Transcriptomic and epigenomic characterization of the developing bat wing

Abstract

The order Chiroptera, commonly known as bats, is the only group of mammals to have evolved the capability of flight. They are estimated to have diverged from their arboreal ancestors ~51 million years ago 1 . Their adaptions for flight include substantial specialization of the forelimb, characterized by the notable extension of digits II-V, a decrease in wing bone mineralization along the proximal-distal axis, and the retention and expansion of interdigit webbing, which is controlled by a novel complex of muscles 2,3 . Bat hindlimbs are comparatively short, with free, symmetrical digits, providing an informative contrast that can be used to highlight the genetic processes involved in bat wing formation. Previous studies that examined gene expression in developing bat forelimbs and hindlimbs reported differential expression of several genes, including Tbx3, Brinp3, Meis2, the 5′ HoxD genes and components of the Shh-Fgf signaling loop, suggesting that multiple genes and processes are involved in generating these morphological innovations [4][5][6][7][8] . Gene regulatory elements are thought to be important drivers of these changes: for example, replacement of the mouse Prx1 limb enhancer with the equivalent bat sequence resulted in elongated forelimbs 9 . However, an integrated understanding of how changes in regulatory elements, various genes and signaling pathways combine to collectively shape the bat wing remains largely elusive.To characterize the genetic differences that underlie divergence in bat forelimb and hindlimb development, we used a comprehensive, genome-wide strategy. We generated a de novo whole-genome assembly for the vesper bat, M. natalensis, for which a well-characterized stage-by-stage morphological comparison between developing bat and mouse limbs is available 10 . In this species, the developing forelimb noticeably diverges from the hindlimb from developmental stages CS15 and CS16, with clear morphological differences seen at a subsequent stage, CS17 (ref. 10). This developmental window is equivalent to embryonic day (E) 12.0 to E13.5 in mouse 4,10 . M. natalensis embryos were obtained and transcriptomic (RNA-seq) data and ChIP-seq data for both an active (acetylation of histone H3 at lysine 27, H3K27ac; refs. 11,12) and a repressive (trimethylation of histone H3 at lysine 27, H3K27me3; ref. 13) mark were generated for these three developmental stages (Fig. 1). Bats are the only mammals capable of powered flight, but little is known about the genetic determinants that shape their wings. Here we generated a genome for Miniopterus natalensis and performed RNA-seq and ChIP-seq (H3K27ac and H3K27me3) analyses on its developing forelimb and hindlimb autopods at sequential embryonic stages to decipher the molecular events that underlie bat wing development. Over 7,000 genes and several long noncoding RNAs, including Tbx5-as1 and Hottip, were differentially expressed between forelimb and hindlimb, and across different stages. ChIP-seq analysis identified thousands of regions that are differentiall...

The origins, scaling and loss of tetrapod digits

“…These results suggest a putative molecular mechanism for the control of differential bone growth and species-specific skeletal scaling [127]. Indeed, pathway analysis of RNASeq data from bat fore-and hindlimbs [130] raises the possibility that Igf-1 may also play a role in morphological divergence of the wing in late fetal development.…”

“…Transcriptome and chromatin modification assays highlighted thousands of loci that are differentially expressed in the fore-versus hindlimb over developmental time including a number of known limb patterning genes (5 0 -Hoxd, Tbx3-5, Pitx1, Msx1 and 2 and Meis2) and genes not previously identified in the limb, including Fam5c, Mllt3 and Lhx8 [130,131]. Speculation on the functions of these genes in bat limb development awaits a genome editing approach in bat or methods to replace homologous stretches of sequence in the mouse.…”

Bat Biology, Genomes, and the Bat1K Project: To Generate Chromosome-Level Genomes for All Living Bat Species

“…Several genomic studies have recently been carried out on developing bat embryonic limbs that identified numerous candidate wing development-associated genes and regulatory elements (87)(88)(89). Importantly, complete genomes are critical to this task, which requires tracking changes in expression encoded by regulatory regions, and not just protein-coding changes.…”

Exploring the bZIP regulatory network in the sponge Amphimedon queenslandica: insights into the ancestral animal regulatory genome

“…Antibodies against these modifications of the histone H3 tail have also successfully been used in a range of eukaryotic species (Barraza et al, 2015;Eckalbar et al, 2016;Harmeyer et al, 2015;Liu et al, 2007;Sebe-Pedros et al, 2016). Since the amino acid sequence of A. queenslandica histone H3 is identical to its bilaterian orthologues (Appendices 5.3, 5.6) and A. queenslandica also have the relevant histone methyltransferases and acetyltransferases (Appendix 5.7), these antibodies are prone to recognise the correct epitopes in this sponge.…”

“…This time point corresponds to larvae that are competent to settle and undergo metamorphosis (10-12 hours post emergence from the brood chamber) . The entire amino acid sequence of histone H3 is perfectly conserved between A. queenslandica and other eukaryotes where these antibodies have been used successfully (Barraza et al, 2015;Eckalbar et al, 2016;Harmeyer et al, 2015;Liu et al, 2007;Sebe-Pedros et al, 2016) (Appendix 5.3). I also used a custom-made affinity-purified rabbit polyclonal antibody, raised by GenScript Biotechnology (NJ, USA), against a peptide located outside the bZIP domain of AqATF4 (CTPKRTPQGRGGRKS).…”