Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Dev Biol. 2014 Jul 22;394(1):170–180. doi: 10.1016/j.ydbio.2014.07.010

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

Nicolas Haupaix 1,2,3,5, Philip B Abitua 4,5, Cathy Sirour 1,2, Hitoyoshi Yasuo 1,2, Michael Levine 4, Clare Hudson 1,2,*
PMCID: PMC4258108  NIHMSID: NIHMS616571  PMID: 25062608

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 30 minutes 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: melanocyte, pigment cell, ascidian, neural crest, oriented cell division

Introduction

The ascidian pigment cells form part of the ocellus and the otolith, light and gravity sensing organs, respectively (Jiang et al., 2005; Tsuda et al., 2003). Previously, the ascidian pigment cells have been proposed as the evolutionary precursors of the vertebrate retinal pigment epithelium or pineal organ, based on expression of gene markers and the probable function of the ocellus pigment cell in shielding the photoreceptors (Lamb et al., 2007; Sato and Yamamoto, 2001). However, recent evidence using a larger panel of genes supports a closer evolutionary relationship to the cephalic neural crest of vertebrates (Abitua et al., 2012; Ivashkin and Adameyko, 2013). This hypothesis is strengthened by the acquisition of migratory properties upon the misexpression of Twist, a cell behavior exhibited by neural crest.

Of the four founder embryonic lineages of Ciona (A- and B- of the vegetal hemisphere and a- and b- of the animal hemisphere), the central nervous system (CNS) derives from three lineages, A-, a- and b- (Nishida, 1987). This study is concerned with the a-line neural lineages, from which the pigment cells arise. At the 64- to 76-cell stage, the a-line neural lineages form one row of six cells (Fig. 1) (Lemaire et al., 2002). These cells then divide twice along the anterior-posterior axis to generate four rows of six cells at the 6-row neural plate stage (Fig. 1). The 6-row neural plate is arranged with row I posterior-most and row VI anterior-most (Nicol and Meinertzhagen, 1988). The a-line cells comprise rows III-VI (pink cells in Fig. 1) and the A-line cells, rows I and II. Of the a-line cells, only rows III and IV will undergo neurulation and contribute to the CNS. These two rows generate the anterior part of the sensory vesicle, which is the ascidian larval brain, and also contribute to the oral siphon primordium (Christiaen et al., 2007; Cole and Meinertzhagen, 2004; Nishida, 1987; Taniguchi and Nishida, 2004; Veeman et al., 2010). The anterior two rows, V and VI, generate the palps, an adhesive organ, as well as non-neural ectoderm. The pigment cell lineage is situated in the lateral-most cell of row III. Between the 6-row neural plate and the 12-row neurula stages, each row of cells divides in a specific sequence, with row II cells dividing first, followed by row I, then row III and V and lastly row IV and VI (Nicol and Meinertzhagen, 1988). In each row, cells in the medial four columns divide prior to those in the lateral columns. At the 12-row neural plate stage, the pigment cell lineage is segregated into the posterior row III (IIIp) cell, a10.97. As we will describe in this manuscript, the a10.97 pigment cell precursors undergo a further and final round of cell division along the anterior-posterior axis, whereby the posterior-positioned daughters, a11.193, intercalate at the midline to become the pigment cells of the otolith and ocellus.

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

Fig. 1

Open in a new tab

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).

FGFs are secreted ligands that bind to a class of receptor tyrosine kinases, the FGF receptors (FGFRs), and predominantly activate the Ras/MEK/ERK cascade. FGF/ERK signaling is implicated in a vast range of processes during vertebrate neural development including the induction of neural crest (Guillemot and Zimmer, 2011; Hébert, 2011; Stuhlmiller and Garcia-Castro, 2012). During ascidian neural development, FGF/ERK signaling is responsible for the specification of different cell types. It is involved in the process of neural induction of a-line neural lineages at the 32-cell stage (Bertrand et al., 2003) and later during patterning of the developing neural plate and neural tube (Abitua et al., 2012; Hudson et al., 2003; Hudson et al., 2007; Squarzoni et al., 2011; Stolfi et al., 2011; Wagner and Levine, 2012). Recently, Eph/ephrin signaling via RasGAP has been shown to limit the spatial extent of activation of ERK1/2 during several developmental cell fate choices in Ciona embryos including during neural development (Haupaix et al., 2013; Ohta and Satou, 2013; Picco et al., 2007; Shi and Levine, 2008; Stolfi et al., 2011). Here we investigated the role of Eph/ephrin signaling and the MEK/ERK pathway during pigment cell specification at neural plate stages of Ciona intestinalis development.

Materials and Methods

Embryo experiments and constructs

Adult Ciona intestinalis were purchased from the Station Biologique de Roscoff (France) or M-Rep (San Diego, CA). Cell nomenclature, lineage and the fate maps are previously described (Cole and Meinertzhagen, 2004; Conklin, 1905; Nishida, 1987). Ascidian embryo culture has been described (Sardet et al., 2011). Electroporation was carried out as described (Christiaen et al., 2009). U0126 (Calbiochem) was added to artificial seawater at 2μM. All data was pooled from at least two independent experiments.

For the electroporation constructs, the upstream sequences ZicL, Tyr, Mitf, Dmrt, Msx and Fog are previously described (Abitua et al., 2012; Haupaix et al., 2013; Rothbächer et al., 2007; Wagner and Levine, 2012). These were used to drive expression of LacZ, mCherry, Eph1ΔC, Histone 2B (H2B), unc76GFP, Eph3ΔC, RG(R818E) (Abitua et al., 2012; Haupaix et al., 2013; Hudson et al., 2007; Stolfi et al., 2011; Wagner and Levine, 2012). Following electroporation, embryos were selected at gastrula stages for correct morphology. In Figure 3, Dmrt∷Eph3ΔC electroporated embryos were co-electroporated with Trp∷mCherry. Only fluorescent (i.e. electroporated) embryos were counted for this experiment, in all other experiments, all embryos were counted. In order to assess ocellus and otolith pigment cell type, embryos were mounted in 80% glycerol and slightly compressed. Ocellus type or otolith type pigment cells were defined based on pigment granule morphology; such that tiny dispersed granules were defined as ocellus type and larger, round or flower shaped granules were defined as otolith. The number of ocellus type pigment cells was likely underestimated since it was more difficult to distinguish individual ocelli from one another. In both control and electroporated embryos, even very large ocelli were counted as one.

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

Fig. 3

Open in a new tab

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.

In situ hybridization and immunofluorescence

In situ hybridization was carried out as previously described (Hudson et al., 2013). Diglabeled probes were synthesized from the following Ciona cDNA clones: Trp (Hudson et al., 2003), FoxC (cilv050a24) (Imai et al., 2004), ZicL (cicl002e04) (Imai et al., 2002b), Mitf (cilv41b12) (Abitua et al., 2012). Six3/6 (cicl021e08), Dll-B (cicl022f04), ephrinAc (ciad074h16), ephrinAb (cieg037l08), ephrinAd (ciad008n17), Eph3 (cieg009e01), FgfR (citb040h06), Fgf9/16/20 (citb007k01) and Fgf8/17/18 (citb002j04) are all described (Imai et al., 2004). Clone numbers refer to clones from the ‘Release 1 Gene Collection Plates’ (Satou et al., 2002). For Dll-B in situ hybridization, DAPI staining was used to verify cell identity. In some batches of embryos, Dll-B was also detected in row IV. These batches of embryos were removed from the analysis. For Trp in situ hybridization in Figure 5, it should be noted that the intensity of the Trp signal increases with developmental time. At the 6-row neural plate stage, the color reaction was developed for approximately 12 hours, whereas at the early tailbud stage, the color reaction time was 1 to 3 hours.

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

Fig. 5

Open in a new tab

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.

dpERK1/2 immunofluorescence is described previously (Haupaix et al., 2013). LacZ immunofluorescence is described previously (Stolfi et al., 2011) except that anti-β-galactosidase (Molecular Probes, A11132) was used at 1/500 and the secondary antibody used was goat anti-rabbit Alexa Fluor 488 (Molecular Probes, A11008) at 1/1000.

In our analysis, we refer to embryos as the 6-row neural plate stage to indicate that no row of a-line cells has divided, though cells in row II have sometimes divided. We refer to the 12-row neurula stage to indicate that row III cells have divided. Embryos in Figs. 2, 6 and Supplementary Fig. 1 were mounted in VECTASHIELD/DAPI (Vector Laboratories). Embryos in Figs. 2,5,6,7 and Supplementary figures were photographed on an Olympus BX51 using Leica DFC310FX camera and those in Figs. 3 and 4G on a Zeiss Axio Imager AZ using a SPOT RT3 camera.

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

Fig. 2

Open in a new tab

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

Fig. 6

Open in a new tab

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.

Fig. 7

Open in a new tab

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.

Fig. 4. A revised pigment cell lineage.

Fig. 4

Open in a new tab

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.

Time-lapse movie

The time-lapse sequence was obtained using a Zeiss LSM 700 microscope using a plan-apochromat 20x objective. Confocal stacks contained 20 optical slices at a thickness of approximately 1μm each and were taken every 3 minutes for 2 hours. Images were rendered in 3D using Volocity 6 with the 3D opacity visualization tool.

Results

Differential activation of ERK1/2 in the neural plate is required for patterning along the A-P axis

It has previously been shown that differential ERK1/2 activity between rows III/IV (CNS) and V/VI (palp) at the 3-row neural plate stage (Fig.1) accounts for acquisition of their distinct genetic programs (Wagner and Levine, 2012). ERK1/2 is subsequently differentially activated between rows III and IV at the 6-row neural plate stage with ERK1/2 active in row III cells (Fig.2A) (Hudson et al., 2007). This pattern of ERK1/2 activation is required for the differential fate specification of these two sister rows (Fig. 2C). We inhibited MEK/ERK signal transduction by applying U0126, an inhibitor of the MAP kinase kinase, MEK1/2, at precise developmental time points. The following markers were used to assess neural plate patterning: FoxC (rows V and VI), Dll-B (epidermis and rows V and VI), ZicL (rows III and IV), Six3/6 (row IV), Mitf and Trp (row III) (Abitua et al., 2012; Irvine et al., 2007; Wagner and Levine, 2012). As reported previously, application of U0126 at the 76-cell stage disrupts the choice between row III/IV and row V/VI fates (Wagner and Levine, 2012). Genes expressed in rows III and IV (ZicL, Six3/6, Trp and Mitf) were lost and genes normally restricted to rows V and VI (FoxC and Dll-B) were ectopically expressed in rows III and IV (Fig. 2B and C). In contrast, application of U0126 after the separation of rows III/IV and V/VI, at the early gastrula 3-row neural plate stage (approximate stage 11+ (Hotta et al., 2007)), resulted in loss of row III gene expression (Trp, Mitf) and ectopic expression of the row IV gene, Six3/6, in row III. Thus, inhibition of MEK1/2 signals at these two successive time points results, in each case, in a distinct posterior to anterior fate transformation of sister cells. In the first instance, differential activation of ERK1/2 specifies row III/IV over V/VI fate and then, following division of row III/IV cells, it promotes row III over row IV fates.

Eph/ephrin signals are not required for neural plate patterning at the 3- and 6-row neural plate stages

There are several examples during Ciona embryogenesis whereby Eph/ephrin signals control the spatial extent of ERK1/2 activation (Haupaix et al., 2013; Ohta and Satou, 2013; Picco et al., 2007; Shi and Levine, 2008; Stolfi et al., 2011). Three ephrin ligands are expressed in the neural lineages during neural plate and neurula stages and the receptor Eph3 is expressed ubiquitously, becoming upregulated in the pigment cell lineage soon after the 6-row neural plate stage (Supplementary Fig. 1) (Imai et al., 2004; Tassy et al., 2010). These gene expression profiles suggest that Eph/ephrin signals could also be involved in the modification of ERK1/2 activity during neural plate patterning. In order to address this possibility, we expressed a dominant negative form of the Eph receptor Eph3, Eph3ΔC (Picco et al., 2007), in the a-line neural lineages. For this, we used the Dmrt promoter which drives expression throughout the a-line neural lineages from the 64-cell stage and has previously been used to disrupt gene function at the 3-row neural plate stage (Ikeda et al., 2013; Wagner and Levine, 2012). Following electroporation of Dmrt∷Eph3ΔC, we observed that expression of FoxC, Dll-B, ZicL, Six3/6, Mitf and Trp were unaffected at the 6-row stage neural plate stage (Fig. 2B and C). Consistently, the spatial pattern of ERK1/2 activation at the 6-row neural plate stage was not perturbed in these embryos (Fig. 2A). We conclude that Eph/ephrin signals are not required to generate the spatial pattern of ERK1/2 activation in the neural plate during the 3-row or 6-row neural plate stages. A similar conclusion was reached analyzing embryos electroporated with an early-active promoter, Fog, to drive Eph3ΔC in all ectodermal cells from the 16-cell stage (Rothbächer et al., 2007) (Supplementary Fig. 2).

An extra cell division in the pigment cell lineage

Despite little effect on the neural plate genes analyzed, we consistently observed ectopic formation of pigment cells in larvae resulting from embryos electroporated with Dmrt∷Eph3ΔC (Fig. 3). In some cases, five or more pigment cells could be observed. This result suggests that Eph/ephrin signals play a role in neural patterning. We thus set out to understand how Eph/ephrin signals are involved in this process.

In order to study the role of Eph/ephrin signals in pigment cell formation, we first observed the pigment cell lineage in detail, revealing a previously unrecognized cell division (Fig. 4; Supplementary movie 1). We analyzed the time-lapse sequence of embryos electroporated with Dmrt∷H2BYFP (Histone 2B fused with YFP, magenta in images) to label rows III through VI and ZicL∷H2BCFP (green in images) to label rows I through IV. These reporters are thus co-expressed in rows III and IV allowing for the delineation of cell lineages. Analyzing the time-lapse sequence revealed a further cell division of the a10.97 cell, which was previously described as a postmitotic pigment cell precursor (Cole and Meinertzhagen, 2004; Whittaker, 1979). The pigment cell lineage and cell positions in the neural tube are thus modified to account for this additional division (Fig. 4G). We also recognized this extra division by analyzing Trp expression every 15 minutes from the 6-row neural plate stage until the early tailbud stage (Fig. 5A-H). Trp expression begins at the 6-row neural plate stage in a9.49 (Fig. 5A). Following cell division of a9.49, Trp expression is observed in both daughter cells, but rapidly becomes stronger in the posterior-positioned a10.97 compared to its anterior sister cell, a10.98 (Fig. 5B-D). Next, a10.97 divides into a11.194 and a11.193, both cells retain Trp expression and converge towards the midline (Figs. 4D and 5E), touch (Figs. 4E and 5F) and begin to intercalate (Fig. 5G). Finally these cells are positioned with the two smaller a11.194 cells side by side anterior to the two larger a11.193 pigment cell precursors intercalated at the midline (Fig. 4F and G; Supplementary movie 1).

MEK1/2 activity is required until approximately 30 minutes after the final division in the pigment cell lineage

Since patterning of the 6-row neural plate was not affected by inhibition of Eph/ephrin signaling (Fig. 2; Supplementary Fig. 2), we hypothesized that the supernumerary pigment cells observed following inhibition of Eph/ephrin signals resulted from a failure to control the spatial extent of ERK1/2 activation after the 6-row neural plate stage. We first analyzed precisely when pigment cell specification is dependent on MEK/ERK signals. We applied U0126 to embryos every 15 minutes from the 6-row neural plate stage to the early tailbud stage, when pigment cell formation has been shown to occur independently of MEK1/2 signaling (Squarzoni et al., 2011). We monitored the precise developmental stage at which embryos were placed in U0126 by analyzing a sample of embryos at each time point for Trp expression (Fig. 5A-I). We then assessed the presence of pigmented cells in resultant larvae (Fig. 5J). This analysis shows that pigment cell formation requires MEK1/2 signaling until approximately 30 minutes after the pigment cell lineage has undergone its final cell division (corresponding approximately to the embryonic stage shown in Fig. 5G). Following this time point, U0126-treatment had little effect on specification of pigment cells, albeit their pigmentation was somewhat reduced.

ERK1/2 is activated in the pigment cell lineage during neurulation

We showed that pigment cell specification requires an intact MEK1/2 signaling pathway until around 30 minutes after the final cell division of this lineage (Fig. 5). Consistent with a direct requirement for the pathway during pigment cell specification, we found that ERK1/2 remains active in the pigment cell lineage following the 6-row neural plate stage. When the neural plate consists of 12-rows of cells (in this analysis all row III cells and medial row IV cells had completed division), ERK1/2 activation was preferentially detected in the posterior daughters of rows III and IV (row IIIp and IVp) (Fig. 6A). At this stage, the pigment cell lineage is segregated into a10.97, the lateral-most cells of row IIIp (Figs. 1 and 6A). At the early tailbud stage (corresponding to the stage in Fig. 5F), we observed higher ERK1/2 activity in a11.193, the posterior daughter of a10.97, compared to the anterior daughter a11.194 (Fig. 6D). Thus, ERK1/2 is continuously differentially activated in the pigment cell lineage following each successive A-P oriented cell division, consistent with an ongoing and direct requirement for MEK/ERK dependent signals for pigment cell specification (Figs. 5 and 6).

Eph/ephrin signals restrict the spatial extent of ERK1/2 activation during the penultimate division of the pigment cell precursors

Differential ERK1/2 activity is involved in differential fate specification at both the 3-row and the 6-row neural plate stages (Fig. 2) (Wagner and Levine, 2012). We showed that Eph/ephrin signals are not involved in the spatial control of ERK1/2 or neural plate patterning at these stages (Fig. 2; Supplementary Fig. 2). However, we found that Eph/ephrin signals are required to restrict the number of pigment cells (Fig. 3). We also found that ERK1/2 is continuously activated in the pigment cell lineage during neurulation, until the early tailbud stage (Fig. 5). Focusing on the 12-row neural plate stage, we found that Eph/ephrin signals are required to restrict ERK1/2 activation to the posterior daughter cells of row III and IV (row IIIp and row IVp) (Fig. 6B). In Dmrt∷Eph3ΔC-embryos, ERK1/2 activation was observed in both the anterior and posterior daughter cells of rows III and IV. This effect was particularly prominent in the pigment cell lineage with strong ectopic ERK1/2 activation in a10.98, the anterior sister cell of a10.97. Consistent with a critical role for Eph/ephrin signals in restricting pigment cell fate to the a10.97 lineage, we observed ectopic expression of Trp in a10.98 in Dmrt∷Eph3ΔC-embryos (Fig. 7). In these experiments, we also occasionally (3% of embryo halves) observed persistence of Trp expression in row IV cells. It is not clear to us why this happens, since we do not see ectopic expression of row III genes in row IV at the 6-row stage in Dmrt∷Eph3ΔC embryos. Similar results were obtained using Fog∷Eph3ΔC (Supplementary Fig. 2). Conversely, treatment of embryos with U0126 from the 6-row neural plate stage resulted in a strong reduction of Trp expression (Fig.7). Thus, Eph/ephrin signals are required to restrict ERK1/2 activity at the 12-row neurula stage, but not at earlier stages (3-row and 6-row).

The spatial control of ERK1/2 activation by Eph/ephrin signals is mediated by p120RasGAP in Ciona (Haupaix et al., 2013). This is also likely to be the case during pigment cell specification since electroporation of a dominant negative version of RasGAP, RG(R818E), resulted in a similar phenotype of supernumerary pigment cells in larvae and ectopic activation of ERK1/2 in 12-row neurula, but not 6-row neural plate stage embryos (Figs. 2A, 3 and 6C).

A role for Eph/ephrin at the 12-row stage is consistent with the formation of more than five pigmented cells that we frequently observed in Dmrt∷Eph3ΔC-larvae, since transformation of a10.98 into a a10.97-like fate should result in a pool of a maximum of eight Trp-positive cells instead of the usual four. The presence of more than four pigmented cells also suggests that the subsequent fate choice between a11.194 and a11.193 is also perturbed in many Dmrt∷Eph3ΔC-larvae.

Finally, we did not find any evidence that the choice between ocellus and otolith fates requires Eph/ephrin signals. In a pool of embryos electroporated with Dmrt∷Eph3ΔC, we counted an average of 1.5 otolith and 1.4 ocellus-type pigmented cells (n=299) compared to control embryos, which exhibited an average of 0.8 otolith and 1.2 ocellus-type pigment cells (n=324). The preferential increase in otolith-type pigment cells is consistent with a previous study where the ectopic activation of Ets results in extra pigments cells of the otolith-type. The choice between ocellus and otolith fate is governed by Wnt7, which serves as a positional cue for specifying the posterior ocellus pigment cell (Abitua et al., 2012).

Discussion

We document an additional division of the pigment cell lineage, which was previously described to become post-mitotic following nine cell divisions after fertilization in Ciona (Whittaker, 1979). We found that disruption of Eph/ephrin signals results in supernumerary pigment cells in larvae. Our results show that ephrin/Eph/RasGAP signaling is required during the 12-row neural plate stage, but not at earlier neural plate stages, to restrict ERK1/2 activation to the posterior daughters of row III (IIIp, as well as row IVp). Removal of this control mechanism results in ectopic activation of ERK1/2 in row IIIa and ectopic Trp expression in a10.98.

Reiterative differential ERK1/2 associated with A-P oriented cell division in the developing nervous system

FGF/MEK/ERK-driven cell fate choices generate cell diversity at multiple steps during ascidian embryogenesis (Bertrand et al., 2003; Davidson et al., 2006; Hudson et al., 2003; Hudson et al., 2007; Imai et al., 2002a; Kim and Nishida, 2001; Picco et al., 2007; Shi and Levine, 2008; Shimauchi et al., 2001; Squarzoni et al., 2011; Stolfi et al., 2011; Yasuo and Hudson, 2007). Even within the a-line neural lineage, there are many examples. At the 32-cell stage, the a5.3 cell divides to produce the a6.6 cell and the a6.5 cell. The a6.5 cell exhibits ERK1/2 activation in response to the neural inducing FGF signal and adopts a neural fate while the a6.6 cell, with no ERK1/2 activation, adopts an epidermal fate (Bertrand et al., 2003; Hudson et al., 2003). Restriction of ERK1/2 activation to the neural precursor depends upon ephrin signals (Ohta and Satou, 2013). At this stage, a-line neural cells contain both CNS and palp lineages. These cells are prevented from precociously activating the CNSspecific program by a time delay mechanism involving a pair of Blimp1-like zinc finger repressors (Ikeda et al., 2013). From the 3-row neural plate stage, differential ERK1/2 activation is used repeatedly to specify the posterior-most cell fates during at least four successive rounds of A-P oriented cell divisions in the a-line neural lineages. During each division, the pigment cell lineage segregates into the posterior cells. At the 3-row neural plate stage, CNS lineages (row III/IV) segregate from palp lineages (row V/VI) with FGF/MEK/ERK promoting CNS over palp fate in row III/IV (Wagner and Levine, 2012). Differential activation of ERK1/2 between rows III/IV and V/VI may be achieved simply by the restricted expression of the inducing ligand, since Fgf9/16/20 is expressed in row I/II cells, adjacent to the row III/IV cells (Supplementary Fig. 1) (Imai et al., 2006). At the 6-row neural plate stage, ERK1/2 activation is restricted to row III of the a-line neural plate. Localized expression of FGF ligands (Fgf9/16/20 and Fgf8/17/18) in row II might also account for the differential activation of ERK1/2 between rows III and IV (Supplementary Fig. 1) (Hudson et al., 2007; Squarzoni et al., 2011). At this stage, differential ERK1/2 activation also drives the cell fate choices between row I and row II sister cells of the A-line neural plate with ERK1/2 activated in row I cells, though it is not clear how this differential activation pattern is established (Hudson et al., 2007).

Here we described an additional differential activation of ERK1/2 in row III and row IV derivatives following their division into rows IIIa, IIIp, IVa and IVp, whereby ERK1/2 activation is again associated with the posterior daughter cells in row IIIp and row IVp (Fig. 6). We show that ERK1/2 activation is spatially restricted to the posterior rows by Eph/ephrin signals. This mechanism is unlikely to be confined to the pigment cell lineage choice between a10.97 and a10.98 fates, since ectopic ERK1/2 activation is seen broadly in rows IIIa and IVa following inhibition of Eph/ephrin signals. It is not yet clear which ephrin ligands are involved in this process. ephrin-Ad is expressed in rows V-VI from the 6-row neural plate stage and may therefore be involved in the restriction of ERK1/2 activity between rows IVa and IVp (Wagner and Levine, 2012) (Supplementary Fig. 1). ephrin-Ac and ephrin-Ab are expressed in discrete domains of the neural plate in close proximity to row III and thus might be involved in the spatial control of ERK1/2 activation between row IIIa and IIIp cells (Supplementary Fig. 1). Early in development the Eph3 receptor is expressed ubiquitously, then shortly after the 6-row stage it is upregulated in the pigment cell lineage. Interestingly, this upregulation depends on MEK/ERK signaling, suggesting that MEK/ERK signals render this cell lineage particularly sensitive to Eph/ephrin signals during subsequent stages (Supplementary Fig. 1). Based on ephrin and Fgf ligand and receptor expression profiles, it is unclear how such a precise spatial pattern of ERK1/2 activation is achieved at the 12-row neural plate stage. It is possible that other mechanisms are also involved, however our data show that Eph/ephrin plays an essential role in establishing this pattern (Figure 6).

Following the MEK/ERK-mediated specification of a10.97 as the pigment cell lineage, these cells divide into a11.194 and a11.193 and the posteriorly positioned a11.193 cells become pigmented. It is not yet clear how this fate choice between a non-pigmented and pigmented cell fate is achieved, although differential ERK1/2 may be involved since ERK1/2 is preferentially activated in a11.193. Differentially-expressed gene markers of a11.193 and a11.194 would be required to address this issue. To determine whether Eph/ephrin signals are directly involved will necessitate a means to block signaling specifically after the 12-row neural plate stage. The fact that five or more pigment cells are frequently observed in Dmrt∷Eph3ΔC larvae, suggests that this fate choice is also perturbed in this experimental context.

Finally, the two a11.193 cells from both sides of the neural plate intercalate at the midline and adopt either an ocellus or otolith fate. The post-mitotic pigment cell precursors have been shown to form an equivalence group in another ascidian species, Halocynthia roretzi. These cells intercalate at random and the posteriorly positioned cell adopts the ocellus fate (Nishida, 1987; Nishida and Satoh, 1989). We did not find any evidence that Eph/ephrin signals are involved in this final fate choice. In Ciona, this fate choice is controlled by Wnt7 signals, whereas BMP-Chordin antagonism has been implicated in Halocynthia (Abitua et al., 2012; Darras and Nishida, 2001).

If the pigment cell lineage resembles the evolutionary precursors of the vertebrate neural crest, it is relatively easy to envisage how these cell types could both increase in number and adopt a migratory capacity. Simple deregulation of signaling pathways leads to quite a dramatic increase in pigment cell number (Fig. 3) (Abitua et al., 2012; Squarzoni et al., 2011). A subset of these cells would then need to acquire the expression of a mesenchyme-promoting factor such as Twist in order for a primordial neural crest to develop (Abitua et al., 2012).

Supplementary Material

01. Supplementary Movie 1.

Time-lapse movie of an embryo electroporated with Dmrt∷H2BYFP to label rows III-VI and ZicL∷H2BCFP to label rows I-IV. The movie begins just after the cells in row III have divided and runs until the pigment cell precursors intercalate at the midline. Image stacks were taken every 3 minutes for 2 hours at 21°C. Annotations refer to the cell lineage nomenclature at the tenth generation except for the final division of the pigment cells. The final division of a10.97 (labeled “97”) gives rise to a11.193 (labeled “193”) and a11.194 (labeled “194”). Similarly, cells a10.68 and a10.67 of row IV, previously described as postmitotic (Cole and Meinertzhagen, 2004), can also be seen dividing.

Download video file (65.9MB, mov)
02. Supplementary Figure 1.

Expression of Fgf, ephrin, FgfR and Eph3 at neural plate and neurula stages. A red dot indicates the pigment cell lineage on the schematics and a-line neural plate cells are colored in pink. The key indicates gene expression in 6-row neural plate cells. ephrinAc is not expressed at the 6-row neural plate stage, but is expressed in all animal cells at the early gastrula stage (Imai et al., 2004). FgfR and Eph3 are ubiquitously expressed during early gastrula and neural plate stages. Broad low levels of expression are also detected at the 12-row neural plate stage, with specific domains of stronger expression also detected, including in the a-line neural lineages. Strong Eph3 expression starts to be detected in the pigment cell lineage between the 6- and 12-row neural plate stage. This expression was dependent upon MEK activity; embryos were treated with U0126 from the 3-row neural plate stage. Arrowheads point to the nucleus of a9.49 in the middle panel and a10.97 in the lower panels. Some of this data overlaps with published studies (http://ghost.zool.kyoto-u.ac.jp/otherfr_kh.html; http://www.aniseed.cnrs.fr/; Hudson et al., 2007; Imai et al., 2004; Imai et al., 2009; Shi et al., 2009; Tassy et al., 2010; Wagner and Levine, 2012).

03. Supplementary Figure 2.

Gene expression and pigment cell formation in embryos electroporated with Fog∷Eph3ΔC. A) Graphs show the expression of the genes indicated (above) in control and electroporated embryos. Embryos were scored following the key (below). The expression domains of each gene are shown in Figure 2. Embryos were scored for levels of expression similar to the majority of controls (normal), reduced compared to the majority of controls (reduced) or ectopic expression in the corresponding sister cell lineage (ectopic). For each gene marker, expression in control and electroporated embryos was similar. At the top of each bar on the graph is the total number of embryos analyzed. B) Trp expression at 12-row neurula stage. Each embryo half was scored independently. Data is presented as percentage of embryos with stronger expression in a10.97 compared to a10.98 (a10.97>a10.98), equal expression between a10.97 and a10.98 (a10.97=a10.98) and expression in row IV cells, which was always in the lateral column. C) The number of pigment cells counted in electroporated embryos following the same scheme as Fig.3.

Highlights.

  • Live-imaging of the melanocyte lineage during Ciona neurulation

  • The melanocyte lineage undergoes a previously unrecognized cell division

    Differential ERK1/2 activity during four successive A-P oriented cell divisions

  • Eph signal inhibition results in ectopic ERK activity and supernumerary pigment cells

Acknowledgments

We thank Nori Satoh and colleagues for the Ciona gene collection plates. We thank Filomena Ristoratore and Lionel Christiaen for communicating data prior to publication. N.H is supported by PhD fellowships from the Ministère de la Recherche (2009-2012) and the Fondation ARC pour la Recherche sur le Cancer (DOC20120604723; 2012-2013). The group of H.Y is supported by the Centre National de la Recherche Scientifique (CNRS), the Université Pierre et Marie Curie, the Fondation ARC pour la Recherche sur le Cancer (1144) and the Agence Nationale de la Recherche (ANR-09-BLAN-0013-01). P.B.A is supported by a graduate fellowship from the US National Science Foundation. Work in the laboratory of M.L is supported by a grant from the US National Institutes of Health (NS 076542).

Footnotes

Note added in proof:

Some of the findings reported in this article have also been reported elsewhere.

Claudia Racioppi, Ashwani K. Kamal, Florian Razy-Krajka, Gennaro Gambardella, Laura Zanetti, Diego di Bernardo, Remo Sanges, Lionel A. Christiaen and Filomena Ristoratore. Fibroblast Growth Factor signals sequentially govern nervous system patterning and pigment cell formation in Ciona intestinalis. Nature Communications. In Press.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Abitua PB, Wagner E, Navarrete IA, Levine M. Identification of a rudimentary neural crest in a non-vertebrate chordate. Nature. 2012;492:104–107. doi: 10.1038/nature11589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bertrand V, Hudson C, Caillol D, Popovici C, Lemaire P. Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors. Cell. 2003;115:615–627. doi: 10.1016/s0092-8674(03)00928-0. [DOI] [PubMed] [Google Scholar]
  3. Christiaen L, Jaszczyszyn Y, Kerfant M, Kano S, Thermes V, Joly J-S. Evolutionary modification of mouth position in deuterostomes. Semin Cell Dev Biol. 2007;18:502–511. doi: 10.1016/j.semcdb.2007.06.002. [DOI] [PubMed] [Google Scholar]
  4. Christiaen L, Wagner E, Shi W, Levine M. Electroporation of transgenic DNAs in the sea squirt Ciona. Cold Spring Harb Protoc. 2009;2009 doi: 10.1101/pdb.prot5345. pdb.prot5345. [DOI] [PubMed] [Google Scholar]
  5. Cole AG, Meinertzhagen IA. The central nervous system of the ascidian larva: mitotic history of cells forming the neural tube in late embryonic Ciona intestinalis. Dev Biol. 2004;271:239–262. doi: 10.1016/j.ydbio.2004.04.001. [DOI] [PubMed] [Google Scholar]
  6. Conklin EG. The organisation and cell lineage of the ascidian egg. J Acad Natl Sci Phila. 1905:1–119. [Google Scholar]
  7. Darras S, Nishida H. The BMP/CHORDIN antagonism controls sensory pigment cell specification and differentiation in the ascidian embryo. Dev Biol. 2001;236:271–288. doi: 10.1006/dbio.2001.0339. [DOI] [PubMed] [Google Scholar]
  8. Davidson B, Shi W, Beh J, Christiaen L, Levine M. FGF signaling delineates the cardiac progenitor field in the simple chordate, Ciona intestinalis. Genes Dev. 2006;20:2728–2738. doi: 10.1101/gad.1467706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guillemot F, Zimmer C. From cradle to grave: the multiple roles of fibroblast growth factors in neural development. Neuron. 2011;71:574–588. doi: 10.1016/j.neuron.2011.08.002. [DOI] [PubMed] [Google Scholar]
  10. Haupaix N, Stolfi A, Sirour C, Picco V, Levine M, Christiaen L, Yasuo H. p120RasGAP mediates ephrin/Eph-dependent attenuation of FGF/ERK signals during cell fate specification in ascidian embryos. Dev Camb Engl. 2013;140:4347–4352. doi: 10.1242/dev.098756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hébert JM. FGFs: Neurodevelopment’s Jack-of-all-Trades - How Do They Do it? Front Neurosci. 2011;5:133. doi: 10.3389/fnins.2011.00133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hotta K, Mitsuhara K, Takahashi H, Inaba K, Oka K, Gojobori T, Ikeo K. A web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva. Dev Dyn Off Publ Am Assoc Anat. 2007;236:1790–1805. doi: 10.1002/dvdy.21188. [DOI] [PubMed] [Google Scholar]
  13. Hudson C, Darras S, Caillol D, Yasuo H, Lemaire P. A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate, the ascidian Ciona intestinalis. Dev Camb Engl. 2003;130:147–159. doi: 10.1242/dev.00200. [DOI] [PubMed] [Google Scholar]
  14. Hudson C, Lotito S, Yasuo H. Sequential and combinatorial inputs from Nodal, Delta2/Notch and FGF/MEK/ERK signalling pathways establish a grid-like organisation of distinct cell identities in the ascidian neural plate. Dev Camb Engl. 2007;134:3527–3537. doi: 10.1242/dev.002352. [DOI] [PubMed] [Google Scholar]
  15. Hudson C, Kawai N, Negishi T, Yasuo H. β-Catenin-Driven Binary Fate Specification Segregates Germ Layers in Ascidian Embryos. Curr Biol CB. 2013;23:491–495. doi: 10.1016/j.cub.2013.02.005. [DOI] [PubMed] [Google Scholar]
  16. Ikeda T, Matsuoka T, Satou Y. A time delay gene circuit is required for palp formation in the ascidian embryo. Dev Camb Engl. 2013;140:4703–4708. doi: 10.1242/dev.100339. [DOI] [PubMed] [Google Scholar]
  17. Imai KS, Satoh N, Satou Y. Early embryonic expression of FGF4/6/9 gene and its role in the induction of mesenchyme and notochord in Ciona savignyi embryos. Dev Camb Engl. 2002a;129:1729–1738. doi: 10.1242/dev.129.7.1729. [DOI] [PubMed] [Google Scholar]
  18. Imai KS, Satou Y, Satoh N. Multiple functions of a Zic-like gene in the differentiation of notochord, central nervous system and muscle in Ciona savignyi embryos. Dev Camb Engl. 2002b;129:2723–2732. doi: 10.1242/dev.129.11.2723. [DOI] [PubMed] [Google Scholar]
  19. Imai KS, Hino K, Yagi K, Satoh N, Satou Y. Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. Dev Camb Engl. 2004;131:4047–4058. doi: 10.1242/dev.01270. [DOI] [PubMed] [Google Scholar]
  20. Imai KS, Levine M, Satoh N, Satou Y. Regulatory blueprint for a chordate embryo. Science. 2006;312:1183–1187. doi: 10.1126/science.1123404. [DOI] [PubMed] [Google Scholar]
  21. Imai KS, Stolfi A, Levine M, Satou Y. Gene regulatory networks underlying the compartmentalization of the Ciona central nervous system. Dev Camb Engl. 2009;136:285–293. doi: 10.1242/dev.026419. [DOI] [PubMed] [Google Scholar]
  22. Irvine SQ, Cangiano MC, Millette BJ, Gutter ES. Non-overlapping expression patterns of the clustered Dll-A/B genes in the ascidian Ciona intestinalis. J Exp Zoolog B Mol Dev Evol. 2007;308:428–441. doi: 10.1002/jez.b.21169. [DOI] [PubMed] [Google Scholar]
  23. Ivashkin E, Adameyko I. Progenitors of the protochordate ocellus as an evolutionary origin of the neural crest. EvoDevo. 2013;4:12. doi: 10.1186/2041-9139-4-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jiang D, Tresser JW, Horie T, Tsuda M, Smith WC. Pigmentation in the sensory organs of the ascidian larva is essential for normal behavior. J Exp Biol. 2005;208:433–438. doi: 10.1242/jeb.01420. [DOI] [PubMed] [Google Scholar]
  25. Kim GJ, Nishida H. Role of the FGF and MEK signaling pathway in the ascidian embryo. Dev Growth Differ. 2001;43:521–533. doi: 10.1046/j.1440-169x.2001.00594.x. [DOI] [PubMed] [Google Scholar]
  26. Lamb TD, Collin SP, Pugh EN., Jr Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nat Rev Neurosci. 2007;8:960–976. doi: 10.1038/nrn2283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lemaire P, Bertrand V, Hudson C. Early steps in the formation of neural tissue in ascidian embryos. Dev Biol. 2002;252:151–169. doi: 10.1006/dbio.2002.0861. [DOI] [PubMed] [Google Scholar]
  28. Nicol D, Meinertzhagen IA. Development of the central nervous system of the larva of the ascidian, Ciona intestinalis L. I. The early lineages of the neural plate. Dev Biol. 1988;130:721–736. doi: 10.1016/0012-1606(88)90363-6. [DOI] [PubMed] [Google Scholar]
  29. Nishida H. Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev Biol. 1987;121:526–541. doi: 10.1016/0012-1606(87)90188-6. [DOI] [PubMed] [Google Scholar]
  30. Nishida H, Satoh N. Determination and regulation in the pigment cell lineage of the ascidian embryo. Dev Biol. 1989;132:355–367. doi: 10.1016/0012-1606(89)90232-7. [DOI] [PubMed] [Google Scholar]
  31. Ohta N, Satou Y. Multiple signaling pathways coordinate to induce a threshold response in a chordate embryo. PLoS Genet. 2013;9:e1003818. doi: 10.1371/journal.pgen.1003818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Picco V, Hudson C, Yasuo H. Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos. Dev Camb Engl. 2007;134:1491–1497. doi: 10.1242/dev.003939. [DOI] [PubMed] [Google Scholar]
  33. Rothbächer U, Bertrand V, Lamy C, Lemaire P. A combinatorial code of maternal GATA, Ets and beta-catenin-TCF transcription factors specifies and patterns the early ascidian ectoderm. Dev Camb Engl. 2007;134:4023–4032. doi: 10.1242/dev.010850. [DOI] [PubMed] [Google Scholar]
  34. Sardet C, McDougall A, Yasuo H, Chenevert J, Pruliere G, Dumollard R, Hudson C, Hebras C, Le Nguyen N, Paix A. Embryological methods in ascidians: the Villefranche-sur-Mer protocols. Methods Mol Biol Clifton NJ. 2011;770:365–400. doi: 10.1007/978-1-61779-210-6_14. [DOI] [PubMed] [Google Scholar]
  35. Sato S, Yamamoto H. Development of pigment cells in the brain of ascidian tadpole larvae: insights into the origins of vertebrate pigment cells. Pigment Cell Res Spons Eur Soc Pigment Cell Res Int Pigment Cell Soc. 2001;14:428–436. doi: 10.1034/j.1600-0749.2001.140602.x. [DOI] [PubMed] [Google Scholar]
  36. Satou Y, Yamada L, Mochizuki Y, Takatori N, Kawashima T, Sasaki A, Hamaguchi M, Awazu S, Yagi K, Sasakura Y, et al. A cDNA resource from the basal chordate Ciona intestinalis. Genes N Y N 2000. 2002;33:153–154. doi: 10.1002/gene.10119. [DOI] [PubMed] [Google Scholar]
  37. Shi W, Levine M. Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo. Dev Camb Engl. 2008;135:931–940. doi: 10.1242/dev.011940. [DOI] [PubMed] [Google Scholar]
  38. Shi W, Peyrot SM, Munro E, Levine M. FGF3 in the floor plate directs notochord convergent extension in the Ciona tadpole. Dev Camb Engl. 2009;136:23–28. doi: 10.1242/dev.029157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shimauchi Y, Murakami SD, Satoh N. FGF signals are involved in the differentiation of notochord cells and mesenchyme cells of the ascidian Halocynthia roretzi. Dev Camb Engl. 2001;128:2711–2721. doi: 10.1242/dev.128.14.2711. [DOI] [PubMed] [Google Scholar]
  40. Squarzoni P, Parveen F, Zanetti L, Ristoratore F, Spagnuolo A. FGF/MAPK/Ets signaling renders pigment cell precursors competent to respond to Wnt signal by directly controlling Ci-Tcf transcription. Dev Camb Engl. 2011;138:1421–1432. doi: 10.1242/dev.057323. [DOI] [PubMed] [Google Scholar]
  41. Stolfi A, Wagner E, Taliaferro JM, Chou S, Levine M. Neural tube patterning by Ephrin, FGF and Notch signaling relays. Dev Camb Engl. 2011;138:5429–5439. doi: 10.1242/dev.072108. [DOI] [PubMed] [Google Scholar]
  42. Stuhlmiller TJ, Garcia-Castro MI. FGF/MAPK signaling is required in the gastrula epiblast for avian neural crest induction. Dev Camb Engl. 2012;139:289–300. doi: 10.1242/dev.070276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taniguchi K, Nishida H. Tracing cell fate in brain formation during embryogenesis of the ascidian Halocynthia roretzi. Dev Growth Differ. 2004;46:163–180. doi: 10.1111/j.1440-169X.2004.00736.x. [DOI] [PubMed] [Google Scholar]
  44. Tassy O, Dauga D, Daian F, Sobral D, Robin F, Khoueiry P, Salgado D, Fox V, Caillol D, Schiappa R, et al. The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program. Genome Res. 2010;20:1459–1468. doi: 10.1101/gr.108175.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tsuda M, Sakurai D, Goda M. Direct evidence for the role of pigment cells in the brain of ascidian larvae by laser ablation. J Exp Biol. 2003;206:1409–1417. doi: 10.1242/jeb.00235. [DOI] [PubMed] [Google Scholar]
  46. Veeman MT, Newman-Smith E, El-Nachef D, Smith WC. The ascidian mouth opening is derived from the anterior neuropore: reassessing the mouth/neural tube relationship in chordate evolution. Dev Biol. 2010;344:138–149. doi: 10.1016/j.ydbio.2010.04.028. [DOI] [PubMed] [Google Scholar]
  47. Wagner E, Levine M. FGF signaling establishes the anterior border of the Ciona neural tube. Dev Camb Engl. 2012;139:2351–2359. doi: 10.1242/dev.078485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Whittaker JR. Quantitative control of end products in the melanocyte lineage of the ascidian embryo. Dev Biol. 1979;73:76–83. doi: 10.1016/0012-1606(79)90139-8. [DOI] [PubMed] [Google Scholar]
  49. Yasuo H, Hudson C. FGF8/17/18 functions together with FGF9/16/20 during formation of the notochord in Ciona embryos. Dev Biol. 2007;302:92–103. doi: 10.1016/j.ydbio.2006.08.075. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplementary Movie 1.

Time-lapse movie of an embryo electroporated with Dmrt∷H2BYFP to label rows III-VI and ZicL∷H2BCFP to label rows I-IV. The movie begins just after the cells in row III have divided and runs until the pigment cell precursors intercalate at the midline. Image stacks were taken every 3 minutes for 2 hours at 21°C. Annotations refer to the cell lineage nomenclature at the tenth generation except for the final division of the pigment cells. The final division of a10.97 (labeled “97”) gives rise to a11.193 (labeled “193”) and a11.194 (labeled “194”). Similarly, cells a10.68 and a10.67 of row IV, previously described as postmitotic (Cole and Meinertzhagen, 2004), can also be seen dividing.

Download video file (65.9MB, mov)
02. Supplementary Figure 1.

Expression of Fgf, ephrin, FgfR and Eph3 at neural plate and neurula stages. A red dot indicates the pigment cell lineage on the schematics and a-line neural plate cells are colored in pink. The key indicates gene expression in 6-row neural plate cells. ephrinAc is not expressed at the 6-row neural plate stage, but is expressed in all animal cells at the early gastrula stage (Imai et al., 2004). FgfR and Eph3 are ubiquitously expressed during early gastrula and neural plate stages. Broad low levels of expression are also detected at the 12-row neural plate stage, with specific domains of stronger expression also detected, including in the a-line neural lineages. Strong Eph3 expression starts to be detected in the pigment cell lineage between the 6- and 12-row neural plate stage. This expression was dependent upon MEK activity; embryos were treated with U0126 from the 3-row neural plate stage. Arrowheads point to the nucleus of a9.49 in the middle panel and a10.97 in the lower panels. Some of this data overlaps with published studies (http://ghost.zool.kyoto-u.ac.jp/otherfr_kh.html; http://www.aniseed.cnrs.fr/; Hudson et al., 2007; Imai et al., 2004; Imai et al., 2009; Shi et al., 2009; Tassy et al., 2010; Wagner and Levine, 2012).

NIHMS616571-supplement-02.jpg (790.5KB, jpg)
03. Supplementary Figure 2.

Gene expression and pigment cell formation in embryos electroporated with Fog∷Eph3ΔC. A) Graphs show the expression of the genes indicated (above) in control and electroporated embryos. Embryos were scored following the key (below). The expression domains of each gene are shown in Figure 2. Embryos were scored for levels of expression similar to the majority of controls (normal), reduced compared to the majority of controls (reduced) or ectopic expression in the corresponding sister cell lineage (ectopic). For each gene marker, expression in control and electroporated embryos was similar. At the top of each bar on the graph is the total number of embryos analyzed. B) Trp expression at 12-row neurula stage. Each embryo half was scored independently. Data is presented as percentage of embryos with stronger expression in a10.97 compared to a10.98 (a10.97>a10.98), equal expression between a10.97 and a10.98 (a10.97=a10.98) and expression in row IV cells, which was always in the lateral column. C) The number of pigment cells counted in electroporated embryos following the same scheme as Fig.3.

NIHMS616571-supplement-03.jpg (760.1KB, jpg)

RESOURCES