Biological Responses of Pacific Herring Embryos to Crude Oil Are Quantifiable at Exposure Levels Below Conventional Limits of Quantitation for PAHs in Water and Tissues.

Pacific herring (Clupea pallasii), a cornerstone of marine food webs, generally spawn on marine macroalgae in shallow nearshore areas that are disproportionately at risk from oil spills. Herring embryos are also highly susceptible to toxicity from chemicals leaching from oil stranded in intertidal and subtidal zones. The water-soluble components of crude oil trigger an adverse outcome pathway that involves disruption of the physiological functions of cardiomyocytes in the embryonic herring heart. In previous studies, impaired ionoregulation (calcium and potassium cycling) in response to specific polycyclic aromatic hydrocarbons (PAHs) corresponds to lethal embryolarval heart failure or subtle chamber malformations at the high and low ends of the PAH exposure range, respectively. Sublethal cardiotoxicity, which involves an abnormal outgrowth (ballooning) of the cardiac ventricular chamber soon after hatching, subsequently compromises juvenile heart structure and function, leading to pathological hypertrophy of the ventricle and reduced individual fitness, measured as cardiorespiratory performance. Previous studies have not established a threshold for these sublethal and delayed-in-time effects, even with total (∑)PAH exposures as low as 29 ng/g of wet weight (tissue dose). Here, we extend these earlier findings showing that (1) cyp1a gene expression provides an oil exposure metric that is more sensitive than typical quantitation of PAHs via GC-MS and (2) heart morphometrics in herring embryos provide a similarly sensitive measure of toxic response. Early life stage injury to herring (impaired heart development) thus occurs below the quantitation limits for PAHs in both water and embryonic tissues as a conventional basis for assessing oil-induced losses to coastal marine ecosystems.

Neurodevelopmental Toxicity of Emamectin Benzoate to the Early Life Stage of Zebrafish Larvae (Danio rerio).

Emamectin benzoate (EMB) is a widely used pesticide and feed additive in agriculture and aquaculture. It easily enters the aquatic environment through various pathways, thus causing adverse effects on aquatic organisms. However, there are no systematic studies regarding the effects of EMB on the developmental neurotoxicity of aquatic organisms. Therefore, the aim of this study was to evaluate the neurotoxic effects and mechanisms of EMB at different concentrations (0.1, 0.25, 0.5, 1, 2, 4 and 8 µg/mL) using zebrafish as a model. The results showed that EMB significantly inhibited the hatching rate, spontaneous movement, body length, and swim bladder development of zebrafish embryos, as well as significantly increased the malformation rate of zebrafish larvae. In addition, EMB adversely affected the axon length of motor neurons in Tg (hb9: eGFP) zebrafish and central nervous system (CNS) neurons in Tg (HuC: eGFP) zebrafish and significantly inhibited the locomotor behavior of zebrafish larvae. Meanwhile, EMB induced oxidative damage and was accompanied by increasing reactive oxygen species in the brains of zebrafish larvae. In addition, gene expression involvement in oxidative stress-related (cat, sod and Cu/Zn-sod), GABA neural pathway-related (gat1, gabra1, gad1b, abat and glsa), neurodevelopmental-related (syn2a, gfap, elavl3, shha, gap43 and Nrd) and swim bladder development-related (foxa3, pbxla, mnx1, has2 and elovlla) genes was significantly affected by EMB exposure. In conclusion, our study shows that exposure to EMB during the early life stages of zebrafish significantly increases oxidative damage and inhibits early central neuronal development, motor neuron axon growth and swim bladder development, ultimately leading to neurobehavioral changes in juvenile zebrafish.

Phenotyping Zebrafish Mutant Models to Assess Candidate Genes Associated with Aortic Aneurysm.

(1) Background: Whole Exome Sequencing of patients with thoracic aortic aneurysm often identifies "Variants of Uncertain Significance" (VUS), leading to uncertainty in clinical management. We assess a novel mechanism for potential routine assessment of these genes in TAA patients. Zebrafish are increasingly used as experimental models of disease. Advantages include low cost, rapid maturation, and physical transparency, permitting direct microscopic assessment. (2) Methods: Zebrafish loss of function mutations were generated using a CRISPRC/CAS9 approach for EMILIN1 and MIB1 genes similar to VUSs identified in clinical testing. Additionally, "positive control" mutants were constructed for known deleterious variants in FBN1 (Marfan's) and COL1A2, COL5A1, COL5A2 (Ehlers-Danlos). Zebrafish embryos were followed to six days post-fertilization. Embryos were studied by brightfield and confocal microscopy to ascertain any vascular, cardiac, and skeletal abnormalities. (3) Results: A dramatic pattern of cardiac, cerebral, aortic, and skeletal abnormalities was identified for the known pathogenic FBN1 and COL1A2, COL5A1, and COL5A2 mutants, as well as for the EMILIN1 and MIB1 mutants of prior unknown significance. Visualized abnormalities included hemorrhage (peri-aortic and cranial), cardiomegaly, reduced diameter of the aorta and intersegmental vessels, lower aortic cell counts, and scoliosis (often extremely severe). (4) Conclusion: This pilot study suggests that candidate genes arising in clinical practice may be rapidly assessed via zebrafish mutants-thus permitting evidence-based decisions about pathogenicity. Thus, years-long delays to clinically demonstrate pathogenicity may be obviated. Zebrafish data would represent only one segment of analysis, which would also include frequency of the variant in the general population, in silico genetic analysis, and degree of preservation in phylogeny.

Highly luminescent gold nanocluster assemblies for bioimaging in living organisms.

Fluorescent gold nanoclusters are promising nanomaterials for biomedical applications but confronted with low emission efficiency and poor surface functionality. Herein, three kinds of highly luminescent and functionalized gold nanocluster nano-assembled structures were fabricated by poly-l-arginine surface engineering for luminescence improvement. The assembly is employed for imaging the glutathione molecule in cells and living organisms with low background and high sensitivity.

E-liquids and vanillin flavoring disrupts retinoic acid signaling and causes craniofacial defects in Xenopus embryos.

Environmental teratogens such as smoking are known risk factors for developmental disorders such as cleft palate. While smoking rates have declined, a new type of smoking, called vaping is on the rise. Vaping is the use of e-cigarettes to vaporize and inhale an e-liquid containing nicotine and food-like flavors. There is the potential that, like smoking, vaping could also pose a danger to the developing human. Rather than waiting for epidemiological and mammalian studies, we have turned to an aquatic developmental model, Xenopus laevis, to more quickly assess whether e-liquids contain teratogens that could lead to craniofacial malformations. Xenopus, like zebrafish, has the benefit of being a well-established developmental model and has also been effective in predicting whether a chemical could be a teratogen. We have determined that embryonic exposure to dessert flavored e-liquids can cause craniofacial abnormalities, including an orofacial cleft in Xenopus. To better understand the underlying mechanisms contributing to these defects, transcriptomic analysis of the facial tissues of embryos exposed to a representative dessert flavored e-liquid vapor extract was performed. Analysis of differentially expressed genes in these embryos revealed several genes associated with retinoic acid metabolism or the signaling pathway. Consistently, retinoic acid receptor inhibition phenocopied the craniofacial defects as those embryos exposed to the vapor extract of the e-liquid. Such malformations also correlated with a group of common differentially expressed genes, two of which are associated with midface birth defects in humans. Further, e-liquid exposure sensitized embryos to forming craniofacial malformations when they already had depressed retinoic acid signaling. Moreover, 13-cis-retinoic acid treatment could significantly reduce the e-liquid induced malformation in the midface. Such results suggest the possibility of an interaction between retinoic acid signaling and e-liquid exposure. One of the most popular and concentrated flavoring chemicals in dessert flavored e-liquids is vanillin. Xenopus embryos exposed to this chemical closely resembled embryos exposed to dessert-like e-liquids and a retinoic acid receptor antagonist. In summary, we determined that e-liquid chemicals, in particular vanillin, can cause craniofacial defects potentially by dysregulating retinoic acid signaling. This work warrants the evaluation of vanillin and other such flavoring additives in e-liquids on mammalian development.

Developmental Toxicity and Biotransformation of Two Anti-Epileptics in Zebrafish Embryos and Early Larvae.

The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5»- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish.

Mechanosensitive Notch-Dll4 and Klf2-Wnt9 signaling pathways intersect in guiding valvulogenesis in zebrafish.

In the zebrafish embryo, the onset of blood flow generates fluid shear stress on endocardial cells, which are specialized endothelial cells that line the interior of the heart. High levels of fluid shear stress activate both Notch and Klf2 signaling, which play crucial roles in atrioventricular valvulogenesis. However, it remains unclear why only individual endocardial cells ingress into the cardiac jelly and initiate valvulogenesis. Here, we show that lateral inhibition between endocardial cells, mediated by Notch, singles out Delta-like-4-positive endocardial cells. These cells ingress into the cardiac jelly, where they form an abluminal cell population. Delta-like-4-positive cells ingress in response to Wnt9a, which is produced in parallel through an Erk5-Klf2-Wnt9a signaling cascade also activated by blood flow. Hence, mechanical stimulation activates parallel mechanosensitive signaling pathways that produce binary effects by driving endocardial cells toward either luminal or abluminal fates. Ultimately, these cell fate decisions sculpt cardiac valve leaflets.

Effect of Maternal Marginal Zinc Deficiency on Development, Redox Status, and Gene Expression Related to Oxidation and Apoptosis in an Avian Embryo Model.

Maternal severe zinc (Zn) deficiency resulted in growth retardation and high mortality during embryonic development in human. Therefore, this study is aimed at evaluating the effect of maternal marginal Zn deficiency on the development and redox status to avoid severe Zn deficiency using an avian model. A total of 324 laying duck breeders at 214 days old were randomly allotted into 3 dietary Zn levels with 6 replicates of 18 ducks per replicate. The birds were fed experimental diets including 3 dietary supplemental Zn levels of 0 mg/kg (maternal Zn-deficient group, 29.2 mg Zn/kg diet), 60 mg/kg (maternal Zn-adequate group), and 120 mg/kg (maternal Zn-high group) for 6 weeks. Dietary Zn levels had on effect on egg production and fertility (P > 0.05), whereas dietary Zn deficiency decreased breeder plasma Zn concentration and erythrocytic alkaline phosphatase activity at week 6 and inhibited erythrocytic 5'-nucleotidase (5'-NT) activity at weeks 2, 4, and 6 (P < 0.05), indicating that marginal Zn-deficient status occurred after Zn depletion. Maternal marginal Zn deficiency increased embryonic mortality and contents of superoxide anion radical, MDA, and PPC and reduced MT content and CuZnSOD activity in duck embryonic livers on E29. The MDA content was positively correlated with embryonic mortality. Maternal marginal Zn deficiency increased BCL2-associated X protein and Caspase-9 mRNA expressions as well as decreased B-cell lymphoma-2 and MT1 mRNA and signal AKT1 and ERK1 protein expressions (P < 0.05). Breeder plasma Zn concentration and erythrocytic 5'-NT activities at week 6 were positively correlated with GSH-Px activity and GPx, MT1, and BCL2 mRNA expressions in embryonic livers on E29. In conclusion, erythrocytic 5'-NT activity could be more rapid and reliable to monitor marginal Zn-deficient status. Marginal Zn deficiency impaired hatchability and antioxidant defense system and then induced oxidative damage and apoptosis in the embryonic liver, contributing to the greater loss of duck embryonic death.

Ttc30a affects tubulin modifications in a model for ciliary chondrodysplasia with polycystic kidney disease.

Skeletal ciliopathies (e.g., Jeune syndrome, short rib polydactyly syndrome, and Sensenbrenner syndrome) are frequently associated with nephronophthisis-like cystic kidney disease and other organ manifestations. Despite recent progress in genetic mapping of causative loci, a common molecular mechanism of cartilage defects and cystic kidneys has remained elusive. Targeting two ciliary chondrodysplasia loci (ift80 and ift172) by CRISPR/Cas9 mutagenesis, we established models for skeletal ciliopathies in Xenopus tropicalis Froglets exhibited severe limb deformities, polydactyly, and cystic kidneys, closely matching the phenotype of affected patients. A data mining-based in silico screen found ttc30a to be related to known skeletal ciliopathy genes. CRISPR/Cas9 targeting replicated limb malformations and renal cysts identical to the models of established disease genes. Loss of Ttc30a impaired embryonic renal excretion and ciliogenesis because of altered posttranslational tubulin acetylation, glycylation, and defective axoneme compartmentalization. Ttc30a/b transcripts are enriched in chondrocytes and osteocytes of single-cell RNA-sequenced embryonic mouse limbs. We identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins and suggest that tubulin modifications and cilia segmentation contribute to skeletal and renal ciliopathy manifestations of ciliopathies in a cell type-specific manner. These findings have implications for potential therapeutic strategies.

Oxidative stress induced antioxidant and neurotoxicity demonstrated in vivo zebrafish embryo or larval model and their normalization due to morin showing therapeutic implications.

AIMS: The study examined that morin as possible antioxidant and neuroprotective due to oxidative stress (H2O2) in zebrafish larval model. MATERIALS AND METHODS: Zebrafish larvae were induced with oxidative stress using H2O2 at 1 mM; their behavioural changes were assessed through partition preference and horizontal compartment test. The head section without eyes and yolk sac of zebrafish larvae were employed for enzyme assays such as SOD, CAT, Thiobarbituric acid reactive substances assay, reduced glutathione, glutathione peroxidase activity, glutathione S transferase, Acetylcholinesterase activity and nitrate levels. Also, intracellular ROS and apoptosis in larval head was detected by DCFDA and acridine orange staining followed by gene expression studies. KEY FINDINGS: Morin exposure was not harmful to the larvae at concentration between 20 and 60 µM, but it caused non-lethal deformity between 80 and 100 µM. In the partition test, zebrafish embryos treated with H2O2 showed cognitive impairment, whereas the morin-treated groups showed an improved behavioural activity. The study also found that restoring antioxidant enzymes and reduced lipid peroxidation which had a neuroprotective impact. Inhibition of NO overproduction and increased AChE activity were also shown to reduce the neuronal damage. Apoptosis and intracellular ROS levels were reduced in larvae when it was co-incubated with morin. Morin treatment up regulated the antioxidant enzymes against oxidative stress. SIGNIFICANCE: Morin provides protection against H2O2 induced oxidative stress through a cellular antioxidant defence mechanism by up-regulating gene expression, thus increasing the antioxidant activity at cellular or organismal stage.

zmiz1a zebrafish mutants have defective erythropoiesis, altered expression of autophagy genes, and a deficient response to vitamin D.

ZMIZ1 is a transcriptional coactivator that is related to members of the protein inhibitor of activated STAT (PIAS) family. ZMIZ1 regulates the activity of various transcription factors including the androgen receptor, p53, and Smad3. ZMIZ1 also interacts with Notch1 and selectively regulates Notch1 target genes relevant for T cell development and leukemogenesis in mammals. Human ZMIZ1 is additionally characterized as a latitude-dependent autoimmune disease (LDAD) risk gene, as it is responsive to vitamin D and has been associated with at least eleven blood cell traits. To address the function of ZMIZ1 in fish, we introduced CRISPR/Cas9 mutations in the zmiz1a gene in zebrafish. We observed that inactivation of zmiz1a in developing zebrafish larvae results in lethality at 15 days post fertilization (dpf) and delayed erythroid maturation. Differential gene expression analysis indicated that 15 dpf zmiz1a-null larvae had altered expression of autophagy genes, and erythrocytes that lacked Zmiz1a function exhibited an accumulation of mitochondrial DNA. Furthermore, we observed that autophagy gene expression was dysregulated at earlier stages of development, which suggests the involvement of Zmiz1a in the regulation of autophagy genes beyond the process of red blood cell differentiation. Finally, we showed that the loss of Zmiz1a decreased the capacity of the embryos to respond to vitamin D, indicating additional participation of Zmiz1a as a mediator of vitamin D activity.

Sub-lethal Camphor Exposure Triggers Oxidative Stress, Cardiotoxicity, and Cardiac Physiology Alterations in Zebrafish Embryos.

Camphor is a terpene ketone with aromatic and volatile properties in nature derived from the bark of Cinnamomum camphora or synthesized from turpentine. Camphor exhibits various biological properties such as anti-microbial, anti-viral, anti-coccidial, and anti-cancer. It is also used as a form of topical medication for skin irritation, joint pain, and as a relief for itching from insect bites. However, even though the high dose of camphor has been documented to be toxic/lethal in humans in different studies, camphor's developmental toxicity has not yet been explored, and its extensive mechanism of action is still unclear. In the present study, we aimed to assess the toxic effects of camphor in zebrafish embryos in the initial developmental stages. The obtained results demonstrated that a sub-lethal dose of camphor caused a decrease in hatching rate, body length, and substantial elevation in malformation rate on zebrafish embryos. On further observation, in the following time frame, curved body and pericardial edema of zebrafish were also observed. Furthermore, exposure to a sub-lethal dose of camphor was also able to trigger cardiotoxicity in zebrafish larvae. Later, on subsequent biochemical analysis, it was found that the antioxidant capacity inhibition and oxidative stress elevation that occurred after camphor exposure might be associated with the inhibition of total superoxide dismutase (SOD) activity and an increase in reactive oxygen species (ROS) and malondialdehyde (MDA) concentration. In addition, compared to the control group, several apoptotic cells in treated zebrafish were also found to be elevated. Finally, after further investigation on marker gene expressions, we conclude that the developmental toxicity of camphor exposure might be associated with apoptosis elevation and oxidative stress. Taken together, the current study provides a better understanding of the developmental toxicity of camphor on zebrafish, a promising alternative animal model to assess the developmental toxicity of chemical compounds.

The toxicity mechanism of toxic compounds from Euphorbiae pekinensis Radix on zebrafish embryos.

Euphorbiae pekinensis Radix (EP) is effective in treating various diseases, but it's toxicity is a major obstacle in use in clinical. Although EP was processed with vinegar to reduce it's toxicity, the detailed mechanism of toxicity in EP have not been clearly delineated. This study investigate the toxicity attenuation-mechanism of Euphorbiae pekinensis after being processed with vinegar (VEP) and the toxic mechanism of four compounds from EP on zebrafish embryos. The contents of four compounds decreased obviously in VEP. Correspondingly, slower development on embryos can be seen as some symptoms like reduction of heart rate, liver area and gastrointestinal peristalsis after exposed to the compounds. Some obvious pathological signals such as pericardial edema and yolk sac edema were observed. Furthermore, the compounds could increase the contents of MDA and GSH-PX and induce oxidative damage by inhibiting the activity of SOD. Also, four compounds could provoke apoptosis by up-regulating the expression level of p53, MDM2, Bax, Bcl-2 and activating the activity of caspase-3, caspase-9. In conclusion, the four compounds play an important role in the toxicity attenuation effects of VEP, which may be related to the apoptosis induction and oxidative damage. This would contribute to the clinical application and further toxicity-reduction mechanism research.

Toxicity of Jegosaponins A and B from Styrax japonica Siebold et al. Zuccarini in Prostate Cancer Cells and Zebrafish Embryos Resulting from Increased Membrane Permeability.

(1) Background: Screening of medicinal herbs is one of the most powerful approaches to identifying novel therapeutic molecules against many human diseases. To avoid potential harmful effects during medicinal use, toxicity testing is necessary in the early stages of drug discovery. The objective of this study was to identify the cytotoxic mechanisms of jegosaponin A and B from Styrax japonica Siebold et al. Zuccarini; (2) Methods: We screened Japanese medicinal herb extracts using PC-3 prostate cancer cells and found that a methanol extract isolated from the unripe fruit of Styrax japonica Siebold et al. Zuccarini (SJSZ) had an inhibitory effect on cell viability. We further performed fractionation assays with PC-3 cells and identified the bioactive compounds using LC/MS and NMR analysis. We clarified the toxic mechanisms of these compounds using PC-3 cells and zebrafish embryos; (3) Results: We identified two active molecules, jegosaponin A and jegosaponin B, in the inhibitory fractions of the methanol extract. These jegosaponins are toxic to zebrafish embryos during the early developmental stage. Jegosaponin A and B showed strong haemolytic activity in sheep defibrinated blood (EC50 = 2.1 µM, and 20.2 µM, respectively) and increased the cell membrane permeability in PC-3 cells and zebrafish embryos, which were identified using a membrane non-permeable DRAQ7, a fluorescent nucleus staining dye; (4) We identified the cytotoxic compounds jegosaponin A and B from SJSZ, which we showed to exhibit cell membrane disruptive properties using cell- and zebrafish-based testing.

Low-level embryonic crude oil exposure disrupts ventricular ballooning and subsequent trabeculation in Pacific herring.

There is a growing awareness that transient, sublethal embryonic exposure to crude oils cause subtle but important forms of delayed toxicity in fish. While the precise mechanisms for this loss of individual fitness are not well understood, they involve the disruption of early cardiogenesis and a subsequent pathological remodeling of the heart much later in juveniles. This developmental cardiotoxicity is attributable, in turn, to the inhibitory actions of crude oil-derived mixtures of polycyclic aromatic compounds (PACs) on specific ion channels and other proteins that collectively drive the rhythmic contractions of heart muscle cells via excitation-contraction coupling. Here we exposed Pacific herring (Clupea pallasi) embryos to oiled gravel effluent yielding ΣPAC concentrations as low as ~ 1 µg/L (64 ng/g in tissues). Upon hatching in clean seawater, and following the depuration of tissue PACs (as evidenced by basal levels of cyp1a gene expression), the ventricles of larval herring hearts showed a concentration-dependent reduction in posterior growth (ballooning). This was followed weeks later in feeding larvae by abnormal trabeculation, or formation of the finger-like projections of interior spongy myocardium, and months later with hypertrophy (overgrowth) of the spongy myocardium in early juveniles. Given that heart muscle cell differentiation and migration are driven by Ca2+-dependent intracellular signaling, the observed disruption of ventricular morphogenesis was likely a secondary (downstream) consequence of reduced calcium cycling and contractility in embryonic cardiomyocytes. We propose defective trabeculation as a promising phenotypic anchor for novel morphometric indicators of latent cardiac injury in oil-exposed herring, including an abnormal persistence of cardiac jelly in the ventricle wall and cardiomyocyte hyperproliferation. At a corresponding molecular level, quantitative expression assays in the present study also support biomarker roles for genes known to be involved in muscle contractility (atp2a2, myl7, myh7), cardiomyocyte precursor fate (nkx2.5) and ventricular trabeculation (nrg2, and hbegfa). Overall, our findings reinforce both proximal and indirect roles for dysregulated intracellular calcium cycling in the canonical fish early life stage crude oil toxicity syndrome. More work on Ca2+-mediated cellular dynamics and transcription in developing cardiomyocytes is needed. Nevertheless, the highly specific actions of ΣPAC mixtures on the heart at low, parts-per-billion tissue concentrations directly contravene classical assumptions of baseline (i.e., non-specific) crude oil toxicity.

Joint development recovery on resumption of embryonic movement following paralysis.

Fetal activity in utero is a normal part of pregnancy and reduced or absent movement can lead to long-term skeletal defects, such as Fetal Akinesia Deformation Sequence, joint dysplasia and arthrogryposis. A variety of animal models with decreased or absent embryonic movements show a consistent set of developmental defects, providing insight into the aetiology of congenital skeletal abnormalities. At developing joints, defects include reduced joint interzones with frequent fusion of cartilaginous skeletal rudiments across the joint. At the spine, defects include shortening and a spectrum of curvature deformations. An important question, with relevance to possible therapeutic interventions for human conditions, is the capacity for recovery with resumption of movement following short-term immobilisation. Here, we use the well-established chick model to compare the effects of sustained immobilisation from embryonic day (E)4-10 to two different recovery scenarios: (1) natural recovery from E6 until E10 and (2) the addition of hyperactive movement stimulation during the recovery period. We demonstrate partial recovery of movement and partial recovery of joint development under both recovery conditions, but no improvement in spine defects. The joints examined (elbow, hip and knee) showed better recovery in hindlimb than forelimb, with hyperactive mobility leading to greater recovery in the knee and hip. The hip joint showed the best recovery with improved rudiment separation, tissue organisation and commencement of cavitation. This work demonstrates that movement post paralysis can partially recover specific aspects of joint development, which could inform therapeutic approaches to ameliorate the effects of human fetal immobility. This article has an associated First Person interview with the first author of the paper.

Sfxn1 is essential for erythrocyte maturation via facilitating hemoglobin production in zebrafish.

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one­carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.

The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor.

Understanding how fate specification of distinct cell-types from multipotent progenitors occurs is a fundamental question in embryology. Neural crest stem cells (NCSCs) generate extraordinarily diverse derivatives, including multiple neural, skeletogenic and pigment cell fates. Key transcription factors and extracellular signals specifying NCSC lineages remain to be identified, and we have only a little idea of how and when they function together to control fate. Zebrafish have three neural crest-derived pigment cell types, black melanocytes, light-reflecting iridophores and yellow xanthophores, which offer a powerful model for studying the molecular and cellular mechanisms of fate segregation. Mitfa has been identified as the master regulator of melanocyte fate. Here, we show that an Mitf-related transcription factor, Tfec, functions as master regulator of the iridophore fate. Surprisingly, our phenotypic analysis of tfec mutants demonstrates that Tfec also functions in the initial specification of all three pigment cell-types, although the melanocyte and xanthophore lineages recover later. We show that Mitfa represses tfec expression, revealing a likely mechanism contributing to the decision between melanocyte and iridophore fate. Our data are consistent with the long-standing proposal of a tripotent progenitor restricted to pigment cell fates. Moreover, we investigate activation, maintenance and function of tfec in multipotent NCSCs, demonstrating for the first time its role in the gene regulatory network forming and maintaining early neural crest cells. In summary, we build on our previous work to characterise the gene regulatory network governing iridophore development, establishing Tfec as the master regulator driving iridophore specification from multipotent progenitors, while shedding light on possible cellular mechanisms of progressive fate restriction.

A deep learning approach for staging embryonic tissue isolates with small data.

Machine learning approaches are becoming increasingly widespread and are now present in most areas of research. Their recent surge can be explained in part due to our ability to generate and store enormous amounts of data with which to train these models. The requirement for large training sets is also responsible for limiting further potential applications of machine learning, particularly in fields where data tend to be scarce such as developmental biology. However, recent research seems to indicate that machine learning and Big Data can sometimes be decoupled to train models with modest amounts of data. In this work we set out to train a CNN-based classifier to stage zebrafish tail buds at four different stages of development using small information-rich data sets. Our results show that two and three dimensional convolutional neural networks can be trained to stage developing zebrafish tail buds based on both morphological and gene expression confocal microscopy images, achieving in each case up to 100% test accuracy scores. Importantly, we show that high accuracy can be achieved with data set sizes of under 100 images, much smaller than the typical training set size for a convolutional neural net. Furthermore, our classifier shows that it is possible to stage isolated embryonic structures without the need to refer to classic developmental landmarks in the whole embryo, which will be particularly useful to stage 3D culture in vitro systems such as organoids. We hope that this work will provide a proof of principle that will help dispel the myth that large data set sizes are always required to train CNNs, and encourage researchers in fields where data are scarce to also apply ML approaches.

Asxl1 C-terminal mutation perturbs neutrophil differentiation in zebrafish.

ASXL1 is one of the most frequently mutated genes in malignant myeloid diseases. In patients with myeloid malignancies, ASXL1 mutations are usually heterozygous frameshift or nonsense mutations leading to C-terminal truncation. Current disease models have predominantly total loss of ASXL1 or overexpressed C-terminal truncations. These models cannot fully recapitulate leukemogenesis and disease progression. We generated an endogenous C-terminal-truncated Asxl1 mutant in zebrafish that mimics human myeloid malignancies. At the embryonic stage, neutrophil differentiation was explicitly blocked. At 6 months, mutants initially exhibited a myelodysplastic syndrome-like phenotype with neutrophilic dysplasia. At 1 year, about 13% of mutants further acquired the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or increased progenitors, which mimics acute myeloid leukemia. These features are comparable to myeloid malignancy progression in humans. Furthermore, transcriptome analysis, inhibitor treatment, and rescue assays indicated that asxl1-induced neutrophilic dysplasia was associated with reduced expression of bmi1a, a subunit of polycomb repressive complex 1 and a reported myeloid leukemia-associated gene. Our model demonstrated that neutrophilic dysplasia caused by asxl1 mutation is a foundation for the progression of myeloid malignancies, and illustrated a possible effect of the Asxl1-Bmi1a axis on regulating neutrophil development.