Conservation of different mechanisms of Hox cluster regulation within chordates

[eng] In this thesis we have covered the importance of finding underlying conservation events to better understand the regulatory mechanisms of important development orchestrators like the Hox cluster. As an example of these non-evident conservation, we have shown two cases, as described below. The first case studied, after developing a software able to detect homologous long noncoding RNAs by means of microsynteny analyses, is the conservation of Hotairm1 in Chordata. For assessing the homology of this lncRNA, first we had to identify the lncRNA fraction within the B. lanceolatum transcriptome. With a reliable lincRNA dataset, we used our pipeline, LincOFinder, to identify orthologs between human and amphioxus through microsynteny. After the identification of Hotairm1 as one of the lincRNAs with conserved microsynteny, we used Xenopus as a proxy to analyse the homologies in the expression and the function. We had to proceed this way due to the difficulties associated with the inhibition of genes in B. lanceolatum, and the unavailability of expression patterns for Hotairm1 in the bibliography. After we successfully characterised Hotairm1 expression in amphioxus and Xenopus, we injected morpholino oligonucleotides to target and inhibit the splicing of Hotairm1 to promote an isoform imbalance. Through the phenotype obtained and the performing of qPCRs, we were able to deduct the mechanism of Hotairm1 and successfully relate this mechanism with the one described in human cells. With all the data obtained we were able to strongly suggest that the amphioxus Hotairm1 is homologous to the Xenopus and human Hotairm1, thus being conserved in most of the lineages within chordates. The second case studied was the conservation of the regulation of the Hox cluster mediated by Cdx. When analysing the B. floridae knockouts of Cdx and Pdx obtained using the TALEN technique, we found a severe phenotype of the developing larvae in Cdx-/- and a mild phenotype in Pdx-/-. The Cdx-/- phenotype consisted in the disruption of posterior gut development, as well as an underdevelopment of the postanal tail, coupled with a non-opening anus. When looking at changes in the expression of the Hox cluster in this Cdx-/- embryos, we found collinear misregulation of the expressed Hox genes, with the most anterior Hox cluster genes upregulated, and the most posterior ones downregulated. This is very similar to findings seen in triple morpholino knockdowns of the Cdx genes in Xenopus, indicating that in both, Xenopus and amphioxus, Cdx is regulating the Hox cluster through a homologous mechanism

New Genes Born-In or Invading Vertebrate Genomes

Which is the origin of genes is a fundamental question in Biology, indeed a question older than the discovery of genes itself. For more than a century, it was uneven to think in origins other than duplication and divergence from a previous gene. In recent years, however, the intersection of genetics, embryonic development, and bioinformatics, has brought to light that de novo generation from non-genic DNA, horizontal gene transfer and, noticeably, virus and transposon invasions, have shaped current genomes, by integrating those newcomers into old gene networks, helping to shape morphological and physiological innovations. We here summarized some of the recent research in the field, mostly in the vertebrate lineage with a focus on protein-coding novelties, showing that the placenta, the adaptative immune system, or the highly developed neocortex, among other innovations, are linked to de novo gene creation or domestication of virus and transposons. We provocatively suggest that the high tolerance to virus infections by bats may also be related to previous virus and transposon invasions in the bat lineage

Mathematical foundations for efficient structural controllability and observability analysis of complex systems

The relationship between structural controllability and observability of complex systems is studied. Algebraic and graph theoretic tools are combined to prove the extent of some controller/observer duality results. Two types of control design problems are addressed and some fundamental theoretical results are provided. In addition new algorithms are presented to compute optimal solutions for monitoring large scale real networks

Mutation of amphioxus Pdx and Cdx demonstrates conserved roles for ParaHox genes in gut, anus and tail patterning

Background: The homeobox genes Pdx and Cdx are widespread across the animal kingdom and part of the small ParaHox gene cluster. Gene expression patterns suggest ancient roles for Pdx and Cdx in patterning the through-gut of bilaterian animals although functional data are available for few lineages. To examine evolutionary conservation of Pdx and Cdx gene functions, we focus on amphioxus, small marine animals that occupy a pivotal position in chordate evolution and in which ParaHox gene clustering was first reported. Results: Using transcription activator-like effector nucleases (TALENs), we engineer frameshift mutations in the Pdx and Cdx genes of the amphioxus Branchiostoma floridae and establish mutant lines. Homozygous Pdx mutants have a defect in amphioxus endoderm, manifest as loss of a midgut region expressing endogenous GFP. The anus fails to open in homozygous Cdx mutants, which also have defects in posterior body extension and epidermal tail fin development. Treatment with an inverse agonist of retinoic acid (RA) signalling partially rescues the axial and tail fin phenotypes indicating they are caused by increased RA signalling. Gene expression analyses and luciferase assays suggest that posterior RA levels are kept low in wild type animals by a likely direct transcriptional regulation of a Cyp26 gene by Cdx. Transcriptome analysis reveals extensive gene expression changes in mutants, with a disproportionate effect of Pdx and Cdx on gut-enriched genes and a colinear-like effect of Cdx on Hox genes. Conclusions: These data reveal that amphioxus Pdx and Cdx have roles in specifying middle and posterior cell fates in the endoderm of the gut, roles that likely date to the origin of Bilateria. This conclusion is consistent with these two ParaHox genes playing a role in the origin of the bilaterian through-gut with a distinct anus, morphological innovations that contributed to ecological change in the Cambrian. In addition, we find that amphioxus Cdx promotes body axis extension through a molecular mechanism conserved with vertebrates. The axial extension role for Cdx dates back at least to the origin of Chordata and may have facilitated the evolution of the post-anal tail and active locomotion in chordates

A combined algorithm for analyzing structural controllability and observability of complex networks

In this paper a combined algorithm for analyzing structural controllability and observability of complex networks is presented. The algorithm addresses the two fundamental properties to guarantee structural controllability of a system: the absence of dilations and the accessibility of all nodes. The first problem is reformulated as a Maximum Matching search and it is addressed via the Hopcroft- Karp algorithm; the second problem is solved via a new wiring algorithm. Both algorithms can be combined to efficiently determine the number of required controllers and observers as well as the new required connections in order to guarantee controllability and observability in real complex networks. An application to a Twitter social network with over 100,000 nodes illustrates the proposed algorithms

The ADAR Family in Amphioxus: RNA Editing and Conserved Orthologous Site Predictions.

RNA editing is a relatively unexplored process in which transcribed RNA is modified at specific nucleotides before translation, adding another level of regulation of gene expression. Cephalopods use it extensively to increase the regulatory complexity of their nervous systems, and mammals use it too, but less prominently. Nevertheless, little is known about the specifics of RNA editing in most of the other clades and the relevance of RNA editing from an evolutionary perspective remains unknown. Here we analyze a key element of the editing machinery, the ADAR (adenosine deaminase acting on RNA) gene family, in an animal with a key phylogenetic position at the root of chordates: the cephalochordate amphioxus. We show, that as in cephalopods, ADAR genes in amphioxus are predominantly expressed in the nervous system; we identify a number of RNA editing events in amphioxus; and we provide a newly developed method to identify RNA editing events in highly polymorphic genomes using orthology as a guide. Overall, our work lays the foundations for future comparative analysis of RNA-editing events across the metazoan tree

Microsyntenic Clusters Reveal Conservation of lncRNAs in Chordates Despite Absence of Sequence Conservation

Homologous long non-coding RNAs (lncRNAs) are elusive to identify by sequence similarity due to their fast-evolutionary rate. Here we develop LincOFinder, a pipeline that finds conserved intergenic lncRNAs (lincRNAs) between distant related species by means of microsynteny analyses. Using this tool, we have identified 16 bona fide homologous lincRNAs between the amphioxus and human genomes. We characterized and compared in amphioxus and Xenopus the expression domain of one of them, Hotairm1, located in the anterior part of the Hox cluster. In addition, we analyzed the function of this lincRNA in Xenopus, showing that its disruption produces a severe headless phenotype, most probably by interfering with the regulation of the Hox cluster. Our results strongly suggest that this lincRNA has probably been regulating the Hox cluster since the early origin of chordates. Our work pioneers the use of syntenic searches to identify non-coding genes over long evolutionary distances and helps to further understand lncRNA evolution

Analysis of Fox genes in Schmidtea mediterranea reveals new families and a conserved role of Smed‑foxO in controlling cell death

The forkhead box (Fox) genes encode transcription factors that control several key aspects of development. Present in the ancestor of all eukaryotes, Fox genes underwent several duplications followed by loss and diversification events that gave rise to the current 25 families. However, few Fox members have been identified from the Lophotrochozoa clade, and specifically from planarians, which are a unique model for understanding development, due to the striking plasticity of the adult. The aim of this study was to identify and perform evolutionary and functional studies of the Fox genes of lophotrochozoan species and, specifically, of the planarian Schmidtea mediterranea. Generating a pipeline for identifying Forkhead domains and using phylogenetics allowed us the phylogenetic reconstruction of Fox genes. We corrected the annotation for misannotated genes and uncovered a new family, the QD, present in all metazoans. According to the new phylogeny, the 27 Fox genes found in Schmidtea mediterranea were classified into 12 families. In Platyhelminthes, family losses were accompanied by extensive gene diversification and the appearance of specific families, the A(P) and N(P). Among the newly identified planarian Fox genes, we found a single copy of foxO, which shows an evolutionary conserved role in controlling cell death

Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations