Zebrafish and medaka as models for biomedical research of bone diseases.

The identification of disease-causing mutations has in recent years progressed immensely due to whole genome sequencing approaches using patient material. The task accordingly is shifting from gene identification to functional analysis of putative disease-causing genes, preferably in an in vivo setting which also allows testing of drug candidates or biotherapeutics in whole animal disease models. In this review, we highlight the advances made in the field of bone diseases using small laboratory fish, focusing on zebrafish and medaka. We particularly highlight those human conditions where teleost models are available.

Mutations in the T (brachyury) gene cause a novel syndrome consisting of sacral agenesis, abnormal ossification of the vertebral bodies and a persistent notochordal canal.

BACKGROUND: The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates. RESULTS: We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1 Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development. CONCLUSIONS: We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.

New tools for studying osteoarthritis genetics in zebrafish.

OBJECTIVE: Increasing evidence points to a strong genetic component to osteoarthritis (OA) and that certain changes that occur in osteoarthritic cartilage recapitulate the developmental process of endochondral ossification. As zebrafish are a well validated model for genetic studies and developmental biology, our objective was to establish the spatiotemporal expression pattern of a number of OA susceptibility genes in the larval zebrafish providing a platform for functional studies into the role of these genes in OA. DESIGN: We identified the zebrafish homologues for Mcf2l, Gdf5, PthrP/Pthlh, Col9a2, and Col10a1 from the Ensembl genome browser. Labelled probes were generated for these genes and in situ hybridisations were performed on wild type zebrafish larvae. In addition, we generated transgenic reporter lines by modification of bacterial artificial chromosomes (BACs) containing full length promoters for col2a1 and col10a1. RESULTS: For the first time, we show the spatiotemporal expression pattern of Mcf2l. Furthermore, we show that all six putative OA genes are dynamically expressed during zebrafish larval development, and that all are expressed in the developing skeletal system. Furthermore, we demonstrate that the transgenic reporters we have generated for col2a1 and col10a1 can be used to visualise chondrocyte hypertrophy in vivo. CONCLUSION: In this study we describe the expression pattern of six OA susceptibility genes in zebrafish larvae and the generation of two new transgenic lines marking chondrocytes at different stages of maturation. Moreover, the tools used demonstrate the utility of the zebrafish model for functional studies on genes identified as playing a role in OA.

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Deciphering the heterogeneity of the Lyve1+ perivascular macrophages in the mouse brain.

Perivascular macrophages (pvMs) are associated with cerebral vasculature and mediate brain drainage and immune regulation. Here, using reporter mouse models, whole brain and section immunofluorescence, flow cytometry, and single cell RNA sequencing, besides the Lyve1+F4/80+CD206+CX3CR1+ pvMs, we identify a CX3CR1- pvM population that shares phagocytic functions and location. Furthermore, the brain parenchyma vasculature mostly hosts Lyve1+MHCII- pvMs with low to intermediate CD45 expression. Using the double Cx3cr1GFP x Cx3cr1-Cre;RosatdT reporter mice for finer mapping of the lineages, we establish that CD45lowCX3CR1- pvMs are derived from CX3CR1+ precursors and require PU.1 during their ontogeny. In parallel, results from the Cxcr4-CreErt2;Rosa26tdT lineage tracing model support a bone marrow-independent replenishment of all Lyve1+ pvMs in the adult mouse brain. Lastly, flow cytometry and 3D immunofluorescence analysis uncover increased percentage of pvMs following photothrombotic induced stroke. Our results thus show that the parenchymal pvM population is more heterogenous than previously described, and includes a CD45low and CX3CR1- pvM population.

Atypical E2Fs inhibit tumor angiogenesis.

Atypical E2F transcription factors (E2F7 and E2F8) function as key regulators of cell cycle progression and their inactivation leads to spontaneous cancer formation in mice. However, the mechanism of the tumor suppressor functions of E2F7/8 remain obscure. In this study we discovered that atypical E2Fs control tumor angiogenesis, one of the hallmarks of cancer. We genetically inactivated atypical E2Fs in epithelial and mesenchymal neoplasm and analyzed blood vessel formation in three different animal models of cancer. Tumor formation was either induced by application of 7,12-Dimethylbenz(a)anthracene/12-O-Tetradecanoylphorbol-13-acetate or by Myc/Ras overexpression. To our surprise, atypical E2Fs suppressed tumor angiogenesis in all three cancer models, which is in a sharp contrast to previous findings showing that atypical E2Fs promote angiogenesis during fetal development in mice and zebrafish. Real-time imaging in zebrafish displayed that fluorescent-labeled blood vessels showed enhanced intratumoral branching in xenografted E2f7/8-deficient neoplasms compared with E2f7/8-proficient neoplasms. DLL4 expression, a key negative inhibitor of vascular branching, was decreased in E2f7/8-deficient neoplastic cells, indicating that E2F7/8 might inhibit intratumoral vessel branching via induction of DLL4.

Mesoderm formation in response to Brachyury requires FGF signalling.

BACKGROUND: The Brachyury (T) gene is required for the formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In these species, and in Xenopus, the gene is expressed transiently throughout the presumptive mesoderm, and transcripts then persiste in notochord and posterior tissues. In Xenopus embryos, expression of the Xenopus homologue of Brachyury, Xbra, can be induced in presumptive ectoderm by basic fibroblast growth factor (FGF) and activin; in the absence of functional FGF or activin signalling pathways, expression of the gene is severely reduced. Ectopic expression of Xbra in presumptive ectoderm causes mesoderm to be formed. As Brachyury and its homologues encode sequence-specific DNA-binding proteins, it is likely that each functions by directly activating downstream mesoderm-specific genes. RESULTS: We show that expression in Xenopus embryos of RNA encoding a dominant-negative FGF receptor inhibits the mesoderm-inducing activity of Xbra. We demonstrate that ectopic expression of Xbra activates transcription of the embryonic FGF gene, and that embryonic FGF can induce expression of Xbra. This suggests that the two genes are components of a regulatory loop. Consistent with this idea, dissociation of Xbra-expressing cells causes a dramatic and rapid reduction in levels of Xbra, but the reduction can be inhibited by addition of FGF. CONCLUSION: Formation of mesoderm tissue requires an intact FGF signalling pathway downstream of Brachyury. This requirement is due to a regulatory loop, in which Brachyury activates expression of a member of the FGF family, and FGF maintains expression of Brachyury.(ABSTRACT TRUNCATED AT 250 WORDS)

A mutation in the zebrafish maternal-effect gene nebel affects furrow formation and vasa RNA localization.

BACKGROUND: In many animals, embryonic patterning depends on a careful interplay between cell division and the segregation of localized cellular components. Both of these processes in turn rely on cytoskeletal elements and motor proteins. A type of localized cellular component found in most animals is the germ plasm, a specialized region of cytoplasm that specifies the germ-cell fate. The gene vasa has been shown in Drosophila to encode an essential component of the germ plasm and is thought to have a similar function in other organisms. In the zebrafish embryo, the vasa RNA is localized to the furrows of the early cellular divisions. RESULTS: We identified the gene nebel in a pilot screen for zebrafish maternal-effect mutations. Embryos from females homozygous for a mutation in nebel exhibit defects in cell adhesion. Our analysis provides genetic evidence for a function of the microtubule array that normally develops at the furrow in the deposition of adhesive membrane at the cleavage plane. In addition, nebel mutant embryos show defects in the early localization of vasa RNA. The vasa RNA localization phenotype could be mimicked with microtubule-inhibiting drugs, and confocal microscopy suggests an interaction between microtubules and vasa-RNA-containing aggregates. CONCLUSIONS: Our data support two functions for the microtubule reorganization at the furrow, one for the exocytosis of adhesive membrane, and another for the translocation of vasa RNA along the forming furrow.

The lymphatic vasculature revisited-new developments in the zebrafish.

The lymphatic system is lined by endothelial cells and part of the vasculature. It is essential for tissue fluid homeostasis, absorption of dietary fats, and immune surveillance in vertebrates. Misregulation of lymphatic vessel formation and dysfunction of the lymphatic system have been indicated in a number of pathological conditions including lymphedema formation, obesity or chronic inflammatory diseases such as rheumatoid arthritis. In zebrafish, lymphatics were discovered about 10years ago, and the underlying molecular pathways involved in its development have since been studied in detail. Due to its superior live cell imaging possibilities and the broad tool kit for forward and reverse genetics, the zebrafish has become an important model organism to study the development of the lymphatic system during early embryonic development. In the current review, we will focus on the key players during zebrafish lymphangiogenesis and compare the roles of these genes to their mammalian counterparts. In particular, we will focus on novel findings that shed new light on the molecular mechanisms of lymphatic cell fate specification, as well as sprouting and migration of lymphatic precursor cells.

The development of the paired fins in the zebrafish (Danio rerio).

In the present study, we describe the structure and normal development of the zebrafish (Danio rerio) paired fins. Particularly, we focus on the structure of the apical epidermis and on endoskeletal morphogenesis. Endoskeletal development proceeds differently in the pectoral and pelvic fins. Whereas in both fins major parts of the endoskeletal girdle develop within the fin bud mesenchyme, the pattern of chondrogenic condensations observed in the pelvic fins directly reflects the adult endoskeletal pattern. In the pectoral fin, a morphogenetic detour is taken via a functional larval endoskeleton, the endoskeletal disc. It arises in the fin bud mesenchyme from a chondrogenic anlage common with the girdle. The disc chondrifies and represents the functional endoskeleton of the larval pectoral fin. The pectoral fin endoskeleton is expanded as well as restructured during larval stages in a process which involves decomposition of cartilage matrix in the endoskeletal disc. Our comparisons of apical fold morphology with reports on other teleosts and tetrapod apical ridges show them to be homologous on the structural level. Comparisons of endoskeletal development of the zebrafish with reports on teleosts, actinopterygians and chondrichthyans show that endoskeletal morphogenesis in the zebrafish pectoral fin follows a morphogenetic process which is wide-spread among actinopterygians.

Zebrafish zic1 expression in brain and somites is affected by BMP and hedgehog signalling.

Here we report the expression of the zebrafish zic1 gene, also known as opl, a homologue to other vertebrate Zic genes and the Drosophila odd-paired gene. zic1 expression starts during epiboly stages in lateral parts of the neural plate and eventually comes to lie in dorsal regions of the developing brain following the morphogenetic movements of neural tube formation. To address the question whether BMP2 signalling affects the extent of zic1 expression, we analysed swirl and chordino mutant embryos. Expanded Zic1 expression in swirl and reduced expression in chordino as well as in bmp2 injected embryos suggest that BMP2 and its antagonists define the extent of zic1 expression in the neural plate. By searching for factors responsible for the dorsal restriction of Zic1 expression, we found zic1 expression is eliminated in sonic hedgehog (shh) injected embryos. The most rostral expression however is not affected by Shh suggesting that Shh plays a different role in dorso-ventral patterning of the future telencephalon. During somitogenesis zic1 is expressed in the dorsal most part of the developing somites. Here zic1 marks cells that are distinct from the main adaxial somite portion, the future myomere. zic1 expression in the somites is expanded in swirl but reduced in shh injected embryos, suggesting these factors have opposing activity in dorsoventral patterning of the somites. Later, a growing mass of zic1 expressing cells occurs in a dorsal mesenchyme that eventually invades the dorsal fin fold, suggesting a somitic contribution to the dorsal fin mesenchyme.

The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

Overlapping and distinct functions provided by fgf17, a new zebrafish member of the Fgf8/17/18 subgroup of Fgfs.

Members of the fibroblast growth factor (Fgf) family are important signaling molecules in several inductive and patterning processes, and act as brain organizer-derived signals during formation of the early vertebrate nervous system. We isolated a new member of the Fgf8/17/18 subgroup of Fgfs from the zebrafish, and studied its expression and function during somitogenesis, optic stalk and midbrain-hindbrain boundary (MHB) development. In spite of a slightly higher aminoacid similarity to Fgf8, expression analysis and mapping to a chromosome stretch that is syntenic with mammalian chromosomes shows that this gene is orthologous to mammalian Fgf17. These data provide a further example of conserved chromosomal organization between zebrafish and mammalian genomes. Using an mRNA injection assay, we show that fgf17 can act similar to fgf8 during gastrulation, when fgf17 is not normally expressed. Direct comparison of the expression patterns of fgf17 and fgf8 suggest however a possible cooperation of these Fgfs at later stages in several tissues requiring Fgf signaling. Analysis of zebrafish MHB mutants demonstrates a gene-dosage dependent requirement of fgf17 expression for the no isthmus// pax2.1 gene, showing that no isthmus/pax2.1 functions upstream of fgf17 at the MHB in a haplo-insufficient manner, similar to what has been reported for mammalian pax2 mutants. In contrast, only maintenance of fgf17 expression is disturbed at the MHB of acerebellar/fgf8 mutants. Consistent with a requirement for fgf8 function, implantation of FGF8-soaked beads induces fgf17 expression, and expression is upregulated in aussicht mutants, which display upregulation of the Fgf8 signaling pathway. Taken together, our results argue that Fgf8 and Fgf17 act as hierarchically organized signaling molecules during development of the MHB organizer and possibly other organizers in the developing nervous system.

Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation.

BACKGROUND: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate myosin light chain kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. METHODS AND RESULTS: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP-tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5-fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet-specific cd41 co-localized in thrombi. Expression of other mlck subtypes was lower in GFP-tagged platelets (mlck1b; 0.77-fold enriched) and absent in thrombi (mlck1b, -2, -3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a-deficient embryos. CONCLUSION: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.

Signals from the yolk cell induce mesoderm, neuroectoderm, the trunk organizer, and the notochord in zebrafish.

We have analyzed the role of the zebrafish yolk cell in the processes of mesoderm induction and establishment of the organizer. By recombining blastomere-free yolk cells and animal cap tissue we have shown that the yolk cell itself can induce mesoderm in neighboring blastomeres. We further demonstrate the competence of all blastomeres to form mesoderm, suggesting the endogenous mesoderm inducing signal to be locally restricted. Ablation of the vegetal third of the yolk cell during the first 20 min of development does not interfere with mesoderm formation in general, but results in completely ventralized embryos. These embryos lack the notochord, neuroectoderm, and the anterior-most 14-15 somites, demonstrating that the ablation affects the formation of the trunk-, but not the tail region of the embryo. This suggests the presence of a trunk organizer in fish. The dorsalized mutant swirl (zbmp-2b) shows expanded dorsal structures and missing ventral structures. In contrast to the phenotypes obtained upon the ablation treatment in wild-type embryos, removal of the vegetal-most yolk in swirl mutants results in embryos which do form neuroectoderm and anterior trunk somites. However, both wild-type and swirl mutants lack a notochord upon vegetal yolk removal. These ablation experiments in wild-type and swirl mutant embryos demonstrate that in zebrafish dorsal determining factors originate from the vegetal part of the yolk cell. These factors set up two independent activities: one induces the notochord and the other is involved in the formation of the neuroectoderm and the trunk region by counteracting the function of swirl. In addition, these experiments show that the establishment of the anteroposterior axis is independent of the dorsoventral axis.

dackel acts in the ectoderm of the zebrafish pectoral fin bud to maintain AER signaling.

Classical embryological studies have implied the existence of an apical ectodermal maintenance factor (AEMF) that sustains signaling from the apical ectodermal ridge (AER) during vertebrate limb development. Recent evidence suggests that AEMF activity is composed of different signals involving both a sonic hedgehog (Shh) signal and a fibroblast growth factor 10 (Fgf10) signal from the mesenchyme. In this study we show that the product of the dackel (dak) gene is one of the components that acts in the epidermis of the zebrafish pectoral fin bud to maintain signaling from the apical fold, which is homologous to the AER of tetrapods. dak acts synergistically with Shh to induce fgf4 and fgf8 expression but independently of Shh in promoting apical fold morphogenesis. The failure of dak mutant fin buds to progress from the initial fin induction phase to the autonomous outgrowth phase causes loss of both AER and Shh activity, and subsequently results in a proximodistal truncation of the fin, similar to the result obtained by ridge ablation experiments in the chicken. Further analysis of the dak mutant phenotype indicates that the activity of the transcription factor engrailed 1 (En1) in the ventral non-ridge ectoderm also depends on a maintenance signal probably provided by the ridge. This result uncovers a new interaction between the AER and the dorsoventral organizer in the zebrafish pectoral fin bud.

Effects of truncated activin and FGF receptors and of follistatin on the inducing activities of BVg1 and activin: does activin play a role in mesoderm induction?

Activin and Vg1, two members of the TGF-beta family, are believed to play roles in mesoderm induction and axis formation in the amphibian embryo. Both molecules are provided maternally, either as protein (activin) or as RNA and protein (Vg1), and experiments with a truncated form of a type IIB activin receptor have led to the conclusion that activin is required for induction of mesoderm in vivo. In this paper we first show that truncated versions of two different Xenopus activin receptors also have severe effects on the activity of the mature region of Vg1, suggesting that such receptors may block the function of several members of the TGF-beta family. We go on to demonstrate that follistatin, a secreted protein which binds activin and blocks its activity, does not interfere with Vg1 signalling. Furthermore, overexpression of follistatin mRNA in Xenopus embryos does not perturb mesoderm formation. Taken together, our data show that the effects of truncated activin receptors on Xenopus development can be explained by the inhibition of Vg1 activity, while the lack of effect of follistatin argues against a function for activin in mesoderm induction.

The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo.

Embryos mutant for the T gene, in mice, make insufficient mesoderm and fail to develop a notochord. We report the cloning and sequencing of the T gene in the zebrafish (Brachydanio rerio) and show the nuclear localization of the protein product. Both RNA and protein are found in cells of the germ ring, including enveloping layer cells, prior to and during gastrulation of zebrafish embryos. Nuclei of the yolk syncytial layer do not express Zf-T. High levels of expression are maintained throughout early development in the notochord, while in paraxial mesoderm cells the gene is turned off during gastrulation. Exposure of animal cap cells to activinA induces Zf-T expression, as does transplantation into the germ ring.