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Adult mammalian hearts show limited capacity to proliferate after injury, while zebrafish are capable to completely regenerate injured hearts through the proliferation of spared cardiomyocytes. BMP and Notch signaling pathways have been implicated in cardiomyocyte proliferation during zebrafish heart regeneration. However, the molecular mechanism underneath this process as well as the interaction between these two pathways remains to be further explored. In this study we showed BMP signaling was activated after ventricle ablation and acted epistatic downstream of Notch signaling. Inhibition of both signaling pathways differentially influenced ventricle regeneration and cardiomyocyte proliferation, as revealed by time-lapse analysis using a cardiomyocyte-specific FUCCI (fluorescent ubiquitylation-based cell cycle indicator) system. Further experiments revealed that inhibition of BMP and Notch signaling led to cell-cycle arrest at different phases. Overall, our results shed light on the interaction between BMP and Notch signaling pathways and their functions in cardiomyocyte proliferation during cardiac regeneration.

Nanomaterials have broad application prospects in human health and diseases, including medical imaging and drug delivery and treatment. With the gradual advent of the nano era, the safety of nanomaterials in biology and the environment must be evaluated more widely and deeply. Herein, we scanned some widely used biomedical nanomaterials toxicity with an in vivo zebrafish model and demonstrated that cadmium selenide (CdSe) quantum dots (QDs) were the most toxic. Embryos exposed to CdSe QDs showed the activation of NADPH oxidase nox4 and an excessive generation of reactive oxygen species (ROS). The expression of antioxidant defense enzyme superoxide dismutase sod1 and glutathione peroxidase gpx1 increased the response to oxidative stress. Further, excessive ROS activated apoptosis by the p53- and caspase-3-mediated signaling pathway. Several modification methods, including silica coating, core–shell structure development, and organic molecular camouflage were applied to reduce the toxicity of the materials, whereby the polyethylene glycol (PEG) camouflage method exhibited better performance for improving the biocompatibility of CdSe QDs, which could effectively reduce ROS and apoptosis induced by CdSe QDs in the zebrafish model. Meanwhile, we employed a series of genetically modified zebrafish models to assess the CdSe QDs toxicity, and the polyethylene glycol (PEG) camouflage showed protective effects in the heart, nerves, blood vessels, and immune system. CdSe QDs disguised by PEG could effectively alleviate the toxicity of CdSe QDs in multiple organs. In summary, our data revealed that CdSe QDs induced an excessive production of ROS, which would activate the apoptosis pathway mediated by p53 and caspase-3 and resulted in cardiovascular system, nervous system, and immune system toxicity. PEG camouflage could effectively decrease ROS generation and apoptosis to improve the biocompatibility of CdSe QDs.

Mammals exhibit limited heart regeneration ability, which can lead to heart failure after myocardial infarction. In contrast, zebrafish exhibit remarkable cardiac regeneration capacity. Several cell types and signaling pathways have been reported to participate in this process. However, a comprehensive analysis of how different cells and signals interact and coordinate to regulate cardiac regeneration is unavailable. We collected major cardiac cell types from zebrafish and performed high-precision single-cell transcriptome analyses during both development and post-injury regeneration. We revealed the cellular heterogeneity as well as the molecular progress of cardiomyocytes during these processes, and identified a subtype of atrial cardiomyocyte exhibiting a stem-like state which may transdifferentiate into ventricular cardiomyocytes during regeneration. Furthermore, we identified a regeneration-induced cell (RIC) population in the epicardium-derived cells (EPDC), and demonstrated Angiopoietin 4 (Angpt4) as a specific regulator of heart regeneration. angpt4 expression is specifically and transiently activated in RIC, which initiates a signaling cascade from EPDC to endocardium through the Tie2-MAPK pathway, and further induces activation of cathepsin K in cardiomyocytes through RA signaling. Loss of angpt4 leads to defects in scar tissue resolution and cardiomyocyte proliferation, while overexpression of angpt4 accelerates regeneration. Furthermore, we found that ANGPT4 could enhance proliferation of neonatal rat cardiomyocytes, and promote cardiac repair in mice after myocardial infarction, indicating that the function of Angpt4 is conserved in mammals. Our study provides a mechanistic understanding of heart regeneration at single-cell precision, identifies Angpt4 as a key regulator of cardiomyocyte proliferation and regeneration, and offers a novel therapeutic target for improved recovery after human heart injuries.

It remains challenging to construct a complete cell lineage map of the origin of vascular endothelial cells in any vertebrate embryo. Here, we report the application ofin totolight-sheet fluorescence imaging of embryos to tracing the origin of vascular endothelial cells (ECs) at single-cell resolution in zebrafish. We first adapted a previously-reported method to mount embryos and light-sheet imaging, created analignment,fusion, andextraction all-in-one software (AFEIO) for processing big data, and performed quantitative analysis of cell lineage relationships using commercially-available Imaris software. Our data revealed that vascular ECs originated from broad regions of the gastrula along the dorsal-ventral and anterior-posterior axes, of which the dorsal-anterior cells contributed to cerebral ECs, the dorsal-lateral cells to anterior trunk ECs, and the ventral-lateral cells to posterior trunk and tail ECs. Therefore, this work, to our knowledge, charts the first comprehensive map of the gastrula origin of vascular ECs in zebrafish, and has potential applications for studying the origin of any embryonic organs in zebrafish and other model organisms.

Airy light-sheet microscopy is rapidly gaining importance for imaging intact biological specimens because of the rapid speed, high resolution, and wide field nature of the imaging method. However, the depth of field (DOF) of the detection objective imposes limitations on the modulation transfer function (MTF) of the light sheet, which in turn affects the size of the field of view (FOV). Here we present an optimized phase modulation model, based on ‘Airy-like’ beam family, to stretch the curved lobes, which brings a wider FOV while maintaining high resolution. In addition, we further develop a planar ‘Airy-like’ light-sheet by two-photon excitation which can avoid the deconvolution process. We validated the new imaging method by performing a real-time monitoring of the dynamic process of cerebral hemorrhage in zebrafish larva. The proposed Airy-like beam-based light-sheet microscopy has great potential to be applied to the precise screening of cerebral hemorrhage-related drugs to help precision medicine in the future.

Background:Nanomaterials are under a wide range of application prospects in human health and disease, such as medical imaging and drug delivery and treatment. With the gradual advent of the nano era, the safety of nanomaterials in biology must be evaluated more widely and deeply. Therefore, superficial safety assessment based on specific cell lines in vitro is difficult to meet the current needs. Multi-organs assessment in vivo will be beneficial in establishing a more comprehensive pathway for the understanding of nanomaterials-induced biotoxicity. Results:In this work, we employed a series of genetically modified zebrafish models for nanomaterials toxicity assessment. The results demonstrated that cadmium selenide (CdSe) quantum dot (QDs) was the most toxic after scanning some popular nanomaterials. Among modified methods of silica coating, core-shell structure development and organic molecular camouflage, the polyethylene glycol camouflage method exhibits a better performance of improving the biocompatibility of CdSe QDs in multiple-organs including hearts, nerves, blood vessels, and immune system. Conclusion:Our work provides an alternative paradigm for more in-depth and sensitive preclinical validation of biocompatibility, especially in neurotoxicity and cardiotoxicity of embryos, and a guidance for reducing the toxicity of biomedical materials.

Conventional genetic screens for recessive mutants are inadequate for studying biological processes in the adult vertebrate due to embryonic lethality. Here, we report that a novel inducible mutagenesis system enables to study gene function in both embryonic and adult zebrafish. This system yields genetic mutants with conditional ectopic over- or under-expression of genes in F1 heterozygotes by utilizing inducible Tet-On transcriptional activation of sense or anti-sense transcripts from entrapped genes by Tol2 transposase-meditated transgenesis. Pilot screens identified 37 phenotypic mutants displaying embryonic defects (34 lines), adult fin regeneration defects (7 lines), or defects at both stages (4 lines). Combination of various techniques (such as: generating a new mutant allele, injecting gene specific morpholino or mRNA etc) confirms that Dox-induced embryonic abnormalities in 10 mutants are due to dysfunction of entrapped genes; and that Dox-induced under-expression of 6 genes causes abnormal adult fin regeneration. Together, this work presents a powerful mutagenesis system for genetic analysis from zebrafish embryos to adults in particular and other model organisms in general.

Novel stem cells expressing stage-specific embryonic antigen 3 (SSEA-3) reside among human dermal fibroblasts and are known as multilineage-differentiating stress-enduring (Muse) cells. They enhance the generation efficiency of induced pluripotent stem cells. However, Muse cells have only been found in humans. We aimed to isolate SSEA3-positive cells from terminally differentiated skin fibroblasts of adult goat and determine their pluripotency. Cell clusters from SSEA3(+) populations possessed stem cell-like morphological features and normal karyotypes, were consistently positive for alkaline phosphatase, and expressed stem cell pluripotency markers. These SSEA3(+) cells remained undifferentiated over eight passages in suspension culture and were able to differentiate into cells of all three germ layers in vitro and in vivo. Our combined findings suggest that a subset of adult stem cells expressing SSEA3 also exist among adult goat skin fibroblasts. We are the first to report that multipotent adult goat cells exist among terminally differentiated goat skin in suspension culture. Our results also provide a promising platform for generation of a transgenic goat, because the undifferentiated state of stem cells was thought to be more efficient as donor cells for somatic cell nuclear transfer.

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

Previous studies have demonstrated that skin stem cells expressing the pluripotency marker stage-specific embryonic antigen 3 (SSEA3) are easier to reprogram into induced pluripotent stem cells (iPSCs) than skin fibroblasts. Furthermore, it is widely speculated that the undifferentiated state may make stem cells more efficient donor cells for somatic cell nuclear transfer (SCNT). In this study, we isolated SSEA3(+) cells from goat skin fibroblast cells (SFCs) using fluorescence-activated cell sorting (FACS) and examined expression of pluripotency markers and in vitro development of cloned embryos following SCNT. Results showed that cell clusters from SSEA3(+) cells were consistently positive for alkaline phosphatase staining and pluripotency markers, Nanog, Oct4, Sox2, and SSEA3. The cleavage rate of cloned embryos derived from SSEA3(+) cells did not differ compared with SFCs (70.5±0.8% and 68.4±2.1%, respectively), but was significantly higher compared with SSEA3(-) cells (64.9±1.6%, p0.05). The blastocyst rate was significantly increased in the SSEA3(+) cell group compared with the SFC and SSEA3(-) cell groups (30.3±1.2% vs. 21.2±0.9 and 19.0±1.0%, respectively, p0.05). The quality of cloned blastocysts from SSEA3(+) cells was higher compared with SFCs and SSEA3(-) cells, based on total cell number and number of apoptotic cells per blastocyst. These findings suggest that using SSEA3(+) cells as donors for SCNT is beneficial for enhancing in vitro development and quality of cloned goat embryos.