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Abstract Fibroblast growth factors (FGFs) signal through high-affinity tyrosine kinase receptors to regulate a diverse range of cellular processes, including cell growth, differentiation and migration, as well as cell death. Here we identify XFLRT3, a member of a leucine-rich-repeat transmembrane protein family, as a novel modulator of FGF signalling. XFLRT3 is co-expressed with FGFs, and its expression is both induced after activation and downregulated after inhibition of FGF signalling. In gain- and loss-of function experiments, FLRT3 and FLRT2 phenocopy FGF signalling in Xenopus laevis. XFLRT3 signalling results in phosphorylation of ERK and is blocked by MAPK phosphatase 1, but not by expression of a dominant-negative phosphatidyl inositol 3-OH kinase (PI(3)K) mutant. XFLRT3 interacts with FGF receptors (FGFRs) in co-immunoprecipitation experiments in vitro and in bioluminescence resonance energy transfer assays in vivo. The results indicate that XFLRT3 is a transmembrane modulator of FGF-MAP kinase signalling in vertebrates.
Figure 1 Structure and expression pattern of XFLRT3. (a), Schematic
representation of XFLRT3. Signal peptide (red box); LRRNT, N-terminal
leucine-rich repeat cysteine flank; LRR, leucine-rich repeats; LRRCT,
C-terminal leucine-rich repeat; FNIII, fibronectin domain type III; TM,
transmembrane domain. (b) FLRT protein homology tree showing relationship
between Xenopus laevis, Fugu rubripes, mouse and human FLRT proteins.
(c) mRNA encoding Myc-tagged XFLRT3 was microinjected animally into all
blastomeres of a four-cell Xenopus embryo. Animal caps of stage-10 embryos
were prepared for immunofluorescence microscopy with an anti-Myc
antibody. Note labelling of the plasma membrane. (d–i) Expression pattern of
XFLRT3 (d–f) and XFGF8 (g–i) in Xenopus gastrulae (d, g, vegetal view),
neurulae, (e, h, anterior view) and tadpoles (f, i). (j, k) Regulation of XFLRT3
by FGF. In j, four-cell embryos were uninjected or microinjected into each
blastomere with 0.025, 0.125 or 0.25 ng XFD mRNA. Ventral marginal zone
(VMZ) fragments were cut from early gastrulae and analysed for expression of
XFLRT3 and Xbra at stage 11 by RT–PCR. −RT, minus reverse transcription
control sample; H4, histone H4 for normalization. In k, eight-cell embryos
were microinjected into all four animal blastomeres with increasing doses of
eFGF mRNA (1.25, 12.5 or 25 pg per blastomere) or FGF8 mRNA (2.5 or
20 pg per blastomere). Animal caps were excised from blastula embryos,
cultivated until stage 10 and analysed by RT–PCR for gene expression. The
‘Embryo’ lane indicates whole embryos.
Figure S4 Expression pattern of XFLRT2 mRNA during Xenopus laevis
development. a-e, Whole-mount in situ hybridization of XFLRT2. Expression
is first seen anteriorly at early neurula stages (a, stage 14, anterior view). b,
Stage 18, animal view. c, Stage 24, lateral view. XFLRT2 is predominantly
expressed in the head. At tailbud stages, stage 32 (d) and stage 36 (e),
XFLRT2 transcripts are detected in the head and in the somites. f, RT-PCR
analysis of XFLRT3 and XFLRT2 expression in embryos of the indicated
stages. -RT, minus reverse transcription control sample; H4, histone H4 for
normalization. g, Sequence of the FLRT2 morpholino target region. An
alignment of Xenopus laevis (X.l. FLRT2) and tropicalis (X. trop. FLRT2)
FLRT2 nucleotide sequences is shown. A XFLRT2 pseudoallel was also
identified (not shown) whose sequence is identical in the XF2(b)-MO target
region but has two base-pair mismatches in the XF2(a)-MO target region.