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 S100β-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 S100β-positive cells, we performed immunohistochemical analysis using several markers in S100β/GFP-TG rats, which express GFP in S100β-expressing cells under control of the S100β 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 S100β-positive cells of extrapituitary origin invade the anterior lobe, undergoing proliferation and diverse transformation during pituitary organogenesis.
Citation: Horiguchi K, Yako H, Yoshida S, Fujiwara K, Tsukada T, Kanno N, et al. (2016) S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland. PLoS ONE 11(10): e0163981. https://doi.org/10.1371/journal.pone.0163981
Editor: Wenhui Hu, Temple University School of Medicine, UNITED STATES
Received: June 23, 2016; Accepted: September 16, 2016; Published: October 3, 2016
Copyright: © 2016 Horiguchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information file.
Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The adenohypophysis, which is composed of anterior and intermediate lobes, develops through invagination of the oral ectoderm under the influence of several growth factors by contacting the diencephalon and both sides of the ectoderm [1–3]. Both the anterior and intermediate lobes contain six types of differentiated cells that play important roles in the synthesis and secretion of several hormones. These endocrine cells are required in all vertebrates for the maintenance of vital functions such as reproduction, metabolism, growth, and homeostasis. Additionally, substantial populations of non-hormone-producing cells exist in the anterior and intermediate lobes and participate in maintaining, assisting, and supplementing hormone-producing cells and the vessel system. For quite some time, the non-endocrine cells that have attracted the most attention are folliculo-stellate (FS) cells, which have a star-like shape . S100β, a Ca2+-binding protein, is a marker for FS cells. S100β-positive cells in the anterior lobe are believed to have several roles, acting as stem cells, phagocytes, cells that regulate hormone release, and cells that participate in cell-cell communication [5–7].
Recently accumulated data indicate that S100β-positive cells are composed of heterogeneous cell populations that are relevant to several functions. Immunohistochemical analysis with stem/progenitor cell markers revealed that S100β-positive cells are composed of at least three groups of cells . S100β-positive cells can also be grouped into two cell types based on their adhesiveness to the extracellular matrix: stellate-shaped cells and dendritic-like cells . As postulated previously, some S100β-positive cells have the ability to differentiate into skeletal muscle cells [10–12]. More recently, we have reported that some S100β-positive cells are able to differentiate into all hormone-producing cell types in the anterior and intermediate lobes . Despite these new findings, it is not yet clear how S100β-positive cells originate and develop into plural states with diverse roles.
Facilitating further investigation of the roles of S100β-positive cells, a transgenic rat that expresses green fluorescent protein (GFP) under the control of the S100β promoter (S100β/GFP-TG rat) has been generated . Using the S100β/GFP-TG rat, we observed that S100β transcripts are present in the embryonic pituitary on embryonic day 21.5 (E21.5) , though it was previously believed that S100β-positive cells do not appear until approximately ten days after birth . In the present study, we examined the appearance of S100β-positive cells in the embryonic pituitary and their characteristics via immunohistochemistry using several marker proteins. As a result, we observed that S100β/GFP-positive cells are present in the prenatal pituitary, appearing by migration from Atwell's recess, an intraglandular fossa that receives several blood vessels . These cells are present with mesenchymal cells and other cell types that surround the pituitary gland. They exhibit proliferative activity and co-expression with several markers of vessels or neural crest cells, and they reflect transient, multipotent, and migratory characteristics. Thus, our results suggest that some S100β-positive cells are extrapituitary in origin and partially participate in vasculogenesis and formation of the pituitary gland.
Materials and Methods
All animal experiments were performed following approval from the Institutional Animal Experiment Committee of Meiji University (IACUC 14–0012) and Jichi Medical University (No. 13004 and 14051) and were conducted in accordance with the Institutional Regulations of Animal Experiments and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Japanese Ministry of Education, Culture, Sports, Science and Technology. All treatments were performed under deep anesthesia and all efforts were made to minimize suffering. All rats did not become severely ill or died at any time prior to the experimental endpoint. Rats were sacrificed by exsanguination from the right atrium under deep pentobarbital anesthesia (40mg/kg) and then perfused with 4% paraformaldehyde in 0.05 M phosphate buffer (pH 7.4) for experiments.
S100β/GFP-TG rats  that express GFP under control of the promoter for the S100βgene, a marker of FS cells, were provided by Professor K. Inoue of Saitama University and bred in our laboratory. Male rats 8–10 weeks old weighing 250–300 g were provided with ad libitum access to food and water and housed under conditions of 12 h light and 12 h darkness.
Heads of S100β/GFP-TG embryonic rats on E15.5, E18.5, E19.5, and E20.5 were fixed in 4% paraformaldehyde buffered with 0.05 M phosphate buffer (PB; pH 7.4) for 20–24 h at 4°C, followed by immersion for more than two days in PB (pH 7.2) containing 30% sucrose at 4°C. This was followed by embedding the samples in optimum cutting temperature (O.C.T.) compound (Sakura Finetek Japan, Tokyo, Japan) at -80°C before sectioning. Frozen sections 10 μm thick in the sagittal plane were mounted on glass slides (Matsunami, Osaka, Japan). Antigen retrieval was performed with an Immunosaver (Nisshin EM, Tokyo, Japan) for 60 min at 80°C. Slides were then washed in 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (pH 7.5) containing 100 mM NaCl (HEPES), followed by blocking in HEPES containing 0.4% Triton X100 and 10% fetal bovine serum (FBS) or 1% bovine serum albumin (BSA). Primary antibodies, antisera, and lectin were reacted overnight at 4°C. The primary antibodies used were chicken immunoglobulin (Ig) Y against GFP (1:250 dilution, Aves Labs, Inc., Tigard, OR, USA), rabbit IgG against cow S100β (1:1000 dilution, Dako, Glostrup, Denmark), rabbit antiserum against rat PRRX1 (1:1,000 dilution, raised in our laboratory and assessed for specificity) , rabbit antiserum against rat PRRX2 (1:1,000 dilution, raised in our laboratory and assessed for specificity) , goat IgG against stem/progenitor cell marker human SOX2 (1:500 dilution, Neuromics, Edina, MN, USA), mouse monoclonal antibody against neural crest cell marker rat p75 (1:100 dilution, Abcom, Plc., Cambridge, UK), mouse monoclonal antibody against smooth muscle cell marker human α-SMA (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse antiserum against pericyte and a neural and mesenchymal stem/progenitor cell marker rat NESTIN (1:250 dilution, BD Biosciences, San Jose, CA, USA), mouse monoclonal antibody against mesenchymal cell marker pig VIMENTIN (1:10,000 dilution, Sigma-Aldrich Corp., St. Louis, MO, USA) and rabbit antibody against dividing cell marker human Ki67 (1:500 dilution, Abcom, Plc.). Afterwards, a cocktail of the following guinea pig antisera against pituitary hormones was used: anti-rat LHβ (1:8,000 dilution), anti-rat FSHβ (1:4,000 dilution), anti-rat TSHβ (1:16,000 dilution), and anti-rat PRL (1:4,000 dilution). The antisera were provided by the National Institute of Diabetes and Digestive and Kidney Disease, courtesy of Dr. A. F. Parlow. Guinea pig anti-human ACTH (1:8,000 dilution) and anti-human GH (1:1,000 dilution) antisera were provided by Dr. S. Tanaka, Shizuoka University, and isolectin B4-conjugated Dyelight 649 (1:100 dilution) was provided by Vector Laboratories (Burlingame, CA, USA). Sections were washed with HEPES, followed by incubation with secondary antibodies, performed with Cy3- or Cy5-conjugated AffiniPure donkey with anti-goat, -mouse, -rabbit, and -guinea pig IgG and fluorescein isothiocyanate (FITC)-conjugated AffiniPure donkey with anti-chicken IgY (1:500 dilution, Jackson ImmunoResearch, West Grove, PA, USA). The sections were again washed with HEPES and then enclosed in VECTASHIELD Mounting Medium with 4',6-diamino-2-phenylindole (DAPI; Vector Laboratories) to stain nuclei. Immunofluorescence was observed under fluorescence microscopy with a BZ-9000 (KEYENCE, Osaka, Japan) and fluorescence confocal microscopy with a FV1000 (OLYMPUS, Tokyo, Japan).
Appearance of GFP-positive cells at Atwell's recess on E15.5
We first examined whether GFP-positive cells were present in the embryonic pituitary on E15.5 by staining for GFP. As shown in Fig 1, GFP-positive cells were observed at Atwell’s recess, while very strong GFP signals were observed beneath the pituitary gland (Fig 1Aʹ). The recess is characterized as an intraglandular fossa that receives several blood vessels ; we have previously suggested that PRRX1-positive cells are present here and invade in order to participate in pituitary vasculogenesis [18,19]. To further characterize the GFP-positive cells, we performed triple immunostaining for GFP, PRRX1, and SOX2. In the enlarged images (Fig 1B–1Bʹʹʹ), it is clear that GFP-positive and PRRX1-positive cells do not overlap, while SOX2-positive cells were not present at the recess, in the brain, or in the anterior pituitary gland (Fig 1Aʹʹ–1Bʹʹ). We analyzed the ratios of GFP- and PRRX1-positive cells to the total number of cells in Atwell’s recess, counted by DAPI staining. GFP-positive cells accounted for approximately 5.7% of cells, while PRRX1-positive cells accounted for 69.8% (Fig 1C).
Using sagittal sections of embryonic pituitaries on E15.5, immunostaining was performed for GFP, PRRX1, and SOX2, which were visualized with FITC (A and B, green), Cy3 (Aʹ and Bʹ, red), and Cy5 (Aʹʹ and Bʹʹ, white), respectively. Cells positive for GFP only are indicated with yellow arrows. The boxed area in Aʹʹʹ is enlarged in B–Bʹʹʹ. Each cell type was counted in four independent pituitaries (n = 4), and results are listed in C. AL anterior lobe; IL intermediate lobe; PL posterior lobe; AR Atwell's recess. Bars = 50 μm (A–Aʹʹʹ) and 10 μm (B–Bʹʹʹ).
GFP-positive cells during pituitary development
We performed the same histochemical analysis for the late embryonic stages (Fig 2). Enlarged images of the rostral part of the anterior pituitary on E18.5 and E19.5 reveal the presence of GFP-single (Fig 2D–2Dʹʹʹ, yellow arrow), PRRX1-single (Fig 2B–2Bʹʹʹ, white open arrowhead), and GFP/PRRX1-double positive cells (Fig 2B–2Bʹʹʹ and 2D–2Dʹʹʹ, yellow open arrowheads). Cells were counted in the anterior and intermediate lobes of four sections each on E18.5, E19.5, and E20.5 (image data not shown), as listed in Fig 2F. Results showed that in the anterior lobe, GFP-single and GFP/PRRX1-double positive cells were present at low frequencies (1.2–2.3%), while PRRX1-single positive cells were more prevalent (18.9–19.8%; Fig 2F). SOX2-positive cells were negative for GFP, and the frequency of SOX2/PRRX1-double cells was 10.7–15.5% (Fig 2F). In the intermediate lobe, GFP/SOX2/PRRX1-triple positive cells (Fig 2E–2Eʹʹʹ) were observed, but GFP- and/or PRRX1-single and -double positive cells were absent on E19.5 and E20.5 (Fig 2F).
Using sagittal sections of embryonic pituitaries on E18.5 (A–Bʹʹʹ) and E19.5 (C–Eʹʹʹ), immunostaining was performed for GFP, PRRX1, and SOX2, which were visualized with FITC (green), Cy3 (red), and Cy5 (white), respectively. GFP/PRRX1/SOX2-triple (yellow arrowheads), GFP/PRRX1-double (yellow open arrowheads), PRRX1/SOX2-double (white arrowheads), GFP-single (yellow arrows) and PRRX1-single (white open arrowheads) positive cells are indicated. The boxed areas in A and C are enlarged in B–Bʹʹʹ, D–Dʹʹʹ, and E–Eʹʹʹ. Each cell type was counted (n = 1 for E18.5, n = 2 for E19.5, and n = 2 for E20.5, with four slices each), and results are shown in F. AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm (A, C) and 10 μm (B–Bʹʹʹ and D–Eʹʹʹ).
We have previously shown that PRRX2, a cognate of PRRX1, is expressed in the mesenchyme cells surrounding the pituitary gland . Triple immunostaining for GFP, SOX2, and PRRX2 was performed on E20.5. As shown in S1 Fig, PRRX2-positive cells were not observed in the pituitary gland, but they were present in the surrounding mesenchyme, especially outside the posterior lobe. GFP/PRRX2-double positive cells, as well as GFP- and PRRX2-single positive cells (S1B–S1Bʹʹʹ Fig), were observed in the caudal and dorsal area outside of the pituitary. As PRRX2 was not present in the pituitary, we did not include PRRX2 in further staining experiments.
Proliferative ability of GFP-positive cells
Immunohistochemical analysis of Ki67, a cell division marker, was performed together with GFP staining to verify the proliferative activity of GFP-positive cells on E20.5. There were GFP-positive cells that were also positive for Ki67, and these were small and elongated (Fig 3). In addition, a number of GFP-positive cells in the intermediate lobe were obviously positive for Ki67. A count of the number of immunopositive cells in the anterior lobe showed that a quarter of GFP-positive cells were also positive for Ki67 (Fig 3C).
Double immunostaining for GFP (green) and Ki67 (red) was performed on a section on E20.5. The boxed area in A is enlarged in B–Bʹʹ. GFP/Ki67-double positive (yellow open arrowheads) and GFP-single positive (yellow arrow) cells are indicated. Each cell type was counted in the anterior lobe (n = 2, with one slice each), and results are shown in C. AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm (A) and 10 μm (B–Bʹʹ).
Absence of pituitary hormones in GFP-positive cells
We recently demonstrated that a subset of S100β-positive cells prepared from adult rat anterior lobes differentiate into hormone-producing cells . To examine whether GFP-positive cells colocalize with pituitary hormones, we used a cocktail of antibodies against LHβ, FSHβ, PRL, TSHβ, ACTH, and GH (HORMONES, Fig 4). Colocalization of HORMONES with GFP-positive cells was not observed (Fig 4B–4Bʹʹʹ), while HORMONES/SOX2-double positive cells were present in the anterior (Fig 4B–4Bʹʹʹ, white arrowhead) and intermediate lobes (Fig 4C–4Cʹʹʹ, white arrowhead). In addition, GFP/SOX2-double positive cells were present in the intermediate lobes (Fig 4C–4Cʹʹʹ, yellow open arrowhead).
Triple immunostaining using a section from E20.5 was performed for GFP (green), SOX2 (red), and pituitary hormones (white) using a cocktail of antibodies against the hormones FSHβ, LHβ, prolactin, TSHβ, ACTH, and GH (HORMONES). The boxed area in A is enlarged in B–Bʹʹʹ and C–Cʹʹʹ. GFP/SOX2-double positive (yellow open arrowheads), SOX2/HORMONES-double positive (white arrowheads), GFP-single positive (yellow arrows), and HORMONES-single positive (white open arrowheads) cells are indicated. AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm (A) and 10 μm (B–Cʹʹʹ).
Characterization of non-endocrine GFP-positive cells
Several studies to date have postulated that extrapituitary cells, which are non-pituitary in origin, such as neural and mesenchymal stem/progenitor cells and vessel precursor cells, are present in the pituitary gland [18–20]. GFP-positive cells can be clearly seen in Atwell’s recess but not in the anterior pituitary at E15.5 (Fig 1B). To further characterize GFP-positive cells, histochemical analysis using several cell markers was performed as follows.
First, as indicated in Fig 5, immunohistochemistry for p75, a neural crest cell marker , together with staining for GFP and PRRX1 showed the presence of GFP/p75/PRRX1-triple and GFP/Ki67-double positive cells, as well as p75/PRRX1-double positive cells. Notably, the p75-positive cells were elongated in shape (Fig 5B–5Bʹʹʹ). Results revealed that 41.2% of GFP-positive cells were GFP/Ki67-double positive cells (Fig 5C).
Triple immunostaining for GFP (green), the neural crest marker p75 (white), and PRRX1 (red) was performed on E20.5. The boxed area in A is enlarged in B–Bʹʹʹ. GFP/p75/PRRX1-triple (yellow arrowheads), GFP/p75-double (yellow open arrowheads), and p75/PRRX1-double (white open arrowheads) positive cells are indicated. Counts of each cell type (n = 2, with two slices each) is listed in C. AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm (A) and10 μm (B–Bʹʹʹ).
Immunohistochemical analysis of NESTIN, a neural and mesenchymal stem/progenitor cell marker [22–25], together with GFP and PRRX1 staining revealed the presence of GFP/NESTIN/PRRX1-triple positive and GFP/PRRX1-double positive cells (Fig 6). Approximately 8.0% of the NESTIN-positive cells were also positive for GFP (Fig 6C). Then, VIMENTIN, a mesenchymal progenitor cell marker , was immunostained together with GFP and PRRX1. The results revealed a heterogeneous population of VIMENTIN-positive cells, with GFP/VIMENTIN/PRRX1-triple positive cells, VIMENTIN/PRRX1-double positive cells, and VIMENTIN-single positive cells (Fig 6E–6Eʹʹʹ). Approximately 15.5% of the VIMENTIN-positive cells were also positive to GFP (Fig 6F).
Triple immunostaining for GFP (green), PRRX1 (red), and NESTIN or VIMENTIN (white) was performed on E18.5. Merged images are shown on the right. The boxed areas in Aʹʹʹ and Dʹʹʹ are enlarged in B–B‴ and E-E‴, respectively. GFP/PRRX1/NESTIN- and GFP/PRRX1/VIMENTIN-triple positive, GFP/PRRX1- and GFP/VIMENTIN-double positive, PRRX1/NESTIN- and PRRX1/VIMENTIN-double positive, and NESTIN- or VIMENTIN-single positive cells are indicated. Each cell type was counted, and the results are listed in C (n = 2 with two slices each) and F (n = 1 with four slices). AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm (Aʹʹʹ and Dʹʹʹ) and 10 μm (Bʹʹʹ and Eʹʹʹ).
More recently, we have demonstrated that PRRX1-positive mesenchymal cells invade through Atwell's recess during pituitary vasculogenesis [18,19]. To further verify the correlation between GFP-positive cells and blood vessels, we performed a histochemical analysis with fluorescence-labeled isolectin B4, a marker of vascular endothelial cells. A few isolectin B4-positive cells were observed in Atwell’s recess and the region surrounding the pituitary gland, but none were observed in the anterior lobe on E15.5 (Fig 7). Notably, GFP/isolectin B4-double positive cells were present at Atwell's recess, in addition to GFP-single and isolectin B4-single positive cells (Fig 7B–7Bʹʹ). GFP-positive and isolectin B4-positive cells were visible in the parenchyma of the anterior pituitary on E18.5 (Fig 7D–7Dʹʹ). GFP-positive cells were likely to enter into the anterior lobe, and some were positive for isolectin B4. Each cell type was then counted (Table 1). The frequency of GFP/isolectin B4-double positive cells in the parenchyma of the anterior lobe on E18.5 (9/26, 34.6%) was slightly higher than that in Atwell’s recess on E15.5 (6/38, 15.8%). In contrast, the frequency of total isolectin B4-positive cells decreased, from 18.0% (38/208) in Atwell’s recess on E15.5 to 10.6% (26/246) in the parenchyma of the anterior lobe on E18.5 (Table 1), reflecting the progress of cell differentiation and scattering in the parenchyma.
Using sagittal sections of embryonic pituitaries on E15.5 (A–Bʹʹ), E18.5 (C–Dʹʹ), and E20.5 (E–Hʹʹ), immunostaining was performed on four slices for GFP and endothelial cell marker isolectin B4 and was visualized with FITC (green) and Cy5 (white), respectively. Merged images are shown on the right. The boxed areas in Aʹʹ, Cʹʹ, and Eʹʹ are enlarged in B–Bʹʹ, D–Dʹʹ, and F–Fʹʹ. GFP/isolectin B4-double (yellow arrowheads), GFP-single (yellow arrows), and isolectin B4-single (open white arrowheads) cells are indicated. The boxed area in Fʹʹ is enlarged in G–Gʹʹ, and its orthogonal projections were analyzed by confocal Z-stack imaging with 0.1-μm slices (H–Hʹʹ). AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm (Aʹʹ, Cʹʹ, Eʹʹ, and Gʹʹ) and 10 μm (Bʹʹ, Dʹʹ, Fʹʹ, and Hʹʹ).
Immunohistochemical analysis of DESMIN, a marker of immature and mature pericytes , was performed together with analysis of GFP and isolectin B4 (Fig 8). DESMIN-positive cells were observed along with isolectin B4-positive cells, some of which colocalized with GFP. In addition, DESMIN/isolectin B4-double and DESMIN-single positive cells were observed. GFP-positive cells were also positive for DESMIN at a frequency of 40.0% (6/15) (Fig 8C). Finally, immunohistochemical analysis of α-SMA, an early vascular marker present in vascular smooth muscle cells and pericytes , was performed. As shown in Fig 8D and 8E, a small number of α-SMA-positive cells was observed and were negative for GFP. PRRX1-positive cells were negative for α-SMA.
Using sagittal sections of embryonic pituitaries on E18.5, staining for vascular endothelial cells with isolectin B4 (Aʹ) or α-SMA (Dʹ) was performed by visualization with Cy5 (white), together with staining of GFP (FITC; green, A and B) and the pericyte marker DESMIN or PRRX1 (Cy3; red, Aʹʹ and Bʹʹ). GFP/DESMIN-double (yellow open arrowheads), DESMIN/isolectin B4-double (white open arrowheads), GFP-single (yellow arrows), and DESMIN- or α-SMA-single (white arrowheads) positive cells are indicated. Merged images (Aʹʹʹ, Bʹʹʹ, Dʹʹʹ, and Eʹʹʹ) are shown on the right. Each cell type (n = 2 with two slices each) was counted, and results are listed in C and E. AL anterior lobe; IL intermediate lobe; PL posterior lobe. Bars = 50 μm (Aʹʹʹ and Dʹʹʹ) and 10 μm (Bʹʹʹ and Eʹʹʹ).
S100β-positive cells play special roles as non-endocrine cells in the anterior lobe of the pituitary gland. In the present study, we examined how S100β-positive cells arrive in the embryonic pituitary. Thus, we have demonstrated for the first time that extrapituitary S100β-positive cells exhibit diverse characteristics, such as those typical of vascular cells, mesenchymal cells, and neural crest cells. They also exhibit proliferation activity and invade the embryonic anterior lobe of the pituitary gland.
The S100β protein is often used as a tumor marker, and it is believed to exhibit diverse biological functions [29–31]. S100β has attracted attention owing to its characteristic presence in non-endocrine cells involved in various pituitary functions. Since the first observation of S100β in the anterior pituitary , many studies have suggested that S100β-positive cells play several distinct roles, such as being involved in phagocytosis, cell-cell communication, hormone release, and the maintenance of cell resources as stem/progenitor cells [5–7]. S100β-positive cells in the anterior pituitary can be grouped into three main types: astrocyte-like cells expressing glial fibrillary acidic protein and/or vimentin , epithelial cell-like cells expressing keratin , and dendritic cell-like cells expressing interleukin-6 [9,35–37]. This might suggest the presence of heterogeneous lineages of S100β-positive cells. Recently, we showed that a subset of S100β-positive cells has the ability to differentiate into hormone-producing cells [12,13], consistent with previous indications . The generation of S100β-positive cells from SOX2-positive cells has been demonstrated using genetic lineage tracing . These studies were conducted with postnatal pituitaries, as it was believed that S100β-positive cells appear approximately ten days after birth . However, our previous study revealed the presence of S100β transcripts in the embryonic pituitary , indicating that S100β-positive cells are already present in the embryonic pituitary. Here, we demonstrated that S100β-positive cells at Atwell's recess and in the embryonic anterior lobe are SOX2-negative, differing from SOX2-lineage S100β-positive cells . These appear by extrapituitary invasion with other mesenchymal cells.
The oral ectoderm, a pituitary primordium, originates from the thickened epithelium of an early neural primordium, the cranial placode of neural plate origin [40,41]. However, our recent results suggest that non-neural-plate originating cells positive for PRRX1 and PRRX2 appear in this tissue during organogenesis [18,19,42]. PRRX1 (also known as MHox) and PRRX2 (also known as S8) are known as mesenchymal markers and modulate, as well as act as, stem/progenitor cells [43–45]. We previously suggested that mesenchymal cells positive for PRRX1, PRRX2, and NESTIN are involved in pituitary vasculogenesis [18,19]. In the present study, we observed that S100β-positive cells are first negative for PRRX1 at Atwell's recess but are later positive for it in the anterior lobe, exhibiting transdifferentiation. Notably, Krylyshkina et al.  reported that some NESTIN-positive cells exhibit pericyte phenotypes and are sporadically positive for S100, exhibiting progenitor characteristics. Some S100β-positive cells were positive for NESTIN or VIMENTIN, which are known to indicate plasticity. Indeed, S100β/PRRX1-positive and S100β-positive cells are similar to vascular cells that are isolectin B4- and DESMIN-positive. However, S100β-positive cells are negative for α-SMA, indicating that a different cell lineage is responsible for generating smooth muscle cells. Accordingly, some S100β-positive cells may participate in vasculogenesis by transdifferentiation.
In the present study, we observed that some PRRX1- and S100β-positive cells are also positive for p75, exhibiting an elongated cell shape similar in appearance to vessels differentiating into pericytes and smooth muscle cells in the anterior lobe (Fig 5). p75 is a receptor for neurotrophin and is known as a neural crest marker . Two decades ago, Borson et al. (1994) reported comparative data that showed that p75-positive cells are present in surrounding mesenchymal cells and blood vessels in the developing macaque pituitary . These observations provide intriguing and suggestive insights for understanding pituitary organogenesis, since the neural crest, now referred to as the fourth germ layer in vertebrates, originates from the border area between the neural plate and non-neural ectoderm. This is followed by delamination and an epithelial-mesenchymal transition (EMT) to produce diverse cell lineage derivatives of the neural crest that then invade several tissues during the embryonic period [48–50]. These derivative lineages include pericytes, smooth muscle cells  and S100β-positive cells [52,53], the latter of which were observed in the present study. More recently, the involvement of neural crest cells in pituitary vasculogenesis has been reported . Motohashi et al. (2014) revealed that neural crest-derived cells sustain their multipotency even after entry into their target tissues . It should also be mentioned that the reverse transition from mesenchyme to epithelium includes the acquisition of stemness  and that neural crest-derived Schwann cells can be reprogrammed to acquire multipotency . The role of neural crest lineage cells and their plasticity in the anterior lobe remain interesting subjects of study.
We previously showed that various types of cells invade into the pituitary gland, in particular S100β-positive cells with differentiation and proliferation abilities , confirming and exploring in further detail the previous results that extrapituitary lineage cells invade the anterior lobe . A previous study that revealed the importance of direct contact between the pituitary primordium and surrounding ventral diencephalon, mesenchyme tissue, and notochord  suggested the partial invasion of surrounding cells, in addition to signals promoting growth and differentiation. In future studies, we intend to investigate whether S100β-positive and other extrapituitary cells maintain their plasticity and/or acquire stemness in the anterior lobe. To accomplish this, lineage tracing of S100β-positive cells will be required.
S1 Fig. Triple-immunostaining for GFP, PRRX2, and SOX2.
Triple-immunostaining for GFP (green), PRRX2 (red), and SOX2 (white) is shown in sections on E20.5. Merged images are shown on the right. The boxed area in Aʹʹʹ is enlarged in B–Bʹʹʹ. GFP/PRRX2-double positive (yellow arrowheads), GFP-single positive (yellow arrows) and PRRX2-single positive (white arrowheads) cells are indicated. IL intermediate lobe; PL posterior lobe. Bars = 50 μm (Aʹʹʹ) and 10 μm (Bʹʹʹ).
- Conceptualization: KH YK.
- Data curation: KH HY KF TT TK YK.
- Formal analysis: HY SY KF TT NK HU HN TY TK YK.
- Funding acquisition: TK YK.
- Investigation: KH HY SY KF TT NK HU HN TK YK.
- Methodology: KH HY SY KF TT NK HU HN TY TK YK.
- Project administration: KH TY YK.
- Resources: KH TY YK.
- Supervision: TY TK YK.
- Validation: KH HY SY KF TT NK HU HN TY TK YK.
- Visualization: HY SY KF TT NK HU HN.
- Writing – original draft: KH HY TK YK.
- Writing – review & editing: KH HY SY KF TT TK YK.
- 1. Zhu X, Rosenfeld MG (2004) Transcriptional control of precursor proliferation in the early phases of pituitary development. Curr Opin Genet Dev 14(5): 567–574. pmid:15380249
- 2. Davis SW, Castinetti F, Carvalho LR, Ellsworth BS, Potok MA, Lyons RH et al. (2010) Molecular mechanisms of pituitary organogenesis: in search of novel regulatory genes. Mol Cell Endocrinol 323(1): 4–19. pmid:20025935
- 3. Vankelecom H (2012) Pituitary stem cells drop their mask. Curr Stem Cell Res Ther 7(1): 36–71. pmid:22023621
- 4. Rinehart JF, Farquhar MG (1953) Electron microscopic studies of the anterior pituitary gland. J Histochem Cytochem 1(2): 93–113. pmid:13035080
- 5. Inoue K, Couch EF, Takano K, Ogawa S (1999) The structure and function of folliculo-stellate cells in the anterior pituitary gland. Arch Histol Cytol 62(3): 205–218. pmid:10495875
- 6. Fauquier T, Guerineau NC, McKinney RA, Bauer K, Mollard P (2001) Folliculostellate cell network: a route for long-distance communication in the anterior pituitary. Proc Natl Acad Sci USA 98(15): 8891–8896. pmid:11438713
- 7. Allaerts W, Vankelecom H (2005) History and perspectives of pituitary folliculo-stellate cell research. Eur J Endocrinol 153(1): 1–12. pmid:15994739
- 8. Yoshida S, Kato T, Yako H, Susa T, Cai LY, Osuna M et al. (2011) Significant quantitative and qualitative transition in pituitary stem/progenitor cells occurs during the postnatal development of the rat anterior pituitary. J Neuroendocrinol 23(10): 933–943. pmid:21815952
- 9. Horiguchi K, Fujiwara K, Yoshida S, Higuchi M, Tsukada T, Kanno N et al. (2014) Isolation of dendritic cell-like S100β-positive cells in rat anterior pituitary gland. Cell Tissue Res 357(1): 301–308. pmid:24737488
- 10. Inoue K, Taniguchi Y, Kurosumi K (1987) Differentiation of striated muscle fibers in pituitary gland grafts transplanted beneath the kidney capsule. Arch Histol Jpn 50(5): 567–578. pmid:3439851
- 11. Mogi C, Miyai S, Nishimura Y, Fukuro H, Yokoyama K, Takaki A et al. (2004) Differentiation of skeletal muscle from pituitary folliculo-stellate cells and endocrine progenitor cells. Exp Cell Res 292(2): 288–294. pmid:14697336
- 12. Osuna M, Sonobe Y, Itakura E, Devnath S, Kato T, Kato Y et al. (2012) Differentiation capacity of native pituitary folliculostellate cells and brain astrocytes. J Endocrinol 213(3): 231–237. pmid:22434586
- 13. Higuchi M, Kanno N, Yoshida S, Ueharu H, Chen M, Yako H et al. (2014) GFP-expressing S100β-positive cells of the rat anterior pituitary differentiate into hormone-producing cells. Cell Tissue Res 357(3): 767–779. pmid:24842050
- 14. Itakura E, Odaira K, Yokoyama K, Osuna M, Hara T, Inoue K (2007) Generation of transgenic rats expressing green fluorescent protein in S-100beta-producing pituitary folliculo-stellate cells and brain astrocytes. Endocrinology 148(4): 1518–1523. pmid:17234709
- 15. Soji T, Herbert DC (1989) Intercellular communication between rat anterior pituitary cells. Anat Rec 224(4): 523–533. pmid:2782632
- 16. Daikoku S, Kawano H, Abe K, Yoshinaga K (1981) Topographical appearance of adenohypophysial cells with special reference to the development of the portal system. Arch Histol Jpn 44(2): 103–116. pmid:6797381
- 17. Higuchi M, Kato T, Chen M, Yako H, Yoshida S, Kanno N et al. (2013) Temporospatial gene expression of Prx1 and Prx2 is involved in morphogenesis of cranial placode-derived tissues through epithelio-mesenchymal interaction during rat embryogenesis. Cell Tissue Res 353(1): 27–40. pmid:23644741
- 18. Susa T, Kato T, Yoshida S, Yako M, Higuchi M, Kato Y (2012) 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. J Neuroendocrinol 24(9): 1201–1212. pmid:22577874
- 19. Higuchi M, Yoshida S, Ueharu H, Chen M, Kato T, Kato Y (2015) PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are involved in vasculogenesis during rat embryonic pituitary development. Cell Tissue Res 361(2): 557–565. pmid:25795141
- 20. Couly GF, Le Douarin NM (1987) Mapping of the early neural primordium in quail-chick chimeras. II. The prosencephalic neural plate and neural folds: implications for the genesis of cephalic human congenital abnormalities. Dev Biol 120(1): 198–214. pmid:3817289
- 21. Bixby S, Kruger GM, Mosher JT, Joseph NM, Morrison SJ (2002) Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 35(4): 643–656. pmid:12194865
- 22. Hunziker E, Stein M (2000) Nestin-expressing cells in the pancreatic islets of Langerhans. Biochem Biophys Res Commun 271(1): 116–119. pmid:10777690
- 23. Shih CC, Weng Y, Mamelak A, LeBon T, Hu MC, Forman SJ (2001) Identification of a candidate human neurohematopoietic stem-cell population. Blood 98(8): 2412–2422. pmid:11588038
- 24. Krylyshkina O, Chen J, Mebis L, Denef C, Vankelecom H (2005) Nestin-immunoreactive cells in rat pituitary are neither hormonal nor typical folliculo-stellate cells. Endocrinology 146(5): 2376–2387. pmid:15677762
- 25. Yoshida S, Kato T, Higuchi M, Yako H, Chen M, Kanno N et al. (2013) Rapid transition of NESTIN-expressing dividing cells from PROP1-positive to PIT1-positive advances prenatal pituitary development. J Neuroendocrinol 25(9): 779–791. pmid:23855824
- 26. Korsching E, Packeisen J, Liedtke C, Hungermann D, Wulfing P, van Diest PJ et al. (2005) The origin of vimentin expression in invasive breast cancer: epithelial-mesenchymal transition, myoepithelial histogenesis or histogenesis from progenitor cells with bilinear differentiation potential? J Pathol 206(4): 451–457. pmid:15906273
- 27. Kurz H, Fehr J, Nitschke R, Burkhardt H (2008) Pericytes in the mature chorioallantoic membrane capillary plexus contain desmin and alpha-smooth muscle actin: relevance for non-sprouting angiogenesis. Histochem Cell Biol 130(5): 1027–1040. pmid:18688635
- 28. Boyd NL, Dhara SK, Rekaya R, Godbey EA, Hasneen K, Rao RR et al. (2007) BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies. Exp Biol Med (Maywood) 232(6): 833–843. pmid:17526776
- 29. Bresnick AR, Weber DJ, Zimmer DB (2015) S100 proteins in cancer. Nat Rev Cancer 15(2): 96–109. pmid:25614008
- 30. Donato R, Cannon BR, Sorci G, Riuzzi F, Hsu K, Weber DJ et al. (2013) Functions of S100 proteins. Curr Mol Med 13(1): 24–57. pmid:22834835
- 31. Salama I, Malone PS, Mihaimeed F, Jones JL (2008) A review of the S100 proteins in cancer. Eur J Surg Oncol 34(4): 357–364. pmid:17566693
- 32. Nakajima T, Yamaguchi H, Takahashi K (1980) S100 protein in folliculostellate cells of the rat pituitary anterior lobe. Brain Res 191(2): 523–531. pmid:6991054
- 33. Tachibana O, Yamashima T (1988) Immunohistochemical study of folliculo-stellate cells in human pituitary adenomas. Acta Neuropathol 76(5): 458–464. pmid:2847475
- 34. Hofler H, Denk H, Walter GF (1984) Immunohistochemical demonstration of cytokeratins in endocrine cells of the human pituitary gland and in pituitary adenomas. Virchows Arch A Pathol Anat Histopathol 404(4): 359–368. pmid:6208678
- 35. Allaerts W, Fluitsma DM, Hoefsmit EC, Jeucken PH, Morreau H, Bosman FT et al. (1996) Immunohistochemical, morphological and ultrastructural resemblance between dendritic cells and folliculo-stellate cells in normal human and rat anterior pituitaries. J Neuroendocrinol 8(1): 17–29. pmid:8932733
- 36. Horiguchi K, Fujiwara K, Higuchi M, Yoshida S, Tsukada T, Ueharu H et al. (2014) Expression of chemokine CXCL10 in dendritic cell-like S100β-positive cells in rat anterior pituitary gland. Cell Tissue Res 357(3): 757–765. pmid:24770897
- 37. Horiguchi K, Higuchi M, Yoshida S, Nakakura T, Tateno K, Hasegawa R et al. (2014) Proton receptor GPR68 expression in dendritic cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification. Cell Tissue Res 358(2): 515–525. pmid:25129106
- 38. Vankelecom H (2007) Non-hormonal cell types in the pituitary candidating for stem cell. Semin Cell Dev Biol 18(4): 559–570. pmid:17509912
- 39. Andoniadou CL, Matsushima D, Mousavy Gharavy SN, Signore M, Mackintosh AI, Schaeffer M et al. (2013) Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell 13(4): 433–445. pmid:24094324
- 40. Couly GF, Le Douarin NM (1985) Mapping of the early neural primordium in quail-chick chimeras. I. Developmental relationships between placodes, facial ectoderm, and prosencephalon. Dev Biol 110(2): 422–439. pmid:4018406
- 41. Kouki T, Imai H, Aoto K, Eto K, Shioda S, Kawamura K et al. (2001) Developmental origin of the rat adenohypophysis prior to the formation of Rathke's pouch. Development 128(6): 959–963. pmid:11222149
- 42. Yako H, Kato T, Yoshida S, Higuchi M, Chen M, Kanno N et al. (2013) Three-dimensional studies of Prop1-expressing cells in the rat pituitary just before birth. Cell Tissue Res 354(3): 837–847. pmid:24026438
- 43. Leussink B, Brouwer A, el Khattabi M, Poelmann RE, Gittenberger-de Groot AC, Meijlink F (1995) Expression patterns of the paired-related homeobox genes MHox/Prx1 and S8/Prx2 suggest roles in development of the heart and the forebrain. Mech Dev 52(1): 51–64. pmid:7577675
- 44. de Jong R, Meijlink F (1993) The homeobox gene S8: mesoderm-specific expression in presomite embryos and in cells cultured in vitro and modulation in differentiating pluripotent cells. Dev Biol 157(1): 133–146. pmid:7683282
- 45. Shimozaki K, Clemenson GDJ, Gage FH (2013) Paired related homeobox protein 1 is a regulator of stemness in adult neural stem/progenitor cells. J Neurosci 33(9): 4066–4075. pmid:23447615
- 46. Ross AH, Grob P, Bothwell M, Elder DE, Ernst CS, Marano N et al. (1984) Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies. Proc Natl Acad Sci USA 81(21): 6681–6685. pmid:6093111
- 47. Borson S, Schatteman G, Claude P, Bothwell M (1994) Neurotrophins in the developing and adult primate adenohypophysis: a new pituitary hormone system? Neuroendocrinology 59(5): 466–476. pmid:8022522
- 48. Trost A, Schroedl F, Lange S, Rivera FJ, Tempfer H, Korntner S et al. (2013) Neural crest origin of retinal and choroidal pericytes. Invest Ophthalmol Vis Sci 54(13): 7910–7921. pmid:24235018
- 49. Liu HX, Komatsu Y, Mishina Y, Mistretta CM (2012) Neural crest contribution to lingual mesenchyme, epithelium and developing taste papillae and taste buds. Dev Biol 368(2): 294–303. pmid:22659543
- 50. Dupin E, Sommer L (2012) Neural crest progenitors and stem cells: from early development to adulthood. Dev Biol 366(1): 83–95. pmid:22425619
- 51. Etchevers HC, Vincent C, Le Douarin NM, Couly GF (2001) The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development 128(7): 1059–1068. pmid:11245571
- 52. Woodhoo A, Dean CH, Droggiti A, Mirsky R, Jessen KR (2004) The trunk neural crest and its early glial derivatives: a study of survival responses, developmental schedules and autocrine mechanisms. Mol Cell Neurosci 25(1): 30–41. pmid:14962738
- 53. Hauser S, Widera D, Qunneis F, Muller J, Zander C, Greiner J, et al. (2012) Isolation of novel multipotent neural crest-derived stem cells from adult human inferior turbinate. Stem Cells Dev 21(5): 742–756. pmid:22128806
- 54. Davis SW, Mortensen AH, Keisler JL, Zacharias AL, Gage PJ, Yamamura K et al. (2016) beta-catenin is required in the neural crest and mesencephalon for pituitary gland organogenesis. BMC Dev Biol 16(1): 16. pmid:27184910
- 55. Motohashi T, Kitagawa D, Watanabe N, Wakaoka T, Kunisada T (2014) Neural crest-derived cells sustain their multipotency even after entry into their target tissues. Dev Dyn 243(3): 368–380. pmid:24273191
- 56. Qiao B, Gopalan V, Chen Z, Smith RA, Tao Q, Lam AK (2012) Epithelial-mesenchymal transition and mesenchymal-epithelial transition are essential for the acquisition of stem cell properties in hTERT-immortalised oral epithelial cells. Biol Cell 104(8): 476–489. pmid:22548277
- 57. Widera D, Heimann P, Zander C, Imielski Y, Heidbreder M, Heilemann M et al. (2011) Schwann cells can be reprogrammed to multipotency by culture. Stem Cells Dev 20(12): 2053–2064. pmid:21466279
- 58. Gleiberman AS, Fedtsova NG, Rosenfeld MG (1999) Tissue interactions in the induction of anterior pituitary: role of the ventral diencephalon, mesenchyme, and notochord. Dev Biol 213(2): 340–353. pmid:10479452