Studies In Zebrafish Embryos Research Articles

Indigo dyes: Toxicity, teratogenicity, and genotoxicity studies in zebrafish embryos

Wastewater released by textile dyeing industries is a major source of pollution. Untreated wastewater released from indigo dyeing operations affects aquatic ecosystems and threatens their biodiversity. We have assessed the toxicity of natural and synthetic indigo dye in zebrafish embryos, using the endpoints of teratogenicity, genotoxicity, and histopathology. The zebrafish embryo toxicity test (ZFET) was conducted, exposing embryos to ten concentrations of natural and synthetic indigo dyes; the 96-hour LC50 values were approximately 350 and 300 mg/L, respectively. Both dyes were teratogenic, causing egg coagulation, tail detachment, yolk sac edema, pericardial edema, and tail bend, with no significant difference in effects between the natural and synthetic dyes. Both dyes were genotoxic (using comet assay for DNA damage). Real-time RT-PCR studies showed upregulation of the DNA-repair genes FEN1 and ERCC1. Severe histological changes were seen in zebrafish larvae following exposure to the dyes. Our results show that indigo dyes may be teratogenic and genotoxic to aquatic organisms, underscoring the need for development of sustainable practices and policies for mitigating the environmental impacts of textile dyeing.

Quantitative Real-Time PCR Method to Evaluate Gene Expression in Zebrafish Embryos.

The fast zebrafish embryonic development offers an opportunity for the study of genes crucial for developmental processes. Several genes associated with human diseases have orthologs in zebrafish. Enhanced comprehension of a gene's function can be achieved by knowing when and where it is expressed. This knowledge also makes it possible to alter a gene-by-gene knockdown in a time- and place-specific manner. Moreover, gene expression analyses contribute greatly to teratogenicity studies in zebrafish embryos. Therefore, the importance of examining the differences between the expressions of these genes has increased day by day. The incorporation of reverse transcription (RT) as the initial step prior to thermal cycling in quantitative real-time polymerase chain reaction (RT-qPCR) has made a significant contribution to RNA research. RT-qPCR is the gold standard and an effective method for gene expression analysis. Quick readout, high sensitivity, reproducibility, as well as high potential throughput, along with reliable quantification, are just a few advantages of RT-qPCR. However, there are drawbacks to its application, such as RNA's inherent variability, impurities during RNA extraction, and variations in reverse transcription and PCR efficiencies. Implementing a precise normalization technique is crucial to account for these inaccuracies. In this chapter, the protocol for gene expression analysis by RT-qPCR for zebrafish embryos is explained.

Assessing developmental toxicity and non-CYP mediated biotransformation of two anti-epileptics and their human metabolites in zebrafish embryos and larvae

Zebrafish embryo-based assays are a promising alternative for animal testing to screen new compounds for developmental toxicity. However, recent studies in zebrafish embryos showed an immature intrinsic cytochrome P450 (CYP)-mediated biotransformation capacity, as most CYPs were only active at the end of the organogenesis period. Data on other phase I enzymes involved in the biotransformation of xenobiotics in zebrafish embryos is limited. This information is pivotal for proteratogens needing bioactivation to exert their teratogenic potential. Therefore, this study aimed to investigate whether carbamazepine (CBZ) and levetiracetam (LTC), two anti-epileptic drugs that require bioactivation to exert their teratogenic potential, are biotransformed into non-CYP mediated metabolites in the zebrafish embryo and whether one or more of these metabolites cause developmental toxicity in this species. In the first step, zebrafish embryos were exposed to LTC and CBZ and their non-CYP mediated human metabolites, etiracetam carboxylic acid (ECA) and 9-acridine carboxaldehyde (9ACA), acridine (AI), and acridone (AO), respectively, from 5.25 to 120 hpf and morphologically evaluated. Next, the uptake of all compounds and the formation of the metabolites were assessed using LC-MS methods. As LTC and ECA were, respectively, poorly or not taken up by zebrafish larvae during the exposure experiments, we could not determine if LTC and ECA are teratogenic. However, biotransformation of LTC into ECA was observed at 24 hpf and 120 hpf, which indicates that the special type of B-esterase is already active at 24 hpf. CBZ and its three metabolites were teratogenic, as a significant increase in malformed embryos was observed for all of them. All three metabolites were more potent teratogens than CBZ, with AI being the most potent, followed by 9ACA and AO. The myeloperoxidase (MPO) homologue is already active at 24 hpf, as CBZ was biotransformed into 9ACA and AO in 24 hpf zebrafish embryos, and into 9ACA in 120 hpf larvae. Moreover, 9ACA was also found to be biotransformed into AI and AO, and AI into AO. As such, one or more of these metabolites probably contribute to the teratogenic effects observed in zebrafish larvae after exposure to CBZ.

A comprehensive untargeted metabolomics study in zebrafish embryos exposed to perfluorohexane sulfonate (PFHxS)

Perfluorohexane sulfonate (PFHxS) is one of the short-chain perfluoroalkyl substances (PFASs), and frequently detected in the environment, humans, and wildlife, but a detailed mechanism of toxicity has been not studied yet. In this study, a comprehensive set of polar metabolites was determined in i) the developing zebrafish embryo (4, 24, 48, 72, and 120 h post fertilization (hpf)), and ii) in the developing zebrafish after exposure to four concentrations of PFHxS (0.3, 1, 3, and 10 μM) from 24to 120 hpf. The temporal (developmental stages) distribution of individual metabolites (541 metabolites) in zebrafish provided comprehensive information about the biological roles of various metabolites in developing vertebrates such as genetic processes, energy metabolism, protein metabolism, and glycerophospholipid metabolism. PFHxS in zebrafish embryo showed time- and concentration- dependent bioaccumulation, and no baseline toxicity was expected at the test concentrations. However, effects on many metabolites were already observed at the lowest tested concentration (0.3 μM), and these effects were more pronounced at later stages of developmental (72 and 120 hpf). In addition to oxidative stress, the effects of PFHxS on zebrafish embryos were related to the disruption of the fatty acid oxidation (FAO), sugar metabolism, and other metabolic pathways. This study gave new and comprehensive information on the underlying mechanism of the toxicity of PFHxS.

A novel hydrazone platform for the recognition of Cd2+ and F− ions: Imaging analysis in Zebrafish embryos

A new salicylhydrazone anchored imidazolyl derivative IHL has been designed and fabricated by simple synthetic route. The probe was functioned as multifunctional receptor for the revelation of cadmium and fluoride ions in the solvent system (DMSO/water, 9:1). The photophysical studies and binding stoichiometry between the probe and Cd2+/F‒ ions were performed via different spectral techniques such as UV–vis, fluorescence titrations, 1H &19F NMR, ESI-MS analysis and by DFT calculations respectively. In-vitro fluorescence imaging studies in Zebrafish embryos was successfully carried out and their result explored the excellent biological potential of the probe in the environment.

In vitro antidiabetic and anti-inflammatory effects of Fe-doped CuO-rice husk silica (Fe-CuO-SiO2) nanocomposites and their enhanced innate immunity in zebrafish

Recent studies are now focussing on synthesis of metal oxide based nanocomposites because of their exceptional physicochemical characteristics. In this study, CuO-SiO2 nanocomposite (CuONC) and Fe-doped CuO-SiO2 nanocomposite (FeCuONC) were synthesized by a simple hydrothermal method. XRD, SEM, EDX, FTIR, UV-vis, PSA, and TGA were utilized to confirm the synthesis of CuONC and FeCuONC. The in vitro biological activities and in vivo toxicity (zebrafish) of CuONC and FeCuONC were explored. The α-amylase assay showed that both CuONC, and FeCuONC exhibit dose-dependent antidiabetic activity. Interestingly, antidiabetic activity of FeCuONC was higher as compared to CuONC. Furthermore, FeCuONC showed higher anti-inflammatory effects (inhibition of denaturation of egg albumin) than those of CuONC. In vivo toxicity study in zebrafish embryos and larvae suggested that FeCuONC induce higher innate immunity response as compared to CuONC. Altogether, we observed that Fe-doped CuO-SiO2 nanocomposites exhibited advance anti-inflammatory effect, sophisticated antidiabetic activity and enhanced innate immunity as compared to CuO-SiO2 nanocomposite. This study warrants further research on therapeutic application of Fe-CuO-SiO2 nanocomposites in suitable mammalian models.

Residues of Cardiovascular and Lipid-Lowering Drugs Pose a Risk to the Aquatic Ecosystem despite a High Wastewater Treatment Ratio in the Megacity Shanghai, China

The residues of pharmaceuticals in surface waters of megacities and ecotoxicological implications are of particular concern. In this study, we combined field investigations and model simulations to explore the contamination of cardiovascular and lipid-lowering drugs, one group of the most prescribed medications globally, in surface waters of a typical megacity, Shanghai, with a high wastewater treatment ratio (≈96%). Among 26 target substances, 19 drugs were detected with aqueous concentrations ranging from 0.2 (ketanserin) to 715 ng/L (telmisartan). Of them, angiotensin II receptor antagonists, telmisartan and irbesartan, were dominant besides β-blockers. Spatial distribution analysis demonstrated their much higher levels in tributaries compared to the mainstream. The results of model simulations and field investigation revealed relatively low concentrations of cardiovascular and lipid-lowering drugs in surface waters of Shanghai compared to other cities in highly developed countries, which is associated with low per capita usage in China. Ecotoxicological studies in zebrafish embryos further revealed developmental effects, including altered hatching success and heart rate, by irbesartan, telmisartan, lidocaine, and their mixtures at ng/L concentrations, which are typical levels in surface waters. Overall, the present results suggest that the high wastewater treatment ratio was not sufficient to protect fish species in the aquatic ecosystem of Shanghai. Exposure to cardiovascular and lipid-lowering drugs and associated risks will further increase in the future due to healthcare improvements and population aging.

Combination of ZnO Nanoparticle with Marine Sponge Derived Dipeptide for Enhanced Anticancer Efficacy in Liver Cancer Cells and their Toxicity Evaluation on Embryonic Zebrafish

Background: Nanomaterials-based cancer therapy plays a significant role in increasing the therapeutic efficiency of anticancer drugs, reducing side effects and targeted delivery of the drug payloads. The present study was aimed to enhance the anticancer effect of a novel dipeptide isolated from marine sponge-associated Bacillus pumilus AMK1 by formulating with Zinc oxide (ZnO) nanoparticles for the effective treatment against HepG2 liver cancer cells. Methods: ZnO nanoparticles were synthesized by chemical method and size of the nanoparticle was characterized by Scanning electron microscope, X-Ray diffraction and Fourier-transform infrared spectroscopy. Furthermore, ZnO nanoparticles were conjugated with the isolated dipeptide and evaluated for anticancer activity. In addition, distinct morphological changes were observed by performing apoptotic staining methods such as propidium iodide staining and acridine orange/ ethidium bromide staining. Furthermore, embryotoxic and teratogenic effects of conjugated dipeptide on the development of zebrafish embryo were investigated in this study. Results: It was observed that conjugated dipeptide showed enhanced cytotoxicity against HepG2 liver cancer cells without any toxic effect on normal liver cells. ZnO with dipeptide showed significant higher apoptosis of liver cancer cells, with around 19% in early apoptosis and 53% in late apoptosis stage. The obtained results suggest that ZnO nanoparticle conjugated dipeptide initiated cytotoxicity through apoptotic death in HepG2 cells. The embryotoxic studies in zebrafish embryos revealed the LC50 197.0 μg/mL. These findings suggest that conjugated dipeptide affected the development of zebrafish embryos only at relatively higher concentrations. Conclusion: The experimental results demonstrate that ZnO nanoparticle conjugated dipeptide has the potential to improve anticancer efficacy against liver cancer cells by inducing apoptosis in cancer cells without any effect on normal liver cells.

Optimized Photoactivatable Lipid Nanoparticles Enable Red Light Triggered Drug Release.

Encapsulation of small molecule drugs in long-circulating lipid nanoparticles (LNPs) can reduce toxic side effects and enhance accumulation at tumor sites. A fundamental problem, however, is the slow release of encapsulated drugs from these liposomal systems at the disease site resulting in limited therapeutic benefit. Methods to trigger release at specific sites are highly warranted. Here, it is demonstrated that incorporation of ultraviolet (UV-A) or red-light photoswitchable-phosphatidylcholine analogs (AzoPC and redAzoPC) in conventional LNPs generates photoactivatable LNPs (paLNPs) having comparable structural integrity, drug loading capacity, and size distribution to the parent DSPC-cholesterol liposomes. It is shown that 65-70% drug release (doxorubicin) can be induced from these systems by irradiation with pulsed light based on trans-to-cis azobenzene isomerization. In vitro it is confirmed that paLNPs are non-toxic in the dark but convey cytotoxicity upon irradiation in a human cancer cell line. In vivo studies in zebrafish embryos demonstrate prolonged blood circulation and extravasation of paLNPs comparable to clinically approved formulations, with enhanced drug release following irradiation with pulsed light. Conclusively, paLNPs closely mimic the properties of clinically approved LNPs with the added benefit of light-induced drug release making them promising candidates for clinical development.

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.

Morphological Abnormalities and Gene Expression Changes Caused by High Incubation Temperatures in Zebrafish Xenografts with Human Cancer Cells

Published studies show that most of the human cancer xenograft studies in zebrafish embryos have used incubation temperatures in the range of 32–34 °C for 3–6 days post-injection, trying to find a compromise temperature between the zebrafish embryos (28 °C) and the human injected cells (37 °C). While this experimental setup is widely used, a question remains: is possible to overcome the drawbacks caused by a suboptimal temperature for the injected cells? To clarify the effect of temperature and injected cells on the host, in this study, we analyzed the development and health of the last in response to different temperatures in the presence or absence of injected human cancer cells. Comparing different incubation temperatures (28, 34 and 36 °C), we determined morphological abnormalities and developmental effects in injected and non-injected embryos at different time points. Besides this, the expression of selected genes was determined by qPCR to determine temperature affected metabolic processes in the embryos. The results indicate that an incubation temperature of 36 °C during a period of 48 h is suitable for xenotransplantation without morphological or metabolic changes that could be affecting the host or the injected cells, allowing them to proliferate near their optimal temperature.

Graphene oxide-silver nanoparticle hybrid material: an integrated nanosafety study in zebrafish embryos

This work reports an integrated nanosafety study including the synthesis and characterization of the graphene oxide-silver nanoparticle hybrid material (GO-AgNPs) and its nano-ecotoxicity evaluation in the zebrafish embryo model. The influences of natural organic matter (NOM) and a chorion embryo membrane were considered in this study, looking towards more environmentally realistic scenarios and standardized nanotoxicity testing. The nanohybrid was successfully synthesized using the NaBH4 aqueous method, and AgNPs (~ 5.8 nm) were evenly distributed over the GO surface. GO-AgNPs showed a dose-response acute toxicity: the LC50 was 1.5 mg L-1 for chorionated embryos. The removal of chorion, however, increased this toxic effect by 50%. Furthermore, the presence of NOM mitigated mortality, and LC50 for GO-AgNPs changed respectively from 2.3 to 1.2 mg L-1 for chorionated and de-chorionated embryos. Raman spectroscopy confirmed the ingestion of GO by embryos; but without displaying acute toxicity up to 100 mg L-1, indicating that the silver drove toxicity down. Additionally, it was observed that silver nanoparticle dissolution has a minimal effect on these observed toxicity results. Finally, understanding the influence of chorion membranes and NOM is a critical step towards the standardization of testing for zebrafish embryo toxicity in safety assessments and regulatory issues.

Vincristine exposure impairs skin keratinocytes, ionocytes, and lateral-line hair cells in developing zebrafish embryos

Environmental contamination by anticancer pharmaceuticals has been widely reported. These drugs are not readily biodegradable, and their parent compounds and/or metabolites have been detected in surface waters and groundwater throughout the world. Adverse effects of anticancer drugs occur frequently in cancer patients, and a large body of clinical knowledge has accumulated. However, the effects of these drugs on aquatic organisms have not been thoroughly studied. This study aimed to investigate the effects of acute exposure to a common anticancer drug, vincristine (VCR), on zebrafish embryonic development and skin function. After 96 h of VCR exposure (0, 1, 10, 15, and 25 mg/L), significant teratogenic effects were observed, including growth retardation, pericardial edema, spine, tail, and yolk sac malformations (VCR ≥ 15 mg/L), a decreased heart rate, and ocular malformations (VCR ≥ 10 mg/L). The value of the half lethal concentration for zebrafish embryos was 20.6 mg/L. At ≥10 mg/L VCR, systemic ion contents and acid secretion in the skin over the yolk-sac decreased, and these findings were associated with decreases in skin ionocytes (H+-ATPase-rich cells and Na+-K+-ATPase-rich cells). Also, the microridge-structure of skin keratinocytes was significantly damaged. The number of lateral line hair cells was reduced when VCR was ≥10 mg/L, and functional impairment was detected when VCR was as low as 1 mg/L. Results of this in vivo study in zebrafish embryos indicate that acute exposure to VCR can lead to developmental defects, impairment of skin functions, and even fish death.

In vivo bio-imaging of sodium meta-arsenite and hydrogen phosphate in zebrafish embryos using red fluorescent zinc complex

The photophysical behaviours of quinoline based red fluorescent zinc complex (QAZn) in the presence of the highly toxic AsO2− and biologically active H2PO4− ions have been explored in aqueous DMSO solution. The sequential detection of both the anions was examined using absorption and fluorescence techniques. As the zinc complex (QAZn) could form strong adducts with AsO2− and H2PO4− ions, it could be easier to recognize the ions. Moreover, successive addition of H2PO4 to QAZn-AsO2 solution caused the dissociation of Zn2+ from parent zinc complex (QAZn) and the liberated free ligand restored its original green fluorescence. Since the probe showed lowest mortality, it has been further exploited for live cell studies in zebrafish embryos. Moreover, the spectroscopic data of probe QAZn were utilized to construct the molecular logic circuits with AND and NOT gates.

Hemodynamic Forces Sculpt Developing Heart Valves through a KLF2-WNT9B Paracrine Signaling Axis

Hemodynamic forces play an essential epigenetic role in heart valve development, but how they do so is not known. Here, we show that the shear-responsive transcription factor KLF2 is required in endocardial cells to regulate the mesenchymal cell responses that remodel cardiac cushions to mature valves. Endocardial Klf2 deficiency results in defective valve formation associated with loss of Wnt9b expression and reduced canonical WNT signaling in neighboring mesenchymal cells, a phenotype reproduced by endocardial-specific loss of Wnt9b. Studies in zebrafish embryos reveal that wnt9b expression is similarly restricted to the endocardial cells overlying the developing heart valves and is dependent upon both hemodynamic shear forces and klf2a expression. These studies identify KLF2-WNT9B signaling as a conserved molecular mechanism by which fluid forces sensed by endothelial cells direct the complex cellular process of heart valve development and suggest that congenital valve defects may arise due to subtle defects in this mechanotransduction pathway.

Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension.

Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Abstract 50: GATA4-Dependent Control of Myocyte Protein Quality Mediated by HSPB7

The transcription factor GATA4 plays essential roles in heart development, including regulation of cardiomyocyte stress response. GATA4 depletion leads to defective cardiogenesis while GATA4 overexpression is protective in stress models including myocardial ischemia and cardiotoxin administration. However, the molecular basis for GATA4 function remains poorly understood. We discovered that expression of small heat shock protein beta 7 (HSPB7) is regulated by GATA4 and that HSPB7 depletion causes defects in cardiomorphogenesis. The goal of our study is to determine the cellular and molecular mechanism of action of HSPB7 in cardiomyogenesis. Through proteomic mass spectrometry and co-immunoprecipitation, we identified Filamin C (FLNC), a large sarcomeric protein, as an HSPB7 binding partner. FLNC mutations lead to pathological aggregate formation and progressive myopathy. Previous work suggests HSPB7 may prevent damaged protein aggregation through autophagic pathways, and our studies in zebrafish embryos suggest HSPB7 depletion alters the morphology and cytoskeletal architecture of ventricular cardiomyocytes, in particular the trabeculae. We hypothesize that HSPB7 facilitates myocyte function by processing damaged FLNC to prevent aggregation. Preliminary data suggest that BAG3 is also a part of this chaperone complex, linking HSPB7 to the process of Chaperone Assisted Selective Autophagy. Interestingly, disturbing autophagic pathways in the chick embryo precipitates dextrocardia, a phenotype observed in HPSB7 morphant embryos. We also generated a spectrum of defined HSPB7 mutant alleles using TALEN technology. Four such alleles have been bred to homozygosity including a suspected N-terminal truncation that deletes a putative regulatory polyserine stretch. Although homozygous mutants do not exhibit an overt cardiac phenotype, we hypothesize that loss of this polyserine domain may result in a hypomorphic HSPB7 protein. Exposure of mutant embryos to various stressors, including increased temperature (32°C) and the autophagy inducer rapamycin (1μM), led to an increase in phenotypes we have observed in HSPB7 morphants, including dextrocardia and the incidence of defects in cardiac morphology.

Monte Carlo Simulation of Wnt Propagation by a Novel Transport Mechanism Complementing a Joint Experimental Study

Tissue development is a key process in living organisms. An essential component for these developmental processes - but also for tissue regeneration and stem cell regulation - is the communication of cells by paracrine signaling. Following the French flag model, these processes are responsive to concentration gradients of signal carrying molecules, so-called morphogens. The highly conserved family of Wnt proteins can act as morphogens and represents important regulators of all these processes. After secretion, specific transport mechanism must ensure proper distribution of the morphogen. Experimental studies in zebrafish embryos and human kidney cells have given first evidence for a novel short-range transport of Wnt morphogens from the Wnt active tissue towards receiving cells using cell protrusions, so-called filopodia, as mediating agent. These specialized filopodia transmit signaling proteins between communicating cells and allow a high degree of control of propagation speed, direction and concentration of the transmitted ligand. The crucial question is how this novel short-range mechanism can result in a long-range gradient of morphogen molecules covering the complete responsive tissue. In order to give an answer to this question and address the theoretical feasibility of the new model we have set up complementary Monte Carlo simulations. The simulation iteratively reproduces ligand production, cell migration, and a slight ligand decay in concordance with experimentally measured boundary conditions. In a filopodia mediated transport system the major parameters are not anymore diffusion rate, cell adhesion, and concentration of the ligand but length, angle distribution, and growth frequency of filopodia. During the simulation we were able to identify key parameters of the underlying mechanism and quantitatively reproduce our experimental data. These results provide evidence that a filopodia based short-range transport system for Wnt has long-range signalling function.

Developmental biology: Cells unite by trapping a signal.

Gradients of fibroblast growth factors often induce cells to adopt different fates. A study in zebrafish embryos reveals another, unexpected role when the factors are trapped in small spaces by a special arrangement of cells. See Letter p.120 The developing embryo assembles complex tissues and organs through the coordinated differentiation of cell groups, a collective process that depends on highly efficient cell communication. How cells in growing tissues exert control on morphogens that direct their fate and are secreted into extracellular spaces has been unclear. Using live imaging Darren Gilmour and colleagues show that cells in the developing lateral line system of zebrafish arrange themselves in shared microenvironments or microlumina so that signals — such as fibroblast growth factor (FGF) — are more concentrated in some areas than in others. This allows a coordinated response from nearby cells according to their position, a feedback process that in turn influences the arrangement of the cells and propels further development.

A Systems Biology Approach for Defining the Molecular Framework of the Hematopoietic Stem Cell Niche

Despite progress in identifying the cellular composition of hematopoietic stem/progenitor cell (HSPC) niches, little is known about the molecular requirements of HSPC support. To address this issue, weused a panel of six recognized HSPC-supportive stromal lines and less-supportive counterparts originating from embryonic and adult hematopoietic sites. Through comprehensive transcriptomic meta-analyses, we identified 481 mRNAs and 17 microRNAs organized in a modular network implicated in paracrine signaling. Further inclusion of 18 additional cell strains demonstrated that this mRNA subset was predictive of HSPC support. Our gene set contains most known HSPC regulators as well as a number of unexpected ones, such as Pax9 and Ccdc80, as validated by functional studies in zebrafish embryos. In sum, our approach has identified the core molecular network required for HSPC support. These cues, along with a searchable web resource, will inform ongoing efforts to instruct HSPC exvivo amplification and formation from pluripotent precursors.