Evolutionarily conserved genetic interactions between nphp-4 and bbs-5 mutations exacerbate ciliopathy phenotypes.

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.

An Adult Zebrafish Model of Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare human skeletal disease caused by constitutively activating mutations in the gene ACVR1, which encodes a type I BMP/TGFß family member receptor. FOP is characterized by progressive heterotopic ossification (HO) of fibrous tissues, including skeletal muscle, tendons, and ligaments, as well as malformation of the big toes, vertebral fusions, and osteochondromas. Surgical interventions in patients often result in enhanced HO, which can exacerbate rather than improve diagnostic outcomes. As a result of these difficulties, a variety of animal models are needed to study human FOP. Here we describe the methods for creating and characterizing zebrafish conditionally expressing Acvr1lQ204D, the first adult zebrafish model for FOP.

Injury of Adult Zebrafish Expressing Acvr1lQ204D Does Not Result in Heterotopic Ossification.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant genetic disorder in humans characterized by the gradual ossification of fibrous tissues, including skeletal muscle, tendons, and ligaments. In humans, mutations in the Type I BMP/TGFß family member receptor gene, ACVR1, are associated with FOP. Zebrafish acvr1l, previously known as alk8, is the functional ortholog of human ACVR1. We previously created and characterized the first adult zebrafish model for FOP by generating animals harboring heat shock-inducible mCherry-tagged constitutively active Acvr1l (Q204D). Since injury is a known trigger for heterotopic ossification (HO) development in human FOP patients, in this study, we investigated several injury models in Acvr1lQ204D-expressing zebrafish and the subsequent formation of HO. We performed studies of Activin A injection, cardiotoxin (CTX) injection, and caudal fin clip injury. We found that none of these methods resulted in HO formation at the site of injury. However, some of the cardiotoxin-injected and caudal fin-clipped animals did exhibit HO at distant sites, including the body cavity and along the spine. We describe these results in the context of new and exciting reports on FOP, and discuss future studies to better understand the etiology and progression of this disease.

Animal models of fibrodysplasia ossificans progressiva.

Fibrodysplasia Ossificans Progressiva is a rare human disease of heterotopic ossification. FOP patients experience progressive development of ectopic bone within fibrous tissues that contributes to a gradual loss of mobility and can lead to early mortality. Due to lack of understanding of the etiology and progression of human FOP, and the fact that surgical interventions often exacerbate FOP disease progression, alternative therapeutic methods are needed, including modeling in animals, to study and improve understanding of human FOP. In this review we provide an overview of the existing animal models of FOP and the key mechanistic findings from each. In addition, we highlight the specific advantages of a new adult zebrafish model, generated by our lab, to study human FOP. Developmental Dynamics 247:279-288, 2018. © 2017 Wiley Periodicals, Inc.

A Zebrafish Model of Human Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare, autosomal dominant genetic disorder in humans characterized by explosive inflammatory response to injury leading to gradual ossification within fibrous tissues, including skeletal muscle, tendons, and ligaments. A variety of animal models are needed to study and understand the etiology of human FOP. To address this need, here we present characterizations of the first adult zebrafish model for FOP. In humans, activating mutations in the Type I BMP/TGFß family member receptor, ACVR1, are associated with FOP. Zebrafish acvr1l, previously known as alk8, is the functional ortholog of human ACVR1, and has been studied extensively in the developing zebrafish embryo, where it plays a role in early dorsoventral patterning. Constitutively active and dominant negative mutations in zebrafish acvr1l cause early lethal defects. Therefore, to study roles for activating acvr1l mutations in adult zebrafish, we created transgenic animals expressing mCherry-tagged, heat-shock-inducible constitutively active Acvr1l, Acvr1lQ204D, to investigate phenotypes in juvenile and adult zebrafish. Our studies showed that adult zebrafish expressing heat-shock-induced Acvr1lQ204D develop a number of human FOP-like phenotypes, including heterotopic ossification lesions, spinal lordosis, vertebral fusions, and malformed pelvic fins. Together, these results suggest that transgenic zebrafish expressing heat-shock-inducible Acvr1lQ204D can serve as a model for human FOP.

CACN-1/Cactin plays a role in Wnt signaling in C. elegans.

Wnt signaling is tightly regulated during animal development and controls cell proliferation and differentiation. In C. elegans, activation of Wnt signaling alters the activity of the TCF/LEF transcription factor, POP-1, through activation of the Wnt/ß-catenin or Wnt/ß-catenin asymmetry pathways. In this study, we have identified CACN-1 as a potential regulator of POP-1 in C. elegans larval development. CACN-1/Cactin is a well-conserved protein of unknown molecular function previously implicated in the regulation of several developmental signaling pathways. Here we have used activation of POPTOP, a POP-1-responsive reporter construct, as a proxy for Wnt signaling. POPTOP requires POP-1 and SYS-1/ß-catenin for activation in L4 uterine cells. RNAi depletion experiments show that CACN-1 is needed to prevent excessive activation of POPTOP and for proper levels and/or localization of POP-1. Surprisingly, high POPTOP expression correlates with increased levels of POP-1 in uterine nuclei, suggesting POPTOP may not mirror endogenous gene expression in all respects. Genetic interaction studies suggest that CACN-1 may act partially through LIT-1/NLK to alter POP-1 localization and POPTOP activation. Additionally, CACN-1 is required for proper proliferation of larval seam cells. Depletion of CACN-1 results in a loss of POP-1 asymmetry and reduction of terminal seam cell number, suggesting an adoption of the anterior, differentiated fate by the posterior daughter cells. These findings suggest CACN-1/Cactin modulates Wnt signaling during larval development.

Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development.

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.