Localization of DLL1- and NICD-positive osteoblasts in cortical bone during postnatal growth in rats Yukihiro Kohara a, b, * , Sohei Kitazawa a , Riko Kitazawa a, c , Ryuma Haraguchi a , Kiyotaka Arai d , Hajime Amasaki b , Satoshi Soeta b a Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan b Laboratory of Veterinary Anatomy, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino City, Tokyo,180-8602, Japan c Division of Diagnostic Pathology, Ehime University Hospital, Shitsukawa, Toon City, Ehime, 791-0295, Japan d Department of Veterinary Surgery, Okayama University of Science, 1-3 Ikoinooka, Imabari City, Ehime, 794-8555, Japan article info Article history: Received 19 May 2020 Accepted 8 June 2020 Keywords: Osteoblasts Primary osteons Cortical bone NOTCH1 NICD DLL1 abstract The long bone midshaft expands by forming primary osteons at the periosteal surface of cortical bone in humans and rodents. Osteoblastic bone formation in the vascular cavity in the center of primary osteons is delayed during cortical bone development. The mechanisms of the formation of primary osteons is not fully understood, however. Focusing on NOTCH1 signaling, an inhibitory signaling on osteoblastic bone formation, our immunohistochemical analysis revealed Delta like1 (DLL1), a ligand of NOTCH1, and the NOTCH1 intracellular domain (NICD, an activated form of NOTCH1) immunoreactivity, in the cuboidal osteoblasts lining the bone surface in the vascular cavity of primary osteons during postnatal growth in rats. Interestingly, five days after treatment of primary osteoblasts with ascorbic acid and b glycer- ophosphate, protein levels of both DLL1 and NICD increased transiently, indicating that DLL1 activates NOTCH1 in primary cultured osteoblasts. Thus, the results imply that DLL1-NOTCH1 signaling in oste- oblasts is associated with primary osteonal bone formation. © 2020 Elsevier Inc. All rights reserved. 1. Introduction The development of cortical bone during growth in rodents is similar to that in humans, but differs in terms of cortical bone remodeling in the adult skeleton [1e3]. Initial cortical bones of long bone midshafts are constructed with primary osteons through intramembranous ossification in humans and rodents [1 ,4]. Although not replaced in the adult rodent, primary osteonal bones are gradually replaced by secondary osteons in humans during maturation [1 ,5]. Although transposing the remodeling of adult rodent cortical bone to humans is notional, it is relevant to the study of cortical bone during growth. The mechanisms of primary osteon architecture remain to be elucidated, however. NOTCH1, a single pass transmembrane receptor that mediates cell fate and embryonic development [6,7] is activated by cell sur- face ligands Serrate/Jagged (JAG) and Delta like (DLL), which cause proteolysis of the NOTCH1 intracellular domain (NICD: an activated form of NOTCH1) by release and translocation to the nucleus [8]. In humans, loss-of-function mutation in NOTCH1 is one of the causes of Adams Oliver Syndrome characterized by congenital incisional dysplasia and terminal limb deficiency [9]. While Col1 (2.3-kb)-Cre mediated Notch1 and Notch2 double deficiency has no impact on the skeletal phenotype in mice [10], osteoblast-targeted over- expression of NICD transgenic mice exhibits severe osteopenia because of reduced bone formation [11e 13], suggesting that NOTCH1 signaling suppresses osteoblastic bone formation. To date however, the relation between NOTCH1 signaling and cortical bone development is not fully understood. In the present study, we demonstrated that DLL1 and NICD are expressed in cuboidal osteoblasts lining the bone surface in the vascular cavity of primary osteons, in rats during growth. On the other hand, no cells expressing these proteins were observed at the end-phases of primary osteonal bone formation, suggesting that DLL1-NOTCH1 signaling in osteoblasts is associated with primary osteonal bone formation. * Corresponding author. Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan. E-mail address: kohara.yukihiro.yu@ehime-u.ac.jp (Y. Kohara). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2020.06.039 0006-291X/© 2020 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 529 (2020) 186e190