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.