Supplemental Materials and Methods
Genetic Mapping
For those mutants where the enhancement bred true, if the enhancement was
inherited with the p[w+] chromosome, this indicated that the enhancing mutation
was on the second chromosome, and male flies of the genotype w-; p[w+], * / +;
Df(3R)p13/ TM6B were selected and bred to w-; Cyo / Sco virgin females. w-;
p[w+], * / Cyo; +/ TM6B flies were selected based on eye color, and self crossed
to create independent stocks.
If the mutation was not inherited with the p[w+], this indicated that the
enhancing mutation was on the third chromosome, and males of the genotype w-;
+/ +; Df(3R)p13, e, */ TM6B were selected by the presence of the ebony marker,
and the absence of the p[w+] marker, and used to create independent stocks.
Those mutants which segregated with the X chromosome were discarded.
Complementation and mapping:
Chromosome 2:
For mutants isolated on the second chromosome, each mutant was
tested for complementation by crossing it to each other mutant on the second
chromosome. Alleles were then separated into different groups based on their
inability to complement one another. To rule out the presence of mutations that
dominantly affect eye development directly, each second chromosome mutation
was also tested individually for failure to complement a known allele of Star,
mutations in which show a dominant rough eye phenotype when heterozygous.
Only one Star allele was isolated in this screen out of 15 total enhancers on the
second chromosome. We also isolated three separate complementation groups
on the second chromosome that consisted of multiple alleles (Group A consisting
of 2 lethal alleles, Group B consisting of 4 lethal alleles, and Group C consisting
of 1 lethal and 1 temperature sensitive lethal allele), 1 allele of Star, and 6
additional enhancers that did not separate into any complementation group, and
were also not alleles of Star. We concluded that these 6 additional mutants on
the second chromosome represent single hit mutants in unidentified loci, and
were not further characterized.
Single lethal representative mutants from each complementation group
were then crossed to the second Chromosome Bloomington Deficiency collection
(http://flystocks.bio.indiana.edu/Browse/df-dp/dfkit.htm), and those deficiencies
that failed to complement these mutants were further analyzed. Through this
deficiency mapping, both alleles of complementation Group A failed to
complement Bloomington Deficiency Df(2L)JS17 (BL#1567), which deletes the
lilliputian gene (among others). Single mutations in lilliputian were obtained
(lilliXS407, lilliXS575, lilliA17-2, lilliS35), and tested for complementation with each of the
alleles of this complementation group, and each failed to complement both
alleles (Table I). We therefore concluded that complementation Group A
represented mutations in the lilliputian gene, and renamed the alleles of this
group as lilliGD17, and lilliAG5.
Concurrent with the second chromosome deficiency mapping, we
attempted mapping each complementation group through meiotic mapping using
molecularly defined P element insertions as described (ZHAI et al. 2003). This
method utilizes two independent alleles within a complementation group, and
maps the lethality associated with these trans-heterozygous combinations to a
number of molecularly characterized P element insertions located at various
intervals along the length of the second chromosome. In this fashion we mapped
the lethality of two of the four alleles of complementation Group B to less than
.01% from P element P{SUPor-P}KG00569 (BL#13139). P{SUPor-P}KG00569
has a cytological position of 21B1 as reported by Flybase
(http://flybase.org/reports/FBti0021228.html), and 21A5 as reported by (ZHAI et
al. 2003). It is reported as having a molecular map position of 66,954 (ZHAI et al.
2003). We therefore tested for complementation of the alleles of this group with
deficiencies that removed genes within these cytological locations, and found
that all four alleles failed to complement Df(2L)ED19 (BL#8901), which deletes
the kismet gene (among others). We obtained single lethal mutations in kismet
(kis1, and kisk13416) and found that all four alleles of complementation Group B
failed to complement both of these alleles. We therefore concluded that
complementation Group B represented mutations in the kismet gene, and
renamed the alleles of this group as kisEC1, kisLM27, kisAS21, and kisAO9.
Concurrent with the second chromosome deficiency and meiotic mapping,
we crossed each second chromosome allele to the candidate gene daughterless,
which is on the second chromosome in Drosophila, and which was previously
shown to affect Atonal protein expression (BROWN et al. 1996). We found that
Complementation Group C failed to complement known alleles of daughterless.
Complementation Group C consists of 1 lethal and 1 temperature sensitive lethal
allele. The lethal allele of this group (which we named daIB21) is fully lethal at
both 25C and 29C. This allele fully fails to complement a null allele of da (daUX,
0% complementation at both 25C and 29C), and partially fails to complement a
temperature sensitive allele at 25C (da1, 45% complementation), and fully fails
to complement a temperature sensitive allele at 29C (da1, 0%
complementation).
The temperature sensitive allele of this group (which we named daAB12) is
partially lethal at 25C (51% viability), and nearly fully lethal at 29C (9%
viability). Trans-heterozygous daAB12/ daIB21 flies are partially viable at both 25C
(27% viability) and 29C (24% viability). daAB12 partially complements a null allele
of da (daUX, 81% complementation at 25C, 51% complementation at 29C).
Chromosome 3:
For mutants isolated on the third chromosome, each mutant was first
tested for complementation to the hedgehog gene as a potential candidate, as
we had established that null alleles of hedgehog were dominant enhancers of our
loss-of-function atonal phenotype (Figure 2J), and the hedgehog gene is on the
third chromosome. We found that of the 33 third chromosome mutations, three
failed to complement the lethality associated with a hedgehog null mutant (hhAC).
We therefore concluded these mutants represented mutations in the hedgehog
gene, and renamed these alleles as hhFS1, hhMM2, and hhRM2.
We next mapped each of the mutations on the third chromosome by
looking for genetic linkage between the enhancement of our atonal loss-offunction eye phenotype, and the presence of either the Df(3R)p13 (cytological
breakpoints 84F1-85B9), or ebony (cytological position 93C7-93D1), both of
which are present on the mutagenized chromosome. Df(3R)p13, e, */ TM6B flies
were crossed to w-; ato1090/ TM6B. Df(3R)p13, e, */ ato1090 display an enhanced
atonal loss-of-function eye phenotype, and virgin females of this genotype were
crossed to Df(3R)p13, e/ TM6B males. F1 genotype ato1090/ Df(3R)p13 flies were
then scored for the presence or absence of the enhanced atonal phenotype in
either a wild type or ebony background. We found that four mutations were
strongly linked to the Df(3R)p13 deficiency, and we tested for complementation
of these alleles with mutants in candidate genes close to the deficiency. We
found that four enhancer mutants showed small rough eyes when transheterozygous with mutations in the roughened eye gene (roe1, and roe3).
Roughened eye (roe) is a differentially expressed transcript that contains part of
the rotund (rn) gene (ST PIERRE et al. 2002), and the roe mutants that we tested
specifically knock out only roe function (ST PIERRE et al. 2002). We therefore
tested mutations that specifically affect only rn gene function (rn19, and rn5) and
found that all four alleles showed normal eyes. Additionally, all four alleles also
show small rough eyes when trans-heterozygous for a mutation that removes
both rn and roe function (rn16) (ST PIERRE et al. 2002). Importantly, the
Df(3R)p13 chromosome shows normal eyes when trans-heterozygous with both
rn specific and roe specific mutants, indicating that the rough eyes observed are
associated with enhancer mutations on the Df(3R)p13 chromosome and not with
the deletion within the chromosome itself. Based on these results, we concluded
these mutants represented mutations in the roughened eye gene, and renamed
these alleles as roeSM8, roeBM10, roeKK16, and roeKM29.
For the remaining 26 alleles, each was recombined on to the ato1090
chromosome, and each was tested for complementation against each other. No
further complementation groups were obtained in this fashion. Thus, we
concluded that each of these mutations represents single hit mutations in
undefined loci. It may be that by performing further mutatgenesis, additional
alleles of these genes may be obtained.