CESNs and Tail Epidermis Medio-Lateral Patterning

<div><p>(A–C) β-tubulin immunostaining showing CESN projections in the fin tunic. Confocal images projection with nuclei in red (propidium iodide) (C).</p> <p>(D and E) Acetylated α-tubulin showing the proximal part of the projections from paired CESNs (Acetylated α-tubulin in green, DAPI in blue, phalloidin in red).</p> <p>(F) 3-D projection extracted from a time lapse movie of an embryo injected with H2B-YFP mRNA and Fast Green (red). Trunk nuclei are green, while tail nuclei are colored to highlight the longitudinal rows made of single cell.</p> <p>(G and H) Schematic representation of the tail medio-lateral patterning (medial row, purple; medio-lateral row, yellow; lateral row, blue).</p> <p>(I–P) Expression patterns of tail epidermis markers at the mid-tailbud stage. Lateral view (I–L and P). Ventral view (M–O). Anterior is to the left. DAPI staining of nuclei in (M–P).</p></div

bFGF and BMP4 Induce Midline Fate and ESN Formation in Isolated b-Line Explants

<div><p>b4.2 Blastomeres were isolated at the eight-cell stage and treated with proteins from the 16-cell stage.</p> <p>(A–D) Acetylated α-tubulin immunostaining at larval stage: CNS and PNS structures are labelled. Arrowheads indicate ESNs.</p> <p>(E–P) Molecular marker expression at early tailbud stages: midline marker <i>KLF1/2/4</i> (E–H), lateral and medio-lateral marker <i>citb003j16</i> (I–L), and tail nerve cord marker <i>cilv038e16</i> (M–P). </p> <p>Top row, whole embryos (A,E,M) lateral view, (I) dorsal view, anterior to the left.</p> <p>Second row, control b4.2 blastomeres treated with BSA.</p> <p>Third row, b4.2 blastomeres treated with bFGF protein.</p> <p>Bottom row, b4.2 blastomeres treated with BMP4 protein.</p></div

Delta/Notch Signalling Controls the Number of CESNs within Neurogenic Midlines

<div><p>(A,B,E,G) Electroporation of the construct pFOG::Venus (a bright YFP) has no effect on the midline domain and the CESNs, respectively, revealed by the <i>KLF1/2/4</i> and <i>β-thymosin-like</i> probes. The expression levels of <i>KLF1/2/4</i> at mid- to late tailbud stages (E) are lower than at mid-tailbud stages (A). </p> <p>(C and D) Electroporation of the dominant-negative construct pFOG::Venus-Su(H)DBM does not affect the expression of <i>KLF1/2/4</i> but leads to an increase in the number of <i>β-thymosin-like-</i>positive cells. </p> <p>(F and H) Electroporation of pFOG::Delta2 results in a loss of <i>β-thymosin-like-</i>positive cells, without affecting <i>KLF1/2/4</i> expression. </p> <p>(I and J) The construct p10.27::LacZ preferentially drives expression of β-galactosidase in the ventral tail epidermis (I), without affecting the development of CESNs (J).</p> <p>(K and L) Electroporation of p10.27::NICD, carrying an active form of Notch, does not affect the formation of the midline domain (K), but leads to a loss of CESNs in the ventral midline (arrow in L).</p> <p>(M–P) Treatment with the γ-secretase inhibitor, DAPT, at various developmental stages results in varying degrees of midline epidermal cell replacement by <i>ETR</i>-positive CESN precursors. e.neur., early neurula; l.neur., late neurula; and e.tbud, early tailbud. </p></div

Cell Lineage of the Tail Epidermis in <i>Ciona</i>

<div><p>(A) Color-coding illustrates the clonal base of dorso–ventral regionalization of tail epidermis. Pink, dorsal midline; light yellow, dorso–lateral domain; blue, lateral domain; yellow, ventro-lateral domain; purple, ventral midline. For a detailed description of each 110-cell stage b–line blastomere's contribution to the tail epidermis, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040225#sg001" target="_blank">Figure S1</a>. </p> <p>(B–E) composition of four representative clones derived from DiI-labelled epidermal precursors.</p> <p>(C) a lateral domain clone consists of 16 epidermal cells.</p> <p>(B and E) Dorsal (B) and ventral (E) midine clones consist of both epidermal cells and CESN pairs (green arrowheads). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040225#st002" target="_blank">Table S2</a> for a quantification of midline clone composition. </p> <p>(B) The progeny of a 110-cell stage dorsal precursor only contributes to the medial domain.</p> <p>(D) The progeny of a 110-cell stage ventral precursor contributes to both medial and medio-lateral domains.</p> <p>(E) A single-cell division segregates the ventral medial and medio-lateral domains.</p></div

The FGF and Nodal Pathways Control Dorsal Midline and CESN Formation

<div><p>(A) bFGF-treated embryos show ectopic expression of the midline marker <i>msxb</i> at the late neurula stage when the treatment starts at the 16-cell or early 32-cell stages. Embryos do not respond to bFGF at the 64-cell stage. </p> <p>(B) Blocking Erk activity with the pharmacological inhibitor U0126 from the 16-cell stage abolishes dorsal <i>KLF1/2/4</i> and <i>ETR</i> expression at tailbud stages, while treatment from the late 32-cell stage has no effect on these markers. FGF9/16/20 is sufficient to induce ectopic midline and ESNs formation (arrow), while Lefty prevents dorsal midline formation. </p> <p>(C) FGF9/16/20 MO–injected embryos show a loss of dorsal <i>KLF1/2/4</i> expression while embryos injected with a control MO are not affected. (Lateral view, anterior to the left, dorsal to the top in all panels). </p></div

Additional file 12: Figure S7. of MAPK signaling is necessary for neurogenesis in Nematostella vectensis

NvashA expression in animals with varied regiments of U0126 treatment. (A) NvashA expression in control animals, or in animals treated with U0126 continuously for 48 hours, or from 24 to 48 hpf. Unlike early stages when no NvashA expression could be detected (Fig. 3), NvashA expression was ultimately detected in U0126-treated animals by 48 hpf. Treatment with U0126 from 24 to 48 hpf reduced NvashA expression, but NvashA could be detected in many cells, albeit at reduced levels. (B) Levels of NvashA and Nvfgfa1 as detected by qPCR at late gastrula stage (48 hpf at 17 °C) in animals injected with the Nvfgfra MO or treated with U0126 or SU5402 from 24 to 48 hpf. Relative expression levels are compared to control MO- or DMSO-treated animals respectively. The red box defines 1.5 to −1.5 fold change region. Error bars are standard error. (TIF 11166 kb

Additional file 3: Figure S2. of MAPK signaling is necessary for neurogenesis in Nematostella vectensis

Changes in NvashA, Nvath-like, and NvsoxB2 expression following drug treatments or in with increased Notch activity. mRNA in situ hybridization for NvsoxB(2) (A) and NvashA (B) in DMSO-treated control animals grown to early planula stages (48 hpf at 22 °C). NvsoxB(2) (A’) and NvashA (B’) expression in same stage animal treated with U0126 from 24 to 48 hpf or treated with SU5402 from 24 to 48 hpf (B”). mRNA in situ expression of Nvath-like (C) and NvsoxB(2) (D) in control embryos injected with the venus mRNA. Expression of Nvath-like (C’) and NvsoxB(2) (D’) in animals injected with NvnotchICD:venus (the intracellular domain of the Notch receptor), which has been previously shown to hyperactivate Notch signaling. Embryos were classified and quantified as the percent having normal expression, weak expression, or no expression. The phenotypic class with the highest percentage of embryos is indicated. In C and D, the main figure panels are ectodermal focal planes, and insets show deeper focal planes used to confirm embryonic stage. All images are of lateral views with the oral side to the left. (TIF 34682 kb

Additional file 13: Figure S8. of MAPK signaling is necessary for neurogenesis in Nematostella vectensis

NvashA regulation of target genes in the embryonic ectoderm. Gene expression in NvashA morphants (A–C), control morpholino (D–F), and NvashA mRNA injected (G, H). Quantification below each image represents percent of embryos in each phenotypic class (see key in figure). All images except C and F are aboral views. C and F are oral views. Embryos were classified and quantified as the percent having normal expression, weak expression, or no expression.. The phenotypic class with the highest percentage of embryos is indicated. (TIF 21122 kb

Additional file 15: Table S7. of MAPK signaling is necessary for neurogenesis in Nematostella vectensis

Primer pairs used in this study for gene cloning. (XLS 73 kb

Godetia amoena Lilja

原著和名: イロマツヨヒ科名: アカバナ科 = Onagraceae採集地: 千葉県 四街道市 千代田 (下総 四街道市 千代田)採集日: 1985/5/27採集者: 萩庭丈壽整理番号: JH026754国立科学博物館整理番号: TNS-VS-97675