Drosophila: eye genetics and mosaic
analysis
Drosophila is arguably the most versatile and one
of the most powerful eukaryotic genetic model systems. Flies are easy to maintain
and breed in large numbers in the lab and have a generation time of 10 days (at
25 C). The genome is relatively compact (1.7 x 108 bp and has been
completely sequenced - the annotated version has been freely available on the
web since March 2000.
In order to illustrate general
principles of Drosophila
research techniques in these lectures I will focus on one particular aspect of
the fly's biology, the development and function of the compound eye,. . Drosophila has a typical insect compound eye. Each
eye is composed of several hundred simple units called ommatidia arranged in an
extremely regular array. In the fly each ommatidium consists of a core of 8
photoreceptor cells (R1-R8) surrounded by 4 cone cells (equivalent to the
vertebrate lens in function) pigment cells and a sensory bristle. The number of
cells, their identities and functions within each ommatidium is invariant.
The eye develops from the
eye-antennal imaginal disc. Not all ommatidia form at the same time.
Differentiation occurs in posterior to anterior sequence: the first ommatidia
to differentiate do so at the posterior pole of the eye disc, the last at the
anterior pole. The morphogenetic furrow marks the boundary between dividing,
non-differentiating cells and differentiating ommatidia. The further posterior
ommatidia are from the furrow the closer they are to having all cell types
(R1-R8 cone cells and pigment cells) differentiated. Cells anterior from the
furrow divide asynchronously, while cells in the furrow are non-dividing. Immediately
after leaving the furrow cells go through two last synchronous divisions. This
strict control of cell division makes the eye an ideal system for analysing the
developmental effects of cell cycle genes. Programmed cell death is another
important component of the developmental process - once all ommatidia have
differentiated any cells which have not been incorporated into ommatidia die.
The cellular composition of
each ommatidium is as invariant as that of an adult C.elegans and even simpler as it consists of even
fewer cells. However unlike C.elegans the lineage of ommatidial cells varies both
between ommatidia and between individuals in a way that suggests that lineage
plays no part in determining ommatidial cell fate.
The eye is an effective model
for processes including cell signalling, neuronal connectivity, control of cell
proliferation and vesicular transport. Many different types of mutant have been
isolated affecting for example, the size, the pigmentation and the morphology
of the eye. The second half of the lecture focuses on mutations that affect the
development of one specific photoreceptor the R7 cell. The R7 photoreceptor is
the only one which is sensitive to UV light. This make it possible to screen
for R7 defects using simple behavioural screen for flies which fail to show a
phototactic response to UV. Two mutants isolated in this way proved to lack the
R7 photoreceptor -it never develops. The relationship between these two
mutants, sevenless (sev) and bride of sevenless (boss), was initially worked out using the
classical genetic technique of mosaic analysis.
Generating genetically
mosaic eyes by X-radiation induced mitotic recombination
Recombination between
homologous chromosomes is well known to occur during meiosis. It can also occur
during mitosis, although usually at a very low frequency. In flies it is
possible to increase the rate of mitotic recombination by several orders of
magnitude through exposing flies to a short pulse of X-irradiation. This can be
used to induce clones of cells which are homozygous for one or more mutations
in flys in which most cells are heterozygous for these mutations. Assume we
have larvae which are heterozygous mutant at a fully recessive marker gene
locus (a good example of such a locus is the eye pigment gene white). Usually
when cell of these larvae divide they give rise to two daughter cells which are
heterozygous like their parents - both daughters can be said to show the
parental combination of alleles. X-ray induced recombination between the chromosome
carrying the wild type and the chromosome carrying the mutant allele of white
can however produce one recombinant daughter cell which is homozygous for the
wild type allele and one recombinant daughter cell which is homozygous for the
mutant allele. Both recombinant cells will go on to produce "clones"
of progeny with the same homozygous genotype in the adult fly. As the marker
gene is recessive, cells of the homozygous wild type clone will be
indistinguishable from the heterozygous cells around it. But cells of the
homozygous mutant clone will be visibly different from their neighbours making
it possible to see what combination of cell types a single progenitor cell can
give rise to.
Induced mitotic recombination
is an important technique because it can not only be used to produce
genetically marked clones of otherwise normal cells, but also genetically
marked clones of mutant cells. This is because recombination involves not just
single genes but entire chromosomal segments distal to the recombination
breakpoint. Somatic recombination can be induced in larvae or embryos that are
heterozygous mutant both for a marker gene and developmentally interesting
genes which map distal to the marker gene. In adults subjected to this
treatment marked clones will be homozygous mutant for the marker gene and for
the developmental gene(s)and the phenotype of the clone can be analysed for
defects (such as never including an R7 cell). Mosaic analysis can be used to:-
Mosaic analysis of sev and boss shows sev is required in the R7 cell and boss in the R8 cell. R7 is normally the last
photoreceptor to develop and R8 the first. R7 and R8 are adjacent in developing
ommatidia. The results of the mosaic analysis imply that the R8 cell is needed
for R7 to form. A simple theory, which has been proved correct, is that R8
produces a signal (coded for by boss) that activates a receptor on a neighbouring cell
(coded for by sev)
instructing that cell to become the R7 cell.
References
http://www.sdbonline.org/fly/aimorph/eye.htm
Thomas BJ and Wassarman DA
(1999)
Trends in Genetics vol 15
pp184-190