MiRNAs have an interesting evolutionary history. While the transcription factor and signaling pathway spectrums are largely conserved from sponges to humans 
, miRNAs have been continuously added during the metazoan lineage 
. The rate of acquisition of new miRNAs has increased at key periods in evolution including the advent of bilaterians, vertebrates, eutherians, and primates 
. This has lead to the hypothesis that miRNA innovation might have contributed to increases in the morphological complexity of metazoans 
. Given that this study is the first investigation of miRNAs in multiple avian species, we began by asking whether any of the 186 miRNAs that we detected in the frontonasal neural crest of the chicken, duck, and quail might be specific to the avian lineage. We identified six mature miRNAs that appear to be specific to the avian lineage which has been evolving for nearly 120 million years () 
. We used PCR to confirm that five of these (miR-1559, miR-2131, miR-2131*, miR-2954, and miR-2954*) are conserved across, but are specific to, the entire avian lineage. These are the first described examples of validated avian-specific miRNAs and join several other examples of miRNAs that have independently evolved within defined vertebrate lineages 
. However, for most species-specific miRNAs it still remains to be determined whether they are an evolutionary dead-ends or have functional roles in development.
Intriguingly, miRNAs might also have a role in species-specific diversification. While humans 
and mice 
show negative selection against mutations that destroy conserved miRNA binding sites, the morphologically divergent cichlids of Lake Malawi have increased levels of polymorphism in predicted miRNA binding sites within 3′ UTRs 
. However, the divergence times within these lineages varies drastically—approximately 370,000 years for humans 
, 23 million years for mice 
, and 1–2 million years for cichlids 
In remarkable contrast to the relatively unchanged pattern of mRNA expression we previously observed in these neural crest samples 
, miRNA expression is dramatically different between developmental stages before (HH20) and after (HH25) morphological variation in the beak is evident. The patterns of differentially expressed miRNAs (Table S7
) are consistent with the following model (summarized in ). At HH20, both the chicken and the duck have a multipotent, proliferative NC population that expresses high levels of the miR-302 family as well as high levels of miR-222 (Table S7
). These miRNAs promote an undifferentiated fate, in the case of miR-302 
, and proliferation via repression of p27KIP1
, in the case of miR-222 
. By HH25, chicken NC cells have adopted molecular signatures of differentiation. At the same time as the miR-302 family and miR-222 are down-regulated, eleven miRNAs related to the let-7 family are up-regulated, as well as 2 additional miRNAs associated with cellular differentiation (Table S7
. By HH26, chicken facial primordia express molecular markers of the bones and skeleton that will eventually form the adult face 
A model of differences in neural crest differentiation and bone formation in duck and chicken.
Duck NC cells at HH25 have down-regulated the miR-302 family and up-regulated some of the miRNAs associated with cellular differentiation (i.e. the let-7 family), though not as many as chicken NC (Table S7
). However, in contrast to the chicken, duck NC still express high levels of miR-222, and this may act to maintain a higher proliferation rate via continued repression of p27KIP1
. The duck also has higher levels of the miR-23a-27a-24-2 cluster (Table S7
). Each of these miRNAs can independently repress the bone-promoting transcription factor SATB2
, and thus the duck may also have a delay in bone formation, as NC cells continue to proliferate.
Taken together, these miRNA changes, including differential expression of let-7, miR-302, and miR-30 families (Table S7
), indicate that the HH20 to HH25 developmental window may be a critical transition phase in which multipotent NC cells begin to differentiate to form the various tissues of the face. In addition, given that a number of miRNAs related to let-7 and cellular differentiation are only up-regulated in the chicken and quail at HH25 (Table S7
), the timing of this transition may be slightly delayed in the morphologically different duck, perhaps allowing a more prolonged period of proliferation. This is consistent with current theories that differential regions and levels of proliferation can influence the depth, width, and curvature of the beak 
and that miRNAs function during the transitions between different cellular states 
We evaluated one miRNA:mRNA target pair. We speculated that differences in miR-222 levels in the duck versus chicken at HH25 could regulate morphological differences in the beak via its target, the cell cycle regulator p27KIP1
. Our hypothesis was that higher levels of miR-222 in HH25 duck, and the resulting decrease of p27 protein, would result in an increased proliferation level. On the other hand, lower miR-222 levels in the beaked chicken and quail could lead to a release of p27 repression and a consequent decrease in proliferation. This model is in agreement with previous analyses that identified higher proliferation levels in HH26-HH31 duck bills compared to chicken beaks 
. Our analyses of p27 protein and mRNA levels agree with this model: p27 protein is expressed at similar levels in the FNP of the chicken and duck while they are morphologically similar. By HH25, when species-specific morphologies are evident, p27 protein levels are dramatically different in the chicken and duck, in patterns consistent with alterations in miR-222 expression levels. These protein changes are not associated with changes in p27
mRNA, indicating that post-transcriptional mechanisms (such as miRNA inhibition) are important for proper regulation of this cell cycle regulator.
While it is clear that changes in mRNA levels of the BMP/TGF-beta, calmodulin, and Wnt signaling pathways influence beak morphology 
, and it is very likely that many more mRNAs differ across this developmental window, miRNAs add another layer to the regulation of species-specific morphogenesis. Our study provides the first insights into which specific miRNAs play roles in facial morphogenesis and the developmental processes that they may regulate.