Evolution of central complex development: Cellular and genetic mechanisms

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Brains are conserved between insect species, as they consist of a set of anatomically similar areas, or neuropils. Simultaneously, these neuropils differ in size, shape, position and developmental timing between insect species, thus reflecting evolutionary adaptations to specific sensory cues and behavioural repertoires. Although divergences in a common framework are intriguing, the developmental mechanisms underlying the evolution of insect brains are hardly understood. One phenomenon in the evolution of development is heterochrony, i.e. a shift in relative developmental timing of morphological structures between species. The central complex, a neuropil in the insect brain that enables spatial orientation, appears at different developmental stages in different species. In this work, I compare central complex development between the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. In Drosophila, the central complex is functionally an adult structure as it only appears during late larval and pupal stages. In Tribolium, however, parts of the central complex are already present at the end of embryogenesis. Here, I show work that establishes, uses and expands a new method to mark and compare homologous neurons throughout development in different species. The main work is presented in manuscript 1, where I used a novel method of marking and comparing developing, homologous cell groups of the central complex of Drosophila and Tribolium. For this, I generated and characterized transgenic lines specific for the conserved transcription factor retinal homeobox (rx). I then determined which Rx-positive cell groups in the adult brain of Drosophila and Tribolium are homologous. These were then followed throughout development. We were able to identify a complex pattern of heterochronic events between Drosophila and Tribolium central complex development. Most importantly, we found that Tribolium precociously acquires a functional central complex neuropil that has distinct anatomical characteristics and thus represents an immature form of the central body. Manuscript 2 describes two ways to construct transgenic lines, like the ones used in manuscript 1, through CRISPR/Cas mediated genome editing. One relies on homology-directed repair of a bicistronic construct, and results in an exact monitoring of a gene of choice, while the other is mediated by non-homologous end-joining to generate a gene-specific enhancer trap. Manuscript 3 describes methods on how to dissect and stain Tribolium castaneum brains of different developmental periods. It describes how to perform in situ hybridisation and immunohistochemistry in adult and larval brains. Generating tools such as genetic neural lineage marking (manuscript 1, 2) and establishing protocols that can be used in alternative model organisms (manuscripts 2 and 3) can facilitate more detailed understandings of the genetic and developmental underpinnings of brain evolution.