Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in theHoxDLocus
Authors: Booker Betty M. , Friedrich Tara , Mason Mandy K. , VanderMeer Julia E. , Zhao Jingjing , Eckalbar Walter L. , Logan Malcolm , Illing Nicola , Pollard Katherine S. , Ahituv Nadav 
Published: December 2015

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that fiveMyotis lucifugusBARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to theHoxDcluster and had robust forelimb expression for theM. lucifugussequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis ofHoxd10-Hoxd13inMiniopterus natalensisbats showed a high-forelimb weak-hindlimb expression forHoxd10-Hoxd11, similar to the expression trend observed forM. lucifugusBAR116 in mice, suggesting that it could be involved in the regulation of the batHoxDcomplex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing.

Author Summary

The limb is a classic example of vertebrate homology and is represented by a large range of morphological structures such as fins, legs and wings. The evolution of these structures could be driven by alterations in gene regulatory elements that have critical roles during development. To identify elements that may contribute to bat wing development, we characterized sequences that are conserved between vertebrates, but changed significantly in the bat lineage. We then overlapped these sequences with predicted developing limb enhancers as determined by ChIP-seq, finding 166 bat accelerated sequences (BARs). Five BARs that were tested for enhancer activity in mice all drove expression in the limb. Testing the mouse orthologous sequence showed that three had differences in their limb enhancer activity as compared to the bat sequence. Of these, BAR116 was of particular interest as it is located near theHoxDlocus, an essential gene complex required for proper spatiotemporal patterning of the developing limb. The bat BAR116 sequence drove robust forelimb expression but the mouse BAR116 sequence did not show enhancer activity. These experiments correspond to analyses ofHoxDgene expressions in developing bat limbs, which had strong forelimb versus weak hindlimb expression forHoxd10-11. Combined, our studies highlight specific genomic regions that could be important in shaping the morphological differences that led to the development of the bat wing.