INFLUENCE OF RATINAL BASEMENT MEMBRANE ON GROWTH OF NERVE FIBER FROM RETINAL EXPLANT OF CHICK EMBRYO
Liu Li (Shanghai Institute of Cell Biology,Academia Sinica)
During the development of chick embryo,the ganglion cells of retina extend out axons with terminal growth cones to their targets.Growth capability of the nerve fibers is likely to be regulated by intrinsic neuronal genetic programs as well as by signals from the ex-tracellular environment.The signals include the soluble components,such as growth fac-tor,and the extracellular matrix.The base-ment membrane is a kind of extracellular matrix,which is widely believed to serve as conduits or pathway to orient outgrowing nerve fibers and guide them to their targets.As yet the research about the growth of ner-ve fiber using basement membrane (BM) as a substrate in vitro has not reported. Our previous study indicated that peanut agglutinin receptor (PNA-R) within the extra-cellular spaces of visual system might be rela-ted to growth and guidance of nerve fibers of the retina.In this paper we attempted to study the growth rate and growth pattern of nerve fibers from retinal explant on BM sub-strate and their relations to PNA-R of BM.The retina was spreaded on the black mem- brane filter,with the ganglion cell layer up-wards.By being obsorbed on the poly-L-lysine coated petriperm dish,BM could be separated from the retina of 6-day old chick embryo.A stripe of retina with membrane filter was explanted and put on BM.Acoord-ing to the explant position and orientation on BM,the explant was divided into two types: V-F type (ventral side of explant stripe towards fissure of BM) and D-F type (dorsal side of explant stripe towards fissure of BM).Schematic drawing of the method was showed in Fig.1. We found that the lengths of the fibers growing from ventral side of retinal explant were longer than those from dorsal side when cultured on collagen gel for 24 hrs (PLATE I,Fig.1; Table 1).The lengths of fibers growing from ventral side of retinal explant on BM in V-F type were longer than those in D-F type.The growth of nerve fibers of retinal explant on BM was much better than that on collagen gel and on poly-L-lysine,which indicated that BM was a optimal sub-strate for the growth of ganglion nerve fi-bers. The length of nerve fiber on BM after cultured for 24 hrs could reach 1 mm long.They changed very little when pretreatment of BM with PNA and anti-PNA antiserum or with anti-laminin antiserum in exception with trypsin.Pretreatment of BM with neuramini-dase decreased the fiber length by 30%. The growing pattern of nerve fiber on BM or on collagen gel was almost parallel to each other.Sometimes the growing of nerve fiber orientated according to centripetal form on whole mount of chick retina (PLATE I,Fig.2).Nerve fiber prefered to grow on BM than to grow on the substrete of poly-L-lysine (PLATE I,Fig.3 and 4). The growth pattern was changed by pre-teatment of BM with PNA and anti-PNA.However no changes could observed when pre-teated with anti-laminin (PLATE Ⅱ.Fig.5,7 and 9).If PNA was added to culture medium (100μg/ml) to bind the PNA-R on the nerve fibers.The growth pattern within 24 hrs changed from a straight bundle form into a network form (PLATE I,Fig.6 and 8).However,WGA of same concentration showed no such effect (PLATE I,Fig.10).And ConA inhibited the nerve fiber growth. Our present results demonstrated that BM promoted and oriented the growth of nerve fiber of retinal explant from chick embryo.Blockade of laminin with antibody did not change the growth length and pattern of nerve fiber,but the growth pattern was changed when adding PNA or anti-PNA in addition.It was suggested that many molecules,which had PNA-R such as laminin,fibronectin,and other glycoproteins as well as proteoglycans,might be involved in the promoting effect of BM on nerve growth,and PNA-R might play a more important role than laminin.