Whole-mount In Situ Hybridization Research Articles

Molecular characterization, spatiotemporal expression, and background adaptation regulation of tyrosinase in loach (Misgurnus anguillicaudatus)

AbstractThe Poyang Lake region is home to large‐blackspot loaches (LBL), small‐blackspot loaches (SBL), and non‐blackspot loaches (NBL), Misgurnus anguillicaudatus. To investigate the impact of tyrosinase on spot development, the complementary DNAs (cDNA) of tyrosinase in M. anguillicaudatus (designated as Matyr) were cloned using the rapid amplification of cDNA ends (RACE)‐PCR method. The full‐length cDNA for Matyr was 2020 bp, and the open‐reading frame comprised 1617 bp, encoding a predicted protein with 538 amino acids. Phylogenetic studies revealed that MaTyr was first grouped with Tyr of Triplophysa tibetana and Leptobotia taeniops, and then Tyr of other cyprinid fish. The quantitative reverse‐transcription‐PCR results show that Matyr was highly expressed in the muscle, caudal fin, and dorsal skin. The Matyr gene's messenger RNA expression pattern steadily increased from the fertilized ovum period to the somitogenesis period, and from the muscle effect stage to 6 days after fertilization, it considerably increased (p < 0.01). The Matyr hybridization signals with similar location could be found in all developmental stages of three kinds of loaches using whole‐mount in situ hybridization (WISH) technology and were the strongest during the organ development period and melanin formation period. Dot hybridization signals in LBLs rapidly spread to the back of the body beginning at the period when the eyes first formed melanin, and their dimensions were larger than those of NBLs during the same time period. The body color of loaches could change reversibly with black/white background adaptation. The α‐msh, mitfa, and tyr are mainly expressed in loaches adapted with a black background. Tyr gene could be involved in the development of blackspots and body color polymorphism, and contribute to organ development in the loach.

Nlrc3 Signaling Is Indispensable for Hematopoietic Stem Cell Emergence Via Notch Signaling in Vertebrates

Hematopoietic stem and progenitor cells (HSPCs) generate all the lineages of blood cells throughout the lifespan of vertebrates. The emergence of HSPCs occurs through the hemogenic endothelial (HE) to hematopoietic transition (EHT) process, which is finely tuned by a variety of signaling pathways. Previous studies have emerged the essential roles of pattern-recognition receptors such as Toll-like receptors and RIG-I-like receptors in EHT. However, whether the nucleotide-binding domain leucine-rich repeat (NLR) containing family members participate in vertebrate embryonic hematopoiesis remains unclear. To explore the potential pathways involved in hematopoietic ontogeny and differentiation in this stage, we screened the dynamic expression of NLR family genes and other genes related to inflammation in the published scRNA-seq data in vertebrates' embryos including zebrafish, mouse and human during HSPC development. And to verify the expression of NLR family genes in vitro by using HSC-like cells differentiation system of mouse and human embryonic stem cells (ESCs), these results suggest that NLRC3 was highly expressed during embryonic HSPC development in vivo and vitro and might play an important role in HSPC emergence in vertebrates. In order to validate this hypothesis, we synthesized probes from the full-length mRNA of nlrc3 and utilized them to observe the in-situ expression of nlrc3 in zebrafish at different developmental stages. The WISH experiment demonstrated that nlrc3 was expressed from the 1-cell stage and importantly, showed specific expression in the AGM region at the 24-28hpf and CHT region at 72hpf, which coincides with the onset and generation of HSPC generation. Then morpholino were utilized to knockdown nlrc3 in transgenic embryos Tg( runx1:EGFP/ kdrl:mCherry), Tg( cmyb:EGFP/ kdrl:mCherry) and Tg( CD41:GFP), and mutants generated by CRISPR-Cas9 were used to perform whole-mount in situ hybridization (WISH) and qPCR to detect specific markers such as runx1, cmyb(HSPCs), rag1(lymphocytes), gata1a(erythrocytes) and l-plastin(myelocytes), etc. The results showed that nlrc3 was required for HSPCs emergence, expansion and differentiation. In primitive hematopoietic wave, nlrc3 signaling may not impact primitive erythropoiesis but required for primitive neutrophils and macrophages. Mechanistically, we sorted EGFP + cells in Tg ( fli1a:EGFP) zebrafish embryos at 28 hpf to perform RNA-Seq, which was the stage where EHT and these EGFP + cells contained hemogenic endothelial cells. After bioinformatics analysis of sequencing data, we found that the effect of nlrc3 in zebrafish hematopoiesis is not based on affecting the development of arterial vessels or causing apoptosis. By using targeted inhibitor such as DAPT and JSH23, conditional rescue model like Tg ( hsp70:GAL4/ UAS:NICD), overexpression expression experiment in Tg ( tp1:EGFP/ kdrl:mCherry) and nlrc3 -/- mutants, etc, through Imaging systems and qPCR we found that NF-kB- nlrc3-Notch- hey1 axis triggers HSPCs emergence and differentiation in zebrafish. To further extend our findings in mammals, we utilized Nlrc3 knockout mice to explore whether Nlrc3 signaling was evolutionarily conserved in HSPCs emergence. Hematogenic endothelial cells (CD31 + CD41 - CD45 - TER119 -) from the embryonic AGM tissues at E10.5 were sorted and cultured in OP9 for 4 days and flow cytometry was performed to assess that c-Kit +CD45 + hematopoietic cells and CD31 +Sca-1 +CD201 + hematopoietic stem cells were notably decreased in these Nlrc3-/- embryos. Colony-forming unit-cell (CFU-C) assay and BM transplantation experiments were used to observe that fetal liver HSCs from E14.5 Nlrc3-/- embryos was decrease in the number and impaired in function in vitro and in vivo. Finally qPCR using mRNA from FL-derived LSK ( Lin-c-Kit+Sca-1+) cells further demonstrated that the NF-kB- Nlrc3-Notch- Hey1 axis orchestrated development of embryonic HSCs was highly conserved in vertebrates. Overall, by summarizing the results of the physiological and developmental roles in vivo and in vitro, we uncover an indispensable role of Nlrc3 signaling in vertebrates' HSPCs emergence, our findings provide new insights into inflammation-related hematopoietic ontogeny and Nlrc3 signal is of major importance since this investigation might be taken into consideration when recapitulation definitive hematopoiesis in vitro.

Picalm/MLLT10 Expression in Zebrafish Is Associated with an Increase of Hematopoietic Stem Cells and Leads to Leukemia

The PICALM/ MLLT10 (C/A)fusion gene is the result of the (10;11)(p13;q14) translocation, and is found in about 1% of all AML patients, 30% of T-cell ALL with a TCR γ/∂ rearrangement and in some patients with lymphoma. AML patients with a t(10;11)(p13;q14) have poor long-term survival. Mouse models of t(10;11)(p13;q14) show that C/A and the C/A minimal fusion (CA/MF) are strong oncogenic drivers, and that C/A must be expressed in the hematopoietic stem cells (HSCs) to cause leukemia. Patient samples and mouse models of C/A leukemia show that these leukemias harbor additional, cooperating mutations. Zebrafish (ZF) have become an attractive animal model for studying hematological disorders. ZF can be easily genetically modified and kept in large numbers. Although T cell ALL models have been successful in ZF, AML models have not been as successful. Most ZF AML models fail to recapitulate the phenotype, especially transplantability. Here we report a C/A transgenic zebrafish AML model (R:CA/MF). We used the murine Runx1 promoter to direct CA/MF expression to zebrafish HSCs using Tol2-mediated transgenesis. We detected the expression of the CA/MF transcript using RT-PCR in 3 dpf (days post fertilisation) heart marker positive transgenic F0 embryos and the expression of FLAG-tagged CA/MF protein with whole mount in situ immuno histochemistry in stable transgenic R:CA/MF embryos at 3 dpf. We performed whole mount in situ hybridisation (WMISH) and qPCR for hematopoietic genes to examine the impact of CA/MF on zebrafish hematopoiesis. Based on qPCR analysis, we observed that the expression of pu1 (p value <0.0120) and gata1 (p value <0.0004) was significantly decreased in 3 dpf R:CA/MF embryos compared to WT, whereas the expression of mpx at 2 dpf (p value <0.0132) and 3 dpf (p value <0.0378) was significantly increased. Our WMISH analysis showed that the expression of lyz was significantly decreased in R:CA/MF compared to WT embryos at 2 dpf. To investigate the effect of CA/MF expression on HSCs, we injected the R:CA/MF construct into fertilised oocytes of R:GFP;kdrl:RFP fish (R:GFP marks HSCs and Kdrl:RFP marks blood vessels). The results from intravital confocal microscopy showed that the 3 dpf R:GFP larvae that had been injected with the R:CA/MF vector had about a 2.5 fold increase (p value <0.0001) of the GFP expressing cells (HSCs) in both the caudal hematopoietic tissue and circulation compared to control (R:Gal4) and un-injected larvae. These results were also confirmed by flow cytometry. CA/MF transgenic zebrafish developed an aggressive hematological malignancy with a latency of about 1 year and 60 to 70% penetrance. 68% of F1 R:CA/MF (15/22), 64% of F2 R:CA/MF (45/70), 62% of F3 R:CA/MF (22/35) and 62% of F4 R:CA/MF (25/40) adult ZF were euthanised or found dead. The median survival for F1 CA/MF transgenic ZF was 65.6 weeks and 69, 78.4, and 79.1 weeks for F2, F3, and F4, respectively. Histology analysis of R:CA/MF leukemic fish showed hypercellularity in kidney, spleen, heart, gills and eyes. Myeloperoxidase (MPO) staining showed that the number of MPO positive cells in kidney marrow was about 50% higher (p value <0.0001) in R:CA/MF compared to WT. Peripheral blood film analysis of R:CA/MF showed the presence of blast-like cells. In addition, R:CA/MF malignancies were associated with an expansion of either the precursor compartment (N=20/31), the myeloid compartment (N=8/31) or the lymphoid compartment (N=3/31) on forward/side scatter flow cytometry analysis. Transplantation assays showed that R:CA/MF leukemias were serially transplantable. The latency to leukemia development was about six weeks in the first, second, and third recipient fish, which was much shorter than the latency for the primary R:CA/MF leukemias. Our preliminary whole-exome sequencing (WES) analysis identified somatically mutated genes in CA/MF zebrafish leukemia with some of these genes having human orthologues that are known to be somatically mutated in human leukemia suggesting that these are cooperating driver mutations. Preliminary RNA-Seq experiments revealed that genes in the KRAS and MTOR pathways had increased expression in the CA/MF ZF leukemia. R:CA/MF leukemia model is a valuable model for understanding the molecular mechanisms of malignant cell transformation of C/A. It allows us to investigate the effects of C/A on HSCs in vivo and to study the fully developed leukemia via serial transplantation, WES, and RNA-Seq.

N-3 Polyunsaturated Fatty Acid (N-3 PUFA) Derived Lipid Mediators Increase Hematopoiesis during Zebrafish Embryogenesis

Polyunsaturated fatty acids (PUFAs), which include n-3 PUFAs and n-6 PUFAs, are essential fatty acids that we can only get from diet. The richest source of PUFAs is fish oil. N-3 PUFAs have been used in the prevention and treatment of coronary artery disease, diabetes, hypertension, arthritis, cancer, and other inflammatory and autoimmune disorders in humans. Lipid mediators derived from n-3 PUFAs are implicated in mediating the resolution of inflammation. However, their effect on hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation and on hematopoiesis has not been studied. Therefore, in this project, we investigated the pro-hematopoietic effects of n-3 PUFA-derived lipid mediators. We exposed wild-type zebrafish embryos to different n-3 PUFA-derived lipid mediators. After fixation, we performed whole mount in situ hybridization (WISH) using antisense RNA probes such as c-myb (hematopoietic stem cell marker), gata1 (erythroid progenitor cell marker), hbbe1 (erythrocyte marker), l-plastin (macrophage and neutrophil cell marker ), mpx (neutrophil cell marker), lyz (neutrophil cell marker), and rag1 (immature lymphocyte marker); reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and fluorescence-activated cell sorting (FACS) to understand the effect of lipid mediator exposure on the proliferation of HSPCs, erythroid progenitor cells, erythrocytes, granulocytic cells, neutrophils, macrophages, and T lymphocytes in different development stages during vertebrate hematopoiesis. In addition, we also used o-dianisidine staining to study red blood cell (RBC) formation and transgenic zebrafish to observe different blood lineages including HSPCs, neutrophils, macrophages, erythroid progenitors, and erythrocytes with fluorescent microscopy. We identified two novel lipid mediators which promote HSPC proliferation, enhance erythroid progenitor cell and erythrocyte production, and increase neutrophil cell number. These two molecules could be applied as novel therapeutics for blood disorders and blood cancers such as anemia and leukemia in humans. These two compounds can be intensely investigated to become candidate supplements to enhance HSPC amplification before HSPC transplantation for leukemia patients.

A Zebrafish Model of Parn-Deficiency Reveals Conserved Mechanisms Underlying Marrow Failure and Opportunities for Therapeutic Amelioration of Hematopoiesis

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome (IBMFS) and a telomere biology disorder that is characterized by physical malformations such as nail dystrophy, oral leukoplakia, reticular hyperpigmentation, multilineage cytopenia and immunodeficiency. Definitive treatment for the hematological complications is hematopoietic stem cell transplantation (HSCT), which is associated with high risk of organ toxicity and engraftment failure in this population. Further, DC patients are prone to developing myelodysplastic syndrome (MDS) and solid tumors. Germline mutations in the poly(A)-specific ribonuclease (PARN) gene are associated with Hoyeraal-Hreidarsson syndrome, the most severe form of DC. PARN encodes a deadenylase enzyme involved in the 3'-end processing of various RNAs including the telomerase RNA component (TERC). The clinical heterogeneity of phenotypes and the paucity of primary human samples necessitate the use of animal models such as zebrafish ( Danio rerio), given their highly conserved genetic program, to elucidate underlying biology. Zebrafish have a single PARN ortholog with 64% sequence identity at the amino acid level. Using CRISPR-Cas9 mutagenesis, we generated a 1.3 kb deletion in the zebrafish parn gene, predicted to result in premature protein truncation. To measure the hematopoietic output of the parn mutants, we performed whole-mount in situ hybridization (WISH) for key hematopoietic lineage markers. At 48 hours post-fertilization (hpf), we found a reduction in lcp1 + total leukocytes, mpx + neutrophils and hbbe3 + mature erythrocytes. Impaired erythroid differentiation was accompanied by an accumulation of gata1 + erythroid progenitors. This finding was further corroborated by o-dianisidine staining, showing reduced hemoglobinized erythrocytes. Thus, erythrocyte and leukocyte differentiation are compromised in our zebrafish parn mutant, phenocopying the multilineage cytopenia observed in DC patients. Flow cytometry of the whole kidney marrow (WKM, human bone marrow equivalent) at 12 months of age revealed an increase in hematopoietic stem and progenitor cells in the parn mutants. Giemsa staining of WKM touchpreps further identified an increase in myeloid-biased progenitors. Next, we investigated the consequences of Parn loss on telomerase activity. Using the telomere repeat amplification protocol, we found reduced telomerase activity in the WKMs of parn mutant zebrafish. Using 3' rapid amplification of cDNA ends, we observed longer ends in the terc amplicons of the parn mutants, reminiscent of defective deadenylation of human TERC RNA in the setting of PARN mutations. Exogenous thymidine supplementation has been shown to support telomere elongation in human cells. Following treatment of zebrafish embryos with thymidine nucleoside, we observed an improvement in erythrocyte numbers in the parn mutants by o-dianisidine staining at 48 hpf. Collectively, our preliminary findings suggest that telomerase-dependent mechanisms may contribute to the erythropenia seen in parn-mutant zebrafish. In summary, we generated a robust zebrafish model carrying a germline parn mutation that phenocopies the molecular and hematologic features seen in DC patients. Our model holds promise as a new in vivo tool for investigating DC-related disease mechanisms and as a platform for preclinical therapeutic screening.

Drug Repurposing in a Zebrafish AML Model

Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy. AML arises from the stepwise accumulation of mutations in hematopoietic stem cells (HSCs). The t(9;11)(p22;q23) translocation is a translocation detected in about 5 to 7% of AML patients, which gives rise to the KMT2A/MLLT3 or MLL/AF9 ( MA9) fusion gene. Zebrafish (ZF) are well-established model organisms for investigating human hematopoiesis. Approximately 70% of human genes have corresponding orthologues in zebrafish, and over 80% of disease-related genes are conserved between the two species. Previously, we established a transgenic MA9 zebrafish AML model (Runx1+23:MA9), in which the AML was serially transplantable. Our investigations revealed that MA9 expression alters hematopoiesis in transgenic embryos before the onset of AML, leading to myeloid expansion. Whole-mount in situ hybridization (WISH) of embryos showed increased expression of granulocyte markers such as lyz 24 and 48 hours after fertilization (hpf). Based on these findings, we developed a Runx1+23:MA9/lyz-EGFP zebrafish leukemia line, in which lyz-expressing myeloid cells are EGFP positive. This model provides a phenotype-based platform to evaluate the therapeutic potential of any drug that can affect the myeloid cell population. We used this ZF model to investigate the therapeutic potential of nine drugs in MA9-driven AML. MA9 transgenic fish (Runx1+23:MA9) were crossed with lyz-EGFP fish to generate a MA9/lyz-EFGP line. In this line, myeloid cells can be visualized with a fluorescent microscope. The expression of lyz in the MA9/lyz-EFGP transgenic embryos was compared to that in the lyz-EGFP line. In our previous study on MA9 transgenic leukemic fish, RNA-seq analysis had revealed higher expression of genes acting in the EGFR, MEK, RAF, KRAS pathways and downregulation of genes in TP53. Based on these results, we selected nine drugs that are known to affect some of these pathways as well as drugs that are used to treat leukemia for screening in our model. Control and MA9/lyz-EFGP ZF 48 hpf embryos were treated with 10 µM of each drug. 24 hrs after drug treatment, the number of lyz expressing cells was analyzed by visualizing the embryos under a fluorescent microscope, and lyz transcript levels were measured by qPCR. Treatment with erlotinib, lapatinib, cytarabine, and gefitinib led to a visible reduction in the number of lyz-expressing cells in the MA9 transgenic embryos but not in the controls (Figure 1). In comparison, treatment with decitabine resulted in a decrease in the number of lyz expressing cells in both the MA9/lyz-EGFP and the control embryos. Treatment with doxorubicin, zoledronic and arsenic trioxide did not have an effect on the number of lyz-expressing population in the transgenic nor in the control embryos. These results were confirmed by qPCR, which showed that expression of lyz was reduced by 33% to 51% in the erlotinib, lapatinib, cytarabine, and gefitinib treated MA9 transgenic embryos (Figure 2). Treatment with these drugs did not significantly change the lyz expression in lyz-EFGP control embryos. Our results provide strong evidence for the successful establishment of a zebrafish model of KMT2A-rearranged AML that can be used for low to medium-throughput drug screening by employing the expression level of an endogenous gene (lyz) as a read-out. This model is a valuable tool to assess the drug's specificity in targeting oncogene-expressing cells and normal hematopoietic cells and, therefore, to identify novel therapeutic agents that can target the cancer cells with fewer side effects on normal hematopoietic cells.

Influence of two anti-tumor drugs, pazopanib, and axitinib, on the development and thyroid-axis of zebrafish (Danio rerio) embryos/larvae.

In recent years, the potential toxicities of different pharmaceuticals toward the thyroid system have received increasing attention. In this study, we aim to evaluate the toxic effects of pazopanib and axitinib, two anti-tumor drugs with widespread clinical use, on thyroid function in the zebrafish model. We measured levels of thyroid-related hormones using the commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. Whole-mount in situ hybridization (WISH) analysis was employed to detect target gene expression changes. Morphology of the thyroid were evaluated by using transgenic Tg (tg: EGFP) fish line under a confocal microscope. The relative mRNA expression of key genes was verified through quantitative real-time polymerase chain reaction (RT‒qPCR). The size and number of the follicles was quantified whereby Hematoxylin-Eosin (H & E) staining under a light microscope. The results revealed that fertilized zebrafish embryos were incubated in pazopanib or axitinib for 96 hours, development and survival were significantly affected, which was accompanied by significant disturbances in thyroid endocrine system (e.g., increased thyroid-stimulating hormone (TSH) content and decreased triiodothyronine (T3) and thyroxine (T4) content, as well as transcription changes of genes associated with the hypothalamus-pituitary-thyroid (HPT) axis. Moreover, based on whole-mount in situ hybridization staining of tg and histopathological examination of zebrafish embryos treated with pazopanib and axitinib, we observed a significantly abnormal development of thyroid follicles in the Tg (tg: EGFP) zebrafish transgenic line. Collectively, these findings indicate that pazopanib and axitinib may have toxic effects on thyroid development and function, at least partially, by influencing the regulation of the HPT axis. Thus, we believe that the potential thyroid toxicities of pazopanib and axitinib in their clinical applications should receive greater attention.

Natural antisense transcription of presenilin in sea urchin reveals a possible role for natural antisense transcription in the general control of gene expression during development

One presenilin gene (PSEN) is expressed in the sea urchin embryo, in the vegetal pole of the gastrula and then mainly in cilia cells located around the digestive system of the pluteus, as we recently have reported. PSEN expression must be accurately regulated for correct execution of these two steps of development. While investigating PSEN expression changes in embryos after expansion of endoderm with LiCl or of ectoderm with Zn2+ by whole-mount in situ hybridization (WISH) and quantitative PCR (qPCR), we detected natural antisense transcription of PSEN. We then found that Endo16 and Wnt5, markers of endo-mesoderm, and of Hnf6 and Gsc, markers of ectoderm, are also sense and antisense transcribed. We discuss that general gene expression could depend on both sense and antisense transcription. This mechanism, together with the PSEN gene, should be included in gene regulatory networks (GRNs) that theorize diverse processes in this species. We suggest that it would also be relevant to investigate natural antisense transcription of PSEN in the field of Alzheimer's disease (AD) where the role of human PSEN1 and PSEN2 is well known.

Mode of action of antifouling compound albofungin in inhibiting barnacle larval settlement

Marine biofouling causes huge economic losses to the marine industry every year. Albofungin is a potential antifoulant showing strong anti-macrofouling activities against larval settlement of major fouling organisms. In the present study, directed RNA-seq and proteomic analyses were used to investigate changes in the transcriptome and proteome of a major fouling barnacle Amphibalanus amphitrite cyprids in response to albofungin treatment. Results showed that albofungin treatment remarkably upregulated the metabolism of xenobiotics by the cytochrome P450 pathway to discharge the compound and downregulated energy metabolic processes. Intriguingly, immunostaining and whole-mount insitu hybridization (WISH) revealed the spatial expression patterns of selected differentially expressed genes (glutathione S-transferase [GST], nitric oxide synthase [NOS], and calmodulin [CaM]) distributed in the thorax and antennule of A.amphitrite. Our study provides new insights into the mechanism of albofungin in interrupting the larval settlement of A.amphitrite and suggests its potential application as an antifouling agent in marine environments.

UV filter ethylhexyl salicylate affects cardiovascular development by disrupting lipid metabolism in zebrafish embryos

Ethylhexyl salicylate (EHS) is an organic UV filter commonly used in sunscreens to protect people from the UV radiation. The widespread use of EHS will enter the aquatic environment along with human activities. EHS readily accumulates in adipose tissue as a lipophilic compound, but its toxic effects on lipid metabolism and cardiovascular system of aquatic organisms have not been studied. This study investigated the effects of EHS on lipid metabolism and cardiovascular development during zebrafish embryogenesis. The results showed that EHS caused defects such as pericardial edema, cardiovascular dysplasia, lipid deposition, ischemia, and apoptosis in zebrafish embryos. In addition, qPCR and whole-mount in situ hybridization (WISH) results indicated that EHS treatment significantly altered the expression of genes related to cardiovascular development, lipid metabolism, erythropoiesis, and apoptosis. The hypolipidemic drug rosiglitazone was able to alleviate the cardiovascular defects caused by EHS, indicating that EHS affected cardiovascular development by disrupting lipid metabolism. In addition, severe ischemia caused by cardiovascular abnormalities and apoptosis were observed in the EHS-treated embryos, which was likely to be the main cause of embryonic mortality. In conclusion, this study shows that EHS has toxic effects on lipid metabolism and cardiovascular formation. Our findings provide new evidence for assessing UV filter EHS toxicity and contribute to raising awareness of the safety risks of EHS.

Transcriptomic and metabolomic analyses revealed epiboly delayed mechanisms of 2,5-dichloro-1, 4-benuinone on zebrafish embryos.

2,5-Dichloro-1,4-benzenediol (2,5-DCBQ) is a putative disinfection by-product that belongs to the halogenated benzoquinone class. However, its developmental toxicity and related mechanism remained unclarified. In our study, we used zebrafish embryos as the model and exposed them to graded concentrations of 2,5-DCBQ (100, 200, 300, 400μg/L). We found that the rate of epiboly abnormalities increased significantly in a concentration-dependent manner. The results of whole-mount in situ hybridization (WISH) indicated that the expression patterns and levels of chordin (dorsoventral marker), foxa2 (endodermal marker), eve1 (ventral mesodermal marker), and foxb1a (ectodermal marker) were altered, suggesting that 2,5-DCBQ might affect the germ layer development of zebrafish embryos. Integrated transcriptomic and metabolomic analyses were adopted to explore the molecular mechanisms of embryonic developmental delays. The results showed that 2,5-DCBQ exposure induced 1163 differentially expressed genes (DEGs) and 37 differential metabolites (DEMs). Bioinformatic analysis enriched the most affected molecular pathways (Wnt signaling pathway, cell adhesion molecules, actin cytoskeleton regulation) and metabolic pathways (purine metabolism, aminoacyl-tRNA biosynthesis, arginine and proline metabolism) in zebrafish embryos. To summarize, our findings broadened the molecular mechanisms of 2,5-DCBQ embryotoxicity through multi-omics and bioinformatic analyses.

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Whole-Mount In Situ Hybridization for Detection of Migrating Zebrafish Endodermal Cells.

One of the most important events in early vertebrate development is the formation and positioning of the endoderm, the embryonic progenitor cell population that gives rise to the internal organs. Recent years have seen renewed interest in the mechanisms underlying the specification and migration of endodermal progenitor cells. The zebrafish is a well-established, accessible, and powerful model to study this cell population. Zebrafish endodermal cells are specified around 4h after fertilization and subsequently migrate as evenly spaced single cells in a stereotypical manner in the next 6h. Given the large numbers of fertilized eggs that can be obtained from a single breeding pair and the ease of chemical and genetic perturbations, the zebrafish is an excellent model to study mechanisms underlying endoderm specification and migration. An easy approach to visualizing and quantitating endodermal cells and their migratory routes is by whole-mount in situ hybridization (WISH) on fixed embryos, collected in time series. This chapter provides basic information on the organization and staging of the embryos, with an emphasis on the migrating endodermal cell population. In addition, optimized protocols for the isolation and fixation of staged embryos are provided as well as detailed probe synthesis and WISH protocols, specific for migrating endoderm. Finally, details are provided on how to approach these experiments quantitatively, and some common pitfalls are discussed.

Determining miRNA Expression Patterns in Xenopus.

Whole-mount in situ hybridization (WISH) is a technique that enables temporal and spatial visualization of RNA molecules in an embryo or whole tissue by using a complementary labelled probe. MicroRNAs are short noncoding RNAs of 20-25nt in length mainly involved in posttranscriptional regulation of gene expression. In this chapter, we describe how to visualize miRNAs in Xenopus laevis or tropicalis by WISH using two different approaches: LNA-WISH to visualize mature miRNAs and pri-miRNA-WISH to visualize the immature form of miRNAs, the pri-miRNAs.

Whole-mount RNA in situ hybridization technique in Torenia ovules.

The expression pattern of an interested gene at a cellular level provides strong evidence for its functions. RNA in situ hybridization has been proved to be a powerful tool in detecting the spatial-temporal expression pattern of a gene in various organisms. However, classical RNA in situ hybridization (ISH) technique is time-consuming and requires sophisticated sectioning skills. Therefore, we developed a method for whole-mount in situ hybridization (WISH) on ovules of Torenia fournieri, which is a model species in the study of plant reproduction. T. fournieri possesses ovules with protruding embryo sacs, making it easy to be observed and imaged through simple manipulation. To determine the effect of classical ISH and our newly established WISH, we detected the expression of a D-class gene, TfSTK3, using both methods. The expression patterns of TfSTK3 are similar in classical ISH and WISH, confirming reliability of the WISH method. Compared with WISH, classical ISH always leads to distorted embryo sacs, hence difficult to distinguish signals within the female gametophyte. To understand whether our WISH protocol also works well in detecting genes expressed within embryo sacs, we further examined the expression of a synergid-enriched candidate, TfPMEI1, and clearly observed specific signals within two synergid cells. To summarize, our WISH technique allows to visualize gene expression patterns in ovules of T. fournieri within one week and will benefit the field of plant reproduction in the future.

Graphene oxide disruption of homeostasis and regeneration processes in freshwater planarian Dugesia japonica via intracellular redox deviation and apoptosis

The aquatic system is a major sink for engineered nanomaterials released into the environment. Here, we assessed the toxicity of graphene oxide (GO) using the freshwater planarian Dugesia japonica, an invertebrate model that has been widely used for studying the effects of toxins on tissue regeneration and neuronal development. GO not only impaired the growth of normal (homeostatic) worms, but also inhibited the regeneration processes of regenerating (amputated) worms, with LC10 values of 9.86 mg/L and 9.32 mg/L for the 48-h acute toxicity test, respectively. High concentration (200 mg/L) of GO killed all the worms after 3 (regenerating) or 4 (homeostasis) days of exposure. Whole-mount in situ hybridization (WISH) and immunofluorescence analyses suggest GO impaired stem cell proliferation and differentiation, and subsequently caused cell apoptosis and oxidative DNA damage during planarian regeneration. Mechanistic analysis suggests that GO disturbed the antioxidative system (enzymatic and non-enzymatic) and energy metabolism in the planarian at both molecular and genetic levels, thus causing reactive oxygen species (ROS) over accumulation and oxidative damage, including oxidative DNA damage, loss of mitochondrial membrane integrity, lack of energy supply for cell differentiation and proliferation leading to retardance of neuron regeneration. The intrinsic oxidative potential of GO contributes to the GO-induced toxicity in planarians. These data suggest that GO in aquatic systems can cause oxidative stress and neurotoxicity in planarians. Overall, regenerated tissues are more sensitive to GO toxicity than homeostatic ones, suggesting that careful handling and appropriate decisions are needed in the application of GO to achieve healing and tissue regeneration.

Combined method of whole mount and block-face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization.

Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole-mount specimens, the gene expression pattern is represented in 2-dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3-dimensional (3D) data of gene expression from conventional ISH, the "serial section method" has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low-cost 3D imaging method that can create 3D gene expression patterns from conventional ISH-labeled specimens. Our method consists of a combination of whole-mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole-mount ISH-labeled specimens were sliced using a microtome or cryostat, and all block-faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.

Gldc Is Essential for Renal Progenitor Patterning during Kidney Development.

The glycine cleavage system (GCS) is a complex located on the mitochondrial membrane that is responsible for regulating glycine levels and contributing one-carbon units to folate metabolism. Congenital mutations in GCS components, such as glycine decarboxylase (gldc), cause an elevation in glycine levels and the rare disease, nonketotic hyperglycinemia (NKH). NKH patients suffer from pleiotropic symptoms including seizures, lethargy, mental retardation, and early death. Therefore, it is imperative to fully elucidate the pathological effects of gldc dysfunction and glycine accumulation during development. Here, we describe a zebrafish model of gldc deficiency that recapitulates phenotypes seen in humans and mice. gldc deficient embryos displayed impaired fluid homeostasis suggesting renal abnormalities, as well as aberrant craniofacial morphology and neural development defects. Whole mount in situ hybridization (WISH) revealed that gldc transcripts were highly expressed in the embryonic kidney, as seen in mouse and human repository data, and that formation of several nephron segments was disrupted in gldc deficient embryos, including proximal and distal tubule populations. These kidney defects were caused by alterations in renal progenitor populations, revealing that the proper function of Gldc is essential for the patterning of this organ. Additionally, further analysis of the urogenital tract revealed altered collecting duct and cloaca morphology in gldc deficient embryos. Finally, to gain insight into the molecular mechanisms underlying these disruptions, we examined the effects of exogenous glycine treatment and observed analogous renal and cloacal defects. Taken together, these studies indicate for the first time that gldc function serves an essential role in regulating renal progenitor development by modulating glycine levels.

Whole-Mount In Situ Hybridization (WISH) for Salamander Embryos and Larvae.

Whole-mount in situ hybridization (WISH) is widely used to visualize transcribed gene sequences (mRNA) in developing embryos, larvae, and other nucleotide probe permeable tissue samples. This methodology involves the hybridization of an antisense nucleotide probe to the target mRNA, followed by chromogen or fluorescence-based detection. Here we describe a protocol for the spatiotemporal analysis of mRNA transcripts in axolotl embryos/larvae using digoxigenin-labeled riboprobes, anti-digoxigenin alkaline phosphatase, Fab fragments antibody, and NBT/BCIP chromogen detection.

Transcriptomic networks of gba3 governing specification of the dopaminergic neurons in zebrafish embryos.

Molecular networks associated with dopaminergic (DA) neurogenesis remain undefined within mammalian models. To address this issue, the transient zebrafish model lmx1al: EGFP was generated, which expresses GFP in the DA precursor cells as well as in the DA neurons of the ventral diencephalon (VD). We found that the novel pseudogene gba3 has not been well studied in zebrafish neurogenesis. Crucial networks associated with gba3 transcripts were investigated because the biological functions of these networks have not been reported in DA neurogenesis in zebrafish. RNA isolation and sequencing were employed with GFP-expressing cells from 20-, 22-, and 24h post-fertilization (hpf), while subsequent transcriptomic analysis generated differentially expressed genes with DA neurogenesis (DEG-DA) list. Hierarchical cluster analysis provided absolute guidance for the collection of gba3, an essential transcript that is strictly spatiotemporally expressed during DA neurogenesis, which was proven with whole-mount in situ hybridization (WISH) and knockdown and rescue of the gba3 transcripts in zebrafish embryos. The gba3 transcripts were restricted to the midbrain at 24 hpf and the midbrain and pectoral fins at 30 hpf in zebrafish embryos. Functional studies with knockdown of gba3 found a diminished state in the midbrain and midbrain-hindbrain boundary (MHB) and an underdeveloped condition in the anteroposterior and dorsolateral axis relative to the wild type (WT) at 24 hpf. However, it was recovered after forced expression of gba3 transcripts at 24 hpf. Molecular markers for the DA precursors and mature neurons lmx1al, nurr1,th, and pitx3 were analyzed in the gba3 MOs. The levels of transcripts lmx1al, nurr1, and th were significantly reduced in the midbrain ventral diencephalon (VD) and hindbrain of gba3 morphants compared to the WT at 24 hpf, while expression patterns of pitx3 transcripts showed no differences in the identical regions between gba3 MOs and the controls. We discuss transcriptional networks in which transcripts of gba3 plausibly govern the specification of dopaminergic neurogenesis in zebrafish embryos.