Biology subjects

Verheyen, E. M.

Publications and source records attributed to Verheyen, E. M..

7 recordsLinked to original sources

bioRxiv2024

Robinow Syndrome DVL1 mutations disrupt morphogenesis and appendage formation in a Drosophila disease model

Robinow Syndrome is a rare developmental syndrome caused by variants in numerous genes involved in Wnt signaling pathways. We previously showed that expression of patient variants in Drosophila and a chicken model disrupts the balance of canonical and non-canonical/PCP Wnt signaling. We also noted neomorphic effects that warranted further investigation. In this study, we examine morphological changes that occur as a result of one variant, DVL11519{Delta}T, that serves as a prototype for the other pathogenic variants. We show that epithelial imaginal disc development is disrupted in legs and wings. Shortened leg segments are reminiscent of shortened limb bones seen in RS patients. We find that imaginal disc development is disrupted and accompanied by increased cell death, without changes in cell proliferation. Furthermore, we find altered dynamics of basement membrane components and modulators. Notably we find increased MMP1 expression and tissue distortion, which is dependent on JNK signaling. We also find enhanced collagen IV (Viking) secreted from cells expressing DVL11519{Delta}T. Through these studies we have gained more insight into developmental consequences of DVL1 variants implicated in autosomal dominant Robinow Syndrome.

developmental biology↗

bioRxiv2024

DVL1 variants and C-terminal deletions have differential effects on craniofacial development and WNT signaling

Robinow Syndrome (RS) is a rare disease characterized by craniofacial malformations and limb shortening linked with mutations in seven WNT pathway genes. Our objective was to investigate the functional effects of frameshift mutations the intracellular adaptor protein, Dishevelled (DVL1; c.1519{Delta}T, p.Trp507Glyfs*142) on chicken craniofacial development. Misexpression of wt (wt) or mutant hDVL1 variants in vivo caused upper beak shortening (wtDVL1 n=8/14; DVL11519{Delta}T 12/13). At early stages of development, the DVL11519{Delta}T inhibited frontonasal mass narrowing, chondrogenesis, and proliferation. To test whether the phenotypes were caused due to the abnormal C-terminal peptide in DVL11519{Delta}T, we designed two additional constructs. The DVL11519* (DVL1507*) retains first 30 amino acids of the C-terminus while DVL11431* (DVL1477*) removes the entire C-terminus. DVL11519* injected embryos had normal beaks while DVL11431* caused high mortality and the phenotypes were like the DVL11519{Delta}T. In frontonasal micromass cultures, both DVL11519{Delta}T and DVL11431* inhibited skeletogenesis while the DVL11519* resembled wtDVL1 and GFP cultures. In luciferase assays DVL11519{Delta}T, DVL11519*and DVL11431* weakly activated the WNT canonical and non-canonical JNK-PCP pathways compared to wtDVL1. Furthermore, we observed that variant DVL1507*fs is stalled in the nucleus similar to hDVL1477*, possibly due to the abnormal C-terminus interfering with the nuclear export sequence. wtDVL1 and DVL1507* were distributed in nucleus and the cytoplasm. Our RS-DVL11519{Delta}T avian model recapitulates the broad face and jaw hypoplasia and demonstrates defects in both branches of WNT signaling. This is the first study to clarify the role of abnormal C-terminus in ADRS and to recognize the importance of an uncharacterized C-terminal sequence. Summary StatementFunctional and biochemical studies on chicken embryos with the Robinow syndrome (RS) DVL1 variant demonstrate defects in skeletogenesis and both branches of WNT signaling. This is the first study to establish a link between the RS facial defects and the mutated C-terminal sequence. We identified first 30 amino acids of the DVL1 C-terminus are sufficient for normal development.

developmental biology↗

bioRxiv2020

Functional characterization of human Homeodomain-interacting protein kinases (HIPKs) in Drosophila melanogaster reveal both conserved functions and differential induction of HOX gene expression

Homeodomain-interacting protein kinases (Hipks) are a family of conserved proteins that are necessary for development in both invertebrate and vertebrate organisms. Vertebrates have four paralogues, Hipks 1-4. Mice lacking Hipk1 or Hipk2 are viable, however loss of both is lethal during early embryonic development, with embryos exhibiting homeotic skeletal transformations and incorrect HOX gene expression. While these results suggest Hipks have a role in regulating HOX genes, a regulatory mechanism has not been characterized, and further comparisons of the roles of Hipks in development has not progressed. One challenge with characterizing developmental regulators in vertebrates is the extensive redundancy of genes. For this reason, we used Drosophila melanogaster, which has reduced genetic redundancy, to study the functions of the four human HIPKs (hHIPKs). In D. melanogaster, zygotic loss of the single ortholog dhipk results in lethality with distinct eye and head defects. We used a dhipk mutant background to compare the ability of each hHIPK protein to rescue the phenotypes caused by the loss of dHipk. In these humanized flies, both hHIPK1 and hHIPK2 rescued lethality, while hHIPK3 and hHIPK4 only rescued minor dhipk mutant patterning phenotypes. This evidence for conserved functions of hHIPKs in D. melanogaster directed our efforts to identify and compare the developmental potential of hHIPKs by expressing them in well-defined tissue domains and monitoring changes in phenotypes. We observed unique patterns of homeotic transformations in flies expressing hHIPK1, hHIPK2, or hHIPK3 caused by ectopic induction of Hox proteins. These results were indicative of inhibited Polycomb-group complex (PcG) components, suggesting that hHIPKs play a role in regulating its activity. Furthermore, knockdown of PcG components phenocopied hHIPK and dHipk expression phenotypes. Together, this data shows that hHIPKs function in D. melanogaster, where they appear to have variable ability to inhibit PcG, which may reflect their roles in development. Author summaryThe redundancy of vertebrate genes often makes identifying their functions difficult, and Hipks are no exception. Individually, each of the four vertebrate Hipks are expendable for development, but together they are essential. The reason Hipks are necessary for development is unclear and comparing their developmental functions in a vertebrate model is difficult. However, the invertebrate fruit fly has a single essential dhipk gene that can be effectively removed and replaced with the individual vertebrate orthologs. We used this technique in the fruit fly to compare the developmental capacity of the four human HIPKs (hHIPKs). We found that hHIPK1 and hHIPK2 are each able to rescue the lethality caused by loss of dhipk, while hHIPK3 and hHIPK4 rescue minor patterning defects, but not lethality. We then leveraged the extensive adult phenotypes associated with genetic mutants in the fruit fly to detect altered developmental pathways when hHIPKs are mis-expressed. We found that expression of hHIPKs 1-3 or dhipk each produce phenotypes that mimic loss of function of components of the Polycomb-group complex, which are needed to regulate expression of key developmental transcription factors. We therefore propose that Hipks inhibit Polycomb components in normal development, though details of this interaction remain uncharacterized.

developmental biology↗

bioRxiv2019

Homeodomain-interacting protein kinase (Hipk) is required for nervous system and muscle structure and function

Homeodomain-interacting protein kinases (Hipk) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. We have used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression we demonstrate that knockdown and over-expression of Hipk produces early adult lethality, partially due to the effects on the nervous system and muscle involvement. We find that optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) and increases motor neuron axonal branching. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hts (adducin) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular function and influence lethality. Hipk also modifies the distribution of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases.

neuroscience↗

bioRxiv2017

Actomyosin contractility modulates Wnt signaling through adherens junction stability

Mechanical forces can influence the canonical Wnt signaling pathway in processes like mesoderm differentiation and tissue stiffness during tumorigenesis, but a molecular mechanism involving both in a developing epithelium and its homeostasis is lacking. We identified that increased non-muscle myosin II activation and cellular contraction inhibited Wnt target gene transcription in developing Drosophila. Genetic interactions studies identified this effect was due to myosin-induced accumulation of cortical F-actin resulting in clustering and accumulation of E-cadherin to the adherens junctions. E-cadherin titrates any available {beta}-catenin, the Wnt pathway transcriptional co-activator, to the adherens junctions in order to maintain cell-cell adhesion under contraction. We show that decreased levels of cytoplasmic {beta}-catenin result in insufficient nuclear translocation for full Wnt target gene transcription. Our work elucidates a mechanism in which the dynamic activation of actomyosin contractility refines patterning of Wnt transcription during development and maintenance of epithelial tissue in organisms.

cell biology↗

bioRxiv2017

Homeodomain interacting protein kinase promotes tumorigenesis and metastatic cell behavior

Aberrations in signaling pathways that regulate tissue growth often lead to tumorigenesis. Homeodomain interacting protein kinase (Hipk) family members are reported to have distinct and contradictory effects on cell proliferation and tissue growth. From these studies it is clear that much remains to be learned about the roles of Hipk family protein kinases in proliferation and cell behaviour. Previous work has shown that Drosophila Hipk is a potent growth regulator, thus we predicted that it could have a role in tumorigenesis. In our study of Hipk-induced phenotypes, we observed the formation of tumor-like structures in multiple cell types in larvae and adults. Furthermore, elevated Hipk in epithelial cells induces cell spreading, invasion and epithelial-to-mesenchymal transition in the imaginal disc. Further evidence comes from cell culture studies in which we expressed Drosophila Hipk in human breast cancer cells and show that it enhances proliferation and migration. Past studies have shown that Hipk can promote the action of conserved pathways implicated in cancer and EMT, such as Wnt/Wingless, Hippo, Notch and JNK. We show that Hipk-phenotypes are not likely due to activation of a single target, but rather through a cumulative effect on numerous target pathways. Most Drosophila tumor models involve mutations in multiple genes, such as the well-known RasV12 model, in which EMT and invasiveness occur after the additional loss of the tumor suppressor gene scribble. Our study reveals that elevated levels of Hipk on their own can promote both hyperproliferation and invasive cell behaviour, suggesting that Hipks could be potent oncogenes and drivers of EMT.\n\nSummary statementThe protein kinase Hipk can promote proliferation and invasive behaviors, as well as synergize with known cancer pathways, in a novel Drosophila model for tumorigenesis.

cancer biology↗

bioRxiv2017

The Protein Phosphatase 4 complex promotes the Notch 1pathway and wg transcription

The Wnt/Wingless (Wg) pathway controls cell fate specification, tissue differentiation and organ development across organisms. Using an in vivo RNAi screen to identify novel kinase and phosphatase regulators of the Wg pathway, we identified subunits of the serine threonine phosphatase Protein phosphatase 4 (PP4). Knockdown of the catalytic and the regulatory subunits of PP4 cause reductions in the Wg pathway targets Senseless and Distal-less. We find that PP4 regulates the Wg pathway by controlling Notch-driven wg transcription. Genetic interaction experiments identified that PP4 likely promotes Notch signaling within the nucleus of the Notch-receiving cell. Although the PP4 complex is implicated in various cellular processes, its role in the regulation of Wg and Notch pathways was previously uncharacterized. Our study identifies a novel role of PP4 in regulating Notch pathway, resulting in aberrations in Notch-mediated transcriptional regulation of the Wingless ligand. Furthermore, we show that PP4 regulates proliferation independent of its interaction with Notch.\n\nSummary statementThe protein phosphatase 4 complex promotes Notch signaling and target gene expression during Drosophila wing development.

developmental biology↗