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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Bmp activity establishes a gradient of positional information throughout the entire neural plate.

Bone morphogenetic proteins (Bmps) are key regulators of dorsoventral (DV) patterning. Within the ectoderm, Bmp activity has been shown to inhibit neural development, promote epidermal differentiation and influence the specification of dorsal neurons and neural crest. In this study, we examine the patterning of neural tissue in mutant zebrafish embryos with compromised Bmp signalling activity. We find that although Bmp activity does not influence anteroposterior (AP) patterning, it does affect DV patterning at all AP levels of the neural plate. Thus, we show that Bmp activity is required for specification of cell fates around the margin of the entire neural plate, including forebrain regions that do not form neural crest. Surprisingly, we find that Bmp activity is also required for patterning neurons at all DV levels of the CNS. In swirl/bmp2b(-) (swr(-)) embryos, laterally positioned sensory neurons are absent whereas more medial interneuron populations are hugely expanded. However, in somitabun(-) (sbn(-)) embryos, which probably retain higher residual Bmp activity, it is the sensory neurons and not the interneurons that are expanded. Conversely, in severely Bmp depleted embryos, both interneurons and sensory neurons are absent and it is the most medial neurons that are expanded. These results are consistent with there being a gradient of Bmp-dependent positional information extending throughout the entire neural and non-neural ectoderm.

Transient establishment of anteroposterior polarity in the zebrafish pectoral fin bud in the absence of sonic hedgehog activity.

Sonic hedgehog (Shh) is expressed in the posterior vertebrate limb bud mesenchyme and directs anteroposterior patterning and growth during limb development. Here we report an analysis of the pectoral fin phenotype of zebrafish sonic you mutants, which disrupt the shh gene. We show that Shh is required for the establishment of some aspects of anteroposterior polarity, while other aspects of anteroposterior polarity are established independently of Shh, and only later come to depend on Shh for their maintenance. We also demonstrate that Shh is required for the activation of posterior HoxD genes by retinoic acid. Finally, we show that Shh is required for normal development of the apical ectodermal fold, for growth of the fin bud, and for formation of the fin endoskeleton.

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.

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.