86 Erika Awata, Akina Chiba, Chisato Yui and Shigeki Hirano 形態・機能 第 11 巻第 2 号

The Development of Hepatic Sinusoids in Mouse Embryos

Erika Awata1), Akina Chiba2)*, Chisato Yui1) and Shigeki Hirano1) 1) ‌Department of Medical Technology, Graduate School of Health Sciences, Niigata University, Niigata, Japan 2) ‌Division of Gross Anatomy and Morphogenesis, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan (Received:25 May 2012, Accepted:23 August 2012)

Abstract The development of hepatic sinusoids is only partially understood. This study investigated the development of hepatic sinusoids in order to clarify the formation process of sinusoidal by focusing on mouse tissue during a period of active hematopoiesis (E11-E16) using hematoxylin and eosin staining on paraffin-embedded liver sections, and toluidine blue staining on epoxy resin- embedded semi-thin sections. Flat cells lining the luminal surface of hepatic sinusoids were identified as endothelial cells with CD-146 immunohistochemical staining. We observed that developing sinusoids had partially naked walls that were not lined by flat cells, and from that hematopoietic cells and/or hepatic cells protruded into the sinusoidal lumen. In addition, CD-146 positive round cells scattering among hematopoietic cells and hepatic cells were observed. These observations suggest that hepatic sinusoids do not arise from the sprouting of intrahepatic vessels but appear to be formed by CD-146 positive endothelial progenitor cells. Key words fetal mouse liver; sinusoid development; endothelial progenitor cells; CD-146

Introduction vascular system in liver, especially its sinusoidal Most liver functions, including the synthesis development, is poorly understood. and excretion of bile, the storage and supply of As the liver develops, intrahepatic capillary energy, and the metabolism and detoxification vessels that are directly connected to the ductus of various substances, arise gradually during venosus1, 2) can be observed in the primitive liver development. These functions occur in hepatic anlage. The vascular system of the liver anlage lobules in three distinct groups of the vascular is then distinguished as a vessel connecting the system (the afferent interlobular vessels via right and left vitelline , with sinusoids the , sinusoids of each lobe, developing among them. Therefore, it is thought and efferent veins connected to the inferior that one source of the vascular system in vena cava). However, the development of the liver is vitelline veins. It is unlikely, however, whether all vascular sets are derived from the 3) intrahepatic capillary vessels. Yokoyama et al. Corresponding author : Akina Chiba, Ph.D. demonstrated that the long-term survival of Division of Gross Anatomy and Morphogenesis, Niigata University Graduate School of Medical and Dental was dependent on a network of blood Sciences, 757 Asahimachi-dori 1-chome, Chuo-ku, Niigata, Niigata 951-8510, Japan vessels that developed before transplantation Tel: +81-25-227-2048 into the host. These blood vessels contributed to Fax: +81-25-227-0752 E-mail: [email protected] supplying hepatocytes with nutrients but did not 2013 年３月 The development of hepatic sinusoids 87

result, themselves, in sinusoidal development. Histology and immunohistochemical staining Sudo et al.4), who constructed a microfluidic Liver sections were prepared for either platform to analyze angiogenesis in 3D cultures immunohistochemistry or hematoxylin of rat primary hepatocytes, showed that these and eosin (H-E) staining. Anti-albumin microvascular networks did not contribute to immunohistochemical techniques were used to sinusoid formation. Chiba et al.5), observing a identify hepatoblasts and hepatocytes in the hematopoietic cell mass that was enveloped liver anlage. Paraffin-embedded liver sections by endothelial cells in the regenerating liver from E11.5, E12, E13 and E15 embryos were on the chorioallantoic membrane of chick pre-incubated with normal rabbit blocking embryos, suggested that the spaces occupied serum for 20 minutes and incubated overnight by the hematopoietic cell mass gave rise to with anti-mouse albumin in 0.01M PBS (Bethyl sinusoids. Sherer6) indicated that the endothelial Laboratories, Montgomery, TX, USA) at 4°C in lining of sinusoids in the developing quail’s a refrigerator. Biotinylated anti-goat IgG (H+L) liver transplanted onto a chick chorioallantoic (Vector Laboratories, Burlingame, CA, USA) membrane was formed by quail cells. Pérez- was used as the secondary antibody, followed Pomares et al.7) suggested that endothelial cells by Vectorstain ABC Kit (Vector Laboratories). of sinusoids were derived from mesothelial cells. Visualization of immuno-complexes was Taken together, these reports indicate that performed by incubation with diaminobenzidine, sinusoids develop from endothelial progenitor and sections were lightly counterstained with cells rather than intrahepatic capillary vessels. hematoxylin. Hepatic sinusoids are unique vessels, that To reveal the presence of endothelial cells of is, they are constructed from fenestrated sinusoids, CD-146 immunohistochemical staining endothelial cells with no basal lamina. Therefore, of E12, E13 and E15 samples was performed. the developmental mechanisms giving rise to CD-146 is a cell adhesion molecule localized at hepatic sinusoids may be distinct from those of the endothelial junction of the whole vascular other blood vessels. system8, 9). We examined several endothelial In the present study, in order to elucidate cell markers, and adopted CD-146 that showed the origin of sinusoids, we detail the endothelial the most intense reaction on the endothelial formation of the mouse liver at a developmental cells of the mouse embryonic . Paraffin stage in which hematopoiesis is in progress. and cryostat sections were pre-incubated with normal goat serum at room temperature for at Materials and methods least 30 minutes and incubated overnight with Pregnant mice (C57BL/6), purchased from anti-CD-146 antibody (Calbiochem, Frankfurt, Japan SLC Inc. (Shizuoka, Japan), were used. Germany) at 4°C in a refrigerator. Sections The presence of vaginal plaques was taken were then incubated with ImmPRESS reagent to represent embryonic day zero (E0). As (Calbiochem, Frankfurt, Germany) and anti- hematopoiesis started at E10 and reached a mouse IgG (Vector Laboratories) at room peak at E13, the mice were sacrificed under temperature for 30 minutes. Diaminobenzidine intraperitoneal anesthesia with pentobarbital was then used as a chromogen. The livers of sodium on days E9.5, 10, 10.25, 10.5, 11, newborn and adult mice were used as controls 11.25, 11.5, 12, 13 and 15, and the fetuses (data not shown). were collected. Fetal liver samples were fixed with 10% formaldehyde in 0.01M phosphate- Semi-thin sections buffered saline (PBS; pH 7.3). Because the livers To observe the discontinuity of endothelial of E9.5-E13 embryos were extremely small, cells on the walls of sinusoidal spaces, E11 and the entire thoracoabdominal region, including E12 livers were fixed with 2.5% glutaraldehyde the liver anlage, was dissected en bloc under a in 0.01M PBS for 3 hours, and then post-fixed stereomicroscope and immediately fixed. For with 1% osmium tetraoxide in 0.01M PBS for 2 cryostat sections, samples were immersed in 30% hours. The tissues were dehydrated through a sucrose at 4°C in a refrigerator. Other samples graded series of ethanols, placed in propylene were embedded in paraffin. oxide twice, and embedded in epoxy resin (Quetol 812). Serial semi-thin sections (1 μm) were cut 88 Erika Awata, Akina Chiba, Chisato Yui and Shigeki Hirano 形態・機能 第 11 巻第 2 号

with a diamond knife and stained with toluidine blue for light microscopic observation.

Results Hepatoblasts and hepatic cells of the fetal livers were stained with H&E and immunostained with anti-albumin antibody (Fig.1). At E11, intrahepatic capillary vessels could already be seen as a relatively wide space (Fig.1, a-1) that had a direct connection to the (refer Kingsbury et al.2)). Thereafter, the primitive sinusoidal spaces diminished (Fig.1, b-1, c-1). Some primitive sinusoids were observed as having a smooth Fig. 2 Light photomicrograph of resin-embedded wall that was lined by flat cells sharing an semi-thin section of liver at E12. asterisk: The denuded position of the hepatic cells and hematopoietic cells into the sinusoid. arrowheads: the endothelial cells lining the wall of the sinusoid. arrows: Large round endothelial progenitor cell spreading their cytoplasmic processes along the wall of the sinusoid. (scale bar 50 μm)

irregular wall and having no epithelial lining from which hematopoietic cells and hepatic cells were denuded (Fig.1d). At E11 and E12, hepatic cells were polygonal in shape and gathered together to form a network of branched-cell cords (Fig.1, a-2, b-2). The wide spaces of the mesh were filled with albumin-negative cells (most likely hematopoietic cells), which had spherical outlines and a large round nucleus. Thereafter, the liver anlage grew rapidly, increasing the number of hepatic cells, while the size of the mesh decreased. By contrast, the hematopoietic cells decreased in number (Fig.1c, e). Fig. 1 Light photomicrographs stained with H&E To confirm the denudation of hepatocytes and (left column) and anti-albumin (right column) in hematopoietic cells into the sinusoidal space the developing liver. (Fig.1d), resin-embedded semi-thin sections and a (E11): The liver has wide vascular spaces maintaining the form of intrahepatic capillary vessels. CD-146 immunohistochemical staining sections Among the vessels, anti-albumin positive hepatic cells were analyzed (Fig.2, 3). In resin-embedded are observed with hematopoietic cells. b (E12), c (E13) sections, some sinusoidal spaces were completely and e (E15): The wide vascular spaces are diminished lined with flat cells, while other lumina had and the parenchymal area becomes compact. Anti- partially naked walls from which hematopoietic albumin positive hepatic cells form nets with their cells and hepatic cells had denuded into the cytoplasmic processes. d: Enlargement of the area that sinusoidal lumen (Fig.2, Fig.3f, h, i asterisk). It is indicated as a rectangle in b-1. asterisk indicates was observed that round cells elongated film-like the position of the hematopoietic cells and hepatic processes along the lumen of the sinusoid (Fig.2 cells denuded into the sinusoid. arrow: flattened arrow). CD-146, a marker of endothelial cells and endothelial cell. arrowhead: a large endothelial cell of endothelial progenitor cells, labeled flat cells in the middle of a depression. The magnification of lining the wall of the sinusoid (Fig.3f-i arrow); all photomicrographs are the same (scale bar 50 μm) except d (scale bar 20 μm). however, no CD-146 immunopositive cells or 2013 年３月 The development of hepatic sinusoids 89

Fig. 3 CD-146 positive cells in the developing liver at E13. Round cells typically are intensely stained with CD-146 in cryosections (a-c), but the evaluation of the position of each CD-146 positive cell is compromised because the hematopoietic cells slough away during the staining processes. In paraffin sections (d-i), histological architecture is kept firm but the staining intensity of round cells becomes very weak, whereas endothelial cells retain their intense staining. a-c: CD-146 immunopositive round cells (arrowheads) are seen among the hepatic cells and hematopoietic cells but in (c) the position of them is seen along the wall of the sinusoid. d, e: CD-146 immunopositive round cells (white arrows) are surrounded hepatocytes and hemopoietic cells. f-i: Photomicrographs show the developing endothelial cells and incomplete wall of sinusoids. Black arrows in each photomicrograph denotes CD-146 immunopositive endothelial cells. It is likely that the round endothelial cells (f) flatten incrementally (g-i). asterisk: a denuded cell mass into the sinusoidal lumen. upper column (a-c): cryosections, (scale bar 20 μm); middle and bottom columns (d-i): paraffin sections, (scale bar 20 μm)

cell processes were observed at loci from which development had continuous endothelial cells hematopoietic cells and hepatic cells protruded without a gap among them10-12). If sinusoids are (Fig.3f, h, i asterisk). The same distribution formed by branches of them, from the beginning pattern of CD-146 positive cells was observed in of the formation of sinusoids, the lumen should E12, E13 and E15 specimens. CD-146 antigen be completely lined with endothelial cells. Our labeled not only thin endothelial cells but also analysis of H-E stained sections, semi-thin small round cells that were distributed on the sections and CD-146 stained sections showed outside of sinusoids (Fig.3a-e). that sinusoids were imperfectly lined by flat cells and that the hematopoietic cells and Discussion hepatic cells were denuded to the lumen of the Intrahepatic capillary vessels in a developing sinusoids. Chick-quail chimera experiments liver receive blood from vitelline veins. However, have indicated that sinusoidal endothelial cells vitelline veins do not give rise to the whole were formed with quail cells6). These findings vascular system in a maturated liver. Capillary suggest that sinusoids develop independent of vessels observed during the initial stages of liver vitelline and umbilical veins. Hammond1) came 90 Erika Awata, Akina Chiba, Chisato Yui and Shigeki Hirano 形態・機能 第 11 巻第 2 号

to the conclusion that major vascular system References development, including sinusoids, occurred 1) Hammond WS (1939) On the origin of the extravascularly in the liver, and the invasion of cells lining the liver sinusoids in the cat and vitelline veins was a secondary phenomenon. the rat. Am J Anat 65:199-227 Chiba et al.5) also showed extravascular 2) Kingsbury JW, Alexanderson M and formation of sinusoids in the regenerated liver on Kornstein ES (1956) The development of the the chorioallantoic membrane of chick embryos. liver in the chick. Anat Rec 124:165-187 Matsumoto et al.13) suggested that endothelial 3) Yokoyama T, Ohashi K, Kuge H, Kanehiro cells, and/or its progenitor cells, promoted liver H, Iwata H, Yamato M and Nakajima Y organogenesis before developing a vascular (2006) In Vivo Engineering of Metabolically system. They also suggested that progenitor Active Hepatic Tissues in a Neovascularized cells existed in the developing liver. Vitelline and Subcutaneous Cavity. Am J Transplant 6:50- umbilical veins invaded the transverse septum; 59 they are therefore unlikely to contribute to the 4) Sudo R, Chung S, Zervantonakis IK, sinusoidal system by the sprouting of capillary Vickerman V, Toshimitsu Y, Griffith LG vessels, but rather give rise to other liver and Kamm RD (2009) Transport-mediated vasculature systems. angiogenesis in 3D epithelial coculture. In the present study, CD-146 immunopositive FASEB 23:2155-2164 round cells were observed. Some were located 5) Chiba A, Yui C and Hirano S (2010) Liver in the walls of sinusoidal spaces, with thin reconstruction on the chorioallantoic cytoplasmic processes spreading along the membrane of jthe chick embryo. Arch Histol sinusoidal wall. Similar features could also be Cytol 73:45-53 observed in resin-embedded semi-thin sections. 6) Sherer GK (1991) Vasuculogenic mechanisms We hypothesized that CD-146 immunopositive and epithelo-mesenchymal specificity in cells with round soma differentiated into flat endodermal organs.In:The Development of endothelial cells of hepatic sinusoids (Fig.1d, the vascular system. Feinberg RN et al. (eds) 2, 3f-i). The mesoderm of the transverse 37-57 Issues in biomedicine 14: Stolte H et septum, in which the liver anlage developed, al. (eds) Karger Basel, Switzerland had various undifferentiated mesenchymal 7) Pérez-Pomares JM, Carmona R, González- cells, including endothelial progenitor cells. Iriarte M, Macías D, Guadix JA and Muñoz- Hammond1) suggested that mesenchymal cells Chápuli R (2004) Contribution of mesothelium- in the transverse septum differentiated into flat derived cells to liver sinusoids in avian endothelial cells, whereas Pérez-Pomares et al.7) embryos. Dev Dynam 229:465-474 thought that mesothelial cells contributed to the 8) Bardin N, George F, Mutin M, Brisson C, endothelial cells of sinusoids. Taken together, Horschowski N, Frances V, Lesaule G and these observations strongly support the notion Sampol J (1996) S-Endo 1, a pan-endothelial that round cells are the progenitor of endothelial monochlonal antibody recognizing a novel cells. However, further study is required to human endothelial antigen. Tissue Antigens determine whether CD-146 positive round cells 48:531-539 correspond to the cells described by Pérez- 9) Anfosso F, Bardin N, Vivier E, Sabatier Pomares et al.7). F, Sampol J and Dignat-George F (2001) Outside-in signaling pathway linked to Acknowledgments CD146 engagement in human endothelial We are grateful to Dr. J. G. McHaffie, cells. J Biol Chem 276:1564-1569 Department of Neurobiology and Anatomy, Wake 10) Enzan H, Hara H, Yamashita Y, Ohkita Forest University School of Medicine, for his T and Yamane T (1983) Fine structure of proof reading, editorial comments and helpful hepatic sinusoids and their development in suggestions in the preparation of the manuscript. human embryos and fetuses. Acta Pathol Jpn This work was kindly supported by Grants-in-Aid 33:447-466 for Challenging Exploratory Research (21659317) 11) Enzan H, Himeno H, Hiroi M, Kiyoku H, from the Ministry of Education, Culture, Sports, Saibara T and Onishi S (1997) Development Science and Technology of Japan. of hepatic sinusoidal structure with special 2013 年３月 The development of hepatic sinusoids 91

reference to the Ito cells. Microsc Res Tech organogenesis in humans. Blood 87:4568- 39:336-349 4580 12) Couvelard A, Scoazec JY, Dauge MC, 13) Matsumoto K, Yoshitomi H, Rossant J Bringuier AF, Potet F and Feldmann G (1996) and Zaret KS (2001) Liver Organogenesis Structural and functional differentiation Promoted by Endothelial Cells Prior to of sinusoidal endothelial cells during liver Vascular Function. Science 294:559-563

＜原著＞

マウス胚子肝臓の洞様血管の発生について

粟田絵里加1）、千葉映奈2）、油井千里1）、平野茂樹1） 1）新潟大学保健学研究科検査技術科学分野 2）新潟大学大学院医歯学総合研究科肉眼解剖学分野

要 旨 肝臓の洞様血管の発生機構は十分明らかになっていない。そこで洞様血管の形成過程を明らかにするため、造血期に あるマウス肝臓を組織学的に観察した。観察にはパラフィン包埋した材料のヘマトキシリン・エオジン染色標本、エポ キシレジン包埋材料の準超薄切片のトルイジン青染色標本、更に洞様血管内皮細胞を認識するため、CD-146 抗体によ る免疫組織化学染色標本を用いた。 本研究で、洞様血管は、その形成過程の中で部分的に内皮細胞を欠いた壁を持ち、そこから肝臓細胞や造血細胞が洞 様血管内腔に裸出している場所のあることが明らかになった。また洞様血管の壁を作るCD-146 陽性細胞が内腔に向かっ て丘状に突出した状態から次第に扁平な内皮細胞に変わって行く様子が観察された。更に肝細胞や造血細胞の間に CD- 146 陽性細胞が散在しているのも観察された。これ等の所見は、洞様血管は発生初期の肝臓内にあった毛細血管網から 出芽して形成されるのでなく、CD-146 陽性の内皮前駆細胞から形成されることを示唆している。 キーワード マウス胎児肝臓、洞様血管発生、内皮前駆細胞、CD-146