Petri N et al. (2024), Molecular anatomy of emerging Xenopus left-righ...

XB-ART-60759

Dev Dyn 2024 Jun 27; doi: 10.1002/dvdy.722.

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Molecular anatomy of emerging Xenopus left-right organizer at successive developmental stages.

Petri N , Vetrova A , Tsikolia N , Kremnyov S .

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BACKGROUND: Vertebrate left-right symmetry breaking is preceded by formation of left-right organizer. In Amphibian, this structure is formed by gastrocoel roof plate, which emerges from superficial suprablastoporal cells. GRP is subdivided into medial area, which generates leftward flow by rotating monocilia and lateral Nodal1 expressing areas, which are involved in sensing of the flow. After successful symmetry breaking, medial cells are incorporated into a deep layer where they contribute to the axial mesoderm, while lateral domains join somitic mesoderm. RESULTS: Here, we performed detailed analysis of spatial and temporal gene expression of important markers and the corresponding morphology of emerging GRP. Endodermal marker Sox17 and markers of superficial mesoderm display complementary patterns at all studied stages. At early stages, GRP forms Tekt2 positive epithelial domain clearly separated from underlying deep layers, while at later stages, this separation disappears. Marker of early somitic mesoderm MyoD1 was absent in emerging GRP and was induced together with Nodal1 during early neurulation. Decreasing morphological separation is accompanied by lateral to medial covering of GRP by endoderm. CONCLUSION: Our data supports continuous link between superficial mesoderm at the start of gastrulation and mature GRP and suggests late induction of somitic fate in lateral GRP.

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Genes referenced: myod1 nodal1 sox17b tekt2

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	FIGURE 1. Molecular patterning of late blastula (stage 9). (A, a′) Nodal1 expression in the vegetal hemisphere. (B, b′) Sox17 expression in the vegetal hemisphere. (C, c′) Foxj1 expression in the superficial mesoderm. (D, d′) Lefty expression in the deep mesoderm. (E–G) Dand5, MyoD1 and Tekt2 expression is still undetectable. An, animal pole; D, dorsal, L, left; R, right; V, ventral; Veg, vegetal pole. Whole‐mount blastulae (A–G) are viewed from dorso‐vegetal side, transverse sections through dorsal side (a′–d′) are sagittal. Asterisks indicate the blastocoel.
	FIGURE 2. Molecular patterning at the beginning of gastrulation (stage 10). (A, a′) Nodal1 expression in the vegetal hemisphere, bottle cells and suprablastoporal endoderm. (B, b′) Sox17 expression in the vegetal hemisphere, bottle cells and suprablastoporal endoderm. (C, c′) Foxj1 expression in the superficial mesoderm. (D, d′) Lefty expression in the deep mesoderm. An, animal pole; D, dorsal, L, left; R, right; V, ventral; Veg, vegetal pole. Whole‐mount gastrulae (A–D) are viewed from dorso‐vegetal side, transverse sections through dorsal blastopore lip (a′–d′) are sagittal. Arrowheads indicate forming archenteron marked by bottle cells.
	FIGURE 3. Molecular patterning of early gastrula (stage 10.5). (A) Scheme of whole‐mount embryo, dorsal side up. Red line indicates the position of the sagittal sections. (a′) Scheme of dorsal explant, dorsal side up. (a″) Scheme of dorsal explant, ventral side up. (B, b′) Nodal1 expression in the vegetal hemisphere, bottle cells and suprablastoporal endoderm. (C, c′) Sox17 expression in the vegetal hemisphere, bottle cells and suprablastoporal endoderm. (D, d′) Foxj1 expression in the superficial mesoderm. (E, e′) Tekt2 expression in the superficial mesoderm. (F, f′) Lefty expression in the deep axial mesoderm. (G, g′) MyoD1 expression in the deep paraxial mesoderm. Yellow arrowheads indicate zones of expression. (H) Scanning electron microscopy image of emerging archenteron roof. An, animal pole; D, dorsal, L, left; R, right; V, ventral; Veg, vegetal pole. Whole‐mount gastrulae (B–G) are viewed from dorso‐vegetal side, transverse sections through dorsal blastopore lip are sagittal (b′–f′) or cross (g′). Red arrowheads indicate forming archenteron marked by bottle cells.
	FIGURE 4. Molecular patterning of mid gastrula (stage 11). (A) Scheme of whole‐mount embryo, dorsal side up. Red line indicates the position of the sagittal sections. (a′) Scheme of dorsal explant, dorsal side up. (a″) Scheme of dorsal explant, ventral side up. (B, b′) Sox17 expression in the yolk cells, bottle cells and suprablastoporal endoderm involuted into the archenteron. (C, c′) Foxj1 expression in the superficial mesoderm. (D, d′) Tekt2 expression in the superficial mesoderm. (E, e′) Lefty expression in the deep axial mesoderm. (F, f′) MyoD1 expression in the deep paraxial mesoderm. Yellow arrowheads indicate zones of expression. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (B–F) are viewed from ventral side, transverse sections through dorsal blastopore lip are sagittal (b′–e′) or cross (f′). Red arrowheads indicate bottle cells forming the front margin of achenteron.
	FIGURE 5. Molecular patterning of mid gastrula (stage 11.5). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, dorsal side up. (a″) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of emerging archenteron roof. (C, c′) Sox17 expression in the yolk cells, bottle cells and suprablastoporal endoderm now marking the marginal zone of archenteron. (D, d′) Foxj1 expression in the superficial mesoderm undergoing involution. (E, e′) Tekt2 expression in the superficial mesoderm undergoing involution. (F, f′) Lefty expression in the deep axial mesoderm. (G, g′) MyoD1 expression in the deep paraxial mesoderm. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (B–G) are viewed from ventral side, transverse sections through dorsal blastopore lip are cross (c′–g′). Arrowheads indicate bottle cells forming the front margin of achenteron.
	FIGURE 6. Molecular patterning of late gastrula (stage 12). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, dorsal side up. (a″) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of emerging archenteron roof. (C, c′) Sox17 expression in the yolk cells and the endoderm marking the marginal zone of archenteron. (D, d′) Foxj1 expression in the superficial mesoderm undergoing involution. (E, e′) Tekt2 expression in the superficial mesoderm undergoing involution. (F, f′) Lefty expression in the deep axial mesoderm. (G, g′) MyoD1 expression in the deep paraxial mesoderm. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (B–G) are viewed from ventral side, transverse sections through dorsal blastopore lip are cross (c′–g′). Arrowheads indicate bottle cells forming the front margin of achenteron.
	FIGURE 7. Molecular patterning at the end of gastrulation (stage 12.5). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, dorsal side up. (a″) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of archenteron roof. (C, c′) Sox17 expression in the yolk cells and the endoderm marking the marginal zone of archenteron. (D, d′) Foxj1 expression in the superficial mesoderm in the archenteron roof. (E, e′) Tekt2 expression in the superficial mesoderm in the archenteron roof. (F, f′) Lefty expression in the deep axial mesoderm. (G, g′) MyoD1 expression in the deep paraxial mesoderm. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (B–G) are viewed from ventral side, transverse sections through dorsal blastopore lip are cross (c′–g′). Red arrowhead indicates bottle cells forming the front margin of achenteron. Yellow arrowhead indicates «hole‐like» structure in the superficial cell layer of archenteron roof. Dot lines indicate cell separation.
	FIGURE 8. Molecular patterning at the beginning of neurulation (stage 13). Formation of gastrocoel roof plate. (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, dorsal side up. (a″) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of archenteron roof. (C) Nodal1 expression is yet undetectable. (D, d′) Dand5 expression emerges in the lateral cells of GRP. (E, e′) MyoD1 expression in the deep paraxial mesoderm. (F, f′) Sox17 expression in the yolk cells and the endoderm marking the marginal zone of archenteron. (G, g′) Foxj1 expression marks the GRP in the archenteron roof and the neural plate. (H, h′) Tekt2 expression marks the GRP in the archenteron roof. (I, i′) Lefty expression in the neural floor plate. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (C–I) are viewed from ventral side, transverse sections through the explants are cross (B, d′–i′). Dot lines indicate cell separation.
	FIGURE 9. Molecular patterning at early neurula (stage 14). Specification of lateral cells in the GRP. (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of GRP. (C, c′) Nodal1 expression emerges in the lateral cells of GRP. (D, d′) Dand5 expression in the lateral cells of GRP. (E, e′) MyoD1 expression marks the deep paraxial mesoderm and emerges in the lateral cells of GRP. (F, f′) Sox17 expression in the endoderm. (G, g′) Foxj1 expression marks the GRP in the archenteron roof and the neural plate. (H, h′) Tekt2 expression marks the GRP in the archenteron roof. (I, i′) Lefty expression in the neural floor plate. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (C–I) are viewed from ventral side, transverse sections through the explants are cross (B, c′–i′). Dot lines indicate cell separation.
	FIGURE 10. Molecular patterning at mid neurula (stage 15). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of GRP. (C, c′) Nodal1 expression in the lateral cells of GRP. (D, d′) Dand5 expression in the lateral cells of GRP. (E, e′) MyoD1 expression in the deep paraxial mesoderm and in the lateral cells of GRP. (F, f′) Sox17 expression in the endoderm. (G, g′) Foxj1 expression marks the medial part of GRP and the neural plate. (H, h′) Tekt2 expression marks the GRP and the neural plate. (I, i′) Lefty expression in the neural floor plate. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (C–I) are viewed from ventral side, transverse sections through the explants are cross (B, c′–i′). Dot lines indicate cell separation.
	FIGURE 11. Molecular patterning at mid neurula (stage 16). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of GRP. (C, c′) Nodal1 expression in the lateral cells of GRP. (D, d′) Dand5 expression in the lateral cells of GRP. (E, e′) MyoD1 expression in the deep paraxial mesoderm and in the lateral cells of GRP. (F, f′) Sox17 expression in the endoderm. (G, g′) Foxj1 expression marks the medial part of GRP, the neural plate and several cells in the ectoderm. (H, h′) Tekt2 expression marks the GRP, the neural plate and several cells in the ectoderm. (I, i′) Lefty expression in the neural floor plate. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (B–I) are viewed from ventral side, transverse sections through the explants are cross (c′–i′). Dot lines indicate cell separation.
	FIGURE 12. Molecular patterning at late neurula (stage 17). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, ventral side up. (B) Scanning electron microscopy image of GRP. (C, c′) Nodal1 expression in the lateral cells of GRP. (D, d′) Dand5 expression in the lateral cells of GRP. (E, e′) MyoD1 expression in the deep paraxial mesoderm and in the lateral cells of GRP. (F, f′) Sox17 expression in endoderm. (G, g′) Foxj1 expression marks the medial part of GRP, the neural plate and several cells in the ectoderm. (H, h′) Tekt2 expression marks the GRP, the neural plate and several cells in the ectoderm. (I, i′) Lefty expression in the neural floor plate. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (C–I) are viewed from ventral side, transverse sections through the explants are cross (B, c′–i′). Dot lines indicate cell separation.
	FIGURE 13. Molecular patterning at late neurula (stage 18). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, ventral side up. (B, b) Scanning electron microscopy image of GRP. (C, c′, c″) Nodal1 expression in the lateral cells of GRP. (D, d′) Dand5 expression in the lateral cells of GRP. (E, e′, e″) MyoD1 expression in the deep paraxial mesoderm and in the lateral cells of GRP. (F, f′) Sox17 expression in the endoderm. (G, g′) Foxj1 expression disappears from the medial part of GRP and marks the neural plate and several cells in the ectoderm. (H, h′) Tekt2 expression marks the GRP, the neural plate and several cells in the ectoderm. (I, i′) Lefty expression in the neural floor plate. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (C–I) are viewed from ventral side, transverse sections through the explants are cross (B, b, c′–i′). Dot lines indicate cell separation, arrowheads indicate the contraction of apical surface of lateral GRP cells.
	FIGURE 14. Molecular patterning at the tailbud stage (stage 23). (A) Scheme of whole‐mount embryo, dorsal side up. (a′) Scheme of dorsal explant, ventral side up. (B, b′, b″) Nodal1 expression in the lateral cells of GRP is gradually covered by endoderm layer. (C, c′, c″) Dand5 expression in the lateral cells of GRP. (D, d′) MyoD1 expression in the deep paraxial mesoderm and in the lateral cells of GRP. (E, e′, e″) The endoderm marked by Sox17 is covering the GRP. (F, f′, f″) Tekt2 expression marks the GRP, the neural plate and several cells in the ectoderm. Ant, anterior; Dors, dorsal, L, left; Post, posterior; R, right; Ventr, ventral. Dorsal explants (B–F) are viewed from ventral side, transverse sections through the explants are cross (b′–f″). Dot lines indicate cell separation.
	FIGURE 15. Expression pattern scheme of the studied genes at successive stages of Xenopus development. (A) Scheme of expression patterns at early gastrula (stage 10.5). Nodal1 and Sox17 are coexpressed in the vegetal hemisphere, bottle cells and suprablastoporal endoderm. Lefty expression is located in the deep axial mesoderm, and MyoD1 expression in the deep paraxial mesoderm. Foxj1 and Tekt2 are coexpressed in the superficial mesoderm. (B) Scheme of expression patterns at late gastrula (stage 12). Sox17 is expressed in the endoderm around the emerging left–right organizer. Lefty expression is in the deep axial mesoderm, and MyoD1 expression in the deep paraxial mesoderm. Foxj1 and Tekt2 expression areas in the superficial mesoderm have undergone involution and are positioned at the ventral side of the gastrocoel roof. Nodal1 expression in the vegetal hemisphere has faded. (C) Scheme of expression patterns at the beginning of neurulation (stage 13). Sox17 is expressed in the endoderm around the emerging left–right organizer. Lefty expression is in the deep axial mesoderm, and MyoD1 expression in the deep paraxial mesoderm. Foxj1 and Tekt2 expression marks the GRP in the archenteron roof, with additional expression of Foxj1 in the neural plate. Dand5 expression emerges in the lateral cells of GRP. (D) Scheme of expression patterns at early neurula (stage 14). Sox17 is expressed in the endoderm, and Lefty is expressed in the neural floor plate. MyoD1 expression marks the deep paraxial mesoderm and emerges in the lateral cells of GRP. Foxj1 expression marks the GRP in the archenteron roof and the neural plate, and Tekt2 expression marks the GRP in the archenteron roof. Dand5 is expressed in the lateral cells of GRP, and Nodal1 starts to be coexpressed with Dand5. (E) Scheme of expression patterns at late neurula (stage 18). Sox17 is expressed in the endoderm, and Lefty is expressed in the neural floor plate. MyoD1 expression is located in the deep paraxial mesoderm and in the lateral cells of GRP. Foxj1 expression disappears from the medial part of GRP and marks the neural plate and several cells in the ectoderm. Tekt2 expression marks the GRP, the neural plate and several cells in the ectoderm. Nodal1 and Dand5 are coxpressed in the lateral cells of GRP. (F) Scheme of expression patterns at tailbud stage (stage 23). The endoderm marked by Sox17 is covering the GRP. MyoD1 is expressed in the deep paraxial mesoderm and in the lateral cells of GRP. Nodal1 and Dand5 are coexpressed in the lateral cells of GRP which are gradually covered by endoderm layer. Tekt2 expression marks the GRP, the neural plate and several cells in the ectoderm. n, notochord; s, presumptive somitic mesoderm.