Abstract
Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.
Highlights
Most metazoans are able to repair injured tissues at least to some extent; the capacity to regenerate whole body parts after injury or amputation is limited to a handful of organisms [1]
One of the most complex types of regeneration is seen in limb regeneration, in which the wound rapidly closes via migration of neighboring epithelial cells and underlying mesenchymal cells change their morphology and proliferate to form a cell mass known as the blastema [12,13]
We show by in vitro luciferase assays that A. mexicanum (‘‘Amex’’) Jag1 is directly targeted by miR-21
Summary
Most metazoans are able to repair injured tissues at least to some extent; the capacity to regenerate whole body parts after injury or amputation is limited to a handful of organisms [1]. Microarray analyses revealed a set of miRNAs that consistently displayed high statistical support and large fold-changes of differential expression between mid-bud regenerating blastemas and mature non-regenerating stump tissue.
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