FGF10 maintains stem cell compartment in developing mouse incisors

Hidemitsu Harada 2 Takashi Toyono 2 Kuniaki Toyoshima 2 Masahiro Yamasaki 1 Nobuyuki Itoh 1 Shigeaki Kato 0 Keisuke Sekine 0 Hideyo Ohuchi 3 0 Institute of Molecular and Cellular Biosciences, The University of Tokyo , Tokyo 113-0032 , Japan 1 Department of Genetic Biochemistry, Graduate School of Pharmaceutical Sciences, Kyoto University , 46-29 Yoshida-shimo- Adachi-cho, Sakyo-ku, Kyoto City, Kyoto, 606-8501 , Japan 2 Second Department of Oral Anatomy and Cell Biology, Kyushu Dental College , 2-6-1, Manazuru, Kokurakita-ku, Kitakyushu, 803- 8580 , Japan 3 Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima , 2-1 Minami-Jyosanjima-cho, Tokushima City, Tokushima 770-8506 , Japan SUMMARY Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the During embryogenesis, a single fertilized oocyte gives rise to a multicellular organism whose cells and tissues adopt differentiated characteristics or fates to perform the specified functions of each organ of the body. Many tissues and organs maintain a process known as homeostasis: as cells die, either by apoptosis or by injury, they are replenished. Additionally, the epidermis, hair, small intestine and hematopoietic system are all examples of adult tissues that exist naturally in a state of dynamic flux. Even in the absence of injury, these structures continually give rise to new cells, and are able to divide transiently, terminally differentiate and die. These regenerative tissues are maintained by adult stem cells, which have both the capacity to self-renew, to divide and create additional stem cells, and also to differentiate along a specified molecular pathway. In recent years tremendous advances have been made absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs. in our understanding of identification of adult stem cells (reviewed by Fuchs and Segre, 2000). However, molecular mechanisms for establishing adult stem cell compartments during embryogenesis have not been examined in detail. The mouse incisor tooth is an excellent model for analyzing certain aspects of stem cell regulation and function (Harada et al., 1999). The continuous eruption of mouse incisors throughout the lifetime of an animal is maintained by the division of germs localized in the cervical loop of the apical region. We have previously shown that stem cells divide rarely and asymmetrically, and one daughter cell remains as an undifferentiated stem cell in the cervical loop, whereas the other cell moves to the incisal end, giving rise to transitamplifying cells. Dental epithelial stem cells in the cervical loop give rise to four cell-lineages inner enamel epithelium (ameloblast cell-lineage), stratum intermedium, stellate reticulum and outer enamel epithelium. 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 express Fgf3 and Fgf10, and their receptors, Fgfr1b and Fgfr2b, are expressed throughout the dental epithelium (Kettunen et al., 1998; Harada et al., 1999). Furthermore, beadimplantation assays have shown that FGF10 signaling from the mesenchyme indirectly regulated cell division and fate decision of epithelial stem cells by modulating the Notch pathway in the cervical loop via stimulation of lunatic fringe expression. The involvement of FGF10 in tooth morphogenesis was further indicated by the hypoplastic tooth organ in FGF10 null mice (Ohuchi et al., 2000). In the present study, which focuses on mesenchymal molecular cues for forming the stem cell compartment during organogenesis, we used the developing mouse incisor germ cell 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 incisor germ of Fgf10-deficient mice showed that FGF10 is not involved in the early signaling networks that regulate tooth initiation at early morphogenesis, but is involved in the establishment of adults stem cells. Other experiments using neutralizing antibody provided direct evidence that FGF10 prevents apoptosis in the stem cell compartment. We propose a model that demonstrates the formation of adult stem cells during organogenesis and shows that stem cells are maintained as undifferentiated cells by mesenchymal signals. MATERIALS AND METHODS Fgf10-deficient mice Heads from Fgf10/ mice were obtained at desired embryonic stages from (...truncated)