Li J , Shi Y , Sun J , Zhang Y , Mao B .

	Figure 1. Temporal and spatial expression pattern of XRFC. (A) XRFC is expressed maternally and throughout the stages examined (St.0 to St.32) as detected by RT-PCR. T, negative control without reverse transcriptase in the RT reaction. (B) Expression of XRFC at the indicated stages revealed by in situ hybridization. (B) The maternal XRFC expression is detected at the animal pole at 2-cell stage. (C) At stage 18, XFRC is expressed at the anterior neural plate and its border. (D) At the late neurula stages, the expression of XRFC is detected in the prospective eye and forming branchial arches. (F) At tailbud stage, XRFC is strongly expressed in the branchial arches, eyes, head mesenchyme and the somites. Br, branchial arch.
	Figure 2. XRFC is required for neural crest induction and migration. (A) XRFC-MO and tracing lacZ mRNA were injected into one cell of four-cell stage embryos, the expression of neural crest makers were examined at stages 157. (A) Knockdown of XRFC by morphoino inhibits the expression of Zic1, Snail2 and FoxD3 in the injected sides (labeled red by staining of the tracing lacZ). (D) Co-injection of XRFC mRNA (800 pg) rescues the expression of Zic1, Snail2 and FoxD3. (G) Overexpression of XRFC expands the expression domains of Zic1, Snail2 and FoxD3. (J, K) XRFC morpholino injection does not affect the expression of Msx1 and Pax3. (L) Twist1 staining of neural crest cells at stage 32 of embryos injected on one side with XRFC-MO or XRFC-MO plus rescue mRNA. In XRFC-MO injected side, the neural crest cells are retained in a dorsal position (arrowhead in L). (P) Quantification of the effect of XRFC-MO on the expression of neural crest markers and the rescue by RFC mRNA. (Q) RT-PCR analysis in animal cap assay to show the effect of XRFC-MO on the expression of the indicated neural crest genes induced by co-injection of tBR and XWnt7b. (R) Transplantation experiment to show the effect of XRFC-MO on neural crest migration. (R) Schematic drawing showing the transplantation procedure. (S) The EGFP-labeled XRFC-MO injected neural crest tissue fails to migrate at tailbud stage after transplantation. (T) Co-injection of XRFC mRNA restores the migration ability of the neural crest cells. inj. side, injected side; con. side, control side.
	Figure 3. Knockdown of XRFC leads to multiple malformations at tadpole stages. (A) Knockdown of XRFC caused craniofacial malformations (arrowheads) with curled trunks. (B) XRFC-MO inhibited the formation of cranial cartilage. Cartilages in late tadpoles were stained with alcian blue. Cartilage on the RFC-MO injected side was often malformed and reduced. M, meckel's cartilage; CH, ceratohyal cartilage; CB, ceratobranchial cartilage. (C) The XRFC-MO injected embryos were hypopigmented at tadpole stage. (G) The XRFC morphants showed enlarged pericardial cavity (arrowhead), indicative of heart defects. (H) The gut coiling in XRFC was malformed at tailbud (H) and tadpole (I) stages compared to the rescued group (K, L). (D, E, F, G, K, L) Co-injection of XRFC mRNA rescued the above mentioned malformations.
	Figure 4. RFC is involved in the neural crest gene regulatory network. (A) The inhibition of Snail2 expression by dominant negative Msx1, Pax3 and Zic1 was rescued by co-injection of XRFC. (G) Snail1 N, Snail2 N and Snail2 Znf inhibited the migration of neural crest (as indicated by the distribution of the Twist1 positive neural crest cells, G), which was rescued by co-injection of XRFC (J). (M) The inhibition of Snail2 expression by XRFC-MO was rescued by Msx1, Pax3 and Zic1 co-expression. (Q) The neural crest migration defects in XRFC morphants were rescued by co-injection of Snail1 or Snail2. (T) Quantification of the rescue effect of RFC on the expression of neural crest markers inhibited by dominant negative Msx1, Pax3, Msx1, Snail1, and Snail2 (left panel) and the rescue of the XRFC-MO effect by Msx1, Pax3, Zic1, Snail1, and Snail2 (right panel). (U) Dominant negative dishevelled (Xdd1) and GSK3β blocked the expression of Snail2 (U, V), which could not be rescued by XRFC (W, X). doi:10.1371/journal.pone.0027198.g004
	Figure 5. The effects of different hRFC constructs on neural crest gene expression. (A) XRFC-MO injection blocked neural crest maker expression which was rescued by the wild type hRFC. (I) Overexpression of hRFC promoted of the expression of neural crest genes. (M) The two mutated form of hRFC (R133C, R373C) failed to rescue the neural crest gene expression in the XRFC morphants. The arrowheads indicate the injected sides. (U) Quantification of the rescue of the XRFC-MO effect by the wild type and mutated hRFC constructs.
	Figure 6. XRFC regulates neural crest gene expression through the one-carbon metabolism pathway. Co-injection of 5-MTHF (20 ng/embryo, E), SAM (10 ng/embryo, I) or Vitamin B12 (50 ng/embryo, M) with XRFC-MO rescued the expression of the neural crest maker genes (compare with A). (Q) Quantification of the rescue of the XRFC-MO effect by 5-MTHF, SAM, and Vitamin B12.
	Figure 7. RFC regulated neural crest early development through histone modification. (A) Levels of different methylation forms of histone 3 in animal caps injected with XWnt7b and tBR with or without XRFC-MO. Knockdown of XRFC by injection XRFC-MO decreased both the mono- and trimethyl-H3-K4 levels but not dimethyl-H3-K4 level (left panel), which was restored by co-injection of XRFC mRNA (middle panel). Injection of 5-MTHF increased the mono- and trimethyl-H3-K4 levels but not dimethyl-H3-K4 level (right panel). (B) hMLL1 plasmid co-injection rescued the effect of XRFC-MO on the expression of Zic1 and FoxD3, and weakly on Snail2 and Twist1. (J-M) Overexpression of hMLL1 alone promoted the expression of Zic1 and FoxD3, but had no clear effects on Snail2 and Twist1. (N) Number of embryos showed reduced expression of Zic1, FoxD3, Snail2 and Twist1 injected with XRFC-MO with or without hMLL1 plasmid (50 pg/embryo).
	Figure S1. RFC is involved in the neural crest gene regulatory network. (A, B) The inhibition of Zic1 expression by dominant negative Msx1 can be rescued by XRFC. (C, D) Msx1 rescues the inhibition of Zic1 expression by XRFC-MO. (E) The inhibition of Snail2 expression by XRFC-MO can be rescued by Msx1, Pax3 and Zic1 co-expression. (I) The inhibition of FoxD3 expression by dominant negative Msx1, Pax3 and Zic1 can be rescued by co-injection of XRFC. (O, P) The inhibition effect on neural crest migration (as indicated by the distribution of the Twist1 positive neural crest cells) of Snail1-Znf can be rescued by co-injection of XRFC. The injected sides are indicated by the red staining of tracing lacZ. (Q) Quantification of the rescue effect of RFC on the expression of Zic1, FoxD3 or Twist1 inhibited by dominant negative Msx1, Pax3, Zic1 and Snail1 and the rescue of the XRFC-MO effect by Msx1, Pax3, and Zic1.
	Figure S2. Components of the folate metabolism pathway regulate neural crest gene expression in Xenopus embryos. (A) Both folate transportation deficient forms of hRFC (R133C/R373C) failed to promote neural crest makers expression (Twist1, FoxD3, Snail2 and Zic1). (I) Injection of 5-MTHF (20ng/embryo, I), Vitamin B12 (50ng/embryo, M) or SAM (10ng/embryo, Q) promoted neural crest maker genes expression (Twist1, FoxD3, Snail2 and Zic1). (U) Quantification of the promoting effect on the expression of neural crest genes by hRFC, hRFC-R133C, hRFC-R373C and 5-MTHF, SAM, and Vitamin B12.
	Figure S3. The folate antagonist MTX blocks neural crest induction. (A) Injection of MTX (10ng/embryo) inhibits the expression of Twist1, Snail2 and Zic1 in the injected sides. (D) Co-injection 5-MTHF (20ng/embryo) with MTX rescues the effects of MTX on neural crest gene expression. (G) Quantification of the inhibitory effect of MTX on the expression of neural crest genes and its rescue by 5-MTHF.
	slc19a1 (solute carrier family 19 (folate transporter), member 1) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, anterior view, dorsal up.
	slc19a1 (solute carrier family 19 (folate transporter), member 1) gene expression in Xenopus laevis embryos, NF stage 20, as assayed by in situ hybridization, anterior view, dorsal up.
	slc19a1 (solute carrier family 19 (folate transporter), member 1) gene expression in Xenopus laevis embryos, NF stage 23, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.
	slc19a1 (solute carrier family 19 (folate transporter), member 1) gene expression in Xenopus laevis embryos, NF stage 29, as assayed by in situ hybridization, lateral view, anterior right, dorsal up.

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