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Figure 1. BMP Antagonists Are Expressed in Spemann’s OrganizerBlastula and gastrula embryos were bisected, stained, and then cleared to visualize expression of follistatin (A, F, K, and P), chordin (B, G, L, and Q), noggin (C, H, M, and R), xnr3 (D, I, N, and S), and cerberus (E, J, O, and T) at midblastula (A–E), late blastula (F–J), early gastrula (K–O), and midgastrula (P–T). Dorsal is to the right with the animal pole toward the top of the figure. St = stage.
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Figure 2. Single Morphants and Double Morphants Still Form a Substantial Neural PlateAll panels show sox2 expression to visualize the neural plate (dorsal views with anterior to the bottom) in neurula embryos (st 14–15). Uninjected embryos are shown (A, D, and G) with sibling noggin morphants (B), follistatin morphants (E), and chordin morphants (H). Double morphants include follistatin/noggin (K), chordin/noggin (N), and chordin/follistatin (Q) and are shown with sibling uninjected embryos (J, M, and P). Morphants were rescued with pufferfish noggin (C, F, I, L, O, and R). Abbreviations are as follows: F = follistatin, C = chordin, N = noggin, UC = uninjected sibling control embryos, MO = morphant, and MO + R = morphant rescued with noggin mRNA.
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Figure 3. FCN Morphants Have a Catastrophic Loss in Dorsal DevelopmentAll panels are dorsal views with anterior to the top of st 14–15 neurula embryos. Expression of multiple dorsal markers (sox2 [A–D], sox3 [E–H], myf5 [I–L], myoD [M–P], shh [Q–T], and xnot [U–X]) are shown in uninjected sibling embryos (A, C, E, G, I, K, M, O, Q, S, U, and W) and follistatin, chordin, and noggin (FCN) triple morphants (B, F, J, N, R, and V) as well as β-catenin morphants (D, H, L, P, T, and X) for comparison.
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Figure 4. The Phenotype of the FCN Triple Morphant Is Specific to a Loss in BMP AntagonismAll panels are dorsal views of neurula embryos (st 14) with anterior to the top. Triple morphants were subsequently injected with pufferfish noggin mRNA (C, F, and I) and show substantial rescue of dorsal structures (sox2 [A–C], myoD [D–F], and shh [G–I]) when compared to triple morphants (B, E, and H) and uninjected sibling embryos (A, D, and G). The distribution of phenotypes seen in these rescue experiments is depicted in a bar graph (J). The vertical axis is the percent of embryos that show a particular phenotype. The horizontal axis shows the experimental groups and the phenotypes seen. The black bar depicts the percentage of embryos with a substantial neural plate, the dark gray bar depicts the percentage of embryos with a minor neural plate, and the light gray bar depicts the percentage of embryos with no neural plate that show only a ring of sox2 expression. We also tested the phenotype of the triple morphant for specificity by subsequently injecting BMP4,7 MOs (BMP MO), which should reduce BMP signaling. A partial rescue of the neural plate is seen (M) compared to triple morphants (L) and uninjected sibling control embryos (K).
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Figure 5. Ventral Tissues Are Expanded in FCN Morphants(A–P) are lateral views with anterior to the left of neurula (st 13–14) embryos, whereas (Q–F′) are dorsal views with anterior to the top. Expression of multiple ventral markers (BAMBI [A–D and Q–T], msx1 [E–H and U–X], sizzled [I–L and Y–B′], and cytokeratin [M–P and C′–F′]) are shown in uninjected sibling embryos (A, C, E, G, I, K, M, O, Q, S, U, W, Y, A′, C′, and E′) and FCN triple morphants (B, F, J, N, R, V, Z, and D′) as well as in β-catenin morphants (D, H, L, P, T, X, B′, and F′) for comparison.
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Figure 6. Spemann’s Organizer Is Specified in FCN MorphantsAll panels are vegetal views of early gastrula embryos (st 10.5 [A–T]) or midgastrula (st 11 [U–X]) with dorsal to the right in embryos where dorsal can be distinguished. Expression of multiple mesodermal markers (brachyury [xbra] [A–D], goosecoid [gsc] [E–H], xnr3 [I–L], siamois [M–P], vent2 [Q–T], and myf5 [U–X]) are shown in uninjected sibling embryos (A, C, E, G, I, K, M, O, Q, S, U, and W) and FCN triple morphants (B, F, J, N, R, and V) as well as β-catenin morphants (D, H, L, P, T, and X) for comparison.
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myod1 (myogenic differentiation 1) is a standard marker for presomitic mesoderm. Note that as somites form in an anterior to posterior direction, myod1 is broadly expressed more posteriorly in this NF stage 14/15 embryo ( dorsal view, anterior up).
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All panels are dorsal views with anterior to the top of st 14â15 neurula embryos. Expression of dorsal markers sox2.
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All panels are dorsal views with anterior to the top of st 14â15 neurula embryos. Expression of dorsal markers sox3.
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shh (sonic hedgehog) expression, a marker for notochord, in a X. laevis embyro, NF stage 17/18 (mid-neurula stage),
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All panels are dorsal views with anterior to the top of st 14â15 neurula embryos. Expression of dorsal markers shh.
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All panels are dorsal views with anterior to the top of st 14â15 neurula embryos. Expression of dorsal markers shh.
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All panels are dorsal views with anterior to the top of st 14â15 neurula embryos. Expression of dorsal markers shh.
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