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). are colored in pink. The key indicates gene expression in 6-row neural plate cells. 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). and 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 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 (791K) GUID:?CC0CB34B-2CE5-4DA3-8CD1-F2ED0633D618 03: Supplementary Figure 2 Gene expression and pigment cell formation in embryos electroporated with expression at 12-row neurula stage. Each NVP-BAG956 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 (760K) NVP-BAG956 GUID:?E1D675FD-D001-4675-B827-4D9C86A8B7BF 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 embryos. The NVP-BAG956 neural precursors undergo NVP-BAG956 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 NVP-BAG956 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. (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. TMOD3 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,.