Biology Reference
In-Depth Information
postembryonic expression of these miRNAs in the nervous system. In many
of these examples, relevant binding sites for transcription factors have been
elucidated. For example, analysis of
mir-1
CRMs indicated direct regulation
by the transcription factors Dorsal, Twist, d-Mef2, and potentially SRF
(
Biemar
et al
., 2005
;
Kwon
et al
., 2005
;
Sokol and Ambros, 2005
).
Even without prior knowledge of an endogenous transcript pattern,
reporters can provide an entry point to elucidate spatial patterns. In this
case, a critical concern is to include sufficient regulatory sequence to encom-
pass relevant inputs. This might be addressed by inserting reporter sequences
into large constructs (such as fosmids or BACs), or better yet, by inserting
within the endogenous locus. One way this can be accomplished is to include
a reporter, such as Gal4, within an HR targeting cassette. Combining a UAS
reporter with a Gal4 knockin allele was first used to demonstrate that
mir-278
was highly active in the fat body, where it regulates energy homeostasis by
limiting insulin pathway activity and levels of circulating sugar (
Teleman
et al
.,
2006
). Subsequent integrations of Gal4 into the
let-7/mir-100/mir-125
locus
(
Sokol
et al
., 2008
) and the
mir-263a
and
mir-263b
loci (
Hilgers
et al
., 2010
)
have been useful to report on their expression, as well as to manipulate gene
expression within miRNA-expressing cells. Finally, it is worth recalling that
as many miRNA loci are
P
element hotspots, many of these were hit as
enhancer traps in the pre-miRNA era. As mentioned in
Section 2.2
,a
lacZ
insertion in
mir-263a/bereft
was critical to reveal its sensory organ-specific
expression (
Hardiman
et al
., 2002
).
5.3. Genetically encoded sensors of miRNA activity
The above methods have collectively been powerful in analyzing miRNA
expression in the
Drosophila
system. However, one potential limitation is
that these strategies do not directly report on miRNA activity. Given the
growing appreciation of posttranscriptional regulation of miRNA proces-
sing and/or function (
Kim
et al
., 2010
;
Siomi and Siomi, 2010
), it is useful
to be able to monitor miRNA activity
in vivo
. Such an approach was
miRNA activity and vice versa. (C) Schematic representation of Drosophila wing imaginal
discs, illustrating how miRNA reporters and sensors reflect miRNA activity in over-
expression and loss-of-function scenarios. (i) Overexpression of the miRNA in a defined
stripe of cells (red) leads to cell-autonomous repression of the reporter (green); neigh-
boring non-miRNA-expressing cells serve as an internal control for the experiment.
(ii) Mutant clones of a miRNA (negatively marked by absence of red staining) derepress
the miRNA sensor (green), indicating loss of an endogenous miRNA:target repression
event. Homozygous mutant (
/
) and wild-type cells (
þ
/
þ
) are generated within
an otherwise heterozygous animal. Again, cell-autonomy of the sensor derepression
provides stringent controls for the experiment.
