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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
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