Characterizing the cardiovascular phenotype of a new zebrafish model of Marfan syndrome

Background Marfan syndrome (MFS) is a rare disease caused by a defect in the fibrillin-1 gene (FBN1), with potentially severe cardiovascular manifestations. MFS patients are particularly susceptible to a progressive aortic dilation leading to potential dissection and wall rupture. No causal treatment for the disease is available and current medical treatment is aimed at slowing aortic disease progression to minimize severe complications. When indicated, surgical repair of the aortic defect is performed. Although these strategies have clearly led to improved survival, some patients still present with fatal complications. Purpose To generate a new flexible zebrafish model of MFS to gain a better understanding of the underlying pathophysiological mechanisms and to find new treatment options. Methods We used the CRISPR/Cas9 method to disrupt the 3 fibrillin genes in zebrafish (fbn1, fbn2a, and fbn2b). The Tg(kdrl:EGFP) reporter was used to visualize cardiovascular structure by fluorescent microscopy up to 8 days post fertilization (dpf). A subset of embryos was treated with the myosin inhibitor 2,3-butanedione monoxime (BDM). Results We found that zebrafish lacking fbn1 and/or fbn2a do not show any detectable phenotype during development. No evidence of induction of genetic compensation was found in these mutant lines. Zebrafish deficient in fbn2b however do show strong phenotypes, including fully penetrant finfold atrophy (Fig. 1A-B and E, arrowhead). On average 50% of homozygous fbn2b mutant (fbn2b−/−) zebrafish embryos show endocardial detachment (Fig. 1C-D; a:atrium, v:ventricle), leading to vascular embolism, pericardial edema (Fig. 1B, arrow), loss of blood flow, and ultimately death at 7–9 dpf. Interestingly, fbn2b−/− without endocardial detachment survive normally, but develop a dilated bulbus arteriosus phenotype during larval stages (Fig. 1F-G, arrow; 1H: diameter during minimal and maximal distension, *: P<0.05 and ***: P<0.001 by Sidak's post-test after Two-Way ANOVA). This anatomical structure is strongly related to the aortic root in humans, which is the predominant location of aortic dilation in MFS. All fbn2b−/− embryos show abnormal early development of the caudal vein as a cavernous structure lacking vessel integrity (Fig. 2, arrowheads). This phenotype resolves in embryos retaining normal blood flow. We found that fbn2b−/− embryos raised in BDM to inhibit blood flow show a more severe caudal vein phenotype than wild-type (WT) controls (Fig. 2, yellow line: severe vascular dilation). Conclusion Loss of fbn2b, but not the other fibrillin genes, in zebrafish results in cardiovascular manifestations overlapping with MFS. These data indicate that fbn2b−/− zebrafish can be a relevant model to explore the mechanisms leading from fibrillin deficiency to the cardiovascular symptoms observed in MFS. Our preliminary results suggest that there is an interplay between fibrillin deficiency and biomechanical signaling. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fund Baillet Latour Grant for Medical Research Figure 1. Phenotype of fbn2b−/− larvae Figure 2. Effect of blood flow in fbn2b−/−

Protective Effects of Sal B on Oxidative Stress-Induced Aging by Regulating the Keap1/Nrf2 Signaling Pathway in Zebrafish

The models of oxidative damage-induced aging were established by adding ethanol (C2H5OH), hydrogen peroxide (H2O2) and 6-hydroxydopamine (6-OHDA) to zebrafish embryos in this research. To find effective protective drugs/foods, Salvianolic acid B (Sal B) was added after the embryos were treated by these oxidative reagents. After being treated with ethanol, H2O2 and 6-OHDA, the morphological changes were obvious and the deformities included spinal curvature, heart bleeding, liver bleeding, yolk sac deformity and pericardial edema, and the expression of oxidative stress-related genes Nrf2b, sod1 and sod2 and aging-related genes myl2a and selenbp1 were significantly up-regulated compared to the control group. While after adding 0.05 μg/mL and 0.5 μg/mL Sal B to the ethanol-treated group, death rates and MDA levels decreased, the activity of antioxidant enzyme (SOD, CAT and GSH-Px) changed and Nrf2b, sod1, sod2, myl2a, selenbp1, p53 and p21 were down-regulated compared to the ethanol-treated group. The bioinformatics analysis also showed that oxidative stress-related factors were associated with a variety of cellular functions and physiological pathways. In conclusion, Sal B can protect against aging through regulating the Keap1/Nrf2 pathway as well as antioxidative genes and enzyme activity.

Developmental toxicity of cryptotanshinone on the early-life stage of zebrafish development

Cryptotanshinone (Cry) has multiple potential functions in treating different diseases. Most studies on Cry focus on its pharmacological effects and mechanisms, but toxicological reports on Cry are rare. Zebrafish is used as a model organism in drug development as it saves costs and time. This work aimed to investigate the toxicity of Cry on zebrafish. Results showed that growth retardation, pericardial edema, and scoliosis occurred when zebrafish embryos were exposed to Cry, indicating its teratogenic effects. Cell apoptosis was observed in the brainstem area of embryos using acridine orange staining, and qPCR showed that caspase-3 was increased in Cry-exposed embryos. The results of locomotor activity and touched-evoke escape reaction experiments showed that Cry significantly reduced the swimming speed and escape reaction time of larvae.

Novel Pyridyl–Oxazole Carboxamides: Toxicity Assay Determination in Fungi and Zebrafish Embryos

Eight novel pyridyl–oxazole carboxamides were evaluated against fungi and displayed good fungicidal activities against Botrytis cinereal and Rhizoctonia solani. Preliminary bioassay results indicated that at 100 mg/L, compounds 6a–6e, 6g and 6h exhibited 100% fungicidal activities against Botrytis cinerea, and the compound 6b to Rhizoctonia solani at 100%. Then, the zebrafish embryo acute toxicity test was performed to assess the toxicity of 6b and 6c. A series of malformations appeared, when the zebrafish embryos were exposed to 6b and 6c, such as delayed yolk sac resorption, significant shortening of body length, pericardial edema, bending spine, lack of melanin, heart hemorrhage, head hemorrhage, delayed swim sac development, yolk malformation and head malformation. In addition, the acute toxicity of 6b to zebrafish embryo is 4.878 mg/L, and 6c is 6.257 mg/L.

Centriolar Protein C2cd3 Is Required for Craniofacial Development

The primary cilium is a ubiquitous, microtubule-based cellular organelle. Primary cilia dysfunction results in a group of disorders termed ciliopathies. C2 domain containing 3 centriole elongation regulator (C2cd3), encodes a centriolar protein essential for ciliogenesis. Mutations in human C2CD3 are associated with the human ciliopathy Oral-Facial-Digital syndrome type 14 (OFD14). In order to better understand the etiology of ciliopathies including OFD14, we generated numerous murine models targeting C2cd3. Initial analysis revealed several tissue-specific isoforms of C2cd3, and while the loss of C2cd3 has previously been reported to result in exencephaly, tight mesencephalic flexure, pericardial edema, abnormal heart looping and a twisted body axis, further analysis revealed that genetic background may also contribute to phenotypic variation. Additional analyses of a conditional allelic series targeting C-terminal PKC-C2 domains or the N-terminal C2CD3N-C2 domain of C2cd3 revealed a variable degree of phenotypic severity, suggesting that while the N-terminal C2CD3N-C2 domain was critical for early embryonic development as a whole, there was also a craniofacial specific role for the C2CD3N-C2 domains. Together, through generation of novel models and evaluation of C2cd3 expression, these data provide valuable insight into mechanisms of pathology for craniofacial ciliopathies that can be further explored in the future.

Di-2-ethylhexyl phthalate induces heart looping disorders during zebrafish development

Di-2-ethylhexyl phthalate (DEHP) is a type of plasticizer widely used in industry. It is well-known for its toxic effects to endocrine and reproductive systems and has been detected in amniotic fluid and placenta. In the present study, we explored the effects of DEHP on heart development by using zebrafish as a model organism. DEHP (0.02 pg) was injected into the yolk sac of zebrafish embryos at the one-cell stage. No significant difference was found in embryonic lethality between control and DEHP groups at 1-day postfertilization (dpf), but mortality significantly increased in DEHP groups at 2 and 3 dpf. The average heart rate was significantly reduced in the surviving DEHP-treated zebrafish larvae at 3 and 4 dpf. In addition, massive pericardial edema was found in DEHP-treated zebrafish (12.6 ± 1.5%), which was significantly higher than that of the control group. Serious heart looping disorder was also observed in DEHP-treated larvae, mainly manifested with an elongated atrial-ventricular distance. Moreover, the expression of heart development transcription factors was affected by DEHP injection. Real-time polymerase chain reaction confirmed that five transcription factors ( hand2, tp53, mef2c, esr1, and tbx18) were significantly downregulated in the DEHP group at 2 dpf, and three transcription factors ( zic3, tcf21, and gata4) were significantly upregulated. Our results emphasize the need for the development of a nontoxic plasticizer to prevent possible deleterious effects on humans and other life-forms.

Cytarabine-induced pericarditis confirmed using cardiac MRI after inconclusive echocardiography: A case report

Pericarditis is a rare but debilitating complication of cytarabine therapy. While echocardiography can aid with the diagnosis, cardiac MRI has superior accuracy in establishing the diagnosis. In this case, we describe a 65-year-old patient receiving cytarabine as part of induction chemotherapy for acute myeloid leukemia who developed acute pericarditis. Her cardiac MRI revealed pericardial edema on T2-weighted STIR imaging and pericardial late gadolinium enhancement which confirmed the diagnosis.

Microtubule Severing Protein Fignl2 Contributes to Endothelial and Neuronal Branching in Zebrafish Development

Previously, fidgetin (fign) and its family members fidgetin-like 1 (fignl1) and fidgetin-like 2 (fignl2) were found to be highly expressed during zebrafish brain development, suggesting their functions in the nervous system. In this study, we report the effects of loss-of-function of these genes on development. We designed and identified single-guide RNAs targeted to generate fign, fignl1, and fignl2 mutants and then observed the overall morphological and behavioral changes. Our findings showed that while fign and fignl1 null mutants displayed no significant defects, fignl2 null zebrafish mutants displayed pericardial edema, reduced heart rate, and smaller eyes; fignl2 null mutants responded to the light-darkness shift with a lower swimming velocity. fignl2 mRNAs were identified in vascular endothelial cells by in situ hybridization and re-analysis of an online dataset of single-cell RNAseq results. Finally, we used morpholino oligonucleotides to confirm that fignl2 knockdown resulted in severe heart edema, which was caused by abnormal vascular branching. The zebrafish fignl2 morphants also showed longer axonal length and more branches of caudal primary neurons. Taken together, we summarize that Fignl2 functions on cellular branches in endothelial cells and neurons. This study reported for the first time that the microtubule-severing protein Fignl2 contributes to cell branching during development.