Project description:Bilaterian animals differ from other metazoans in their apparent bilateral symmetry and the development of a third germ layer. Both might have facilitated the evolution of the diverse and complex bilaterian body plans. The first cnidarian genome sequence revealed that despite their morphological simplicity, this sister group to all bilaterians shares an immense genomic complexity with vertebrates. This suggested that it might have been the complexity of gene regulation which increased during the evolution of bilaterians. We compared the gene regulatory landscape of cnidarians and bilaterians. To this end we generated the first genome-wide prediction of gene regulatory elements and profiled five epigenetic marks in a non-bilaterian animal, the cnidarian Nematostella vectensis. We found that the location of chromatin modifications relative to genes and distal enhancers is conserved among eumetazoans. Surprisingly, the genomic landscape of gene regulatory elements is highly similar between Nematostella and bilaterian model organisms. This suggests that complex regulation of developmental gene expression evolved in eumetazoans without a major increase in complexity in bilaterians. ChIP-seq of p300, RNA Pol2, and five histone modifications in Nematostella vectensis.
Project description:While the vertebrate body plan is highly conserved amongst all species of this taxon, extreme variations thereof can be documented in snakes, which display both an absence of limbs and an unusually elongated trunk. As Hox genes are strong candidates both for the making and the evolution of this body plan, their comparative study in such a morphologically diverged group is informative regarding their potential causative importance in these processes. In this work we use an interspecies comparative approach where different aspects of regulation at the HoxD locus are investigated. We find that although spatial collinearity and associated epigenetic mark dynamics are conserved in the corn snake, other regulatory modalities have been largely restructured. A BAC transgenic approach indeed revealed that, while the majority of mesodermal enhancers in vertebrates appear to be mostly located outside of the cluster, the corn snake contains most mesodermal trunk enhancers within the HoxD cluster. We also find that, despite the absence of limbs and an altered Hoxd gene regulation in external genitalia, the bimodal chromatin structure at the corn snake HoxD locus is maintained. The analysis of particular enhancer sequences initially defined in the mouse and further isolated at the snake orthologous locus showed differences in their specificities for the limb and genital bud expression. Of particular interest, a snake counterpart of a mouse limb-only enhancer sequence evolved into a genital-only enhancer. Such a regulatory exaptation suggests that enhancer versatility may have been an important factor to accompany the transition towards the snake body plan. These results show that vertebrate morphological evolution is likely to have been associated with extensive reorganization at the HoxD regulatory landscapes while respecting a very conserved general regulatory framework.
Project description:We generated RNA-seq data of Drosophila simulans and Drosophila mauritiana developing male genitalia in order to identify expression level differences between these species. These species are closely related, yet have dramatic differences in their male genital morphologies. Three independent RNA-seq library replicates were generated for Dsim w501 and Dmau D1 developing male genitalia. Flies were reared under the above conditions, and white pre-pupae collected. Males were selected using gonad size and allowed to develop in a humid container at 25ºC until stages 2 and 4.5 (see staging guide in (Hagen et al., 2019); tartan underlies the evolution of Drosophila male genital morphology). Between these stages, the claspers develop from a ridge structure to a distinct appendage separate from the surrounding tissue, and the posterior lobe has begun to extend outwards from the lateral plate primordia (Hagen et al., 2019). The heads of pupae were impaled with a needle onto a charcoal agar plate and submerged in 1xPBS. Dissection scissors were used to remove the distal tip of the pupal case and the outer membrane, and pressure applied to the abdomen to allow the developing genitalia to be quickly expelled from the pupal case and dissected away from the abdomen. Note that the entire genital arch, including internal genital organs (but not including abdominal tissue), was isolated for RNA extraction. The genitalia from fifteen males from each stage were collected and placed directly into TRIzol. RNA was then extracted using standard procedures. Quality and quantity of RNA was verified using a Qubit, and samples were sent to the Centre for Genomic Research at the University of Liverpool where dual-indexed, strand-specific RNA-seq libraries were prepared using NEBNext polyA selection and Ultra Directional RNA preparation kits. Samples were then sequenced using Illumina HiSeq 4000 (paired-end, 2x150 bp sequencing). Dsim w501 and Dmau D1 reads were mapped against reannotated reference coding sequences (Torres-Oliva et al., 2016).
Project description:The existence of homologies between fins and tetrapod limb skeletal parts as well as of the nature of those mechanisms underlying the transition of the former towards the latter, have been a rich source of discussion for more than a century. While the recent use of gene expression patterns to try and infer evolutionary scenarios has been a popular and successful approach, in particular the distribution of Hox transcript domains, it has failed to provide clearcut evidence as to whether fishes do have bony elements related by ancestry to tetrapod hands and feet. In tetrapods, posterior Hoxd genes transcription in digits is controlled by a well-characterized series of enhancers forming a large regulatory landscape, which has its syntenic counterpart in zebrafish. We show here that the deletion of the orthologous landscape in fishes does not affect the transcription of these genes in fin buds. Instead, it abrogates hoxd expression in the cloaca, an essential structure related to the mammalian uro-genital sinus. We also report that Hoxd gene function in the mammalian uro-genital sinus depends on enhancers located in the same regulatory landscape and thus conclude that an ancestral Hox ‘cloacal’ regulation was co-opted, in tetrapod, as a playground to subsequently accompany the evolution of both external genitals and digits regulatory landscapes, along with the emergence of these developmentally and phylogenetically related structures.
Project description:The existence of homologies between fins and tetrapod limb skeletal parts as well as of the nature of those mechanisms underlying the transition of the former towards the latter, have been a rich source of discussion for more than a century. While the recent use of gene expression patterns to try and infer evolutionary scenarios has been a popular and successful approach, in particular the distribution of Hox transcript domains, it has failed to provide clearcut evidence as to whether fishes do have bony elements related by ancestry to tetrapod hands and feet. In tetrapods, posterior Hoxd genes transcription in digits is controlled by a well-characterized series of enhancers forming a large regulatory landscape, which has its syntenic counterpart in zebrafish. We show here that the deletion of the orthologous landscape in fishes does not affect the transcription of these genes in fin buds. Instead, it abrogates hoxd expression in the cloaca, an essential structure related to the mammalian uro-genital sinus. We also report that Hoxd gene function in the mammalian uro-genital sinus depends on enhancers located in the same regulatory landscape and thus conclude that an ancestral Hox ‘cloacal’ regulation was co-opted, in tetrapod, as a playground to subsequently accompany the evolution of both external genitals and digits regulatory landscapes, along with the emergence of these developmentally and phylogenetically related structures.