Trudi A. Westfall

Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/β-catenin activity

We provide genetic evidence defining a role for noncanonical Wnt function in vertebrate axis formation. In zebrafish, misexpression of Wnt-4, -5, and -11 stimulates calcium (Ca2+) release, defining the Wnt/Ca2+ class. We describe genetic interaction between two Wnt/Ca2+ members, Wnt-5 (pipetail) and Wnt-11 (silberblick), and a reduction of Ca2+ release in Wnt-5/pipetail. Embryos genetically depleted of both maternal and zygotic Wnt-5 product exhibit cell movement defects as well as hyperdorsalization and axis-duplication phenotypes. The dorsalized phenotypes result from increased β-catenin accumulation and activation of downstream genes. The Wnt-5 loss-of-function defect is consistent with Ca2+ modulation having an antagonistic interaction with Wnt/β-catenin signaling.

Requirement for intracellular calcium modulation in zebrafish dorsal–ventral patterning

The phosphoinositide (PI) cycle is an important signal transduction pathway that, upon activation, generates intracellular second messengers and leads to calcium release. To determine whether PI cycle-mediated intracellular calcium release is required for body plan formation, we systematically dissect PI cycle function in the zebrafish (Danio rerio). We inhibit PI cycle function at three different steps and deplete internal calcium stores, demonstrating an impact on endogenous calcium release and Wnt/beta-catenin signaling. Inhibition of endogenous calcium modulation induces hyperdorsalized phenotypes in a dose-dependent manner. Ectopic dorsal-signaling centers are generated in PI cycle-inhibited embryos as demonstrated by altered beta-catenin subcellular localization and ectopic expression of beta-catenin target genes. These results provide evidence that modulation of calcium release is critical for early embryonic patterning and acts by influencing the stabilization of beta-catenin protein.

Zebrafish Nkd1 promotes Dvl degradation and is required for left-right patterning

The establishment of the left-right (LR) axis in zebrafish embryos relies on signals from the dorsal forerunner cells (DFC) and the Kupffers vesicle (KV). While the Wnt signaling network influences many aspects of embryonic development, its precise role in LR patterning is still unclear. One branch of the Wnt network leads to stabilization of β-catenin and activation of downstream target genes. Other Wnt ligands appear to act independently of β-catenin to modulate calcium release and influence cell polarity. Central to regulation of β-catenin and coordination of convergent extension (CE) movements is Dishevelled (Dvl). Naked Cuticle (Nkd) binds Dvl and modulates β-catenin-dependent and independent Wnt signaling. Here, we analyze the expression patterns of three zebrafish Nkd homologs and find enriched expression of nkd1 in DFCs and KV. Dvl is degraded upon Nkd1 overexpression in zebrafish. Knockdown of Nkd1 specifically in the DFC results in β-catenin nuclear localization and transcriptional activation as well as alterations to DFC migration, KV formation, ciliogenesis and LR patterning. Furthermore, we identify asymmetric expression of the Nodal antagonist charon around the KV and show that Nkd1 knockdown impacts asymmetric charon expression. Our findings show that Nkd1 acts as a β-catenin antagonist in the DFCs necessary for LR patterning.

Calcium dynamics integrated into signalling pathways that influence vertebrate axial patterning

Many aspects of animal development including fertilization as well as organ formation and function are dependent upon the dynamic release of calcium (Ca2+) ions. Although the controlled release and/or accumulation of Ca2+ ions has been extensively studied, how the release dynamics produce a specific biological output in embryonic development is less clear. We will briefly summarize Ca2+ sources, highlight data on endogenous Ca2+ release in vertebrate embryos relevant to body plan formation and cell movement, and integrate pharmacological and molecular-genetic studies to lend insight into the signalling pathways involved. Finally, based on in vivo imaging in zebrafish genetic mutants, we will put forward the model that distinct Ca2+ release dynamics lead to antagonism of the developmentally important Wnt/β-catenin signalling pathway, while sustained Ca2+ release modulates cell polarization or directed migration.

Hypomorphic mutations in TRNT1 cause retinitis pigmentosa with erythrocytic microcytosis

Retinitis pigmentosa (RP) is a highly heterogeneous group of disorders characterized by degeneration of the retinal photoreceptor cells and progressive loss of vision. While hundreds of mutations in more than 100 genes have been reported to cause RP, discovering the causative mutations in many patients remains a significant challenge. Exome sequencing in an individual affected with non-syndromic RP revealed two plausibly disease-causing variants in TRNT1, a gene encoding a nucleotidyltransferase critical for tRNA processing. A total of 727 additional unrelated individuals with molecularly uncharacterized RP were completely screened for TRNT1 coding sequence variants, and a second family was identified with two members who exhibited a phenotype that was remarkably similar to the index patient. Inactivating mutations in TRNT1 have been previously shown to cause a severe congenital syndrome of sideroblastic anemia, B-cell immunodeficiency, recurrent fevers and developmental delay (SIFD). Complete blood counts of all three of our patients revealed red blood cell microcytosis and anisocytosis with only mild anemia. Characterization of TRNT1 in patient-derived cell lines revealed reduced but detectable TRNT1 protein, consistent with partial function. Suppression of trnt1 expression in zebrafish recapitulated several features of the human SIFD syndrome, including anemia and sensory organ defects. When levels of trnt1 were titrated, visual dysfunction was found in the absence of other phenotypes. The visual defects in the trnt1-knockdown zebrafish were ameliorated by the addition of exogenous human TRNT1 RNA. Our findings indicate that hypomorphic TRNT1 mutations can cause a recessive disease that is almost entirely limited to the retina.

Functional characterization of Prickle2 and BBS7 identify overlapping phenotypes yet distinct mechanisms

Ciliopathies are genetic disorders that are caused by dysfunctional cilia and affect multiple organs. One type of ciliopathy, Bardet-Biedl syndrome, is a rare disorder characterized by obesity, retinitis pigmentosa, polydactyly, mental retardation and susceptibility to cardiovascular diseases. The Wnt/Planar cell polarity (PCP) has been associated with cilia function and ciliogenesis in directing the orientation of cilia and basal bodies. Yet the exact relationship between PCP and ciliopathy is not well understood. Here, we examine interactions between a core PCP component, Prickle2 (Pk2), and a central BBS gene, Bbs7, using gene knockdown in the zebrafish. pk2 and bbs7 knockdown both disrupt the formation of a ciliated organ, the Kupffer׳s vesicle (KV), but do not display a synergistic interaction. By measuring cell polarity in the neural tube, we find that bbs7 activity is not required for Pk asymmetric localization. Moreover, BBS protein complex formation is preserved in the Pk2-deficient (Pk2(-/-)) mouse. Previously we reported an intracellular melanosome transport delay as a cardinal feature of reduced bbs gene activity. We find that pk2 knockdown suppresses bbs7-related retrograde transport delay. Similarly, knockdown of ift22, an anterograde intraflagellar transport component, also suppresses the bbs7-related retrograde delay. Notably, we find that pk2 knockdown larvae show a delay in anterograde transport. These data suggest a novel role for Pk2 in directional intracellular transport and our analyses show that PCP and BBS function independently, yet result in overlapping phenotypes when knocked down in zebrafish.

High-throughput behavioral assay to investigate seizure sensitivity in zebrafish implicates ZFHX3 in epilepsy

Abstract Epilepsy, which affects ∼1% of the population, is caused by abnormal synchronous neural activity in the central nervous system (CNS). While there is a significant genetic contribution to epilepsy, the underlying causes for the majority of genetic cases remain unknown. The NIH Undiagnosed Diseases Project (UDP) utilized exome sequencing to identify genetic variants in patients affected by various conditions with undefined etiology, including epilepsy. Confirming the functional relevance of the candidate genes identified by exome sequencing in a timely manner is crucial to translating exome data into clinically useful information. To this end, we developed a high throughput version of a seizure-sensitivity assay in zebrafish (Danio rerio) to rapidly evaluate candidate genes found by exome sequencing. We developed open access software, Studying Epilepsy In Zebrafish using R (SEIZR), to efficiently analyze the data. SEIZR was validated by disrupting function of a known epilepsy gene, prickle 1. Next, using SEIZR, we analyzed a candidate gene from the UDP screen (Zinc Finger Homeobox 3, ZFHX3), and showed that reduced ZFHX3 function in zebrafish results in a significant hyperactive response to the convulsant drug pentylenetetrazol (PTZ). We find that ZFHX3 shows strong expression in the CNS during neurogenesis including in the pallium, thalamus, tegmentum, reticular formation, and medulla oblongata – all regions which have roles in motor control and coordination. Our findings in the zebrafish confirm human ZFHX3 is a strong candidate for further neurological studies. We offer SEIZR to other researchers as a tool to rapidly and efficiently analyze large behavioral data sets.

The Nkd EF-hand domain modulates divergent wnt signaling outputs in zebrafish

Wnt proteins regulate diverse biological responses by initiating two general outcomes: β-catenin-dependent transcription and β-catenin-independent activation of signaling cascades, the latter including modulation of calcium and regulation of cytoskeletal dynamics (Planar Cell Polarity, PCP). It has been difficult to elucidate the mechanisms by which Wnt signals are directed to effect one or the other outcome due to shared signaling proteins between the β-catenin-dependent and -independent pathways, such as the Dishevelled binding protein Naked. While all Naked paralogs contain a putative calcium-binding domain, the EF-Hand, Drosophila Naked does not bind calcium. Here we find a lineage-specific evolutionary change within the Drosophila Naked EF-hand that is not shared with other insects or vertebrates. We demonstrate the necessary role of the EF-hand for Nkd localization changes in calcium fluxing cells and using in vivo assays, we identify a role for the zebrafish Naked EF-hand in PCP but not in β-catenin antagonism. In contrast, Drosophila-like Nkd does not function in PCP, but is a robust antagonist of Wnt/β-catenin signaling. This work reveals that the zebrafish Nkd1 EF-hand is essential to balance Wnt signaling inputs and modulate the appropriate outputs, while the Drosophila-like EF-Hand primarily functions in β-catenin signaling.