Biology subjects

Jusuf, P. R.

Publications and source records attributed to Jusuf, P. R..

9 recordsLinked to original sources

bioRxiv2026

Optimisation of OptoDrum protocol for measuring optomotor response in juvenile & adult zebrafish

Zebrafish (Danio rerio) are an important vertebrate model for vision and neuroscience research. In the larval stages, the aquatic species begins to elicit the optomotor response (OMR) to stabilize themselves in water -- a behaviour that may be exploited in the laboratory to measure visual acuity. However, up to now, the measurement of the OMR in juvenile and adult zebrafish has been limited due to their behavioural complexity. Here, we optimize a protocol to assay zebrafish aged between 4 and 9 weeks-post-fertilization, by displaying sinusoidal gratings parallel to the zebrafish eye to elicit a robust OMR. We assessed the visual spatial-frequency tuning function of an environmentally induced myopia model to confirm the sensitivity and robustness of the protocol. Additionally, we show the OMR is sensitive to the contrast and temporal resolution of the sinusoidal gratings. Furthermore, we found that the time between stimulus presentations impact the spatial-frequency tuning function likely as time is required for zebrafish to return to baseline swimming after eliciting the OMR. Finally, we found that the OMR after ten versus twenty seconds of stimulus onset appears comparable; indicating that robust OMR responses in zebrafish can be elicited through relatively short stimulus presentations. Through the experiments conducted, we present an optimized protocol specific to zebrafish. The protocol may be used to follow the progression or treatment efficacy of progressive neurological disorders including specific visual disorders and higher brain functions with visual endophenotypes. Ultimately, this protocol allows for high-throughput robust measures of visual and neural function in zebrafish.

neuroscience↗

bioRxiv2026

Phytochemical profiling, antioxidant capacity, and in vivo safety assessment of Warrigal spinach (Tetragonia tetragonioides) and Kensington Pride mango (Mangifera indica) extracts using Zebrafish larvae

Plant-derived bioactive compounds are recognised for their antioxidant potential and benefit for human diseases, including age-related diseases caused by oxidative stress. However, their antioxidant composition and safety profiles remain insufficiently understood. This study integrates phytochemical profiling, antioxidant evaluation, and in vivo toxicological assessment of Warrigal spinach (Tetragonia tetragonioides) and Kensington Pride mango (Mangifera indica). Spinach exhibited greater antioxidant capacity, and higher total phenolic and flavonoid content than mango: TPC (14.2 {+/-} 0.6 mg GAE/g vs 1.30 {+/-} 0.07 mg GAE/g) and TFC (9.61 {+/-} 0.39 mg QE/g vs 0.08 {+/-} 0.0 mg QE/g). LC-ESI-QTOF-MS/MS identified 187 metabolites dominated by flavonoids (53.5%) and phenolic acids (16%), with spinach showing greater chemical diversity. Quantitative analysis revealed higher levels of hydroxycinnamic acids and flavonoid glycosides in spinach, whereas mango contained distinct metabolites, including mangiferin and pyrogallol. Zebrafish embryo / larval assays demonstrated high safety margins, with LC50 values of 478.8 mg/L (spinach) and >480 mg/L (mango). At 480 mg/L spinach displayed developmental abnormalities and malformations. These findings demonstrate that antioxidant capacity is linked to phenolic composition, but does not predict toxicity. Thus, integrated phytochemical and safety evaluation for extracts with complex compound mixtures are critical to identify botanicals suitable for future drug development. HighlightsO_LIWarrigal spinach exhibited >10-fold higher phenolic content and antioxidant capacity than Kensington Pride mango. C_LIO_LILC-ESI-QTOF-MS/MS identified 187 metabolites, with flavonoids as the dominant phytochemical class. C_LIO_LIZebrafish assays confirmed high safety margins, demonstrating no direct correlation between antioxidant capacity and toxicity. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/720960v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f82bbeorg.highwire.dtl.DTLVardef@ffd6d4org.highwire.dtl.DTLVardef@711481org.highwire.dtl.DTLVardef@1f94d7d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

pharmacology and toxicology↗

bioRxiv2023

EFEMP1 contributes to light-dependent ocular growth in zebrafish

Myopia (short-sightedness) is the most common ocular disorder. It generally develops after over-exposure to aberrant visual environments, disrupting emmetropization mechanisms that should match eye growth with optical power. A pre-screening of strongly associated myopia-risk genes identified through human genome-wide association studies implicates efemp1 in myopia development, but how this gene impacts ocular growth remains unclear. Here, we modify efemp1 expression specifically in the retina of zebrafish. We found that under normal lighting, efemp1 mutants developed axial myopia, enlarged eyes, reduced spatial vision and altered retinal function. However, under myopia-inducing dark-rearing, compared to control fish, mutants remained emmetropic and showed changes in retinal function. Efemp1 modification changed the expression of efemp1, egr1, tgfb1a, vegfab and rbp3 genes in the eye, and changes the inner retinal distributions of myopia-associated EFEMP1, TIMP2 and MMP2 proteins. Efemp1 modification also impacted dark-rearing-induced responses of vegfab and wnt2b genes and above-mentioned myopia-associated proteins. Together, we provided robust evidence that light-dependent ocular growth is regulated by efemp1. SummaryThis study shows that retina-specific modification of efemp1 expression in zebrafish results in myopic eye, and impacts responses to myopia-inducing dark-rearing in eye growth, retinal function, and myopia-associated molecular expression and distribution, implicating light-dependent regulation of efemp1 in ocular development.

neuroscience↗

bioRxiv2023

Establishment and comprehensive characterization of a novel dark-reared zebrafish model for myopia studies

PurposeMyopia is predicted to impact approximately 5 billion people by 2050, necessitating mechanistic understanding of its development. Myopia results from dysregulated genetic mechanisms of emmetropization, caused by over-exposure to aberrant visual environments; however, these genetic mechanisms remain unclear. Recent human genome-wide association studies have identified a range of novel myopia-risk genes. To facilitate large-scale in vivo mechanistic examination of gene-environment interactions, this study aims to establish a myopia model platform that allows efficient environmental and genetic manipulations. MethodsWe established an environmental zebrafish myopia model by dark-rearing. Ocular biometrics including relative ocular refraction were quantified using optical coherence tomography images. Spatial vision was assessed using optomotor response (OMR). Retinal function was analyzed via electroretinography (ERG). Myopia-associated molecular contents or distributions were examined using RT-qPCR or immunohistochemistry. ResultsOur model produces robust phenotypic changes, showing myopia after 2 weeks of dark-rearing, which were recoverable within 2 weeks after returning animals to normal lighting. 2-week dark-reared zebrafish have reduced spatial-frequency tuning function. ERG showed reduced photoreceptor and bipolar cell function (a- and b-waves) after only 2 days of dark-rearing, which worsened after 2 weeks of dark-rearing. We also found dark-rearing-induced changes to expression of myopia-risk genes, including egr1, vegfaa, vegfab, rbp3, gjd2a and gjd2b, inner retinal distribution of EFEMP1, TIMP2 and MMP2, as well as transiently reduced PSD95 density in the inner plexiform layer. ConclusionsCoupled with the gene editing tools available for zebrafish, our environmental myopia model provides an excellent platform for large-scale investigation of gene-environment interactions in myopia development.

neuroscience↗

bioRxiv2023

Her6 and Prox1 are novel regulators of photoreceptor regeneration in the zebrafish retina

Damage to light-sensing photoreceptors (PRs) occurs in highly prevalent retinal diseases. As humans cannot regenerate new PRs, these diseases often lead to irreversible blindness. Intriguingly, animals, such as the zebrafish, have the ability to regenerate PRs efficiently and restore functional vision. Upon injury, mature Muller glia (MG) undergo reprogramming to adopt a stem cell-like state. This process is similar to cellular dedifferentiation, and results in the generation of progenitor cells, which, in turn, proliferate and differentiate to replace lost retinal neurons. In this study, we tested whether factors involved in dedifferentiation of Drosophila CNS are implicated in the regenerative response in the zebrafish retina. We found that hairy-related 6 (her6) negatively regulates of PR production by regulating the rate of cell divisions in the MG-derived progenitors. prospero homeobox 1 (prox1) is expressed in differentiated PRs, and likely promotes PR differentiation through phase separation. Interestingly, upon Her6 downregulation, Prox1 is precociously upregulated in the PRs, to promote PR differentiation; conversely, loss of Prox1 also induces a downregulation of Her6. Together, we identified two novel candidates of PR regeneration that cross regulate each other, and may be exploited to promote human retinal regeneration and vision recovery.

genetics↗

bioRxiv2023

Heterogeneity in quiescent Müller glia in the uninjured zebrafish retina drive differential responses following photoreceptor ablation

Loss of neurons in the neural retina is a leading cause of vision loss. While humans do not possess the capacity for retinal regeneration, zebrafish can achieve this through activation of resident Muller glia. Remarkably, despite the presence of Muller glia in humans and other mammalian vertebrates, these cells lack an intrinsic ability to contribute to regeneration. Upon activation, zebrafish Muller glia can adopt a stem cell-like state, undergo proliferation and generate new neurons. However, the underlying molecular mechanisms of this activation subsequent retinal regeneration remains unclear. To address this, we performed single-cell RNA sequencing (scRNA-seq) and report remarkable heterogeneity in gene expression within quiescent Muller glia across distinct dorsal, central and ventral retina pools of such cells. Next, we utilised a genetically driven, chemically inducible nitroreductase approach to study Muller glia activation following selective ablation of three distinct photoreceptor subtypes: long wavelength sensitive cones, short wavelength sensitive cones, and rods. There, our data revealed that a region-specific bias in activation of Muller glia exists in the zebrafish retina, and this is independent of the distribution of the ablated cell type across retinal regions. Notably, gene ontology analysis revealed that injury-responsive dorsal and central Muller glia express genes related to dorsal/ventral pattern formation, growth factor activity, and regulation of developmental process. Through scRNA-seq analysis, we identify a shared genetic program underlying initial Muller glia activation and cell cycle entry, followed by differences that drive the fate of regenerating neurons. We observed an initial expression of AP-1 and injury-responsive transcription factors, followed by genes involved in Notch signalling, ribosome biogenesis and gliogenesis, and finally expression of cell cycle, chromatin remodeling and microtubule-associated genes. Taken together, our findings document the regional specificity of gene expression within quiescent Muller glia and demonstrate unique Muller glia activation and regeneration features following neural ablation. These findings will improve our understanding of the molecular pathways relevant to neural regeneration in the retina.

neuroscience↗

bioRxiv2022

Cell cycle and temporal transcription factors regulate proliferation and neuronal diversity of dedifferentiation-derived neural stem cells

Dedifferentiation is the reversion of differentiated cells to a stem cell like fate, whereby, the gene expression program of mature cells is altered and genes associated with multipotency are expressed. Appropriate terminal differentiation of NSCs is essential for restricting the overall number of neurons produced; in addition, faithful production of neuronal subtypes that populate the brain is important for NSC function. Both characteristics of NSCs are specified through temporal patterning of the NSCs driven by the successive expression of temporal transcription factors (tTFs). In this study, we found that ectopic NSCs induced via bHLH transcription factor Deadpan (Dpn) expression fail to undergo timely expression of temporal transcription factors (tTFs), where they express mid-tTF, Sloppy-paired 1 (Slp-1) and fail to express late-tTF Tailless (Tll); consequently generating an excess of Twin of eyeless (Toy) positive neurons at the expense of Reversed polarity (Repo) positive glial cells. In addition to disrupted production of neuronal/glial progeny, Dpn overexpression also resulted in stalled progression through the cell cycle, and a failure to undergo timely terminal differentiation. Mechanistically, DamID studies demonstrated that Dpn directly binds to both Dichaete (D), a Sox-box transcription factor known to repress Slp-1, as well as a number of cell cycle genes. Promoting cell cycle progression or overexpression of D were able to re-trigger the progression of the temporal series in dedifferentiated NBs, restoring both neuronal diversity and timely NB terminal differentiation.

developmental biology↗

bioRxiv2021

A tripartite structure, the complex nuclear receptor element (cNRE), is a cis-regulatory module of viral origin required for atrial chamber preferential gene expression

Optimal cardiac function requires appropriate contractile proteins in each heart chamber. Atria require slow myosins to act as variable reservoirs, while ventricles demand fast myosin for swift pumping functions. Hence, myosin is under chamber-biased cis-regulatory control to achieve this functional distribution. Failure in proper regulation of myosin genes can lead to severe congenital heart dysfunction. The precise regulatory input leading to cardiac chamber-biased expression remains uncharted. To address this, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives specific gene expression to the atria to uncover the regulatory information leading to chamber expression and understand their evolutionary origins. We show that SMyHC III gene states are autonomously orchestrated by a complex nuclear receptor cis-regulatory element (cNRE), a 32- bp sequence with hexanucleotide binding repeats. Using in vivo transgenic assays in zebrafish and mouse models, we demonstrate that preferential atrial expression is achieved by the combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Through comparative genomics, we provide evidence that the cNRE emerged from an endogenous viral element, most likely through infection of an ancestral host germline. Our study reveals an evolutionary pathway to cardiac chamber-specific expression.

developmental biology↗

bioRxiv2020

Altered visual function in a larval zebrafish knockout of neurodevelopmental risk gene pdzk1

The human PDZK1 gene is located in a genomic susceptibility region for neurodevelopmental disorders. A genome-wide association study (GWAS) identified links between PDZK1 polymorphisms and altered visual contrast sensitivity, an endophenotype for schizophrenia and autism spectrum disorder. The PDZK1 protein is implicated in neurological functioning, interacting with synaptic molecules including post-synaptic density 95 (PSD-95), N-methyl-D-aspartate receptors (NMDAR), corticotropin-releasing factor receptor 1 (CRFR1) and serotonin 2A receptors. To elucidate the role of PDZK1, we generated pdzk1-knockout (pdzk1-KO) zebrafish using CRISPR/Cas-9 genome editing. Visual function of 7-day-old fish was assessed at behavioural and functional levels using the optomotor response (OMR) and scotopic electroretinogram (ERG). We also quantified retinal morphology and densities of PSD-95, NMDAR1, CRFR1 and serotonin in the synaptic inner plexiform layer at 7 days, 4 weeks and 8 weeks of age. Relative to wild-type, pdzk1-KO larvae showed spatial-frequency tuning functions with increased amplitude (likely due to abnormal gain control) and reduced ERG b-waves (suggestive of inner retinal dysfunction). However, these functional differences were not associated with gross synaptic or morphological retinal phenotypes. The findings corroborate a role for pdzk1 in visual function, and our model system provides a platform for investigating other genes associated with abnormal visual behaviour.

neuroscience↗