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enhancement of
GMR
>
reaper
was reverted by precise excision of the
P
insertion, recapitulated in a specific deletion of the
mir-14
locus, and rescued
by reintroduction of
mir-14
genomic DNA. In fact, overexpression of
miR-14 potently suppressed
GMR
>
reaper
, rescuing eyes almost back to
wild type. This was not specific to reaper, since ectopic miR-14 also
strongly rescued the small, rough eyes induced by other proapoptotic
transgenes such as
GMR-hid
and
GMR-grim
(
Xu
et al
., 2003
). These genetic
tests established an intimate connection between miR-14 and suppression
of proapoptotic factors.
Another
Drosophila
locus identified on the basis of forward loss-of-
function screening of
P
insertions was
mir-279
(
Cayirlioglu
et al
., 2008
).
This mutant was identified by clonal screening for mutations that affected
CO
2
-sensing neurons in the olfactory system. These neurons normally arise
only in the antenna, but a
P
element insertion near
mir-279
caused ectopic
CO
2
sensing to be specified in the maxillary palp. As with
mir-14
, the
mir-279
insertion acted as a loss-of-function mutant, since these phenotypes
were recapitulated by independent deletions of the
mir-279
locus. There-
fore, miR-279 suppresses target genes in the maxillary palp to prevent the
specification of CO
2
-sensitive neurons. One of its more critical direct
targets is the zinc finger transcription factor encoded by
nerfin-1
, since
heterozygosity for this gene substantially decreased the ectopic CO
2
-sensing
neurons in
mir-279
mutant palps (
Cayirlioglu
et al
., 2008
). This implied that
derepression of nerfin-1 was critical for generating these neurons; never-
theless, there must be other relevant miR-279 targets since misexpression of
nerfin-1 was not sufficient to generate CO
2
-sensing neurons in palps.
These examples highlight how the context of forward genetics provides
insight regarding the
in vivo
function of miRNAs. Even to this day, despite
great improvements in our understanding of parameters underlying
miRNA target regulation and comprehensive set of sequenced Drosophilid
genomes (
Bartel, 2009
;
Stark
et al
., 2007
), and substantial additional study of
miR-14 genetics (
Varghese and Cohen, 2007
;
Varghese
et al
., 2010
), the
direct connection of miR-14 to the apoptosis pathway remains unclear.
Nevertheless, the dose sensitivity of
mir-14
for reaper-mediated cell death,
as well as the potent capacity of miR-14 to suppress the activity of multiple
other proapoptotic triggers, supports an intimate connection of miR-14 and
cell death. This remains to be deciphered in the future. Likewise,
mir-279
was known for some time to be expressed specifically in the nervous system
from embryo studies (
Aboobaker
et al
., 2005
), but a specific and essential
functional connection to a relatively obscure population of CO
2
-sensing
neurons in the brain certainly could not have been anticipated from target
predictions. Therefore, as was so elegantly illustrated by studies of
C. elegans
miRNAs to emerge from forward genetics—lin-4 and let-7 that control
heterochronic timing (
Lee
et al
., 1993
;
Reinhart
et al
., 2000
) and lsy-6 that
controls left-right asymmetry of ASE neurons (
Johnston and Hobert,
