Luo YJ , Su YH .

	Figure 1. Expression patterns of bmp genes and pSmad staining during sea urchin development.(A) ISH of bmp2/4 and bmp5–8 in the mesenchyme blastula (MB), early gastrula (EG), late gastrula (LG), and early pluteus (EPL). Immunostaining of pSmad in the LG (B and C) and EPL (D–I) stages. In (A–G), the arrowheads indicate the bilateral signals in the aboral skeletogenic cells, and the arrows indicate signals in the CPs. (H–I) Higher magnifications of the left CP show pSmad staining in the ciliated HC (arrow) but not in the base (arrowhead). An antibody against acetylated α-tubulin antibody (AcTubulin) was used to stain cilia. The observed view is indicated at the bottom right corner of each panel (Bp, blastopore view), and the axes are labeled as O, oral; A, aboral; L, left; and R, right.
	Figure 2. Cell lineages and gene expression domains in the archenteron tip and the left CP.BrdU-labeled Smm and pSmad staining at the LG (A) and EPL stage (B; higher magnification of the left CP). (C) Double staining for soxE transcripts and pSmad in the archenteron tip (marked by the dotted line) at the LG stage. ISH of soxE and Vasa protein staining at the LG (D) and EPL stages (E). (F–I) Double staining of Vasa protein and ISH of pax6, six1/2, eya, and dach at the EPL stage. Expression patterns of germline marker genes at the LG (J and K) and EPL (L–N) stages. ISH of foxC (O–R) and foxF (S–V) at the LG, the intermediate LG-EPL, and EPL stages. Illustrations of gene expression domains in the archenteron tip at the LG stage (W), the intermediate LG-EPL stage showing the CP dividing process (X), and the left CP at the EPL stage (Y). The embryos were viewed from the left (Wa), apical (Wb), or oral side (X and Y). The arrows indicate veg2 descendants localized at the aboral tip of the archenteron at the LG stage and the HC at the EPL stage. The open arrowheads denote veg2 lineage at the oral tip of the archenteron at the LG stage and the esophageal domain at the EPL stage and the solid arrowheads indicate the Smm.
	Figure 3. BMP is required for left-sided structure formation and gene expression in the aboral veg2 descendants.ISH of bmp2/4 and pSmad immunostaining in control and DM-treated embryos at the LG (A, treated from 24 to 48 hpf) or EPL stage (B, treated from 42 to 48 hpf). The arrowheads in (B) indicate the single left HC. The sides of the rudiments (arrows) were observed at the advanced rudiment (AR) stage (B, bottom panels). (C) The ratios of rudiment sidedness in control and DM-treated larvae (χ2 test). (D) Immunostaining for pSmad or acetylated α-tubulin and ISH for soxE in mBMP4-treated embryos. The open arrowheads denote the single undivided CP, and the arrowheads indicate bilateral HCs. (E) The ratios of soxE expression patterns (χ2 test). (F–H) ISH analyses of LR marker genes in BMP signaling-perturbed embryos. The arrows in (H) indicate right-sided gene expression. (I) Nodal expression in BMP signaling-perturbed embryos (treated from 24 to 48 hpf) at the LG stage. (J) pSmad staining and gene expression patterns in BMP2/4 vMO-treated embryos. The arrow indicates the pSmad-positive HC. (K) The sides of the rudiments (arrows) at the AR stage in BMP2/4 vMO-treated embryos. The numbers in the bottom left-hand corners indicate the ratios of the displayed phenotypes.
	Figure 4. Nodal signaling blocks BMP signaling on the right side.(A) Nodal transcripts, pSmad staining, and soxE expression in 42, 48, and 54 hpf embryos. The arrows indicate nodal expression shifted from oral to the right lateral ectoderm. The arrowheads denote pSmad signal and soxE expression that changed from symmetric to the left side at the archenteron tip. (B) pSmad staining in Nodal signaling-perturbed embryos. The arrows indicate the single left HC in control embryos and bilateral HCs in SB-505124-treated embryos. (C and D) LR gene expression patterns after Nodal signals were perturbed. The arrowheads indicate soxE, pax6, six1/2, eya, and dach expression in the left CP in control and in both CPs in SB-505124-treated embryos. (E) Effects of Nodal vMO on LR marker genes. Note that in 80% of the Nodal vMO-treated embryos, the left and right CPs were not properly divided, and the cells still accumulated on the tip of the archenteron, presumably due to the developmental delay. The numbers in the bottom left-hand corners indicate the ratios of the displayed phenotypes.
	Figure 5. Nodal controls the asymmetric separation and apoptosis of small micromeres.ISH of nodal (A), pitx2 (B), and not (C) demonstrate that they are expressed in Smm on the right side (arrowheads) that are marked by Vasa antibody (arrows). (D) Percentages of Vasa-positive Smm in the left and right CPs. (E) The asymmetric and symmetric separation of Vasa-positive Smm (a) and the percentages (χ2 test) in control and SB-505124-treated embryos (b). (F) TUNEL staining revealed one (# = 1) to three (# = 3) apoptotic cells in the right CP. (G) TUNEL staining after perturbing Nodal signaling. The arrows in (F) and (G) indicate apoptotic cells in the CPs.
	Figure 6. Summary of gene expression domains and molecular pathways involved in LR patterning.(A) Illustration of gene expression domains in the early pluteus larva (oral view). Different colors represent different domains (I) that express a different set of genes (II), as indicated. (B) Schematic representation of the molecular pathways in LR patterning, created using the BioTapestry program [69]. BMP signal from the aboral skeletogenic mesenchyme cells (aboral PMC) is required for nodal expression in the right lateral ectoderm (RLE) through an unknown mechanism (red dashed line). Nodal signal at the RLE activates its downstream genes in the RLE and right CP (RCP). Nodal signal also induces apoptosis in the right-sided Smm and presumably does so by repressing nanos2 expression. Right-sided BMP signaling is blocked by Nodal and only functions in aboral veg2 descendants in the left CP (LCP) to activate its downstream genes. The genes shown in black, light gray, or dark gray are expressed, not expressed, or expressed weakly, respectively, in their specific domains at the early pluteus stage. The arrows do not necessarily indicate direct interactions.

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