Wahl SE , Kennedy AE , Wyatt BH , Moore AD , Pridgen DE , Cherry AM , Mavila CB , Dickinson AJ .

R01 DE023553 NIDCR NIH HHS, K12 GM093857 NIGMS NIH HHS , P30CA16059 NCI NIH HHS , RDE023553A PHS HHS , P30 CA016059 NCI NIH HHS

Xla Wt + BMS453 + Methotrexate Hydrate (fig.1.l)
Xla Wt + BMS453 + Methotrexate Hydrate (fig.1.m)
Xla Wt + BMS453 + leucovorin (fig.1.d)
Xla Wt + BMS453 + leucovorin (fig.1.g)
Xla Wt + Methotrexate Hydrate (fig.3.c, e)
Xla Wt + Methotrexate Hydrate (fig.3.g)
Xla Wt + Methotrexate Hydrate (fig.4.c, d)
Xla Wt + Methotrexate Hydrate (fig.4.f, g)
Xla Wt + Methotrexate Hydrate (fig.4.k)
Xla Wt + Methotrexate Hydrate (fig.4.m)
Xla Wt + Methotrexate Hydrate (fig.5.b. ii, iii)
Xla Wt + Methotrexate Hydrate (fig.6.b. ii, iii)
Xla Wt + Methotrexate Hydrate (fig.S3.b)
Xla Wt + Methotrexate Hydrate (fig.S4.ii-iv)
Xla Wt + Methotrexate Hydrate (fig.S7.c. ii, iv)
Xla Wt + Methotrexate Hydrate (fig.S7.e. ii, iii)
Xla Wt + dhfr MO (fig.2.a.iii)
Xla Wt + dhfr MO (fig.2.a.vii)
Xla Wt + dhfr MO (fig.2.a.viii)
Xla Wt + dhfr MO (fig.6.b. v, vi)
Xla Wt + dhfr MO (fig.S3.a)
Xla Wt + dhfr MO (fig.S7.e. v, vi)
Xla Wt + dhfr MO (fig.S8.a^1, a^2)
Xla Wt + dhfr MO (fig.S8.b^1, b^2)
Xla Wt + dhfr MO (fig.S8.c^1, c^2)

	Fig. 1. Folate and retinoic acid signaling. (A) Schematic of the experimental design for (B–G) (2 experiments and n=20). (B–C) Representative frontal views of faces at stage 42–43 treated with DMSO (B) or folinic acid (C) RAR inhibitor (D), or RAR inhibitor and folinic acid (E). (F) Schematic of the facial dimensions measured. (G) Bar graphs summarizing quantification of facial dimensions corresponding to (B–E). (H) Schematic of experimental design for (I–M) (2 experiments, n=20). (I–L). Representative frontal views of faces at stage 42 treated with DMSO (I) or MTX (J) RAR inhibitor (K), or RAR inhibitor and MTX (L). (M) Bar graphs summarizing quantification of facial dimensions corresponding to (I–L). Mouth is outlined with red dots in all frontal views and asterisks designate statistical significance with all p values<0.001. All images are at the same magnification, scale bar=175 μm. Abbreviations: C=control, R=RAR inhibitor, F=folinic acid, M=methotrexate.
	Fig. 2. DHFR morpholino knockdown results in abnormal orofacial development. (A) Whole DHFR morphant analysis. (i) Schematic showing the experimental design. (ii) and (iii) Representative frontal views of faces injected with control (ii) or DHFR (iii) morpholinos. (iv) Schematic of facial dimensions measured. (v) Bar graph showing quantification of facial dimensions normalized to the controls (set to 100), asterisk designates statistical significance for 2 experiments (n=60) p value<0.001. (vi) Schematic showing the experimental design for (vii). (vii) Representative frontal view of the face of an embryo injected in one cell as shown in (vi). The injected side is indicated by the green triangle. (viii) and (ix) rescue experiment showing representative frontal views of faces injected with DHFR MO (viii) or DHFR MO and treated with folinic acid (FA) (ix). (B) Localized DHFR morpholinos using face transplants. (i) Schematic of the experimental design. (ii)–(v′) Representative frontal views of embryos that received DHFR morphant tissue. Prime roman numerals are composites of the unprimed counterpart with fluorescent overlays showing the location of morpholino containing tissue (n=2 experiments, n=11). All embryos were imaged at same magnification, scale bar=200 μm. The mouth is outlined in red dots.
	Fig. 3. Pharmacological inhibition of DHFR alters size and shape of the face. (A) Schematic of the experimental design. (B–E) Representative frontal views of faces treated with DMSO (B), 220 μM MTX (C) and (E) and 220 μM MTX+folinic acid (FA) (D). All images were taken at same magnification (scale bar=150 μm). Mouth is outlined in red dots. (F) Schematic of the facial dimensions measured. (G) Bar graphs of the quantification of facial dimensions for 2 experiments (n=20). Asterisks designate statistical difference when compared to control (all p values≤0.001). (H) Landmark locations for morphometric analysis. (I) Canonical variate analysis of landmark coordinates. Statistically significant for 2 experiments, n=10, p value≤0.05). (J) Transformation grids showing the change in landmark position in MTX treated embryos compared to controls (top grid) and controls compared to rescued embryos (bottom grid).
	Fig. 4. Inhibition of DHFR alters jaw muscle and cartilage, gene expression and total histone levels. (A) Schematic of the experimental design. (B–D) Representative ventral views of embryos labeled with phalloidin to mark muscle. Images are all at the same magnification (scale bar=500 μm), 2 experiments (n=14). (E–G) Representative dorsal views of embryos labeled with Alcian blue to show cartilage. Images are all at the same magnification (scale bar =500 μm), 2 experiments (n=20). (H) in-situ hybridization for neural crest marker, AP-2, frontal views (i, ii) and lateral views with anterior to the left (iii, iv). All images are at the same magnification (scale bar=175 μm). Asterisk designates the region measured and graphed in I. (I) Bar graph showing quantification of the AP-2 domain in the face. There is no statistical significance in 2 experiments (n=12, p value=0.64). (J) Bar graph showing quantification of AP-2 mRNA levels by qRT-PCR. Statistical significance designated by asterisk, 2 experiments. (K) mRNA levels of genes expressed in the face relative to GAPDH. Asterisks designate statistical significance (p values for significance are all≤0.005). (M–N) Immunohistochemistry of H3K4me3 in representative transverse sections of the face (2 experiments, n=10). Images are at the same magnification (scale bar=100 μm). (N) Representative western blot of H3K4me3, total H3 and actin (n=2 experiments). (P) The ratio of H3k4me3 to H3 from 2 experiments is not statistically different (p value=0.589). Abbreviations; gh=Geniohyoideus, ih=Interhyoideus, so=Subarcualis obliquus II, oh=Orbitohyoideus, qh=Quadrato-hyo-angularis, cb=Constrictores branchialium, Mk=Meckel's cartilage, ch=ceraohyal cartilage, bh=basihyal cartilage, pc=parachordal cartilage.
	Fig. 5. Cell proliferation is decreased in MTX treated embryos. (A) Schematic of the experimental design. (B) Immunohistochemistry of pH3 in representative transverse sections of the face from embryos treated with DMSO (i), or MTX (ii, iii). Images are all at the same magnification, scale bar=100 μm. (C) FACS analysis of the cell cycle. (i) Representative cell cycle profiles. (ii) The average percentages of cells in each fraction from 4 experiments. Statistical significance was determined using Mann Whitney U test and designated by asterisks (p values≤0.028).
	Fig. 6. DHFR inhibition results in increased apoptosis and DNA damage. (A) Schematic of the experimental design. (B) Immunohistochemistry of cleaved caspase-3 (green) in transverse sections of the face from embryos treated with DMSO (i), or MTX (ii,iii) or injected with control morpholino (CMO)(iv) or DHFR morpholinos (DHFR MO) (v,vi). Images are all at the same magnification (scale bar=100 μm) and counterstained with propidium iodide. (C) Imagestream flow cytometry to analyze cell morphology. (Ci) Schematic showing cellular measurements used in the analysis. Cii) Bar graph showing average perimeter (Per.) and Aspect ratio (Aspect) (major/minor axis). Statistical significance designated by asterisks (p values≤0.05). (Ciii) Representative images of single cells acquired by the ImageStream. (D) Comet assay for DNA damage. (Di) Representative cell to show the comet tail region where fluorescence was quantified. (Dii) Bar graph showing quantification of fluorescence in comet tails from cells isolated in two experiments. Statistical significance is designated by an asterisk (n=40 cells, p value=6.8E–06). (Diii,iv) Representative images of cells from comet assay. Scale bar=50 μm.
	Fig. 7. Mechanism of folate and retinoic acid signaling interaction. (A, B) Representative transverse sections through the face labeled with cleaved caspase-3 (green) and counterstained with propidium iodide. This was consistent in at least 10 embryos at equivalent locations in the face All images were taken at the same magnification, scale bar=175 μm. (D) Model of how folinic acid supplementation prevents median orofacial clefts. (E) Model of the mechanism by which RAR and DHFR inhibition could synergize.
	Supplemental Figure 1: in situ hybridization of DHFR in Xenopus laevis. A) Antisense probe targeting DHFR revealed expression in the neural tube, future eyes and face surrounding the presumptive embryonic mouth in stages 22-30. B) The sense probe was devoid of staining. C) Antisense probe targeting reduced folate carrier (RFC) revealed overlapping expression domains with DHFR, however, DHFR appeared to be more ubiquitously expressed than this gene. These results suggest the possibility that reduced folate carrier is only necessary in some cells and could also perform different/additional functions than simply transporting folate into the cell.
	Supplemental Figure 2: Characterization of DHFR morpholino and . A) DHFR Morpholino sequence. B) Schematic of DHFR exons (E1-E6) to show where the primer sequences and DHFR MO targeted. C) Representative RT-PCR to show that DHFR MO resulted in smaller abnormally spliced DHFR mRNA and reduced levels of normal DHFR. D) Summary of protein sequence comparisons. Full length DHFR protein sequences for selected organisms were retrieved from NCBI as follows: H. sapiens (NP00782.1), M. musculus (NP034179.1), R. norvegicus (NP569084.1), G. gallus (NP001006584.2), X. laevis (NP001088506.1), X. tropicalis (XP004919578.1), and D. rerio (NP571850.1). These full-length protein sequences were compared to humans determine their percent identity using NCBI protein blast (blast.ncbi.nlm.nih.gov). To determine the percent identity for the folate and NADP+ binding sites of DHFR, the residues integral to each site were elucidated using NCBI conserved domain search for each binding site (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Full-length DHFR sequences were aligned using UniProt sequence alignment (http://www.uniprot.org/align/) and the percent identity of all folate binding and NADP+ binding sites compared to H. sapiens was calculated manually. E) The consensus sequence determined from (D) was inputted into the bioinformatics program, Perl, to determine the amount of information at each residue. Shannon’s information equation defines information as H = -  , where H is entropy, or the predictability of a sequence of data (1). When applied to the consensus sequence, a high information value represents amino acid residues that are conserved, while low information values represent those that are not. The consensus sequence was superimposed onto the known three-dimensional structure of human DHFR in JMol. Residues were color-coded based on information values in bits. Residues with a bit value of 3.5 or higher were colored red and are the most conserved. Residues with a bit value between 3.0 and 3.4 were colored yellow, those with a bit value between 2.5 and 2.9 were colored green, and those with bit values lower than 2.4 were colored light purple and considered the least conserved. The ligands, NADPH and folate, were also visualized in JMol and colored dark purple and were well conserved. The folate/MTX binding domain (white line) is surrounded almost entirely by red residues.
	Supplemental Figure 3: Other phenotypes in DHFR morphants and MTX treated embryos. A) DHFR morphants have an enlarged pericardial sac, lack of gut coiling and shorter length. B) MTX treated embryos also have an enlarged pericardial sac, lack of gut coiling and shorter length.
	Supplemental Figure 4: Variability in the MTX phenotype. i-iv) Representative frontal views of the typical facial phenotypes observed in any given concentration from 100-500uM. There are several possible reasons for this variability such as decreased folate transporter function or increased DHFR expression potentially as a result of gene amplification. We routinely saw about 25% of embryos with a very mild or normal phenotype which suggests the possibility that our frogs are carriers for a mutation in a gene that could render them less susceptible to MTX treatment.
	Supplemental Figure 5: Global DNA methylation. DNA was extracted using the DNAeasy tissue kit (Qiagen, 69504) from embryos treated with DMSO or MTX. The optional RNAse treatment was performed. Restriction digests were performed using the Epijet DNA methylation kit (Thermo, K1441) which uses the isoschizomers HpaII and MspI which have different sensitivities to CpG methylation. When the internal CpG in the 5’-CCGG-3’ tetranucleotide sequence is methylated, cleavage with HpaII is blocked, but cleavage with MspI is not affected. Therefore, modifications in global methylation levels would be revealed by a change in the ratio of HpaII to MspI. Digest products were run on a 2% agarose gel and no obvious difference was observed between treatments in 6 experiments. The uppermost band was quantified and the ratio of HpaII to MspI also showed no statistical difference.
	Supplemental Figure 6: Supplemental cell cycle analysis. A) Tables showing percentage cells in each phase at stage 31/32 and 35. B) Representative cell cycle profiles at stage 31/32 for control and MTX treated. C) Western blot for PCNA shows no difference between treated and control groups.
	Supplemental Figure 7. DHFR inhibition resulted in disrupted orofacial development at Stage 40 (66 hpf). A. Schematic for experimental design for B-E. B. left: Schematic of facial dimensions measured (top), quantification of face height and intercanthal distance (bottom). right: Morphometric landmarks (top) and transformation grid (bottom) for control vs. MTX treated embryos at St. 40 (66 hpf) (n=37, *p<0.05)., C. Representative images of in situ hybridization for AP-2 in control (left) and MTX- treated (right) embryos. Top: frontal view, bottom: side view. cg= cement gland (n=20). D. Representative cell cycle profiles at st. 40 (66 hpf, top). The average percentages of cells in each fraction from 2 experiments (bottom, n=8 from 2 different experiments). E. top: representative images of immunohistochemistry in control (i) and MTX-treated (ii,iii) embryos for cleaved caspase-3 (red) (n=10, 1 experiment). bottom: immunohistochemistry in control and DHFR MO-injected embryos for cleaved caspase-3 (red). FITC-tagged MO shown in green. (n=10, 2 experiments).
	Supplemental Figure 8. DHFR MO injections into one cell at the 2-cell stage show specific effects where the morpholino was present. A total of 30 embryos were injected and fixed at stage 28-30. Embryos were sectioned and labeled for H3K4me3, ph3 and cleaved caspase-3. A. Representative image of H3K4Me3 (A) and DHFR MO-FITC (A’), merge (A”). B-B”: Representative image of pH3 (B) DHFR MO FITC (B’) and merge (B”). C-C” and D-D”: Representative image of cleaved caspase-3 (C, D), DHFR MO FITC (C’), control MO FITC (D’) and merges (C”, D”).
	dhfr (dihydrofolate reductase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
	dhfr (dihydrofolate reductase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.

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