Larval Eye Research Articles

Ultrastructure of pigmented eyes in Onuphidae and Eunicidae (Annelida: Errantia: Eunicida) and its importance in understanding the evolution of eyes in Annelida

Annelida displays enormous eye and photoreceptor cell (PRC) diversity. In polychaetes, larval and adult eyes can be readily distinguished as the former are small, inverse and comprised of only two or three cells, and the latter are usually everse and multicellular. However, there are some species in which adult eyes are small, fewer in number or even absent. Recent studies show that two pairs of multicellular adult eyes belong to the ground pattern of a clade comprising Amphinomida/Sipuncula and Pleistoannelida with Errantia and Sedentaria. As ultrastructural data in one higher taxon of Errantia, Eunicida, are scarce or completely lacking for certain subgroups, we investigated the structure of pigmented eyes in three species of eunicidan bristle worms: Aponuphis bilineata (Onuphidae), Paucibranchia bellii (Eunicidae) and Leodice cf. torquata (Eunicidae). All had two pairs of pigmented eyes possessing typical adult structures: rhabdomeric PRCs (rPRC), pigmented supportive cells (PSCs) and additional cell types (in some species). The PSCs formed a shading pigment cup housing the sensory processes of the PRCs in everse orientation. Both Eunicidae species examined possess a lens-like structure formed by extensions of the PSCs as typical for members of Errantia, suggesting that lens-like structures formed by PSC processes were acquired in the stem lineage of Errantia and represent an autapomorphy of this clade. Our observations provide further evidence for the presence of two pairs of adult eyes in the ground pattern of Amphinomida/Sipuncula and Pleistoannelida.

Long-Wavelength Reflecting Filters Found in the Larval Retinas of One Mantis Shrimp Family (Nannosquillidae).

Both vertebrates and invertebrates commonly exploit photonic structures adjacent to their photoreceptors for visual benefits. For example, use of a reflecting structure (tapetum) behind the retina increases photon capture, enhancing vision in dim light [1-5]. Colored filters positioned lateral or distal to a photoreceptive unit may also be used to tune spectral sensitivity by selective transmission of wavelengths not absorbed or scattered bythe filters [6-8]. Here we describe a new category of biological optical filter that acts simultaneously as both a transmissive spectral filter and narrowband reflector. Discovered in the larval eyes of only one family of mantis shrimp (stomatopod) crustaceans (Nannosquillidae), each crystalline structure bisects the photoreceptive rhabdom into two tiers and contains an ordered array of membrane-bound vesicles with sub-wavelength diameters of 153± 5nm. Axial illumination of the intrarhabdomal structural reflector (ISR) invivo produces a narrow band of yellow reflectance (mean peak reflectivity, 572± 18nm). The ISR is similar to several synthetic devices, such as bandgap filters, laser mirrors, and (in particular) fiber Bragg gratings used in optical sensors for awide range of industries. To our knowledge, thestomatopod larval ISR is the first example of a naturally occurring analog to these human-made devices. Considering what is known about these animals' visual ecology, we propose that these reflecting filters may help improve the detection of pelagic bioluminescence in shallow water at night. VIDEO ABSTRACT.

Functional role of pax6 during eye and nervous system development in the annelid Capitella teleta

The transcription factor Pax6 is an important regulator of early animal development. Loss of function mutations of pax6 in a range of animals result in a reduction or complete loss of the eye, a reduction of a subset of neurons, and defects in axon growth. There are no studies focusing on the role of pax6 during development of any lophotrochozoan representative, however, expression of pax6 in the developing eye and nervous system in a number of species suggest that pax6 plays a highly conserved role in eye and nervous system formation. We investigated the functional role of pax6 during development of the marine annelid Capitella teleta. Expression of pax6 transcripts in C. teleta larvae is similar to patterns found in other animals, with distinct subdomains in the brain and ventral nerve cord as well as in the larval and juvenile eye. To perturb pax6 function, two different splice-blocking morpholinos and a translation-blocking morpholino were used. Larvae resulting from microinjections with either splice-blocking morpholino show a reduction of the pax6 transcript. Development of both the larval eyes and the central nervous system architecture are highly disrupted following microinjection of each of the three morpholinos. The less severe phenotype observed when only the homeodomain is disrupted suggests that presence of the paired domain is sufficient for partial function of the Pax6 protein. Preliminary downstream target analysis confirms disruption in expression of some components of the retinal gene regulatory network, as well as disruption of genes involved in nervous system development. Results from this study, taken together with studies from other species, reveal an evolutionarily conserved role for pax6 in eye and neural specification and development.

Serial electron microscopic reconstruction of the drosophila larval eye: Photoreceptors with a rudimentary rhabdomere of microvillar-like processes

Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a “beaded” shape, with thick segments (0.2–0.3 μm diameter) alternating with thin segments (>0.1 μm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.

Orthogonal Drug Pooling Enhances Phenotype-Based Discovery of Ocular Antiangiogenic Drugs in Zebrafish Larvae.

Unbiased screening of large randomized chemical libraries in vivo is a powerful tool to find new drugs and targets. However, forward chemical screens in zebrafish can be time consuming and usually >99% of test compounds have no significant effect on the desired phenotype. Here, we sought to find bioactive drugs more efficiently and to comply with the 3R principles of replacement, reduction, and refinement of animals in research. We investigated if pooling of drugs to simultaneously test 8–10 compounds in zebrafish larvae can increase the screening efficiency of an established assay that identifies drugs inhibiting developmental angiogenesis in the eye. In a phenotype-based screen, we tested 1,760 small molecule compounds from the ChemBridge DIVERSet™ chemical library for their ability to inhibit the formation of distinct primary hyaloid vessels in the eye. Applying orthogonal pooling of the chemical library, we treated zebrafish embryos from 3 to 5 days post fertilization with pools of 8 or 10 compounds at 10 μM each. This reduced the number of tests from 1,760 to 396. In 63% of cases, treatment showed sub-threshold effects of <40% reduction of primary hyaloid vessels. From 18 pool hits, we identified eight compounds that reduce hyaloid vessels in the larval zebrafish eye by at least 40%. Compound 4-[4-(1H-benzimidazol-2-yl)phenoxy]aniline ranked as the most promising candidate with reproducible and dose-dependent effects. To our knowledge, this is the first report of a self-deconvoluting matrix strategy applied to drug screening in zebrafish. We conclude that the orthogonal drug pooling strategy is a cost-effective, time-saving, and unbiased approach to discover novel inhibitors of developmental angiogenesis in the eye. Ultimately, this approach may identify new drugs or targets to mitigate disease caused by pathological angiogenesis in the eye, e.g., diabetic retinopathy or age-related macular degeneration, wherein blood vessel growth and leaky vessels lead to vision impairment or clinical blindness.

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode.

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

Lic regulates JNK-mediated cell death in Drosophila.

ObjectivesThe evolutionary conserved JNK pathway plays crucial role in cell death, yet factors that modulate this signalling have not been fully disclosed. In this study, we aim to identify additional factors that regulate JNK signalling in cell death, and characterize the underlying mechanisms.Materials and Methods Drosophila were raised on standard media, and cross was carried out at 25°C. The Gal4/UAS system was used to express proteins or RNAi in a specific temporal and spatial pattern. Gene expression was revealed by GFP fluorescence, X‐gal staining or immunostaining of 3rd instar larval eye and wing discs. Cell death was visualized by acridine orange (AO) staining. Images of fly eyes and wings were taken by OLYMPUS microscopes.ResultsWe found that licorne (lic) encoding the Drosophila MKK3 is an essential regulator of JNK‐mediated cell death. Firstly, loss of lic suppressed ectopic Egr‐triggered JNK activation and cell death in eye and wing development. Secondary, lic is necessary for loss‐of‐cell polarity‐induced, physiological JNK‐dependent cell death in wing development. Thirdly, Lic overexpression is sufficient to initiate JNK‐mediated cell death in developing eyes and wings. Furthermore, ectopic Lic activates JNK signalling by promoting JNK phosphorylation. Finally, genetic epistatic analysis confirmed that Lic acts in parallel with Hep in the Egr‐JNK pathway.ConclusionsThis study not only identified Lic as a novel component of the JNK signalling, but also disclosed the crucial roles and mechanism of Lic in cell death.

Establishment of correctly focused eyes may not require visual input in arthropods.

For proper function, vertebrate and invertebrate visual systems must be able to achieve and maintain emmetropia, a state where distant objects are in focus on the retina. In vertebrates, this is accomplished through a combination of genetic control during early development and homeostatic visual input that fine-tunes the optics of the eye. While emmetropization has long been researched in vertebrates, it is largely unknown how emmetropia is established in arthropods. We used a micro-ophthalmoscope to directly measure how the lens projects images onto the retina in the eyes of small, live arthropods, allowing us to compare the refractive states of light-reared and dark-reared arthropods. First, we measured the image-forming larval eyes of diving beetles (Thermonectus marmoratus), which are known to grow rapidly and dramatically between larval instars. Then, we measured the image-forming principal anterior-median eyes of jumping spiders (Phidippus audax) after emergence from their egg cases. Finally, we measured individual ommatidia in the compound eyes of flesh flies (Sarcophaga bullata) that had developed and emerged under either light or dark conditions. Surprisingly, and in sharp contrast to vertebrates, our data for this diverse set of arthropods suggest that visual input is inconsequential in regard to achieving well-focused eyes. Although it remains unclear whether visual input that is received after the initial development further improves focusing, these results suggest that at least the initial coordination between the lens refractive power and eye size in arthropods may be more strongly predetermined by developmental factors than is typically the case in vertebrates.

Fate mapping during regeneration: Cells that undergo compensatory proliferation in damaged Drosophila eye imaginal discs differentiate into multiple retinal accessory cell types

Regeneration of tissues that have been damaged by cell loss requires new growth, often via proliferation of precursor cells followed by differentiation to replace loss of specific cell types. When regeneration occurs after normal differentiation of the tissue is complete, developmental pathways driving differentiation must be re-activated. How proliferation and differentiation are induced and balanced during regeneration is not well understood. To investigate these processes, we utilized a paradigm for tissue damage and regeneration in the developing Drosophila melanogaster eye. Previous studies have demonstrated that tissue damage resulting from extensive cell death stimulates quiescent, undifferentiated cells in the developing larval eye to re-enter the cell cycle and proliferate. Whether these cells are restricted to certain fates or can contribute to all retinal cell types and thus potentially be fully regenerative is not known. Here we found by fate mapping experiments that these cells are competent to differentiate into all accessory cell types in the retina but do not differentiate into photoreceptors, likely because cell cycle re-entry in response to damage occurs after photoreceptor differentiation has completed. We conclude that the ability to re-enter the cell cycle in response to tissue damage in the developing Drosophila eye is not restricted to precursors of a specific cell type and that cell cycle re-entry following damage does not disrupt developmental programs that control differentiation.

CtBP represses Dpp-dependent Mad activation during Drosophila eye development

Complex networks of signaling pathways maintain the correct balance between positive and negative growth signals, ensuring that tissues achieve proper sizes and differentiation pattern during development. In Drosophila, Dpp, a member of the TGFβ family, plays two main roles during larval eye development. In the early eye primordium, Dpp promotes growth and cell survival, but later on, it switches its function to induce a developmentally-regulated cell cycle arrest in the G1 phase and neuronal photoreceptor differentiation. To advance in the identification and characterization of regulators and targets of Dpp signaling required for retinal development, we carried out an in vivo eye-targeted double-RNAi screen to identify punt (Type II TGFβ receptor) interactors. Using a set of 251 genes associated with eye development, we identified CtBP, Dad, Ago and Brk as punt genetic interactors. Here, we show that downregulation of Ago, or conditions causing increased tissue growth including overexpression of Myc or CyclinD-Cdk4 are sufficient to partially rescue punt-dependent growth and photoreceptor differentiation. Interestingly, we show a novel role for the transcriptional co-repressor CtBP in inhibiting Dpp-dependent Mad activation by phosphorylation, downstream or in parallel to Dad, the inhibitory Smad. Furthermore, CtBP downregulation activates JNK signaling pathway, implying a complex regulation of signaling pathways by CtBP during eye development.

The Larval Development of the Chiton Deshayesiella curvata (Carpenter in Dall, 1879) (Mollusca: Polyplacophora)

Chronological study of the larval development of the chiton Deshayesiella curvata with sketching of the first stages of egg division and embryos up to the seven-valved shell stage is presented for the first time. D. curvata exhibits pelagic development with lecithotrophic larvae. Larval eyes form at the age of 3 days; at the age of 6 days, a Schwabe organ appears near the eyes in the form of two pigmented spots. Chitons at the stage of seven-valved shell have larval eyes, as well as the Schwabe organ.

Correlative XRF, ptychography and light microscopy on shielding pigments of Clunio larval ocelli

The nature of retinal shielding pigment in the larval eyes of the midge Clunio marinus was studied by high resolution scanning X-ray fluorescence (XRF) analysis correlated with light microscopy (LM) and scanning coherent X-ray diffractive imaging (ptychography). Clunio is known as a unique model system to decipher mechanisms underlying the complex moon-related rhythms. Shielding pigment granules within the photoreceptor cells of their primitive eyes (ocelli) have been shown to change appearance in the same rhythm. We compared the relative amount of trace metals (Ca, Ni, Zn, Cu), obtained by XRF, within single melanosomes (diameter about 500 nm) and the surrounding photoreceptor cells with their optical density in the light microscope and—for the first time—the electron density derived from ptychography images. Though individual pigment granules can be identified and correlated in images of all three techniques the optical density, electron density and especially element concentrations within individual granules seem to vary independently. We discuss that the state of melanogenesis is not synchronized inside the ocellus.

Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode.

The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. Here, we characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define PR subtype identity. Our model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning.

Unexpected ultrastructure of an eye in Spiralia: the larval ocelli of Procephalothrix oestrymnicus (Nemertea)

The evolution of eyes and their constituent photoreceptor cells in Metazoa in general and in Protostomia in particular remains unresolved with present morphological and developmental genetic data. This is mainly due to the lack of comprehensive ultrastructural data in some lineages, such as in the spiralian taxon Nemertea. The eyes of the derived Neonemertea possess rhabdomeric photoreceptor cells, considered typical of the protostome lineage. In the more basally branching palaeonemertean lineages, ultrastructural data on the eyes are wanting. Ultrastructural investigation of the eyes of the larva of the palaeonemertean Procephalothrix oestrymnicus reveals that, although in a similar position as the eyes of adult neonemertean species, the eyes in palaeonemertean larvae differ fundamentally from the expected protostomian type: They consist of one shading-pigment cell that forms a closed optical cavity embedded in the epidermis. Apart from basally distributed shading-pigment vesicles, the pigment cell apically possesses epidermal cilia and microvilli as well as sub-apical, tubular lens vesicles. Two ciliary photoreceptor cells project flattened ciliary membranes into the optical cavity formed by the pigment cell, whereas their basal portions are situated outside of the optical cavity, next to the shading-pigment cell. Although the structure of the eye in P. oestrymnicus is unparalleled in Nemertea, ciliary photoreceptor cells have been found in larval eyes of several other spiralian lineages. Occurrence of additional ciliary-type eyes in Spiralia deepens the doubts regarding the validity of the hypothesis of an exclusively rhabdomeric line of visual photoreceptor cell evolution in Protostomia.

Chronic exposure to acephate triggers ROS-mediated injuries at organismal and sub-organismal levels of Drosophila melanogaster.

The present study demonstrates ROS-mediated organismal and sub-organismal injuries in Drosophila melanogaster following chronic acephate exposure. Larvae and adults of Drosophila were reared on food supplemented with sub-lethal concentrations (1-6 μg mL-1) of acephate (LC50 8.71 μg mL-1). The longevity of the treated adults was reduced to half at 6 μg mL-1 exposure along with declined neuromuscular coordination and physical activities. Apparent developmental defects in the compound eyes were confirmed through the detection of apoptotic lesions in larval eye imaginal discs. The larval gut manifested tissue damage at various sites. Neural and fat cell viability was reduced by ∼1.89- and ∼3.38-fold at 6 μg mL-1 acephate treatment, respectively. A significant reduction in hemocyte viability confirmed the immunotoxic potential of acephate. Nearly 1-3-fold enhancement in the expression of OS markers (MDA, protein carbonyl contents, SOD, catalase and HSP70) in the treated larvae served as evidence of ROS production. The post-treatment increase in CYP450 and GST activities reflects the 'switch-on' states of the phase-I and phase-II detoxification mechanism. The genotoxic potential of acephate was confirmed through alkaline single cell gel electrophoresis. Thus, the findings of the present study validate the fact that besides traditional cholinesterase inhibition, chronic sub-lethal exposure to acephate potentially induces ROS-mediated toxic responses in Drosophila.

Development Of Larvae Rainbow Fish Boesemani (Melanotaenia boesemani): Phase Depreciation of Yellow Egg, Forming Eyes and Opening of Mouth

&lt;p&gt;&lt;em&gt;Abstract &lt;/em&gt;&lt;strong&gt;– One of the factors that caused larval mortality is the availability of food which is not in accordance with the mouth opening. At the same time the larvae require considerable energy to the development of organs. The Completeness of organs used in feed searching as the eyes is also becoming important. The larvae use eyes to see the feed material in their surrounding waters. This study aimed to measure the volume of egg yolk, eye diameter and wide mouth opening during larval development rainbow fish boesemani (&lt;em&gt;Melanotaenia boesemani&lt;/em&gt;). The stages of the research include larval maintenance, depreciation egg yolk observations, calculate the size of the increase in larval eye and mouth openings. The results of this study showed that newly hatched rainbow boesemani larvae have egg yolks, eyes and mouth. The newly hatched larvae have a yolk volume average of 0.053 mm&lt;sup&gt;3&lt;/sup&gt;. Egg yolks will expire at the time of 2 days old larvae. The formation of the eye, has occurred inside the eggs and newly hatched larvae have an average diameter of eye of 0.459 mm. The newly hatched larvae have a wide mouth opening average of 0.229 mm. An larvae older than 21 days and 30 days has a wide mouth opening average of 0.82 mm and 1.725 mm.&lt;/strong&gt;&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt; &lt;/p&gt;&lt;p&gt;&lt;strong&gt;&lt;em&gt;Keywords&lt;/em&gt;&lt;/strong&gt; – &lt;em&gt;Rainbow fish Melanotaenia boesemani, larval development, egg yolks, eyes, mouth&lt;/em&gt;&lt;/p&gt;

Two visual systems in one eyestalk: The unusual optic lobe metamorphosis in the stomatopod Alima pacifica.

The compound eyes of adult stomatopod crustaceans have two to six ommatidial rows at the equator, called the midband, that are often specialized for color and polarization vision. Beneath the retina, this midband specialization is represented as enlarged optic lobe lamina cartridges and a hernia-like expansion in the medulla. We studied how the optic lobe transforms from the larvae, which possess typical crustacean larval compound eyes without a specialized midband, through metamorphosis into the adults with the midband in a two midband-row species Alima pacifica. Using histological staining, immunolabeling, and 3D reconstruction, we show that the last-stage stomatopod larvae possess double-retina eyes, in which the developing adult visual system forms adjacent to, but separate from, the larval visual system. Beneath the two retinas, the optic lobe also contains two sets of optic neuropils, comprising of a larval lamina, medulla, and lobula, as well as an adult lamina, medulla, and lobula. The larval eye and all larval optic neuropils degenerate and disappear approximately a week after metamorphosis. In stomatopods, the unique adult visual system and all optic neuropils develop alongside the larval system in the eyestalk of last-stage larvae, where two visual systems and two independent visual processing pathways coexist. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 3-14, 2018.

Trans-generational transmission of altered phenotype resulting from flubendiamide-induced changes in apoptosis in larval imaginal discs of Drosophila melanogaster

The eye and wing morphology of Drosophila melanogaster maintain unique, stable pattern of genesis from larval eye and wing imaginal discs. Increased apoptosis in cells of eye and wing discs was found to be associated with flubendiamide (fluoride containing insecticide) exposure (at the range 0.25–10μg/mL) in D. melanogaster larvae. The chemical fed larvae on attaining adulthood revealed alterations in morphology and symmetry of their compound eyes and wings through scanning electron microscopy. Nearly 40% and 30% of flies (P generation) demonstrated alterations in eyes and wings respectively. Transmission electron microscopic study (at the range 1–20μg/mL) also established variation in the rhabdomere and pigment cell orientation as well as in the shape of the ommatidium. Subsequent SEM study with F1 and F2 generation flies also revealed structural variation in eye and wing. Decrease in percentage of altered eye and wing phenotype was noted in subsequent generations (P> F1>F2). Thus, the diamide insecticide, flubendiamide, expected to be environmentally safe at sub-lethal concentrations was found to increase apoptosis in larvae and thereby cause morphological alteration in the adult D. melanogaster. This study further demonstrated trans-generational transmission of altered phenotype in three subsequent generations of a non-target insect model, D. melanogaster.

Zebrafish: A Visual Model To Evaluate the Biofate of Transferrin Receptor-Targeted 7Peptide-Decorated Coumarin 6 Micelles.

In the present study, the zebrafish was explored as an in vivo model to assess the biofate of transferrin receptor (TfR)-targeted coumarin 6 (C6) micelles across various biological barriers. Three 7peptide (7pep)-decorated poly(ethylene glycol)-block-poly(ε-caprolactone) micelles loaded with fluorescence coumarin 6 (7pep-M-C6) with different ligand densities were constructed with particle sizes between 30 and 40 nm. Whole-mount immunostaining revealed that the expression level of TfR in the retina, brain, and intestine increased along with development stage. Compared to unmodified micelles, 7pep-M-C6 demonstrated higher uptake efficiency in the larval zebrafish. Preinhibition of TfR with 7pep implicated the TfR-mediated endocytosis pathway in the uptake of 7pep-M-C6. Confocal images of the larval zebrafish eye and brain showed the efficient delivery of C6 across the retinal pigment epithelial to the ganglion cell layer and the significant accumulation of C6 in all brain tissues, respectively, which plateaued when the ligand density was 10%. To investigate the intestinal distribution of C6, micelles were administered to adult zebrafish via gavaging. Notably, 7pep-M-C6 enhanced the transport of C6 across the villi and increased its aggregation into the basolateral membrane of the intestine. After the oral administration of 7pep-M-C6, C6 accumulated in the eye and brain. Förster resonance energy transfer analysis suggested that intact 7pep-modified micelles could enter the epithelial cells of the intestine, brain, and eye after oral administration in adult zebrafish. In conclusion, zebrafish could be used as a model for in vivo visual assessment of the biofate of TfR-targeted drug delivery systems.

Osmotic Concentration of Zebrafish (Danio rerio) Body Fluids Is Lower in Larvae than in Adults.

We intended to perform optical and structural measurements on larval zebrafish eyes at 5 days post fertilization, that is, the earliest age at which zebrafish show visually guided behavior. However, excised larval crystalline lenses deteriorated quickly if immersed in a medium that gives good results with adult lenses from a variety of fish species. We suspected that the larvae have body fluids of lower osmolality and tested a medium with 240 mOsm, which is 75% of the established adult value of 320 mOsm. The optical quality of freshly excised and immersed lenses was used to judge the osmotic matches. In addition, we tested how well the shape of the eye is preserved in fixatives of different osmolalities. In both cases, 240 mOsm produced the best results. Immersed lenses performed better and the fixed eyes had a more natural shape. Our findings indicate that zebrafish body fluids have lower osmolality in larvae than in adults. This is probably due to an unfavorable body surface-to-volume ratio and incompletely developed regulatory mechanisms. Body fluid osmolality deviating from the adult value has to be taken into account in optical and histological work.