Sonic hedgehog (Shh) plays an integral role in both the anteriorposterior (A-P) patterning and expansion of developing vertebrate limbs through a feedback loop involving Fgfs, Bmps, and Gremlin. In bat limbs A-P patterning and the size of the digital field are unique. The posterior digits of the forelimb are elongated and joined by tissue, whereas the thumb is short. The hindlimb digits often are uniform in length. Here, we reveal novel expression patterns for Shh and its target, Patched 1 (Ptc1), during limb development in two bat species. Early Shh expression in the zone of polarizing activity is wider in the bat forelimb than in the mouse forelimb, correlating with the reported expansion of Fgf8 expression in the apical ectodermal ridge and the early loss of symmetry in the bat forelimb. Later in limb development, Shh and Ptc1 expression is reinitiated in the interdigital tissue. Shh is graded along the A-P axis in forelimb and is expressed uniformly at a lower level across the hindlimb interdigital tissue. We also show that the reported Fgf8 expression in the interdigital tissue precedes the expression of Shh. We propose that the reinitiation of Shh and Fgf8 expression in bat limbs reactivates the Shh-Fgf feedback loop in the interdigital tissue of stage 16 bat embryos. The cell survival and proliferation signals provided by the Shh-Fgf signaling loop probably contribute to the lengthening of the posterior forelimb digits, the survival of the forelimb interdigital webbing, and the extension of the hindlimb digits to a uniform length.Miniopterus natalensis ͉ Carollia perspicillata ͉ Patched1 ͉ Fgf8 ͉ evo-devo
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 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.
Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus , revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis ( Mtb ) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.
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