Revinski DR et al. (2010), Delta-Notch signaling is involved in the segreg...

XB-ART-41000

Dev Biol 2010 Mar 15;3392:477-92. doi: 10.1016/j.ydbio.2010.01.010.

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Delta-Notch signaling is involved in the segregation of the three germ layers in Xenopus laevis.

Revinski DR , Paganelli AR , Carrasco AE , López SL .

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In vertebrates, the induction of the three germ layers (ectoderm, mesoderm and endoderm) has been extensively studied, but less is known about how they segregate. Here, we investigated whether Delta-Notch signaling is involved in this process. Activating the pathway in the marginal zone with Notch(ICD) resulted in an expansion of endodermal and neural ectoderm precursors, leaving a thinner mesodermal ring around the blastopore at gastrula stage, when germ layers are segregated. On the other hand, when the pathway was blocked with Delta-1(STU) or with an antisense morpholino oligonucleotide against Notch, the pan-mesodermal brachyury (bra) domain was expanded and the neural border was moved animalwards. Strikingly, the suprablastoporal endoderm was either expanded when Delta-1 signaling was blocked, or reduced after the general knock-down of Notch. In addition, either activating or blocking the pathway delays the blastopore closure. We conclude that the process of delimiting the three germ layers requires Notch signaling, which may be finely regulated by ligands and/or involve non-canonical components of the pathway. Moreover, Notch activity must be modulated at appropriate levels during this process in order to keep normal morphogenetic movements during gastrulation.

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Species referenced: Xenopus laevis
Genes referenced: chrd myc myf5 myod1 notch1 slc5a5 sox17a sox2 tbxt tub tubb2b
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	Fig. 1. Effects of NotchICD on neural and paraxial mesodermal markers at the neural plate stage. Embryos were injected with 1 ng of notchICD mRNA in one cell at the 2- or 4-cell stage and fixed at neural plate stage (B,C,E–K), when sibling control embryos reached stages 13–14 (A,D). They were revealed by ISH with the following probes: myoD (purple staining in A–E,H,I), myf5 (purple staining in F–G) and sox2 (turquoise staining in F–G; purple staining in J,K). Brown dots correspond to immunolocalization of the Myc-tag epitope fused to NotchICD. All photographs were oriented with the injected side towards the right. (D) Transverse section of the sibling control embryo shown in (A). Two layers are apparent in the paraxial mesoderm: one dorsal, expressing higher levels of myoD transcripts (dl, green arrow) and the other ventral, expressing lower levels of myoD transcripts (vl, yellow arrow). The dotted red line depicts the boundary between both layers and arrowheads point to their lateral limits (green: dl; yellow: vl). (E) Transverse section of an embryo injected with notchICD mRNA. The dorsal layer of paraxial mesoderm appears to be missing from the injected side (right), while the ventral layer remains. (F′) Transverse section of the embryo shown in F. (G) Transverse section of an embryo similar to the one shown in (F). The same references as in (D) are used for the dorsal and ventral layers of the paraxial mesoderm as revealed by myf5 expression. (H) Transverse section of the embryo shown in (B). (I) Transverse section of the embryo shown in (C). White bars in (E,H,I) indicate the thickness of the neural ectoderm. no, notochord. Black arrowheads in (F′,G,K) point to the lateral limits of the sox2 domain.
	Fig. 2. Effects of NotchICD on markers of the three germ layers at gastrula stage. Embryos were injected with 1 ng of notchICD mRNA in one cell at the 4-cell stage and fixed when sibling control embryos reached stage 11. They were revealed by ISH with the following probes: bra (purple staining in A,B,C′,D,E,F′), chd (turquoise staining in A,B,D,E), sox17α (purple staining in G–H′) and sox2 (purple staining in I). Green fluorescence corresponds to immunolocalization of the Myc-tag epitope fused to NotchICD (C,F and insets in G,H,I). (A–C,D–F) Lateral views of notchICD-injected embryos to show changes in the bra domain. The dorsal side (up) is marked by chd staining. Photographs were inverted to facilitate comparisons between the injected- and the non-injected sides. (G,H) Vegetal views of embryos injected with notchICD mRNA to show changes in sox17α expression. (I) Dorsal view of an embryo injected with notchICD mRNA to show changes in sox2 expression. (C′,F′,H′) Sections in the planes shown by the yellow dotted lines in (C,F,H) respectively; animal is up, vegetal is down. Photographs in (C′,F′,G–I) were oriented with the injected side towards the right. nis: non-injected side; is: injected side; spe: subprablastoporal prospective endoderm; bpe: blastoporal prospective endoderm. Arrowheads in (C′) point to the animal border of the bra domain in the pre-involuted mesoderm. Arrows in (G–H′) point to the spe. The dotted red line in (G,I) demarcates the blastopore.
	Fig. 3. NotchICD alters the expression domain of myf5 and impairs morphogenetic movements during gastrulation. (A–I) Embryos were injected with 1 ng of notchICD mRNA in one cell at the 2- or 4-cell stage and fixed when sibling controls reached stage 11. They were revealed by ISH with myf5 (purple staining) and sox2 probes (turquoise staining). Dorsal–vegetal views of whole embryos (A,D,G) and a corresponding pair of contralateral parasagittal sections (B,C; E,F; H,I, respectively) are shown, to compare changes between the injected- (is) and the non-injected- (nis) sides. The blastopore is at the upper right angle on the sections. Yellow dotted lines in (A,D,G) indicate the planes of the sections. Brown dots correspond to immunolocalization of the Myc-tag epitope fused to NotchICD. Although the BCIP staining (turquoise) is not as sensitive as the NBT + BCIP staining, it is possible to observe that the sox2 domain is shifted towards the blastopore on the injected side in the embryo shown in (A). (J–N) Wild type sibling embryos were injected with 1 ng of notchICD + 1 ng of GFP mRNA as tracer or with 2 ng of GFP mRNA as negative control at the 1-cell stage. The progress of the blastopore was recorded through gastrulation by scanning the vegetal sides at stage 10 (J), 10.25 (K), 11 (L), 11.5 (M) and 12 (N). Data are represented as the ratio between the perimeter of the blastopore and the embryo's perimeter in notchICD- (gray bars) and GFP- (white bars) injected embryos. Bars represent mean ± sem. An embryo illustrating the mean of the group is shown above each bar. Notice that at early gastrula, the relative perimeter of the blastopore is lower in notchICD-injected embryos than in GFP-injected siblings because of the delay in the appearance and progress of the slit, which is shorter in the former group. At later stages, when the slit depicts a full circumference, the blastopores are larger in notchICD-injected embryos and thus, the relative perimeter of the blastopore is higher than in GFP-injected siblings, also reflecting a delay in its progress. For additional details, see legend to Table 3. This figure illustrates experiment No. 1.
	Fig. 4. Blocking Notch signaling produces complementary changes in the expression of markers of the three germ layers at gastrula. Embryos were injected in one cell at the 4-cell stage with 1 ng of deltaSTU mRNA (A–B′,G–G′,L), 20 ng of Control morpholino (Control Mo; C,H–H′,M), 20 ng of Notch Mo (D–E,I–J′,N), or with 20 ng of Notch Mo + 1 ng of notchICD mRNA (F,K,O). They were fixed when sibling control embryos reached stage 11 and were revealed by ISH with the following probes: bra(purple staining, A–F), sox2 (purple staining, G–K), sox17α (purple staining, L–O). When possible, embryos hybridized with bra were also revealed with a chd probe to readily identify the dorsal side (turquoise staining in A′–B′). All photographs of whole embryos were oriented with the injected side towards the right. is: injected side; nis: non-injected side. Dotted red lines delineate the blastopore. Embryos are shown with the corresponding fluorescence image at the left (A,D for A′,D′, respectively) or in the insets, to identify the injected side (green fluorescence), which was revealed by immunofluorescence of GFP (A,G,L), DOG (C,D,E,H,I,M,N) or the Myc-tag epitope fused to NotchICD (F,K,O). (B,C–D′,L–O) Vegetal views, with the dorsal side up. (A,A′,E,G,H,I,J,K) Dorsal views. (B′,B′) Lateral views of the non-injected- and the injected side, respectively, of the embryo shown in (B). In this case, photographs were inverted to facilitate comparisons between both sides. This embryo was co-injected with 1 ng of GFP mRNA as tracer, and the injected side was determined by the fluorescence of GFP before the ISH procedure. (D,D′ and E) Two different embryos injected with Notch Mo. (G′,G′) Contralateral parasagittal sections of an embryo injected with delta-1STU mRNA + BDA as tracer, which was revealed with BCIP (turquoise staining). (H′,H′) Contralateral parasagittal sections of the embryo injected with Control Mo shown in (H). (J′,J′) Contralateral parasagittal sections of the embryo shown in (J), which was injected with Notch Mo and revealed by immunostaining of the fluorescein epitope with BCIP (turquoise staining). Arrows in (A′,B′,E) point to the animal expansion of the bra domain. The asterisk in (B) indicates an expansion of the gap of unstained cells between the bra+ ring and the blastopore, which correlates with an expansion of the suprablastoporal endoderm (see L for comparison). Arrows in (D′) and the green arrowhead in (E) point to the vegetal expansion of the bra domain. Light blue bars in (D′,E,F) indicate the difference in the width of the bra domain between the injected- and the non-injected side. Notice the stretch of thinner and lower bra staining between the red arrowheads in (F), coinciding with the green fluorescence on the bra domain on the inset, indicating that notchICD reversed the effect of Notch Mo. The black arrowheads in (G′,G′,H′,H′,J′,J′) indicate the distance between the blastopore and the vegetal border of the sox2 domain. The black arrowhead in (K) points to supernumerary sox2+ cells in the marginal zone on the injected side that are not present on the non-injected side (white arrowhead), indicating that notchICD mRNA reversed the effect of Notch Mo on the location of the vegetal boundary of sox2.
	Fig. 5. Blocking Notch signaling impairs morphogenetic movements during gastrulation. (A) Wild type embryos of the same batch and derived from eggs of the same female were injected with 20 ng of Control Mo or with 20 ng of Notch Mo before the first mitotic division. The progress of the blastopore was recorded through gastrulation by scanning the vegetal sides at stages 10.25, 10.5, 11.5 and 12. Data are represented as the ratio between the perimeter of the blastopore and the embryo's perimeter in Notch Mo- (gray bars) and Control Mo- (white bars) injected embryos. Above each bar, an embryo representing the mean of the group is shown. For additional details, see legend to Table 4. This figure illustrates experiment No. 2. (B–E) Embryos of the same batch and derived from eggs of the same female were treated from the 1-cell stage with 100 μM of the γ-secretase inhibitor DAPT or with vehicle alone (DMSO) and fixed at stage 11 (B,C) for ISH of chd (turquoise staining) or at stage 14 for ISH of chd (turquoise staining) and N-tubulin (N-tub, purple staining).
	Fig. 6. Effects of blocking Notch signaling on neural and paraxial mesodermal markers at the late gastrula and neural plate stage. Embryos were injected in one cell at the 4-cell stage with 0.5 ng of delta-1STU mRNA (A,B,D,D′,E,E′), 1 ng of delta-1STU mRNA (C,F,F′) or 20 ng of Notch Mo (G–L). They were fixed when sibling controls reached stage 12.5 (G,J), 14 (A–F,K) or 15 (H,I,I′,L) and were revealed by ISH of myf5 (purple staining in A,D), myoD (purple staining in B,E,G,H,I) or sox2 (purple staining in C,F,J–L). Embryos injected with delta-1STU mRNA and the embryo shown in (K) were co-injected with BDA as tracer, which was revealed after a chromogenic reaction with Magenta Phos (A,B,D,E) or BCIP (turquoise staining, C,F,K). In the rest of the embryos, the injected side was determined by the fluorescence of the morpholino before the ISH procedure. Whole embryos are shown in dorsal views, anterior up, except in (A), which shows a dorsal-posterior view. All photographs were oriented with the injected side towards the right. The horizontal yellow dotted lines in (A,B) show the plane of transverse sections shown in the inset in (A) and in the photograph in (E), respectively. (D,D′) Transverse section at the level of the neurenteric canal of an embryo similar to the one shown in (A). (I) Transverse section at the mid-trunk level of an embryo similar to the one shown in (H). (D′,E′,F′,I′) Nuclear Hoescht staining of the sections shown in (D,E,F,I), respectively. Green arrow in (D): prospective dorsal layer (dl) of the paraxial mesoderm. Yellow arrow in (D): prospective ventral layer (vl) of the paraxial mesoderm. Green arrow in (E): dorsal layer (dl) of the paraxial mesoderm. Yellow arrow in (E): ventral layer (vl) of the paraxial mesoderm. nc: neurenteric canal; no: notochord. The vertical yellow dotted line in F depicts the embryo's midline, and yellow arrowheads point to the lateral borders of the sox2 domain, showing the reduction of the neural plate on the medio-lateral axis on the injected side. Double arrows in (L) show the difference in the width of the sox2 domain between the injected- and the non-injected side.