Fig. 2. Differentiation of the MP4-6 and MNB lineages in the presence of a glial surround. (A-D) 31% neuromere. MP4-6 progeny, MNB progeny, the MNB, and glial nuclei are shown in dorsal, intermediate and ventral levels of a confocal series (A-C). Labeling is by propidium iodide (blue) to reveal nuclei, anti-HRP (green) and anti-Lachesin (red). A schematic shows projected outlines of midline neuronal progeny and of glial nuclei (D). One sibling each (4d, 5d) of the MP4 and MP5 progeny is dorsal and anterior to MP6 progeny (6, 6) (A). The other two (4v, 5v) are ventrally adjacent to their sibling partners (B). The four MP4 and MP5 progeny have punctate HRP labeling (A,B). The more ventral siblings will remain En positive, and mature slightly before their more dorsal siblings, and have more conspicuous HRP labeling. MP6 progeny (6, 6) are apposed with Lachesin label between, and have the first signs of HRP labeling (A). The MNB (C) has produced three GMCs (A,B). The first GMC has produced two GCs, which are apposed and surrounded by Lachesin label (stars, A,B). The second and third GMCs (GMC2, GMC3) are more ventral (B), but dorsal to the MNB (C). The MNB is surrounded by glia (g), including the cap cell (cc) (C). Relative positions of cells of A-C are shown outlined (D). (E-H) 32% (E,F) and 33% neuromeres (G,H). En is downregulated in one sibling each of the MP4, MP5 and MP6 progeny, whereas En is upregulated in GMCs from the MNB. Labeling was by anti-En (red) and anti-HRP (green). In a 32% neuromere, one sibling each (4d, 5d) of the MP4 and MP5 progeny is dorsal, and anterior to MP6 progeny (6, 6) (E). The other two (4v, 5v) are ventrally adjacent to their sibling neurons (F). The more dorsal neurons (4d, 5d) have lowered En expression (E) but the more ventral siblings (4v, 5v) maintain high levels (F). MP4 and MP5 progeny have punctate HRP labeling (E,F), and growth cones of the MP4 progeny (4d, 4v) are evident at the anterior of the group (arrow, E). MP6 progeny (6, 6) show the first signs of HRP-labeling and have slight differences in En, with one (right) having the lower level (E). The first three GMCs of the MNB lineage are posterior to MP6 progeny and also relatively dorsal (E). GMC1 is about to divide, evinced by chromosomal shape, and has the highest level of En of the GMCs (E). The younger GMCs (GMC2, GMC3) each have yet lower levels of En. The MNB is ventral to the GMCs (F). En-positive glia nuclei (g) surround the MNB (F). In a 33% neuromere, the six MP progeny have punctate HRP labeling, and En downregulation is evident in the more dorsal neuron of each sibling pair (G,H). The 4d neuron has attained its final En-negative state, the 5d neuron has marginal En expression and the 6d neuron has conspicuously lower En expression than does its 6v sibling. The ventral siblings, 4v, 5v and 6v, maintain a high level of En expression (G,H). Of the MNB progeny, GMC1 has divided to produce two En-positive GCs (star), and the younger GMCs (GMC2, GMC3) are also En-positive (G). Glia nuclei (g) surround the MNB (H). (I-L) En is upregulated as GMCs mature during early (I-K) and late (L) embryogenesis. Labeling is by anti-En (red) and propidium iodide (blue) or anti-HRP (green). In a 36% neuromere, the dorsal siblings (4d, 5d, 6d) are En negative (I,J) but the ventral siblings (4v, 5v, 6v) continue to express En (J,K). Of the MNB progeny, two GCs more dorsal are En-negative (star, I), whereas their more ventral sibling GCs are En positive (star, J). Six GMCs (two marked with white dots) show variable levels of En expression (I,J) with the oldest and most dorsal GMCs having the highest En expression (I). MNB is ventral to its GMC progeny and is En negative, whereas surrounding glial nuclei (g) are En-positive (K). In a 55% neuromere, upregulation of En is evident in GMCs (white dots) in the MNB lineage (L). The MNB, which is more ventral to the GMCs, is En negative. (M-O) Glia surround the MP lineages and the MNB lineages. Labeling was by anti-Annulin (red), anti-HRP (green) and propidium iodide (blue). In a 27.5% neuromere, Annulin, a glial marker, surrounds the MP4 neurons (4, 4), MP5 and MP6 (M). In a 31.5% neuromere, Annulin labeling is extensive, and surrounds the dividing MNB (N). Other NBs and their associated GMCs are also surrounded by Annulin, only a few of which are seen at this dorsoventral level [e.g. NB 1-1 clusters; MP3 neurons (3, 3)]. In a 43% neuromere, the Annulin surround includes the MNB group as well as its mature neuronal progeny (O). Scale bars: 10 µm.