Saving zebrafish mutants

At present, the zebrafish Danio rerio is the only vertebrate species for which a large-scale mutagenesis effort to identify developmental genes has been reported. Several laboratories are now intensely pursuing the molecular characterization of the genes affected by these mutations. One important criterion for the identity of the mutated gene is the rescue of the mutant phenotype by a wild-type (wt) copy of the gene. Until recently, most rescue attempts were carried out by injecting wt messenger RNA (mRNA) into fertilized eggs. A report by Yan and collaborators shows the partial rescue of floatinghead mutants by injection of genomic fragments cloned in either bacterial artificial chromosomes or bacteriophage lambda vectors. Combined with other ongoing efforts to characterize the zebrafish genome, this approach of mutant rescue opens interesting avenues for a systematic functional analysis of vertebrate genes.     

Identification of zebrafish insertional mutants with defects in visual system development and function

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.     

SPARC/osteonectin in mineralized tissue

Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM40) is one of the most abundant non-collagenous protein expressed in mineralized tissues. This review will focus on elucidating functional roles of SPARC in bone formation building upon results from non-mineralized cells and tissues, the phenotype of SPARC-null bones, and recent discoveries of human diseases with either dysregulated expression of SPARC or mutations in the gene encoding SPARC that give rise to bone pathologies. The capacity of SPARC to influence pathways involved in extracellular matrix assembly such as procollagen processing and collagen fibril formation as well as the capacity to influence osteoblast differentiation and osteoclast activity will be addressed. In addition, the potential for SPARC to regulate cross-linking of extracellular matrix proteins by members of the transglutaminase family of enzymes is explored. Elucidating defined biological functions of SPARC in terms of bone formation and turnover are critical. Further insight into specific cellular mechanisms involved in the formation and homeostasis of mineralized tissues will lead to a better understanding of disease progression.     

Identification of a new mineralized tissue in the notochord of reared Siberian sturgeon (Acipenser baerii)

In a study aiming to improve knowledge on the mineralization of the axial skeleton in reared Siberian sturgeon (Acipenser baerii Brandt, 1869), we discovered a new mineralized tissue within the notochord. To our knowledge, such a structure has never been reported in any vertebrate species with the exception of the pathological mineralization of the notochord remains in degenerative intervertebral disks of mammals. Here, we describe this enigmatic tissue using X‐ray microtomography, histological analyses and solid state NMR‐spectroscopy. We also performed a 1‐year monitoring of the mineral content (MC) of the notochord in relation with seasonal variations of temperature. In all specimens studied from 2‐year‐old juveniles onwards, this mineralized structure was found within a particular region of the notochord called funiculus . This feature first appears in the abdominal region then extends posteriorly with ageing, while the notochord MC also increases. The mineral phase is mainly composed of amorphous calcium phosphate, a small amount of which changes into hydroxyapatite with ageing. The putative role of this structure is discussed as either a store of minerals available for the phosphocalcic metabolism, or a mechanical support in a species with a poorly mineralized axial skeleton. A pathological feature putatively related to rearing conditions is also discussed.     

Dystrophin in adult zebrafish muscle

Mutations in the human dystrophin gene are implicated in the fatal muscle wasting disease Duchenne Muscular Dystrophy (DMD). This gene expresses a sarcolemmal-associated protein that is evolutionarily conserved, underpinning its important role in the architecture of muscle. In terms of DMD modelling, the mouse has served as a suitable vertebrate species but the pathophysiology of the disease in the mouse does not entirely mimic human DMD. We have examined the zebrafish in order to expand the repertoire of vertebrate species for muscle disease modelling, and to dissect further the functional interactions of dystrophin. We report here the identification of an apparent zebrafish orthologue of the human dystrophin gene that expresses a 400-kDa protein that is localised to the muscle membrane surface. These data suggest that the zebrafish may prove to be a beneficial vertebrate model to examine the role and functional interactions of dystrophin in disease and development.     

Three-dimensional neurophenotyping of adult zebrafish behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior.     

成年斑马鱼OKR行为学分析

作为视功能检测和与视觉有关突变体筛选的方法, 眼动(Optokinetic response, OKR)和视动(Optomoter response, OMR)行为学是简单有效的视功能检测手段, 广泛用于幼年斑马鱼研究中, 而成年斑马鱼OKR的分析方法却很少有报道。文章介绍了成年斑马鱼眼动反应诱导方式, 以及使用模板匹配(Pattern match)的方法程序跟踪眼部运动, 实现了成年斑马鱼OKR的定量分析。使用该方法, 检测到斑马鱼双眼视觉区对OKR行为的产生具有一定的贡献作用, 并且成年斑马鱼单眼对运动光栅表现出一定的方向敏感性。同样的方法也可适用于幼年斑马鱼的OKR行为学分析。利用此方法初步检测到了钟基因period1b突变体幼鱼的OKR异常。     

Microanatomy of adult zebrafish extraocular muscles

Binocular vision requires intricate control of eye movement to align overlapping visual fields for fusion in the visual cortex, and each eye is controlled by 6 extraocular muscles (EOMs). Disorders of EOMs are an important cause of symptomatic vision loss. Importantly, EOMs represent specialized skeletal muscles with distinct gene expression profile and susceptibility to neuromuscular disorders. We aim to investigate and describe the anatomy of adult zebrafish extraocular muscles (EOMs) to enable comparison with human EOM anatomy and facilitate the use of zebrafish as a model for EOM research. Using differential interference contrast (DIC), epifluorescence microscopy, and precise sectioning techniques, we evaluate the anatomy of zebrafish EOM origin, muscle course, and insertion on the eye. Immunofluorescence is used to identify components of tendons, basement membrane and neuromuscular junctions (NMJs), and to analyze myofiber characteristics. We find that adult zebrafish EOM insertions on the globe parallel the organization of human EOMs, including the close proximity of specific EOM insertions to one another. However, analysis of EOM origins reveals important differences between human and zebrafish, such as the common rostral origin of both oblique muscles and the caudal origin of the lateral rectus muscles. Thrombospondin 4 marks the EOM tendons in regions that are highly innervated, and laminin marks the basement membrane, enabling evaluation of myofiber size and distribution. The NMJs appear to include both en plaque and en grappe synapses, while NMJ density is much higher in EOMs than in somatic muscles. In conclusion, zebrafish and human EOM anatomy are generally homologous, supporting the use of zebrafish for studying EOM biology. However, anatomic differences exist, revealing divergent evolutionary pressures.     

A study of dense mineralized tissue by neutron diffraction

Neutron diffraction studies of mineralized tissue show a close relationship between the wet state equatorial diffraction spacing and wet tissue density expressable as a second-order polynomial. The molecular fractional shrinkage when the tissue is dried shows a straight line dependence on wet tissue density with a correlation of 0.98. Since the dry state equatorial diffraction spacing is much less than for the corresponding wet state, even in fully mineralized bone, the collagen molecules must be displaced through a mineral-free volume while drying. The mineral can only be located within the available volume of the dried tissue whether intra- or extrafibrillar. The dimension of the dry state equatorial spacing for each of the tissues examined is close to that of dried tendon collagen. It appears unlikely that hydroxyapatite crystallites can be accommodated radially between collagen molecules in bone if the packing is like that of dried tail tendon collagen. The only mineral within the fibrils must be in the intermolecular gaps. It is estimated on the basis of the volume of the axial intermolecular gaps and the minimum extrafibrillar volume that the intrafibrillar mineral can be no more than 20% of the total mineral and may be less than 10%.     

Molecular-genetic mapping of zebrafish mutants with variable phenotypic penetrance

Forward genetic screens in vertebrates are powerful tools to generate models relevant to human diseases, including neuropsychiatric disorders. Variability in phenotypic penetrance and expressivity is common in these disorders and behavioral mutant models, making their molecular-genetic mapping a formidable task. Using a 'phenotyping by segregation' strategy, we molecularly map the hypersensitive zebrafish houdini mutant despite its variable phenotypic penetrance, providing a generally applicable strategy to map zebrafish mutants with subtle phenotypes.     

Hypoxia-induced retinopathy model in adult zebrafish

Hypoxia-induced vascular responses, including angiogenesis, vascular remodeling and vascular leakage, significantly contribute to the onset, development and progression of retinopathy. However, until recently there were no appropriate animal disease models recapitulating adult retinopathy available. In this article, we describe protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1:EGFP zebrafish are placed in hypoxic water for 3-10 d and retinal neovascularization is analyzed using confocal microscopy. It usually takes 11 d to obtain conclusive results using the hypoxia-induced retinopathy model in adult zebrafish. This model provides a unique opportunity to study kinetically the development of retinopathy in adult animals using noninvasive protocols and to assess therapeutic efficacy of orally active antiangiogenic drugs.     

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Summary Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CM^R filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.     

Microcracks colocalize within highly mineralized regions of cortical bone tissue

While much work has been performed to quantify the extent of bone damage, its effects on the mechanical integrity of the tissue and its biological impact, the set of factors which gives forth to microdamage are nebulous, particularly the compositional properties local to microdamage. In this context, the current study tested the hypothesis that microcracks initiate within more mineralized regions of bone. Cortical bone specimens were taken from human male donors aged 31, 38, 53, 64, 71, and 84 years at the mid femoral diaphysis in a plane parallel to the osteonal orientation. The mineralization was assessed in a spatially resolved manner using Raman microspectroscopy. Arrays of measurements were taken over the entire area (i.e. global scans) of each sample followed by measurements in the vicinity of microcracks (i.e. local scans). Histograms of mineralization were constructed for global and local scans to determine whether the mineralization of damaged loci differed from the mean overall mineralization. Statistical analysis of this data revealed that the mean mineralization of damaged loci was significantly greater (P < 0.05) than the overall mineralization for each donor, indicating that there exists a highly-mineralized 'brittle volume' in bone. The presence of this damage prone 'brittle volume' has future implications for the assessment of fracture susceptibility.     

Gastrulation in zebrafish: what mutants teach us.

A major approach to the study of development is to compare the phenotypes of normal and mutant individuals for a given genetic locus. Understanding the development of a complex metazoan therefore requires examination of many mutants. Relatively few organisms are being studied this way, and zebrafish is currently the best example of a vertebrate for which large-scale mutagenesis screens have successfully been carried out. The number of genes mutated in zebrafish that have been cloned expands rapidly, bringing new insights into a number of developmental pathways operating in vertebrates. Here, we discuss work on zebrafish mutants affecting gastrulation and patterning of the early embryo. Gastrulation is orchestrated by the dorsal organizer, which forms in a region where maternally derived beta-catenin signaling is active. Mutation in the zygotic homeobox gene bozozok disrupts the organizer genetic program and leads to severe axial deficiencies, indicating that this gene is a functional target of beta-catenin signaling. Once established, the organizer releases inhibitors of ventralizing signals, such as BMPs, and promotes dorsoanterior fates within all germ layers. In zebrafish, several mutations affecting dorsal-ventral (D/V) patterning inactivate genes functioning in the BMP pathway, stressing the central role of this pathway in the gastrula embryo. Cells derived from the organizer differentiate into several axial structures, such as notochord and prechordal mesoderm, which are thought to induce various fates in adjacent tissues, such as the floor plate, after the completion of gastrulation. Studies with mutants in nodal-related genes, in one-eyed pinhead, which is required for nodal signaling, and in the Notch pathway reveal that midline cell fate specification is, in fact, initiated during gastrulation. Furthermore, the organizer coordinates morphogenetic movements, and zebrafish mutants in T-box mesoderm-specific genes help clarify the mechanism of convergence movements required for the formation of axial and paraxial mesoderm.     

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.     

The identification of zebrafish mutants showing alterations in senescence-associated biomarkers

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated beta-galactosidase (SA-beta-gal) in our current study. We validated the use of embryonic SA-beta-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-beta-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-beta-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-beta-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and senescence mechanisms.     

Issues related to mineralized tissue biology in human evolutionary research

This comunication has two primary aims concerned with mineralized tissue biology (e.g. hard tissue biology of bone and tooth) research in human evolutionary studies: First, to introduce the literature and the methods (at the time of this symposium) so that one has an idea of the nature of this research and where one can go for details of the methodologies, etc; Second — and of primary concern here — to discuss issues that have come to light as a result of these studies mainly because of its recent beginnings as a subfield within paleoanthropology. Issues related to skeletal studies include; 1) whether different cortical surface pattens and bone tissue types influence the appearance and interpretation of bone growth activity states; 2) if SEM analyses of cortical surfaces in fossil hominids allow one to construct meaningful representations of remodeling patterns; 3) whether these representations can be used in phylogenetic arguments; and 4) how intraspecific variability would affect these issues. Issues related to dental studies include: 1) the relationship between the rate and pattern of eraly hominid dental development; 2) experimental support for the calibration of eraly hominid dental developmental rates; and 3) whether replica techniques are suitable for microanatomical studies of these sorts.     

Clastic cells: mineralized tissue resorption in health and disease

Clastic cells are responsible for mineralized tissue resorption. Bone resorbing cells are called osteoclasts; however, they are able to resorb mineralized dental tissues or calcified cartilage and then they are called odontoclasts and chondroclasts, respectively. They derive from mononuclear precursors of the monocyte-macrophage lineage from hemopoietic tissue, reach target mineralized tissues and degrade them under many different physiologic or pathologic stimuli. Clastic cells play a key role in calcium homeostasis, and participate in skeletal growth, tooth movement, and other physiological and pathological events. They interact tightly with forming cells in bone and dental hard tissues; their unbalance may result in disturbed resorptive activity thus, causing local or systemic diseases.     

Kidney stem cells found in adult zebrafish

Recently in Nature, Davidson and coworkers (Diep et al., 2011) identified nephron progenitors/stem cells located at the point of fusion with the pronephric tubules in adult zebrafish. Clumps of progenitors give rise to functional nephrons after serial transplantation, demonstrating the ability of tissue stem cells to regenerate damaged kidney structures.