thermore, this binding ability is conserved in the 
corresponding two repeats from the Xenopus Notch 
homologue. These resuhs raise the possibility that 
Notch may act as a multifunctional receptor whose 
36 EGF repeats form a tandem array of discrete 
ligand-binding units, each of which may potentially 
interact with several different proteins during 
development. 
Recent efforts have been directed toward address- 
ing the in vivo relevance of this system by introduc- 
ing various Notch deletion constructs into flies via 
P-element-mediated transformation and then study- 
ing their biological activity. All constructs were 
placed under the control of two promoters: the regu- 
lar Notch promoter, in order to be able to study the 
effects of mutant Notch proteins expressed at the 
appropriate times and places during development, 
and the hsp70 promoter, in order to look at the ef- 
fects of overexpression of these constructs in tissues 
where Notch is not normally expressed. 
Examination of cell aggregates expressing Notch 
and Delta showed that the proteins are colocalized 
at regions of cell contact and in vesicles within the 
7Vo?cyb-expressing cells. Similar vesicles are also ob- 
served in imaginal discs stained for Notch and exam- 
ined by light or electron microscopy. The internal- 
ization of Delta into A^ofc^-expressing cells was 
further investigated by performing live-cell anti- 
body stainings on the S2 cell lines. Within 5 minutes 
of the mixing of the two cell lines. Notch and Delta 
become colocalized at adhesion interfaces, and 
within 15 minutes both proteins are internalized 
into the A^o^c^-expressing cell. Identical internaliza- 
tion occurs in S2 cells, which express only the ex- 
tracellular portion of Notch, indicating that this pro- 
cess does not require the Notch intracellular 
domain. 
The notion that Notch and Delta may engage in a 
receptor-ligand relationship is supported by genetic 
mosaic experiments in Dr. Pat Simpson's laboratory. 
It was found that cells mutant for Notch, but not 
Delta, display cell autonomy. 
Genetic Interactions Involving Notch 
To identify other genes involved in Notch func- 
tion. Dr. Artavanis-Tsakonas and his co-workers have 
initiated two genetic screens for loci that interact 
with Notch in a dosage-dependent manner. The first 
screen is based on the observation that N'^'/fa^^ 
transheterozygous females exhibit a strong tempera- 
ture sensitivity with respect to their fa^^ eye pheno- 
type yet remain fully viable and fertile at all tempera- 
tures from 18°C to 30°C. So far ~30 mutant 
candidates have been obtained from among 
~ 30,000 N*^^ Ifa^^ female progeny. By searching for 
loci that display haplo-insufficient enhancement of 
a sensitized Notch mutant phenotype, it will be pos- 
sible to identify genes not previously associated 
with Notch function as a result of their embryonic 
lethality and/or lack of a neurogenic phenotype in 
the null state. 
The second genetic screen was designed to isolate 
enhancers of notchoid {nd). This is a hypomorphic 
Notch allele that affects wing morphology, has been 
shown to interact with several Notch group 
members, and is associated with a missense muta- 
tion in the intracellular domain of the protein. A 
screening of 120,000 flies for modification of the 
wrfwing phenotype yielded 140 mutants with modi- 
fied wings. The complementation analysis carried 
out so far has revealed approximately 10 comple- 
mentation groups. As expected, some are members 
of the Notch group as defined now, but more impor- 
tantly, new genes whose relationship with Notch 
was hitherto unknown have also been detected. 
The Notch Group in Mammals 
In an attempt to examine the analogies that may 
exist between the invertebrate experimental model 
and vertebrates, as well as to take advantage of the 
sophisticated cell culture systems that the verte- 
brate model offers, the Artavanis-Tsakonas group 
started to isolate and study the human counterparts 
of the Notch group of genes. So far, human counter- 
parts of two Notch group members have been iso- 
lated: Notch and groucho (the latter, a gene of the 
Enhancer of split complex) . 
While the fly appears to have a single Notch gene, 
vertebrates have at least two diff'erent genes encod- 
ing A^o^c^-like proteins. In humans the Artavanis- 
Tsakonas group has demonstrated the existence of 
two Notch homologues. Interestingly, other work- 
ers have associated rearrangements of one of the hu- 
man A^o^c^-like genes with a particular neoplasm, 
suggesting an important developmental role for the 
human Notch. 
The Drosophila m9/10, or groucho, gene is 
characterized by the presence of a tandem array of 
so-called WD-40 repeats at its carboxyl-terminal 
half. Such a motif was first identified in the /? sub- 
unit of transducin, but has since been found in a 
growing number of proteins associated with diverse 
cellular functions. By screening a cDNA library of 
fetal human brain, four groucho homologous genes 
encoding a family of proteins have been isolated and 
designated TLE (transducin-Iike Enhancer of split) . 
The TLE genes were shown to be expressed differ- 
entially and to encode nuclear proteins. This attri- 
NEUROSCIENCE 391 
