Rodney M. Dale

The emerging role of Wnt/PCP signaling in organ formation.

Over the last two decades zebrafish has been an excellent model organism to study vertebrate development. Mutant analysis combined with gene knockdown and other manipulations revealed an essential role of Wnt signaling, independent of beta-catenin, during development. Especially well characterized is the function of Wnt/planar cell polarity (PCP) signaling in the regulation of gastrulation movements and neurulation, described in other reviews within this special issue. Here, we set out to highlight some of the new and exciting research that is being carried out in zebrafish to elucidate the role that Wnt/PCP signaling plays in the formation of specific organs, including the lateral line, craniofacial development, and regeneration. We also summarized the emerging connection of the Wnt/PCP pathway with primary cilia function, an essential organelle in several organ activities.

Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations

Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue.

Myoferlin regulation by NFAT in muscle injury, regeneration and repair

Ferlin proteins mediate membrane-fusion events in response to Ca2+. Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.

Zebrafish: Modeling for Herpes Simplex Virus Infections

For many years, zebrafish have been the prototypical model for studies in developmental biology. In recent years, zebrafish has emerged as a powerful model system to study infectious diseases, including viral infections. Experiments conducted with herpes simplex virus type-1 in adult zebrafish or in embryo models are encouraging as they establish proof of concept with viral-host tropism and possible screening of antiviral compounds. In addition, the presence of human homologs of viral entry receptors in zebrafish such as 3-O sulfated heparan sulfate, nectins, and tumor necrosis factor receptor superfamily member 14-like receptor bring strong rationale for virologists to test their in vivo significance in viral entry in a zebrafish model and compare the structure-function basis of virus zebrafish receptor interaction for viral entry. On the other end, a zebrafish model is already being used for studying inflammation and angiogenesis, with or without genetic manipulations, and therefore can be exploited to study viral infection-associated pathologies. The major advantage with zebrafish is low cost, easy breeding and maintenance, rapid lifecycle, and a transparent nature, which allows visualizing dissemination of fluorescently labeled virus infection in real time either at a localized region or the whole body. Further, the availability of multiple transgenic lines that express fluorescently tagged immune cells for in vivo imaging of virus infected animals is extremely attractive. In addition, a fully developed immune system and potential for receptor-specific knockouts further advocate the use of zebrafish as a new tool to study viral infections. In this review, we focus on expanding the potential of zebrafish model system in understanding human infectious diseases and future benefits.

A role of glypican4 and wnt5b in chondrocyte stacking underlying craniofacial cartilage morphogenesis.

The Wnt/Planar Cell Polarity (PCP) pathway controls cell morphology and behavior during animal development. Several zebrafish mutants were identified as having perturbed Wnt/PCP signaling. Many of these mutants have defects in craniofacial formation. To better understand the role that Wnt/PCP plays in craniofacial development we set out to identify which of the mutants, known to be associated with the Wnt/PCP pathway, perturb head cartilage formation by disrupting chondrocyte morphology. Here we demonstrate that while vang-like 2 (vangl2), wnt11 and scribbled (scrib) mutants have severe craniofacial morphogenesis defects they do not display the chondrocyte stacking and intercalation problems seen in glypican 4 (gpc4) and wnt5b mutants. The function of Gpc4 or Wnt5b appears to be important for chondrocyte organization, as the neural crest in both mutants is specified, undergoes migration, and differentiates into the same number of cells to compose the craniofacial cartilage elements. We demonstrate that Gpc4 activity is required cell autonomously in the chondrocytes and that the phenotype of single heterozygous mutants is slightly enhanced in embryos double heterozygous for wnt5b and gpc4. This data suggests a novel mechanism for Wnt5b and Gpc4 regulation of chondrocyte behavior that is independent of the core Wnt/PCP molecules and differs from their collaborative action of controlling cell movements during gastrulation.

Iroquois Proteins Promote Skeletal Joint Formation by Maintaining Chondrocytes in an Immature State

An early event in skeletal joint development is the specification of articular chondrocytes at the joint surface. Articular chondrocytes are distinct in producing lower levels of cartilage matrix and not being replaced by bone, yet how they acquire these properties remains poorly understood. Here, we show that two members of the Iroquois transcriptional repressor family, Irx7 and Irx5a, function to block chondrocyte maturation at the developing hyoid joint of zebrafish. These Irx factors suppress the production of cartilage matrix at the joint in part by preventing the activation of a col2a1a enhancer by Sox9a. Further, both zebrafish Irx7 and mouse IRX1 are able to repress cartilage matrix production in a murine chondrogenic cell line. Iroquois proteins may therefore have a conserved role in keeping chondrocytes in an immature state, with the lower levels of cartilage matrix produced by these immature cells contributing to joint flexibility.

Planar cell polarity signaling in craniofacial development

Out of the several signaling pathways controlling craniofacial development, the role of planar cell polarity (PCP) signaling is relatively poorly understood. This pathway, originally identified as a mechanism to maintain cell polarity within the epithelial cells of the Drosophila wing, has been linked to the proper development of a wide variety of tissues in vertebrates and invertebrates. While many of the pathway members are conserved, it appears that some of the members of the pathway act in a tissue-specific manner. Here, we discuss the role of this pathway in vertebrate craniofacial development, highlighting cranial neural crest migration, skull and palate formation and the role of non-traditional modulators of PCP signaling within this developmental process.

Direct activation of chordoblasts by retinoic acid is required for segmented centra mineralization during zebrafish spine development

ABSTRACT Zebrafish mutants with increased retinoic acid (RA) signaling due to the loss of the RA-inactivating enzyme Cyp26b1 develop a hyper-mineralized spine with gradually fusing vertebral body precursors (centra). However, the underlying cellular mechanisms remain incompletely understood. Here, we show that cells of the notochord epithelium named chordoblasts are sensitive to RA signaling. Chordoblasts are uniformly distributed along the anteroposterior axis and initially generate the continuous collagenous notochord sheath. However, subsequently and iteratively, subsets of these cells undergo further RA-dependent differentiation steps, acquire a stellate-like shape, downregulate expression of the collagen gene col2a1a, switch on cyp26b1 expression and trigger metameric sheath mineralization. This mineralization fails to appear upon chordoblast-specific cell ablation or RA signal transduction blockade. Together, our data reveal that, despite their different developmental origins, the activities and regulation of chordoblasts are very similar to those of osteoblasts, including their RA-induced transition from osteoid-producing cells to osteoid-mineralizing ones. Furthermore, our data point to a requirement for locally controlled RA activity within the chordoblast layer in order to generate the segmented vertebral column. Highlighted Article: Retinoic acid induces locally controlled morphological and transcriptional changes in chordoblasts that lead to segmental notochord sheath mineralization, identifying chordoblasts as key skeletogenic cells during chordacentrum development in zebrafish.

Wilms Tumor 1b defines a wound-specific sheath cell subpopulation associated with notochord repair

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments.

Expression and characterization of the zebrafish orthologue of the human FOLR1 gene during embryogenesis

It has been well established that many types of rapidly dividing normal and diseased cells require an increased amount of folate for DNA replication and repair as well as cellular metabolism. Thus one of folates cognate receptors, Folate Receptor 1 (FOLR1) is usually up-regulated in rapidly dividing cells, including many types of cancerous tumors. Because zebrafish have become a model organism for understanding conserved vertebrate cellular pathways and human disease, there has been an increased need to identify and elucidate orthologous zebrafish genes that are central to known human maladies. The cells of all early animal embryos go through a phase of rapid division (cleavage) where particular cell cycle checkpoints are skipped until a specification event occurs directing these embryonic stem cells to their fated germ layer cell type. Interestingly, this rapid cell division that ignores cell cycle checkpoints is also observed in many cancers. Developing blastula and tumor cells both require folr1 expression to obtain folate. In this report we have identified the expression pattern of the zebrafish gene zgc:165502, located on chromosome 15. Using computational and comparative methods and molecular biology techniques such as reverse transcription polymerase chain reaction (RT-PCR) and whole mount in situ hybridization (WISH) during embryogenesis, we demonstrate that zgc:165502 is the zebrafish orthologue of the human FOLR1 gene. Understanding when and where FOLR1 orthologues are expressed in different biomedical model organisms such as the zebrafish will help researchers design better experiments to study the endogenous FOLR1 activity.