Evolution of limb development in mammals: miRNAs and lncRNAs ASSOCTIATED with the regulation of HOX genes expression during marsupial limb development

Abstract

Limbs have been extensively studied to understand the developmental basis of morphological evolution, since they are extremely variable structures crucial for adaption to different habitats. Marsupials present unique limb characteristics, such as highly modified feet in Peramelemorpha and Diprotodontia orders, whose digits I is absent, II and III are syndactylous, and IV is elongated, and heterochronic development, since the forelimbs are formed much before the hindlimbs. HOX genes are crucial to the patterning of morphological structures and their roles and genetic regulatory mechanisms during limb development is well described in mouse, the traditional model organism among eutherian mammals. Various enhancers, as well as non-coding RNAs (ncRNAs), play intricate finely coordinated roles of HOX expression regulation during limb development. Some ncRNAs were already identified that target tammar wallaby Macropus eugenii HOX genes, but very little is known about regulatory mechanisms of HOX expression in marsupial. Here, we propose to investigate limb microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) associated to M. eugenii HOX clusters to define whether differential regulatory elements and their specific spatio-temporal expression account for marsupial digit modifications. miRNAs and lncRNAs will be annotated by phylogenetic footprinting to define conserved regions and their transcription will be validated by reverse transcriptase PCR (RT-PCR). Investigation of the spatio-temporal expression of the ncRNAs in the developing tammar limb will also be performed by quantitative real-time PCR (qPCR) and whole mount and section in situ hybridization. (AU)