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In-Depth Information
miR-196
in posterior derivatives of all three germ layers. However, detailed
section
in situ
analysis remains to be performed. While the paraxial expres-
sion limits of Hox genes are quite dynamic over time, the anterior limit of
miR-196a
defined by these approaches at embryonic day 9.5 (E9.5) (
Asli
and Kessel, 2010
;
Kloosterman
et al
., 2006
) is quite caudal when compared
with genomically adjacent Hox genes (e.g.,
Hoxb-9
expression up to pre-
vertebra 3;
Chen and Capecchi, 1997
). This could reflect differential
upstream regulation or, alternatively, differential stability of the miRNA
relative to Hox transcripts, a point to consider in understanding the com-
plexity of target regulation. Microarray analysis of the developing mouse
limb buds identified a more than 20-fold enrichment of
miR-196a
in the
hindlimb when compared to forelimb (
Hornstein
et al
., 2005
). High-
throughput direct sequencing at three developmental stages (
Chiang
et al
.,
2010
) showed quite striking differences in the relative expression levels of
individual
miR-196
family members.
miR-196a
was more abundant at
E12.5 than earlier stages, while
miR-196b
exhibits very high relative levels
of expression at E7.5 (
Chiang
et al
., 2010
). These data suggest that
miR-196
can potentially act at presomite stages, at least in mouse.
6.3. Function
Contrary to loss-of-function observations for
miR-10
and
miR-iab-4/8
,accu-
mulating evidence supports a dramatic, nonredundant developmental role for
the
miR-196
family of miRNAs. Much of this work has centered on the
regulation of one key gene,
Hoxb-8
, which inmouse has a single target binding
site exhibiting extensive complementarity to
miR-196
within its 3
0
UTR
(
Yekta
et al
., 2004
). Akin to plant miRNA regulation, this suggests
miR-196
binding will initiate endonucleolytic cleavage between nt 10 and 11 and
subsequent mRNA degradation in a RNAi-like manner. Importantly, such
Hoxb-8
cleavage products have been identified in the early mouse embryo
(
Mansfield
et al
., 2004
;
Yekta
et al
., 2004
) confirming this mode of regulation
in vivo
. Genetic manipulation of
Hoxb-8
by both gain- and loss-of-function
highlights the capability of this gene to affect patterning of the skeleton,
nervous system, and A-P symmetry in the forelimb (
Charite
et al
., 1994
;
Fanarraga
et al
., 1997
;
Greer and Capecchi, 2002
;
Holstege
et al
., 2008
;
van
den Akker
et al
., 1999
), and recent evidence suggests a functional interaction
between
miR-196
and
Hoxb-8
in each of these developmental contexts.
Recent studies indicate that
miR-196
acts to clear or repress unwanted
Hoxb-8
activity from both neural and mesodermal tissue caudal to the domain
of
Hoxb-8
expression (
Asli and Kessel, 2010
;
Hornstein
et al
., 2005
). In the
lumbar region of the chick CNS, enforced
Hoxb-8
expression caudal to
its normal domain compromises motor neuron differentiation, highlighting
the need to tightly regulate posterior
Hoxb-8
boundary formation.
In ovo miR-
196
antisense oligo electroporation phenocopies these defects; however, no