EXTRACELLULAR FACTORS AFFECTING NEURON DEVELOPMENT 
Louis F. Reichardt, Ph.D., Investigator 
Dr. Reichardt and his colleagues have continued 
to examine molecules in the extracellular environ- 
ment of neurons that direct their development. 
L Neurotrophic Factors. 
Work in Dr. Reichardt's laboratory has focused 
on nerve grov^^h factor (NGF). In previous studies, 
Dr. Reichardt shov^^ed that target organs of sympa- 
thetic and sensory neurons, neurons that are regu- 
lated by NGF, express the NGF gene at low, but bio- 
logically significant, levels. NGF mRNA and protein 
were also detected in the central nervous system 
(CNS), particularly in the target areas of cholinergic 
neurons in the basal forebrain. 
To study the role of NGF and its receptor in early 
embryonic development, where there are indica- 
tions that NGF may affect cell determination and 
axon guidance. Dr. Reichardt and his colleagues 
have spent the past year studying expression of 
both the NGF and NGF receptor genes in the devel- 
oping chick embryo. A baculovirus expression sys- 
tem has been used to express the extracellular do- 
mains of both the chick and rat NGF receptors. 
These truncated proteins have been purified and 
used to prepare antibodies that interfere with re- 
ceptor-ligand interactions. These antibodies are 
being used to study receptor distribution and the 
consequences of inhibiting receptor function dur- 
ing early development of the avian embryo. 
II. Neuronal Adhesion and Process Extension on 
Extracellular Matrix Constituents. 
Work in previous years has indicated that cell sur- 
face and extracellular matrix (ECM) constituents 
are important in regulation of the extent and direc- 
tion of axon growth in vitro. Several of these ECM 
and cell surface glycoproteins have been shown to 
be expressed in embryos at positions appropriate 
for influencing neuronal growth cone behavior. 
The glycoprotein laminin (LN), one of the ECM 
constituents in the targets innervated by the pe- 
ripheral nervous system, has dramatic stimulatory 
effects on neuronal survival, process outgrowth, 
and expression of neurotransmitters. The primary 
receptors used by neurons to interact with LN are 
members of the integrin superfamily of cell surface 
heterodimers. One integrin heterodimer with an a- 
subunit oiM 180,000 and B,-subunit of M 120,000 
r ' "1 r ' 
was previously purified and shown to be an LN re- 
ceptor. In the past year, Dr. Michael Ignatius and 
Laura Goetzl showed that this purified integrin a/p^ 
heterodimer functions as a dual receptor for LN 
and collagen, but not fibronectin (FN). Clones en- 
coding this receptor a-subunit are being character- 
ized. 
In previous years, PC 12 cells have been used as a 
neuronal model to study interactions with ECM 
constituents. These cells were shown to express 
two integrin a/p^ heterodimers with a-subunits of 
M 180,000 and 140,000. The immunopurified in- 
tegrin heterodimers were shown to bind LN and 
collagen strongly and FN only weakly, mimicking 
the behavior of the PC 12 cells. In the past year, the 
ligands of the individual integrin a/(3j heterodimers 
have been identified. In collaboration with Drs. 
David Turner (State University of New York at Buf- 
falo) and Salvatore Carbonetto (McGill University), 
Drs. Kevin Tomaselli and Deborah Hall were able to 
show that the larger heterodimer is the rat homo- 
logue of ttj/Pj. This heterodimer binds to collagen 
and to the El-4 fragment of LN (a fragment contain- 
ing most of the three short arms of the cruciform 
structure of LN). PC 12 cells also were shown to in- 
teract with a distinct site in the long arm of LN, 
using a different integrin. Dr. Tomaselli showed that 
the second a-subunit expressed by PC12 cells is a^. 
Integrin ctj/Pj heterodimers have been shown by 
Drs. Kurt Gehlsen and Erkki Ruoslahti to function 
as LN and FN receptors, binding LN in the long arm 
of the LN cruciform structure (isolated as fragment 
E8). In summary, this work showed that PC 12 cells 
interact with two distinct sites on LN. Integrin a^/p^ 
heterodimers mediate binding to LN fragment El-4 
and coUagens; integrin ot^/Pj heterodimers appear 
to mediate binding to LN fragment E8 and the weak 
observed binding to FN. These two receptors can 
account for all observed interactions of PC 12 cells 
with the ECM. 
An additional LN-binding integrin a/p^ heterodi- 
mer has been characterized by Dr. Hall in collabo- 
ration with Drs. Caroline Damsky (University of Cal- 
ifornia at San Francisco) and Arnoud Sonnenberg 
(Amsterdam). With a human cell line that does not 
express the integrin a^/Pj heterodimer it was possi- 
ble to show that these cells also interact with at 
least two sites on LN. Interactions with the E8 frag- 
ment of LN are mediated by integrin ct^/p^ 
heterodimers; interactions with the El-4 fragment 
Continued 
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