Development of the Drosophila Peripheral 
Nervotis System 
Hugo J. Bellen, D.V.M., Ph.D. — Assistant Investigator 
Dr. Bellen is also Assistant Professor in the Institute for Molecular Genetics, the Division of Neuroscience, 
the Department of Cell Biology, and the Program in Developmental Biology at Baylor College of Medicine. 
Educated in Belgium, he received a degree in commercial engineering and began research in sociometry, 
but decided to pursue a career in medical science. He obtained a D.V.M. degree from the University of 
Ghent and a Ph.D. degree in genetics from the University of California, Davis, where, in John Kiger's 
laboratory, he studied mutations that affect behavior in the fruit fly. Later, in Basel, Switzerland, as 
a postdoctoral fellow with Walter Gehring, he helped to develop the enhancer detection system. 
OUR research is centered on the development 
of the peripheral nervous system (PNS) of 
the fruit fly Drosophila melanogaster. The re- 
sults will help us analyze the development of the 
nervous system of many eukaryotic species. We 
have focused on the PNS of Drosophila because 
its cells are relatively easy to study and because 
sophisticated genetics can be applied to this 
model organism. 
We are presently studying the couch potato 
(cpo) and neuromusculin (nrm) genes, which 
are expressed at the onset of embryonic nervous 
system development and later in embryogenesis 
in most cells of the PNS. These genes were identi- 
fied in enhancer detector screens in which regula- 
tory sequences of genes are identified by means 
of a ;8-galactosidase reporter. Here we describe 
the preliminary characterization of these essen- 
tial genes and the development of a novel tech- 
nique to ablate nervous system cells. 
The cpo gene was so named because homozy- 
gous mutant flies are viable but have poor jump 
responses, poor flight abilities, slow recovery 
after ether anesthesia, and are generally hypoac- 
tive. More than 10 insertional cpo mutations 
were recovered from several enhancer detector 
screens; none of the insertions caused a complete 
loss of function of the gene. Some insertional al- 
leles cause recessive embryonic lethality, but no 
defects were observed in the PNS of the mutant 
embryos. 
A molecular analysis of cpo was initiated with 
the isolation of cpo cDNAs. In situ hybridizations 
to whole-mount embryos showed that the gene is 
expressed in the precursor cells of the PNS as 
well as glia of the PNS and CNS (central nervous 
system). Sequencing data show that the gene en- 
codes at least three different proteins that contain 
many stretches of the same amino acids. Two of 
these proteins contain a domain that may allow 
binding to RNA molecules. This binding domain 
is found in many proteins that are involved in 
processing immature RNA in a form that can be 
readily translated into a protein. The CPO protein 
has most significant homology with two other 
RNA-binding proteins that are found in nervous 
tissue: HuD, a human brain paraneoplastic en- 
cephalomyelitis antigen, and elav, the protein 
from a Drosophila nervous system-specific gene 
{embryonic lethal abnormal vision). 
The cpo gene sequence was used to produce a 
polyclonal antibody to CPO. The antibody allows 
the localization of this nuclear protein in the de- 
veloping embryo, in essentially the same cells as 
the transcript. In addition, the antibody recog- 
nizes a protein that is associated with polytene 
chromosomal bands isolated from the salivary 
gland of third instar larvae. This association may 
be mediated through the binding of CPO to na- 
scent RNA that is being synthesized from the DNA 
of the giant polytene chromosomes. Thus it is pos- 
sible that CPO acts as an essential PNS diff^erentia- 
tion factor by controlling some aspects of RNA 
maturation specific to the cells in which CPO is 
expressed. 
In addition to its biological functions, the cpo 
gene is also a hot spot for P-element enhancer 
detector insertions. Ten insertions that cause a 
variety of phenotypes were mapped at the nu- 
cleotide level, and molecular defects in cpo mu- 
tant strains that carry an enhancer detector were 
determined. All insertions recovered so far are 
clustered in a 200-bp genomic fragment that con- 
tains key regulatory regions of the cpo gene. Most 
insertions are integrated upstream of the first nu- 
cleotide of the longest cDNA but downstream of 
the consensus binding sites of three known Dro- 
5op^«7a transcription factors. Thus they are proba- 
bly located between the enhancer-binding sites 
and the promoter of cpo. Five insertions causing 
different phenotypes have inserted at exactly the 
same location. There is a perfect correlation be- 
tween the observed defects (or their lack) in em- 
bryos or adults and the size and the orientation of 
these insertions — e.g., large insertions are less 
detrimental than smaller insertions. These data 
suggest that the distance between the binding 
sites of the transcription factors and the transcrip- 
tion initiation site is critical for proper cpo regu- 
lation. In addition, insertions with the /«cZ gene 
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