S100β-Positive Cells of Mesenchymal Origin Reside in the Anterior Lobe of the Embryonic Pituitary Gland

The anterior and intermediate lobes of the pituitary gland develop through invagination of the oral ectoderm and as they are endocrine tissues, they participate in the maintenance of vital functions via the synthesis and secretion of numerous hormones. We recently observed that several extrapituitary cells invade the anterior lobe of the developing pituitary gland. This raised the question of the origin(s) of these 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.


Introduction
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 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.

Rats
S100β/GFP-TG rats [14] 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.

Results
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 [16]; 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).

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 observed, but GFP-and/or PRRX1-single and -double positive cells were absent on E19.5 and E20.5 ( Fig 2F).
We have previously shown that PRRX2, a cognate of PRRX1, is expressed in the mesenchyme cells surrounding the pituitary gland [19]. Triple immunostaining for GFP, SOX2, and PRRX2 was performed on E20.5. As shown in S1 Fig 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).

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 [13]. 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).  [18][19][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 [21], 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 was counted (n = 1 for E18.5, n = 2 for E19.5, and n = 2 for E20. 5  p75-positive cells were elongated in shape (Fig 5B-5Bʹʹʹ). Results revealed that 41.2% of GFPpositive cells were GFP/Ki67-double positive cells (Fig 5C).
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   (Table 1), reflecting the progress of cell differentiation and scattering in the parenchyma.
Immunohistochemical analysis of DESMIN, a marker of immature and mature pericytes [27], 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 [28], 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.

Discussion
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][30][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 [32], 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][6][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 [33], epithelial cell-like cells expressing keratin [34], and dendritic cell-like cells expressing interleukin-6 [9,[35][36][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 [38]. The generation of S100β-positive cells from SOX2-positive cells has been demonstrated using genetic lineage tracing [39]. These studies were conducted with postnatal pituitaries, as it was believed that S100β-positive cells appear approximately ten days after birth [15]. However, our previous study revealed the presence of S100β transcripts in the embryonic pituitary [8], 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 [39]. 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][44][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. [24] 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 [46]. 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 [47]. 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][49][50]. These derivative lineages include pericytes, smooth muscle cells [51] 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 [54]. Motohashi et al. (2014) revealed that neural crest-derived cells sustain their multipotency even after entry into their target tissues [55]. It should also be mentioned that the reverse transition from mesenchyme to epithelium includes the acquisition of stemness [56] and that neural crest-derived Schwann cells can  be reprogrammed to acquire multipotency [57]. 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 [19], confirming and exploring in further detail the previous results that extrapituitary lineage cells invade the anterior lobe [58]. A previous study that revealed the importance of direct contact between the pituitary primordium and surrounding ventral diencephalon, mesenchyme tissue, and notochord [58] 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.