NB 2-2 Details
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NB 2-2 delaminates as an S2 NB.
In grasshopper, at least two of the motoneurons that are derived from NB 2-2 may have been described in grasshopper studies of neuromuscular pathfinding (Ball et al, 1985). Ball et al (1985) examined the role of muscle pioneers in specifying target selection; they identified two excitatory motoneurons that innervate the simple coxal muscle 133a, Df (fast) and Ds (slow). Initially, they used Lucifer Yellow to back-fill Df growth cones at 40% embryonic development; after identifying Df and Ds in this way, they looked in progressively younger embryos, working backward to a time point when these neurons were still dye-coupled to the NB that gave rise to them. They identified this NB as NB 2-2. Df and Ds were observed to be sibling motoneurons derived from the first GMC in the 2-2 lineage. However, it is not clear that the motoneurons Df and Ds, derived from Schistocerca NB 2-2 are the homologs of the motoneurons derived from Drosophila NB 2-2 (see below).
In Drosophila, NB 2-2 delaminates from a medial Row 2 cluster of neurectodermal cells that expresses huckebein (hkb); it is itself hkb+ until at least embryonic stage 12 (Broadus et al, 1995; Bossing et al, 1995; McDonald and Doe, 1997). It also expresses mirror-lacZ (mrr-lacZ) as it delaminates (Broadus et al, 1995; McNeill et al, 1997), and adds castor (cas), Klumpfuss (Klu) and seven-up-lacZ (svp-lacZ) expression only after at least two rounds of cell division have been completed (Cui and Doe, 1992, 1995; Yang et al, 1997; Broadus et al, 1995). We scored runt expression in S1, S2, and S3 neuroblast stages (but not S4 and S5), and found that NB 2-2 does not express runt (Doe, 1992). A recent paper reports NB 2-2 to be runt-positive as it delaminates, and throughout the rest of neurogenesis (Dormand and Brand, 1998). Each study used a different antibody, and thus different expression patterns may be due to each antibody recognizing different runt epitopes. Differences may also be due to a greater sensitivity of one antiserum compared to the other, or due to mistakes in scoring neuroblast identities.
The 2-2 lineage has been thoroughly described in a number of Drosophila studies. Bossing et al, 1995 first published the lineages of NB1-1 and NB 2-2 in order to examine the role of huckebein in glial development. The lineage was also described by Bossing et al (1996) as consisting of 2 or 3 Segmental Nerve motoneurons and 10 to 12 interneurons; they also described thoracic 2-2 clones as producing the SPG-A cell, produced in the abdomen by 1-1 clones.
A. Motoneurons:
In both abdominal and thoracic segments we detect 3-4 motoneurons projecting ipsilaterally out the SNa to innervate muscles 21 and 22 (consistent with Landgraf et al., 1997, but unlike Bossing et al., 1995, 1996). In addition, we usually see a projection out the SNd, innervating muscle 17 (Fig. 2-2G). We also confirm the results of Sink and Whitington (1991a) showing that motoneurons projecting to muscles 21 and 22 have substantial dendritic arborizations in the ipsilateral longitudinal connectives (Fig. 2-2 B,D small arrows), and that all abdominal NB 2-2 clones include these motoneurons. Thus, if these Drosophila motoneurons are homologs of the Schistocerca Df and Ds motoneurons, they either die or acquire functions independent of leg muscle innervation in abdominal segments.
B. Interneurons:
In 25% of NB 2-2 clones, 2 "spacer" cells were seen; they separated themselves from the remaining cells of the cluster and migrated medially toward the midline. In another 25%, there was only 1 such spacer cell. In the remaining 50% of these clones, we were not able to distinguish these cells. Sibling interneuronal axons crossing the midline in the posterior fascicles of the anterior commissure contacted these cells as they entered the commissure (see Fig 2-2). We believe this arrangement to reflect a pathfinding technique for navigating the midline and find it commonly (see Fig 1-2, 2-4,2-5, 3-4,4-2, 5-2, 5-3, 6-2, and 7-1).
Until stage 16, interneuronal projections from this population of interneurons appeared to be fairly uniform in all segments, except that anteriorly projecting intersegmental projections weren't seen in thoracic segments (see Fig 2-2). At stage 17, local and intersegmental interneuronal projections separate themselves from one another. Intersegmental interneurons send axons in both anterior and posterior directions, in the medial fascicles of the contralateral longitudinal connectives, very rapidly. At stage 16, only local projections are apparent; two hours later, these projections have extended three segments (see Fig 2-2). At stage 17, we also observed a complex meshwork of interneuronal endings to form in the contralateral longitudinal connectives. (see Fig 2-2). We found short ipsilateral projections to be present in every segment. Bossing et al (1995, 1996) described these as being specific to abdominal clones. We also found substantial dendritic arborizations from NB 2-2 motoneurons in the ipsilateral longitudinal connectives (see Fig 2-2).
C. Glia:
Consistent with previous descriptions of this lineage, we too observed a single sub-perineurial glial cell in thoracic 2-2 clones (see Fig 2-2, inset). Bossing et al(1996) are able to distinguish this sub-perineurial cell as being SPG-A, as SPG-B is absent in the thorax (Klaembt and Goodman, 1991).
D. Leg Innervation in the Fly:
In the grasshopper metathoracic ganglion, there are nine motoneurons innervating the musculature of the leg. Two of these motoneurons are derived from NB 2-2. They are known as Df and Ds and innervate the coxal muscle 133a (Ball et al 1985). Truman et al (1993) summarized several anatomical and BrdU labeling studies in the fly and observe that the adult fly's leg motoneurons are born during embryonic development, but then wait for larval leg-disk eversion before pioneering leg innervation during later development. This suggests that in the embryonic fly, several large cells are born that do not extend axons. We believe three such cells to derive from NB 2-2's direct neighbor, NB 2-3, which we find to generate three large cells in thoracic segments only. Other candidates for leg motoneurons derive from thoracic 1-1 lineages, which generate two large axonless cells adjacent to aCC and pCC in thoracic segments (see Fig 1-1). Because we don't know what complement of cells derives from the grasshopper NB 2-3, we cannot really speculate on whether or not the motoneurons derived from Drosophila NB 2-2 correspond to the leg motoneurons Df and Ds. There is, as yet, no data on the fate of Drosophila embryonic leg motoneurons during metamorphosis; we don't know whether they are born in every segment and degenerate in abdominal segments, or whether they are generated in a segment specific manner. It is also possible that the NB 2-2 and the NB 2-3 lineages both generate leg motoneurons by different mechanisms.
References:
Ball, E. E., Ho, R. K., and Goodman, C.S. (1985). Development of neuromuscular specificity in the grasshopper embryo: guidance of motoneuron growth cones by muscle pioneers. J. Neurosci 5(7): 1808-19.
Broadus, J., Skeath, J.B., Spana, E. P., Bossing, T., Technau, G.M., and Doe, C.Q. (1995). New neuroblast markers and the origin of the aCC/pCC neurons in the Drosophila central nervous system. Mech Dev 53: 393-402.
Bossing, T., Technau, G. M., and Doe, C.Q. (1995). Huckebein is required for glial development and axon pathfinding in the NB 1-1 and NB 2-2 lineages in the Drosophila central nervous system. Mech Dev 55: 53-64.
Bossing, T., Udolph, G., Doe, C. Q., and Technau, G. M. (1996). The Embryonic CNS lineages of Drosophila melanogaster I. Neuroblast lineages derived from the ventral half of the neurectoderm. Dev Biol 179: 41-64.
Cui, X., and Doe, C.Q. (1992). ming is expressed in neuroblast sublineages and regulates gene expression in the Drosophila central nervous system. Development 116(4): 943-52.
Cui, X., and Doe, C.Q. (1995). The role of the cell cycle and cytokinesis in regulating neuroblast sublineage gene expression in the Drosophila CNS. Development 121(10): 3233-43.
Doe, C. Q. (1992). Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system. Development 116: 855-863.
Dormand, E.L., and Brand, A.H. (1998). Runt determines cell fate in the Drosophila embryonic CNS. Development 125(9):1659-67.
Klaembt, C., and Goodman, C.S. (1991). The diversity and pattern of glia during axon pathway formation in the Drosophila embryo. Glia 4(2): 205-13.
Landgraf, M., Bossing, T., Technau, G. M., and Bate, M. (1997). The origin, location and projections of the embryonic abdominal motoneurons of Drosophila melanogaster. J. Neurosci 17(24): 9642-55.
McDonald, J., and Doe, C. Q. (1997). Establishing NB specific gene expression in the Drosophila CNS: huckebein is activated by wingless and hedgehog and repressed by engrailed and gooseberry. Development 124: 1079-87.
McNeill, H., Yang, C.H., Brodsky, M., Ungos, J., and Simon, M.A. (1997). Mirror encodes a novel PBX-class ofhomeoprotein that functions in the definition of the dorsal-ventral border in the Drosophila eye. Genes Dev 11(8): 1073-82.
Sink, H., and Whitington, P. (1991a). Location and connectivity of abdominal motoneurons in the embryo and larvae of Drosophila melanogaster. J. Neurobiol 22: 298-311.
Sink, H., and Whitington, P. (1991b). Pathfinding in the central nervous system and periphery by identified embryonic Drosophila motor axons. Development 112(1): 307-16.
Truman, J., Taylor, B. J., and Awad, T. A. (1993). " Formation of the Adult Nervous System." in The Development of Drosophila Melanogaster Cold Spring Harbor Laboratory Press: pages 1245-1276.
Udolph, G., Prokop, A., Bossing, T., and Technau, G.M. (1993). A common precursor for glia and neurons in the embryonic CNS of Drosophila gives rise to segment specific lineage variants. Development 118: 765-775.
Weiss, J., VonOhlen, T., Mellerick, D., Dressler, G., Doe, C. Q., and Scott, M.P. (1998). Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity. Genes Dev 12:3591-3602.
Yang, X., Bahri, S., Klein, T., and Chia, W. (1997). Klumpfuss, a putative Drosophila zinc finger transcription factor, acts to differentiate between the identities of two secondary precursor cells within one neuroblast lineage. Genes Dev 11(11):1396-1408.