Whole-mount In Situ Hybridization Research Articles

Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis

The Janus kinase (JAK)–signal transducer and activator of transcription (STAT) (JAK–STAT) pathway is shown to restrain the hair follicles in catagen and telogen and prevent anagen reentry in murine hair follicle cycling. The early roles of JAK-STAT pathway genes in skin development remain uncharacterized in mouse and chicken models. Here, we revealed the expression patterns of three JAK-STAT pathway genes (JAK1, JAK2, and TYK2) in chicken embryonic skin at E6–E10 stages which are key to feather follicle morphogenesis. Multiple sequence alignment of the three genes from chicken and other species all showed a closely related homology with birds like quail and goose. Whole mount in situ hybridization (WISH) revealed weak expression of JAK1, JAK2, and TYK2 in chicken skin at E6 and E7, and followed with the focally restricted signals in the feather follicles of neck and body skin located dorsally at E8 for JAK1, E9 for TYK2 and E10 for JAK2 gene. All three genes displayed stronger expression in feather follicles of neck skin than that of body skin. The expression levels of JAK1 and TYK2 were much stronger than those of JAK2. Quantitative real-time PCR (qRT-PCR) analysis revealed the increased expression tendency for JAK2 both in the neck and body skin from E6 to E10, and the much stronger expression in neck and body skin at later stages (E8-E10) than earlier stages (E6 and E7) for JAK1 and TYK2. Overall, these findings suggest that JAK1 and TYK2, not JAK2 are important to specify the feather follicle primordia, and to arrange the proximal–distal axis of feather follicles, respectively, during the morphogenesis of feather follicles in embryonic chicken skin.

Selenomethionine exposure affects chondrogenic differentiation and bone formation in Japanese medaka (Oryzias latipes)

Excess selenium entering the aquatic environment from anthropogenic activities has been associated with developmental abnormalities in fish including skeletal deformities of the head and spine. However, mechanisms of this developmental toxicity have not been well-characterized. In this study, Japanese medaka (Oryzias latipes) embryos were exposed to seleno-l-methionine (Se-Met) in a range of concentrations. Gene expression was evaluated for sex-determining region Y (SRY)-related box (Sox9a and Sox9b), runt-related transcription factor 2 (Runx2), and melatonin receptor (Mtr). Alterations in the length of Meckel’s cartilage, tail curvature, and decreased calcification were observed in skeletal stains at 10- and 22-days post-fertilization (dpf). Embryonic exposure of Osterix-mCherry transgenic medaka resulted in fewer teeth. Sox9a and Sox9b were up-regulated, while Runx2 and Mtr were down-regulated by Se-Met prior to hatch. Whole mount in situ hybridization (WISH) localized gene expression to areas observed to be affected in vivo. In addition, Se-Met exposures of a Mtr morpholino (Mtr-MO) as well as Luzindole exposed embryos developed similar skeletal malformations, supporting involvement of Mtr. These findings demonstrate that Se-Met modulates expression of key genes involved in chondrogenic differentiation and bone formation during development.

Ezh1 Inhibits Commitment to Hemogenic Fate and HSPC Formation during Vertebrate Development

Understanding how hematopoietic stem cells (HSCs) are specified from mesodermal precursors is essential to the goal of generating patient-specific HSCs capable of multi-potent long-term function. HSCs are born from hemogenic endothelium in select arterial niches during embryonic development through a transdifferentiation process turned endothelial-to-hematopoietic transistion (EHT). Despite increasing efforts to recapitulate this process in vitro, current differentiation protocols largely fail to produce long-lived multi-lineage progenitors from human induced pluripotent stem cell (iPSC) sources. Recently, an in vitro loss-of-function screen in human hematopoietic progenitors identified the Polycomb group protein, Enhancer of Zeste Homolog 1 (EZH1), as a regulator of definitive hematopoietic commitment, as assayed by acquisition of lymphoid competence. To determine the mechanism by which Ezh1 regulates HSPC fate in vivo we employed functional knockdown and epistasis investigations using the zebrafish model. Morpholino-mediated knockdown of ezh1 promoted expression of the conserved HSC markers runx1 and c-myb in the ventral wall of the dorsal aorta (VDA) at 36 hours post fertilization (hpf), as assessed by whole mount in situ hybridization (WISH); additionally, expression of the lymphoid marker rag1 was found to be enhanced at 120 hpf, as assayed by WISH and fluorescent activated cell sorting (FACS), in line with our in vitro observations. An impact on HSPCs was confirmed and quantified by qPCR for runx1 (**p < 0.01) and FACS using the CD41:GFP reporter line (**p < 0.01), indicating significantly increased HSPC number. Importantly, this enhancement in HSPC production had no effect on gross vascular morphology of the niche as determined by confocal microscopy for flk1. Assessment of arterial versus venous fate indicated that while the latter was unchanged in morphant embryos, expression of the arterial markers, epbrinb2a, dll4, dlc and tbx20, was strongly reduced by WISH and qPCR (**p < 0.01, *p < 0.05, **p < 0.01, and **** p < 0.001, respectively). In contrast, markers of hemogenic commitment, gata2b, and scl/flk1, were significantly increased, suggesting that loss of ezh1 enhanced hematopoietic potential at the expense of maintaining arterial fate. Profiling of single-cell RNA-sequencing data obtained from sorted populations of E10.5 mouse embryos revealed EZH1 to be more highly expressed in cells undergoing the endothelial-to-hematopoietic transition, consistent with a role of EZH1 in regulating arterial verses hematopoietic fate. Gene set enrichment analysis (GSEA) from our prior in vitro studies revealed the Notch pathway to be significantly altered following EZH1 knockdown. As Notch signaling has been implicated in both arterial specification and HSC emergence, we next examined the potential role of Notch signaling in ezh1 knockdown-mediated HSPC expansion. Consistent with a hypothesized interaction, differential regulation of Notch ligands and receptors was observed in ezh1 morphants compared to wild-type siblings; specifically, expression of arterial ligands, dll4 and dlc were decreased, while hematopoietic ligands and receptors, jag1a and notch1a were enhanced. Notably, the effect on Notch signaling was specific to ezh1 knockdown, as ezh2 loss shows a distinct pattern and temporal impact, reducing HSC production rather than enhancing it, consistent with recent reports. The strong conservation of ezh1-mediated regulation of HSC number, and our identification of its mechanistic role at the level of Notch receptor/ligand interactions, position zebrafish as a platform to identify chemical mediators that can be used to regulate ezh1 function during in vitro differentiation to unlock multi-lineage HSC commitment of human iPSC for therapeutic application. Disclosures Daley: Epizyme, Inc: Other: Equity & Consulting Fees; 28/7 Therapeutics: Other: Equity & Consulting Fees.

Characterizing Dyskeratosis Congenita Caused By Parn Mutations in the Zebrafish

Inherited bone marrow failure syndromes (IBMFs) are a group of rare genetic disorders characterized by deficient hematopoiesis and extra-hematologic traits. Most known entities are related to a specific gene or group of genes, but others still remain unclassified. In many cases, the involved proteins are required for critical processes involved in cell survival, such as ribosome biogenesis, maintenance of telomere length, and DNA repair. Importantly, patients with IBMFs have higher risks of developing a variety of cancers from leukemia to solid tumours of the head and neck. In 2015, we published four patients from three different families with mutations in the Poly(A)-specific ribonuclease (PARN) gene. This gene encodes a ribonuclease which is involved in degradation of the poly(A) tails, which regulate mRNA turnover, and thus gene expression. Three of the patients presented with several degrees of mental illness and/or developmental delay. The fourth patient, harbored both a monoallelic deletion and a point mutation at the catalytic domain of the protein, and presented with bone marrow failure and hypomyelination, similar to a severe form of dyskeratosis congenital (DC) known as Hoyeraal-Hreidarsson syndrome. In the last two decades, zebrafish (Danio rerio) has emerged as an excellent animal model for human disease, and is especially relevant in hematology, since many of the transcription factors and cell types are highly conserved. Zebrafish have a single PARN ortholog,with 64% sequence identity to the human gene. Using CRISPR-Cas9 genome editing and a combination of six sgRNAs, we generated a 1.2 kb deletion in the zebrafish parn ortholog extending from exon 5 to 13, causing a premature stop codon. Homozygous fish were generated by incrossing to replicate the complete loss-of-function observed in the patient with the DC-like phenotype. Using whole-mount in situ hybridization (WISH) at 48 hours post-fertilization (hpf), we observed a decrease in the number of several mature myeloid cell lineages including neutrophils (labeled with mpx; p<0.0001), macrophages (lcp1; p=0.0005) and mast cells (cpa5; p=0.0005). We also observed a decrease in the amount of hemoglobin (o-dianisidine staining; p<0.0001). However, the number of hematopoietic stem cells (HSCs) was unchanged in parn mutants. This data parallels similar findings using parn directed splice site and translation start site morpholinos. PARN is described as a protein involved in RNA processing, but has also been associated with telomere maintenance. This latter process is crucial for cell senescence and genome integrity, and is a known cause of several IBMFSs. The telomerase ribonucleoprotein complex is highly active in hematopoietic stem and progenitor cells (HSPCs) and plays a role in cell differentiation. This complex is composed of a reverse transcriptase (TERT), RNA template (TERC), and the dyskerin protein complex (DKC1), mutations of which represent a common cause of DC. qPCR analysis in zebrafish parn mutants revealed a 2.98-fold reduction in tert expression compared with the wild type fish. Combined, these findings suggest that PARN plays an important role in HSC differentiation into myeloid and erythroid lineages, resulting in a bone marrow failure phenotype. Our model provides a unique in vivo platform to study the role of PARN in hematopoiesis and for identifying compounds that restore normal blood cell ratios, which may have the potential to prevent future leukemic transformation. Disclosures No relevant conflicts of interest to declare.

Mutation-Specific Dominant Negative Effects Determine the Phenotype in SRP54 Deficiency

Whole exome sequencing analyses are increasingly performed on patients presenting with suspected inherited disease but lacking classical mutations linked to presented phenotypes. Using whole-exome sequencing in SBDS-negative Shwachman-Diamond Syndrome (SDS) families, we recently identified three independent patients, each of whom carried a heterozygous de novo missense variant of SRP54 (encoding signal recognition particle 54 kDa). The SRP54 protein is a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). Whilst two of the identified patients were carrying nucleotide transversion in SRP54 (p.T115A and p.G226E), which manifested in typical SDS features like neutropenia and exocrine pancreatic insufficiency, the third patient was carrying a nucleotide deletion (p.T117Δ), which only manifested in mild neutropenia without additional SDS features (Carapito et al. 2017, JCI). Here, we describe a zebrafish knock-out (KO) mutant as the very first transgenic in vivo model of SRP54 deficiency, translate our previous findings into living organisms and propose disease-driving mechanisms. We show that homozygous srp54 mutant zebrafish are suffering not only from severe neutropenia as shown by flow cytometry and Whole-Mount-In-Situ Hybridization (WISH), but also from gross developmental defects leading to early embryonic lethality. In fact, srp54-/- zebrafish did not survive more than 72 hours post fertilization, indicating that complete loss of Srp54 is not compatible with life. Injection with wild-type human SRP54 mRNA induced transient restoration of SRP54 protein expression and slightly enhanced the survival of the homozygous mutants. However, long-term viability could not be restored, revealing that srp54 is not only critically required during early embryogenesis but also at later stages of development. Heterozygous siblings on the other hand are viable and display only mild neutropenia but no pancreas defects. Interestingly however, injection of mutant mRNAs of human SRP54 (p.T115A, p.T117Δ, p.226E) into heterozygous srp54 KO mutants aggravated the phenotype inducing more profound neutropenia and pancreas changes similar to those observed in classical SDS patients. Of note, these effects were more severe for the transversions p.T115A and p.G226E. Mutation p.T117Δ only caused a minor reduction in the number of neutrophils, without affecting the pancreas. To further investigate SRP54 driven neutrophil defects, we used lentiviral transduction to exogenously express human SRP54 mutant variants in promyelocytic HL-60 cells. When stimulating these cells to differentiate by ATRA treatment, we found significantly impaired morphologic differentiation and CD11b surface induction compared to control cells. The severity of these effects was again specific to the three different identified mutations, with p.T115A and p.G226E being more severe than p.T117Δ. These findings confirm the type-specific effects of SRP54 mutations and indicate that SRP54 defects interfere with neutrophil differentiation and thus ultimately lead to neutropenia. Collectively, we here describe a novel zebrafish disease model of SDS and congenital neutropenia founding on SRP54 as molecular driver. Our model demonstrates that at least one healthy allele of srp54 is pivotal for survival, which is in line with the findings in humans, where homozygous mutations in SRP54 have never been detected. We reveal that the phenotypic manifestation of heterozygous SRP54 mutations strongly depends on the type of mutation: while mutations likely causing a simple SRP54 loss of function (e.g. p.T117Δ) induce a rather mild phenotype characterized by moderate neutropenia only (analogous to the heterozygous fish mutant), more severe SDS-like phenotypes involve SRP54 mutations that exert dominant negative effects (e.g. p.T115A and p.G226E). Ultimately, we make use of the promyelocytic cell line HL-60 to propose neutrophil differentiation defects as the underlying cause of SRP54 driven neutropenia. At the time being, RNA sequencing and protein expression analyses are performed in our laboratory, which will add to the understanding of the mechanistical background of the neutrophilic differentiation blockage and eventually uncover novel treatment strategies for SRP54 deficiency. Disclosures No relevant conflicts of interest to declare.

The regulation of rapamycin in planarian Dugesia japonica Ichikawa & Kawakatsu, 1964 regeneration according to TOR signaling pathway

The freshwater planarian mostly lives in the upper reaches of springs and rivers. Generally, it is realized as a suitable warning indicator of environmental toxicants. The freshwater planarian Dugesia japonica has a powerful regenerative capability and can regenerate a new individual including a complete central nervous system in one week. Rapamycin is an inhibitor of mammalian TORC1 (target of rapamycin complex-1) and used in the treatment of some diseases like cancer, cardiovascular and neurological diseases. However, the roles of rapamycin in the regulation of planarian regeneration remain to be elucidated. In present study, freshwater planarians D. japonica were firstly treated with 1 μM rapamycin for 18 h exposures and the expression patterns of Djtor was analyzed by the whole-mount in situ hybridization (WISH). Our results indicated rapamycin could strongly inhibit Djtor expression in planarian D. japonica and cause asymmetric blastemas and neuronal defects in planarians. Furthermore, knockdown of Djtor gene in planarians using RNA interference resulted in the suppression of downstream autophagy genes. These findings suggested that rapamycin might regulate freshwater planarian regeneration via Djtor signaling pathway.

Spatiotemporal expression profile of embryonic and adult ankyrin repeat and EF-hand domain containing protein 1-encoding genes ankef1a and ankef1b in zebrafish

Recent human next-generation sequencing (NGS) studies indicate a correlation between ANKEF1 (ankyrin repeat and EF-hand domain containing protein 1) expression and cilia formation or function. Additionally, a single study conducted in the African clawed frog (Xenopus laevis) showed ankef1 is down-regulated after pharmacological fibroblast growth factor (FGF) inhibition and plays a role in protocadherin-mediated cell protrusion and adhesion. That study also revealed a critical role for ankef1 in the embryonic development of the frog, with morphants exhibiting phenotypes including spina bifida and a shortened body axis. Interestingly, while little is known about ANKEF1 function in other vertebrate systems, recent proteomic data has shown ANKEF1 enriched in ciliated cells. Likewise, publicly available EST profile databases imply ANKEF1 expression in multiple human tissues, including high levels in the testes. Together, these previous studies suggest an important role for ANKEF1 in ciliated tissues and during embryonic development. Here, we report cloning of zebrafish (Danio rerio) ankef1a, as well as its paralog, ankef1b, and expression analyses by whole-mount in situ hybridization (WISH) and quantitative polymerase chain reaction (qPCR) during embryonic development and in adult tissues. WISH shows both forms are ubiquitously expressed early in development, with more discrete expression of both transcripts in embryonic tissues known to precede or possess motile cilia, including dorsal forerunner cells (DFC) and the otic vesicles, respectively. Additionally, both transcripts are enriched in the developing pharynx and swim bladder. Our qPCR results indicate enhanced expression in the testes, along with increased expression in brain. Certainly, our experiments in the zebrafish model system with ankef1a and ankef1b provide a solid foundation for future studies to uncover the molecular pathways through which Ankef1 acts in both healthy and disease states.

Developmental architecture of the nervous system in Themiste lageniformis (Sipuncula): New evidence from confocal laser scanning microscopy and gene expression.

Sipuncula is a clade of unsegmented marine worms that are currently placed among the basal radiation of conspicuously segmented Annelida. Their new location provides a unique opportunity to reinvestigate the evolution and development of segmented body plans. Neural segmentation is clearly evident during ganglionic ventral nerve cord (VNC) formation across Sedentaria and Errantia, which includes the majority of annelids. However, recent studies show that some annelid taxa outside of Sedentaria and Errantia have a medullary cord, without ganglia, as adults. Importantly, neural development in these taxa is understudied and interpretation can vary widely. For example, reports in sipunculans range from no evidence of segmentation to vestigial segmentation as inferred from a few pairs of serially repeated neuronal cell bodies along the VNC. We investigated patterns of pan-neuronal, neuronal subtype, and axonal markers using immunohistochemistry and whole mount in situ hybridization (WMISH) during neural development in an indirect-developing sipunculan, Themiste lageniformis. Confocal imaging revealed two clusters of 5HT+ neurons, two pairs of FMRF+ neurons, and Tubulin+ peripheral neurites that appear to be serially positioned along the VNC, similar to other sipunculans, to other annelids, and to spiralian taxa outside of Annelida. WMISH of a synaptotagmin1 ortholog in T. lageniformis (Tl-syt1) showed expression throughout the centralized nervous system (CNS), including the VNC where it appears to correlate with mature 5HT+ and FMRF+ neurons. An ortholog of elav1 (Tl-elav1) showed expression in differentiated neurons of the CNS with continuous expression in the VNC, supporting evidence of a medullary cord, and refuting evidence of ontogenetic segmentation during formation of the nervous system. Thus, we conclude that sipunculans do not exhibit any signs of morphological segmentation during development.

Estrogen Receptor β2 Oversees Germ Cell Maintenance and Gonadal Sex Differentiation in Medaka, Oryzias latipes

SummaryIn vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor β2 (ERβ2) during embryogenesis of medaka, and here we investigated the functional importance of ERβ2 in female gonad development and maintenance using a transgenerational ERβ2-knockdown (ERβ2-KD) line and ERβ2-null mutants. We found that ERβ2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERβ2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERβ2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

PF243 AN MLL‐AF9 ZEBRAFISH LEUKEMIA MODEL

Background:Acute Leukaemia (AL) is a genetically heterogeneous disease caused by somatic mutations and chromosomal translocations. Translocations can lead to the formation of fusion genes such as the MLL‐AF9 fusion, which results from the t (9;11)(p22;q23). MLL‐AF9 is one of the most common fusions involving the MLL (KMT2A) gene. MLLAF9 leukemias have an intermediate to poor prognosis. The MLL‐AF9 fusion is frequently used to establish murine leukemia models. However, to date, no MLL‐AF9 leukemia model in zebrafish has been reported.Aims:Our aim was to establish a transgenic zebrafish leukemia model using the MLL/AF9fusion gene.Methods:We cloned two transgenic constructs (pTol2‐Runx1+23_MLL‐AF9_IRES‐EGFP and pTol2‐Runx1+23_MLL‐AF9_IRES‐mCherry). The Runx1+23 murine promoter was used to target MLL‐AF9 expression to zebrafish hematopoietic stem cells, while EGFP or mCherry were used as fluorescent markers. The final transgenic constructs were co‐injected with Tol2 transposase mRNA into fertilized embryos at the one cell stage. The embryos were sorted 24 hours post fertilization based on the expression of the heart marker.Results:About 40% of the transgenic embryos died in the first week after injection but the remaining 60% reached adulthood. After 6 to 12 months, 30% (30 out of 100) fish showed signs of disease. They became less active, sat at the bottom of the tank and, in some cases, they had splayed eyes. When this happened, the fish were euthanized and dissected. The autopsies showed enlarged kidneys, splenomegaly and in some cases a pale spleen. Histological sections revealed increased cellularity of the kidney and liver with massive infiltration of hematopoietic cells in these organs. In flow cytometry, kidney marrow cells from the transgenic fish showed a different forward scatter (FSC) and side scatter (SSC) profile compared to that of the normal zebrafish kidney marrow cells. The FSC/SSC profile in the transgenic F0 zebrafish showed an expansion of the hematopoietic progenitor cell population. These findings strongly suggest that the fish had developed a leukemia‐like disease. We performed whole mount in situ hybridization (WISH) with early hematopoietic markers in 24 hpf embryos and detected reduced expression of gata1, myb and tal1as well as increased expression of lyz in transgenic embryos. Currently, transplantation experiments are being performed to determine whether the disease is transplantable which is a sign of leukemia.Summary/Conclusion:Although our flow cytometry, histology and WISH data strongly suggest that we have established an MLL/AF9‐driven leukemia model in zebrafish, additional experiments are needed to further characterize our model and confirm the malignant nature of the observed disease.Our MLL/AF9 zebrafish leukemia model will be a helpful tool to study leukemia biology and test new therapeutic strategies.

Identification of a potential tissue-specific biomarker cathepsin L-like gene from the planarian Dugesia japonica: Molecular cloning, characterization, and expression in response to heavy metal exposure

Heavy metal pollution is a global health issue affecting people worldwide, and the exploration of sensitive biomarkers to assess the toxicity of heavy metals is an important work for researchers. Cathepsin L, role as a tissue-specific biomarker to assess the biological effects of environmental pollutants, has not received much attention. In this work, the full-length cDNA of cathepsin L gene from the planarian Dugesia japonica (designated DjCatL) was cloned by rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of DjCatL is 1161 bp, which encodes a protein of 346 amino acids with a molecular weight of 39.03 kDa. Sequence analysis revealed that DjCatL contains highly conserved ERF/WNIN, GNFD, and GCXGG motifs, which are the features of the cathepsin L protein family. Whole-mount in situ hybridization (WISH) results revealed that the transcripts of DjCatL are specifically distributed in the intestinal system, suggesting that this gene is related to food digestion in planarians. Both quantitative polymerase chain reaction (qPCR) and WISH results revealed that the transcriptional levels of DjCatL are inhibited significantly by heavy metal (Cd2+, Hg2+, and Cu2+) exposure in a dose-dependent manner. Therefore, we proposed that cathepsin L can be used as a tissue-specific biomarker to assess the heavy metal pollution in the aquatic environment.

Silver nanoparticles induce abnormal touch responses by damaging neural circuits in zebrafish embryos

Although silver nanoparticles (AgNPs) are used in various commercial products, the biological effects of AgNPs on fish embryogenesis and the underlying molecular mechanisms are still poorly understood. In this study, both touch responses and neuron membrane potential were found to be abnormal in AgNPs-stressed embryos. Moreover, neurogenesis genes were unveiled to be down-regulated and were enriched in ligand-gated ion channel activity, dopamine receptor signaling pathway, etc. in AgNPs-stressed embryos by microarray assays. Additionally, the down-regulated expression of otpa/sncgb - gad1b/gad2 dopaminergic neurotransmitter genes, robo2 - vim and glrbb synaptic transmission genes, and motor neuron genes isl1 &isl2a was further identified in both AgNPs- and Ag+-stressed embryos by qPCR, whole-mount in situ hybridization (WISH), and by using specific promoter-derived GFP fluorescence transgenic zebrafish. Moreover, the reduced expression of gad1b, gad2, and isl1 could be recovered by adding Ag+ chelating compound l-cysteine in AgNPs stressed embryos. Our results reveal for the first time that it is through damaging the formation of neural circuits, including dopaminergic neurotransmitter, synaptic transmission, and motor activities, that AgNPs induce abnormal electrical membrane properties, leading to dysfunctional touch responses and locomotor escape responses mostly via their released Ag+ during embryogenesis.

Molecular mechanisms and tissue targets of brominated flame retardants, BDE-47 and TBBPA, in embryo-larval life stages of zebrafish (Danio rerio)

Brominated flame retardants are known to disrupt thyroid hormone (TH) homeostasis in several vertebrate species, but the molecular mechanisms underlying this process and their effects on TH-sensitive tissues during the stages of early development are not well characterised. In this study, we exposed zebrafish (Danio rerio) embryo-larvae to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and tetrabromobisphenol A (TBBPA) via the water for 96 h from fertilisation and assessed for lethality, effects on development and on the expression of a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis via both real time quantitative PCR (qRT-PCR) on whole body extracts and whole mount in situ hybridisation (WISH) to identify tissue targets. The 96-h lethal median concentration (96h-LC50) for TBBPA was 0.9 μM and mortality was preceded by retardation of development (smaller animals) and morphological deformities including, oedemas in the pericardial region and tail, small heads, swollen yolk sac extension. Exposure to BDE-47 did not affect zebrafish embryo-larvae survival at any of the concentrations tested (1–100 μM) but caused yolk sac and craniofacial deformities, a curved spine and shorter tail at the highest exposure concentration. TBBPA exposure resulted in higher levels of mRNAs for genes encoding deiodinases (dio1), transport proteins (ttr), the thyroid follicle synthesis protein paired box 8 (pax8) and glucuronidation enzymes (ugt1ab) and lower levels of dio3b mRNAs in whole body extracts, with responses varying with developmental stage. BDE-47 exposure resulted in higher levels of thrb, dio1, dio2, pax8 and ugt1ab mRNAs and lower levels of ttr mRNAs in whole body extracts. TBBPA and BDE-47 therefore appear to disrupt the TH system at multiple levels, increasing TH conjugation and clearance, disrupting thyroid follicle development and altering TH transport. Compensatory responses in TH production/ metabolism by deiodinases were also evident. WISH analyses further revealed that both TBBPA and BDE-47 caused tissue-specific changes in thyroid receptor and deiodinase enzyme expression, with the brain, liver, pronephric ducts and craniofacial tissues appearing particularly responsive to altered TH signalling. Given the important role of TRs in mediating the actions of THs during key developmental processes and deiodinases in the control of peripheral TH levels, these transcriptional alterations may have implications for TH sensitive target genes involved in brain and skeletal development. These findings further highlight the potential vulnerability of the thyroid system to disruption by BFRs during early developmental windows.

Whole-Mount In Situ Hybridization of Mouse Embryos Using DIG-Labeled RNA Probes.

Whole-mount in situ hybridization (WMISH) is a commonly used technique for visualizing the expression profile of mRNAs in embryos. Unlike traditional in situ hybridization techniques, which require thin tissue sections, the WMISH technique allows gene expression patterns to be assessed over the entire embryo and structure. Here, we describe the detailed procedural steps of WMISH, including probe production, embryo fixation and staining, and post-hybridization signal detection. Using this protocol, we visualized highly specific expression patterns of Sonic hedgehog and Bmp4 mRNAs in E12.5 mouse embryos.

Determining Temporal and Spatial Expression of Calpains in Amphibians.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that Calpains have been identified in the Xenopus genome, their expression patterns and role have not been characterized. Therefore, herein, we describe two methods to determine temporal and spatial expression of Calpain 2 during Xenopus development, namely, RT-PCR and whole-mount in situ hybridization (WISH). In addition, indirect immunofluorescence (IF) is described to determine translocation to the plasma membrane, which correlates with activity levels of Calpain 2.

A Zebrafish Model of Cooperating C and N Terminal CEBPA Mutations Reveals Defects in Early Myelopoeisis and HSPCs Leading to Leukaemogenesis

Acute Myeloid Leukaemia (AML) is thought to occur due to stepwise accumulation of mutations in haematopoietic stem and progenitor cells (HSPCs) or early myeloid progenitor cells (EMPs) to either block differentiation or increase proliferation. In around 10% of sporadic AML cases and several known germline predisposition kindreds the myeloid transcription factor CEBPA is mutated. 50% of cases possess biallelic mutations in both C and N terminae (dm) and 50% monoallelic mutation (sm). Using TALENs we have created both C and N terminal mutants in zebrafish to study their cooperating effects in the development of AML.Our first observation was of striking defects in mature myelocytes and monocytes in all double mutants (cebpamut/mut) as assessed by Sudan black staining at 5 days post fertilisation (dpf) and apoeb whole mount in situ hybridisation (WISH) at 3dpf. In situ hybridisation revealed defects in myelopoeisis in all cebpamut/mut fry as early as 28hpf, with markedly decreased expression of coronin and l-plastin.We then interrogated the model further to assess when and where the differentiation block was occurring. We utilised the transcription factor pu.1 both as an in vivo transgenic marker Tg(pu.1:GFP) and WISH probe at earlier time points. In primitive haematopoiesis (until 22hpf) pu.1 expression showed no defects in dm mutants. However, pu.1 expressing cells were markedly reduced in dm mutants in establishment of myelopoeisis in the caudal haematopoietic tissue (CHT), fetal liver equivalent. Following formation of the CHT at around 30hpf, differing patterns of pu.1 expression were seen in sm mutants, with cebpaWT/Cterm having significantly increased staining, which was reduced in cebpaWT/Nterm (see figure 1).HSPC numbers in cebpamut/mut were seen to be normal by WISH for gata2b and runx1 and flow cytometry in Tg(CD41:GFP) fry during early haematopoeisis (2-5dpf). However, myb expression was markedly increased at 36hpf and 3 days but only in cebpaCterm/Nterm and cebpaNterm/Nterm dm mutants (see figure 2). This suggests an accumulation of early progenitors with myeloid potential but not commitment, as implied by absence of pu.1 expression but normal CD41lo numbers and other stem cell marker expression.Absence of MCherry expression in the transgenic LysC:MCherry, which highlights mature myeloid cells in vivo, was sufficiently reliable to genotype all cebpamut/mut fry and observe them for impaired survival compared to wild-type and heterozygous siblings and for the development of leukaemia. Similarly to the increase in myb staining, leukaemic transformation was only observed in dm mutants with at least one N terminal mutation, as assessed by development of anaemia and florid Tg(CD41:GFP) expression. Flow cytometry in juvenile fish (4-6weeks) also identifies a developing pre-leukaemic phenotype with moderate yet significant expansion of HSPCs and continuing absence LysC expression in cebpaNterm/Cterm and cebpaNTerm/NTerm. However, survival is poor in all dm fish, likely due to the metabolic role of cebpa and vulnerability to infection secondary to the severe myelomonocytic defect.Our results show that absence of WT cebpa has dramatic effects myelopoiesis from early stages of differentiation in definitive haematopoiesis. In addition, accumulation of myb expressing progenitors occurs as they arise from the aorta in dm mutants, identifying this as a sub-population of HSPC vulnerable to further leukaemogenic hits. Ongoing work will define the mechanism of these effects in sm and dm mutants and include comparative expression profiling in myb and pu.1 positive cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Zebrafish JAK1-A634D Gain-of-Function Model Provides New Insights into the Pathogenesis of Familial Hypereosinophilia

Introduction: JAK/STAT signaling is of major importance for hematopoiesis and immune function. A heterozygous JAK1 c.1901C>A de novo point mutation was identified in three members of a family (a mother and two male sons). These patients presented with a unique clinical phenotype of severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, and failure to thrive. Treatment with ruxolitinib, a JAK1/2 inhibitor, resulted in reduced eosinophilia and improved growth (Del Bel et al. JACI 2017). However, the role of this JAK1 mutation in hematopoiesis remains unclear.Methods: ubi-JAK1-WT or A634D-P2A-sfGFP-pA zebrafish transgenic lines were generated incorporating the human genes. Both of these constructs were injected into embryos, but the survival rate for the JAK1-A634D embryos was very low. Six hour post-fertilization (hpf) embryos expressing the mutant transgene were treated with 1 µM ruxolitinib for 24h, which dramatically increased survival. Whole-mount in situ hybridization (WISH) was used to assess changes in hematopoietic stem cells/blood cell populations due to wild-type or JAK1-A634D expression at 24, 28, 36 and 48 hpf using a battery of blood lineage specific probes. O-dianisidine staining was employed to evaluate hemoglobin levels. ImageJ cell counter pluggin was used for cell quantification. Gata1 and o-dianisidine staining intensity was analyzed using Ilastik and CellProfiler. Six groups of 30-50 embryos were analyzed by RNA sequencing (RNAseq): control, JAK1-WT and JAK1-A634D at 28 and 36 hpf. RNA was extracted using Trizol. RNAseq data was analyzed with edgeR and enrichment analysis was performed using DAVID 6.8.Results: JAK1-A634D was toxic to zebrafish embryos but they survived to sexual maturity post-ruxolitinib therapy. F1 embryos from JAK1-A634D founders with high expression of the transgene exhibited abnormal development (70% of embryos) which could be reduced to 12% of embryos following 24h exposure to 1 µM ruxolitinib. This rescue suggests that JAK1-A634D is highly active in zebrafish and its activity needs to be under tight regulation for normal development. Both JAK1-WT and A634D mutant fish demonstrated increased HSPCs (c-myb/runx1 expression) at 36 hpf (p<0.0001) through 48 hpf (A634D>WT>controls; p=0.0308 and p=0.0045, respectively). pu.1/spi1 early myeloid expression showed a similar expression pattern at 28 hpf, A634D>WT>controls; p<0.0001 for both). At 24 and 28 hpf, granulocytes expressing myeloperoxidase (mpx) were increased in both JAK1-WT and JAK1-A634D mutants, but particularly high in JAK1-A634D embryos at 48 hpf (p<0.0001). Macrophages marked by l-plastin (lcp) expression, showed similar cell numbers in JAK1-WT and controls at 24 and 28 hpf, but a significant decrease in JAK1-A634D embryos (p<0.0001). An increase of carboxypeptidase A5 positive mast cells was observed in both JAK1-WT and JAK1-A634D embryos at 48 hpf compared with controls (p<0.0001). We observed an increase of gata1a expression at 24 hpf in A634D embryos (p=0.0058, p=0.0065), but o-dianisidine staining at 48 hpf revealed a decrease in hemoglobin in both JAK1 lines (p<0.0001). The latter data is supported by RNAseq results revealing decreased expression of hemoglobin-associated genes at 36 hpf in both transgenic lines. RNAseq revealed a total of 365 genes differentially expressed, with enrichment for genes coding for proteosomal and ribosomal proteins. In the JAK-1WT we found 79 genes upregulated and 115 downregulated; in the JAK1-A634D, 94 genes were upregulated and 77 were downregulated. JAK-STAT pathway genes were upregulated in the A634D mutant (p<0.05), as well as a trend for genes associated with hematopoiesis (p=0.0633) and negative regulation of cell proliferation (p=0.0658).Conclusions: JAK1-A634D plays an important role in zebrafish development and specifically impacts the myeloid lineage, particularly the granulocyte branch, from which eosinophils arise. Ruxolitinib can revert the arrested development phenotype, demonstrating the specificity of this targeted therapy. Our model provides the first in vivo evidence of the effects of the JAK1-A634D mutation on hematopoiesis and will provide further insight in understanding the cause of familial hypereosinophilia. DisclosuresNo relevant conflicts of interest to declare.

Stress-immune responses and DNA protection function of thioredoxin domain containing 12 in zebrafish (Danio rerio)

Proteins with dithiol-disulfide oxidoreductase catalytic domain are well known for their capacity in the cellular redox homeostasis. In this study, we characterized the zebrafish thioredoxin domain containing 12 (Zftxndc12) gene, analyzed the transcriptional responses and studied the functional properties of its recombinant protein. Full-length cDNA of Zftxndc12 consists 519 bp coding region encoding 172 amino acids (AA) including the signal peptide. Highly consensus active motif (65WCGAC69) and probable ER retrieval motif (169GDEL172) were identified. Ubiquitous expression of Zftxndc12 mRNA was observed from one cell to juvenile stage as well as different organs of adult zebrafish. Moreover, whole mount in situ hybridization (WISH) results showed a higher expression of Zftxndc12 in primordial gills, central nerves system and eye. The tissue specific expression analysis (by qRT-PCR) also showed the highest expression in gills followed by brain in adult zebrafish. In larvae, up-regulated Zftxndc12 mRNA expression upon exposure to H2O2,Edwardsiella tarda and Saprolegnia parasitica suggests that it may involve in both stress and immune responses. Moreover, transcriptional expression of Zftxndc12 was up-regulated upon Streptococcus iniae challenge in gills of adult zebrafish. The recombinant ZfTxndc12 (rZfTxndc12) was overexpressed, purified and tested for its biological activities. Results revealed that rZfTxndc12 is able to reduce the DNA damage and detoxify the H2O2 toxicity in concentration dependent manner. Overall results suggest that Zftxndc12 is important antioxidant and immune functional member of the host defense system in zebrafish.

Whole Mount in situ Localization of miRNAs and mRNAs During Somatic Embryogenesis in Arabidopsis.

Somatic embryogenesis (SE) results from the transition of differentiated plant somatic cells into embryogenic cells that requires the extensive reprogramming of the somatic cell transcriptome. Commonly, the SE-involved genes are identified by analyzing the heterogeneous population of explant cells and thus, it is necessary to validate the expression of the candidate genes in the cells that are competent for embryogenic transition. Here, we optimized and implemented the whole mount in situ hybridization (WISH) method (Bleckmann and Dresselhaus, 2016; Dastidar et al., 2016) in order to analyze the spatiotemporal localization of miRNAs (miR156, miR166, miR390, miR167) and mRNAs such as WOX5 and PHABULOSA-target of miR165/166 during the SE that is induced in Arabidopsis explants. This study presents a detailed step-by-step description of the WISH procedure in which DIG-labeled LNA and RNA probes were used to detect miRNAs and mRNAs, respectively. The usefulness of the WISH in the functional analysis of the SE-involved regulatory pathways is demonstrated and the advantages of this method are highlighted: (i) the ability to analyze intact non-sectioned plant tissue; (ii) the specificity of transcript detection; (iii) the detection of miRNA; and (iv) a semi-quantitative assessment of the RNA abundance.

Identification and expression of transcription factor sox2 in large yellow croaker Larimichthys crocea

As an important transcription and pluripotency factor, Sox2 plays its functions essentially in the regulation of self-renewal and pluripotency of embryonic and neural stem cells, as well as embryogenesis, organogenesis, neurogenesis and regeneration. The data is lacking on Sox2 in large yellow croaker (Larimichthys crocea) (Lc-Sox2) which is a limitation on the generation of induced pluripotent stem cells (iPSCs). In this study, Lc-sox2 was cloned by RACE (rapid amplification of cDNA ends) and analyzed by Bioinformatics. The quantitative real-time PCR (qRT-PCR) and whole mount in situ hybridization (WISH) were used to detect the expression of Lc-sox2. The full-length cDNA sequence of Lc-sox2 is 2135 bp and encodes a 322-aa (amino acids). Lc-Sox2 possesses a highly conserved HMG-box as DNA-binding domain, maintains highly conserved with vertebrates, particularly with teleosts. In tissues, Lc-sox2 was expressed with gender difference in brain and eye (female > male), in embryos, Lc-sox2 was expressed with a zygotic type that the high level expression began to appear in the gastrula stage. The spatio-temporal expression patterns of Lc-sox2 suggested the potential involvement in embryogenesis, neurogenesis, gametogenesis and adult physiological processes of large yellow croaker. Our results contributed to better understanding of Sox2 from large yellow croaker.