EFFECTS OF MONOCLONAL ANTIBODIES TO JUNCTIONAL PROTEIN ON DEVELOPMENT OF LENS IN CHICKEN EMBRYO
Li Xin-ren,Lin Wen-na,Yu Qi-xiang,Qin Guo-qiang,Zhang Fei-chang,Jiang Jian-ming,Yao Zhong-jie,Liu Jun-hong,Steven McLoon and Ross Johnson (Dept.of Biology,Nanjing Univ.,Nanjing,P.R.of China,Dept.of Cell Biology and Neuroanatomy,and Dept,of Genetics and Cell Biology,Univ.of Minnesota,Twin Cities,USA)
We are interested in using specific im-
munological probes to examine the role of gap
junctions in development.In this study,three
monoclonal antibodies (mcAbs),termed ND 6,
NC 6 and NB 2,were produced by immuniza-
tion with chicken lens fiber cell membranes.
These membranes,rich in the junctional pro-
tein MP 28,had first been proteolyzed to re-
move the cytoplasmic determinants and then
acid treated to expose antigenic sites on the
extracellular surface.Western Transfer Ana-
lysis shows that these mcAbs bind speci-
fically to the major 28-KD protein (i.e.
MP 28) and also recognize the 24-KD and
21-KD fragments of MP 28 (in addition,NC6
recognizes 16-KD fragments too) (Fig.1.).
The epitope(s) for the three antibodies,which
appear to be similar,can not be distinguished
from the epitope for another mcAb,A 4,
which is believed to be specific for an extra-
cellular domain on MP 28.Antibodies of
this type could conceivably block the invol-
vement of MP 28 in the formation of junc-
tions between lens fiber cells,with subse-
quent effects on cell function.
Injections of the ND 6 or NC 6 mcAbs
(ca.1—2 μl of the conditioned hybridoma
media with mcAb) into the eyes of 20th
stage chicken embryos were carried out and
heads were fixed after 24 or 36 hours.The
effects of the mcAb injections have been eva-
luated by examining sections with routine
light microscopy.Two types of results have
been obtained.First,lens size has been deter-
mined with respect to three dimensions (i.e.
lens diameter and the length and width of
cross-sectional profiles).In all cases,lenses
from injected eyes have been compared with
lenses from uninjected eyes in the same em-
bryos.With NC 6,it was found that the len-
ses were significantly larger (P0.01) in all
three respects than the opposite,uninjected
control lenses.In lenses from eyes injected
with control media (media without mcAb),
no significant change was detected in any of
the three dimensions (Table 1.).Second,it
was found that the lenses were marked by
loose cell organization (Plate Ⅰ,Figs.1 and
??2) in many profiles of eyes injected with ND
6 or NC 6.In addition,there was a tendency
for the nuclei to lack the typical elongated
shape of the controls,and to display instead
round nuclear profiles (Plate Ⅰ.Figs.3 and
4).Studies with electron microscopy are in
progress and preliminary observations are
similar to those with the light microscopy.
Since the intracellular injection of gap
junction antibodies has been reported to affect
the formation of junctions between embryonic
cells,it is possible that junction assembly
has been inhibited in the studies reported here.
Work is in progress to answer this question.
If the antibodies reduce the formation of jun-
ctions,a resulting change in intercellular co-
mmunication could influence various cellular
processes.One example is cellular differentia-
tion.The loose organization of the fiber
cells in lenses treated with antibodies in this
study could reflect another change of this
type.Alterations in cell growth could also
occur,relating to the variations in lens size
observed here.It appears that this embryonic
system will permit a detailed analysis of im-
portant questions regarding the role of gap
junctions in growth and development.
CAJViewer7.0 supports all the CNKI file formats; AdobeReader only supports the PDF format.