Wyatt BH , Amin NM , Bagley K , Wcisel DJ , Dush MK , Yoder JA , Nascone-Yoder NM .

R01 HD095937 NICHD NIH HHS , P30 ES025128 NIEHS NIH HHS

Xla Wt + pitx2 MO (Fig. 2 I. )

	Fig. 1. Left versus right stomach transcriptome analyses identify sim2. (A) Left lateral views of Lepidobatrachus laevis embryos at stages before (GS18), during (GS19-GS20) and after (GS21) overt stomach curvature; the stomach region is indicated by the white dashed box. Color-coding of transverse sections indicates the left (L; blue) and right (R; yellow) stomach tissues dissected for transcriptome analyses at each phase of curvature. (B) Heat map of 278 L. laevis transcripts with significant left (blue; 179) or right (yellow; 99) enrichment at each stage of curvature (q-value≤0.01). (C) Venn diagram showing the number of transcripts with left- or right-enrichment at each stage (q-value≤0.01), as well as those asymmetrically expressed at more than one stage. Although no transcripts are consistently right-sided across all four stages, two transcripts are left-sided throughout stomach curvature: pitx2 and sim2. (D) Left (blue) and right (yellow) expression of L. laevis pitx2 and sim2 at each stage of stomach curvature. FPKM, fragments per kilobase of transcript per million mapped reads. Significant differences between sides and stages are indicated by lower case letters, used to label means such that bars bearing different letters are statistically different from one another (P<0.05). (E) Diagrams of Xenopus embryos (left lateral and transverse views) at equivalent phases (NF35, NF37, NF39, NF40) of stomach curvature illustrate the approximate planes of section (red dotted lines), and the position of the left (blue) and right (yellow) stomach walls shown in the corresponding tissue sections in F and G. (F,G) The spatial expression patterns of pitx2 (F) and sim2 (G) were validated by RNA ISH. Images in G are neighboring sections from the same embryo shown in F; asterisks demarcate expression in the stomach dorsal midline. See Fig. S1 for additional sim2 expression patterns. Scale bar: 1 mm (A); 500 μm (F,G).
	Fig. 2. Sim2 expression is regulated by LR patterning. (A-F) The expression patterns of Xenopus sox2 (A,D), pitx2 (B,E) and sim2 (C,F) were determined by RNA ISH on anterior and posterior stomach sections from DMSO (A-C; two examples are shown) and two different SB505124-exposed embryos (D-F; NF39) with straightened (Example 1) and reversed (Example 2) stomach curvatures. In A-C and D-F, serial sections from the same individual were hybridized with each probe. Black asterisks and arrowheads in F indicate sim2 expression retained in the stomach dorsal midline and pronephric tubules, respectively. (G-J) Whole mount RNA ISH with a probe for sim2 was also performed on embryos injected with either a control (CoMO; G) or pitx2 morpholino (pitx2-MO; I), targeted to the embryo's left side, or injected with mRNA encoding a dexamethasone-inducible pitx2 construct (pitx2-GR) on the right side and exposed to ethanol control (EtOH; H) or dexamethasone (Dex; J). Each embryo in G-J is shown in right and left views, with a representative transverse section through the stomach (NF40) (dotted lines indicate location of lumen). Arrows indicate sim2 expression evident in the left and/or right stomach walls; red asterisks in I indicate reduction or absence of the expected left side sim2 expression domains.
	Fig. 3. sim2 is required for cellular events underlying stomach curvature. (A-T′) Xenopus embryos were injected with sim2 guide RNA alone (sim2-gRNA; A,C,E,G,G′), sim2 gRNA plus Cas9 RNA (sim2-gRNA+Cas9; B,D,F,H,H′), control morpholino (CoMO; I,K,M-M′,O,Q,Q′,S,S’), sim2 morpholino (sim2-MO; J,L,N-N′,P,R,R′,T,T′) and mRNA encoding GFP (K-T′). The greater curvature of the stomach (NF42) is indicated by arrows in control embryos (A,I; ventral views); asterisks indicate the absence of the greater curvature in Sim2 loss-of-function embryos (B,J). Sections through the stomach of sim2-gRNA or sim2-gRNA+Cas9 embryos (NF39) were stained to reveal Integrin (Int; magenta in C,D, white in E,F) and nuclei (nuc; blue in C,D, white in G-H′). The slit of the gut lumen dividing the right (R) and left (L) stomach walls is demarcated by yellow lines. The boxed areas in C, D, G and H are shown in magnified view in E and G, F and H, G′, and H′, respectively. In the left wall of control stomachs (G,G′), endoderm nuclei are aligned (red arrowheads, G′) and accumulating in close proximity to the basement membrane (BM, red line in G′). However, in the left wall of sim2-CRISPR stomachs (H,H′), endoderm nuclei remain broadly distributed between the apical surface (yellow line) and BM (red line). Sections through the stomach region of CoMO or sim2-MO embryos (NF39) were stained to reveal β-catenin (βcat, red; K-N′), α-tubulin (αTub, red in O-R, white in Q′,R′), E-cadherin (Ecad, magenta in S,T, white in S′,T′), GFP (green; K-N,O-R,S,T) and/or nuclei (nuc; blue in K-N′; white in M′,N′). The boxed areas in K, L, O and P are shown in magnified view in M-M′, N-N′, Q,Q′ (with a neighboring section shown in S,S’) and R,R′ (with a neighboring section shown in T,T′), respectively. In the left wall of CoMO stomachs, endoderm cells are columnar (M,Q,S), with consistently basolaterally localized βcat (arrowheads, M′) and E-cad (arrowheads, S′), and apicobasally polarized microtubules (αTub; arrowheads, Q′). In contrast, endoderm cells in sim2-deficient stomachs are rounded (N,R,T), with irregular βcat (arrowheads, N′) and E-cad (arrowheads, T′) distribution, and sparse, randomly-oriented microtubules (arrowheads, R′). sim2-MO injected (GFP-positive) apoptotic cells, devoid of tubulin staining, are evident in the gut lumen (asterisks in R,R′). In the left wall of CoMO stomachs (M′), endoderm nuclei are radially aligned (arrowheads) and accumulating near the BM (red line). However, in the left wall of sim2-MO stomachs (N′), the nuclei of the disorganized endoderm layer remain broadly and randomly distributed. See Fig. S2 for sim2 CRISPR-induced indels, Fig. S3 for sim2 morpholino validation and Fig. S4 for additional characterization/validation of sim2-MO cellular phenotypes. Scale bars: 500 µm (A,B,I,J); 100 µm (C,D,K,L,O,P); 50 µm (E,F,G-H′,M-N′,Q-T′).
	Fig. S1. Sim2 is expressed in both symmetrical and asymmetrical patterns in different tissues. The expression of Xenopus sim2 was determined by RNA in situ hybridization on whole embryos (A-B, NF32; C-D, NF35) or on sections through the foregut (E, NF35; F, NF40). In A-D, each embryo is shown in right (A,C) and left (B, D) views; the insets show higher magnification views of bilateral expression in craniofacial structures, with red arrowheads indicating asymmetric sim2 expression, which first becomes visible in the prospective stomach region of the left foregut. In sections (E-F), stomach-specific expression is visible only in the left (L) wall of the stomach; the right (R) side is largely devoid of appreciable sim2 expression. Asterisks indicate expression in the dorsal midline of the foregut. Arrowheads in E indicate bilateral expression in both left and right pronephric tubules (NF35), while arrows in F indicate expression in both left and right lung buds (NF40).
	Fig. S2. Indels produced by sim2 CRISPR. A gRNA targeting the conserved HLH domain of Xenopus laevis sim2 (top) was injected into 1-cell Xenopus embryos along with Cas9 mRNA. Pooled genomic DNA from 10 neurulae was PCR amplified using exon 1-specific primers. Sequencing of a subset of individual clones validated the presence of deleterious mutations (red) corresponding with the sim2 gRNA target sequence (highlighted in blue). Of these, 62.5% (5/8; asterisks, *) are expected to generate null alleles (frameshifts) and/or compromise function (non-conservative amino acid substitution, e.g., Q to K).
	Fig. S3. Specificity and efficacy of the sim2 morpholino A) The graph indicates the frequency (percentage) of embryos in which the greater curvature of the stomach was normal (WT, blue) or reduced/absent (red), as elicited by injection of different concentrations of Control MO (CoMO), sim2-MO #1 (targeting the ATG start site of Xenopus sim2 mRNA) or sim2-MO #2 (targeting the boundary between intron 1 and exon 2 of Xenopus sim2); n= 20 embryos, on average (range 7-38), per condition. As expected, some embryos began to exhibit evidence of non-specific toxicity (severe edema, green) at higher concentrations. The two independent morpholinos displayed similar results, but MO #2 had lower efficacy and higher toxicity, so MO #1 was used for further experiments. B) Xenopus embryos (8-cell stage) were injected with CoMO or sim2-MO plus mRNA encoding membrane-tethered Red Fluorescent Protein (memRFP), and mRNA encoding GFP fused with the sim2 #1 MO target sequence (sim2_GFP), and cultured until NF24. Translation of memRFP (red), which does not contain the sim2-MO target sequence, remains unaffected by the presence of CoMO or sim2-MO. In contrast, while embryos injected with CoMO are able to effectively translate the sim2_GFP mRNA, as indicated by the GFP fluorescence (green), translation of this protein is completely knocked down in embryos injected with sim2-MO (as indicated by the lack of GFP fluorescence), demonstrating the efficacy of the sim2-MO reagent. Western blotting also confirms drastic reduction of GFP protein levels in extracts from sim2c-MO- but not CoMO- injected embryos. GAPDH, loading control. C) Frequency (percentage) of embryos in which the greater curvature of the stomach is normal or reduced/absent when CoMO or sim2-MO #1 are co-injected with exogenous, MO-resistant sim2 mRNA (n= 5-13 per condition). The frequency of sim2-MO induced stomach phenotypes was partially rescued (i.e., reduced) by the presence of sim2 mRNA, confirming the specificity of the MO reagent. D) Frequency (percentage) of embryos in which the greater curvature of the stomach is normal or reduced/absent when CoMO or sim2-MO #1 are targeted to the left (L) versus right (R) side of the stomach (n=3-16 per condition, 3 trials); right side injections have no effect on stomach curvature.
	Fig. S4. The sim2-MO induced cellular phenotype is independent of proliferation, precedes stomach curvature, and is specific to Sim2 function A-B) Xenopus embryos were injected with mRNA encoding membrane-localized GFP (mGFP) and either control morpholino (CoMO; A) or sim2 morpholino (sim2-MO; B), targeting the left stomach wall. Sections through the stomach (NF39) were stained to reveal Integrin (Int; blue), phoshphohistone H3 (pHH3; mitotic cells; red) and mGFP (green), showing that some sim2-MO injected (green) cells are also pHH3-positive; thus, Sim2 activity is not required for endoderm cells to remain proliferative. C-J) Xenopus embryos were injected with mRNA encoding nuclear GFP (GFP) and either control morpholino (CoMO; C-F) or sim2 morpholino (sim2-MO; G-J), targeting the left stomach wall, and harvested several hours prior to stomach curvature (NF37). Sections through the still-straight stomach were stained to reveal nuclear GFP (green; C-D, G-H), β−catenin (βcat, red; C, E, G, I), and alpha-tubulin (αTub, blue; C, F, G, J). The boxed areas in C and G are shown in magnified view in D-F and H-J, respectively. In the left wall of CoMO stomachs, endoderm cells are columnar, with consistently membrane-localized βcat (E) and apicobasally polarized microtubules (arrows, F). In contrast, endoderm cells in sim2-deficient stomachs are already rounded (dotted outlines in H and I), with irregularly distributed βcat (I; arrowheads, abnormal accumulation; asterisks, low/absent levels; dotted outlines, nuclear localization), and sparse or randomly-oriented (arrowheads, J) microtubules. K-V) Xenopus embryos were injected with mRNA encoding nuclear GFP plus CoMO (K- N), sim2-MO (O-R), or sim2-MO plus a MO-resistant mRNA encoding Xenopus sim2 (S- V) targeting the left stomach wall. Sections through the stomach (NF39) were stained to reveal nuclear GFP (green; K, L, O, P, S, T), β−catenin (βcat, red; K, M, O, Q, S, U), and alpha-tubulin (αTub, blue; K, N, O, R, S, V). The boxed regions in K, O and S are shown in magnified view in L-N, P-R, and T-V, respectively. In the left wall of control stomachs, CoMO-injected endoderm cells are columnar, with consistently membrane localized βcat (M) and apicobasally polarized microtubules (arrows, N). As expected, endoderm cells in sim2-deficient (sim2-MO injected) stomachs are rounded (dotted outlines in P and Q), with irregularly distributed βcat (Q; arrowheads, abnormal accumulation; asterisks, low/ absent levels; dotted outlines, nuclear localization), and sparse or randomly-oriented (arrowheads, R) microtubules. In contrast, consistent membrane-localized βcat (U) and apicobasally polarized microtubules (arrows, V) are restored when sim2 mRNA is co- injected with the sim2-MO, confirming the cellular effects of the morpholino reagent are specific to loss of Sim2 function. Scale bars for A, B, C, G, K, O, and S = 100 μm; scale bars for all other panels = 50 μm.
	Fig. S5. Over-expression of sim2 does not alter stomach curvature. Xenopus embryos were injected with mRNA encoding a dexamethasone-inducible pitx2 construct (pitx2-GR) or a dexamethasone-inducible sim2 construct (sim2-GR) on the right side and exposed to ethanol control (EtOH) or dexamethasone (Dex). The graph indicates the frequency (average percentage) of embryos in which the greater curvature of the stomach was reduced/absent, as elicited by injection of pitx2-GR (n=4-21 per condition, 2 trials) or sim2-GR (n=7-16 per conditions, 4 trials). While right side induction of pitx2- GR by Dex elicited a high percentage of abnormal stomach phenotypes compared to controls, ectopic induction of sim2-GR had no effect on stomach curvature.

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