Biology Reference
In-Depth Information
approach using chemical mutagenesis or transposon screening has yet to be
applied to
Drosophila
miRNAs, but there is no particular reason to believe
that it would not be profitable.
A quick-and-dirty version of the dominant screen utilizes the
Drosophila
deficiency collection, which allows one to assess genome-wide, and within
only a few hundred crosses, the effects of removing one gene copy on a
genetically sensitized phenotype. This strategy was recently used to great
effect with the
Drosophila
ortholog of
mir-1
(
King
et al
., 2011
), a miRNA
that is deeply conserved across the animals both in sequence and in specific
expression in heart and muscle. Previously, misexpression of
mir-1
in the
center of the developing wing, using
dpp-Gal4
, was found to generate
patterning and morphogenesis defects including cell-autonomous loss of
wing tissue (
Kwon
et al
., 2005
). As this presumably resulted from the
ectopic repression of miR-1 target genes, such as the Notch ligand
Delta
,
it was hypothesized that further reducing the dosage of relevant target genes
should enhance the severity of wing phenotypes. Therefore, a
dpp-
Gal4
>
UAS-mir-1
stock was crossed to the deficiency kit and scored for
dominant enhancers that exhibited greater loss of wing tissue.
Out of 284 deficiency lines covering about 70% of the euchromatic
genome, 32 enhancers were recovered (
King
et al
., 2011
). These included
two conserved targets of miR-1,
Delta
and
mirror
, and novel interacting
partners such as
kayak
, encoding a leucine-zipper transcription factor and a
component of the planar cell polarity (PCP) pathway. Although the wing is
clearly not a physiological location of miR-1 function, these interactors
could be examined in the relevant tissues that endogenously express miR-1.
Indeed, alteration of miR-1 dosage by null mutants or by overexpression
disrupted cardioblast cell polarity in the fly heart, similar to
kayak
mutants.
Importantly, heterozygosity of
kayak
could mitigate the loss of miR-1,
suggesting that dysregulation of Kayak partly accounts for these defects in
mir-1
null mutants. These findings illustrate the power of
Drosophila
genetics
to uncover unexpected functional connections, in an unbiased and system-
atic manner.
5. Detecting miRNA Expression and
Activity In Vivo
Temporal-, tissue-, or cell-specificity of miRNA expression can be
assessed by several strategies. These include Northern analysis or small
RNA library cloning from different sources (
Berezikov
et al
., 2011
;
Leaman
et al
., 2005
;
Ruby
et al
., 2007b
),
in situ
hybridization to primary
miRNA transcripts (
Aboobaker
et al
., 2005
;
Kosman
et al
., 2004
) or mature
miRNAs (
Li and Carthew, 2005
;
Sokol and Ambros, 2005
). We focus this
