Goto T, Fukui A, Shibuya H, Keller R, Asashima M.

R37 HD025594 NICHD NIH HHS

	Fig. 1. Characterization of Xfurry. (A) Temporal expression of Xfurry detected by RT-PCR. Numbers indicate developmental stages. U, unfertilized eggs. (B) Regional RT-PCR of Xfurry at stage 10. (C–F) Whole-mount in situ hybridization. (C) Arrowhead indicates the dorsal lip (stage 10). (D) Arrowhead indicates the chordamesodermal region (stage 12). (E and F) Strong expression in the notochord was maintained through stage 25 (E) and stage 33 (F). (G) Control embryo (stage 33). (H) Xfurry (2 ng/blastomere) injected into ventral blastomeres of the four-cell embryo induced a secondary axis without anterior structures (arrowhead) in 63% of embryos (n = 60). (I) Expression of the chordamesodermal genes was induced in animal caps (stage 10) that were dissected from embryos injected with Xfurry (2 ng/blastomere). Gsc, goosecoid; Pint, pintallavis; Chd, chordin; ODC, Ornithine decarboxylase. (J) Control embryo (stage 26). (K) Injection of Xfurry-MO (5 ng/blastomere) into dorsal blastomeres of four-cell embryos shortened the dorsal axis in all embryos (n = 58). (L) Expression of chordamesodermal marker genes was reduced in Xfurry-MO–injected embryos, but transcripts of Xfurry were increased by Xfurry-MO (stage 10).
	Fig. 2. Function of Xfurry as a transcriptional corepressor. (A) Fluorescent signals of Xfurry constructs in stage-10 mesodermal cells (Fig. S5). Red signals indicate cell nuclei stained with DAPI. (Top Row) Xfurry+GFP. (Second Row) FD+LZ+GFP construct. (Third Row) LZ+GFP. (Bottom Row) FD+GFP. Arrowheads show colocalization of GFP and DAPI signals. Arrows indicate noncolocalized signals. (B) Control embryo (stage 27). (C) Dorsal injection of FD+SiaHD interfered with head development. (D) Expression of FD+SiaHD reduced transcripts of goosecoid (Gsc) and Cerberus (Cer). (E–G) Phenotypes of injected stage-29 embryos. (E) Control embryo. (F) Ventral injection of enR+LZ (400 pg/blastomere) induced secondary axes in all embryos (n = 59). (G) Dorsal injection of VP16+LZ (400 pg/blastomere) shortened the dorsal axis in all embryos (n = 60). (H) Dorsal expression of enR+LZ induced chordamesodermal and head organizer genes, and expression of VP16+LZ reduced those genes (stage 10).
	Fig. 3. Target genes of Xfurry and their superinduction. (A) Early inducers β-catenin, VegT, and Xnr5 induced transcripts of Xfurry (stage 10). (B) enR+LZ induced chordamesodermal genes in the pre-MBT cycloheximide-treated animal caps, and VP16+LZ reduced or did not alter expression of those genes (stage 10). (C) Superinduction in the animal caps treated with both cycloheximide (CHX) (Left) and anisomycin (ANI) (Right) (stage 10). (D) Injection of pintallavis-MO induced expression of pintallavis (stage 10). (E) Injection of goosecoid-MO induced expression of goosecoid (stage 10). (F) Injection of both pintallavis-MO and goosecoid-MO induced expression of Xfurry (stage 10).
	Fig. 4. The role of small RNAs in gene expression. (A–D) Phenotypes of small RNA-injected embryos (stage 30). (A) Control embryo. (B–D) Small RNAs in the range of 20–60 (B), 60–100 (C), and 100–120 (D) bases were injected into dorsal blastomeres of four-cell embryos. (C) Injection of small RNAs (60–100 bases) shortened the dorsal axis in all embryos (n = 56). (E) Injection of small RNAs (60–100 bases) into all blastomeres of four-cell embryos markedly reduced the expression of chordamesodermal genes but increased expression of Xfurry (stage 10). (F) Injection of small RNAs (60–100 bases) into four animal blastomeres of 32-cell embryos in solution containing cycloheximide decreased expression of chordamesodermal genes in the animal caps and slightly reduced the expression of Xfurry (stage 10). (G) Expression of enR+LZ reduced pri-mir-15, and expression of VP16+LZ slightly up-regulated pri-mir-15 in the cycloheximide-treated animal caps (stage 10). (H) Overexpression of mir-15 in the cycloheximide-treated animal caps reduced expression of chordamesodermal genes and did not alter expression of Xfurry. (I) Cycloheximide treatment from stage 7 reduced expression of pri-mir-15 in the treated animal caps (stage 10). (J) Injection of pintallavis-MO (Pint-MO) into all blastomeres of four-cell embryos increased expression of goosecoid and Xnot, did not alter the expression of chordin, and reduced the expression of pri-mir-15 (stage 10). (K) Model of the proposed Xfurry signaling pathway. Xfurry represses expression of mir-15 and microRNAs. Several chordamesodermal genes are repressed by mir-15 and microRNAs. Xfurry also is induced by goosecoid as a downstream gene. Then this feedback loop signaling pathway together with Xfurry regulates expression of chordamesodermal genes. Moreover, low protein levels of several genes activate a system that senses the low-protein condition, thereby inducing Xfurry expression and activating the function of Xfurry as a corepressor. Pin, pintallavis.
	Fig. S2. Dorsal overexpression of Xfurry. (A) Control (stage 28). (B) Injection of Xfurry (2 ng/blastomere) into dorsal blastomeres of four-cell embryos had no effect on axis formation (n = 60).
	Fig. S3. Confirmation of the morpholino specificity. A tracer, Alexa 594-dextran (300 pg), GFP constructs, and morpholinos were injected in the animal pole of four-cell embryos and were observed at stage 10 to show the injected embryos (Top Row), the fluorescent Alexa 594-dextran tracer (Middle Row), and GFP fluorescence (Bottom Row). Xfurry-MO specifically reduced translation of sequences containing Xfurry-GFP targeted with Xfurry-MO at the 5′-region but did not reduce translation of 5-mis-Xfurry-GFP, which has no targeted sequence.
	Fig. S4. Rescue experiments among Xfurry, Xfurry-MO, enR+LZ, and VP16+LZ. (A) Control (stage 33). (B) Injection of Xfurry-MO (5 ng/blastomere) into dorsal blastomeres of four-cell embryos interfered with head formation and shortened the axis of all embryos, as shown also in Fig. 1K (n = 40). (C) The cement gland was rescued, and the axis was slightly elongated by coinjection of Xfurry (2 ng/blastomere) and Xfurry-MO (5 ng/blastomere) into dorsal blastomeres of fourcell embryos (57.9%; n = 57). (D) Dorsal expression of enR+LZ (400 pg/blastomere) also rescued the phenotype of Xfurry-MOnjected embryos (78.6%; n = 56). (E) Dorsal injection of VP16+LZ (400 pg/blastomere) interfered with head formation and shortened the axis of all embryos, as in Fig. 2G (n = 40). (F) Dorsal expression of Xfurry (2 ng/blastomere) rescued the phenotype of VP16+LZ-injected embryos (52.5%, n = 59).
	Fig. S7. Overexpression of nuclear DBF2-related 1 (NDR1) and NDR2. (A) Control (stage 33). (B) Overexpression of NDR1 (1 ng/blastomere) into ventral blastomeres of four-cell embryos had no effect on axis formation (n = 60). (C) Ventral overexpression of NDR2 (1 ng/blastomere) at the four-cell embryo stage also had no effect on axis formation (n = 60).
	fry (FRY microtubule binding protein) gene expression in Xenopus laevis embryos, NF stage 10, as assayed by in situ hybridization, vegetal view, dorsal up.
	fry (FRY microtubule binding protein ) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 12, dorsal left, animal pole up.
	fry (FRY microtubule binding protein) gene expression in Xenopus laevis embryo, NF stage 25, as assayed by in situ hybridization, lateral view, dorsal up, anterior left.
	fry (FRY microtubule binding protein) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.

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