Investigating the gene regulatory network underlying the segmentation clock of the flour beetle Tribolium castaneum

This research will identify important similarities and differences in the way animals as diverse as arthropods (i.e. fruit flies, beetles and spiders) and vertebrates (i.e. fish, mice and humans) develop their body during embryogenesis. This work is crucial for understanding how the genetic and developmental mechanisms forming animal body plans changed and diverged over evolutionary time to produce the diverse types of animal body plans we see in nature today. It is also important for understanding how the common ancestor of all animals developed its body plan, deep in evolutionary history, over 550 million years ago.
Humans, and other vertebrates, possess a segmented body plan: internal parts of our body are divided into separated and repeated structures, for example our vertebrae and ribs. Vertebrate embryos grow during their development, with head structures formed first and the trunk grown in an anterior to posterior sequence. During this process, the cell populations that will later give rise to ribs/vertebrae are determined early, and one-by-one, in an anterior to posterior sequence. This process is controlled by a network of genes that are repeatedly turned on and off to define each group of cells (i.e. each future rib/vertebra). This complex oscillating network of genes is called the vertebrate segmentation clock.
Arthropods, including fruit flies, beetles and spiders, also have a visibly segmented body plan. The abdominal body segments of the red flour beetle Tribolium castaneum form sequentially, one-by-one, in an anterior to posterior progression during beetle development, in a process similar to that described above for vertebrates. I have recently shown that this developmental process is also controlled by a network of genes that oscillate to sequentially pattern abdominal segments - the arthropod segmentation clock. Interestingly, the fruit fly Drosophila melanogaster has evolved to speed up its development, such that all body segments form simultaneously in the egg in a process that is already well understood at the genetic level by biologists.
The first objective of this work programme is to determine whether the gene network underlying the arthropod segmentation clock is organized in a similar way to the vertebrate segmentation clock. If striking similarities are found, it could suggest that the segmentation clock was a feature of the common ancestor of arthropods and vertebrates. Alternatively, if there are striking differences, it could suggest that disparate animal groups have evolved similar genetic mechanisms independently and in parallel. Either way, this research promises to reveal important information about our evolutionary history.
The second objective of this work programme is to determine how a sequential segmentation mechanism involving a segmentation clock was modified in evolution to produce the mechanism controlling the simultaneous formation of segments in Drosophila. Identifying the changes in gene regulation that facilitated this transition could reveal general principles by which developmental mechanisms evolved to produce the wide variety of animal body plans we see in nature today.
The third objective of this work programme is to develop new genetic techniques that will advance Tribolium castaneum as a cheap, amenable, ethically acceptable, invertebrate model with which to study segmentation clocks.
The general public will benefit from this research via an increase in our understanding of human and animal evolution, through the development of a powerful invertebrate genetic model for studying the genetic principles underlying segmentation clocks thus reducing the current dependence on less ethically acceptable vertebrate models, and through better value for money from future research on segmentation clocks via the use of Tribolium, a cheaper and potentially more amenable alternative to vertebrate models.

Established Tribolium bioinformatics and transgenic techniques will be used to address the first two work programme objectives: 1. Determine whether intracellular negative regulatory feedback loops (involving Tc-eve, Tc-odd and/or Tc-run) are a feature of the Tribolium segmentation clock. 2. Determine whether the homologues of Drosophila gap genes (i.e. Tc-kni/Tc-giant), act downstream of the segmentation clock (i.e. are regulated by Tc-Odd or Tc-Eve) in Tribolium. First, embryonic germline transformation will be used to establish Tribolium transgenic lines carrying reporter genes (destabilized GFP) for the Tribolium primary pair-rule genes Tc-eve, Tc-odd & Tc-run (Obj.1), and the Tribolium gap gene homologues Tc-kni and Tc-gt (Obj. 2) that contain sufficient regulatory sequence to recapitulate wildtype expression patterns. Additional transgenic reporter lines will then be established carrying smaller stretches of regulatory sequence to dissect the regulatory regions of the pair-rule genes (Obj.1) and identify the minimal enhancers of the gap gene homologues (Obj.2). Whether regulatory interactions are direct will be established via examining lines carrying reporter genes in which specific putative transcription factor binding sites have been mutated, or via Chromatin-IP using Tc-Eve, Tc-Odd and Tc-Run antibodies (both Objectives). The design of reporter gene constructs, and the stretches of endogenous regulatory sequences included, will be informed by bioinformatics binding site cluster analyses of genomic regions surrounding these five genes using the MSCAN, MCAST, ClusterBuster, SWAN and/or Stubb algorithms (both Objectives). Objective 3 is to adapt the state-of-the-art CRISPR RNA/Cas9 system for genome editing in Tribolium. This will be achieved using a similar cloning and transgenic strategy to that described for Drosophila in Gratz et al. 2013 (Genetics. 194, 1029), except that the Drosophila promoters will be replaced by endogenous Tribolium promoters.

This work programme will demonstrate impact in four distinct areas: 1) It will further advance the beetle, Tribolium castaneum, as a powerful genetic invertebrate model for studying segmentation clocks. 2) It will generate data that will inform on the evolutionary origin of segmentation clocks; i.e. whether the arthropod and vertebrate segmentation clocks share significant similarities in their genetic organization that suggest homology and their existence in the common ancestor of all bilaterally symmetrical (bilaterian) animals, or alternatively show significant differences, making it more likely that they evolved independently in the lineages leading to arthropods and vertebrates. The answer to this question is crucial to our understanding of the direction of evolution change in developmental gene networks in all bilaterian lineages, and therefore of widespread relevance to a large number of evolutionary biologists. 3) It will provide important insights into how complex developmental gene networks evolve; i.e. by providing an understanding of how the hierarchical Drosophila segmentation gene cascade evolved from an ancestral oscillating segmentation gene network. The principles revealed could also be of widespread relevance to evolutionary developmental biologists, a growing international research community. 4) It will develop two highly skilled members of the UK workforce.
The work programme outlined in this application constitutes basic research addressing fundamental questions in developmental biology and the genetic basis to evolution in developmental mechanisms. Nonetheless, the work does offer large potential for impact beyond academia in the medium to long-term. This potential impact is significant, but either qualitative in nature or difficult to quantify. The ultimate beneficiaries beyond academia are the general public at large. These benefits fall into four categories of impact: 1) Addressing concerns within the public at large about the routine use of vertebrate animals in research by providing the first powerful invertebrate genetic model for studying the fundamental genetic principles underlying segmentation clocks, thus reducing the dependence on vertebrate models in this area of research. 2) Increasing the 'value-for-money' from present and future publically funded research by; i) improving and promoting an invertebrate model for studying segmentation clocks that could provide significant savings when compared to established vertebrate models; e.g. with respect to the cost of animal husbandry, the maintenance of transgenic lines, and/or the potential ease and speed of experimentation (i.e. knowledge progression), ii) developing a cheap, quick, powerful and widely applicable state-of-the-art transgenic technique (the CRISPR RNA/Cas9 system) for use by the growing Tribolium research community and iii) by supporting a synergistic interdisciplinary collaboration between a bioinformatician (Erik Clark) and developmental biologists (the PDRA employed on the grant and myself). 3) Increasing our understanding of animal evolution, thus contributing to both the public acceptance and understanding of a controversial area of scientific theory and research, and enhancing cultural enrichment by revealing more about our evolutionary roots as a species, and our similarities to, and shared history with, other animals. 4) Develop two highly skilled members of the UK workforce; both researchers funded on the grant (the PDRA and myself) will develop transferrable and scientific skills appropriate to their background and existing skills level, such as time and personnel management, planning, data collection and archiving, experimental design and statistics, as well as various forms of science communication, from informal lab meetings and theme seminars, to drafting press releases, open days, external talks to the general public, and scientific conferences.