Schacht, M. I. | Search bioRxiv

bioRxiv2018

Clonal analysis by tunable CRISPR-mediated excision

Clonal marking techniques based on the Cre/lox and Flp/FRT systems are widely used in multicellular model organisms to mark individual cells and their progeny, in order to study their morphology, growth properties and developmental fates. The same tools can be adapted to introduce specific genetic changes in a subset of cells within the body, i.e. to perform mosaic genetic analysis. Marking and manipulating distinct cell clones requires control over the frequency of clone induction, which is sometimes difficult to achieve. Here we present Valcyrie, a new method that replaces the conventional Cre or Flp recombinase-mediated excision of a marker cassette by CRISPR-mediated excision. A major advantage of this approach is that CRISPR efficiency can be tuned in a predictable fashion by manipulating the degree of sequence complementarity between the CRISPR guide RNA and its targets. We establish the method in the beetle Tribolium castaneum. We demonstrate that clone marking frequency can be tuned to generate embryos carrying single marked clones. The Valcyrie approach can be applied to a wide range of experimental settings, for example to modulate clone frequency with existing tools in established model organisms and to introduce clonal analysis in emerging experimental models.

genetics↗

bioRxiv2016

foxQ2 evolved a key role in anterior head and central brain patterning in protostomes

Anterior patterning of animals is based on a set of highly conserved transcription factors but the interactions within the protostome anterior gene regulatory network (aGRN) remain enigmatic. Here, we identify the foxQ2 ortholog of the red flour beetle Tribolium castaneum as novel upstream component of the insect aGRN. It is required for the development of the labrum and higher order brain structures, namely the central complex and the mushroom bodies. We reveal Tc-foxQ2 interactions by RNAi and heat shock-mediated misexpression. Surprisingly, Tc-foxQ2 and Tc-six3 mutually activate each other forming a novel regulatory module at the top of the insect aGRN. Comparisons of our results with those of sea urchins and cnidarians suggest that foxQ2 has acquired functions in head and brain patterning during protostome evolution. Our findings expand the knowledge on foxQ2 gene function to include essential roles in epidermal development and central brain patterning.\n\nAuthor summaryThe development of the anterior most part of any animal embryo - for instance the brain of vertebrates and the head of insects - depends on a very similar set of genes present in all animals. This is true for the two major lineages of bilaterian animals, the deuterostomes (including sea urchin and humans) and protostomes (including annelids and insects) and the cnidarians (e.g. the sea anemone), which are representatives of more ancient animals. However, the interaction of these genes has been studied in deuterostomes and cnidarians but not in protostomes. Here, we present the first study the function of the gene foxQ2 in protostomes. We found that the gene acts at the top level of the genetic network and when its function is knocked down, the labrum (a part of the head) and higher order brain centers do not develop. This is in contrast to the other animal groups where foxQ2 appears to play a less central role. We conclude that foxQ2 has acquired additional functions in the course of evolution of protostomes.

developmental biology↗