April 1, 1999;
A new secreted protein that binds to Wnt proteins and inhibits their activities.
The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1
), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1
is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm
, branchial arches
and neural crest
derivatives. We use Xenopus embryos to show that WIF-1
overexpression affects somitogenesis (the generation of trunk mesoderm
segments), in agreement with its normal expression in paraxial mesoderm
. In vitro, WIF-1
binds to Drosophila Wingless and Xenopus Wnt8
produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled
-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.
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FIGURE 2. Expression of WIF-1 in the adult mouse and during Xenopus and zebrafish development.a, RNase protection assay using a mWIF-1 probe (top) or a mouse RNA polymerase II large-subunit probe (bottom); B, brain; E, eye; H; heart; K, kidney; Li, liver; Lu, lung; S, spleen; T, testis; Y, yeast tRNA. b–j, WIF-1 mRNAs detected by in situ hybridization. b–f, Xenopus; g–j, zebrafish. Expression is first detectable at the onset of somitogenesis in paraxial mesoderm (b, g). Expression is prominent in unsegmented presomitic mesoderm (psm), and is much weaker in newly formed somites (som; c, d, h, i). WIF-1 is expressed in the notochord (not) of 15-somite-stage zebrafish embryos (h) and stage-24 Xenopus embryos (d) in register with mature somites; no expression is detected in notochord regions flanked by unsegmented paraxial mesoderm. This pattern persists until the late stages of somitogenesis (e, i). In Xenopus tadpoles, strong WIF-1 expression is seen in visceral arches (va), and also in otic vesicles (ov), nasal placodes (npl) and several regions of the anterior brain (f). In the 24-h zebrafish embryo, expression is seen in the epiphysis (ep) and ventral midbrain (vmb) (j).
FIGURE 3. WIF-1 inhibits Wnt signaling and affects somite formation in Xenopus embryos. a–f, WIF-1 dorsalizes Xenopus embryos. hWIF-1 RNA (500 pg per blastomere) was injected into dorsal blastomeres (a, c, e) or ventral blastomeres (b, f) at the 4-cell stage. Dorsal injections led to anteriorization and hyperdorsalization of the embryos, as revealed by the formation of an enlarged head at the tadpole stage (a), an expanded cement gland (cg) field at the neurula stage (c), and an enlarged notochord visible in sections (e; control embryo in d). Ventral injections led to the formation of a partial secondary axis in 20% of the embryos (b) containing muscle (mus) and neural tissue (nt) (f). g–i, WIF-1 synergizes with chordin to generate a complete secondary axis (Table 1). g, Co-injection of chordin and Xenopus sFRP-3/Frzb RNAs gave rise to embryos with a complete but cyclopic secondary axis. h, By contrast, co-injection of chordin and hWIF-1 gave rise to embryos with a complete secondary axis; two separated eyes are evident. i, Co-injection of chordin and WD RNA does not promote two-eyed ectopic heads. j–p, WIF-1 affects the process of somite formation. 4-cell embryos were injected on one side with 100 pg pCS2+/hWIF-1 DNA and beta-galactosidase RNA. beta-Galactosidase activity is seen as brown dots and embryos were probed for XMyoD (j–n). The posterior domain of XMyoD expression in presomitic mesoderm was suppressed by hWIF-1 injection at the late neurula stage (j, dorsal view; k, posterior view). Segmentation is impaired by hWIF-1 injection, as revealed by XMyoD expression in mature somites of a stage-20 embryo (l) and a stage-26 embryo (m, n), and by a Hoechst-stained horizontal section of a stage-26 embryo (o, p). Note the disorganization of somites with occasional fusions (arrows in o). The process of somite rotation (rot) is delayed and myotome nuclei do not align properly (p).