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Midline lineages in grasshopper produce neuronal siblings with asymmetric expression of Engrailed

Department of Biology and Graduate Program in Neuroscience, Emory University, Atlanta, GA 30322, USA

View larger version (131K): [in a new window]   Fig. 1. Midline precursors MP4, MP5 and MP6, and the MNB originate sequentially from the En-positive midline regions. Labeling was by anti-En (red) and anti-HRP (green). (A) 28% embryo shows the pattern of En expression and the progression of neuronal differentiation across neuromeres. Neuromeres posterior to T3 are successively younger by about 1%. (B-G) Sequence of MP and MNB differentiation in 25-29% neuromeres. Stars indicate MP2 progeny. (B,C) 25% neuromere with MP4 and incipient MP5. (B) A relatively dorsal scan; (C) The scan in B together with underlying En-positive region. MP4 is the largest and most strongly En-positive cell at the midline. The incipient MP5 (arrow) is just posterior to MP4. (D) 26% neuromere. MP4 and MP5 express high levels of En. The incipient MP6 (arrow) lies ventral to MP5. (E) 27% neuromere. MP4 has divided to form two progeny (4, 4), anterior to MP5. (F) 28% neuromere. MP4 progeny (4, 4) have strong HRP label, indicating neuronal differentiation and one of the two siblings (on left) has decreased En expression. MP5 has divided to give rise to two progeny (5, 5). MP6 and the MNB with associated cap cell (cc) are side-by-side and posterior in the cluster. (G) 29% neuromere. MP6 has divided to give rise to two progeny (6, 6). One of the two MP4 progeny (4d) is more dorsal than its sibling and has decreased En expression. MP5 progeny (5, 5) are beginning neuronal differentiation judged by HRP-labeling, but MP6 progeny (6, 6) are yet unlabeled. The MNB is ventral to MP6 progeny. Scale bar: 100 µm in A; 15 µm in B-G.

View larger version (103K): [in a new window]   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.

View larger version (27K): [in a new window]   Fig. 4. Midline precursors and GMCs each produce one En-positive and one En-negative neuron. (A) En expression in neurons from MP4, MP5 and MP6, in 28% to 35% neuromeres (n=36). Vertical bars show the number of neurons (left y-axis) in individual neuromeres as the sum of neurons with no detectable En (white) and at least some En expression (gray). Downward arrows indicate neuromeres having one additional MP sibling that had not yet differentiated neuronal markers (not included in bar total). Chart bars are rank-ordered, first by the total neurons present, and then by the number of En-positive neurons. Filled circles above each bar indicate percent development of neuromere in which neurons were tallied (right y-axis). (B,C) Population curves for midline neurons in T3 (B) and A1 (C) neuromeres, including neurons from MP4-6 and MNB lineages. En-positive neurons (black circles) and En-negative neurons (white circles) are equal in number through 45% embryogenesis. After 45%, the number of En-positive neurons continues to increase, but the number of En-negative neurons remains steady or decreases. (D,E) Embryos injected with BrdU at 35% (D) or 55% (E) were allowed to develop a further 48 hours (see text). Data obtained from T3 and A1 neuromeres were pooled, as there was no population difference. For injections at 35%, n=22 neuromeres counted, of which 11 were T3, and 11 were A1. For injections at 55%, n=17 neuromeres counted, of which 10 were T3, and 7 were A1. BrdU-labeled neurons were counted by type, and the number of En-positive neurons in each neuromere (x-axis) was plotted against the number of En-negative neurons in the same individual neuromere (y-axis). Where more than one neuromere had identical counts of the two types, the points are annotated by that number.

View larger version (110K): [in a new window]   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.

View larger version (102K): [in a new window]   Fig. 5. Birth and death of En-positive and En-negative neurons. (A,B) Birth of neurons. The embryo was injected with BrdU at 35% and dissected after a further 48 hours (45% embryogenesis). Labeling was by anti-HRP (green), anti-BrdU (blue) and anti-En (red). The MNB, GMCs, GCs and the neurons born during the injection period are BrdU positive. In A, a T3 neuromere, examples of BrdU-positive neurons that have downregulated En (En-negative) are marked by single stars; those that have some level of En are marked by double stars. Two neurons born prior to BrdU injection are also marked: one En negative (En-) and the other En positive (En+). The horizontal arrow indicates a dividing GMC. In B, an A1 neuromere, sibling BrdU-positive neurons are marked: one is En negative (single arrow, left); the other is En positive (double arrow, right). (C,D) Death of neurons. Pyknotic profiles shown in DIC images, with superimposed labeling from anti-HRP (green) and anti-En (red). (C) Pyknotic profiles at 70% in T3 (En positive, black arrows; En negative, white arrow); (D) pyknotic profiles at 55% in A1 (En negative, white arrows). Scale bar: 20 µm.

View larger version (18K): [in a new window]   Fig. 6. Summary of MP4, MP5, MP6 and MNB lineages.