Understanding Images: Challenging the Wnt signaling dogma
Author: Alexandra Franz, Institute of Molecular Life Sciences, University of Zurich, Switzerland
Competing Interests: Alexandra Franz is an author of the article discussed in this blog.
Image Caption: The full cover image shows the animals Armadillo and Pangolin on top of nucleotide sequences, illustrating their control over Wingless-responsive genes and enhancers.
Acknowledgements: I thank George Hausmann for his valuable comments on a draft of this blog.
Image Credit: Alexandra Franz
Underlying the diversity of life are genetic programs – sets of genes whose orchestrated expression control metazoan development and tissue homeostasis. A small cohort of conserved signaling pathways controls these genetic programs. One of the conserved signaling cascades is the so-called canonical Wnt signaling pathway, triggered by a family of secreted proteins called Wnts.
Almost 40 years have passed since Wnt proteins were identified. The first Wnt protein (Int1, now Wnt1) was originally described as a proto-oncogene in mouse, and found to be a homologue of the product of the Drosophila segment-polarity gene wingless (wg). The intimate involvement of Wnt signaling in both disease (especially cancer) and development was thus established long ago. In the intervening years, further genetic and molecular studies have revealed an ever wider spectrum of biological processes to be controlled by Wnts; these include orchestrating early embryonic development, maintaining adult tissue homeostasis and, when deregulated, leading to diseases such as cancer. Intensive studies of the mechanisms underpinning the Wnt signal transduction cascade are encapsulated by the current dogma of Wnt signaling: the transcriptional induction of Wnt target genes is mediated by two proteins – beta-catenin and the transcription factor TCF. In Drosophila, these two proteins are called Armadillo and Pangolin, respectively. However, there are some examples in the literature of beta-catenin and TCF interacting with other proteins, causing speculation that there may exist an alternative Wnt signaling machinery.
In the work described in the associated featured article from PLOS Genetics’ April 2017 issue, we addressed the question of whether the two main, and, according to the dogma, obligate components – Armadillo and Pangolin – can be bypassed to execute the Wnt signaling response in Drosophila cells.
Drosophila was chosen as the initial model system to test this idea for practical reasons. Pangolin is the only TCF homolog, and is also widely used to study Wnt/Wg signaling. Indeed, Armadillo and Pangolin were discovered in Drosophila, and their names derive from the phenotype of the mutant Drosophila embryos, which have a scaly appearance reminiscent of the animals depicted in the cover image.
To elucidate whether Armadillo and Pangolin are absolutely required for achieving a Wnt/Wg response, we first determined a high-confidence set of Wnt/Wg target genes in Drosophila Kc cells by RNA-sequencing. After establishing the Wnt/Wg signature, we generated cells lacking either of the two signal-transducing components using the genome-editing tool CRISPR/Cas9. With these cells, and again using RNA-sequencing as a functional read-out, we subsequently determined whether a Wnt/Wg response could be executed in the absence of Armadillo or Pangolin. Unexpectedly, we found that both Armadillo and Pangolin are indeed absolutely required to induce the transcription of all Wnt/Wg target genes.
To obtain a more comprehensive understanding of the transcriptional regulation of the Wnt/Wg signature, we next carried out STARR-sequencing to identify Wnt/Wg-responsive enhancers, and determine their dependency on Pangolin. STARR-sequencing is a technique developed in the Stark lab that allows the isolation of enhancers responsive to different stimuli. Consistent with our previous results, in the STARR-sequencing experiments all Wnt/Wg-responsive enhancers are entirely dependent on Pangolin.
Collectively our study demonstrates that in Drosophila cells, the Wnt/Wg signals proceed obligatorily through Armadillo and Pangolin – validating the veracity of the Wnt signaling dogma, at least in this system. The critical next step is to extend these studies to other systems – in mammalian cell lines, in developing tissues, and in disease models. In mammals, the situation is complicated by the fact that there are four TCFs, as opposed to one in Drosophila. These studies would also be of great importance for understanding the different roles of Wnt signaling in several malignant pathomechanisms of human cancers.
Franz A, Shlyueva D, Brunner E, Stark A, Basler K (2017) Probing the canonicity of the Wnt/Wingless signaling pathway. PLoS Genet 13(4): e1006700. https://doi.org/10.1371/journal.pgen.1006700