Biodistribution of surfactant-free poly(lactic-acid) nanoparticles and uptake by endothelial cells and phagocytes in zebrafish: Evidence for endothelium to macrophage transfer.

In the development of therapeutic nanoparticles (NP), there is a large gap between in vitro testing and in vivo experimentation. Despite its prominence as a model, the mouse shows severe limitations for imaging NP and the cells with which they interact. Recently, the transparent zebrafish larva, which is well suited for high-resolution live-imaging, has emerged as a powerful alternative model to investigate the in vivo behavior of NP. Poly(D,L lactic acid) (PLA) is widely accepted as a safe polymer to prepare therapeutic NP. However, to prevent aggregation, many NP require surfactants, which may have undesirable biological effects. Here, we evaluate 'safe-by-design', surfactant-free PLA-NP that were injected intravenously into zebrafish larvae. Interaction of fluorescent NPs with different cell types labelled in reporter animals could be followed in real-time at high resolution; furthermore, by encapsulating colloidal gold into the matrix of PLA-NP we could follow their fate in more detail by electron microscopy, from uptake to degradation. The rapid clearance of fluorescent PLA-NP from the circulation coincided with internalization by endothelial cells lining the whole vasculature and macrophages. After 30 min, when no NP remained in circulation, we observed that macrophages continued to internalize significant amounts of NP. More detailed video-imaging revealed a new mechanism of NP transfer where NP are transmitted along with parts of the cytoplasm from endothelial cells to macrophages.

AS1411-conjugated gold nanoparticles affect cell proliferation through a mechanism that seems independent of nucleolin.

G-rich oligonucleotide, AS1411, has been shown to interact with nucleolin and to inhibit cancer cell proliferation and tumor growth. This antiproliferative action is increased when AS1411 is conjugated to different types of nanoparticles. However, the molecular mechanisms are not known. In this work, we show in several cell lines that optimized AS1411-conjugated gold nanoparticles (GNS-AS1411) inhibit nucleolin expression at the RNA and protein levels. We observed an alteration of the nucleolar structure with a decrease of ribosomal RNA accumulation comparable to what is observed upon nucleolin knock down. However, the expression of genes involved in cell cycle and the cell cycle blockage by GNS-AS1411 are not regulated in the same way as that in cells where nucleolin has been knocked down. These data suggest that the anti-proliferative activity of GNS-AS1411 is not the only consequence of nucleolin targeting and down-regulation.

Specific and Efficient Uptake of Surfactant-Free Poly(Lactic Acid) Nanovaccine Vehicles by Mucosal Dendritic Cells in Adult Zebrafish after Bath Immersion.

Activation of mucosal immunity is a key milestone for next-generation vaccine development. Biocompatible polymer-based nanoparticles (NPs) are promising vectors and adjuvants for mucosal vaccination. However, their in vivo uptake by mucosae and their biodistribution in antigen-presenting cells (APCs) need to be better understood to optimize mucosal nanovaccine designs. Here, we assessed if APCs are efficiently targeted in a spontaneous manner by surfactant-free poly(lactic acid) nanoparticles (PLA-NPs) after mucosal administration. Combining histology and flow imaging approaches, we describe and quantify the mucosal uptake of 200 nm PLA-NPs in adult zebrafish. Following bath administration, PLA-NPs penetrated and crossed epithelial barriers from all exposed mucosae. In mucosae, PLA-NPs accumulated in APCs, which were identified as dendritic cells (DCs), macrophages, and IgZ+ B cells in gills and skin. PLA-NP uptake by phagocytes was specific to these cell types, as PLA-NPs were not detected in neutrophils. Importantly, quantitative analyses in gills revealed that DCs take up PLA-NPs with specifically high efficiency. This study shows that surfactant-free PLA-NPs, which display optimal biocompatibility, can spontaneously target DCs with high efficiency in vivo following mucosal administration, and highlights PLA-NPs as powerful platforms for mucosal vaccine delivery in the medical and veterinary fields, and particularly in aquaculture.

Multiscale investigation of USPIO nanoparticles in atherosclerotic plaques and their catabolism and storage in vivo.

The storage and catabolism of Ultrasmall SuperParamagnetic Iron Oxide (USPIO) nanoparticles were analyzed through a multiscale approach combining Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM) at different times after intravenous injection in an atherosclerotic ApoE(-/-) mouse model. The atherosclerotic plaque features and the USPIO heterogeneous biodistribution were revealed down from organ's scale to subcellular level. The biotransformation of the nanoparticle iron oxide (maghemite) core into ferritin, the non-toxic form of iron storage, was demonstrated for the first time ex vivo in atherosclerotic plaques as well as in spleen, the iron storage organ. These results rely on an innovative spatial and structural investigation of USPIO's catabolism in cellular phagolysosomes. This study showed that these nanoparticles were stored as non-toxic iron compounds: maghemite oxide or ferritin, which is promising for MRI detection of atherosclerotic plaques in clinics using these USPIOs. From the Clinical Editor: Advance in nanotechnology has brought new contrast agents for clinical imaging. In this article, the authors investigated the use and biotransformation of Ultrasmall Super-paramagnetic Iron Oxide (USPIO) nanoparticles for analysis of atherosclerotic plagues in Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM). The biophysical data generated from this study could enable the possible use of these nanoparticles for the benefits of clinical patients.

The development of the myotendinous junction. A review.

The myotendinous junction (MTJ) is a complex specialized region located at the muscle-tendon interface that represents the primary site of force transmission. Despite their different embryologic origins, muscle and tendon morphogenesis occurs in close spatial and temporal association. After muscle attachment, muscle and tendon constitute a dynamic and functional integrated unit that transduces muscle contraction force to the skeletal system. We review here the current understanding of MTJ formation describing changes during morphogenesis and focusing on the crosstalk between muscle and tendon cells that leads to the development of a functional MTJ. Molecules involved in the formation of the linkage, both at the tendon side and at the muscle side of the junction are described. Much of this knowledge comes from studies using different animal models such as mice, zebrafish and Drosophila where powerful methods for in vivo imaging and genetic manipulations can be used to enlighten this developmental process.

Development of the zebrafish myoseptum with emphasis on the myotendinous junction.

Zebrafish myosepta connect two adjacent muscle cells and transmit muscular forces to axial structures during swimming via the myotendinous junction (MTJ). The MTJ establishes transmembrane linkages system consisting of extracellular matrix molecules (ECM) surrounding the basement membrane, cytoskeletal elements anchored to sarcolema, and all intermediate proteins that link ECM to actin filaments. Using a series of zebrafish specimens aged between 24 h post-fertilization and 2 years old, the present paper describes at the transmission electron microscope level the development of extracellular and intracellular elements of the MTJ. The transverse myoseptum development starts during the segmentation period by deposition of sparse and loosely organized collagen fibrils. During the hatching period, a link between actin filaments and sarcolemma is established. The basal lamina underlining sarcolemma is well differentiated. Later, collagen fibrils display an orthogonal orientation and fibroblast-like cells invade the myoseptal stroma. A dense network of collagen fibrils is progressively formed that both anchor myoseptal fibroblasts and sarcolemmal basement membrane. The differentiation of a functional MTJ is achieved when sarcolemma interacts with both cytoskeletal filaments and extracellular components. This solid structural link between contractile apparatus and ECM leads to sarcolemma deformations resulting in the formation of regular invaginations, and allows force transmission during muscle contraction. This paper presents the first ultrastructural atlas of the zebrafish MTJ development, which represents an useful tool to analyse the mechanisms of the myotendinous system formation and their disruption in muscle disorders.

Characterization of spatial and temporal expression pattern of Col15a1b during zebrafish development.

In mammals, collagen XV is primarily expressed in skeletal and cardiac muscles, and loss of its expression in mice results in a mild skeletal myopathy. We recently identified Col15a1a, a zebrafish ortholog of the human collagen XV gene which expression was restricted to notochord in embryos. Col15a1a knockdown led to defects in muscle maintenance via Shh signaling. Here we report that zebrafish express a second ortholog Col15a1b. The identification of its complete primary sequence showed that the overall structure of collagen XV is well conserved between vertebrates. Whole mount in situ hybridization and RT-PCR analysis revealed that at 12hpf Col15a1b is mainly expressed in slow muscle cell lineage and in nervous tissues, and, at later stages transcripts are detected in eyes, otic placodes and aortic arches. Based on the expression pattern of col15a1b, sequence alignments and synteny comparisons, we conclude that, contrary to collagen XVa, the zebrafish collagen XVb likely displays the same or similar function than the mammalian orthologs.

Craniofacial cartilage morphogenesis requires zebrafish col11a1 activity.

The zebrafish ortholog of the human COL11A1 gene encoding the cartilage collagen XI proalpha1 chain was characterized to explore its function in developing zebrafish using the morpholino-based knockdown strategy. We showed that its expression in zebrafish is developmentally regulated. A low expression level was detected by real-time PCR during the early stages of development. At 24 hpf, a sharp peak of expression was observed. At that stage, in situ hybridization indicated that col11a1 transcripts are restricted to notochord. At 48 hpf, they were exclusively detected in the craniofacial skeleton, endoskeleton of pectoral fins and in otic vesicles. Collagen XI alpha1-deficient zebrafish embryos developed defects in craniofacial cartilage formation and in notochord morphology. Neural crest specification and mesenchymal condensation occurred normally in morpholino-injected embryos. Col11a1 depletion affected the spatial organization of chondrocytes, the shaping of cartilage elements, and the maturation of chondrocytes to hypertrophy. Knockdown of col11a1 in embryos stimulated the expression of the marker of chondrocyte differentiation col2a1, resulting in the deposit of abnormally thick and sparse fibrils in the cartilage extracellular matrix. The extracellular matrix organization of the perichordal sheath was also altered and led to notochord distortion. The data underscore the importance of collagen XI in the development of a functional cartilage matrix. Moreover, the defects observed in cartilage formation resemble those observed in human chondrodysplasia such as the Stickler/Marshall syndrome. Zebrafish represent a novel reliable vertebrate model for collagen XI collagenopathies.

Morpholino knockdown of lysyl oxidase impairs zebrafish development, and reflects some aspects of copper metabolism disorders.

Lysyl oxidase (LOX), a copper-dependent amine oxidase known in mammals to catalyze the cross-linking of collagen and elastin in the extracellular matrix, is a member of a multigenic family. Eight genes encoding lysyl oxidase isoforms have been identified in zebrafish. Recent studies have revealed a critical role for two zebrafish lysyl oxidases-like in the formation of the notochord. We now present the role of Lox in zebrafish development. lox morpholino-mediated knockdown results in a mildly undulated notochord, truncated anterior-posterior axis, tail bending and smaller head. Analyses of morphants show a complete disorganization of muscle somites and neural defects, in accordance with the lox expression pattern. Lox inhibition also induces pigment defects and pharyngeal arch deformities consistent with neural crest dysfunction. Taken together, these data reveal a role for Lox in early morphogenesis, especially in muscle development and neurogenesis, and resume some aspects of physiopathology of copper metabolism.

Collagen XV, a novel factor in zebrafish notochord differentiation and muscle development.

Muscle cells are surrounded by extracellular matrix, the components of which play an important role in signalling mechanisms involved in their development. In mice, loss of collagen XV, a component of basement membranes expressed primarily in skeletal muscles, results in a mild skeletal myopathy. We have determined the complete zebrafish collagen XV primary sequence and analysed its expression and function in embryogenesis. During the segmentation period, expression of the Col15a1 gene is mainly found in the notochord and its protein product is deposited exclusively in the peri-notochordal basement membrane. Morpholino mediated knock-down of Col15a1 causes defects in notochord differentiation and in fast and slow muscle formation as shown by persistence of axial mesodermal marker gene expression, disorganization of the peri-notochodal basement membrane and myofibrils, and a U-shape myotome. In addition, the number of medial fast-twitch muscle fibers was substantially increased, suggesting that the signalling by notochord derived Hh proteins is enhanced by loss of collagen XV. Consistent with this, there is a concomitant expansion of patched-1 expression in the myotome of morphant embryos. Together, these results indicate that collagen XV is required for notochord differentiation and muscle development in the zebrafish embryo and that it interplays with Shh signalling.

Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).

The first part of this article is a review of the current status of knowledge of the fish skin, with particular attention to its development. In the second part we present original results obtained in zebrafish (Danio rerio), with particular emphasis on the deposition and organisation of the dermal collagenous stroma. Using a series of zebrafish specimens aged between 15 hours postfertilization (hpf) and 4.5 years old, we have combined Transmission Electron Microscopy (TEM) observations and in situ hybridisation using type I collagen a2 chain (Col1a2) probe. Collagen fibrils, with a diameter of 22 nm, appear first in an acellular subepidermal space at 24 hpf, are first all oriented in the same direction, and form the primary dermal stroma. Subsequently, three events occur. (1) From 5-7 days pf (dpf) onwards the collagen fibrils self-organise into several lamellae arranged in a plywood-like structure, starting in the upper layers and progressing throughout the entire thickness of the dermis. (2) At 20-26 dpf, fibroblasts of unknown origin progressively invade the acellular collagenous stroma, some of them accumulating below the epidermis. (3) Concomitant with the invasion of fibroblasts, the collagen fibrils increase progressively in diameter to reach 160 nm towards the end of the fish life. In situ hybridisation experiments reveal that, between 24 and 48 hpf, the collagen matrix is produced by the epidermis only. From 72 hpf to 20-26 dpf, both the basal epidermal cells and the dermal cells bordering the deep region of the dermis are involved in the production of collagen. When the fibroblasts invade the plywood-like structure, the epidermal cells progressively cease to synthesise collagen, which from this point is produced only by the fibroblasts. This suggests that the fibroblasts secrete a still unidentified signalling molecule that downregulates collagen production by the epidermis.

Sequence and embryonic expression of collagen XVIII NC1 domain (endostatin) in the zebrafish.

Endostatin, located in the NC1 domain of the collagen XVIII, is believed to inhibit the migration and proliferation of endothelial cells (Fed. Am. Soc. Exp. Biol. J. 15 (2001) 1044) and to play a role in axon guidance in Caenorhabditis elegans (J. Cell Biol. 152 (2001) 1219). Zebrafish is an attractive vertebrate model to determine the role of endostatin and the entire molecule of collagen XVIII during vertebrate development. Therefore, we have investigated the expression pattern of COL18A1 in zebrafish embryos from the segmentation to the hatching period stages.

Phylogenetic analysis of vertebrate fibrillar collagen locates the position of zebrafish alpha3(I) and suggests an evolutionary link between collagen alpha chains and hox clusters.

Type I collagen in tetrapods is usually a heterotrimeric molecule composed of two alpha1 and one alpha2 chains. In some teleosts, a third alpha chain has been identified by chromatography, suggesting that type I collagen should also exist as an alpha1(I)alpha2(I)alpha3(I) heterotrimer. We prepared, from zebrafish, three distinct cDNAs identified to be those of the collagen alpha1(I), alpha2(I), and alpha3(I) chains. In this study on the evolution of fibrillar collagen alpha chains and their relationships, an exhaustive phylogenetic analysis, using vertebrate fibrillar collagen sequences, showed that each alpha chain constitutes a monophyletic cluster. Results obtained with the newly isolated sequences of the zebrafish showed that the alpha3(I) chain is phylogenetically close to the alpha1(I) chain and support the hypothesis that the alpha3(I) chain arose from a duplication of the alpha1(I) gene. The duplication might occur during the duplication of the actinopterygian genome, soon after the divergence of actinopterygians and sarcopterygians, a hypothesis supported by the demonstration of a syntenic evolution between a set of fibrillar collagen genes and Hox clusters in mammals. An evolutionary scenario is proposed in which phylogenetic relationships of the alpha chains of fibrillar collagens of vertebrates could be related to Hox cluster history.

Structure and spatio temporal expression of the full length DNA complementary to RNA coding for alpha2 type I collagen of zebrafish.

Twenty distinct genetic types of collagen have been identified up to now. Their structure and function are not completely elucidated. We have chosen zebrafish as a model to bring information about the role of collagen during embryogenesis. In the present study, we isolated four overlapping DNA complementary to RNA clones covering the 4879 nucleotides of a zebrafish messenger RNA (mRNA) encoding a fibrillar procollagen chain. The comparison of its primary structure with known other vertebrate collagens allowed to conclude that it encodes collagen pro-alpha2(I) chain. The 5' untranslated region showed a typical stem-loop structure with three ATG codons which is found in mammals types I and III collagen chains (but not in type II), which are expressed in the same tissues. This suggests that the supposed regulatory role of the stem loop structure could be tissue specific. The comparison of the Gly-Gly doublets found along the helical domain of several species allowed to speculate that the Gly-Gly repeats could be a poikilotherm feature. Expression of pro-alpha2(I) was examined during zebrafish development by reverse transcriptase-polymerase chain reaction and in situ hybridization on whole embryo and tissue section. Col1a2 was expressed as early as stage10 h post fertilization (hpf) and two peaks of expression were observed at 20 and 48 hpf. alpha2 mRNAs, whose presence suggests a collagen synthesis, were detected principally in the superficial cell layers surrounding 20-72 hpf embryos which are characterized by an acellular collagen stratum. At 26-30 days, fibroblasts invade the dermis and take over from the epithelial cells to synthesize collagen. This suggests a fine regulation of collagen synthesis in these cells that remains to be elucidated. alpha2 mRNA were also detected in other tissues such as the tail fin primordium and the notochord primordium suggesting a participation of type I collagen in a pathway for notochord and tail formation.