High affinity choline uptake by spinal cord neurons in dissociated cell culture.
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Acetylcholine↗
Biomedical subjects
K F Barald
Publications and source records attributed to K F Barald.
Monoclonal antibodies made to chick mesencephalic neural crest cells and to ciliary ganglion neurons identify a common antigen on the neurons and a neural crest subpopulation.
We previously reported the production of monoclonal antibodies (Mabs) that identified cell surface components of cultured chick and quail ciliary ganglion (CG) neurons and of a subpopulation of neural crest (NC) cells from 31-hr chick embryos (stage 9). Here we demonstrate that another Mab, CG-14, which was prepared to nitrocellulose-immobilized, lightly fixed (0.125% paraformaldehyde) mesencephalic NC cells from 31-hr (stage 9) chick embryos, labels the same antigen(s) recognized by CG-1 and CG-4 on both the CG neurons and the subpopulation of NC cells. All three Mabs label a polypeptide of 75 kD on Western blots of one-dimensional SDS-polyacrylamide gels. CG-14 blocked the binding of CG-1 and/or CG-4 to the 75 kD band on Western blots and blocked the binding of CG-1 and CG-4 to CG and NC cells. CG-1 and/or CG-4 antibodies, in turn, blocked the binding of CG-14 to Western blots, as well as NC and CG cells. We had previously shown that antibodies CG-1 and CG-4 were synergistically cytotoxic for the majority (95%) of cultured CG neurons in vitro in the presence of guinea pig complement. Here we show that the antibodies, which are both of the gamma 2a subclass, are also cytotoxic for the NC cells that they label in vitro. After the cells are ablated in culture, no other cells bearing the antigen(s) recognized by any of the three Mabs appear over a 2.5-week period. CG-14, however, is not cytotoxic for either the CG or NC cell populations alone or in combinations with CG-1 or CG-4. These results confirm our original observation that cultured CG neurons and NC cells share cell-surface antigen(s). The antigen recognized by all three Mabs appears to be the same whether the immunogen used to produce the antibodies was CG neurons or NC cells. This finding encourages us to continue tests of the hypothesis that the subpopulation of mesencephalic neural crest cells contributes to the formation of the ciliary ganglion in the embryo. Further characterization of the antigen appears in the accompanying paper.
Animals↗
Antigen recognized by monoclonal antibodies to mesencephalic neural crest and to ciliary ganglion neurons is involved in the high affinity choline uptake mechanism in these cells.
High-affinity choline uptake mechanisms are among the characteristics of cholinergic neurons such as the ciliary and choroid subpopulations in the ciliary ganglion (Barald and Berg, 1979). We have produced three monoclonal antibodies (Mabs), two of which were made to 8-day embryonic chick ciliary ganglion (CG) neurons (CG-1, CG-4) (Barald, 1982) and one of which was made to cultured mesencephalic neural crest (NC) cells (CG-14) removed from the embryo 31 hr after incubation. We have shown that all three Mabs label a common 75 kD antigen present on the cell surface of both CG neurons and NC cells (Barald, 1988). Here we report that the CG-1 and CG-4 antibodies, used in the same ratios in which they are synergistically cytotoxic for both the CG and NC cells (Barald, 1988), and Mab CG-14 alone, have specific effects on the high-affinity choline uptake mechanism (HACU) of CG neurons and isolated antigen-positive NC cells in the absence of complement. CG-1 and CG-4 in ratios of 8/1 (the same ratios that are used to kill the CG and the NC subpopulation), but neither singly, inhibit the HACU of CG neurons by 40% and that of isolated antigen-positive NC cells by 75%. However, CG-14 alone, at 1 microgram/ml, inhibits the HACU of both CG neurons and isolated NC cells by 95%. None of the antibodies had an effect on numbers of ouabain binding sites (a measure of the Na+/K+ ATPase) or cell surface acetylcholinesterase (AChE) of CG neurons or NC cells isolated by "no-flow" fluorescence cytometry with a Meridian Instruments ACAS470 cytometer. CG or NC cells grown in the presence of the antibodies without complement grow and remain healthy for many weeks. They exhibit no difference in morphology, protein content, lactate dehydrogenase activity (LDH), or division time from untreated sister cultures. Therefore, the antigen recognized by all three Mabs may be involved in a high-affinity choline uptake mechanism, a common characteristic of cholinergic neurons. The Mabs themselves may possibly label some element of the high-affinity transporter or a proximal membrane component. This implies that such a high-affinity uptake mechanism is present in the subpopulation of NC cells at early times in development. If these cells in fact are destined to contribute to the avian CG, these characteristics are present in the subpopulation before the NC cells take on a neuronal morphology.
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Expression of an engrailed-like gene during development of the early embryonic chick nervous system.
The engrailed gene has been identified in Drosophila as an important developmental gene involved in the control of segmentation. Here we describe the embryonic expression of a chicken gene, ChickEn (Darnell et al.: J Cell Biol 103(5):311a, 1986), which contains homology to the Drosophila engrailed gene. Northern blots of early chick embryo tissue poly(A)+ RNA resulted in hybridization to at least three bands expressed predominantly in the brain/head region when probed with ChickEn genomic fragments. Eight cDNA clones generated from embryonic day 6 (stage 29-30) chick brain poly(A)+ RNA are identical in their nucleotide sequence with the ChickEn genomic clone. In situ hybridization to sections of 4-day (stage 24) embryos indicated that ChickEn transcripts were concentrated in the posterior mesencephalon and anterior metencephalon. In cultures of chick cranial neural crest cells (eight to nine somites; stage 9) ChickEn transcripts were localized in a subset (approx. 8%) of cells examined after 2 days in culture. A mouse monoclonal antibody, inv-4D9D4, made by Coleman and Kornberg recognizes the engrailed-like homeo domain of the engrailed and invected proteins (Martin-Blanco, Coleman, and Kornberg, personal communication). Patel, Coleman, Kornberg and Goodman (unpublished) have shown that this antibody binds to the hindbrain of 2-day-old chick embryos. We have confirmed these results and shown that this antibody binds to the same region of 4-day (stage 24) chick brains that in situ hybridization showed contained ChickEn transcripts. This antibody also recognizes a homeo domain-containing ChickEn peptide expressed as a beta-galactosidase fusion protein in Drosophila cell culture. We have not detected ChickEn protein in any tissue prior to eight to nine somites (stage 9). These results delineate the major expression pattern of the ChickEn gene during early (prior to stage 30) embryonic development in the chick.
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Improved culture of individual muscle fibres with and without spinal cord explants in a collagen gel.
Suspension culture of single adult rat flexor digitorum brevis (FDB) muscle fibres in Vitrogen, a purified collagen, on tissue culture plastic or glass with mesh ring supports is superior to culture upon other substrates including collagen-, laminin-, or Vitrogen-coated tissue culture plastic. The Vitrogen gel-fibre mixture which attaches to glass or plastic provides at least 10 times more fibres per dish than does plating fibres on other substrates. Use of Vitrogen gel permits variable plating densities and the production of adequate numbers of cultures for long-term experimental comparisons of acetylcholinesterase (AChE) and rhodamine-alpha-bungarotoxin (RBTX) distribution on muscle fibres. Use of 40 micrograms/ml ovotransferrin (OT) instead of chick embryo extract in the culture medium significantly improves long-term survival. Cultured fibres, with or without the addition of ventral spinal cord explants. may also be examined with electrophysiological techniques.
Acetylcholinesterase↗
Expression of Pax2 and patterning of the chick inner ear.
Early regionalized gene expression patterns within the otocyst appear to correlate with and contribute to development of mature otic structures. In the chick, the transcription factor Pax2 becomes restricted to the dorsal and entire medial side of the otocyst by stage 16/17. The dorsal region of the otocyst forms the endolymphatic duct and sac (ED/ES), and the cochlear duct is derived from the ventromedial region. In the mouse, however, Pax2 expression is reported only in the ventromedial and not the dorsal otocyst. In Pax2 null mice, the cochlea is missing or truncated, but vestibular structures differentiate normally. Here we demonstrate that in the chick, the emerging ED/ES express high levels of Pax2 even when the position of the emerging ED is altered with respect to its environment, either by 180 degrees otocyst rotations about the anterior/posterior axis or transplantation of the otocyst into the hindbrain cavity. However, the Pax2 expression pattern is plastic in the rest of the otic epithelium after 180 degrees rotation of the otocyst. Pax2 is upregulated on the medial side (formerly lateral), and downregulated on the lateral side (formerly medial and expressing Pax2) indicating that Pax2 expression is influenced by the environment. Although Pax2 is upregulated in the epithelium after 180 degrees rotations in the region that should form the cochlear duct, cochlear ducts are truncated or absent, and the ED/ES emerge in a new ventrolateral position. Ablation of the hindbrain at the placode or early otic pit stage alters the timing of regionalized Pax2 expression in the otocyst. The resulting otocysts and ears are generally smaller, vestibular structures are abnormal, ED/ES are missing but cochlear ducts are of normal length. The hindbrain and dorsal periotic mesenchyme provide unique trophic and patterning information to the dorsal otocyst. Our results demonstrate that the ED is the earliest structure patterned in the inner ear and that the hindbrain is important for its specification. We also show that, although normal Pax2 expression is required for cochlear duct development, it is downstream of ventral otocyst patterning events.
Animals↗