Transgenic Reporter Lines Research Articles

'Iterative Bleaching Extends Multiplexity' facilitates simultaneous identification of all major retinal cell types.

To understand the multicellular composition of tissues, and how it is altered during development, ageing and/or disease, we must visualise the complete cellular landscape. Currently, this is hindered by our limited ability to combine multiple cellular markers. To overcome this, we adapted a highly multiplexed immunofluorescence (IF) technique called 'Iterative Bleaching Extends Multiplexity' (IBEX) to the zebrafish retina. We optimised fluorescent antibody micro-conjugation to perform sequential rounds of labelling on a single tissue to simultaneously visualise all major retinal cell types with 11 cell-specific antibodies. We further adapted IBEX to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and whole-mount immunofluorescence (WMIF). We applied IBEX at multiple stages to study the spatial and temporal relationships between glia and neurons during retinal development. Finally, we demonstrate the utility of IBEX across species by testing it on the turquoise killifish (Nothobranchius furzeri) and African clawed frog (Xenopus laevis) to glean large amounts of information from precious tissues. These techniques will revolutionise our ability to visualise multiple cell types in any organism where antibodies are readily available.

Conserved glucokinase regulation in zebrafish confirms therapeutic utility for pharmacologic modulation in diabetes

Glucokinase (GCK) is an essential enzyme for blood glucose homeostasis. Because of its importance in glucose metabolism, GCK is considered an attractive target for the development of antidiabetic drugs. However, a viable therapeutic agent has still to emerge, prompting efforts to improve understanding of the complex regulation and biological effects of GCK. Using the vertebrate organism zebrafish, an attractive model to study metabolic diseases and pharmacological responses, we dissected the complexities of gck regulation and unraveled effects of Gck modulation. We found that while gck expression in zebrafish islet cells is constitutive, gck expression in the liver is regulated by nutritional status, confirming similarity to the mammalian system. A combination of transgenic gck reporter lines and our diabetes model, the pdx1 mutant, allowed monitoring of gck expression under pathological conditions, revealing reduced gck expression and activity in the liver, which was unresponsive to nutrient stimulation, and decreased expression in the islet due to the reduced number of β-cells. Gck activation substantially ameliorated hyperglycemia in pdx1 mutants, without inducing oxidative stress responses in liver or islet. In-depth characterization of Gck activity and regulation at the cellular level in a whole-organism diabetes model clarifies its applicability as a drug target for therapies.

The Wnt1-Cre2 transgene causes aberrant recombination in non-neural crest cell types.

The Wnt1-Cre2 driver, designed to address the effect of Wnt1 overactivation in the ventral neural tube in the original Wnt1-Cre line, was recently shown to have ectopic expression in the male germline. When crossed with a reporter mouse, we observed fluorescent protein expression in non-neural-crest cell types in the gut. Here, we characterize the pattern of Cre-mediated recombination in the Wnt1-Cre2 driver using three transgenic reporter lines. We find aberrant reporter activation in the gut endoderm in embryonic and postnatal timepoints, starting as early as E8.5. This pattern of recombination was independent of the age, sex, and type of reporter line used, with the Wnt1-Cre2 allele inherited from either sires or dams resulting in ectopic fluorescence in the intestinal epithelium. We also detect reporter activity in the ventral neural tube. However, expression in the neural crest and its derivatives remained consistent with previous studies. We further quantify differences in the non-specific recombination observed across reporter lines using flow cytometry. Interestingly, the penetrance of reporter activation between reporter lines was different, with R26R mTmG showing less ectopic activation than the R26R tdTom and R26R eYFP lines. Finally, we propose a potential mechanism whereby genes surrounding the Wnt1-Cre2 insertion site on mouse chromosome 2 contribute to its Wnt1-independent activation in the endoderm. Taken together, our results suggest that users should exercise caution when using the Wnt1-Cre2 driver line for neural crest studies in the mouse.

Identification of a Specific Granular Marker of Zebrafish Eosinophils Enables Development of New Tools for Their Study.

Eosinophils control many aspects of the vertebrate innate immune response. They contribute to homeostasis, inflammatory conditions and defense against pathogens. With the varied functions of eosinophils, they have been found to play both protective and pathogenic roles in many diseases. The zebrafish (Danio rerio) has emerged as a useful model organism for human diseases but tools to study eosinophils in this model are severely limited. Here, we characterize a new and highly specific marker gene, embp, for eosinophils in zebrafish and report a new transgenic reporter line using this gene to visualize eosinophils invivo. In addition, we created an Embp-specific polyclonal Ab that allows the identification of eosinophils ex vivo. These new tools expand the approaches for studying eosinophils in the zebrafish model. Using these reagents, we have been able to identify Embp as a constituent of eosinophil granules in zebrafish. These advances will allow for the investigation of eosinophil biology in the zebrafish model organism, allowing researchers to identify the contribution of eosinophils to the many diseases that are modeled within zebrafish and also shed light on the evolution of eosinophils within vertebrates.

Exposure effects of synthetic glucocorticoid drugs on skeletal developmental and immune cell function in zebrafish

Synthetic glucocorticoids (GCs) are used to treat a wide range of human health conditions and as such are frequently detected in the aquatic environment. This, together with the highly conserved nature of the glucocorticoid system across vertebrates means that the potential for biological effects of GCs in fish is relatively high. Here, we found that exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of 4 of the most widely used synthetic GCs (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone), from 0 to 4 days post fertilisation (dpf), resulted in no effects on embryo-larval development or bone and cartilage formation. However, after exposure to equivalents of human therapeutic plasma levels, developmental abnormalities were observed that included pericardial oedema, blood pooling and alterations in jaw cartilage. Furthermore, using a double transgenic zebrafish osteoblast and chondrocyte reporter line, exposure up to 10 dpf resulted in alterations to lower jaw cartilage and bone development for all compounds at, and above, human therapeutic plasma concentrations. In the case of beclomethasone dipropionate, a reduction in lower jaw intercranial distance was observed at the environmentally relevant concentration of 0.1 μg/L. Using further transgenic reporter lines with fluorescently tagged neutrophils and macrophages, we also show exposure of embryo-larvae (0–4 dpf) to the GCs tested resulted in altered immune cell migration, but only at relatively high exposure concentrations. Collectively, our findings show GC exposure impacts embryo-larval zebrafish development, immune function, and skeletal formation, but predominantly at concentrations greater than those currently reported for the aquatic environment. Despite this, however, it is suggested that studies with longer exposure times, and to mixtures of multiple GCs (many GCs act via the same mechanism of action) are warranted before we can confidently assert that these commonly detected contaminants do not pose a risk to fish in the wild.

A population of Vasa2 and Piwi1 expressing cells generates germ cells and neurons in a sea anemone

Germline segregation, essential for protecting germ cells against mutations, occurs during early embryogenesis in vertebrates, insects and nematodes. Highly regenerative animals (e.g., cnidarians), however, retain stem cells with both germinal and somatic potentials throughout adulthood, but their biology and evolution remain poorly understood. Among cnidarians (e.g., sea anemones, jellyfish), stem cells are only known in few hydrozoans (e.g., Hydra). Here, we identify and characterize a rare, multipotent population of stem and/or progenitor cells expressing the conserved germline and multipotency proteins Vasa2 and Piwi1 in the sea anemone Nematostella vectensis. Using piwi1 and vasa2 transgenic reporter lines, we reveal that the Vasa2+/Piwi1+ cell population generates not only gametes, but also a diversity of proliferative somatic cells, including neural progenitors, in juveniles and adults. Our work has uncovered a multipotent population of Vasa2+/Piwi1+ stem/progenitor cells that forms the cellular basis to understand body plasticity and regenerative capacities in sea anemones and corals.

Identification of the Novel Small Compound Stress Response Regulators 1 and 2 That Affect Plant Abiotic Stress Signaling.

Abiotic stresses, such as drought, salinity, and extreme temperatures, limit plant growth and development, reducing crop yields. Therefore, a more comprehensive understanding of the signaling mechanisms and responses of plants to changing environmental conditions is crucial for improving sustainable agricultural productivity. Chemical screening was conducted to find novel small compounds that act as regulators of the abiotic stress signaling pathway using the ABA-inducible transgenic reporter line. Small molecules called stress response regulators (SRRs) were isolated by screening a synthetic library composed of 14,400 small compounds, affecting phenotypes such as seed germination, root growth, and gene expression in response to multiple abiotic stresses. Seeds pretreated with SRR compounds positively affected the germination rate and radicle emergence of Arabidopsis and tomato plants under abiotic stress conditions. The SRR-priming treatment enhanced the transcriptional responses of abiotic stress-responsive genes in response to subsequent salt stress. The isolation of the novel molecules SRR1 and SRR2 will provide a tool to elucidate the complex molecular networks underlying the plant stress-tolerant responses.

Smooth muscle Cxcl12 activation is associated with vascular remodeling in flow-induced pulmonary hypertension.

Patients with congenital heart disease (CHD) resulting in significant left-to-right shunting of blood are at risk for the development of pulmonary arterial hypertension (PAH). The underlying mechanism by which pulmonary overcirculation and shear stress lead to vascular remodeling remains unclear. Our study established a new "two-hit" murine model of severe pulmonary hypertension (PH) by combining left pneumonectomy and exposure to hypoxia (LP/Hx). Utilizing transgenic reporter lines, immunofluorescence staining, and advanced microscopy, we conducted cell-lineage tracing experiments for endothelial cells (ECs), smooth muscle cells (SMCs), and pericytes. We identified that SMCs is a primary contributor to distal arteriolar remodeling after LP/Hx. Subsequent qPCR analysis on isolated cells demonstrated that Cxcl12 was upregulated in both ECs and SMCs from LP/Hx animals. Likewise, CXCL12 was overexpressed in the SMC layer of arterioles in patients with acyanotic PAH-CHD. These findings provide novel insights into the contribution of SMCs and Cxcl12 to pulmonary flow-induced vascular remodeling. This newly established murine model of PH will serve as a new tool for research and targeted therapeutics for patients with PAH.

Doublecortin-like kinase is required for cnidocyte development in Nematostella vectensis

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.

Innervation of adipocytes is limited in mouse perivascular adipose tissue.

Perivascular adipose tissue (PVAT) regulates vascular tone by releasing anticontractile factors. These anticontractile factors are driven by processes downstream of adipocyte stimulation by norepinephrine; however, whether norepinephrine originates from neural innervation or other sources is unknown. The goal of this study was to test the hypothesis that neurons innervating PVAT provide the adrenergic drive to stimulate adipocytes in aortic and mesenteric perivascular adipose tissue (aPVAT and mPVAT), and white adipose tissue (WAT). Healthy male and female mice (8-13 wk) were used in all experiments. Expression of genes associated with synaptic transmission were quantified by qPCR and adipocyte activity in response to neurotransmitters and neuron depolarization was assessed in AdipoqCre+;GCaMP5g-tdTf/WT mice. Immunostaining, tissue clearing, and transgenic reporter lines were used to assess anatomical relationships between nerves and adipocytes. Although synaptic transmission component genes are expressed in adipose tissues (aPVAT, mPVAT, and WAT), strong nerve stimulation with electrical field stimulation does not significantly trigger calcium responses in adipocytes. However, norepinephrine consistently elicits strong calcium responses in adipocytes from all adipose tissues studied. Bethanechol induces minimal adipocyte responses. Imaging neural innervation using various techniques reveals that nerve fibers primarily run alongside blood vessels and rarely branch into the adipose tissue. Although nerve fibers are associated with blood vessels in adipose tissue, they demonstrate limited anatomical and functional interactions with adjacent adipocytes, challenging the concept of classical innervation. These findings dispute the significant involvement of neural input in regulating PVAT adipocyte function and emphasize alternative mechanisms governing adrenergic-driven anticontractile functions of PVAT.NEW & NOTEWORTHY This study challenges prevailing views on neural innervation in perivascular adipose tissue (PVAT) and its role in adrenergic-driven anticontractile effects on vasculature. Contrary to existing paradigms, limited anatomical and functional connections were found between PVAT nerve fibers and adipocytes, underscoring the importance of exploring alternative mechanistic pathways. Understanding the mechanisms involved in PVAT's anticontractile effects is critical for developing potential therapeutic interventions against dysregulated vascular tone, hypertension, and cardiovascular disease.

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo.

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

Macrophages modulate fibrosis during newt lens regeneration

BackgroundPrevious studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated.MethodsHere we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery.ResultsMacrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process.ConclusionsTogether, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales.

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new therapeutics that could ease the burden on healthcare systems. As these diseases are complex, it is difficult and expensive to accurately model bone pathophysiology in a lab setting. The challenge for the field is to establish a cost-effective, biologically relevant platform for modeling bone disease that can be used to test potential therapeutic compounds. Such a platform should ideally allow dynamic visualization of cell behaviors of bone-building osteoblasts and bone-degrading osteoclasts acting in their mineralized matrix environment. Zebrafish are increasingly used as models due to the availability of genetic tools, including transgenic reporter lines, and the fact that some skeletal tissues (including the scales) remain translucent to adulthood, allowing dynamic imaging options. Since zebrafish scales have both osteoblasts and osteoclasts and are highly abundant, they provide an easily accessible and abundantly available resource of independent bone units. Moreover, once removed, adult zebrafish scales fully regenerate, therefore offering a way to study the spatiotemporal growth of mineralized tissue in vivo. Here, we detail protocols for harvesting and tracking the regeneration of the scales. Lastly, a protocol for stable culture of scales ex vivo for a week and following the healing response after controlled damage to the mineralized matrix of the scale over time is also presented.

Tissue-specific silencing of integrated transgenes achieved through endogenous RNA interference in Caenorhabditis elegans

ABSTRACT Transgene silencing is a common phenomenon observed in Caenorhabditis elegans, particularly in the germline, but the precise mechanisms underlying this process remain elusive. Through an analysis of the transcription factors profile of C. elegans, we discovered that the expression of several transgenic reporter lines exhibited tissue-specific silencing, specifically in the intestine of C. elegans. Notably, this silencing could be reversed in mutants defective in endogenous RNA interference (RNAi). Further investigation using knock-in strains revealed that these intestine-silent genes were indeed expressed in vivo, indicating that the organism itself regulates the intestine-specific silencing. This tissue-specific silencing appears to be mediated through the endo-RNAi pathway, with the main factors of this pathway, mut-2 and mut-16, are significantly enriched in the intestine. Additionally, histone modification factors, such as met-2, are involved in this silencing mechanism. Given the crucial role of the intestine in reproduction alongside the germline, the transgene silencing observed in the intestine reflects the self-protective mechanisms employed by the organisms. In summary, our study proposed that compared to other tissues, the transgenic silencing of intestine is specifically regulated by the endo-RNAi pathway.

What can we learn about fish neutrophil and macrophage response to immune challenge from studies in zebrafish

Fish rely, to a high degree, on the innate immune system to protect them against the constant exposure to potential pathogenic invasion from the surrounding water during homeostasis and injury. Zebrafish larvae have emerged as an outstanding model organism for immunity. The cellular component of zebrafish innate immunity is similar to the mammalian innate immune system and has a high degree of sophistication due to the needs of living in an aquatic environment from early embryonic stages of life. Innate immune cells (leukocytes), including neutrophils and macrophages, have major roles in protecting zebrafish against pathogens, as well as being essential for proper wound healing and regeneration. Zebrafish larvae are visually transparent, with unprecedented in vivo microscopy opportunities that, in combination with transgenic immune reporter lines, have permitted visualisation of the functions of these cells when zebrafish are exposed to bacterial, viral and parasitic infections, as well as during injury and healing. Recent findings indicate that leukocytes are even more complex than previously anticipated and are essential for inflammation, infection control, and subsequent wound healing and regeneration.

Contrasting cytosolic glutathione redox dynamics under abiotic and biotic stress in barley as revealed by the biosensor Grx1-roGFP2.

Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness.

Biallelic loss-of-function variants in CACHD1 cause a novel neurodevelopmental syndrome with facial dysmorphism and multisystem congenital abnormalities

PurposeWe established the genetic etiology of a syndromic neurodevelopmental condition characterized by variable cognitive impairment, recognizable facial dysmorphism, and a constellation of extra-neurological manifestations. MethodsWe performed phenotypic characterization of 6 participants from 4 unrelated families presenting with a neurodevelopmental syndrome and used exome sequencing to investigate the underlying genetic cause. To probe relevance to the neurodevelopmental phenotype and craniofacial dysmorphism, we established two- and three-dimensional human stem cell-derived neural models and generated a stable cachd1 zebrafish mutant on a transgenic cartilage reporter line. ResultsAffected individuals showed mild cognitive impairment, dysmorphism featuring oculo-auriculo abnormalities, and developmental defects involving genitourinary and digestive tracts. Exome sequencing revealed biallelic putative loss-of-function variants in CACHD1 segregating with disease in all pedigrees. RNA sequencing in CACHD1-depleted neural progenitors revealed abnormal expression of genes with key roles in Wnt signaling, neurodevelopment, and organ morphogenesis. CACHD1 depletion in neural progenitors resulted in reduced percentages of post-mitotic neurons and enlargement of 3D neurospheres. Homozygous cachd1 mutant larvae showed mandibular patterning defects mimicking human facial dysmorphism. ConclusionOur findings support the role of loss-of-function variants in CACHD1 as the cause of a rare neurodevelopmental syndrome with facial dysmorphism and multisystem abnormalities.

Automated, high-throughput quantification of EGFP-expressing neutrophils in zebrafish by machine learning and a highly-parallelized microscope.

Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM™), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM™ we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species.

Populus trichocarpa PtHSFA4a Enhances Heat Tolerance by Regulating Expression of APX1 and HSPs

Heat stress can severely inhibit plant growth and reproduction, resulting in heavy financial and crop yield losses. Heat shock transcription factors (HSFs) play an important role in regulating plant responses to abiotic stress. However, compared with the in-depth study of HSF gene function in herbaceous species, reports on the regulatory mechanism of the response of HSFs to heat stress in trees are scarce. Here, we demonstrated that PtHSFA4a is induced by high temperatures in Populus trichocarpa leaves. Intense GUS activity was detected in the leaves of PtHSFA4a promoter-GUS reporter transgenic line under heat stress. Ectopic expression of PtHSFA4a in Arabidopsis thaliana enhanced heat stress tolerance, which reduced malondialdehyde and reactive oxygen species levels. RT-qPCR revealed that the expression of key heat stress-related genes (that is, AtMBF1c, AtZAT12, AtAPX1, AtHSA32, and AtHSPs) was upregulated in PtHSFA4a transgenic plants. Additionally, PtHSFA4a directly bind to the promoters of AtAPX1 and AtHSPs under heat stress to enhance heat tolerance by upregulating the antioxidant defense system and maintaining protein folding homeostasis in A. thaliana leaves. These findings provide novel insights into the molecular mechanisms underlying PtHSFA4a-mediated regulation of plant responses to heat stress.

Plasticity of cone photoreceptors in adult zebrafish revealed by thyroid hormone exposure

Vertebrate color vision is predominantly mediated by the presence of multiple cone photoreceptor subtypes that are each maximally sensitive to different wavelengths of light. Thyroid hormone (TH) has been shown to be essential in the spatiotemporal patterning of cone subtypes in many species, including cone subtypes that express opsins that are encoded by tandemly replicated genes. TH has been shown to differentially regulate the tandemly replicated lws opsin genes in zebrafish, and exogenous treatments alter the expression levels of these genes in larvae and juveniles. In this study, we sought to determine whether gene expression in cone photoreceptors remains plastic to TH treatment in adults. We used a transgenic lws reporter line, multiplexed fluorescence hybridization chain reaction in situ hybridization, and qPCR to examine the extent to which cone gene expression can be altered by TH in adults. Our studies revealed that opsin gene expression, and the expression of other photoreceptor genes, remains plastic to TH treatment in adult zebrafish. In addition to retinal plasticity, exogenous TH treatment alters skin pigmentation patterns in adult zebrafish after 5 days. Taken together, our results show a remarkable level of TH-sensitive plasticity in the adult zebrafish.