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Neurofilament number and subunit composition, which are highly regulated during development, have been proposed to help regulate axonal diameter and stability. From experiments on dissociated cell cultures of Xenopus laevis embryonic spinal cord, we have obtained direct evidence that neurofilaments help maintain the structural integrity of newly developing axons. An anti-neurofilament monoclonal antibody specific for Xenopus NF-M and the cell lineage tracer, lysinated FITC-dextran, were coinjected into a single blastomere of 2-cell stage embryos. Within neurons descended from the injected blastomere, this antibody specifically confined neurofilaments to the cell body for the first two days of culture, as assayed by immunocytochemical staining with antiserum against the low molecular weight neurofilament protein XNIF. Although whole IgGs and Fab fragments both affected neurofilament distribution, the whole IgGs were more effective. For the first 9 hr of culture, neurites containing anti-NF-M developed normally. By 21 hr, they were shorter than those of sibling control neurons within the same dish, and many became morphologically abnormal. Defects included large variations in diameter, poorly defined separations between the growth cone and neurite, and more collateral branching. Despite these abnormal features, neurons containing anti-NF-M had normal distributions of alpha-tubulin immunoreactivity and phalloidin-stained F-actin. These latter observations argued that defects resulted from the absence of neurofilaments rather than from interference of the movement of other structural materials essential for axonal growth. These results support the hypothesis that neurons use neurofilaments to help maintain the characteristic shapes of axons against the increasing structural demands placed upon the elongating process.
Figure 1. SDS-polyacrylamidgee l electrophoresoisf anti-NF-M antibody
solutions. Lane I shows 18 kg of purified XClOC6 whole IgG
run under nonreducing conditions. Lane 2 shows 7 p,g of total protein
containing Fab fragments under nonreducing conditions. Although in
addition to the Fab frament some residual Fc is visible, the solution is
devoid of any detectable levels of either whole IgG or Fab,. The positions
of molecular weight standards are indicated on the left. They
are: myosin, 200 kDa; P-galactosidase; 116 kDa; phosphorylase B, 97
kDa; bovine serum albumin, 66 kDa; ovalbumin, 43 kDa; and carbonic
anhydrase, 31 kDa.
Figure 2. Tdentification of antibody-containing neurons in cultures, 21 hr after plating. Examples are shown of neurons descended from blastomeres
injected with anti-NF-M (A-C), anti-l3 tubulin (D-F), and anti-p galactosidase (G-I) monoclonal antibodies. The left three panels (A, 0, G) show
cells viewed by phase contrast microscopy. The middle three panels (B, E, H) show the same cells as in A, 0, and G, respectively, but viewed by
fluorescein fluorescence to visualize the FITC-dextran. The right three panels (C, F, I) were the same neurons as in B, E, and H, respectively, but
stained by rhodamine conjugated anti-mouse IgG and viewed by rhodamine fluorescence to visualize the distribution of the injected antibodies. The
scale is the same for all panels.
Figure 3. Accumulation of neurofilament protein immunoreactivity in neuronal cell bodies after injection of anti-NF-M monoclonal antibody. The
left three panels are cells seen through phase contrast opaics and the adjacent right three panels show the same cells stained by the anti-XNIF
antibody and viewed by immunofluorescence. A and B show a neuron descended from a blastomere injected with BSA. The XNIF immunoreactivity
in the cell body and neurite is distributed as in uninjected neurons. C-F show cells descended from blastomeres injected with the anti-NF-M
antibody. In these neurons, the XNIF immunoreactivity has accumulated in the soma (arrowheads in D and F) and is depleted from the neurites.
The scale bar in F indicates the scale for all of the panels.
Figure 4. The distribution of XNIF immunoreactivity in control antibody-injected neurons, 21 hr after plating. The left panels show phase contrast
views of neurons containing either anti-l3 tubulin (A) or anti-p galactosidase (C) monoclonal antibodies. The right panels show the same cell as in
A and C, respectively, stained by the anti-XNIF antibody and viewed by immunofluorescence. The XNIF immunostaining is distributed within the
cell bodies and neurites as in uninjected neurons. The scale, as indicated in D, is the same for all panels.
Figure 5. The effect of injection of Fab fragments of the anti-NF-M
antibody. B shows the distribution of Fab fragments in a neuron, 21 hr
after plating, that was descended from a blastomere injected with Fab
fragments. The distribution of Fab fragments is visualized by immunofluorescence
after the neuron was stained with anti-mouse IgG-Fab.
D shows the distribution of XNIF immunoreactivity in a neuron descended
from a blastomere injected with Fab fragments. XNIF immunoreactivity
is concentrated in the cell body (arrowhead) and absent
from the neurite. A and C show the same neurons as in B and D,
respectively, but viewed under phase contrast conditions. The scale bar
in D applies to all panels.
Figure 6. The distribution of a-tubulin immunoreactivity in neurons descended from an uninjected blastomere (A, B) andf romb lastomereinsj ected
with eithera nti-NF-M (C-F) or anti-P-tubulin(G , H) monoclonaal ntibodiesC. ells are from cultures,2 1 hr after platidg.A , C, E, andG show
neuronsp hotographetdh roughp hasec ontrasto ptics.B , D, F, andH showt heses amen euronss tainedw ith anti-cw-tubulainn dv iewedb y immunofluorescencTeh.
e scaleb ar in H indicatesth e scalef or all of the panels.
Figure 7. The distribution of F-a&n in a neuron descended from an
uninjected blastomere (A, B) and from a blastomere injected with anti-
NF-M antibody (C, D). A and C are photomicrographs taken with phase
contrast optics; B and D show the same neurons, respectively, stained
for F-actin with rhodamine-phalloidin and viewed by fluorescence.
Phalloidin staining of F-actin is most abundant in the growth cone (arrowhead).
Patches of staining seen along the neurites may indicate positions
where neurites are attached to the culture substrate. The scale,
indicated in D, is the same for all panels.
Figure 8. Effects of anti-NF-M antibody injection on neurite appearance. The left five panels (A-E) show neurons from control groups. These
include two uninjected neurons (A, B), and neurons descended from blastomeres injected with BSA (C), anti-e-tubulin (D) or anti-P-galactosidase
(E). The adjacent right panels (F-J) show neurons representative of the range of morphologies seen in cells containing anti-NF-M antibody. Whereas
neurons from control groups had long uniform neurites with well-defined growth cones, neurons filled with neurofilament antibody had neurites
exhibiting a variety of shapes. For example, the neuron in F is normal. The neurites of the neurons in G and H have uneven diameters and
abnormally long minor collateral processes emanating from the middle of the neurite. The neurites of the cells in I and J are aberrantly broad with
collateral processes ending in extensive growth cones. The scale bar in J applies to all panels.
