Fig. 3. Development of midline bundles. (A-E) 32% neuromere. Labeling is by propidium iodide (blue), anti-HRP (green) and anti-8B7 (red). Two midline growth cones extend anteriorly between two glia (g), one growth cone from 4v (left) and one from 4d (right) (A-C). Continuous and strong 8B7 label occurs at the core of the growth cones and around cell bodies, whereas HRP-label also marks the entirety of the growth cones from the 4d and 4v neurons (A,B). 8B7 label is faint and discontinuous on neuron 5d and HRP label is only weakly developed (A,B), as is also true for sibling neuron 5v (ventral to plane of scan). 8B7 labeling of 4d and 4v neurons was traced through all sections as shown on the schematic (C). 8B7 labeling of the 4v neurite is traced in white (left) and labeling of the 4d neurite is traced in magenta (right). The MNB and the MNB-associated glia are ventral to the plane of section. A lateral neuron has strong 8B7 label around its cell body (vertical arrow) and at the core of its relatively short growth cone (horizontal arrow) (D). HRP label (not shown) was comparable to that of the 4d neuron in A. Another and older lateral neuron has a well developed primary neurite (arrows), shown in both upper and lower panels (E). Upper panel shows all three labels used; lower panel shows confocal channels for anti-8B7 and PI labels only. 8B7 label is strong within the core of the neurite, but barely evident around the cell body (upward arrows). HRP label is strongly maintained on both the cell body and the primary neurite. (F) 31.5% neuromere. The midline fiber tract is just forming. The 4v neuron has an anteriorly extended growth cone (gc), with broad lamellipodium and filopodia. Its trajectory is towards other growth cones (upward oblique arrow) from lateral neurons that pioneer the posterior commissure (PC). HRP-label also shows part of the incipient anterior commissure (AC) and progeny of MP3 (3, 3). Annulin labeling reveals the glial surround; some glial nuclei (g) are indicated. Labeling is by propidium iodide (blue), anti-HRP (green) and anti-Annulin (red). (G-K) Neurites of MP4-6 progeny comprise two bundles, one dorsal, which branches at the AC, and one ventral, which branches at the PC. Panels show dorsal neurons (G,I), ventral neurons (H,J) or both (K). In a 35.5% neuromere (G,H), the three dorsal MP neurons are En-negative, and have midline neurites that cross the PC and extend anteriorly to branch bilaterally (oblique arrows) at the AC (G). The three ventral MP neurons are En positive, and have midline neurites that branch bilaterally (oblique arrows) at the PC (H). Labeling is by anti-8B7 (green) and anti-En (red). In a 38% neuromere (I-K), the dorsal and ventral bundles also have different mediolateral positions. The 8B7 label is shown by turquoise in the more dorsal panel (I) and by magenta in the more ventral panel (J), or combined (K). In the more dorsal panel (I), neurites (horizontal arrows) of the dorsal neurons (4d, 5d, 6d) extend to the AC, where they branch bilaterally (oblique arrows). In the more ventral panel (J), neurites of the ventral neurons (4v, 5v, 6v) extend only to the PC, where they branch bilaterally (oblique arrows). In both panels, the more posterior cells are MNB progeny. (L-N) The median nerve develops separately. Labeling was by anti-8B7. All panels show the encompassing neuronal framework in flat gray; each panel also shows in black the 8B7 labeling at only one dorsoventral level. In a 38% neuromere, the most dorsal panel shows the developing median nerve (L). The Y-shaped figure at the anterior (oblique arrows) is formed by neurites of spiracle motoneurons. The neurites meet at the midline just posterior to the PC and travel posteriorly in a bundle that is dorsal to, and separate from, the midline fiber tract. The horizontal neurites at the posterior (upward vertical arrows) comprise the peripheral portions of the incipient median nerve. The neurites have reached the midline, and in later stages will grow anteriorly to meet the neurites of the spiracle motoneurons and form the median nerve. Two successively more ventral panels show the dorsal bundle of the midline fiber tract (horizontal arrows) (M), and the ventral bundle of the midline fiber tract with bilateral branches in the PC (downward vertical arrows) (N). Scale bar: 10 µm in A-K, 20 µm in L-N.