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Fig 1. The pre-signaling state of most of the ectoderm is neurogenic and the region is patterned along the dorsal-ventral axis after restricted anteriorly.(A) A brief summary of the early cWnt-deficient embryonic phenotype, in which the initial (default) neuroectoderm covers most of the embryo and a number of serotonergic neurons are differentiated. In the drawings, the pink field and green spots indicate neuroectoderm and serotonergic neurons, respectively. (B-F) foxQ2 patterns during the embryogenesis of the sea urchin, Hemicentrotus pulcherrimus. foxQ2 is initially expressed at the anterior half (B) and gradually restricted to the anterior end by the blastula/gastrula stages (C-E). (F) foxQ2 is expressed ventrally in the restricted AP region in the early pluteus stage. Left is ventral (V) and right is the dorsal (D) side. Arrowhead indicates the position where foxQ2 gene expression is missing in the AP region. (G, H) FoxQ2 is required for the development of serotonergic neurons. Without FoxQ2, serotonergic neurons are not differentiated at 48 h (H) compared to control (G). (I-L) Sagittal section of the neuroectoderm field in a prism larva. Serotonergic neuron is differentiated at the dorsal edge of the AP and never includes FoxQ2 protein in its nucleus. Bar in (F) is 20 μm.
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Fig 2. Homeobrain is required for the development of serotonergic neurons.(A-K’) Spatiotemporal expression pattern of hbn. Left is ventral (V) and right is the dorsal (D) side. Compared with foxQ2 expression, the expression of hbn is ‘shifted’ towards the dorsal side of AP. nodal is the ventral marker. Arrows in (E, F) indicate hbn expression in the stomodeum. Dot-lined circle in (K’) indicates the AP region. Serotonergic neurons in control embryos (L, M) are missing in Hbn morphants (N, O). (P, Q) Serotonergic neurons are not differentiated in the Δcad and Hbn-MO co-injected embryos. Hbn is an upstream gene of tph, tryptophan 5-hydroxylase (R, S), zfhx (T, U), and fez (V, W). Arrowheads in (R, T, V) indicate the precursors of serotonergic neurons. Arrows in (U) indicate lateral ganglion neurons.
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Fig 3. Nodal maintains foxQ2 expression.foxQ2 mRNA and FoxQ2 protein patterns in control embryos (A-E), Nodal morphants (F-J) and Lefty morphants (K-O). Arrows in (C, H, M) and (D, I, N) indicate the edge of the foxQ2-mRNA and FoxQ2-protein region, respectively. (P) The image shows how the foxQ2-expressing area was measured with the angle (θ) from the posterior end. (Q) Quantification of the size of the foxQ2 region in the control (A, B), Nodal morphants (F, G) and Lefty morophants (K, L). The foxQ2 region in Nodal morphants and Lefty morphants is significantly narrower and wider, respectively, than that in the control at 18 h, whereas they are all similar at 12 h. (R) Quantification of the number of FoxQ2-protein positive cells in the control (E), Nodal morphants (J) and Lefty morphants (O). n.s. not significant, ***P<0.001, Student’s t-test. foxQ2 expression pattern is almost asymmetrical during all stages shown in this figure.
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Fig 4. TGF-ß signals regulate the dorsoventral pattern of hbn.(A-E) hbn is shifted toward the dorsal side, where serotonergic neurons are differentiated (E) in control embryos. (F-J) hbn surrounds the foxQ2 region and the serotonergic neurons are differentiated at the same region (J). Clear shifting toward the edge of the AP and strongly localized expression at later stages is not observed in Lefty morphants (K-O) or BMP2/4 morphants (P-T). Both have no serotonergic neurons. Asterisks indicate the precursors of serotonergic neurons, which express zfhx1.
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Fig 5. Canonical Wnt signal regulates the patterning of anterior neuroectoderm.In contrast to control embryos (A), LRP6 morphant had a wider foxQ2 region (B). (C) Quantification of the size of the foxQ2 region in control (A) and LRP6 morphants (B). ***P<0.001, Student’s t-test. LRP6 morphants retain the apical tuft at 72 h (E) and foxQ2 (G) at 96 h, whereas both are diminished in control embryos at those times (D, F). The differentiation of serotonergic neurons is delayed and not observed at 48 h in LRP6 morphants (H, I). Arrows in (H) indicate serotonergic neurons. Asterisk shows foxQ2 protein. This delay is independent of the presence or absence of Δcad mRNA injection (J, K). (L, M) foxQ2 is maintained in Fz5/8 morphants. (N) hbn pattern in Nodal morphants. (O-R) Spatiotemporal expression pattern of hbn in Δcad-injected embryos. hbn mRNA is maintained at the anterior end until at least 24 h. The clearance occurs without early cWnt and TGF-ß signals even though the timing is delayed. hbn clearance is not observed in LRP6 morphant at 24 hr (S, T), but it occur by 50 h (U, V). (W) Double fluorescent in situ hybridization with hbn and foxQ2 to confirm hbn clearance from the anterior end. White arrowheads indicate the location of hbn clearance.
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Fig 6. Wnt7 is a ligand for cWnt pathway that regulates the patterning of anterior neuroectoderm.(A) Quantitative PCR data revealing wnt3, wnt6 and wnt7 during sea urchin embryogenesis. The data from two independent batches are shown. (B-E) wnt7 expression patterns during sea urchin embryogenesis. In the Wnt7 morphants, foxQ2 mRNA (H) and protein (I) are remained until late stages, e.g., 96 h, whereas they are not expressed in the control (F, G). The number of serotonergic neurons is significantly smaller in Wnt7 morphants at 72 h (J) and 96 h (I) than in controls (G, J). ***P<0.001, Student’s t-test. (K) Top-Flash assay revealed that LRP6 and Wnt7 are involved in the canonical Wnt pathway at the AP region. (L, M) hbn clearance occurs in Wnt7 morphant, indicating that hbn is not a strong target of the cWnt pathway. White arrowheads indicate the location of hbn clearance.
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Fig 7. Wnt6 is a ligand for non-cWnt pathway that regulates the patterning of hbn expression.(A, C) foxQ2 expression in the control (DMSO-treated) and JNK inhibitor treated embryos at 24 h. (B, D) foxQ2 expression in the control (B) and in JNK inhibitor treated embryo (D) at 60 h. A trace level of foxQ2 was expressed in JNK inhibitor treated embryos, whereas foxQ2 is completely diminished in the control at this stage. (E, F) The serotonergic neurons are differentiated at 48 h with or without JNK function. The AP region is magnified. (G-J) hbn clearance does not occur in JNK inhibitor-treated embryos. (K, L) hbn remains at the anterior end of AP in Fzl5/8 morphants at 56 h. (M-P) wnt6 was strongly expressed in the veg2 endoderm region at 20 h, and afterward it was maintained in the vegetal plate until at least 30 h. (Q, S) hbn does not disappear from the anterior end and is not expressed in the non-AP region in Wnt6 morphants, and this is independent of the presence or absence of Nodal (R, T). (U-X) Without Wnt6 function, serotonergic neurons are differentiated at the anterior end of the AP. To visualize the phenotype more clearly during early stages, Nodal-MO was simultaneously injected. Arrow in (U, W) indicates the anterior end that misses serotonergic neurons. Asterisk in (V, X) shows that the serotonergic neurons are present at the central part of AP. White and black arrowheads indicate the location of hbn absence and presence, respectively.
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Fig 8. Models for anterior neuroectoderm patterning.(A) Schematic images of signaling pathways regulating foxQ2 and hbn expression. Anterior views. foxQ2 (green) is suppressed by cWnt mediated by Wnt7/LRP6 from the blastula to gastrula stages. Nodal inhibits the signal from the ventral side. Serotonergic neurons (blue) begin to be differentiated in the foxQ2-missing area, i.e., the dorsal/lateral edge of the AP. hbn (magenta) expression shifts towards the dorsal/lateral edge of the AP. Nodal and non-cWnt mediated by Wnt6/JNK signals regulate the suppression, whereas BMP2/4 promotes its expression. The dotted line indicates the dorsal border of the AP. MBL, mesenchyme blastula; early G, early gastrula; late G, late gastrula. (B) Summary of signaling pathways regulating the serotonergic neural fate at the dorsal/lateral edge of the AP. Before receiving extrinsic signals beginning from early cWnt, the cell fate of most cells in sea urchin embryos are the AP, which differentiates a number of serotonergic neurons. However, under normal conditions, five signals restrict its fate to a small region.
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