Zebrafish Embryonic Development Research Articles

Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells.

MicroRNAs (miRNAs) play vital roles in various biological processes under multiple stress conditions by leading to mRNA cleavage or translational repression. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the target genes of known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, both miR-100-3p inhibitor and miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. Further study showed that miR-100-3p and miR-16b could regulate inversely the expression of their target gene (atad5a, cyp2ae1, lamp1, rilp, atxn7, tnika, btbd9), and that overexpression of miR-100-3p disturbed the early embryonic development of zebrafish. In summary, the present data show that miRNAs are closely involved in cold acclimation in zebrafish ZF4 cells and provide information for further understanding of the roles of miRNAs in cold acclimation in fish.

Lasting alterations induced in glial cell phenotypes by short exposure to alcohol during embryonic development in zebrafish.

Despite the known teratogenic effects of alcohol (ethanol) on the developing human fetus, the prevalence of fetal alcohol spectrum disorder (FASD) is not decreasing. Appropriate treatment for this life-long disease has not been developed, and even diagnostic biomarkers are unavailable. FASD remains a large unmet medical need. Numerous animal models have been developed to mimic FASD and study potential underlying biological mechanisms. However, most of these models focused on neuronal phenotypes. Given that glial cells represent the majority of cells in the vertebrate brain, and given the increasingly appreciated roles they play in a myriad of neuronal functions as well as CNS disorders, we decided to investigate potential embryonic alcohol exposure induced changes in them. Building upon a previously introduced zebrafish model of milder and most prevalent forms of FASD, we investigated the effect of a 2-hour-long exposure to alcohol (1% vol/vol bath concentration) employed at the 24th hour postfertilization stage of development of zebrafish on a number of glial cell-related phenotypes. We studied oligodendrocyte, astrocyte as well as microglia-related phenotypes using immunohistochemistry, lipid, and enzyme activity analyses. We report significant changes in wide-spread glial cell phenotypes induced by embryonic alcohol exposure in the zebrafish brain and conclude that the zebrafish will advance our understanding of the mechanisms of this devastating disorder.

Evaluation of developmental toxicity and genotoxicity of aqueous seed extract of Croton tiglium L. using zebrafish

Croton tiglium L. has been used in Ayurvedic and Chinese herbal medicinal formulations from ancient times. Although its seeds are widely prescribed as traditional medicine, there is a dearth of information, regarding its toxic effects, and the mechanisms underlying its toxicity. This study aims to investigate the developmental toxicity and genotoxicity of the aqueous seed extract of C. tiglium L. (AECT) in zebrafish. We have examined the effects of AECT on the early embryonic development of zebrafish. Zebrafish embryos, treated with different concentrations of the AECT, suffered embryonic lethality and displayed various developmental defects. The 96 h-LC50 of AECT was found to be 162.78 µg/ml. Interestingly, the developmental abnormalities observed, such as pericardial edema (PE), yolk sac edema (YSE), spinal curvature (SC), and delayed hatching, varied in severity, in a dose-dependent manner. Zebrafish embryos, treated with different concentrations of AECT, exhibited exaggerated cell death in the anatomical regions of brain, heart, and trunk. Our data suggest that the phenomenon of apoptosis is probably responsible for both embryonic lethality and developmental toxicity in zebrafish embryos. Furthermore, the genotoxic potential of the AECT, in vivo, was evaluated using micronucleus assay and comet assay, on the peripheral blood of zebrafish. The results suggest that AECT has the potential to cause genotoxicity in the peripheral blood of zebrafish.

Expression patterns of pcbp gene family members during zebrafish embryogenesis

The poly(C)-binding protein (PCBP) family members belong to a subtype of RNA-binding proteins that are ubiquitously expressed with diverse functions. In mammals, PCBP family, also known as hnRNP E family, is composed of four proteins, namely PCBP1, PCBP2, PCBP3 and PCBP4. So far, no study has been documented on the physiological roles of each member in vertebrate development. Here we analysed the spatiotemporal expression patterns of zebrafish (Danio rerio) pcbp2 (identical to pcbp1 and pcbp2 in mammals), pcbp3 and pcbp4 at various stages of zebrafish embryonic development by whole-mount in situ hybridization. Our results revealed that all pcbp genes are maternally expressed, especially pcbp2, which is strongly expressed from the embryogenetic stage to larva. The expression patterns of PCBP members are similar to each other at the very early developmental stage sharing with common strong expression in the intestine, otic vesicle, retina and brain of zebrafish. Subsequently, the messenger RNAs of PCBP members are gradually constrained and highly expressed in intestines of the larvae. Collectively, our study figured out the expression pattern of each PCBP member in diverse organogenesis during embryo development, indicating that PCBP members may play predominant roles in the development of neural and digestive systems to maintain their normal physiological functions. Moreover, the similar expression patterns at the developmental stages and organ types among this family suggest that the aberrant expression of these genes would lead to the neural or intestinal diseases.

The role of chorion around embryos in toxic effects of bisphenol AF exposure on embryonic zebrafish (Danio rerio) development

Bisphenol AF (BPAF) has been found ubiquitously in the environment and attributed to the population degradation of the fish community. It was proved that the susceptibilities of fish to BPAF exposure are diverse in different developing stages. However, the role of embryonic chorion under BPAF exposure remains unknown. In the study, model organism zebrafish (Danio rerio) was adopted to carry out the embryo acute toxicity tests (OECD No. 236) combined with microinjection (MI, at the blastula) and dechorionation (DC, at the pharyngula) techniques and non-treatment (NT-1, NT-2) for investigating the role of chorion around embryos. The results showed that the BPAF exposure could considerably contribute to the growth-retardation, delayed hatching, pericardial edema, yolk sac edema, non-inflated swimming bladder and death. The embryos in the MI-ZR group which soaked to ZR embryo medium after microinjected with BPAF solutions were hardly affected. While the mortalities and malformation rates in the MI-BPAF (the embryos were injected with BPAF and soaked in BPAF solution) and NT-1 (the embryos were not injected but soaked in BPAF solution) increased significantly with time and dose. Under BPAF soaking exposure, the dechorionated embryos in the DC (the embryos were dechorionated and soaked in BPAF solution) were significantly sensitive in comparison with the parallel undechorionated embryos in the NT-2 (the embryos were not dechorionated but soaked in BPAF solution). The 96 h LC50 values of MI-BPAF, NT-1, DC and NT-2 were 1.61, 1.68, 0.27, 1.30 mg L−1, respectively. The results indicate that the chorion has good permeability for BPAF passing through it at the earlier stage of zebrafish embryonic development, but becomes an effective barrier for protecting embryos over time. Moreover, zebrafish are more sensitive to BPAF exposure at embryos-to-larvae development. Thus, BPAF exposure hazards the development of zebrafish embryos and larvae. Waterborne BPAF exposure might pose serious risks to aquatic organisms and break the balance of aquatic ecosystem.

Expression analysis of Rab11 during zebrafish embryonic development

BackgroundRab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate vesicular recycling during embryonic development. In zebrafish, there are 3 rab11 paralogues, known as rab11a, rab11ba and rab11bb, sharing high identity with each other. However, the expression analysis of rab11 is so far lacking.ResultsHere, by phylogeny analysis, we found the three rab11 genes are highly conserved especially for their GTPase domains. We examined the expression patterns of rab11a, rab11ba and rab11bb using RT-PCR and in situ hybridization. We found that all the three genes were highly enriched in the central nervous system, but in different areas of the brain. Apart from brain, rab11a was also expressed in caudal vein, pronephric duct, proctodeum, pharyngeal arches and digestive duct, rab11ba was detected to express in muscle, and rab11bb was expressed in kidney, fin and spinal cord. Different from rab11a and rab11ba, which both have maternal expressions in embryos, rab11bb only expresses during 24hpf to 96hpf.ConclusionsOur results suggest that rab11 genes play important but distinct roles in the development of the nervous system in zebrafish. The findings could provide new evidences for better understanding the functions of rab11 in the development of zebrafish embryos.

Rice Husk Silica Enhances Innate Immune in Zebrafish (Danio rerio) and Improves Resistance to Aeromonas hydrophila and Streptococcus iniae Infection

Rice husk (RH) contains abundant silica such that RH silica (RHS) may be useful for possible industrial exploitation. Here, amorphous silica nanoparticles with multiple pore structures were acquired from RH by simple thermochemical processes. RHS antimicrobial activity and effects on zebrafish innate immunity against pathogen infections were evaluated. A toxicity assay showed that zebrafish exposed to an RHS dose lower than 200 μg/mL did not exhibit damage to zebrafish embryonic development or juvenile survival. RHS showed a wide spectrum of bacteriostatic activity against a variety of pathogens including antibiotic-resistant pathogens, implying its potential application as an antimicrobial agent in diverse industries. Fish exposed to 20 or 200 μg/mL RHS exhibited significantly increased mRNA expression of immune-related genes, including IL-1β, IL-6, IL-15, TNF-α, COX-2a, TLR-4a, lysozyme, and complement C3b. RHS-treated zebrafish exhibited a higher cumulative survival compared to that in control fish after infecting with Aeromonas hydrophila and Streptococcus iniae. The present results showed that a safe RHS dose enhanced innate immunity against infections without toxic effects in healthy fish, suggesting that RHS may be developed as an immunostimulant for improving health status in aquaculture.

The food preservative ethoxyquin impairs zebrafish development, behavior and alters gene expression profile

In the present study, we investigated the detrimental effects of ethoxyquin (EQ) on zebrafish embryonic development using different endpoints including lethality, malformations, locomotion and gene expression. EQ is primarily used as a preservative in animal feed and it has been shown to have negative impacts on different laboratory animals. However, studies on the adverse effects of EQ in aquatic animals are still limited. In this study, zebrafish eggs were exposed to different concentrations of EQ ranging from 1 to 100 μM for six days. In the 100 μM treated groups 95 and 100% mortality was observed at 24 and 48 h, respectively. Delayed development, decreased pigmentation and pericardial edema were observed in larvae. Behavioral analysis of larvae demonstrated a distinct locomotive pattern in response to EQ both in light and dark indicating a possible developmental neurotoxicity and deficits in locomotion. The expression levels of genes involved in several physiological pathways including stress response, cell cycle and DNA damage were altered by EQ. Our results demonstrate that EQ could cause developmental and physiological toxicity to aquatic organisms. Hence, its toxic effect should be further analyzed and its use and levels in the environment must be monitored carefully.

Adverse effect of cylindrospermopsin on embryonic development in zebrafish (Danio rerio).

Eutrophication of freshwater bodies increases the occurrence of toxic cyanobacterial blooms. The cyanobacterial toxin cylindrospermopsin (CYN) is receiving great interest due to its increasing presence in waterbodies. However, the toxic effects of CYN on zebrafish development are poorly understood, especially the toxicological mechanism, which is still unclear. In this study, we examined the adverse effects of CYN on embryonic development in zebrafish. CYN (2-2000 nM) exposure decreased embryos survival rate, hatching rate, body length and eye size in a concentration-dependent manner and caused abnormalities in embryo morphology, including pericardial edema, spinal curvature, tail deformity, uninflated swim bladder, cardiac and vascular defects. CYN at concentrations of 20 nM or higher significantly increased ROS level and promoted cell apoptosis in zebrafish embryos. To preliminarily elucidate the potential mechanism of zebrafish developmental toxicity caused by CYN, we examined the expression of oxidative stress- and apoptotic-related genes. CYN could promote the expression of oxidative stress-related genes (SOD1, CAT and GPx1) and induce changes in transcriptional levels of apoptotic-related genes (p53, Bax and Bcl-2). Taken together, CYN induced adverse effects on zebrafish embryos development, which may associate with oxidative stress and apoptosis. These outcomes will advance our understanding of CYN toxicity, environmental problems and health hazards caused by climate changes and eutrophication.

Effects of Five Substances with Different Modes of Action on Cathepsin H, C and L Activities in Zebrafish Embryos.

Cathepsins have been proposed as biomarkers of chemical exposure in the zebrafish embryo model but it is unclear whether they can also be used to detect sublethal stress. The present study evaluates three cathepsin types as candidate biomarkers in zebrafish embryos. In addition to other functions, cathepsins are also involved in yolk lysosomal processes for the internal nutrition of embryos of oviparous animals until external feeding starts. The baseline enzyme activity of cathepsin types H, C and L during the embryonic development of zebrafish in the first 96 h post fertilisation was studied. Secondly, the effect of leupeptin, a known cathepsin inhibitor, and four embryotoxic xenobiotic compounds with different modes of action (phenanthrene—baseline toxicity; rotenone—an inhibitor of electron transport chain in mitochondria; DNOC (Dinitro-ortho-cresol)—an inhibitor of ATP synthesis; and tebuconazole—a sterol biosynthesis inhibitor) on in vivo cathepsin H, C and L total activities have been tested. The positive control leupeptin showed effects on cathepsin L at a 20-fold lower concentration compared to the respective LC50 (0.4 mM) of the zebrafish embryo assay (FET). The observed effects on the enzyme activity of the four other xenobiotics were not or just slightly more sensitive (factor of 1.5 to 3), but the differences did not reach statistical significance. Results of this study indicate that the analysed cathepsins are not susceptible to toxins other than the known peptide-like inhibitors. However, specific cathepsin inhibitors might be identified using the zebrafish embryo.

“Ecological risk assessments and eco-toxicity analyses using chemical, biological, physiological responses, DNA damages and gene-level biomarkers in Zebrafish (Danio rerio) in an urban stream”

We conducted the ecological risk assessment in an urban stream by using multiple-level approaches ranging from community level, chemical analyses in water and sediments, physiological assays of DNA biomarkers, embryonic development tests, and gene-level marker analyses of cyp1a, c-Fos, CRH, transgenic fli1:GFP and HuC:eGFP in zebrafish (Danio rerio). In water, the chemical perturbations based on nutrients (N,P), organic matter, ionic contents and metals identified in downstream zone. Analogous corroborations verified in sediment samples having hazardous metals (Zn, Pb, Cu, Ni, As, Cd). The chemical contaminations reflected significant damages in fish DNA, based on tDNA, tail length (TL), and tail extent moment (TEM). Zebrafish embryonic development experiments significantly enlightened the chemical contaminants in downstream compared to those in control and reference conditions. Hatching and survival rates rigorously declined in downstream region. Embryonic development delayed and followed by death in the downstream substantiated by the above-mentioned findings. Similar were the findings on heart rate and pigmentation largely affected in the contaminated zone. Pollutants in urban stream reflected significantly at the gene level, and were corroborated through experiments using transgenic zebrafish strains that were influenced by pollutants during the process of occurrence. In conclusion, these studies illuminate the community to gene-level ecological health assessment that could be useful for ecological risk assessments of urban streams and rivers. Further, the gene-level biomarkers and transgenic zebrafish experiments combination propose the procedures could be effectively used as sensitive and efficient biomarkers of ecological health and risk assessment in urban streams from community to gene-level assessments.

Tris(1,3-dichloro-2-propyl) phosphate disturbs mouse embryonic development by inducing apoptosis and abnormal DNA methylation.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is a kind of additive flame retardants (FRs) and was found to affect early embryonic development in zebrafish; however, there are few studies to investigate whether TDCPP will disturb the development of early mouse embryos. In our studies, we used mouse embryos as models to study the toxicology of TDCPP on the early embryos. The results showed that TDCPP disturbed the development of early mouse embryos in a dose-dependent manner. 10 μM TDCPP decreased the blastocyst formation and 100 μM TDCPP was a lethal concentration for the mouse embryos. We proved that TDCPP was detrimental to embryonic development potential by increasing the reactive oxygen species level and inducing early apoptosis. Furthermore, TDCPP changed the DNA methylation patterns of imprinted genes in treated blastocysts. The methylation of H19 and Snrpn promoter regions was increased from 37.67% to 46.00% and 31.56% to 44.38% in treated groups, respectively. In contrast, Peg3 promoter region methylation was declined from 86.55% to 73.27% in treated embryos. Taken together, our results demonstrated that TDCPP could adversely impair the early embryonic development in mouse. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Di-butyl phthalate (DBP) induces craniofacial defects during embryonic development in zebrafish.

Di-butyl phthalate (DBP) is commonly added to make plastics softer and more pliable and is found in a variety of consumer and industrial products. Alarmingly high levels of DBP have been detected in water and sediment as DBP leaches from products. These levels are concerning and have led the Environmental Protection Agency to label DBP as a priority environmental pollutant and the European Commission to label DBP as a priority substance. Given the ubiquitous presence of DBP globally and continuous exposure to DBP, studies on the developmental toxicity of DBP are needed. The endocrine disrupting effects of DBP are well documented, but developmental toxicity of DBP during critical developmental time windows is understudied. Here, we investigate the developmental effects of DBP exposure during early development. We find defects in craniofacial development including a decrease in overall cranial size in DBP treated embryos, but the intraocular distance was increased compared to controls. Further investigation of jawbone development demonstrated loss of and disorganization of cartilage development. Defects in vascular innervation and neuronal patterning were also noted. Here we conclude that exposure to DBP during crucial time windows of embryonic development is toxic to craniofacial development.

FoxH1 represses miR-430 during early embryonic development of zebrafish via non-canonical regulation

BackgroundFoxH1 is a forkhead transcription factor with conserved key functions in vertebrate mesoderm induction and left-right patterning downstream of the TGF-beta/Nodal signaling pathway. Binding of the forkhead domain (FHD) of FoxH1 to a highly conserved proximal sequence motif was shown to regulate target gene expression.ResultsWe identify the conserved microRNA-430 family (miR-430) as a novel target of FoxH1. miR-430 levels are increased in foxH1 mutants, resulting in a reduced expression of transcripts that are targeted by miR-430 for degradation. To determine the underlying mechanism of miR-430 repression, we performed chromatin immunoprecipitation studies and overexpression experiments with mutant as well as constitutive active and repressive forms of FoxH1. Our studies reveal a molecular interaction of FoxH1 with miR-430 loci independent of the FHD. Furthermore, we show that previously described mutant forms of FoxH1 that disrupt DNA binding or that lack the C-terminal Smad Interaction Domain (SID) dominantly interfere with miR-430 repression, but not with the regulation of previously described FoxH1 targets.ConclusionsWe were able to identify the distinct roles of protein domains of FoxH1 in the regulation process of miR-430. We provide evidence that the indirect repression of miR-430 loci depends on the connection to a distal repressive chromosome environment via a non-canonical mode. The widespread distribution of such non-canonical binding sites of FoxH1, found not only in our study, argues against a function restricted to regulating miR-430 and for a more global role of FoxH1 in chromatin folding.

Abstract 5232: PES1 is highly expressed and its biological function in gastrointestinal tumor

Abstract Nucleolus, one of the most important organelles in cell nuclear of eukaryotic cells, is made of rRNA, rDNA and nucleoproteins. Its main function is rRNA synthetic. PES1 is part of nucleolar protein, and was identified in the mutational study of the embryonic development of Zebrafish. Its main biological functions include forming nucleolus, promoting cell proliferation and regulating cell cycle. A few researches indicate the close relationship between PES1 and tumorigenesis. The purpose of this study is to explore the role of PES1 in tumorigenesis and the possible mechanism. Three specific monoclonal antibodies against PES1 were made through hybridoma technique. Using these antibodies, it is found that PES1 had significantly higher expression in gastric and colon carcinoma than in tumor-adjacent and -distant tissues. Immunohistochemical assay on gastric carcinoma specimens and colon carcinoma specimens indicated that PES1 had significantly higher expression in carcinoma than in tumor-adjacent tissues. Moreover, we demonstrated the localization of PES1 was changed from nucleolus to nucleoplasm significantly under the stimulation of chemotherapeutic drugs and UV. The phenomenon of PES1 translocation in nucleus was weaken in drug-resistant cancer cell lines. These results suggest that the translocation of PES1 in nucleus might be related to DNA damage repair and drug resistance mechanism of cancer cells. In summary, PES1 expression is significantly higher in gastric and colon carcinoma tissue. And dynamic changes of PES1 localization in the cell nucleus is an important biological function and is worth further research. Note: This abstract was not presented at the meeting. Citation Format: Qin Feng, Shenyi Lian, Ping Wang, Yue Wang, Zhongwu Li, Chengchao Shou, Tingting Ren, Dongmei Lin. PES1 is highly expressed and its biological function in gastrointestinal tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5232.

Fibroblast Growth Factor Receptors Function Redundantly During Zebrafish Embryonic Development.

Fibroblast growth factor (Fgf) signaling regulates many processes during development. In most cases, one tissue layer secretes an Fgf ligand that binds and activates an Fgf receptor (Fgfr) expressed by a neighboring tissue. Although studies have identified the roles of specific Fgf ligands during development, less is known about the requirements for the receptors. We have generated null mutations in each of the five fgfr genes in zebrafish. Considering the diverse requirements for Fgf signaling throughout development, and that null mutations in the mouse Fgfr1 and Fgfr2 genes are embryonic lethal, it was surprising that all zebrafish homozygous mutants are viable and fertile, with no discernable embryonic defect. Instead, we find that multiple receptors are involved in coordinating most Fgf-dependent developmental processes. For example, mutations in the ligand fgf8a cause loss of the midbrain-hindbrain boundary, whereas, in the fgfr mutants, this phenotype is seen only in embryos that are triple mutant for fgfr1a;fgfr1b;fgfr2, but not in any single or double mutant combinations. We show that this apparent fgfr redundancy is also seen during the development of several other tissues, including posterior mesoderm, pectoral fins, viscerocranium, and neurocranium. These data are an essential step toward defining the specific Fgfrs that function with particular Fgf ligands to regulate important developmental processes in zebrafish.

Cul4a promotes zebrafish primitive erythropoiesis via upregulating scl and gata1 expression

CUL4A and CUL4B are closely related members in Cullin family and can each assemble a Cullin-RING E3 ligase complex (Cullin-RING Ligase 4A or 4B, CRL4A, or CRL4B) and participate in a variety of biological processes. Previously we showed that zebrafish cul4a, but not cul4b, is essential for cardiac and pectoral fin development. Here, we have identified cul4a as a crucial regulator of primitive erythropoiesis in zebrafish embryonic development. Depletion of cul4a resulted in a striking reduction of erythroid cells due to the inhibition of erythroid differentiation. Transcript levels for early hematopoietic regulatory genes including scl, lmo2, and gata1 are significantly reduced in cul4a-deficient embryos. Mechanistically, we demonstrated that scl and gata1, the central regulators of primitive hematopoiesis for erythroid determination, are transcriptionally upregulated by cul4a. These findings demonstrate an important role for cul4a in primitive erythropoiesis and may bear implications in regeneration medicine of anemia and related diseases.

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Teratogenic effects of aqueous extract of Ficus glomerata leaf during embryonic development in zebrafish (Danio rerio)

Over past few decades, many reports published in scientific journals describing medicinal properties of Ficus glomerata. However, its effects on embryonic development and its safety characteristics have not been studied. The purpose of this investigation was to determine LC50 value and study the effect of aqueous extract of Ficus glomerata leaf on developmental abnormalities in zebrafish embryos. LC50 value of aqueous extract of Ficus glomerata leaf was calculated by using probit analysis. Effect on percentage hatchability, heartbeat rate, total body length and developmental morphological abnormalities i.e delayed growth, abnormal movement, tail detachment, abnormal head trunk angle, scoliosis/ flexure, yolk sac edema were recorded. Aqueous extract of Ficus glomerata leaf revealed LC50 of 239.88 ppm. The result showed a significant reduction in percent hatchability (p

Phthalimide Derivative Shows Anti-angiogenic Activity in a 3D Microfluidic Model and No Teratogenicity in Zebrafish Embryos.

Angiogenesis is a crucial event for tumor progression and metastasis. It is the process through which new blood vessels are formed and has become a therapeutic target in many cancer therapies. However, current anti-angiogenic drugs such as Thalidomide still have detrimental teratogenic effects. This property could be caused by the presence of chiral carbons, intrinsic to such compounds. We synthesized four different phthalimide derivatives that lack chiral carbons in their chemical structure. We hypothesized that these achiral carbon compounds would retain similar levels of anti-angiogenic activity whilst reducing teratogenic effects. We tested for their anti-angiogenic functions using an in vitro 3D microfluidic assay with human endothelial cells. All four compounds caused a drastic inhibition of angiogenesis at lower effective concentrations compared to Thalidomide. Quantification of the blood vessel sprouting in each condition allowed us to classify compounds depending on their anti-angiogenic capabilities. The most effective identified compound (C4), was tested in vivo on a zebrafish embryo model. Blood vessel development was measured using number and lengths of the stalks visible in the fli1a:EGFP transgenic line. Potential teratogenic effects of C4 were monitored over zebrafish embryonic development. The in vivo results confirmed the increased potency of C4 compared to Thalidomide demonstrated by results in embryos exposed to concentrations as low as 0.02 μM. The teratogenic analysis further validated the advantages of using C4 over Thalidomide in zebrafish embryos. This study highlights how the use of in vitro 3D model can allow rapid screening and selection of new and safer drugs.