Endocrine-Related Cancer (2001) 8 211–217 Fibroblast growth factor signalling in mouse mammary gland development B Spencer-Dene, C Dillon, V Fantl, K Kerr, A Petiot and C Dickson Laboratory of Viral Carcinogenesis, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK (Requests for offprints should be addressed to C Dickson; Email: [email protected]) Abstract Fibroblast growth factors (Fgfs) and their receptors are important intercellular signalling molecules involved in many aspects of animal development. The aberrant expression of the Fgfs or the inappropriate activation of their cell surface receptors have been implicated in tumorigenesis. Here, we describe the evidence that as well as playing a critical role in the formation of the mammary primordia during embryogenesis, signalling by Fgfs is necessary for optimal lobuloalveolar development of the mouse mammary gland during pregnancy. Endocrine-Related Cancer (2001) 8 211–217 Introduction apoptosis of the epithelial component of the tissue as it remodels to a resting state similar in structure to the virgin The mouse mammary gland has been extensively studied as gland. Hence the mammary gland is particularly useful for a model for breast cancer, as well as a system to investigate the study of branching morphogenesis and functional the mechanisms underlying branching morphogenesis differentiation in tissues. The timing of pregnancy can be (reviewed in Cunha & Hom 1996, Daniel & Smith 1999, easily controlled. Moreover, there are ten glands that can Robinson et al. 1999). Development of the mouse mammary be analysed and, importantly, the mammary gland can be gland is usually considered to occur in three separate stages: physically and genetically manipulated without seriously embryonic, postnatal and postpubertal. Embryonic affecting the health of the animal. development is such that by birth a very rudimentary Mammary tissues show a remarkable regenerative branched ductal structure is present under the epidermis that capacity, a process that was recently demonstrated by lies adjacent to the presumptive mammary fat pad. During Kordon & Smith (1998). By using genetically marked the 6–7 weeks of postnatal development the rudimentary epithelial cells and serial transplantation they were able to ducts extend and branch into the underlying fat pad to form show that a single cell could eventually generate an entire a simple tree-like structure. After puberty, the virgin gland mammary gland. Hence an understanding of the properties of may undergo small changes with the appearance of a few this potential mammary stem cell, and its more differentiated alveoli stimulated by the hormonal fluctuations of the daughters may have important implications regarding the oestrous cycle. However, a substantial part of mammary characteristics of cells that become targets for neoplastic gland development occurs in the adult female. This can be transformation. essentially subdivided into four phases (reviewed in Daniel & In mammals, fibroblast growth factors (Fgfs) are a large Silberstein 1987). The first, which proceeds through early to multigene family that transduce signals by activating the mid-pregnancy is characterised by epithelial cell proliferation cytoplasmic tyrosine kinase of transmembrane receptors and results in further branching of the ductal tree and the (reviewed in McKeehan et al. 1998, Ornitz 2000). Although appearance along the ducts of alveoli. During the second their name implies that they can induce the proliferation of phase, from mid- to late pregnancy, the alveoli increase in fibroblasts, collectively Fgfs are broad-spectrum mitogens density to form lobules, and the systemic rise in lactogenic acting on cells of ectodermal, endodermal and mesenchymal hormones initiates their functional differentiation into origin. They also act as cell survival factors and motogens as milk-producing units. At parturition, the rapid drop in well as inducing or inhibiting cell differentiation depending progesterone levels allows both synthesis and secretion of on context (Basilico & Moscatelli 1992, McKeehan et al. milk to reach maximum levels. Finally, after the young are 1998). The extracellular domain of the receptors (FgfR) for weaned, the glands involute. This is characterised by these ligands is composed of two or three Ig-like loops, with Endocrine-Related Cancer (2001) 8 211–217 Online version via http://www.endocrinology.org 1351-0088/01/008–211 2001 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 12:41:43PM via free access Spencer-Dene et al.: Fgfs and mammary gland development loops II and III configured to form the Fgf-binding domain. behaviours. In vivo studies suggest these Fgfs must function However, signal transduction is complicated by the need for in concert with other secreted factors to co-ordinate the a co-receptor in the guise of a proteoglycan, where the growth and differentiation of cell masses during organ glucosaminoglycan side chains function to form a morphogenesis as recently reviewed (Hogan & Yingling heteromeric complex with the Fgf and FgfR (reviewed in 1998). Johnson & Williams 1993, Ornitz 2000). There are four receptor genes that encode, by means of alternative splicing, FgfR2-IIIb signalling is implicated in a number of different receptor isoforms. For example, FgfR1, lobuloalveolar development FgfR2 and FgfR3 each encode two isoform sets that show distinct Fgf binding properties. Moreover, the different A crucial part of FgfR signal transduction is the receptor isoforms are expressed in different cell lineages ligand-mediated dimerisation of the receptor. This leads to (Ornitz 2000). Each of these sets is designated after the an activating transphosphorylation of the kinase domain and isoform specific exon which it encodes, IIIb and IIIc (Fig. concomitant signalling through associated second 1). The receptor FgfR2-IIIb is expressed on many types of messengers. Thus if kinase deleted receptor mutants are epithelium, while FgfR2-IIIc occurs on cells of mesenchymal introduced into cells that constitutively express the wild-type origin (Peters et al. 1992, Orr-Urtreger et al. 1993). The Fgfs receptor, the latter can be sequestered into inactive that signal through the epithelial receptor FgfR2-IIIb are heteromeric complexes with the truncated receptor. predominantly expressed in the adjacent mesenchyme, Therefore, by expressing an excess of mutant receptor in a thereby providing the basis for instructive paracrine target cell population, signalling through the wild-type signalling (Finch et al. 1989). This process is of paramount receptor can be inhibited. To assess the role of Fgf signalling importance during organ morphogenesis. Therefore, the Fgfs in postpubertal mammary gland development, dominant can be considered as intercellular signalling molecules negative forms of two different FgfRs were expressed in the capable of mediating a variety of properties and cell mammary epithelium (Jackson et al. 1997). The aim was to Figure 1 Schematic depiction of Fgf signalling and its abrogation by dominant negative receptors. On the left is the structure of an Fgf receptor, indicating the three Ig-like loops, and the region of alternative splicing in the third Ig-loop which confers ligand specificity. In conjunction with its interaction with the glucosaminoglycan side chains of proteoglycan, ligand binding causes heteromeric complex formation, autophosphorylation and subsequent signal transduction. On the right the depiction shows how the tyrosine kinase deleted receptor is able to sequester functional receptor into inactive complexes, thus reducing signal transduction in the targeted cells. 212 Downloadedwww.endocrinology.org from Bioscientifica.com at 09/29/2021 12:41:43PM via free access Endocrine-Related Cancer (2001) 8 211–217 inhibit autocrine signalling within the epithelium as well as Fgf10 as the prime candidate. However, the lack of a to disrupt paracrine signalling from the mammary mammary gland phenotype in the absence of Fgf7, might mesenchyme (see Fig. 1 and Jackson et al. 1997). As the merely reflect functional redundancy among the Fgfs. Both formation of wild-type and mutant receptor dimers requires Fgf7 and Fgf10 are normally expressed in mesenchymal receptor interaction with ligand, dominant negative tissue such as the dermis, so that both these growth factors constructs were selected for their ability to bind a wide range would have the potential to signal to the mammary of Fgfs. Accordingly, FgfR1-IIIc, which can be activated by epithelium in a paracrine manner. Nevertheless, it is not clear Fgf1, Fgf2, Fgf4, Fgf6, Fgf8, and FgfR2-IIIb, which can be what function such signalling would play in the adult activated by Fgf1, Fgf3, Fgf7 and Fgf10, were chosen (Ornitz mammary gland during pregnancy, nor is it clear how this et al. 1996). These mutant receptors, lacking sequences signalling might be regulated. Fgf signalling may act to encoding the tyrosine kinase domain, were each introduced stimulate the proliferation of the mammary epithelium, into the mouse germ line as transgenes expressed from a contributing directly to lobuloalveolar growth, however it mouse mammary tumor virus promoter (Jackson et al. 1997). could also function as a cell survival stimulus. For example, Examination of mammary glands from mice expressing a this latter property of FgfR2-IIIb signalling is particularly dominant negative FgfR1-IIIc receptor showed no discernible important in early limb