October 1, 2000;
The Toll/IL-1 receptor binding protein MyD88 is required for Xenopus axis formation.
The Toll/Dorsal pathway regulates dorsoventral axis formation in the Drosophila embryo
. We had previously obtained evidence that a homologous pathway exists in Xenopus, however, its role during normal frog development had not been established. Here we report the cloning of Xenopus MyD88
), whose mammalian homologs are adaptor proteins linking Toll/IL-1 receptors and IRAK
kinases. We show that in the frog embryo
overexpression of a dominant-negative form of XMyD88
blocked Toll receptor activity, specifically inhibited axis formation and reduced expression of pivotal organizer
genes. The observed stage-dependency of interference suggests a function for maternal XMyD88
soon after fertilization. We conclude that XMyD88
activity is required for normal Spemann organizer
formation, implying an essential role for maternal Toll/IL-1 receptors in Xenopus axis formation.
[+] show captions
Fig. 1. Sequence and expression profile of Xenopus MyD88. Multiple sequence alignment (A) of frog, mouse and human MyD88 (ClustalW software); light shading – isofunctional amino acids; dark shading – identical residues. Red underlining marks the Death Domain, green underlining the Toll/IL-1 receptor (TIR) homology domain. Developmental expression of XMyD88 by Northern blot (B); in comparison to histone H4 levels, used as a loading control, oocytes contain high amounts of maternal XMyD88 mRNA. Whole-mount RNA in situ hybridization at initial gastrula stage (9 hours post fertilization [hpf]; animal pole up); (C) antisense probe; (D) sense probe.
Fig. 2. δNXMyD88 blocks axis rescue in UV-ventralized Xenopus embryos through the Drosophila Toll receptor and its ligand Spätzle, but not through the Drosophila Rel protein Dorsal. Embryos were scored on the dorsoanterior index scale (DAI; Kao and Elinson, 1988), on which 5 represents wildtype, and 0, fully ventralized embryos (for statistical analysis see Table 1). (A) Uninjected wild-type embryos; UV-ventralized siblings (B) were injected into two opposing blastomeres at 4-cell stage to generate two dorsalizing centers with: (C) Spätzle mRNA (500 pg); (D) Spätzle and δNXMyD88 mRNA (500 pg/100 pg). (E) Toll mRNA (500 pg); (F) Toll and δNXMyD88 mRNA (500 pg/100 pg). (G) Dorsal mRNA (500 pg); (H) Dorsal and δNXMyD88 mRNA (500 pg/100 pg). (I) Comparison of vertebrate and Drosophila Toll-like signaling pathways and their epistatic hierarchies.
Fig. 3. Phenotypic effects of δNXMyD88 mRNA on axis formation: embryos were injected dorsally into both blastomeres at two-cell stage, based on pigmentation differences between the prospective dorsal and ventral sides (see Section 4.2 for details). (A) Series of typical axis truncations induced by injection of δNXMyD88 mRNA (50 pg). From top to bottom: no effect (DAI 5), reduced forehead (DAI 4), cyclopia (DAI 3), microcephalic and headless (DAI 2). (B) Phenotypic spread of embryos: black, uninjected control embryos (n=234); red, embryos injected with 50 pg (n=162) or blue, injected with 1000 pg (n=210) δNXMyD88 RNA; green, rescue experiment with embryos coinjected with 50 pg δNXMyD88 RNA and 100 pg XMyD88 (n=117). (C) Phenotypic penetrance as a function of injection timing. X-axis indicates the developmental stage, at which δNXMyD88 RNA was injected. (D,E) δNXMyD88-injected embryos lacked notochord (MZ15 staining): (D) Control embryo; (E) δNXMyD88-injected embryo (DAI 2). (F–H) Sagittal sections through δNXMyD88-injected embryos at the level of the otic vesicle (ot, otic vesicle; nt, neural tube; not, notochord, so, somites): (F) Control embryo; (G) microcephalic embryo without notochord, impaired neural tube and somite structure; (H) cyclopic embryo with slightly aberrant somites. N≥3 independent experiments.
Fig. 4. Selective repression of organizer genes by dorsally injected δNXMyD88 RNA. (A–E) Normal expression in control embryos; (F–K) representative δNXMyD88-injected (300 pg) embryos, coinjected with 100 pg nuclear β-galactosidase RNA as lineage tracer; numbers to the right indicate phenotypic penetrance. (L) Quantitative RT-PCR analysis for direct target genes of maternal β-catenin; histone H4 serves as a control for the template amounts. (M) Listing of analyzed genes, which were found to be unaffected (sample size ≥20 embryos/gene). N=2–4 independent experiments.
Fig. 5. Model of the contribution of XMyD88-mediated signaling to early embryonic pattern in relation to other key regulatory pathways.
myd88 (myeloid differentiation primary response 88 ) gene expression in xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, lateral view, animal pole up.