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Abstract:
Paired related homeobox 1 gene (Prrx1) encodes for a transcrip- tional coactivator critical for skeletal development, particularly in the limbs, craniofacial region and the inner ear. An enhancer element located upstream of Prrx1 was shown to drive the expression of the reporter gene in the limb mesenchyme. The replacement of the mouse enhancer by the orthologous region from bats caused the elongation of mouse forelimbs, suggesting that variations in Prrx1 regulation may play a role in the evolutionary differences between the mouse and the bat limbs. How- ever, this enhancer alone does not account for the entire Prrx1 expression pattern and little more is known about its regulation. To decipher Prrx1 cis-regulation, we cloned individual enhancer candidates based on limb-specific p300 sites and histone modifi- cation marks upstream of a lacZ reporter gene driven by a mini- mal b-globin promoter. We plan to improve the existing transgenic reporter assay in the mouse by using phiC integrase- mediated site-specific integration at the Hipp11 locus. Site-specific integration eliminates variations arising from positional effects and transgene copy number. In parallel, we will examine large genomic regions by testing lacZ-tagged mouse- and bat-derived bacterial artificial chromosomes (BACs) within the Prrx1 topo- logical domain. Additionally, we have access to DNA from multiple individuals of two related bat species, the Greater and the Lesser mouse-eared bats (Myotis myotis and Myotis blythii oxy- gnathus). Taking advantage of the available genomic data on mouse fore- and hindlimb-specific active chromatin marks and transcription factor binding profiles, we will perform deep sequencing of the homologous regions in bat to identify sequence differences between mouse and bat in putative limb regulators. We will carry out further transgenic reporter assays on limb enh- ancers showing major sequence dissimilarities between bats and mice, especially those near Prrx1, to determine if such mutations lead to expression variations. In summary, the results of trans- genic experiments will shed light on the cis-regulation of Prrx1, while the data obtained from the deep sequencing may indicate how such differences could have evolved. This will in turn open a way to better understanding of how cis-regulation of genes is achieved and modulated during the evolution of various morphological forms.
