Molecular Genetics of Limb Development in Drosophila 
why Do Insects Have Six Legs? 
Insects develop legs only in their thoracic seg- 
ments, while more primitive arthropods may de- 
velop abdominal legs as well. A class of genes 
known collectively as homeotic genes distin- 
guish segments from one another. The homeotic 
genes of the Bithorax complex act as negative reg- 
ulators of thoracic segment identity. The Bithorax 
genes repress Distal- less expression and block 
leg development in abdominal segments. Accord- 
ing to our model, the intercellular signal re- 
sulting from the intersection of wingless- and 
decapentaplegic-express'mg cells should in prin- 
ciple be capable of eliciting limb development in 
the abdomen. The signaling sources are ex- 
pressed in precisely the same pattern in thoracic 
and abdominal segments. We have found that this 
response is blocked by the Bithorax genes. Re- 
moving Bithorax genes permits Distal-less ex- 
pression in the abdominal segments and leads to 
ectopic limb development in the abdomen. 
Therefore, the signal to specify leg primordia in 
the abdominal segments exists, but the conse- 
quences of signaling are overridden. 
Molecular analysis of the control elements that 
regulate Distal-less expression in the embryo 
provides compelling evidence that the intercel- 
lular signal is actually received and correctly in- 
terpreted by cells in the abdominal segments, just 
as in the thorax. We have identified a small piece 
of DNA, known as an enhancer, that acts as the 
target of this signal. By functional dissection of 
this enhancer we have identified a regulatory ele- 
ment that mediates the repression of limb devel- 
opment in the abdominal segments. Removing a 
small piece of DNA renders the enhancer element 
insensitive to the repressor genes of the Bithorax 
complex but has no effect on its response to the 
activation signal. Gene expression driven by this 
mutant version of the enhancer element is there- 
fore constitutive ly "de-repressed" in the abdo- 
men. These observations demonstrate that the 
wingless- and decapentaplegic-dependent acti- 
vation signal is both received and correctly inter- 
preted by the would-be abdominal leg cells. Re- 
pression of leg development must therefore lie in 
the prevention of Distal- less expression by these 
cells. These observations strongly suggest that 
turning on the Distal-less gene in these cells is 
the only signal necessary to specify their identity 
as leg cells. 
Proximal-Distal Pattern Formation 
in the Leg 
As mentioned above, flies that lack Distal- less 
gene activity do not develop any leg structures. 
Flies in which Distal-less gene activity is im- 
paired, but not eliminated, develop abnormal 
legs. Distal-less mutant legs are foreshortened 
along the proximal-distal axis. The characteris- 
tics of different Distal-less mutations tell us that 
the amount of activity of the gene is important in 
controlling the range of structures that the leg 
can develop. Distal parts of the leg require more 
Distal-less gene activity than do proximal parts to 
develop normally. These observations suggest 
that Distal- less may play an important role in or- 
ganizing the proximal-distal axis of the leg. 
Early in development, presumptive leg cells 
express Distal-less, whereas presumptive body 
wall cells do not. As the leg matures, this simple 
pattern transforms into a graded distribution of 
Distal-less RNA across the developing limb. The 
distal-most region of the limb expresses a high 
level of the gene product, intermediate levels ex- 
press lower levels of Distal- less RNA, and proxi- 
mal regions express little or no Distal- less RNA. 
These observations are particularly intriguing in 
view of the regional differences in the require- 
ment for the activity of the gene along the leg. We 
are interested in understanding the transition 
from the initial simple, two-state system of the 
embryo to the complex, graded distribution of 
the gene product that we see at later stages. 
Understanding pattern formation in the devel- 
oping fly leg will teach us about fundamental 
mechanisms important for the development of 
vertebrate embryos. We are studying a system in 
which intercellular signaling molecules are used 
to assign cells to functional developmental units. 
Although the particular details of the systems 
vary, the fundamental principles and molecular 
mechanisms will prove to be of wider applicabil- 
ity. In this context we are intrigued by the role 
that Distal-less plays as a pattern organizer at the 
genetic level and its prospective role as a regula- 
tor of gene expression at the molecular level. 
80 
