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reminiscent of various syndromes in which nuclear receptors behave as
constitutive repressors because of ligand insufficiency. Impenetrant hetero-
chronic phenotypes for null mutants are not unique to
daf
-
12
. Most other
heterochronic mutants that affect L2/L3 temporal programs also display
impenetrant phenotypes, reflecting functional redundancy or multiple layers
of regulation of microRNA transcription, maturation, and activity (
Ding,
Spencer, Morita, & Han, 2005; Grishok et al., 2001; Hammell, Lubin,
Boag, Blackwell, & Ambros, 2009; Morita & Han, 2006; Pepper et al.,
2004; Xia, Huang, &Zhang, 2009
). In support of this idea, the heterochronic
phenotypes of
daf
-
12
nulls are strongly enhanced in double mutant combi-
nations with these loci.
An important insight into how DAF-12 regulates these transitions
came with the observation that mutants showed reduced RNA levels of a
number of microRNAs of the
let
-
7
family (
Bethke, Fielenbach, Wang,
Mangelsdorf, & Antebi, 2009; Esquela-Kerscher et al., 2005; Hammell,
Karp, & Ambros, 2009
). Additionally, a triple deletion of the
let
-
7
related
microRNAs,
mir
-
48
,
mir
-
84
,
mir
-
241
gave phenotypes similar to
daf
-
12
mutants, in which seam cells repeat L2 programs at the L3 stage giving rise
to extra cells (
Abbott et al., 2005
). This led to the hypothesis that DAF-12
directly regulates these microRNAs. Indeed, DA/DAF-12 were shown
to potently activate
mir
-
84
and
mir
-
241
in human cell culture and bind directly
to the microRNA promoters. Similarly in worms,
mir
-
84
and
mir
-
241
expres-
sion showed DA and DAF-12 dependence in specific tissues (
Bethke et al.,
2009
). Notably,
mir
-
84
expression in seam cells was abrogated in
daf
-
12
null
mutants, suggesting that heterochronic phenotypes in the epidermis arise in
part from a failure to activate this microRNA. Consistent with a downstream
role, the microRNA target, HBL-1/hunchback, was inappropriately elevated
in
daf
-
12
mutants, particularly in
daf
-
12
(
rh61
) LBD truncation mutants and to
a lesser extent in null mutants (
Bethke et al., 2009; Hammell, Karp, &Ambros,
2009
). Moreover loss of
hbl
-
1
completely suppressed the extra seam cell
phenotype seen in
daf
-
12
mutants, showing that
daf
-
12
works through the
let
-
7
related microRNAs and
hbl
-
1
(
Fig. 7.1
A).
The interactions described above reveal DAF-12's influence on micro-
RNA expression during reproductive development, but what about during
dauer arrest? In DA-deficient
daf
-
9
mutants, DAF-12 switches to repressor
mode, animals enter the dauer stage, and overall expression of the
let
-
7
fam-
ily microRNAs in most tissues is dramatically repressed (
Bethke et al., 2009;
Hammell, Karp, & Ambros, 2009
). In fact, DAF-12's function as a repressor
in these circuits is more potent than its role as activator. Similarly, repression
