|
FIGURE 1. Spatiotemporal pattern of serotonergic and cholinergic neuron in the brain. (A,A′) Expression of serotonin (asterisks) begins around at 36 h prism larva in the brain. (A′) Magnified image of the square in (A). (B) Expression of non-serotonergic neurons (Serotonin -/SynB +, arrowheads) begins at 48 h pluteus larva in the brain. (C,C′) Expression pattern of Serotonin and SynB at 96 h (C′) A magnified image of the rectangle in (C). (D)
chat mRNA expression pattern at 72 h pluteus larva. (E)
chat (white arrowheads) and tph (asterisks) expression patterns at 72 h pluteus larva in the brain. (F–F′′) Chat (white arrowheads) and Serotonin (asterisks) expression pattern in brain. (F′,F′′) are ventral and dorsal side of (F), respectively. Yellow arrowheads in (E,F,F′) show cholinergic neurons in the edge of the upper lip. There are only Chat positive cells at ventral side and there are only serotonergic neurons at dorsal side. (G–L) Serotonin (asterisks) and non-serotonergic neurons (white arrowheads) expression pattern at 60 h, 72 h and 96 h (G′), (I′) and (K′) are ventral side images of (G), (I) and (K), respectively, and (G′′), (I′′) and (K′′) are dorsal side images of (G), (I) and (K), respectively. (H′), (J′) and (L′) are magnified images of squares in (H), (J), and (L), respectively. (H′′), (J′′) and (L′′) are schematic images of (H′), (J′) and (L′), respectively. (H′) is a stacked image of 8 sections (1 µm interval) and the insertion diagrams of (H′), (H′-1) and (H′-2) show stacked images of the front and back 4 sections, respectively. These images show that there is a non-serotonergic neuron in (H′-1), and, on the other hand, there is a serotonergic neuron in (H′-2). A, anterior. P, posterior. D, dorsal. V, ventral. (M–O) The detailed expression pattern of non-serotonergic neurons in brain (white arrowheads) and in the edge of the upper lip (yellow arrowheads). (M) A magnified image of the square in (K). (N) A stacked image of ventral side of (M) (12 sections). A stacked image of the most ventral 4 sections (N′), the middle 4 sections (N′′) and the most dorsal 4 sections (N‴). (O) A schematic image of (N). (P) Schematic image of neurons in brain and the edge of the upper lip. Bars in (A) and (A′) are 50 µm and 20 μm, respectively. Bars in (H) and (M) are 10 µm.
|
|
FIGURE 2. Rx is required for development of cholinergic neurons. (A,B)
foxQ2 and rx expression patterns at 18 h and 24 h. White dotted circle in (B,B′) show foxQ2 positive region. (C)
hbn and rx expression patterns in 24 h (D)
lefty and rx patterns in 24 h. The rx expressing region shift to dorsal side. The inserts in A-D are bright field images. V, ventral, D, dorsal. (E)
rx and tph are co-expressed in brain region of 36 h larvae. (F,J,N) Bright field images viewed from lateral in control, Rx MO-1 and Hbn MO injected embryos. (G,G′,K,K′,O,O′) Almost all non-serotonergic neurons (cholinergic neurons; white arrowheads) are disappeared in the brain of Rx morphants, whereas in Hbn morphants, serotonergic neurons (asterisks) are mostly disappeared. (G′,K′,O′) Magnified images of the squares in (G), (K) and (O). (H,L,P) Neuro-plexus from cholinergic neurons are disappeared in both Rx and Hbn morphants (white dotted lined circles). (I,M,Q) There are few chat positive cells (white arrowheads) at brains in Rx morphant, but there are in Hbn morphants as same as controls. Green dotted rectangles show brain region. Chat cells in upper lips (yellow arrowheads) and ciliary bands (orange arrowheads) are invariant in control and these morphants. Bars in insets of (A) and (B), and (F) and (G), are 50 µm.
|
|
FIGURE 3. Msi is downstream factor of Rx during brain formation. (A–C) In brain region, msi begins to express by 48 h pluteus larvae. (D,E,F)
msi is co-expressed with foxQ2 but neither rx nor tph at 48 h (G) msi express at chat expressing region in the brain at 72 h. White arrowheads and yellow arrowhead show cholinergic neurons in brain and at upper lip region, respectively. (H,I)
msi in brain (arrow) is missing in Rx morphants.
|
|
FIGURE 4. Msi1 is required for the expression of chat. (A–D) Control. (E–H) Msi1 morphants. (A,E) Bright field images of lateral view. (B–B‴,F–F‴) Both serotonergic (asterisks) and non-serotonergic neurons (white arrowheads) are present in Msi1 morphants as same as control. Magnified images of the square in (B) and (F), respectively. (C,G) Neuro-plexus from non-serotonergic neurons are absent at 96 h in Msi1 morphant (white dotted-line circle). (D,H)
chat and tph expression patterns. chat positive cells in the brain are missing in Msi1 morphants (white arrowheads). Green dot-lined rectangles show the brain region. There are chat positive cells of the edge of upper lip (yellow arrowheads) and ciliary band (orange arrowheads) in Msi1 morphants. (I,J)
ngn positive cells (white arrowheads) are missing in the brain of Rx morphants, but not in Msi1 morphants (K).
|
|
FIGURE 5. Formation of bilateral clusters on the ventral side is regulated by Rx. (A,B,C)
rx expression pattern of bilateral clusters with chat, zfhx1 and SynB. rx expressing at bilateral clusters of neurons (arrows) is not co-localized with chat.
(D,E,F)
Go-Opsin and rx are co-expressed in the bilateral clusters at 84 h (yellow arrows). (G,H,I) Bilateral clusters (yellow asterisks) is disappeared in Rx morphants but not in Msi1 morphants. Neural processes from bilateral clusters extend towards the brain neurons (arrowheads). (J) Schematic image of neurons in brain, the edge of the upper lip, and the bilateral clusters. (K) Molecular pathway for the development of cholinergic neurons in and around sea urchin brain.
|
