Bradley Yoder

University of Alabama at Birmingham; Birmingham AL, United States of America

MH2211

Caenorhabditis elegans

LaFayette, Cameron et al. (2019) International Worm Meeting "Exploring cca-1 as a novel regulator of primary cilia function in C. elegans."

Yoder, Bradley, LaBonty, Melissa, LaFayette, Cameron

[International Worm Meeting, 2019]

Primary cilia are cell surface-localized, microtubule-based organelles that are involved in various signal transduction pathways. The transition zone (TZ) of the primary cilia houses numerous proteins that are critical to proper ciliary function. When TZ proteins are mutated, they can cause large-scale ciliary dysfunction, and can lead to disorders known as ciliopathies in humans. One such ciliopathy is Nephronophthisis (NPHP), a cystic kidney disease that is associated with mutations found in the TZ protein NPHP4. Although it is known for its link to cystic kidney disease, NPH has additional variable phenotypes. We hypothesize that the variable phenotypes seen in ciliopathy patients are aggregate effects of several mutations. Due to the similarity in primary cilia form and function between humans and nematodes, we are using C. elegans as a model to explore nphp-4 gene interactions. The objective of my project is to validate cca-1 as a novel regulator of nphp-4 for proper primary cilia function. Previously, our lab conducted an EMS mutagenesis screen in C. elegans to identify potential genes that may interact with nphp-4. One such gene that was identified was cca-1, which encodes a voltage-gated calcium channel subunit. We have confirmed the genetic interaction between cca-1 and nphp-4 through the use of the DiI dye-filling assay for ciliary integrity. Moving forward, we plan to investigate changes in ciliary structure and sensory behaviors in cca-1, nphp-4, and double mutants. We also plan to experiment with live imaging of calcium dynamics to determine whether cca-1's known functions are related to its role in primary cilia. We hope that this work done in C. elegans demonstrates how the interaction of cca-1 and nphp-4 is critical for normal primary cilia function, ultimately illustrating how a cca-1 mutation can be linked to NPH patients.

MH2294

Caenorhabditis elegans

Sengupta P et al. (2014) Traffic "New insights into an old organelle: meeting report on biology of cilia and flagella."

Barr MM, Sengupta P

[Traffic, 2014]

The rising interest of the scientific community in cilia biology was evident from the fact that registration for the third FASEB conference on 'The Biology of Cilia and Flagella' closed out before the early bird deadline. Cilia and flagella are organelles of profound medical importance; defects in their structure or function result in a plethora of human diseases called ciliopathies. 240 clinicians and basic scientists from around the world gathered from 23 June 2013 to 28 June 2013 at Sheraton at the Falls, Niagara Falls, NY to present and discuss their research on this intensely studied subcellular structure. The meeting was organized by Gregory Pazour (University of Massachusetts Medical School), Bradley Yoder (University of Alabama-Birmingham), and Maureen Barr (Rutgers University) and was sponsored by the Federation of American Societies for Experimental Biology (FASEB). Here, we report highlights, points of discussion, and emerging themes from this exciting meeting.

LaBonty, Melissa et al. (2019) International Worm Meeting "Identifying novel regulators of primary cilia function in C. elegans."

LaBonty, Melissa, LaFayette, Cameron, Yoder, Bradley

[International Worm Meeting, 2019]

Primary cilia are microtubule-based structures that extend from nearly all mammalian cell types and play a role in cellular sensing and signal transduction. Ciliopathies are a spectrum of human disorders associated with defects in primary cilia formation and function. Mutations in the cilia-associated gene MKS1 cause three different ciliopathies, Meckel-Gruber Syndrome, Joubert Syndrome, and Bardet-Biedl Syndrome with varying severity. It is not well understood why mutations in the same human gene, MKS1, can result in multiple disorders with varying clinical features. We hypothesize that this phenotypic variability may stem from the presence of modifier alleles causing different degrees of primary cilia dysfunction. Therefore, the objective of this work is to identify novel genetic mutations that can exacerbate the effects of loss of MKS1 gene function. To identify these novel modifiers, we are using the tractable invertebrate model organism, C. elegans, which exhibits conservation of most aspects of primary cilia form and function when compared to mammals. We conducted an ethyl methanesulfonate (EMS) mutagenesis screen on mks-1(yhw146) mutants to identify secondary mutations that, when combined with an mks-1 mutation, cause defects in primary cilia formation. These defects were identified by looking for loss of normal DiI dye filling of ciliated sensory neurons. We isolated 10 strains carrying homozygous recessive gene mutations that combine with mks-1(yhw146) to result in defects in cilia function. Next generation sequencing is underway to identify the causal mutations. The isolation of multiple secondary mutations from our mutagenesis screen suggests that we will be able to identify and characterize novel genes influencing primary cilia formation and function in C. elegans. These discoveries could translate into new diagnostic and therapeutic possibilities for human ciliopathy patients.

Williams, Corey L et al. (2009) International Worm Meeting "B9 domain proteins are required for the regulation of ciliary membrane composition."

Williams, Corey L, Yoder, Bradley K, Winkelbauer, Marlene E

[International Worm Meeting, 2009]

Cilia are microtubule-based and membrane-bound organelles that serve diverse functions in various organisms and tissues. The ciliary membrane is a tightly regulated region that is enriched with many proteins not found throughout the rest of the cell membrane. Likewise, many proteins found in the cell membrane are excluded from the cilium. The mechanisms by which the cell membrane and ciliary membrane are differentially regulated are unknown. It is proposed that proteins at the base of the cilium participate in cell/ciliary membrane protein sorting, but their molecular identity has not been uncovered. Here, we demonstrate that the B9 domain proteins, TZA-1(Y38F2AL.2) and TZA-2(K03E6.4), function in such a capacity. MKS-3(F35D2.4), a transmembrane protein implicated in the human developmental disorder Meckel-Gruber Syndrome (MKS), normally localizes specifically to the ciliary base in C. elegans. In the absence of TZA-1 or TZA-2, MKS-3 freely enters the cilium, suggesting a requirement of these proteins in restricting ciliary access. Additionally, the transmembrane protein TRAM-1a (C24F3.1a), which normally concentrates directly adjacent to the ciliary base at the dendritic tip, is allowed ciliary access in the absence of TZA-1 or TZA-2. These data provide important insight into a mechanism by which ciliary membrane composition is regulated.

Masyukova, Svetlana et al. (2011) International Worm Meeting "A new osm-3 missense allele is a specific genetic modifier of nphp-4 cilia phenotypes."

Masyukova, Svetlana, Ptacek, Travis, Yoder, Bradley, Roszczynialski, Kelly, Williams, Corey

[International Worm Meeting, 2011]

Primary cilium dysfunctions cause complex oligogenic disorders called the ciliopathies. Among the ciliopathies are Nephronophthisis (NPHP), and Meckel-Gruber syndrome (MKS). MKS/NPHP patients exhibit multiple phenotypes including midgestation lethality, left-right body asymmetry defects, skeletal abnormalities, cystic kidney disease, retinal degeneration and CNS malformation. In NPHP4 patients phenotypes can be limited to renal dysfunction or may include retinal degeneration, with no apparent genotype-phenotype correlation. Many of the genes identified in NPHP and MKS patients encode proteins that localize to the base of the cilium. Most of these proteins are conserved in Caenorhabditis elegans. Compound mutations in C.elegans nphp and mks genes result in the appearance of more severe cilia phenotypes that are not observed in any of the single mutants, for example inability to absorb lipophilic dye DiI into the sensory neurons (Dyf phenotype). The goal of this study was to identify mutations in new genes producing synergistic Dyf phenotypes with nphp-4(tm925) null mutation using C. elegans. These genes would be potential targets for analysis in human patients. For this we performed a large scale EMS mutagenesis screen on nphp-4 (tm925) mutants and isolated 40 synthetic Dyf mutant lines. SNP mapping was done to place new mutations on chromosomes. Interval mapping and gene rescue experiments performed for one of the lines (YHW66) identified a new missense mutation in osm-3 gene encoding kinesin motor protein required for building of ciliary distal segments in subset of sensory neurons. Similarly, Zebrafish homolog of osm-3, Kif17 is essential for vertebrate photoreceptor sensory outer segment development. Multispecies alignment revealed that the position of the new mutation is highly conserved in humans. Dye-filling and osmotic avoidance experiments showed that new osm-3 allele does not produce noticeable cilia defects compared to wild type worms. In contrast, when combined with nphp-4(tm925) mutation, but not mutations in other transition zone proteins, it caused severe dye-filling and osmotic avoidance defects comparable to osm-3 null allele. Further, OSM-3::GFP containing the new missense mutation properly localizes to the cilium in wild type background. Interestingly, it abnormally accumulates at the base of the cilium in YHW66 mutant background. Together our data suggest that NPHP-4 may be involved in controlling OSM-3 entry to the cilium. This can potentially explain retinal degeneration in a subgroup of NPHP4 human patients. Collectively, our studies identify a new genetic modifier of nphp-4 and help to understand complex genetic interactions contributing to the diversity of phenotypes associated with cilia disorders.

LaBonty, Melissa et al. (2021) International Worm Meeting "Investigating the Genetic Interaction Between Ciliary bbs-5 and nphp-4"

LaBonty, Melissa, Bentley-Ford, Melissa, Yoder, Bradley, LaFayette, Cameron, Croyle, Mandy, Scott, Mikyla, Parant, John

[International Worm Meeting, 2021]

Primary cilia are critical sensing and signaling hubs that extend from nearly all mammalian cell types. Ciliopathies are a spectrum of human disorders associated with defects in cilia formation and function that result in a wide and variable range of clinical features, often with low genotype to phenotype correlation. This phenotypic variability may stem from the presence of multiple modifier alleles causing different degrees of primary cilia dysfunction. To identify modifier alleles, we conducted a mutagenesis screen in C. elegans with a primary mutation in the ciliary transition zone component, nphp-4(tm925), and looked for secondary mutations that caused ciliary dysfunction. From this screen, we identified a mutation in the BBSome component, bbs-5(yhw62), as a genetic modifier of nphp-4(tm925) mutants. By assessing a variety of behaviors associated with ciliated sensory neuron function, including chemotaxis and egg laying, we find that our novel bbs-5(yhw62) mutation causes enhanced ciliary defects when compared to an existing mutation, bbs-5(gk537). In addition, we observe that bbs-5; nphp-4 double mutants carrying either bbs-5 mutation display dye filling defects in ciliated sensory neurons and defects in dauer formation not seen in single mutants alone. We have also investigated whether the genetic interaction between bbs-5 and nphp-4 is conserved in two vertebrate models, zebrafish and mouse. In adult Bbs5; Nphp4 zebrafish, we observe scoliosis and disorganization of the outer nuclear layer of the retinal photoreceptor cells, although these phenotypes are also observed in adult Bbs5 single mutant animals. In contrast to zebrafish, mice with congenital expression of both Bbs5 and Nphp4 do not survive to weaning age. Juvenile induction of Bbs5 loss in an Nphp4 mutant background is associated with seizure-like activity and early mortality. These results suggest that the genetic interaction between bbs-5 and nphp-4 identified in C. elegans that causes enhanced ciliary dysfunction may be conserved in mice, but not in zebrafish. The mammalian conservation of this genetic interaction provides excellent rationale to pursue further mechanistic studies in C. elegans.

Li, Chunmei et al. (2011) International Worm Meeting "Sensing the environment: novel molecular pathway for the formation of sensory cilia."

Kida, Katarzyna, Blacque, Oliver E., Williams, Corey L., Leroux, Michel R., Yoder, Bradley K., Li, Chunmei, Jensen, Victor L.

[International Worm Meeting, 2011]

C. elegans perceives its environment mainly by way of sensory neurons which have cilia at the distal ends of dendrites, much like mammals can smell with the use of olfactory cilia or can see with photoreceptor cilia. In studying several human disorders involving cilia dysfunction (ciliopathies), we have uncovered a novel molecular pathway necessary for ciliogenesis. The disorders in question-nephronophthisis (NPHP), Meckel syndrome (MKS), Joubert syndrome (JBTS), Senior-Loken syndrome (SLSN), and Leber congenital amaurosis (LCA)-present with overlapping ailments, such as retinopathy, kidney disease, liver fibrosis and brain malformations. They also show considerable allelism between at least twelve causative genes, suggesting a common molecular aetiology that remains unexplained. We demonstrate using C. elegans that the recently-identified MKS-6 and MKS-2 proteins, together with MKS-1, MKSR-1, MKSR-2, MKS-5, NPHP-1 and NPHP-4, collectively function at the base of cilia, in a region termed transition zone (TZ), to orchestrate cilium formation. Specifically, the proteins act as two distinct modules, which we term MKS and NPHP, to facilitate basal body-transition zone anchoring to the membrane; disruption of the TZ proteins results in defects in prominent ciliary TZ and axoneme formation defects, and thus, chemosensory anomalies. This first pathway is independent of a second pathway specifically required for the formation and function of the ciliary organelles, involving intraflagellar transport (IFT) and Bardet-Biedl syndrome (BBS) proteins. Our genetic and cell biology analyses reveal a hierarchical organisation of the TZ proteins, with MKS-5 as the central anchor, followed by B9 domain-containing proteins (MKS-1, MKSR-1, MKSR-2). Together, our findings expand the interaction network of ciliopathy-associated proteins and suggest a two-stage ciliogenic pathway that first involves transition zone proteins, followed by an intraflagellar transport (IFT)-dependent formation of the remaining axoneme.