MOLECULAR GENETICS OF LIMB DEVELOPMENT IN DROSOPHILA 
Stephen M. Cohen, Ph.D., Assistant Investigator 
The organization of a complex three-dimensional 
body plan in a developing embryo requires that indi- 
vidual cells acquire distinct developmental fates. 
Cells become progressively distinguished from one 
another by alterations in their pattern of gene ex- 
pression. These changes must be integrated so that 
neighboring cells adopt compatible developmen- 
tal programs. Intercellular signaling systems are 
thought to play a fundamental role in the allocation 
of groups of cells into functional developmental 
units. Research in Dr. Cohen's laboratory is focused 
on one such system, the specification of the limb 
primordia in the Drosophila embryo. Drosophila 
provides an amenable system in which to study the 
genetic signals required to allocate a population of 
founder cells as the progenitors of limbs. 
Origin of the Embryonic Limbs 
Expression of the Distal- less gene provides the 
first indication that the limb developmental field 
has been established in the embryo. The Distal-less 
gene is expressed in the limb primordia prior to any 
overt morphological sign of limb development. This 
observation supports the suggestion that Distal- less 
expression might be an early cue that initiates com- 
mitment of embryonic cells to limb development. 
Genetic evidence indicates that the product of Dis- 
tal-less is required for the specification of these em- 
bryonic cells as limb precursors. Embryos that lack 
Distal-less gene activity fail to develop any limb 
structures. Distal-less encodes a protein with fea- 
tures expected of a transcription factor. Conse- 
quently the gene is expected to exert its function by 
regulating additional subordinate target genes. The 
Distal-less protein is nuclear, binds to DNA in a se- 
quence-specific manner, and activates transcription 
in a heterologous system (yeast) . 
In the Drosophila embryo. Distal-less is required 
at two stages. First, it must specify development of 
the larval limbs and, subsequently, the progenitor 
cells of the adult limbs. The discrete requirements 
for Distal-less activity in these processes have been 
shown to be controlled independently by different 
cis-regulatory elements that direct Distal-less ex- 
pression in appropriate cells at the appropriate 
time. 
Spatial and Temporal Control 
of Distal-less Expression 
The leg primordia arise as distinct clusters of cells 
at well-defined positions in the embryo. It is of obvi- 
ous importance that the positioning of the limbs be 
carefully regulated with respect to the rest of the 
body pattern. Both the establishment and position- 
ing of the limb primordia were shown to depend on 
spatial cues provided by the segmentation genes. In 
particular, the wingless gene was implicated as the 
source of a positional cue locating the limb primor- 
dia with respect to the anterior-posterior axis of the 
embryonic segments. 
An additional source of information is required to 
locate the limb primordia with respect to dorsal- 
ventral position in the segment. Preliminary studies 
implicate the decapentaplegic gene, which is in- 
volved in the organization of dorsal-ventral pattern 
in the embryo, where it is expressed on the flank in 
longitudinal stripes of cells. These stripes intersect 
the stripes of wingless gene expression at the pre- 
cise location where the limb primordia develop. 
The embryo locates its limbs at the intersections 
of lines of positional information drawn by the seg- 
mentation and dorsal-ventral genes. Interestingly, 
both wingless and decapentaplegic encode se- 
creted signaling molecules. It seems probable that 
cells receiving both signals may be instructed to be- 
come limb cells. An early acting cis-regulatory en- 
hancer element responsible for directing Distal-less 
expression in response to these intercellular induc- 
tive signals has been defined. 
Repression of Abdominal Limb Development 
Insects evolved from more primitive arthropods 
in which legs develop in all body segments. Limb 
development in the abdominal segments of insects 
is suppressed through the activity of the homeotic 
genes of the Bithorax complex. These genes have 
been shown to act by repressing expression of the 
Distal-less gene in the abdominal segments. The 
Bithorax complex genes encode homeodomain 
transcriptional regulatory proteins that have been 
shown to function as repressors. These proteins 
bind to the cis-regulatory enhancer element that 
controls Distal-less in the early embryo and thereby 
repress Distal-less expression in the abdominal 
segments. 
The binding sites in the Distal-less enhancer to 
which these proteins bind in vitro are essential for 
the activity of the repressor element in vivo. En- 
hancers in which the sites have been specifically 
mutated to preclude binding are unable to confer 
repression by the Bithorax genes in the embryo. 
Therefore Distal-less acts as a direct downstream 
GENETICS 171 
