DIFFERENTIATION OF THE DROSOPHILA NERVOUS SYSTEM 
Hugo J. Bellen, D.V.M., Ph.D., Assistant Investigator 
Dr. Bellen and his colleagues study the molecular, 
cellular, and genetic mechanisms by which neural 
cells differentiate in the peripheral nervous system 
(PNS) during embryonic development. 
RNA Processing May Require couch-potato 
in the PNS 
In order to isolate PNS differentiation genes, 
whose expression may be controlled by proneural 
genes, about 4,400 enhancer detector strains were 
screened for /3-galactosidase expression in the sen- 
sory motor cells (SMC) or PNS precursor cells. Fif- 
teen enhancer detector strains that express lacZ in 
the SMC and map at cytological subdivision 90D 
were identified and genetically characterized. The 
gene adjacent to these insertions was shown to be 
essential and was named couch potato (cpo) be- 
cause of severe hypoactive behavior of some adult 
mutant flies. 
The cpo gene has now been cloned and character- 
ized at the molecular level. It is complex and has 
two (or more) promoters and two transcripts. One 
of the transcripts is differentially spliced and forms 
two messages. Some of the messages, characterized 
by their cDNAs, are expressed in different cells of 
the PNS during development. Three different types 
of cDNAs were isolated, sequenced, and mapped. 
None of the three cDNAs contains a standard AUG 
initiation codon. Two of the three cDNAs encode 
putative proteins that are similar. Both proteins con- 
tain a carboxyl-terminal motif that is found in many 
RNA-binding proteins, termed the RNA-binding con- 
sensus. These domains are most homologous to simi- 
lar domains of two proteins that are also expressed 
in the nervous system. One of these proteins has 
been implicated in a human sensory neuropathy. In 
collaboration with Dr. David Anderson (HHMI, Cali- 
fornia Institute of Technology) , Dr. Bellen's labora- 
tory has cloned and sequenced a mammalian cpo 
cDNA homologue. Both proteins contain very simi- 
lar RNA-binding consensus sites, and it will soon be 
established whether these proteins are expressed in 
similar tissues in vertebrates. 
Based on these and other genetic data, it is pro- 
posed that cpo expression is regulated by the pro- 
neural genes and that it carries out an essential dif- 
ferentiation function in the PNS. The Cpo protein 
probably modifies some of the RNA species that are 
produced in the cells in which it is expressed. These 
modifications may produce a number of proteins 
that are specific to the PNS. These proteins seem to 
be required mainly for a normal function of the PNS, 
as no obvious morphological defects have been ob- 
served in embryos that lack Cpo protein. (The proj- 
ect described above was supported by a grant from 
the Muscular Dystrophy Association.) 
Neuromusculin, a Cell Adhesion Molecule 
The neuromusculin {nrni) gene was identified 
in the same enhancer detector screen as cpo. It is 
also expressed in the SMC and the more differen- 
tiated cells of the PNS. However, in contrast to cpo, 
its expression in the embryonic cells of the PNS is 
transient. It is essentially absent in the PNS of em- 
bryos at stage 15 and older. At stages 15 and 16, 
prior to the formation of the neuromuscular junc- 
tion, muscles start expressing nrm. 
Sequencing of a number of nrm cDNAs showed 
that it encodes a molecule of the immunoglobulin- 
like (Ig-like) family with a signal peptide, nine Ig- 
like domains, a transmembrane domain, and a very 
short cytoplasmic domain. In addition, it contains 
20 potential glycosylation sites and a single protease 
cleavage site in the membrane-proximal Ig-like do- 
main. These observations suggest that Nrm is an ex- 
tracellular protein that is membrane anchored and 
possibly secreted. Immunoblots with polyclonal an- 
tibodies against Nrm show that it is both associated 
with membranes and secreted by S2-expressing 
cells. 
Expression of Nrm in S2 cells indicates that it is a 
weak homophilic cell adhesion molecule. How- 
ever, mixing assays with Fasciclin 11-expressing S2 
cells and Nrm-expressing cells suggests that Nrm 
and Fasciclin II are homophilic and heterophilic 
cell adhesion molecules. Interestingly, Fasciclin II 
is abundantly expressed on the growth cones of 
many motoneurons prior to the formation of the neu- 
romuscular junctions. 
It is proposed that Nrm and Fasciclin II interact in 
vivo and that both proteins may play a role in setting 
up the neuromuscular junction. In addition, Nrm 
may function in growth cone guidance, as nrm is 
expressed in the PNS prior to the initiation of axon- 
ogenesis of motoneurons and sensory neurons. Since 
motoneurons express Fasciclin II and sensory neu- 
rons Nrm, and since both types of neurons fascicu- 
late in the PNS, it is possible that their interactions 
play a role in peripheral axonogenesis. 
GENETICS 157 
