Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS - PubMed (original) (raw)

Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS

Nicolas Haupaix et al. Dev Biol. 2014.

Abstract

Recent evidence suggests that ascidian pigment cells are related to neural crest-derived melanocytes of vertebrates. Using live-imaging, we determine a revised cell lineage of the pigment cells in Ciona intestinalis embryos. The neural precursors undergo successive rounds of anterior-posterior (A-P) oriented cell divisions, starting at the blastula 64-cell stage. A previously unrecognized fourth A-P oriented cell division in the pigment cell lineage leads to the generation of the post-mitotic pigment cell precursors. We provide evidence that MEK/ERK signals are required for pigment cell specification until approximately 30min after the final cell division has taken place. Following each of the four A-P oriented cell divisions, ERK1/2 is differentially activated in the posterior sister cells, into which the pigment cell lineage segregates. Eph/ephrin signals are critical during the third A-P oriented cell division to spatially restrict ERK1/2 activation to the posterior daughter cell. Targeted inhibition of Eph/ephrin signals results in, at neurula stages, anterior expansion of both ERK1/2 activation and a pigment cell lineage marker and subsequently, at larval stages, supernumerary pigment cells. We discuss the implications of these findings with respect to the evolution of the vertebrate neural crest.

Keywords: Ascidian; Melanocyte; Neural crest; Oriented cell division; Pigment cell.

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Figures

Fig. 1. The a-line neural lineages of Ciona

Throughout the indicated stages, the a-line derived neural plate cells are colored pink in the illustrated embryos. Below the embryo illustrations are simplified schematics of the a-line derived neural plate, with the row III-IV CNS lineages in black and the row V-VI palp lineages in grey. Bars indicate sister cell relationships. A red dot indicates the pigment cell lineage at each stage with the corresponding cell nomenclature indicated. After segregation from the non-neural ectoderm, the a-line neural lineage is a single row of six cells at the 76-cell stage. During gastrulation, the six a-line neural precursors divide along the anterior-posterior axis, making two rows of six cells. Together with the single row of A-line neural precursors, this makes a 3-row neural plate. All cells of the 3-row stage divide in a specific order to generate the 6-row stage (Nicol and Meinertzhagen, 1988). The 6-row neural plate then divide in an asynchronous but predictable order (see text for details) to produce 12-rows of neural plate cells in neurula embryos. At this stage the pigment cell lineage is positioned in the lateral column in row IIIp. This cell undergoes one final division to produce a11.194 and a11.193 prior to their intercalation at the midline (see also Supplementary movie 1 and Fig. 4).

Fig. 2. Role of MEK1/2 and Eph/ephrin signals at the 3-row and 6-row neural plate stage

Experimental conditions are indicated above each column of panels. A) dpERK1/2 immunofluorescence at the 6-row neural plate stage showing ERK1/2 activation in row III (arrowhead). B-C) Analysis of gene expression following various treatments. U0126 was added to embryos at the 76-cell stage (76c), or at the late 3-row neural plate stage when endoderm invagination is well underway (3row+). An embryo illustration on the right shows the control expression domains. Percentages indicate the proportion of embryos that the panel represents. For the Dll-B U0126 3row+ panel, the remaining embryos showed Dll-B expression in rows III and IV, although this was often weaker than in rows V and VI, and/or patchy. The red brackets indicate ectopic expression.

Fig. 3. Inhibition of Eph/ephrin signaling results in excess pigment cells

Embryos were electroporated with the constructs indicated on the panels. Dmrt∷Eph1ΔC and Dmrt∷LacZ were used as controls for electroporation, showing that the results were specific to disruption of Eph3 and RasGAP. Co-electroporation with Tyr∷mCherry (red) shows that the pigment cells derive from Tyrosinase positive cells. The graph (E) shows the proportion of embryos with the number of pigment cells indicated in the key, following electroporation. In embryos exhibiting five or more pigment cells, it was difficult to distinguish each pigment cell, so these embryos were scored as 5+. Scale bars, 50μm.

Fig. 4. A revised pigment cell lineage

This figure accompanies Supplementary movie 1. A-F) Embryos were electroporated with ZicL∷H2BCFP (green) to label rows I-IV and Dmrt∷H2BYFP (magenta) to label rows III-VI. Still images from the movie are shown on the left. On the right are the same still images with false-labeled nuclei to highlight the positions of row III and IV derivatives in orange and blue respectively. The a10.97 lineage is marked in pink, highlighting the extra division (D). G) An embryo that has been electroporated with Mitf∷Unc76GFP (green), Msx∷H2BmCherry (red) to show the lineage derivatives of a10.97 and a9.49 respectively. The position of each cell is labeled on the tailbud stage embryo.

Fig. 5. Pigment cell formation depends on MEK1/2 signals until early tailbud stage

A-H) Expression of Trp at different stages of the pigment cell lineage as indicated by the colored boxes. See text for details. Time points are hours post fertilization (hpf) at 18°C, the time indicated reflects the embryological stage seen for the majority of embryos at each time point (as shown in I). I-J) In these experiments, one half of the embryos were fixed for Trp in situ hybridization (I) to determine the precise stage of U0126 treatment (n= minimum 36), the other half were treated with U0126 until larval stages in order to monitor pigment cell formation (J) (n= minimum 34). I) The color code of the graph represents the stage of the pigment cell lineage, defined as a9.49 (blue), a10.97 (red) and a11.193 (green). Bars are further broken down into the proportion of embryos representing the precise embryological stage, as represented in A-H. J) Bars represent the proportion of embryos with any pigmented cells at each time point. Representative resultant larvae after treatment with U0126 from the time points indicated are shown.

Fig. 6. Eph/ephrin signals control the spatial extent of ERK1/2 activation at the 12-row neurula stage

A-C) Experimental conditions are indicated on the top panels. Embryos were stained for activated ERK1/2 (dpERK1/2) in red, with nuclei (DAPI) in white. For this analysis embryos were also co-electroporated with Msx∷LacZ or Dmrt∷LacZ followed by LacZ immunofluorescence to ensure our cell identification was correct (not shown). The top panel shows the entire embryo and the bracket indicates the annotated region in the lower panels. The divided rows III and IV are outlined in white and yellow respectively in the middle panel. At the stage of analysis, the lateral precursors of row IV have not yet divided. In the lower panel, only the dpERK immunofluorescence is shown for clarity. The interpretation of the dpERK immunofluorescence intensity is depicted by red and pink dots on the schematic of divided rows III and IV. The percentage of dpERK1/2 detection in each cell of row IIIa and IIIp is indicated on the lower schematic, representing independent scoring for each embryo half. n= number of half embryos scored. D) Early tailbud stage embryo stained for dpERK1/2 (red) and DAPI (white). Enlarged region is indicated by bracket on whole embryo panel.

Fig. 7. Ectopic Trp expression in a10.98 following inhibition of Eph/ephrin signals

Expression of Trp at 12-row neurula stage in control embryos, embryos electroporated with Dmrt∷Eph3ΔC or embryos treated with U0126 just after the 6-row stage (U0126 6row+). In these experiments, U0126-treatment began once embryos started to leave the 6-row neural plate stage and the cells in row II begin to divide. Each embryo half was scored independently and the quantification is shown in the graph.

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Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS - PubMed (original) (raw)