Receptor binding and tortuosity explain morphogen local-to-global diffusion coefficient transition.

Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and receptor binding within these spaces are sufficient to reproduce experimentally measured morphogen dynamics. Importantly, this work demonstrates that hindered diffusion is a viable mechanism for gradient formation, without requiring additional regulatory control.

Wnt3 Is Lipidated at Conserved Cysteine and Serine Residues in Zebrafish Neural Tissue.

Wnt proteins are a family of hydrophobic cysteine-rich secreted glycoproteins that regulate a gamut of physiological processes involved in embryonic development and tissue homeostasis. Wnt ligands are post-translationally lipidated in the endoplasmic reticulum (ER), a step essential for its membrane targeting, association with lipid domains, secretion and interaction with receptors. However, at which residue(s) Wnts are lipidated remains an open question. Initially it was proposed that Wnts are lipid-modified at their conserved cysteine and serine residues (C77 and S209 in mWnt3a), and mutations in either residue impedes its secretion and activity. Conversely, some studies suggested that serine is the only lipidated residue in Wnts, and substitution of serine with alanine leads to retention of Wnts in the ER. In this work, we investigate whether in zebrafish neural tissues Wnt3 is lipidated at one or both conserved residues. To this end, we substitute the homologous cysteine and serine residues of zebrafish Wnt3 with alanine (C80A and S212A) and investigate their influence on Wnt3 membrane organization, secretion, interaction and signaling activity. Collectively, our results indicate that Wnt3 is lipid modified at its C80 and S212 residues. Further, we find that lipid addition at either C80 or S212 is sufficient for its secretion and membrane organization, while the lipid modification at S212 is indispensable for receptor interaction and signaling.

Cancer-Cell-Activated in situ Synthesis of Mitochondria-Targeting AIE Photosensitizer for Precise Photodynamic Therapy.

Maximization of phototoxic damage on tumor with minimized side effect on normal tissue is essential for effective anticancer photodynamic therapy (PDT). This requires highly cancer-cell-specific or even cancer-cell-organelle-specific synthesis or delivery of efficient photosensitizers (PSs) in vitro and in vivo, which is difficult to achieve. Herein, we report a strategy of cancer-cell-activated PS synthesis, by which an efficient mitochondria-targeting photosensitizer with aggregation-induced-emission (AIE) feature can be selectively synthesized as an efficient image-guided PDT agent inside cancer cells. MOF-199, a CuII -based metal-organic framework, was selected as an inert carrier to load the PS precursors for efficient delivery and served as a CuI catalyst source for in situ click reaction to form PSs exclusively in cancer cells. The in situ synthesized PS showed mitochondria-targeting capability, allowing potent cancer-cell-specific ablation under light irradiation. The high specificity of PSs produced in cancer cells also makes it safer post-treatment.

Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions.

Wnt3 proteins are lipidated and glycosylated signaling molecules that play an important role in zebrafish neural patterning and brain development. However, the transport mechanism of lipid-modified Wnts through the hydrophilic extracellular environment for long-range action remains unresolved. Here we determine how Wnt3 accomplishes long-range distribution in the zebrafish brain. First, we characterize the Wnt3-producing source and Wnt3-receiving target regions. Subsequently, we analyze Wnt3 mobility at different length scales by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. We demonstrate that Wnt3 spreads extracellularly and interacts with heparan sulfate proteoglycans (HSPG). We then determine the binding affinity of Wnt3 to its receptor, Frizzled1 (Fzd1), using fluorescence cross-correlation spectroscopy and show that the co-receptor, low-density lipoprotein receptor-related protein 5 (Lrp5), is required for Wnt3-Fzd1 interaction. Our results are consistent with the extracellular distribution of Wnt3 by a diffusive mechanism that is modified by tissue morphology, interactions with HSPG, and Lrp5-mediated receptor binding, to regulate zebrafish brain development.

miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/ß-catenin Signaling Regulation.

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/ß-catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease.

Cancer-Cell-Activated Photodynamic Therapy Assisted by Cu(II)-Based Metal-Organic Framework.

Activation of photosensitizers (PSs) in targeted lesion and minimization of reactive oxygen species (ROS) depletion by endogenous antioxidants constitute promising approaches to perform highly effective image-guided photodynamic therapy (PDT) with minimal non-specific phototoxicity. Traditional strategies to fabricate controllable PS platforms rely on molecular design, which requires specific modification of each PS before PDT. Therefore, construction of a general tumor-responsive PDT platform with minimum ROS loss from endogenous antioxidant, typically glutathione (GSH), is highly desirable. Herein, MOF-199, a Cu(II) carboxylate-based metal-organic framework (MOF), is selected to serve as an inert carrier to load PSs with prohibited photosensitization during delivery. After cellular uptake, Cu (II) in the MOFs effectively scavenges endogenous GSH, concomitantly induces decomposition of MOF-199 to release the encapsulated PSs, and recovers their ROS generation. In vitro and in vivo experiments demonstrate highly effective cancer cell ablation and anticancer PDT with diminished normal cell phototoxicity. This strategy is generally applicable to PSs with both aggregation-induced emission and aggregation-caused quenching to implement activatable and enhanced image-guided PDT.

Visualize Embryogenesis and Cell Fate Using Fluorescent Probes with Aggregation-Induced Emission.

Horseradish peroxidase (HRP) and fluorogen-dextran conjugate are tracers extensively used for injection-based lineage tracing. However, HRP is sensitive to proteolytic digestion, whereas the high-molecular-weight dextran may have antigenicity. Small molecular tracers can overcome these problems, but they usually diffuse from labeled cells, causing inaccurate information. Herein, we developed a small-molecular-weight fluorogen with aggregation-induced emission (AIEgen) for embryonic cell tracing with strong signals against tracer dilution caused by cell division. Once injected into the ancestor cells, the AIEgen can be entrapped in the cells without leakage because of the two hydrophilic and neutral arms. Consequently, it can specifically trace the progenies of the treated ancestor cells. More importantly, the operating concentration of AIEgen can be much higher than that of fluorogens with aggregation-caused quenching, which provides bright signals in daughter cells during embryonic cell tracing, thus overcoming the problem of fast signal degradation typically encountered with the use of traditional cell tracers.

J-Aggregation of Perylene Diimides in Silica Nanocapsules for Stable Near-Infrared Photothermal Conversion.

A novel near-infrared-responsive (NIR-responsive) photothermal therapy (PTT) agent based on perylene-diimide-encapsulated (PDI-encapsulated) PEGylated silica nanocapsules (SNCs) is developed. Dicyclohexylamino-PDI (DCAPDI) with electron-donating cyclohexylamino substitutes at bay positions aggregates into J-aggregation in the core of SNCs, and their electronic coupling interactions are strengthened because of the spatial confinement of SNCs, resulting in strong NIR absorption but negligible fluorescence emission which is crucial for NIR-responsive PTT. Based on our knowledge, this is the first example of generating NIR photothermal conversion by means of molecular aggregation derived from spatial confinement. Unprecedented photostability is achieved with the DCAPDI-encapsulated SNCs in response to more than 60 runs of cyclic NIR exposure with each run exposed to the 808 nm, 1 W cm-2 laser for 10 min. It overcomes the common photodegradation problem of small organic NIR dyes under continuous high-power laser irradiation thanks to the robust molecular skeleton of PDIs and their formation of structurally stable J-aggregates in SNCs. The DCAPDI-encapsulated SNCs demonstrate low cellular cytotoxicity and excellent in vivo photothermal efficacy in tumor ablation in a tumor-bearing zebrafish model, and thus allow the practical employment of a stable photothermal agent in clinical applications.

Visualizing Photodynamic Therapy in Transgenic Zebrafish Using Organic Nanoparticles with Aggregation-Induced Emission.

Photodynamic therapy (PDT) employs accumulation of photosensitizers (PSs) in malignant tumor tissue followed by the light-induced generation of cytotoxic reactive oxygen species to kill the tumor cells. The success of PDT depends on optimal PS dosage that is matched with the ideal power of light. This in turn depends on PS accumulation in target tissue and light administration time and period. As theranostic nanomedicine is driven by multifunctional therapeutics that aim to achieve targeted tissue delivery and image-guided therapy, fluorescent PS nanoparticle (NP) accumulation in target tissues can be ascertained through fluorescence imaging to optimize the light dose and administration parameters. In this regard, zebrafish larvae provide a unique transparent in vivo platform to monitor fluorescent PS bio-distribution and their therapeutic efficiency. Using fluorescent PS NPs with unique aggregation-induced emission characteristics, we demonstrate for the first time the real-time visualization of polymeric NP accumulation in tumor tissue and, more importantly, the best time to conduct PDT using transgenic zebrafish larvae with inducible liver hyperplasia as an example.

A membrane film sensor with encapsulated fluorescent dyes towards express freshness monitoring of packaged food.

A new Membrane Film Sensor (MFS) has been developed to measure pH of fluids. MFS comprises a polyelectrolyte multilayer film with uniformly distributed compartments (microchambers) where a fluorescent sensing dye is encapsulated. Fabricated film is sealed onto a polyethylene film for a future use. MFS was applied to report changes in golden pomfret fillet upon its storage at 5 °C. MFS pH readings were correlated to bacteriological analysis of fish samples. A hike in pH of fish juices happens after 10 days of storage signaling bacterial spoilage of fish. The design of developed MFS allows easy integration with transparent packaging materials for future development of "SMART" packaging sensing food freshness.

Microencapsulated fluorescent pH probe as implantable sensor for monitoring the physiological state of fish embryos.

In vivo physiological measurement is a major challenge in modern science and technology, as is environment conservation at the global scale. Proper toxicological testing of widely produced mixtures of chemicals is a necessary step in the development of new products, allowing us to minimize the human impact on aquatic ecosystems. However, currently available bioassay-based techniques utilizing small aquatic organisms such as fish embryos for toxicity testing do not allow assessing in time the changes in physiological parameters in the same individual. In this study, we introduce microencapsulated fluorescent probes as a promising tool for in vivo monitoring of internal pH variation in zebrafish embryos. The pH alteration identified under stress conditions demonstrates the applicability of the microencapsulated fluorescent probes for the repeated analysis of the embryo's physiological state. The proposed approach has strong potential to simultaneously measure a range of physiological characteristics using a set of specific fluorescent probes and to finally bring toxicological bioassays and related research fields to a new level of effectiveness and sensitivity.

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

While the cytosolic events of Wnt/ß-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/ß-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity.

Responsive mesoporous silica nanoparticles for sensing of hydrogen peroxide and simultaneous treatment toward heart failure.

Chronic heart failure is often characterized by the elevated amounts of reactive oxygen species such as hydrogen peroxide (H2O2) in the heart. Thus, it is of importance that selective release of therapeutic drugs occurs at the heart failure site to maximize therapeutic effects. In this work, functional mesoporous silica nanoparticles (MSNPs) were developed for detection of H2O2, selective drug release and controlled treatment toward heart failure. The H2O2-sensitive probe was attached to the surface of the MSNPs, and a therapeutic drug of heart failure, captopril, was loaded within the pores of the MSNPs and retained by the binding of α-cyclodextrin to the probe. H2O2 present in tissue could react with the probe and enable the dissociation of α-cyclodextrin present on the nanoparticle surface, so that captopril could be successfully released along with "turn-on" of the probe fluorescence. In vivo experiments using the KillerRed heart failure transgenic zebrafish model demonstrated that this therapeutic system is physiologically responsive. Captopril-loaded MSNPs showed high therapeutic efficacy, improving the heartbeat rate and cardiac output in zebrafish experiencing acute KillerRed-induced heart failure.

Redox and pH Dual Responsive Polymer Based Nanoparticles for In Vivo Drug Delivery.

Responsive nanomaterials have emerged as promising candidates as drug delivery vehicles in order to address biomedical diseases such as cancer. In this work, polymer-based responsive nanoparticles prepared by a supramolecular approach are loaded with doxorubicin (DOX) for the cancer therapy. The nanoparticles contain disulfide bonds within the polymer network, allowing the release of the DOX payload in a reducing environment within the endoplasm of cancer cells. In addition, the loaded drug can also be released under acidic environment. In vitro anticancer studies using redox and pH dual responsive nanoparticles show excellent performance in inducing cell death and apoptosis. Zebrafish larvae treated with DOX-loaded nanoparticles exhibit an improved viability as compared with the cases treated with free DOX by the end of a 3 d treatment. Confocal imaging is utilized to provide the daily assessment of tumor size on zebrafish larva models treated with DOX-loaded nanoparticles, presenting sustainable reduction of tumor. This work demonstrates the development of functional nanoparticles with dual responsive properties for both in vitro and in vivo drug delivery in the cancer therapy.

Development of Circumventricular Organs in the Mirror of Zebrafish Enhancer-Trap Transgenics.

The circumventricular organs (CVOs) are small structures lining the cavities of brain ventricular system. They are associated with the semitransparent regions of the blood-brain barrier (BBB). Hence it is thought that CVOs mediate biochemical signaling and cell exchange between the brain and systemic blood. Their classification is still controversial and development not fully understood largely due to an absence of tissue-specific molecular markers. In a search for molecular determinants of CVOs we studied the green fluorescent protein (GFP) expression pattern in several zebrafish enhancer trap transgenics including Gateways (ET33-E20) that has been instrumental in defining the development of choroid plexus. In Gateways the GFP is expressed in regions of the developing brain outside the choroid plexus, which remain to be characterized. The neuroanatomical and histological analysis suggested that some previously unassigned domains of GFP expression may correspond to at least six other CVOs-the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), paraventricular organ (PVO), pineal (epiphysis), area postrema (AP) and median eminence (ME). Two other CVOs, parapineal and subcommissural organ (SCO) were detected in other enhancer-trap transgenics. Hence enhancer-trap transgenic lines could be instrumental for developmental studies of CVOs in zebrafish and understanding of the molecular mechanism of disease such a hydrocephalus in human. Their future analysis may shed light on general and specific molecular mechanisms that regulate development of CVOs.

Quantitative imaging reveals real-time Pou5f3-Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish.

Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3-Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3-Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3-Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis.

A Porphyrin-Based Conjugated Polymer for Highly Efficient In Vitro and In Vivo Photothermal Therapy.

Conjugated polymers have been increasingly studied for photothermal therapy (PTT) because of their merits including large absorption coefficient, facile tuning of exciton energy dissipation through nonradiative decay, and good therapeutic efficacy. The high photothermal conversion efficiency (PCE) is the key to realize efficient PTT. Herein, a donor-acceptor (D-A) structured porphyrin-containing conjugated polymer (PorCP) is reported for efficient PTT in vitro and in vivo. The D-A structure introduces intramolecular charge transfer along the backbone, resulting in redshifted Q band, broadened absorption, and increased extinction coefficient as compared to the state-of-art porphyrin-based photothermal reagent. Through nanoencapsulation, the dense packing of a large number of PorCP molecules in a single nanoparticle (NP) leads to favorable nonradiative decay, good photostability, and high extinction coefficient of 4.23 × 104 m-1 cm-1 at 800 nm based on porphyrin molar concentration and the highest PCE of 63.8% among conjugated polymer NPs. With the aid of coloaded fluorescent conjugated polymer, the cellular uptake and distribution of the PorCP in vitro can be clearly visualized, which also shows effective photothermal tumor ablation in vitro and in vivo. This research indicates a new design route of conjugated polymer-based photothermal therapeutic materials for potential personalized theranostic nanomedicine.

The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study.

Wnt3 is a morphogen that activates the Wnt signaling pathway and regulates a multitude of biological processes ranging from cell proliferation and cell fate specification to differentiation over embryonic induction to neural patterning. Recent studies have shown that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, plays a significant role in the intracellular membrane trafficking of Wnt3 and subsequently, its secretion in live zebrafish embryos, where chemical inhibition of Porcupine reduced the membrane-bound and secreted fractions of Wnt3 and eventually led to defective brain development. However, the membrane distribution of Wnt3 in cells remains not fully understood. Here, we determine the membrane organization of functionally active Wnt3-EGFP in cerebellar cells of live transgenic zebrafish embryos and the role of palmitoylation in its organization using single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS), a multiplexed modality of FCS, which generates maps of molecular dynamics, concentration, and interaction of biomolecules. The FCS diffusion law was applied to SPIM-FCS data to study the subresolution membrane organization of Wnt3. We find that at the plasma membrane in vivo, Wnt3 is associated with cholesterol-dependent domains. This association reduces with increasing concentrations of Porcupine inhibitor (C59), confirming the importance of palmitoylation of Wnt3 for its association with cholesterol-dependent domains. Reduction of membrane cholesterol also results in a decrease of Wnt3 association with cholesterol-dependent domains in live zebrafish. This demonstrates for the first time, to our knowledge, in live vertebrate embryos that Wnt3 is associated with cholesterol-dependent domains.

Modulating the expression level of secreted Wnt3 influences cerebellum development in zebrafish transgenics.

The boundaries of brain regions are associated with the tissue-specific secretion of ligands from different signaling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. Using light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics, we demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP is secreted from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porcn inhibitor Wnt-C59 (C59), which, when applied early, reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work, supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane-localized and secreted Wnt3 during cerebellum development.

Bayesian model selection applied to the analysis of fluorescence correlation spectroscopy data of fluorescent proteins in vitro and in vivo.

Fluorescence correlation spectroscopy (FCS) is a powerful technique to investigate molecular dynamics with single molecule sensitivity. In particular, in the life sciences it has found widespread application using fluorescent proteins as molecularly specific labels. However, FCS data analysis and interpretation using fluorescent proteins remains challenging due to typically low signal-to-noise ratio of FCS data and correlated noise in autocorrelated data sets. As a result, naive fitting procedures that ignore these important issues typically provide similarly good fits for multiple competing models without clear distinction of which model is preferred given the signal-to-noise ratio present in the data. Recently, we introduced a Bayesian model selection procedure to overcome this issue with FCS data analysis. The method accounts for the highly correlated noise that is present in FCS data sets and additionally penalizes model complexity to prevent over interpretation of FCS data. Here, we apply this procedure to evaluate FCS data from fluorescent proteins assayed in vitro and in vivo. Consistent with previous work, we demonstrate that model selection is strongly dependent on the signal-to-noise ratio of the measurement, namely, excitation intensity and measurement time, and is sensitive to saturation artifacts. Under fixed, low intensity excitation conditions, physical transport models can unambiguously be identified. However, at excitation intensities that are considered moderate in many studies, unwanted artifacts are introduced that result in nonphysical models to be preferred. We also determined the appropriate fitting models of a GFP tagged secreted signaling protein, Wnt3, in live zebrafish embryos, which is necessary for the investigation of Wnt3 expression and secretion in development. Bayes model selection therefore provides a robust procedure to determine appropriate transport and photophysical models for fluorescent proteins when appropriate models are provided, to help detect and eliminate experimental artifacts in solution, cells, and in living organisms.