Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus

Abstract

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 five Myotis lucifugus BARs 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 the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing.

Fig 1. Computational pipeline to identify bat accelerated regions.

Limb ChIP-seq peaks were unified, then overlapped with conserved regions and then scored with PhyloP values (0 to 20) by comparing Myotis lucifugus, Pteropus vampyrus, Myotis davidii, and Pteropus alecto to 48 available vertebrate genomes. A total of 166 BAR elements were identified as accelerated regions in bats [false discovery rate (FDR) < 0.05].

Fig 2. UCSC Genome Browser snapshots showing the location of each mouse BAR (BAR2, BAR4, BAR61, BAR97, BAR116).

The BAR4 browser snapshot displays the ‘TAD Domain’ [70] track (in green) containing a cluster of five Spry1 BARs. The bottom panel displays the mouse HoxD locus and a zoom-in of the overlapping regions between mouse BAR116 (~1.9kb in length) and mouse CNS9 (~700 bp in length) [75]. Both sequences were negative for limb enhancer activity in mouse assays. The HoxD ‘Telomeric Domain’ track is also shown in green.

Fig 3. M. lucifugus BARs are active enhancers in the developing mouse limb.

A representative mouse embryo (E12.5) showing the limb enhancer expression pattern for each M. lucifugus BAR. Nearby limb-associated gene names are written in parenthesis with the number of embryos showing a limb expression pattern given below. M. lucifugus BARs were scored by the number of transgenic LacZ positive limb/ LacZ positive expressing embryos. All five M. lucifugus BARs have LacZ expression in the limbs.

Fig 4. Comparison of enhancer expression patterns for bat and mouse sequences in forelimb and hindlimb.

Representative mouse (E12.5) forelimbs (FLs) and hindlimbs (HLs) showing both M. lucifugus BAR and mouse BAR expression pattern. Three M. lucifugus BAR sequences (BAR4, 97, and 116) show differences in expression patterns as compared to the mouse BAR sequence. BAR61 (Shh) retains a similar expression pattern for both the bat and the mouse BAR sequences. Nearby limb-associated gene names are written in parenthesis next to the BAR ID.

Fig 5. The bat-mouse BAR116 composite sequence displays a loss of tissue specificity in the limbs.

The M. lucifugus sequence (blue) was replaced with the mouse (red) PhastCons sequence encompassing the BAR element. While the M.lucifugus sequence drove consistent enhancer activity in the limbs at E12.5, both the mouse sequence and the synthesized bat-mouse BAR116 composite sequence failed to drive consistent enhancer activity, suggesting that the M. lucifugus BAR116 sequence is essential for limb enhancer expression.

Fig 6. HoxD gene expression patterns in bats and mice.

Hoxd10-13 bat embryonic forelimb (A-L) and hindlimb (A’-L’) expression pattern at CS15-CS17 compared equivalently staged mouse (E12.0-E13.5) forelimb (M-X) and hindlimb (M’-X’) expression (scale bar represents 500 μm).