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Dev Dyn
2001 May 01;2211:14-25. doi: 10.1002/dvdy.1121.
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A novel POZ/zinc finger protein, champignon, interferes with gastrulation movements in Xenopus.
Goto T
,
Hasegawa K
,
Kinoshita T
,
Kubota HY
.
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We have cloned a novel krüppel-like transcription factor of Xenopus that encodes POZ/zinc finger protein by expression cloning. Overexpression of mRNA resulted in interference with gastrulation. Because the injected embryo looks like a mushroom in appearance at the neurula stage, we have named this gene champignon (cpg). In cpg-injected embryos, the blastopore appeared normally, but regressed thereafter. The injected embryos then elongated along the primary dorsoventral axis during the tailbud stage. Histologic sections and reverse transcription-polymerase chain reaction analysis showed that cpg had no effect on the cell differentiation. The animal pole region of cpg-injected embryos was thick during the gastrula stage, and mesodermal cells remained in the marginal zone. Furthermore, neither Keller-sandwich explants nor activin-treated animal cap explants excised from cpg-injected embryos elongated. These results suggest that cpg acts as a potent inhibitor of cell migration and cell intercalation during gastrulation.
Figure 2. Temporal and spatial expression of cpg during Xenopus development. A: Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed by using 1 mu g of total RNA extracted from Xenopus embryos at different stages. Oo indicates the oocyte stage and numbers indicate the developmental stages. Maternal transcripts decreased gradually until the mid-blastula stage (st. 8), and zygotic expression increased after the initial gastrula stage (st. 10). B: Quantitative RT-PCR to determine spatial expression was performed by using total RNA extracted from different dissections of embryos at the mid-blastula stage (st. 8) and the initial gastrula stage (st. 10). cpg was expressed ubiquitously during these stages. C-F: Localization of cpg transcripts in Xenopus embryos after the neurula stage by whole-mount in situ hybridization. At the neurula stage (st. 16), cpg was expressed in the cement gland precursor region in C (ventral view) and D (anterior view). After the tailbud stage, cpg was expressed in the anterior region especially in the cement gland (arrowheads) in E (st. 25) and F (st. 30).
Figure 3. Overexpression of cpg interfered with gastrulation. A,C,E,G,I: Neurula stage embryos (st. 20). B,D,F,H,J: Tailbud stage embryos (st. 30). A,C,E: Upper, lateral view; anterior is to the left. Lower, blastopore view. G,I: Left, lateral view, anterior is to the left. Right, blastopore view. cpg mRNA (1 ng) was injected into the lateral equatorial regions of the two-cell embryo (C-F). cpg mRNA (1 ng) was injected into two dorsal (G,H) or two ventral (I,J) equatorial regions of the four-cell embryo. A,B: Control embryos. C,D: Embryos showing slightly defective phenotypes. Some of the injected embryos had a slightly opened blastopore in C and formed partial axial structures, including the head region in D. E,F: Embryos showing severely defective phenotypes. The blastopore of most of the injected embryos did not close but regressed during the neurula stage in E. The injected embryos elongated along the initial dorsoventral axis gradually during the tailbud stage and looked like flat layers of double sheets of the ectodermal and endodermal tissues in F. G,H: The morphology of the embryos that were injected into two dorsal regions at the four-cell stage was similar to that seen in E and F. I,J: Only ventral gastrulation was inhibited by overexpression of cpg in the ventral region.
Fig. 1. A: Alignment of the cpg, human c-krox, and mouse c-krox amino acid sequences. The amino acid residues that are identical between the proteins are shaded gray. The putative nuclear localization signals identified by using PSORT are boxed. The POZ domain at the N-terminus is indicated by an interrupted underline. The zinc finger domains at the C-terminus are underlined. B: Homology of the zinc finger domains of cpg to those of other kru ̈ ppel-like zinc finger proteins. The zinc finger domains are boxed. Asterisks indicate the cysteines and histidines of the zinc fingers.
Fig. 4. Midsagittal sections (A-D) and revers transcriptase chain reaction (RT-PCR) (E,F) of the control and cpg-injected em- bryos. cpg mRNA (1 ng) was injected into two lateral equatorial regions of the two-cell embryo. A: The control embryo (stage 11.5). B: A cpg- injected embryo at stage 11.5. Initial invagination occurred (arrowhead), but gastrulation did not proceed thereafter. Although cpg overexpression interfered with invagination, the yolky endodermal cells were displaced toward the animal pole region. The border between the ectodermal layer and inner cells was not clear. C: A cpg-injected embryo at stage 20 looks like a mushroom. The blastocoelic cavity has disappeared, and the three germ layers are clearly defined. D: A cpg-injected embryo at stage 30. Neural tube (Ne) and notochord (No) are shown apparently at the site where the blastopore first appeared. E,F: RT-PCR showed no significant effect of overexpression of cpg on the expression of the early mesoder- mal (Xbra, goosecoid, noggin, and Xwnt-8) and mesendodermal (siam- ois) marker genes at the gastrula stage (stage 11) in E, and the late neural (N-CAM and Otx-2) and mesodermal (MyoD and GATA-2) marker genes at the late neurula stage (stage 23) in F.
Fig. 5. Scanning electron microscopic images of cpg-injected em- bryos. cpg mRNA (1 ng) was injected into two lateral equatorial regions of the two-cell embryo, which was then fixed at the mid-gastrula stage (stage 11.5). A: Epiboly of the animal cap was inhibited, and the ecto- dermal layer of the animal pole was thick. B: The ectodermal layer of the lateral region became thin after lined with the yolky endodermal cells. C: Endodermal cells were scattered and did not closely adhere to each other. D: Normal-shaped bottle cells were formed.
Fig. 7. Both the POZ and zinc finger domains of cpg were necessary to function as a transcription factor. cpg- POZ (four independent experiments, n 80) and cpg- Zn (n 80), which lack a part of the POZ domain and zinc finger domain, respectively, did not interfere with gastrulation. cpg- (POZ-Zn) interfered with gastrulation and the phe- notype of cpg- (POZ-Zn)-injected embryos (n 80) was similar to that of cpg-injected embryos (n 80).
Fig. 6. cpg inhibited extension of Keller sandwich explants and the animal cap explants treated with activin. A: Keller sandwich explants from control embryos (four independent experiments, n 31). B: Keller sand- wich explants from cpg-injected embryos. cpg mRNA (1 ng) was injected into two lateral equatorial regions of the two-cell embryo. Dorsal sectors of cpg-injected embryos were excised at stage 10 and sandwiched. Overexpression of cpg caused a defect in the elongation (n 28). C: Animal cap explants from control embryos. The animal caps were ex- cised from stage 8 embryos and incubated for 6 hr with recombinant activin protein (10 ng/ml) and cultured in MSS until control embryos reached stage 20. A high dose of activin induced elongation of the animal cap (n 68). D: Animal cap explants from cpg-injected embryos. cpg mRNA (1 ng) was injected into the animal pole regions of both blas- tomeres at the two-cell stage, then explanted at stage 8, and treated with a high dose of activin (10 ng/ml) for 6 hr. cpg completely interfered with the elongation of the explants treated with a high dose of activin (n 73). E: Control animal cap explants without any treatment (n 63). F: cpg alone did not induce any morphologic changes and resulted in formation of spherical masses similar to those in E (n 25). G: RT-PCR of RNA extracted from the animal caps (stage 11) showed no significant effect of the overexpression of cpg on the expression of the mesodermal marker genes induced by activin (noggin, MyoD, and Xbra). H: cpg mRNA (1 ng) was injected into the lateral equatorial region of the two-cell embryo. RT-PCR showed no significant effect of overexpression of cpg on the expression of genes C-cadherin and PAPC, which are known to be related to the gastrulation movements.
Fig. 8. Expression of Xenopus fibronectin, biglycan, and COL1A1 in cpg-injected embryos. cpg mRNA was injected into two lateral equatorial region of the two-cell embryo. A: Reverse transcriptase polymerase chain reaction at the gastrula stage (stage 11) shows that fibronectin is not affected by overexpression of cpg. B: The expression of Xenopus biglycan and COL1A1 was remarkably repressed, but fibronectin was not affected by overexpression of cpg at the tailbud stage (stage 30).
