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The Spemann organizer secretes several antagonists of growth factors during gastrulation. We describe a novel secreted protein, Mig30, which is expressed in the anterior endomesoderm of the Spemann organizer. Mixer-inducible gene 30 (Mig30) was isolated as a target of Mixer, a homeobox gene required for endoderm development. The Mig30 gene encodes a secreted protein containing a cysteine-rich domain and an immunoglobulin-like domain that belongs to the insulin-like growth factor-binding protein family. Overexpression of Mig30 in the dorsal region results in the retardation of morphogenetic movements during gastrulation and leads to microcephalic embryos. Overexpression of Mig30 also inhibits activin-induced elongation of ectodermal explants without affecting gene expression patterns in mesoderm and endoderm. These results suggest that Mig30 is involved in the regulation of morphogenetic movements during gastrulation in the extracellular space of the Spemann organizer.
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11850177
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Fig. 3. Patterns of expression of Mig30 mRNA and Mig30 protein. (A–F) Mig30 expression during early Xenopus development. (A) Dorsal view of the late blastula (stage 9). No blastopore pigment is seen. (B) Vegetal view of early gastrula (stage 10). Dorsal is up. (C) Vegetal view of early gastrula (stage 10+). Dorsal is up. (D) Dorsal view of late gastrula (stage 12). Anterior is up. (E) The same embryo in (D) was hemisectioned sagittally. Dorsal is to the left. Anterior is up. (F) Lateral view of the tailbud (stage 31). (G–I) Mig30 expression of manipulated gastrula embryos (stage 10+). Embryos were cleared in Murray's clearing solution. (G) Vegetal view of an untreated control embryo. Dorsal is up. (H) An embryo dorsalized by LiCl treatment (I). An embryo ventralized by UV irradiation. (J–L) Localization of Mig30 protein. (J) Dorsal view of an early gastrulaembryo (stage 10+). (K) An embryo incubated with normal rabbit IgG as primary antibody (stage 10+). (L) Vegetal view of an embryo coincubated with anti-Mig30 antibody and antigen peptide. Dorsal is up.
Fig. 7. Effects of overexpression of Mig30 on gene expression during gastrulation. Mig30 mRNA, 500 pg, was injected into two dorsal blastomeres at the four-cell stage. Gene expression was analyzed by in situ hybridization. Left panels show control embryos. Right panels show Mig30-injected embryos. The stage is indicated at the bottom right of each panel. (A–D) Xbra expression. (E–H) chordin expression. (I–L) Frzb expression. The arrowhead indicates the position of the dorsal blastopore lip.
Fig. 1. Isolation of genes activated by Mixer-GR. (A) Screening procedure for genes activated by Mixer-GR in the presence of DEX. The details are described in Section 4. (B) Screening of Mixer-GR-inducible genes by virtual Northern blot analysis. cDNA, 0.5 μg, was loaded in each lane. (C) Mig30 expression by Mixer does not require protein synthesis. Mixer-GR mRNA, 50 pg, was injected into the animal pole region at the one-cell stage. Ectodermal explants were prepared at the late blastula stage. To inhibit protein synthesis, CHX was added to the medium 30 min before addition of activin (100 ng/ml) or DEX, and the explants were then cultured for 2.5 h. Expression of Mig30 and Sox17alpha was analyzed by RT-PCR. ODC serves as a loading control. WE10.5, sibling whole embryo; WE-RT, control reaction without reverse transcription.
Fig. 2. Mig30 belongs to the IGFBP family. (A) Amino acid sequence alignment of Mig30 and mouse IGFBP-like protein. There is 32% similarity. Boxes show identical residues. Asterisks show conserved cysteine residues. (B) IGD possesses similarity to the IGD of the axon guidance receptor, human roundabout 1. (C) A schematic drawing of the structure of Mig30 protein. SP, signal peptide. (D) Mig30 protein is secreted. Mig30 mRNA was injected into oocytes, and the medium or cell extract was analyzed by Western blot using anti-Mig30 antibody. Mig30 protein was detected in the cell extract (lane 5) and in the medium (lane 6). Mig30-δSS was detected in the cell extract (lane 3), not in the medium (lane 4). Secreted Mig30 protein under non-reducing conditions is shown in lane 7. No signals were detected in the cell extract (lane 1) or in the medium (lane 2) of uninjected oocytes.
Fig. 5. Overexpression of Mig30 inhibits head development. (A) Embryos (stage 40) injected with 500 pg Mig30 mRNA into two dorsal blastomeres at the four-cell stage. Anterior structures are affected. (B) Expression pattern of Xotx5 in embryos (stage 31) injected with 500 pg Mig30 mRNA into all blastomeres at the four-cell stage. The embryo at the top was not injected. (C) An embryo injected with 250 pg IGD mRNA into two dorsal blastomeres at the four-cell stage, showing the APDI 1 phenotype. (D) Enlarged view of the bottom embryo in (E), showing abnormal retina formation. (E) Embryos were injected with 250 pg CRD mRNA into two dorsal blastomeres at the four-cell stage. The head is slightly enlarged compared with the control embryo. The embryo at the top was not injected. (F) Coinjection of Mig30 mRNA abolished the effect of CRD mRNA.
Fig. 6. Positional relationship between midline structures and eye field in Mig30-injected embryos. After injecting 500 pg Mig30 mRNA into two dorsal blastomeres at the four-cell stage, the embryos were analyzed by in situ hybridization for expression of shh and Rx1. shh and Rx1 demarcate the midline structures and eye field, respectively. (A–D) Control embryos. (E–H) Mig30-injected embryos. (A,E) Stage 13. (B,F) Stage 15. (C,G) Stage 17. (D,H) Stage 19.
Fig. 8. Overexpression of Mig30 can inhibit convergent extension movements. (A–G) Mig30 mRNA was injected into all blastomeres in the animal pole region at the four-cell stage. The animal caps were cut at late blastula stage, exposed to activin (5 ng/ml) for 3 h. Elongation of the animal caps was observed 15–17 h after dissection. (A) Uninjected animal caps were left untreated. (B) Those treated with activin showed elongation. (C–F) Animal caps injected with 250 pg Mig30 mRNA (C), 500 pg Mig30 mRNA (D), 1 ng Mig30 mRNA (E) or 1 ng GFP mRNA (F) were treated with activin. The caps injected with Mig30 mRNA showed no elongation, whereas those injected with GFP mRNA elongated. (G) RT-PCR analysis of expression of mesodermal or endodermal markers in animal caps injected with Mig30 mRNA. Markers were analyzed at stage 10. (H,I) Cell lineage analysis. (H) Embryos were injected with 200 pg of β-gal mRNA into two dorsal blastomeres at the four-cell stage. The embryos were fixed at early neurula stage (stage 16) and β-gal activity was visualized. The β-gal-positive cells are concentrated on the dorsal midline. (I) Embryos coinjected with 200 pg of β-gal and 500 pg of Mig30 mRNA. β-gal-positive cells spread mediolaterally and showed little elongation along the anteroposterior axis. Bottom is an embryo in which the blastopore failed to close.
