Single-minded 2 is required for left-right asymmetric stomach morphogenesis

Abstract

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects.

Keywords: Down syndrome; Frog; Left-right asymmetry; Morphogenesis; Pitx2; Sim2; Stomach.

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′).