Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis - PubMed (original) (raw)

Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis

Claudia Racioppi et al. Nat Commun. 2014.

Abstract

During the development of the central nervous system (CNS), combinations of transcription factors and signalling molecules orchestrate patterning, specification and differentiation of neural cell types. In vertebrates, three types of melanin-containing pigment cells, exert a variety of functional roles including visual perception. Here we analysed the mechanisms underlying pigment cell specification within the CNS of a simple chordate, the ascidian Ciona intestinalis. Ciona tadpole larvae exhibit a basic chordate body plan characterized by a small number of neural cells. We employed lineage-specific transcription profiling to characterize the expression of genes downstream of fibroblast growth factor signalling, which govern pigment cell formation. We demonstrate that FGF signalling sequentially imposes a pigment cell identity at the expense of anterior neural fates. We identify FGF-dependent and pigment cell-specific factors, including the small GTPase, Rab32/38 and demonstrated its requirement for the pigmentation of larval sensory organs.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. The Ciona neural plate and cell lineage of the PCPs.

(a, left) Schematic representations of the bilaterally symmetrical embryo at the late gastrula stage with higher magnification of neural plate scheme from row II to V; the PCP a9.49 cell pairs are indicated in pink while their sister cells (a9.50s) are in blue. (a, right) Pigmented cell lineage, right side is shown: letters and numbers indicate cell names according to the referred cell lineages. The approximate timeline applies to the diagrams and lineage tree (hpf, hours post fertilization). Cells with the same colours are derived from a single cell at the late gastrula stage; only the a11.193 cell pair (in black) differentiates into pigment cells at larval stage. Merged bright field/fluorescent images of embryos from early neurula (b) to tailbud stages (c–e) electroporated with ptyrp1/2a>2xGFP cells expressing the GFP (green) and correspond to pigment cell lineage-specific expression. Oc, ocellus, ot, otolith.

Figure 2

Figure 2. Pigment cell lineage-specific transcription profile.

Phenotype of larvae electroporated with ptyrp1/2a>LacZ (a), ptyrp1/2a>FGFRDN (b), ptyrp1/2a>Ets:Vp16 (c) observed with transmitted light. Lateral view, anterior is on the left. (d) Schematic representation illustrating the microarray experimental design in two developmental stages, neurula (8 hpf) and tailbud stage (12 hpf); PCPs-lineage cells for microarray analysis were sorted based on their GFP fluorescence. tyrp1/2a enhancer drives target expression of FGFRDN or Ets:Vp16 in PCPs; FGFRDN and Ets:Vp16 conditions mimic regression (light pink) or expansion (dark pink) of the pigmented cells, respectively; (e) FACS plots: ptyrp1/2a>2xGFP (8 hpf, right), ptyrp1/2a>GFP (12 hpf, left) and pMyoD>YFP-expressing cells are distinguished by their green or yellow fluorescence, respectively (hpf, hours post fertilization). The GFP-only quadrant (GFP2) was used to sort PCPs. (f) Hierarchical clustering and heat map: training data set expression values (log2 FC) consisting of known PCP markers (Ci-tyr, Ci-tyrp1/2a, Ci-Tcf/Lef, Ci-Mitf, Ci-bmp5/7, Ci-msxb and Ci-FoxD). Clustering was performed taking in account all three conditions (control, FGFRDN, Ets:Vp16) at the two developmental stages (8 and 12 hpf). Expression levels are colour coded as represented by the colour range below. (g) Identification of 912 probe sets differentially expressed when comparing the two perturbated conditions with each other and with control samples (FGFRDN versus Control, Ets:Vp16 versus Control and FGFRDN versus Ets:Vp16) at 8 and 12 hpf (for more details on transcript selection, see the text); (h) Log2FC of 912 probe sets showing an increased (upregulated) or decreased (downregulated) expressions when FGFRDN is compared with control or Ets:Vp16 conditions. Colours indicate fold change in pairwise comparisons, for example, dnFGFr-EtsVp 12, means differential expression in ptyrp1/2a>FGFRDN compared with ptyrp1/2a>Ets:VP16 samples sorted at 12 hpf; red indicate upregulation, green downregulation, respectively. See also Supplementary Fig. 1.

Figure 3

Figure 3. FGF signalling imposes anterior neural plate fate in PCPs.

Double fluorescent whole-mount in situ hybridization (WMISH) with tyramide staining for Ci-Six3/6 (red) and Ci-tyrp1/2a (green) at neurula stage of Ciona wild-type embryo (a) and at tailbud stage of Ciona electroporated embryos with ptyrp1/2a>LacZ (b), ptyrp1/2a>FGFRDN (c) and ptyrp1/2a>Ets:Vp16 (d) plasmids. Zoom on the expression domains, dashed line indicate the border among Ci-Six3/6 and _Ci-tyrp1/2a-_positive cells. Embryos showing separate channels for tyrp1/2a (green) that marks a10.97 derivatives (green asterisks) and very faintly a10.98s progenies (white asterisks) and Six3/6 (red) expression. On the right, schematic illustration of Ciona neural plate and neural tube cells at tailbud stage showing Ci-Six3/6 (red) and Ci-tyrp1/2a (green) expressing cells, adapted from Cole and Meinertzhagen, 2004. (a)Vegetal view, anterior is on the top; (b–d) lateral view, anterior is on the left. Scale bar, 50 μm; for zoomed embryos, scale bar, 20 μm; nuclear staining by DAPI (blue). Number (n) of embryos showing the observed phenotype out of the total counted embryos, referred to one experiment that was repeated at least three times. See also Supplementary Fig. 3.

Figure 4

Figure 4. Sequential induction mediated gradually by FGF/MAPK/ERK on PCPs.

Activation of ERK1/2 visualized by dpERK1/2 antibody staining. Ciona embryos were electroporated with ptyrp1/2a>2xGFP and stained using anti-GFP antibody (green) to label the PCPs from early neurula (a) (a10.97s and a10.98s), late neurula (b,c) (a10.97s and a10.98s in division) to early tailbud stage (d) (a11.194s, a11.195s and a11.196s). Note that at early and late neurula stages, nuclear dpERK is detected in the a10.97s and their progenies but not in a10.98s. (a,b,d) Scale bar, 50 μm; (c) scale bar, 20 μm; nuclear staining is visualized by DAPI (blue). White asterisks and arrowhead indicate a10.98s and a10.97s and their cell progenies, respectively. Number of embryos showing dpERK1/2 staining out of the total embryos scored. Each experiment was repeated at least three times.

Figure 5

Figure 5. Sequential FGF-MAPK signalling is required to inhibit expression of anterior CNS markers in PCP territories.

(a) Summary model of the timed inhibition of MAPK signalling using UO126 treatment. Wild-type in vitro fertilized embryos were treated with U0126 at the gastrula, neurula and early tailbud stages for 30 min at room temperature. Ciona embryos were electroporated with ptyrp1/2a>2xGFP to label the PCPs and stained with anti-GFP (green) antibody. (b) Control embryos incubated with dimethylsulphoxide showing Ci-Six3/6 (yellow) and Ci-tyrp1/2a (red) expression at tailbud stage. Dashed line indicates the border among Ci-Six3/6 and Ci-tyrp1/2a endogenous expression pattern. (c) Sequential treatment with UO126 at 5h 30′ (a9.49 PCPs), at 6h 20′ (d) (a10.97s and a10.98s) and 6h 50′ (e) (a11.193s, a11.194s, a11.195s and a11.196s) showing alteration in Ci-tyrp1/2a (red) and _Ci-Six3/_6 (yellow) expression in PCPs. Lateral view, anterior towards the left. Scale bar, 50 μm; scale bar, 20 μm, for zoomed embryos. Number (n) of embryos showing the observed phenotype out of the total counted embryos, referred to one experiment that was repeated at least three times. Nuclear staining is visualized by DAPI (blue).

Figure 6

Figure 6. Expression of Ciona candidate PCP genes.

Expression pattern at tailbud stage of PCP candidate genes (red): Ci-bzrap,(a), Ci-Slc45a2 (b), Ci-Lrp4 (c), Ci-Mnt (d), Ci-Casz1(e), Ci-Piwi (f) and Ci-doc2a (g); co-localization (merge) of these PCP markers with Ci-tyrp1/2a (green),higher magnification on the right. Schematic representation of a9.49-derived cells in the neural tube of Ciona tailbud stage showing Ci-tyrp1/2a + cells (green) and PCP candidates expressing cells (red), adapted from Cole and Meinertzhagen, 2004.tyrp1/2a transcripts are accumulated mostly in a10.97 progeny (a11.193s/a11.94s, green) than a10.98s derivatives (a11.195s/a11.96s, light green) that maintains only a residual amount of tyrp1/2a RNAs. Lateral view, anterior is on the left. Scale bar, 50 μm; scale bar, 20 μm for zoomed embryo; nuclear staining by DAPI (blue). Number of embryos scored was higher than 30; each experiment was repeated at least two times.

Figure 7

Figure 7. FGF signalling controls the expression of new PCP marker genes.

Double fluorescent WMISH of Ci-Casz1, Ci-Slc45a2 and Ci-Bzrap1 (red) with Ci-tyrp1/2a (green) coupled with immunostaining of GFP (light blue, in zoomed embryos). Embryos are electroporated both with ptyrp1/2a>LacZ (a–c) and ptyrp1/2a>FGFRDN (d–f) and ptyrp1/2a>GFP. Lateral view, anterior is on the left. Scale bar, 50 μm; scale bar, 20 μm, for zoomed embryos; nuclear staining by DAPI (blue); number (n) of embryos showing the observed phenotype out of the total counted embryos, referred to one experiment, that was repeated at least three times.

Figure 8

Figure 8. Rab-type small GTPases as ‘hub’ gene in Ciona pigment cell formation.

(a) Early PCP-specific subnetwork inferred from the analysis of microarrays. ‘Hub’ genes are represented as grey spots, numbered from 1 to 5, by ranking them according to the number of neighbours. Ci-Rab32/38 is evidenced in red. (b) Ci-Rab32/38 expression (green) in PCPs during Ciona neurulation: neurula, initial TB, early TB, middle and late TB stages. (c) Double WMISH for Ci-Rab32/38 (green) and Ci-tyrp1/2a (red) from neurula to middle TB stage; nuclear staining by DAPI (blue); _n_>50 embryos scored for gene expressions.

Figure 9

Figure 9. Role of Rab32/38 in Ciona pigment cell biogenesis.

Histograms showing the percentage of larvae with two pigment cells (red), one pigment cell (violet) and no pigment cells (orange) after electroporation of dominant-negative forms of Ci-Rab32/38 under control of PCP enhancer (ptyr>Rab32/38G19V or ptyr>Rab32/38G19V+T36N) (a) or short hairpin inhibiting construct against Ci-Rab32/38 mRNA (U6>shRab32/38-C-D and U6>shRab32/38-E-F) (b) compared with control larvae electroporated with ptyr>LacZ.Merged bright field/fluorescent images of transgenic larvae. (c,d) Double fluorescent WMISH was performed to test the ability of U6>shRab32/38 used to knock down the endogenous Ci-Rab32/38. The expression of Ci-Rab32/38 (red) is compared with Ci-tyrp1/2a (green) and with anti-GFP antibody to follow expression of the electroporated constructs. Embryos were electroporated with ptyrp1/2a>GFP plus ptyrp1/2a>LacZ (c) and ptyrp1/2a>GFP plus U6>shRab32/38-C-D (d). U6>shRab32/38-C–D is able to knock down the endogenous Ci-Rab32/38 transcripts (_n_=33/59, d) and to induce defects in pigmented sensory organ melanization (a,b), while Ci-tyrp1/2a expression resulted not affected (c) as compared with the control embryos (_n_=73/80, c). Anterior to the left; nuclear staining by DAPI (blue). Scale bar, 50 μm; scale bar, 20 μm for zoomed embryos. Within each histogram is the combined number of larvae counted during at least three trials; _n_>150 embryos scored for transgene expression; error bar, s.e.m.; oc, ocellus, ot, otolith.

Figure 10

Figure 10. FGF/MAPK inputs sequentially govern PCP versus anterior CNS formation.

Summary of pigmented cell development within CNS; embryonic stage drawings are indicated below the time line (hpf, hours post fertilization). Black bars connect sister cells. At 6 hpf embryo, the bilateral a9.50s are marked in blue; a9.49 cell pair (in pink) are in close proximity to two sources of FGF signalling molecules, Ci-FGF8/17/18 (light green) and Ci-FGF9/16/20 (dark green) in the underneath row, respectively. Cell lineage scheme indicate one side of the embryo and the colour code of cells remains the same used in the upper drawings. Red arrows indicate FGF/MAPK signalling mediated inductions. a8.25-derived cells with specific gene expression patterns: boxes represent a9.50- and a9.49-derived cells with the same colour code used in the upper drawings. FGFRDN: dominant-negative form of FGF receptor; Ets:Vp16: constitutive active form of Ets1/2.

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