proteins, especially PRX2, play a role in the morphogenesis of the primordial tissues formed by the epithelio-mesenchymal interaction and that neural crest cells contribute to the morphogenesis of tissues derived from the cranial placode.

Regulatory System for Stem/Progenitor Cell Niches in the Adult Rodent Pituitary

The anterior lobe of the pituitary gland is a master endocrine tissue composed of five types of endocrine cells. Although the turnover rate of pituitary endocrine cells is as low as about 1.6% per day, recent studies have demonstrated that Sex-determining region Y-box 2 (SOX2)+-cells exist as pituitary stem/progenitor cells in the adult anterior lobe and contribute to cell regeneration. Notably, SOX2+-pituitary stem/progenitor cells form two types of niches in this tissue: the marginal cell layer (MCL-niche) and the dense cell clusters scattering in the parenchyma (parenchymal-niche). However, little is known about the mechanisms and factors for regulating the pituitary stem/progenitor cell niches, as well as the functional differences between the two types of niches. Elucidation of the regulatory mechanisms in the niches might enable us to understand the cell regeneration system that acts in accordance with physiological demands in the adult pituitary. In this review, so as to reveal the regulatory mechanisms of the two types of niche, we summarize the regulatory factors and their roles in the adult rodent pituitary niches by focusing on three components: soluble factors, cell surface proteins and extracellular matrixes.

Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland

S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-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 S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β 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 S100β-positive cells by means of their property of adhering to laminin.

PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are involved in vasculogenesis during rat embryonic pituitary development

We have recently shown that cells positive for the paired-related homeobox transcription factors PRRX1 and PRRX2 occur in the rat pituitary, and that they are derived from two different origins: pituitary-derived cells positive for stem cell marker SOX2 and extra-pituitary-derived cells negative for SOX2. In this study, we have further characterized the PRRX1- and PRRX2-positive cells that originate from extra-pituitary cells. Immunohistochemical analyses were performed with specific antibodies against PRRX1 and PRRX2 in order to clarify their roles in pituitary vasculogenesis. PRRX1- and PRRX2-positive cells were found in Atwell’s recess and at the periphery of the pituitary on embryonic day 15.5 (E15.5). Several PRRX1-positive cells then invaded the anterior lobe, together with a few PRRX2-positive cells, on E16.5. Some PRRX1-positive cells were also positive for mesenchymal stem cell marker NESTIN. Moreover, some PRRX1/NESTIN double-positive cells showed characteristics of vascular endothelial cells with an Isolectin-B4-binding capacity. PRRX1 co-localized with vascular smooth muscle cell/pericyte marker α-smooth muscle actin in the deep area of Atwell’s recess. We confirmed the presence of PRRX2/NESTIN double-positive cells at an entry area in Atwell’s recess and at the periphery of the pituitary, but PRRX2 did not co-localize with Isolectin B4 or α-smooth muscle actin. These data suggest that PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are present at the periphery of the embryonic pituitary and at the entry from Atwell’s recess and participate in pituitary vasculogenesis by differentiation into vascular endothelial cells and pericytes, whereas the presence of PRRX2 indicates much higher stemness than PRRX1.