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Development 127, 2563-2572 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 DEV4343
FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development Helen P. Makarenkova1,*, Masataka Ito1,*, Venkatesh Govindarajan2, Sonya C. Faber, Li Sun3, Gerald McMahon3, Paul A. Overbeek2 and Richard A. Lang1,‡ 1Skirball
Institute for Biomolecular Medicine, Developmental Genetics Program, Cell Biology and Pathology Departments, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA 3SUGEN, Inc., 230 East Grand Ave., South San Francisco, CA 94080-4811, USA *These authors have contributed equally to this publication ‡Author for correspondence (e-mail:
[email protected])
Accepted 11 April; published on WWW 23 May 2000
SUMMARY We investigated the mechanism of tissue induction and specification using the lacrimal gland as a model system. This structure begins its morphogenesis as a bud-like outgrowth of the conjunctival epithelium and ultimately forms a branched structure with secretory function. Using a reporter transgene as a specific marker for gland epithelium, we show that the transcription factor Pax6 is required for normal development of the gland and is probably an important competence factor. In investigating the cell-cell signaling required, we show that fibroblast growth factor (FGF) 10 is sufficient to stimulate ectopic lacrimal bud formation in ocular explants. Expression of FGF10 in the mesenchyme adjacent to the presumptive lacrimal bud and absence of lacrimal gland development in
FGF10-null mice strongly suggest that it is an endogenous inducer. This was supported by the observation that inhibition of signaling by a receptor for FGF10 (receptor 2 IIIb) suppressed development of the endogenous lacrimal bud. In explants of mesenchyme-free gland epithelium, FGF10 stimulated growth but not branching morphogenesis. This suggested that its role in induction is to stimulate proliferation and, in turn, that FGF10 combines with other factors to provide the instructive signals required for lacrimal gland development.
INTRODUCTION
(FGF) families (Mason et al., 1994). In particular, FGF10, also known as keratinocyte growth factor-2 (KGF2), has been implicated in budding outgrowth of epithelia (Bellusci et al., 1997b; Ohuchi et al., 1997; Sekine et al., 1999). Both FGF7 and FGF10 can bind to and signal through fibroblast growth factor receptor-2 (FGFR2 (Ornitz et al., 1996)). The mouse FGFR2 gene has alternative splicing for exons 8 and 9 and this results in an alternative amino-acid sequence in the C-terminal half of the third immunoglobulin fold of the extracellular domain. The IIIb (exon 8) isoform is known as the KGF receptor and the IIIc isoform as bek (Johnson and Williams, 1993; Xu et al., 1998). bek binds with high affinity to FGF1, FGF2, FGF4 and FGF5 but not FGF7 (Ornitz et al., 1996). In contrast, the KGFR binds FGF7 and FGF10 as well as FGF1; the binding of FGF2 to the KGFR is at very low affinity (Miki et al., 1992; Lu et al., 1999). The observation that expression of the KGFR is restricted to epithelial lineages and that bek is found only in mesenchymal cells (Orr-Urtreger et al., 1993b; Iseki et al., 1997; Xu et al., 1998) indicates that tissue-specific alternative splicing is one way to provide FGF signaling specificity. The Pax6 gene is expressed in many tissues of the eye
Development of the lacrimal gland is an example of an epithelial-mesenchymal interaction. In the mouse, a single budlike invagination of the conjunctival epithelium at the temporal extremity of the eye is the initial sign of lacrimal gland formation (Kammandel et al., 1999). The mesenchymal cells that surround the point of epithelial budding are the periocular cells, of neural crest origin (Johnston et al., 1979). The tubular invagination of the lacrimal gland extends and branches multiple times to give the lobular structure of the mature gland (Kammandel et al., 1999). The type of morphogenesis (Hogan, 1999) that accompanies development of the lacrimal gland has been studied in detail in several other organ systems, including the limb (Martin, 1998), the lung (Peters et al., 1994; Hogan and Yingling, 1998; Weaver et al., 1999) and teeth (Peters and Balling, 1999). As a result of these analyses, a selection of soluble signaling molecules has been implicated in generating morphology of this type. These include sonic hedgehog (Bellusci et al., 1997a; Pepicelli et al., 1998) and members of the bone morphogenetic protein (BMP) (Graff, 1997) and fibroblast growth factor
Key words: FGF, FGF10, Budding morphogenesis, Paracrine induction, Lacrimal gland, Pax6
2564 H. P. Makarenkova and others (Grindley et al., 1995). Recent analysis has identified a conserved transcriptional enhancer that, in the context of reporter transgenes, is necessary and sufficient for directing expression to the lens placode, the lens and corneal epithelium and, subsequently, to the epithelium of the lacrimal gland (Williams et al., 1998; Kammandel et al., 1999). Pax6 is a member of the group of transcription factors that has both paired and homeodomain DNA binding motifs (Mansouri et al., 1994). It has been broadly implicated in eye development with the demonstration that Drosophila, mouse and human eye defects are a consequence of mutations in Pax6 (Quiring et al., 1994). Furthermore, in both invertebrates (Halder et al., 1995) and vertebrates (Chow et al., 1999), Pax6 can direct the formation of ectopic eyes. In this study, we have taken advantage of a Pax6-based reporter transgene (Williams et al., 1998) to study the mechanism of lacrimal gland development. We have shown, using the Small eye mouse, that normal lacrimal gland development requires a wild-type level of Pax6 expression in conjunctival epithelium. In addition, we demonstrate that signaling through FGFR2IIIb is necessary for lacrimal gland induction and in the context of ventral periocular mesenchyme, is sufficient. This is consistent with the localized expression of FGF10 in the periocular mesenchyme adjacent to Pax6expressing presumptive lacrimal gland epithelium, and suggests a model in which FGF10 provides a paracrine stimulus for outgrowth of the lacrimal gland bud. MATERIALS AND METHODS Histological analysis Paraffin sections were prepared and stained either with Hematoxylin and Eosin or only Hematoxylin, using conventional methods. Explant cultures Explant cultures were prepared from embryonic day (E)12.5-14.5 fetuses of the P6 5.0 lacZ reporter line. The whole eye, together with ectoderm and mesenchyme from one side of the head, was excised and placed on a filter (0.8 µm pore, Millipore) supported by a stainless steel grid. Explants were cultured in CMRL-1066 medium (Gibco BRL) supplemented with heat-inactivated 10% fetal calf serum, 2% rat serum, glutamine, non-essential amino acids and an antibioticantimycotic (Gibco BRL). After culture for 48 hours, tissues were fixed and stained with X-gal according to established protocols (Song et al., 1996). Mesenchyme-free explants were generated from E16.5E17.0 lacrimal glands according to established procedures (Shannon et al., 1999). The tips of developing buds were cultured in defined medium (Zuniga et al., 1999) alone or defined medium supplemented with FGF2 or FGF10 at 80 ng/ml. FGF-loaded beads were used as previously described (Cohn et al., 1995). Briefly, heparin acrylic beads (Sigma) of 80-120 µm diameter were washed in PBS and immersed in PBS containing BSA with or without recombinant FGFs. Beads were incubated with BSA or FGF solution (1 mg/ml) at 4°C overnight. Human recombinant FGF7 and 10 were obtained from R&D systems while human recombinant FGF2 was a gift from D. Rifkin. For bead implantation, an explant was punctured through the ectoderm near the conjunctival epithelium with a tungsten needle, and a bead was inserted into the mesenchyme using blunt forceps. Antisense experiments The sense and antisense oligonucleotides used have been described previously (Miralles et al., 1999) and were used at final concentration
10 µM in the presence of Lipofectamine-2000 (Gibco BRL) in a 30% F 127 pluronic gel (Makarenkova and Patel, 1999). An 8 µl drop of oligonucleotide/Lipofectamine/pluronic gel was placed on the surface of the ocular region explants and at the temporal edge of the eye. In each experiment, one side of embryo was treated with sense and the other side with antisense. The statistical significance of the difference in control and experimental explant responses was assessed using the nonparametric Wilcoxon signed-rank test (Ostle and Mensing, 1975). P
