Development 129, 1533-1541 (2002) 1533 Printed in Great Britain © The Company of Biologists Limited 2002 DEV14510 DEVELOPMENT AND DISEASE FGF10 maintains stem cell compartment in developing mouse incisors Hidemitsu Harada1,*, Takashi Toyono1, Kuniaki Toyoshima1, Masahiro Yamasaki2, Nobuyuki Itoh2, Shigeaki Kato3, Keisuke Sekine3 and Hideyo Ohuchi4 1Second Department of Oral Anatomy and Cell Biology, Kyushu Dental College, 2-6-1, Manazuru, Kokurakita-ku, Kitakyushu, 803- 8580, Japan 2Department of Genetic Biochemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimo- Adachi-cho, Sakyo-ku, Kyoto City, Kyoto, 606-8501, Japan 3Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan 4Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minami-Jyosanjima-cho, Tokushima City, Tokushima 770-8506, Japan *Author for correspondence (e-mail: [email protected]) Accepted 14 December 2001 SUMMARY Mouse incisors are regenerative tissues that grow absence of the cervical loop was due to a divergence in continuously throughout life. The renewal of dental Fgf10 and Fgf3 expression patterns at E16. Furthermore, epithelium-producing enamel matrix and/or induction of we estimated the growth of dental epithelium from incisor dentin formation by mesenchymal cells is performed by explants of FGF10-null mice by organ culture. The dental stem cells that reside in cervical loop of the incisor apex. epithelium of FGF10-null mice showed limited growth, However, little is known about the mechanisms of stem cell although the epithelium of wild-type mice appeared to compartment formation. Recently, a mouse incisor was grow normally. In other experiments, a functional disorder used as a model to show that fibroblast growth factor (FGF) of FGF10, caused by a neutralizing anti-FGF10 antibody, 10 regulates mitogenesis and fate decision of adult stem induced apoptosis in the cervical loop of developing mouse cells. To further illustrate the role of FGF10 in the incisor cultures. However, recombinant human FGF10 formation of the stem cell compartment during tooth protein rescued the cervical loop from apoptosis. Taken organogenesis, we have analyzed incisor development in together, these results suggest that FGF10 is a survival Fgf10-deficient mice and have examined the effects of factor that maintains the stem cell population in developing neutralizing anti-FGF10 antibody on the developing incisor germs. incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later Key words: Stem cells, FGF10, Incisor development, Epithelial- stage, the cervical loop was not formed. We found that the mesenchymal interactions, Mouse INTRODUCTION in our understanding of identification of adult stem cells (reviewed by Fuchs and Segre, 2000). However, molecular During embryogenesis, a single fertilized oocyte gives rise to mechanisms for establishing adult stem cell compartments a multicellular organism whose cells and tissues adopt during embryogenesis have not been examined in detail. differentiated characteristics or fates to perform the specified The mouse incisor tooth is an excellent model for analyzing functions of each organ of the body. Many tissues and organs certain aspects of stem cell regulation and function (Harada maintain a process known as homeostasis: as cells die, either et al., 1999). The continuous eruption of mouse incisors by apoptosis or by injury, they are replenished. Additionally, throughout the lifetime of an animal is maintained by the the epidermis, hair, small intestine and hematopoietic system division of germs localized in the cervical loop of the apical are all examples of adult tissues that exist naturally in a state region. We have previously shown that stem cells divide rarely of dynamic flux. Even in the absence of injury, these structures and asymmetrically, and one daughter cell remains as an continually give rise to new cells, and are able to divide undifferentiated stem cell in the cervical loop, whereas the transiently, terminally differentiate and die. These regenerative other cell moves to the incisal end, giving rise to transit- tissues are maintained by adult stem cells, which have both the amplifying cells. Dental epithelial stem cells in the cervical capacity to self-renew, to divide and create additional stem loop give rise to four cell-lineages – inner enamel epithelium cells, and also to differentiate along a specified molecular (ameloblast cell-lineage), stratum intermedium, stellate pathway. In recent years tremendous advances have been made reticulum and outer enamel epithelium. 1534 H. Harada and others The ability to select among multiple terminal differentiation pathways and to generate cells that can migrate in the selected direction are characteristics that parallel those of the hair follicle bulge or of crypt cells of the small intestine (Rochat et al., 1994; Fuchs and Segre, 2000). These stem cells seem to rely upon mesenchymal cues for their survival and differentiation (Korinek et al., 1998; DasGupta and Fuchs, 1999; Millar et al., 1999). Epithelial-mesenchymal interactions play an essential role in regulating a wide variety of developmental processes, including those of the teeth (Thesleff and Sharpe, 1997; Peters and Balling, 1999; Hogan, 1999). Tissue recombination experiments have shown that the mesenchymal tissue controls advancing morphogenesis after initiation of tooth development and regulates the continuous growth of epithelium in the mouse incisor (Koller and Baird, 1969). Fibroblast growth factors (FGFs) play a crucial role in the developing tooth (Kettunen and Thesleff, 1999). Our recent studies have shown that mouse incisor mesenchymal cells Fig. 1. The developing mouse incisor during the initiation (E11), bud express Fgf3 and Fgf10, and their receptors, Fgfr1b and (E13), cap (E14), early bell (E16) and late bell (E19.5) stages. The Fgfr2b, are expressed throughout the dental epithelium initial stages of morphogenesis are very similar in all teeth. The first (Kettunen et al., 1998; Harada et al., 1999). Furthermore, bead- morphological sign of incisor development is a thickening of the oral implantation assays have shown that FGF10 signaling from the epithelium (E11), which subsequently buds into the underlying mesenchyme indirectly regulated cell division and fate mesenchyme (E13, bud stage). At later stages, the developing incisor decision of epithelial stem cells by modulating the Notch rotates anteroposteriorly (E14, cap stage) and becomes parallel to the pathway in the cervical loop via stimulation of lunatic fringe long axis of the incisors (E16, early bell stage). At early bell stage expression. The involvement of FGF10 in tooth morphogenesis (E16), the cervical loop is seen at the apical end of the labial epithelium. Only the labial epithelium gives rise to the enamel- was further indicated by the hypoplastic tooth organ in FGF10 forming ameloblasts (E19.5, late bell stage). Epithelium in dark blue, null mice (Ohuchi et al., 2000). dental mesenchyme in light blue dots, enamel in red and dentin in In the present study, which focuses on mesenchymal green. cl, cervical loop; cm, condensed mesenchyme; d, dentine; dp, molecular cues for forming the stem cell compartment during dental papilla; e, enamel; eo, enamel organ; m, molar; oe, oral organogenesis, we used the developing mouse incisor germ cell epithelium; p, pulp; vl, vestibular lamina. as a model of regenerative tissues. To further examine the role of FGF10 during incisor development, we have designed two loss-of-function experimental lines. Analysis of developing Organ culture experiments Culture methods were as described previously (Harada et al., 1999). incisor germ of Fgf10-deficient mice showed that FGF10 is not +/+ +/– involved in the early signaling networks that regulate tooth Dissected incisors from the mandible of Fgf10 or Fgf10 (n=8) and Fgf10–/– mice (n=7) at E19 were cultured for 20 days. All pictures initiation at early morphogenesis, but is involved in the were digitized and the growth length of dental epithelium was establishment of adults stem cells. Other experiments using estimated at intervals of about 5 days by using NIH image 1.62 (public neutralizing antibody provided direct evidence that FGF10 domain program, US National Institute of Health). Averaged growth prevents apoptosis in the stem cell compartment. We propose rate was plotted using Microsoft Excel 98 (Microsoft Corporation, a model that demonstrates the formation of adult stem cells Redmond, WA). during organogenesis and shows that stem cells are maintained For culture of the cervical loop, the mesenchyme or tissue as undifferentiated cells by mesenchymal signals. recombinants the incisors were incubated for 4 hours in 2% collagenase (GibcoBRL) in DMEM at 4°C, and the epithelia were mechanically separated from mesenchyme in PBS. The cervical loops were cut from the epithelial sheets with 26G needle. On tissue recombination MATERIALS AND METHODS experiments the cervical loop was placed in contact with mesenchyme. These explants were cultured for 48 hours on the filters coated matrigel Fgf10-deficient mice (BD Biosciences, MA). The cervical loops were cultured in the medium Heads from Fgf10–/– mice were obtained at desired embryonic stages supplemented with human recombinant FGF10 (R&D systems Inc, from intercrosses of heterozygous breeding pairs. Genotyping was Minneapolis, MN) varying