Development 126, 221-228 (1999) 221 Printed in Great Britain © The Company of Biologists Limited 1998 DEV2331 Conserved regulation of mesenchymal gene expression by Fgf-8 in face and limb development Abigail S. Tucker1, Abdul Al Khamis1, Christine A. Ferguson1, Ingolf Bach2, Michael G. Rosenfeld2 and Paul T. Sharpe1,* 1Department of Craniofacial Development, GKT Dental Institute, Kings College, Guy’s Hospital, London SE1 9RT, UK 2Howard Hughes Medical Institute, Department and School of Medicine, University of California at San Diego, La Jolla, California, USA *Author for correspondence (e-mail: [email protected]) Accepted 5 November; published on WWW 14 December 1998 SUMMARY Clim-2 (NLI, Lbd1) is one of two related mouse proteins show that in the mandibular arch, as in the limb, Fgf-8 that interact with Lim-domain homeoproteins. In the functions in combination with CD44, a cell surface binding mouse, embryonic expression of Clim-2 is particularly protein, and that blocking CD44 binding results in pronounced in facial ectomesenchyme and limb bud inhibition of Fgf8-induced expression of Clim-2 and Lhx-6. mesenchyme in association with Lim genes, Lhx-6 and Regulation of gene expression by Fgf8 in association with Lmx-1 respectively. We show that in common with both CD44 is thus conserved between limb and mandibular arch these Lim genes, Clim-2 expression is regulated by signals development. from overlying epithelium. In both the developing face and the limb buds we identify Fgf-8 as the likely candidate Key words: LIM homeodomain, Clim-2, NLI, Ldb-1, Fgf-8, CD44, signalling molecule that regulates Clim-2 expression. We Branchial arch, Limb, Mouse INTRODUCTION thought to be similar to, or the same as Lhx-8 (L3)), are largely restricted to anterior mesenchymal cells of the mandibular and Early embryonic development of the facial processes and limb maxillary arches (Grigoriou et al., 1998; Wanaka et al., 1997; buds has been considered to share some common basic Kitanaka et al., 1998). Removal of the oral epithelium results mechanisms despite the mesenchymal cells having different in a downregulation of Lhx-6/-7 expression, which can be embryonic origins. The mesenchymal cells that contribute to rescued by the addition of Fgf-8 protein on beads (Grigoriou et oral and facial hard tissues are derived from cranial neural crest al., 1998). Since Fgf-8 is normally expressed in oral epithelium cells whereas limb mesenchyme cells are derived from axial it is an ideal candidate for the endogenous inducer of Lhx-6/-7. mesoderm. The outgrowth of facial processes has been Limb bud epithelium, a source of Fgf-8, is capable of inducing compared with limb bud outgrowth, and the tooth bud enamel Lhx-6/-7 expression in branchial arch mesenchyme cells but not knot has been identified as a signalling centre with similarities in limb bud mesenchyme cells, indicating that the neural crest to both the limb ZPA and AER signalling centres (Koyama, et origin of the mesenchymal cells determines their ability to al., 1996; Jernvall et al., 1994; Vaahtokari et al., 1996). In activate Lhx-6/-7 expression in response to Fgf-8 (Tucker et al., addition, several homeobox-containing genes have been 1999). The expression of Lmx-1 is restricted to the dorsal implicated in both branchial arch and limb development, such mesenchymal cells of the limb bud. Removal of the dorsal limb as members of the Msx, Dlx and Lim-homeobox families (Chen bud epithelium results in downregulation of Lmx-1 expression, et al., 1996; 1997; Bulfone et al., 1993; Ferrari et al., 1995; Qiu which can be rescued by ectopic expression of Wnt-7a, a gene et al., 1995; Grigoriou et al., 1998; Riddle et al., 1995). Msx-1 normally expressed in dorsal epithelium (Riddle et al., 1995). expression, for example, has been shown to be maintained in Overexpression of Lmx-1 results in limbs with a double dorsal face to limb mesenchyme grafts (Brown et al., 1993). phenotype, while loss of Lmx-1 expression, as seen in Wnt-7a The Lim domain is a specialised double-zinc finger motif mutant embryos, results in limbs with a double ventral found in a variety of proteins. Lim domains are thought to phenotype (Vogel et al., 1995; Cygan et al., 1997). Restricted function as protein interaction modules, and have important expression of both Lhx-6/-7 in facial mesenchyme and Lmx-1 functions in cell lineage determination and pattern formation in limb bud mesenchyme are thus under the control of specific (Dawid et al., 1998). Regulation of the Lim-homeobox genes but different signals from the overlying epithelium. in the face and limb has been shown to be under the control of In the mouse, the Clim family of proteins have been the epithelium. The expression of the Lim-domain gene Lhx-6, identified as interacting (co-factors) with Lim-domain proteins and its closely associated family member Lhx-7 (which is (Bach et al., 1997). Clim-2 (cofactor of Lim homeodomain 222 A. S. Tucker and others proteins 2) was identified by its ability to bind to the Lim were detected was considered as E0.5. Cultures were carried out at domain of Lhx-3 (Bach et al., 1997). It is also known in the E10.5. Mandibular arch and forelimb buds were dissected in D-MEM. mouse as Ldb-1 and NLI (Agulnick et al., 1996; Jurata et al., The epithelium was removed using Dispase in calcium- and 1996). Homologues are found in Xenopus (Xldb-1), Zebrafish magnesium-free PBS at 2 units per ml, for 10 minutes at 37°C. After (Zfldb-1-4) and Drosophila (Chip) (Agulnick et al., 1996; incubation the tissues were washed in D-MEM with 10% fetal calf Toyama et al., 1998; Morcillo et al., 1997), all of which have serum (FCS), and the epithelium was dissected using fine tungsten needles. The mesenchyme was placed on membrane filters supported been characterised by their interactions with Lim proteins. α by metal grids according to the Trowel technique as modified by Clim-2 is required for the synergistic activation of the GSU Saxén (Trowel, 1959; Saxén, 1966). For the recombinations, gene by other Lim homeodomain genes, but is unable to epithelium and mesenchyme were aligned in the correct orientation activate transcription itself (Bach et al., 1997). Thus Clim-2 is on top of transparent Nucleopore membrane filters (0.1 µm pore believed to be an essential co-activator of Lim protein function. diameter; Costar). For the bead experiments, limb buds and From work in Xenopus, it has been shown that Xldb-1 binds mandibular arches were placed on top of Millipore filters (0.1 µm pore the Lim domains through its carboxyl-terminal group and diameter) and beads added. For both these experiments it was forms homodimers via its amino-terminal region. Both regions important that the mandibular arch mesenchyme was facing oral side are necessary for the synergism of Ldb1 with Xlim-1 in the up. activation of downstream genes (Breen et al., 1998). For the Fgf and BSA control protein heparin, acrylic beads (Sigma) were used. These were washed several times in PBS, then incubated Misexpression of Clim-2 alone when injected into Xenopus overnight at 4°C in 1 µg/µl Fgf-8b, or Fgf-9 (R & D systems). For embryos had no obvious effect on development, whereas co- the BMP2 and 4 proteins, Affi-Gel-blue beads (Bio Rad) were used injection with Lhx-1 resulted in partial axis duplication, (Genetics Institute). These were washed and dried out before being indicating again that Clim-2 acts as a co-factor for LIM- placed in the protein for 1 hour at 37°C. The BMPs were used at a domain proteins (Agulnick et al., 1996). Similarly in chick concentration of 100 µg/ml, a concentration which has been shown to limb development, misexpression of Clim-2 using retroviruses induce Msx-1 and repress Pax-9 expression in a similar assay (Tucker has no effect on development (Bach et al., unpublished). et al., 1998; Neubüser et al., 1997). Protein-soaked beads were stored In the mouse, Clim-2 is detected from E8.5, at the time when at 4°C for up to 3 weeks. the Lim homeodomain genes are first starting to be expressed. The explants were cultured for 4-24 hours in D-MEM with 10% At E9.0-E9.5, high expression is seen in the brain, neural tube, FCS. A standard incubator was used at 37°C with an atmosphere of 5% CO2 in air and 100% humidity. All solutions used contained the forming branchial arches and limbs, overlapping with the penicillin and streptomycin at 20 IU/ml. After the period of culture, expression patterns of several Lim domain genes including, cultures were washed in ice-cold methanol for 1 minute then fixed in Lhx-2 (LH-2), Lhx-3 (P-Lim), Lhx-6, Lhx-7 (Lhx-8) and Lmx1 fresh 4% paraformaldehyde for 1 hour at RT. Cultures were then (Bach et al., 1997, Grigoriou et al., 1998; Wanaka et al., 1997; prepared for whole-mount digoxigenin in situ hybridisation. Kitanka et al., 1998; Riddle et al., 1995). We show that in common with Lhx-6 and Lmx-1, the epithelium is required for CD44 blocking experiments Clim-2 expression in underlying mesenchyme. However Clim- Digested oligosaccharide fragment preparations of hyaluronic acid 2 differs from the Lim-domain genes in that its expression is (HAOS) were prepared by treating hyaluronan with testicular regulated by the same signal, Fgf-8, in both the limb buds and hyaluronidase (40U/mg hyaluronan) for 4 hours in 0.2 M sodium mandibular arches.