Mok, J.-W. | bioRxiv Science

bioRxiv2025

Hippo signaling regulates cuticle pigmentation and dopamine metabolism in Drosophila

Pigmentation plays multiple important roles in development, physiology and evolution. Melanization of the insect cuticle requires dopamine as a precursor of melanin and involves key enzymes in dopamine biosynthesis including Tyrosine hydroxylase (TH) and Dopa decarboxylase (Ddc). Some studies have hinted that disruption of the evolutionarily conserved Hippo signaling pathway, which has been primarily studied in the context of tissue growth, may lead to changes in cuticle pigmentation in the fruit fly Drosophila melanogaster. However, to our knowledge, there have not been any systematic investigations into their potential mechanistic links. In this study, we identified that all genes that comprise the canonical Hippo signaling pathway [hippo (hpo), salvador (sav), mats, warts (wts), yorkie (yki) and scalloped (sd)] are involved in cuticle pigmentation in the fly notum based on tissue-specific gene knock-down/out experiments and epistatic analysis. Despite the notable divergence of pigmentation mechanisms between invertebrates and vertebrates, these phenotypes can often be rescued by the human orthologs of corresponding fly genes. While we find that manipulation of Hippo signaling in dopaminergic neurons does not affect global dopamine levels in the fly brain, developmental inhibition of this pathway can increase dopamine levels in the fly head, indicating that the mechanism by which dopamine levels are regulated in the nervous system is distinct from that in epithelial cells. Through single nuclei RNA sequencing of the developing fly nota and subsequent functional studies of differentially expressed genes that are altered upon inhibition of Hippo signaling, we found many genes that contribute to cuticle pigmentation downstream of Hippo signaling. We conclude that regulation of cuticle pigmentation by canonical Hippo signaling acts through multiple downstream genes rather than directly through TH and Ddc. We also propose that the Drosophila melanogaster cuticle may serve as a useful platform to identify previously uncharacterized mediators of Hippo signaling as well as an in vivo experimental system to test the functionality of rare genetic variants found in human Hippo signaling orthologs associated with a variety of diseases.

genetics↗

bioRxiv2022

De Novo Variants in MRTFB have gain of function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features.

Myocardin-Related Transcription Factor B (MRTFB) is an important transcriptional regulator which promotes the activity of an estimated 300 genes during different stages of development. Here we report two pediatric probands with de novo variants in MRTFB (R104G and A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. As the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, we generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Expression of the human MRTFBR104G variant using a mrtf-T2A-GAL4 line proved to be embryonic lethal. Additional phenotypes were also identified by expressing the MRTFBR104G and MRTFBA91P variant in a subset of Drosophila tissues. Notably, expression within wing tissues resulted in an expansion of intervein tissue, wing vein thickening, shortening or loss of wing veins, and blistering. The MRTFBR104G and MRTFBA91P variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the Actin cytoskeleton. These changes were not observed in flies expressing two additional candidate variants, MRTFBN95Sand MRTFBR109Q, highlighting that the location of the mutation within the 2nd RPEL domain is critical to the pathogenicity of the variant. These changes suggest that the MRTFBR104G and MRTFBA91P alleles we have identified affect the regulation of the protein and that these variants in MRTFB underly a novel neurodevelopmental disorder.

genetics↗