NEURODEVELOPMENT IN DROSOPHILA 
Flora Katz, Ph.D., Assistant Investigator 
Dr. Katz is interested in the mechanisms by 
which cell surface interactions in the nervous sys- 
tem govern differentiation and the establishment of 
neural networks. 
The nac {neurally altered carbohydrate) muta- 
tion in Drosophila alters a neural-specific glycan 
carried by multiple cell surface neuronal proteins. 
The nac glycan, which was originally identified as 
an epitope that is expressed on growth cones and 
their target cells, was highly correlated with grov^^h 
cone pathfinding activity in the grasshopper. A bio- 
chemical analysis revealed that the epitope was imi- 
tated by a common asparagine-linked glycan struc- 
ture found in plants, which contains unusual 
xylose and fucose linkages. Lectins and antibodies 
generated against plant glycoproteins were used to 
analyze the nature of this structure in Drosophila. 
Both xylose-containing and fucose-containing 
glycans appear to be present and highly enriched in 
the nervous system of wild-type flies, but absent in 
nac. The expression patterns (stable and specific 
expression in the nervous system; transient expres- 
sion during development in other cell types), evo- 
lutionary conservation, and inclusion on multiple 
proteins suggested that the presence of this carbo- 
hydrate might be more important as a marker of 
neuronal cell surfaces than as a functional unit for 
the individual proteins that carried the modification 
and might serve as one model for the extensive use 
of tissue-specific glycosylation throughout the ani- 
mal kingdom. 
An extensive phenotypic analysis has been car- 
ried out. nac is a maternal-effect cold-sensitive le- 
thal mutation that arrests early in embryonic devel- 
opment. Although it is 100% penetrant, it shows 
some variability in the time of arrest, and a small 
number of animals persist through hatching. Using 
fluorescently labeled phalloidin [which binds to F- 
actin and clearly highlights the structure of the em- 
bryonic central nervous system (CNS)], Dr. Katz 
and her colleagues studied the morphology of the 
CNS in the embryos that achieve neuralization. Al- 
though these embryos were often arrested early in 
the process of creating the commissural network 
that forms the backbone of the CNS, they were 
grossly normal in structure until the time of arrest. 
It is not known whether the arrest is caused di- 
rectly by altered glycan structure or is a secondary 
consequence. To assess whether the nac alteration 
is acting as a signal in neural assembly in conjunc- 
tion with other redundant clues, Dr. Katz and her 
colleagues constructed double mutants with other 
candidate mutants that affect neural cell surface 
proteins; these proteins have mild effects on CNS 
structure but may cause concerted disruptions 
with nac. 
The zygotic cold-sensitive effect of nac is to alter 
the assembly of the eye facets and the morphology 
of the wing. The eye is mildly altered in both the 
identities and the relationships of its cells. How- 
ever, when maternal-effect embryos are allowed to 
begin development at the permissive temperature 
and then shifted to the nonpermissive temperature, 
severe and extensive alterations of the eye occur. 
This suggests that maternal product contributes to 
imaginal differentiation. 
To explore further the role of the glycan in these 
phenotypes. Dr. Katz and her colleagues have de- 
veloped a tissue culture system. Cells derived from 
dissociated cellular blastoderm embryos can be 
grown in defined media and survive for long peri- 
ods of time. Extensive differentiation of these cells 
into recognizable neural and myotube-like struc- 
tures occurs within 24 h, which correlates with the 
cessation of mitosis, as judged by [^H]thymidine 
uptake experiments. Conditions can be adjusted to 
prevent this differentiation indefinitely in normal 
cells and prolong the period of mitosis. This system 
is useful to study the cellular phenotype of nac, 
both with regard to its ability to differentiate neu- 
rons and to sustain mitosis. It also provides an in 
vitro system to study cellular neural interactions. 
An ELISA assay has been used to search for addi- 
tional alleles of nac and other members of neural 
glycan pathways, so far unsuccessfully. In the ab- 
sence of additional alleles, small deficiencies were 
induced in the mapped area by 7-irradiation of a 
marked P element that maps close to nac. Some of 
the 10 new deficiencies uncover nac\ some do not. 
These deficiencies, along with other recombinants 
recovered during meiotic mapping, have been used 
for a cosegregation analysis to demonstrate that all 
the phenotypes of the mutant segregate together 
and hence constitute a single mutation. 
A molecular analysis of nac is in progress. The 
mapped area was entered from a clone provided by 
Dr. Richard Garber (Seattle). As glycan expression is 
recovered in nac maternal-effect embryos injected 
with wild-type cytoplasm, maternal RNA hybrid se- 
lection is being used as a transient assay for the 
Continued 
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