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upregulation of Hoxb-8 could be identified in this context, and whether
additional target derepression contributes to the phenotype remains unclear
( Asli and Kessel, 2010 ). Given this potential role in spinal cord generation, it is
interesting to note that miR-196 has also been shown to regulate key stages of
the spinal cord regenerative process in the axolotl, Ambystoma mexicanum ,
however, potentially not through Hox gene regulation in this case ( Sehm
et al ., 2009 ). In the developing forelimb, Hoxb-8 acts downstream of retinoic
acid (RA) and can act upstream of the key A-P patterning molecule Sonic
hedgehog ( Charite et al ., 1994 ; Stratford et al ., 1999 ). Interestingly, this
genetic hierarchy is not conserved in the hindlimb ( Stratford et al ., 1999 )
given the absence of Hoxb-8 expression at this site and the inability of ectopic
RA to induce hindlimb Hoxb-8 expression. The tapering of Hoxb-8 expres-
sion caudally is achieved primarily by transcriptional mechanisms; however,
the presence of mir-196 -directed Hoxb-8 cleavage products in the hindlimb
indicates the incomplete nature of this regulation and confirms an endoge-
nous role for this miRNA interaction in reinforcing repression at this site
( Hornstein et al ., 2005 ). In this context, miR-196 apparently acts in a failsafe
mode: it is redundant with transcriptional repression because Hoxb-8 does not
accumulate in dicer mutant hindlimbs unless exogenous RA is added.
These failsafe mechanisms are intuitive with the genomic positioning of
miR-196 and parallel the molecular repression of Ubx by miR-iab-4/8 ;
however, a more direct role for miR-196 in defining functional levels of
Hoxb-8 within its normal domain of expression has been suggested
( McGlinn et al ., 2009 ). miR-196 knockdown studies in chick, utilizing
antagomiR injection into precursors of the axial skeleton at stage 10-12
of development, resulted in an anteriorly expanded domain of Hoxb-8 and
homeotic transformation of the last cervical vertebrae toward a thoracic
identity. This suggests that miR-196 acts to raise the threshold of Hoxb-
8 expression required to induce morphological change, thereby preventing
small fluctuations in gene expression and increasing the robustness of this
genetic program. No alterations were observed at more posterior transition
points. This is likely to be due to a technical reason, since dilution of the
antagomiR is possibly below the threshold of functional activity by the time
more posterior vertebrae are being specified. Alternatively, in this context,
ectopic expression of a more anterior Hox gene may be silenced by
additional mechanisms reinforcing posterior prevalence.
7. Summary and Conclusions
The efforts over recent years to integrate a whole new layer of
regulatory control within the previously established Hox genetic hierarchies
have yielded fascinating insight into the complexity of these processes. The
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