Reciprocal signaling between the ectoderm and a mesendodermal left-right organizer directs left-right determination in the sea urchin embryo

Abstract

During echinoderm development, expression of nodal on the right side plays a crucial role in positioning of the rudiment on the left side, but the mechanisms that restrict nodal expression to the right side are not known. Here we show that establishment of left-right asymmetry in the sea urchin embryo relies on reciprocal signaling between the ectoderm and a left-right organizer located in the endomesoderm. FGF/ERK and BMP2/4 signaling are required to initiate nodal expression in this organizer, while Delta/Notch signaling is required to suppress formation of this organizer on the left side of the archenteron. Furthermore, we report that the H(+)/K(+)-ATPase is critically required in the Notch signaling pathway upstream of the S3 cleavage of Notch. Our results identify several novel players and key early steps responsible for initiation, restriction, and propagation of left-right asymmetry during embryogenesis of a non-chordate deuterostome and uncover a functional link between the H(+)/K(+)-ATPase and the Notch signaling pathway.

Figure 1. Establishment of left-right asymmetry in echinoderms.

Left-right asymmetry in echinoderms is characterized by the asymmetric positioning of the imaginal rudiment on the left side of the bilateral pluteus larva. The adult emerges from this imaginal rudiment through metamorphosis. Formation of the rudiment is intimately linked to development of two mesodermal derivatives, the coelomic pouches, that form from an unpaired coelomic sac that budds off from the tip of the archenteron. The coelomic pouches are bilateral structures, but only the coelomic pouch located on the left side of the larva proliferates and differentiates to form the rudiment. Precursors of the coelomic pouches have a double origin. Part of these precursors derives from the small micromeres that form by asymmetric division of the large micromeres at 5th cleavage. These cells are thought to contribute to the germ line. Another population of coelomic pouch precursors derives from the non-skeletogenic mesoderm that is induced during blastula stages by Delta signals emanating from the skeletogenic mesenchymal cell precursors.

Figure 2. Left-right asymmetric expression of nodal is initiated in a discrete endodermal territory.

A, Whole-mount in situ hybridizations with a nodal probe. Asymmetric expression of nodal is first detected in the endoderm, several hours before the onset of asymmetric nodal expression in the ectoderm. AV, view from the animal pole; DV, view from the dorsal side; L, left; R, right. The black arrowheads highlight the beginning of nodal expression on the right side of the tip of the archenteron and in the ectoderm. The inset shows a high magnification view of the nodal expressing cells arranged in a rosette. B, Double fluorescent in situ hybridization with nodal (red) and either the endodermal marker gene foxA or the coelomic pouch marker gene foxF (green). The early expression of nodal is initiated in the endoderm territory underlying the coelomic pouch precursors that express foxF. The schemes on the right depict the territories expressing nodal (red) with respect to the mesodermal territory that expresses foxF and the endodermal territory that expresses foxA (green).

Figure 3. Both inhibition of Notch signaling and inhibition of the H^+/K⁺-ATPase cause bilateral expression of nodal in the endomesoderm and randomize nodal expression in the ectoderm.

A, nodal expression in control embryos and in embryos treated with DAPT or omeprazole starting after fertilization or injected with a morpholino oligonucleotide against Delta. Black arrowheads show an ectopic expression of nodal on the left side. The percentages indicate the proportion of embryos showing the same sidedness of nodal expression as that showed in the panel. B, At pluteus stage, nodal and univin expression in DAPT-treated embryos or in Delta-morpholino injected embryos is randomized. C, The expression of pitx2, sox9 and foxF in the coelomic pouch precursors as well as the expression of tropomyosin in the muscle cell precursors is strongly reduced or absent in DAPT-treated embryos. D, Time course of DAPT treatments. Embryos were treated with DAPT starting at different stages and nodal expression was scored at pluteus stages. DAPT treatments perturb left-right asymmetry only when performed before hatching. VEB, very early blastula; EB, early blastula; SB, swimming blastula; MB, mesenchyme blastula. E, The window during which DAPT treatments interfere with left-right asymmetry coincides with the period during which non skeletogenic mesoderm precursors are induced by Delta/Notch signaling. AV, Animal views; DV, Dorsal views; L, Left; R, Right.

Figure 4. Treatments with the H⁺/K⁺ATPase inhibitor omeprazole phenocopy inhibition of Notch signaling.

A, Phenotype of control embryos and of embryos treated as indicated at late gastrula and at pluteus stages. DAPT, omeprazole-treated embryos and Delta morpholino injected embryos develop with a smooth archenteron that lacks delaminating secondary mesenchymal cells at late gastrula stage and that are albinos at pluteus stage. The black arrowhead shows pigment cells embedded in the ectoderm of control embryos. B, Whole mount in situ hybridization with mesodermal or endodermal molecular markers. The expression of the Delta target genes gcm, papss, and gata1/2/3 in the non-skeletogenic mesoderm territory is abolished in DAPT-treated and in omeprazole-treated embryos. In contrast, Delta, msp130 and foxA are expressed at normal levels in the skeletogenic primary mesenchymal cell precursors or endodermal precursors in DAPT-treated, omeprazole-treated or Delta morpholino injected embryos. Vegetal pole views of control and treated embryos are shown in the upper corners. Note, that the expression of foxA in the Delta Morpholino injected embryos and in the DAPT or omeprazole treated embryos, has expanded towards the vegetal pole, consistent with the absence of mesodermal precursors in these embryos. SB, swimming blastula; MB, mesenchyme blastula. Embryos are in frontal views. C, Luciferase assays with the Notch reporter RBP-JK. Omeprazole strongly inhibits Notch signaling induced by overexpression of Delta but has no effect on Notch signaling induced by overexpression of NEXT or NICD.

Figure 5. FGF/MAP kinase signaling is required for nodal expression in the left-right organizer.

A, The early asymmetrical expression of nodal in the endoderm is lost following inhibition of FGFR/MAPK signaling. In embryos treated with the FGF receptor inhibitor SU5402 or the MEK inhibitor U0126 from fertilization onwards, nodal expression is not initiated in the endomesoderm. DAPT treated embryos show bilateral expression of nodal in the endomesoderm but embryos treated with DAPT followed by U0126 treatment at mesenchyme blastula stage do not express nodal in the endomesoderm. B, Inhibition of ERK or FGF signaling randomizes nodal and univin expression in the ectoderm as well as pitx2 and sox9 expression in the coelomic pouch precursors at pluteus stage. C, Inhibition of MAPK signaling randomizes the positioning of the rudiment. D, Time course of U0126 treatments. Embryos were treated with U0126 starting at the indicated stages and the sidedness of nodal expression was scored at pluteus stage. The efficiency of U0126 treatment on left-right asymmetry drops after 22 hpf, which coincides with the onset of asymmetrical expression of nodal in the endoderm. The percentages and total number of embryos in each experiment are indicated at the bottom of pictures. E, The drop in the ability of U0126 to block nodal expression coincides with the onset of asymmetrical expression of nodal in the endoderm. AV, animal views; DV, dorsal views; L, left; R, right.

Figure 6. BMP signaling is required for nodal expression in the left-right organizer.

A, nodal expression in the endomesoderm requires BMP signaling. Both treatments with BMP4 protein and injection of bmp2/4 or alk3/6 morpholinos abolish nodal expression in the endoderm at gastrula stage. B, nodal expression at pluteus stage in the bmp2/4 or alk3/6 morphants. Following inhibition of bmp2/4 or alk3/6 by morpholino injection into the egg, nodal expression occurs randomly on the left or right sides. The arrowheads point to the expansion of nodal to the dorsal part of the ciliary band. C,D, Alk3/6 function is required in the endomesoderm for nodal expression in the left-right organizer. Following injection of alk3/6 morpholino into the four vegetal blastomeres, nodal expression is not inititated in the endomesoderm and is randomized at pluteus stage. E–G, Effects of inhibiting BMP signaling on either the left or the right side on nodal expression. Embryos were injected with the bmp2/4 morpholino together with FLDX (green) into one blastomere at the two-cell stage, the position of the lineage tracer was revealed after in situ hybridization (red in pictures). F Injection of the bmp2/4 morpholino targeting the left side more strongly affected nodal expression in the organizer at gastrula stage than injections targeted to the right side. Injection of the bmp2/4 morpholino on the left but not on the right side randomizes nodal expression at pluteus stage. H, BMP signaling in the endomesoderm is biased towards the left side at late gastrula stage. Fluorescence microscope images (3 middle images) and confocal microscope image (lower picture) of embryos at the late gastrula stage immunostained with an anti phospho Smad1/5/8 reveal a domain of strong BMP signaling near the tip of the archenteron. This staining was asymmetric in about two third of the embryos, with stronger staining in the dorsal-left sector of the archenteron opposite to the nodal expressing territory. AV, animal view; DV, dorsal view; plut, pluteus larva; R, right side; L, left side.

Figure 7. Establishment of left-right asymmetry requires reciprocal Nodal signaling between the ectoderm and endomesoderm.

A,B ectodermal Nodal signals are required for nodal expression in the endomesoderm. A, Experimental design. The four animal blastomeres of a 8-cell stage embryo were injected with a nodal morpholino and nodal expression was analyzed at the gastrula stage. B, DIC and Fluorescent images of injected larvae and whole mount in situ hybridization of injected embryos with the nodal probe. Blocking nodal mRNA translation in the ectoderm abolishes nodal expression in the endomesoderm at gastrula stage and radializes the embryos. C,D, nodal signaling in the endomesoderm is required for establishment of left-right asymmetry in the ectoderm. C, Experimental design. The vegetal half (four blastomeres) of embryos at the eight-cell stage were injected with the alk4/5/7 morpholino. D, DIC and Fluorescent images of injected larvae and whole mount in situ hybridization of embryos with the nodal probe. nodal is expressed on the right side of the ectoderm in control embryos but in 9 out of 19 injected embryos, nodal expression is detected on the left side. pitx2 is expressed on the right side of the endomesoderm in control embryos but in 5 out of 6 alk4/5/7 morpholino injected embryos, pitx2 expression is lost. E,F, Mosaic analysis using chimeric embryos produced by microsurgery. E, Experimental design. The vegetal half of a nodal morphant (green) was combined with a control animal half (grey). F, Fluorescent and DIC images of a chimeric larvae and whole mount in situ hybridization of chimeric embryos with a nodal probe. nodal is expressed on the right side of the ectoderm in control embryos but in 5 out of 12 chimeric embryos, nodal expression is detected on the left side. V, ventral; D, dorsal; LV, lateral view; AV, animal pole view; L, left; R, right; An, animal pole; Veg, vegetal pole.

Figure 8. Long-range Nodal signaling within the ectoderm and between the ectoderm and the endodermal left-right organizer requires Univin.

A, Experimental design to test the role of Univin in long-range signaling of Nodal during establishment of left-right asymmetry. Following injection of the nodal morpholino into the egg, a synthetic nodal mRNA (immune against the morpholino) alone or a mixture of nodal+univin mRNAs were injected into one animal blastomere (presumptive ectoderm) of embryos at the 8-cell stage B, Experimental results. Injection of the nodal morpholino alone abolishes pitx2 expression and causes bilateral expression of sox9. In this nodal morpholino background, local injection of nodal mRNA alone into one blastomere at the 8-cell stage does not rescue pitx2 expression but co-injection of nodal and univin mRNAs efficiently induces pitx2 at a long distance from the injection clone. The fluorescent images show a Nodal morpholino injected larva (RLDX fluorescence) or larvae rescued by injection of nodal mRNA or by a combination of both nodal and univin mRNAs into one blastomere at the 8-cell stage (merged images of RLDX and FLDX fluorescence).

Figure 9. Model for establishment of left-right asymmetry by reciprocal signaling between the ectoderm and the endomesoderm.

A, At midgastrula stage, nodal is expressed asymmetrically in endodermal cells on the right side under the influence of both positive (FGF and BMP signaling) and negative (signal X) inputs. At this stage, nodal is also expressed symmetrically in the ventral ectoderm. In contrast, univin is expressed more laterally in the presumptive ciliary band ectoderm and throughout the endoderm. As a consequence, the nodal and univin territories only partially overlap in the archenteron and in two ectodermal regions flanking the presumptive stomodeum (purple color). At this stage, while expression of nodal in the ventral ectoderm vanishes, asymmetrical Nodal+Univin signaling on the right side of the archenteron induces nodal expression in the lateral right ectoderm that expresses univin creating a novel Nodal+Univin expressing signaling center on the right side. The reaction-diffusion mechanism between Nodal and Lefty stabilizes nodal expression on the right side and prevents its expansion to the rest of the embryo. B, Summary diagrams of the experiments. Both the loss of nodal expression in the endoderm caused by inhibition of the FGF or BMP pathways or the bilateralisation of nodal expression in the endoderm caused by inhibition of Notch signaling, randomize nodal expression in the ectoderm at pluteus stage. In the endoderm, FGF/ERK positively regulates nodal expression on the right side while unidentified signals coming from the mesoderm induced by Delta/Notch signaling negatively regulate nodal expression on the left side of the archenteron. The window during which SU5402 and UO126 are effective at perturbing left-right asymmetry extends from fertilization to mesenchyme blastula/gastrula stage (green shading). H⁺/K⁺-ATPase acts on Delta/Notch signaling to regulate left-right asymmetry. The window during which DAPT (red shading) and omeprazole (blue shading) are effective on left-right asymmetry extends from egg up to the early blastula stage. Inhibition of Nodal or BMP signaling in the endomesoderm randomizes nodal expression in ectoderm. Injection of nodal mRNA alone fails to rescue left-right asymmetry and pitx2 expression in embryos previously injected with a nodal morpholino but coinjection of nodal+univin mRNAs efficiently rescues pitx2 expression in the coelomic pouch and ciliary band. In most of these embryos, pitx2 is expressed more strongly on the right side. An, animal pole; Veg, vegetal pole.

Figure 10. Multiple signals cooperate to establish a left-right endodermal organizer in the sea urchin embryo.

Biotapestry diagram describing the gene regulatory interactions identified in this study that specify the left-right endodermal organizer. During gastrulation, Nodal and Univin signals produced by the ventral ectoderm induce nodal and univin expression on the right side of the endoderm territory while non-skeletogenic mesodermal cells specified by Delta/Notch signaling send a putative inhibitory signal (signal X) that represses nodal expression on the left side of the adjacent endodermal territory. At gastrula stage, BMP signals emanating from the ventral ectoderm that expresses bmp2/4 are received on the dorsal sector of the archenteron where they induce an unknown signal (signal Y) that cooperates with FGF signaling to induce nodal expression on the ventral-right side of the endoderm territory. Cell interactions between the left-right organizer located in the ventral-right sector and the dorsal endoderm further pattern this region causing BMP signaling to be restricted to the left portion of archenteron. Note that production of signal X may involve several steps and that the origin of the FGF signal is not known. Nodal and Univin signals emanating from the ventral-right endodermal region in turn promote nodal, univin and pitx2 expression on the right side of the ciliary band ectoderm and prevent rudiment formation on the right side in part by repressing the expression of genes encoding germ line specific factors such as Sox9 .

Figure 11. Comparison between vertebrates and echinoderms: the laterality information emanating from a mesendodermal left-right organizer propagates to distant tissues through Nodal/Univin signaling.

In vertebrates, the asymmetric expression of nodal is initiated in a discrete region on the midline that plays the role of a left-right organizer (the node in mammals, the Küpffer vesicle in zebrafish or the gastrocoel roof in amphibians). H⁺/K⁺-ATPases, Delta/Notch and FGF signaling as well as cilia driven leftward flow have been implicated in the symmetry breaking process. Nodal expression subsequently propagates to the lateral regions of the embryos by mechanisms that involve long-range signaling by Nodal and GDF1 as well as communication with endodermal cells through gap junctions. In the sea urchin embryo, H⁺/K⁺-ATPase, Delta/Notch and FGF signaling are involved in the initiation of asymmetrical expression of nodal in a discrete region of the archenteron that appears to play the role of a left-right organizer. In the sea urchin, the laterality information is relayed from the left-right organizer to more distant regions through long range Nodal-Univin signaling.