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Protein expression of the transcription factor genes mix1 and vegt characterized the presumptive endoderm in embryos of the frogs Engystomops randi, Epipedobates machalilla, Gastrotheca riobambae, and Eleutherodactylus coqui, as in Xenopus laevis embryos. Protein VegT was detected in the animal hemisphere of the early blastula in all frogs, and only the animal pole was VegT-negative. This finding stimulated a vegt mRNA analysis in X. laevis eggs and embryos. vegt mRNA was detected in the animal region of X. laevis eggs and early embryos, in agreement with the VegT localization observed in the analyzed frogs. Moreover, a dorso-animal relocalization of vegt mRNA occurred in the egg at fertilization. Thus, the comparative analysis indicated that vegt may participate in dorsal development besides its known roles in endoderm development, and germ-layer specification. Zygotic vegt (zvegt) mRNA was detected as a minor isoform besides the major maternal (mvegt) isoform of the X. laevis egg. In addition, α-amanitin-insensitive vegt transcripts were detected around vegetal nuclei of the blastula. Thus, accumulation of vegt mRNA around vegetal nuclei was caused by relocalization rather than new mRNA synthesis. The localization of vegt mRNA around vegetal nuclei may contribute to the identity of vegetal blastomeres. These and previously reportedly localization features of vegt mRNA and protein derive from the master role of vegt in the development of frogs. The comparative analysis indicated that the strategies for endoderm, and dorsal specification, involving vegt and mix1, have been evolutionary conserved in frogs.
Fig. S1. Phylogenetic tree of genera and comparisons of the X. laevis epitope sequence for anti-VegT polyclonal ab with the vegt sequences of other frogs. (A) Phylogenetic tree of genera from maximum likelihood analysis of mitochondrial DNA using MEGA8. The sequences included were ribosomal RNA genes (S16 and 12S) and tRNA-Val gene. In case of G. riobambae, only the 16S ribosomal RNA gene was used, modified from ref. 19. (B) Phylogenetic tree of VegT epitopes using the MEGA8, mitochondrial DNA method. The vegt sequence of E. randi is unknown. (C) Sequence aligment of E. coqui (Ec), E. machalilla (Em), and G. riobambae (Gr) against X. laevis (Xl). Sequences were aligned by using the ClustalW multiple sequence alignment. Identical residues are boxed. *Epitope sequences analyzed in this study.
Fig. 1. Immune localization of Mix1 in frog embryos. The animal hemisphere is oriented toward the top. Darkly stained Mix1-positive nuclei were detected in the vegetal hemisphere in embryos of all species. (A, C, E, G, and H) Blastula sections and a bisected blastula in H. (B, D, and F) Sagittal sections of gastrulae with the dorsal side oriented to the right. (A and B) X. laevis embryos. (C and D) E. randi embryos. (E and F) E. machalilla embryos. (G) Vegetal cells of a G. riobambae stage 9.75 blastula. (H) Bisected blastula of E. coqui stage 9.75. The blastocoel roof collapsed during fixation. Cleavage and gastrula embryos of G. riobambae and E. coqui were not analyzed. b, blastocoel; bf, blastocoel floor; dl, dorsal blastopore lip. (Scale bars: A, B, E, and F, 200 µm; C and D, 150 µm; G, 100 µm; H, 400 µm.)
Fig. 2. Immune localization of VegT in frog embryos. The animal hemisphere is oriented toward the top. Darkly stained VegT-positive nuclei were detected in the animal and vegetal hemispheres in the embryos of all species and only the uppermost area of the animal pole was VegT-negative. (A and B) Embryos of X. laevis. (A) Parasagittal section of a st 5 embryo. VegT-positive nuclei were observed in animal and vegetal cells. (B) VegT immune localization and Hoechst 33258 fluorescence of a bisected blastula. The dark color of VegT-positive reaction quenched Hoechst 33258 nuclear fluorescence in animal and vegetal cells, whereas VegT-negative nuclei were brightly fluorescent. (C and D) E. randi embryos. (E and F) Embryos of E. machalilla. The blastocoel roof collapsed during embryo fixation in F. (G–H′) An embryo of G. riobambae. (G) Panoramic view of a st-8.5 embryo section. The figure is a composite of multiple images. Areas of the marginal and vegetal regions are boxed and shown at higher magnification in H and H′ to show VegT-positive nuclei. (H) Marginal zone. (H′) Vegetal region. (I and J) E. coqui embryos. (I) Bisected blastula. VegT-positive nuclei were observed in the marginal zone and vegetal region. (J) Vegetal region from a st-8.5 embryo at higher magnification. VegT-positive nuclei are visible. Black arrows in A, B, H, H′, I, and J point to VegT-positive nuclei. White arrow in B points to a VegT-negative nucleus. b, blastocoel; bf, blastocoel floor; br, blastocoel roof. (Scale bars: A, B, and F, 200 µm; C and D, 150 µm; E, 250 µm; G and I, 300 µm; H, H′, and J, 100 µm.)
Fig. S2. vegt mRNA and protein localization in the X. laevis blastula. (A and A′) Immune localization of VegT in a hemibisected blastula. (B and C) In situ hybridization of vegt mRNA in blastula hemibisections. (A) VegT immune localization. The boxed area is shown at higher magnification in A′. (A′) VegT immune localization and DAPI nuclear staining of the embryo in A. The color reaction of VegT quenched the DAPI fluorescence in VegT-positive nuclei. In contrast, VegT-negative nuclei were brightly fluorescent. (B) Higher magnification of the boxed area of Fig. 3H. vegt transcripts were detected around less color-dense round structures, probably cell nuclei. (C) DAPI nuclear staining of a blastula after vegt mRNA in situ hybridization. Nuclei were brightly fluorescent in vegt-negative and positive blastomeres. vegt mRNA was detected around the brightly fluorescent nuclei. White arrows indicate vegt-negative nuclei. Black arrow points to the accumulation of VegT protein inside a nucleus. Black arrowhead indicates the accumulation of vegt transcripts around a cell nucleus. Black arrow with white outline points to the accumulation of vegt mRNA around a brightly fluorescent DAPI stained nucleus.
Fig. S3. mix1 mRNA localization in X. laevis embryos. The animal hemisphere is oriented toward the top, and the dorsal side is oriented to the right. Embryos were dorsally hemibisected before in situ hybridization. (A–C) Blastulae. (D–F) Gastrulae. White arrowheads mark the position of the dorsal blastopore lip.
Fig. 3. vegt mRNA localization in X. laevis embryos. The animal hemisphere is oriented toward the top. In all embryos, dorsal is oriented to the right. Embryos were dorsally hemibisected before in situ hybridization. The row of diagrams summarizes the vegt expression of the unfertilized egg and st 1–9.5 embryos. The animal hemisphere of the egg and the blastocoel roof (shown in gray) were vegt-negative and the vegetal region (in orange) was vegt-positive. (A) The unfertilized egg. vegt mRNA was found in the vegetal hemisphere with a lower concentration toward the animal region. (B) The egg 1 h after fertilization. vegt mRNA was detected in the vegetal hemisphere with a displacement toward the dorsal-animal side. In addition, vegt transcripts were detected in irregular areas located at the vegetal pole and colocalized with the germ plasm. Germ plasm and mitochondria localization are shown in Fig. S4 E–G. (C) The four-cell embryo. (D) The eight-cell embryo. (E–K) Blastulae. (L–N) Gastulae. (E–J) Lightly stained half–blastulae after vegt in situ hybridization. vegt mRNA localized mainly around the nuclei of vegetal cells. The boxed area in H is shown at higher magnification in Fig. S2B. (E′–J′) Darkly stained half-blastulae after in situ hybridization. The dark color of the embryos partially masked the vegt mRNA signal around nuclei in E′–G′ in comparison with the lightly stained half blastulae in E–G. Yellow arrows indicate the dorso–anterior limit of vegt mRNA localization. White arrows at the vegetal region mark the accumulation of vegt transcripts in the germ plasm. Black arrowheads point to the accumulation of vegt transcripts in the central region of vegetal blastomeres. White arrowheads indicate the position of the dorsal blastopore lip.
Fig. 4. vegt mRNA isoforms of the X. laevis unfertilized egg and embryos. (A) vegt mRNA isoforms of the unfertilized egg amplified by RT-PCR and separated in an agarose gel. (B–D) Relative expression of mvegt and zvegt by RT-qPCR and δδCt analysis normalized by histone H4 expression. (B) Unfertilized egg. (C) Embryo st 7. (D) Early st 9 embryo. The ratios of zvegt mRNA to mvegt mRNA were 2.8% in B, 4.0% in C, and 5.2% in D. The efficiency of mvegt and zvegt primer sets is almost the same. The results are the means of three independent experiments.
Fig. 5. Dorsoventral relocalization of vegt mRNA in animal blastomeres of eight-cell stage embryos (st 4) of X. laevis. (A) Outline of the methods. (B) Quantitation of mvegt and zvegt expression by RT-qPCR in animal blastomeres, normalized by the expression of histone H4. The data show the results of three independent experiments. dor, dorsal; P, statistics P value; vent, ventral.
Fig. 6. Effect of α-amanitin on the expression of vegt, mix1, and Xnr5 mRNA in X. laevis embryos. milliQ-water or α-amanitin (100 pg per cell) were injected into each blastomere at the two-cell stage followed by in situ hybridization of hemibisected embryos or by RT-qPCR at the indicated stages. (A, E, and H) In situ hybridization of embryos treated with milliQ-water. (B, F, and I) In situ hybridization of embryos treated with α-amanitin. (C, D, and G) Relative gene expressions of mvegt, zvegt, and Xnr5 detected by RT-qPCR normalized by histone H4 expression. (A–D) vegt mRNA expression of st-7 embryos. (A and B) vegt mRNA in situ hybridization. (C) mvegt relative gene expression. (D) zvegt relative gene expression. (E–G) Xnr5 mRNA expression in st-9 embryos. (E and F) Xnr5 mRNA in situ hybridization. (G) Xnr5 relative gene expression. (H and I) mix1 mRNA expression of st-9 embryos detected by in situ hybridization. The data show the results of three independent experiments. Black arrowheads in A and B point to the accumulation of vegt transcripts around nuclei of vegetal blastomeres. a-Am, α-amanitin; mQ, milliQ water; ns, nonsignificant; P, statistics P value.
Fig. S4. Mitochondria and germ plasm identification in the X. laevis blastula. The immune detection methods are given in the SI Materials and Methods. The mitochondrial marker ab14730 (Abcam) was used for the immune detection of mitochondria. Incubation of embryos with only the secondary antibody gave negative results (not shown). (A) Immune detection of mitochondria without heat treatment in a hemibisected embryo. Mitochondria were detected inside blastomeres, likely around nuclei. (B) DAPI nuclear staining of a blastula section. (C) Immune fluorescent detection of mitochondria in the same section shown in B. (D) Merged B and C images indicates the localization of mitochondria around nuclei. (E) Immune localization of mitochondria in a hemibisected embryo after an overnight heat treatment at 65 °C in TE buffer, pH 9.0, as explained in the SI Materials and Methods. Mitochondria were detected around nuclei, and in vegetal islands, considered to be the germ plasm. Mitochondria associated with the germ plasm were not detected in embryos that did not receive the heat treament. (F) The germ plasm of a hemibisected embryo detected by the localization of vegt mRNA by in situ hybridization. (G) The germ plasm of a hemibisected embryo detected by the localization of VegT protein by immune localization with anti-VegT. Black arrows point to mitochondria around cell nuclei. White arrows indicate the germ plasm. Blue arrowhead points to a DAPI-stained nucleus. Red arrowhead signals the mitochondria around the nucleus. Black arrowhead signals the merged image of the nucleus surrounded by mitochondria. a-mit, anti-mitochondria treatment without heat treatment; a-mit-heat, antimitochondria treatment after heat treatment; a-VegT, anti-VegT treatment.
Fig. S5. Vegetal views of X. laevis embryos after vegt mRNA in situ hybridization. Arrowheads point to vegt mRNA localization in the germ plasm islands of the vegetal pole. (A) Two-cell stage embryo. (B) Four-cell stage embryo. (C) Eight-cell stage embryo.
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