Mapping the chromatin accessibility landscape of zebrafish embryogenesis at single-cell resolution by SPATAC-seq.

Currently, the dynamic accessible elements that determine regulatory programs responsible for the unique identity and function of each cell type during zebrafish embryogenesis lack detailed study. Here we present SPATAC-seq: a split-pool ligation-based assay for transposase-accessible chromatin using sequencing. Using SPATAC-seq, we profiled chromatin accessibility in more than 800,000 individual nuclei across 20 developmental stages spanning the sphere stage to the early larval protruding mouth stage. Using this chromatin accessibility map, we identified 604 cell states and inferred their developmental relationships. We also identified 959,040 candidate cis-regulatory elements (cCREs) and delineated development-specific cCREs, as well as transcription factors defining diverse cell identities. Importantly, enhancer reporter assays confirmed that the majority of tested cCREs exhibited robust enhanced green fluorescent protein expression in restricted cell types or tissues. Finally, we explored gene regulatory programs that drive pigment and notochord cell differentiation. Our work provides a valuable open resource for exploring driver regulators of cell fate decisions in zebrafish embryogenesis.

ULI-ssDRIP-seq revealed R-loop dynamics during vertebrate early embryogenesis.

R-loop, a chromatin structure containing one RNA:DNA hybrid and one unpaired single-stranded DNA, plays multiple biological roles. However, due to technical limitations, the landscapes and potential functions of R-loops during embryogenesis remain elusive. Here, we developed a quantitative and high-resolution ultra-low input R-loop profiling method, named ULI-ssDRIP-seq, which can map global R-loops with as few as 1000 cells. By using ULI-ssDRIP-seq, we reveal the R-loop dynamics in the zebrafish from gametes to early embryos. In oocytes, the R-loop level is relatively low in most regions of the nuclear genome, except maternal-inherited rDNA and mitochondrial genome. The correlation between R-loop and CG methylation dynamics during early development is relatively weak. Furthermore, either up- or down-regulation of global R-loops by knockdown or overexpression of RNase H1 causes a delay of embryonic development with dramatic expression changes in zygotic and maternal genes. This study provides comprehensive R-loop landscapes during early vertebrate embryogenesis and demonstrates the implication of R-loops in embryonic development.

LINE-1 transcription activates long-range gene expression.

Long interspersed nuclear element-1 (LINE-1 or L1) is a retrotransposon group that constitutes 17% of the human genome and shows variable expression across cell types. However, the control of L1 expression and its function in gene regulation are incompletely understood. Here we show that L1 transcription activates long-range gene expression. Genome-wide CRISPR-Cas9 screening using a reporter driven by the L1 5' UTR in human cells identifies functionally diverse genes affecting L1 expression. Unexpectedly, altering L1 expression by knockout of regulatory genes impacts distant gene expression. L1s can physically contact their distal target genes, with these interactions becoming stronger upon L1 activation and weaker when L1 is silenced. Remarkably, L1s contact and activate genes essential for zygotic genome activation (ZGA), and L1 knockdown impairs ZGA, leading to developmental arrest in mouse embryos. These results characterize the regulation and function of L1 in long-range gene activation and reveal its importance in mammalian ZGA.

Aagab is required for zebrafish larval development by regulating neural activity.

Clathrin-mediated endocytosis has been implicated in various physiological processes, including nutrient uptake, signal transduction, synaptic vesicle recycling, maintenance of cell polarity, and antigen presentation. Despite prior knowledge of its importance as a key regulator in promoting clathrin-mediated endocytosis, the physiological function of α- and γ-adaptin binding protein (aagab) remains elusive. In this study, we investigate the biological function of aagab during zebrafish development. We establish a loss-of-function mutant of aagab in zebrafish, revealing impaired swimming and early larval mortality. Given the high expression level of aagab in the brain, we probe into its physiological role in the nervous system. aagab mutants display subdued calcium responses and local field potential in the optic tectal neurons, aligning with reduced neurotransmitter release (e.g., norepinephrine) in the tectal neuropil of aagab mutants. Overexpressing aagab mRNA or nervous stimulant treatment in mutants restores neurotransmitter release, calcium responses, swimming ability, and survival. Furthermore, our observations show delayed release of FM 1-43 in AAGAB knockdown differentiated neuroblastoma cells, pointing towards a probable link to defective clathrin-mediated synaptic vesicle recycling. In conclusion, our study underscores the significance of Aagab in neurobiology and suggests its potential impacts on neurological disorders.

Temporospatial inhibition of Erk signaling is required for lymphatic valve formation.

Intraluminal lymphatic valves (LVs) and lymphovenous valves (LVVs) are critical to ensure the unidirectional flow of lymphatic fluid. Morphological abnormalities in these valves always cause lymph or blood reflux, and result in lymphedema. However, the underlying molecular mechanism of valve development remains poorly understood. We here report the implication of Efnb2-Ephb4-Rasa1 regulated Erk signaling axis in lymphatic valve development with identification of two new valve structures. Dynamic monitoring of phospho-Erk activity indicated that Erk signaling is spatiotemporally inhibited in some lymphatic endothelial cells (LECs) during the valve cell specification. Inhibition of Erk signaling via simultaneous depletion of zygotic erk1 and erk2 or treatment with MEK inhibitor selumetinib causes lymphatic vessel hypoplasia and lymphatic valve hyperplasia, suggesting opposite roles of Erk signaling during these two processes. ephb4b mutants, efnb2a;efnb2b or rasa1a;rasa1b double mutants all have defective LVs and LVVs and exhibit blood reflux into lymphatic vessels with an edema phenotype. Importantly, the valve defects in ephb4b or rasa1a;rasa1b mutants are mitigated with high-level gata2 expression in the presence of MEK inhibitors. Therefore, Efnb2-Ephb4 signaling acts to suppress Erk activation in valve-forming cells to promote valve specification upstream of Rasa1. Not only do our findings reveal a molecular mechanism of lymphatic valve formation, but also provide a basis for the treatment of lymphatic disorders.

Protocol for imaging nuclear pore complexes on isolated nuclei of zebrafish early embryos by field emission scanning electron microscopy.

Here, we present a protocol to observe the three-dimensional surface of nuclear pore complexes (NPCs) of vertebrate early embryos by field emission scanning electron microscopy (FESEM). We describe steps from zebrafish early embryo collection and nuclei exposure to FESEM sample preparation and final NPC state analysis. This approach provides an easy way to observe surface morphology of NPCs from the cytoplasmic side. Alternatively, additional purification steps after nuclei exposure supply intact nuclei for further mass spectrometry analysis or other utilization. For complete details on the use and execution of this protocol, please refer to Shen et al.1.

Comprehensive maturity of nuclear pore complexes regulates zygotic genome activation.

Nuclear pore complexes (NPCs) are channels for nucleocytoplasmic transport of proteins and RNAs. However, it remains unclear whether composition, structure, and permeability of NPCs dynamically change during the cleavage period of vertebrate embryos and affect embryonic development. Here, we report that the comprehensive NPC maturity (CNM) controls the onset of zygotic genome activation (ZGA) during zebrafish early embryogenesis. We show that more nucleoporin proteins are recruited to and assembled into NPCs with development, resulting in progressive increase of NPCs in size and complexity. Maternal transcription factors (TFs) transport into nuclei more efficiently with increasing CNM. Deficiency or dysfunction of Nup133 or Ahctf1/Elys impairs NPC assembly, maternal TFs nuclear transport, and ZGA onset, while nup133 overexpression promotes these processes. Therefore, CNM may act as a molecular timer for ZGA by controlling nuclear transport of maternal TFs that reach nuclear concentration thresholds at a given time to initiate ZGA.

Regulatory factor identification for nodal genes in zebrafish by causal inference.

Activation of nodal genes is critical for mesoderm and endoderm induction. Our previous study reported that zebrafish nodal genes ndr1/squint and ndr2/cyclops are coordinately regulated by maternal Eomesa, Hwa-activated ß-catenin (Hwa/ß-catenin) signaling, and Nodal autoregulation (Nodal/Smad2) signaling. However, the exact contribution and underlying mechanisms are still elusive. Here, we applied "causal inference" to evaluate the causal between the independent and dependent variables, and we found that Hwa/ß-catenin and Smad2 are the cause of ndr1 activation, while Eomesa is the cause of ndr2 activation. Mechanistically, the different cis-regulatory regions of ndr1 and ndr2 bound by Eomesa, ß-catenin, and Smad2 were screened out via ChIP-qPCR and verified by the transgene constructs. The marginal GFP expression driven by ndr1 transgenesis could be diminished without both maternal Eomesa and Hwa/ß-catenin, while Eomesa, not ß-catenin, could bind and activate ndr2 demonstrated by ndr2 transgenesis. Thus, the distinct regulation of ndr1/ndr2 relies on different cis-regulatory regions.

The second polar body contributes to the fate asymmetry in the mouse embryo.

The polar bodies (PBs) are extruded microcells during oocyte meiosis and generally regarded as inessentials for embryonic development. Therefore, PBs have been widely used as important materials for pre-implantation genetic diagnosis in human. Here we report that the second PB (PB2) in the mouse zygote may play roles in cell-fate specification and post-implantation development. A subset of mRNAs encoding pluripotency-related factors are enriched in PB2. Nascent proteins may be synthesized in PB2 after fertilization and transport from PB2 to the zygote before the two-cell stage. The PB2-attached blastomere (pbB) at the two-cell stage, compared to the other blastomere (npbB), likely contributes more descendants to the inner cell mass (ICM) lineage in the blastocyst. Removal of PB2 from the zygote or transient blockage of material exchange between PB2 and the zygote by nocodazole treatment appears to cause a loss of the ICM fate bias of pbB. PB2 removal or nocodazole treatment also results in abnormal post-implantation development. Injection of PB2 lysate into pbB of PB2-removed two-cell-stage embryos may reset the cell-fate preference and rescue post-implantation development. Our data collectively suggest that PB2 would demarcate the earliest cell-fate asymmetry of the mouse zygote and be required for post-implantation development.

Maternal Factors and Nodal Autoregulation Orchestrate Nodal Gene Expression for Embryonic Mesendoderm Induction in the Zebrafish.

Nodal proteins provide crucial signals for mesoderm and endoderm induction. In zebrafish embryos, the nodal genes ndr1/squint and ndr2/cyclops are implicated in mesendoderm induction. It remains elusive how ndr1 and ndr2 expression is regulated spatiotemporally. Here we investigated regulation of ndr1 and ndr2 expression using Mhwa mutants that lack the maternal dorsal determinant Hwa with deficiency in ß-catenin signaling, Meomesa mutants that lack maternal Eomesodermin A (Eomesa), Meomesa;Mhwa double mutants, and the Nodal signaling inhibitor SB431542. We show that ndr1 and ndr2 expression is completely abolished in Meomesa;Mhwa mutant embryos, indicating an essential role of maternal eomesa and hwa. Hwa-activated ß-catenin signaling plays a major role in activation of ndr1 expression in the dorsal blastodermal margin, while eomesa is mostly responsible for ndr1 expression in the lateroventral margin and Nodal signaling contributes to ventral expansion of the ndr1 expression domain. However, ndr2 expression mainly depends on maternal eomesa with minor or negligible contribution of maternal hwa and Nodal autoregulation. These mechanisms may help understand regulation of Nodal expression in other species.

The rise of developmental biology in China.

Developmental biology research in China started from experimental embryology, in particular from studies on aquatic and reptile animals. The recent growth of the developmental biology community in China parallels the increased governmental funding support and the recruitment of overseas talents. This flourishing field in China embraces the activities of developmental biology-related societies, national meetings, key research initiatives and talented scientists. The first Development paper from China, published in 2000, marked the beginning of a new era. More recently, the second decade in the 21st century witnessed the blossoming of developmental biology research in China. Significant research spotlights, technical advances, and up-and-coming areas will be discussed in this overview.

Understanding recurrent pregnancy loss: recent advances on its etiology, clinical diagnosis, and management.

Recurrent pregnancy loss (RPL) has become an important reproductive health issue worldwide. RPL affects about 2%-3% of reproductive-aged women, and makes serious threats to women's physical and mental health. However, the etiology of approximately 50% of RPL cases remains unknown (unexplained RPL), which poses a big challenge for clinical management of these patients. RPL has been widely regarded as a complex disease where its etiology has been attributed to numerous factors. Heretofore, various risk factors for RPL have been identified, such as maternal ages, genetic factors, anatomical structural abnormalities, endocrine dysfunction, prethrombotic state, immunological factors, and infection. More importantly, development and applications of next generation sequencing technology have significantly expanded opportunities to discover chromosomal aberrations and single gene variants responsible for RPL, which provides new insight into its pathogenic mechanisms. Furthermore, based upon patients' diagnostic evaluation and etiologic diagnosis, specific therapeutic recommendations have been established. This review will highlight current understanding and recent advances on RPL, with a special focus on the immunological and genetic etiologies, clinical diagnosis and therapeutic management.

Methylome inheritance and enhancer dememorization reset an epigenetic gate safeguarding embryonic programs.

Marked epigenetic reprogramming is essential to convert terminally differentiated gametes to totipotent embryos. It remains puzzling why postfertilization global DNA reprogramming occurs in mammals but not in nonmammalian vertebrates. In zebrafish, global methylome inheritance is however accompanied by extensive enhancer "dememorization" as they become fully methylated. By depleting maternal dnmt1 using oocyte microinjection, we eliminated DNA methylation in early embryos, which died around gastrulation with severe differentiation defects. Notably, methylation deficiency leads to derepression of adult tissue­specific genes and CG-rich enhancers, which acquire ectopic transcription factor binding and, unexpectedly, histone H3 lysine 4 trimethylation (H3K4me3). By contrast, embryonic enhancers are generally CG-poor and evade DNA methylation repression. Hence, global DNA hypermethylation inheritance coupled with enhancer dememorization installs an epigenetic gate that safeguards embryonic programs and ensures temporally ordered gene expression. We propose that "enhancer dememorization" underlies and unifies distinct epigenetic reprogramming modes in early development between mammals and nonmammals.

5' Half of specific tRNAs feeds back to promote corresponding tRNA gene transcription in vertebrate embryos.

5'tRFls are small transfer RNA (tRNA) fragments derived from 5' half of mature tRNAs. However, it is unknown whether 5'tRFls could feed back to regulate tRNA biogenesis. Here, we show that 5'tRFlGly/GCC and 5'tRFlGlu/CTC function to promote transcription of corresponding tRNA genes and are essential for vertebrate early embryogenesis. During zebrafish embryogenesis, dynamics of 5'tRFlGly/GCC and 5'tRFlGlu/CTC levels correlates with that of tRNAGly/GCC and tRNAGlu/CTC levels. Morpholino-mediated knockdown of 5'tRFlGly/GCC or 5'tRFlGlu/CTC down-regulates tRNAGly/GCC or tRNAGlu/CTC levels, respectively, and causes embryonic lethality that is efficiently rescued by coinjection of properly refolded corresponding tRNA. In zebrafish embryos, tRNA:DNA and 5'tRFl:DNA hybrids commonly exist on the template strand of tRNA genes. Mechanistically, unstable 5'tRFl:DNA hybrid may prevent the formation of transcriptionally inhibitory stable tRNA:DNA hybrids on the same tRNA loci so as to facilitate tRNA gene transcription. The uncovered mechanism may be implicated in other physiological and pathological processes.

A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission.

Endosome fission is essential for cargo sorting and targeting in the endosomal system. However, whether organelles other than the endoplasmic reticulum (ER) participate in endosome fission through membrane contacts is unknown. Here, we characterize a Golgi-derived vesicle, the SEC14L2 compartment, that plays a unique role in facilitating endosome fission through ternary contacts with endosomes and the ER. Localized to the ER-mediated endosome fission site, the phosphatidylinositol transfer protein SEC14L2 promotes phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 3-phosphate (PtdIns3P) conversion before endosome fission. In the absence of SEC14L2, endosome fission is attenuated and more enlarged endosomes arise due to endosomal accumulation of PtdIns4P and reduction in PtdIns3P. Collectively, our data suggest roles of the Golgi network in ER-associated endosome fission and a mechanism involving ER-endosome contacts in the regulation of endosomal phosphoinositide conversion.

TGFß family signaling and development.

The transforming growth factor ß (TGFß) signaling family is evolutionarily conserved in metazoans. The signal transduction mechanisms of TGFß family members have been expansively investigated and are well understood. During development and homeostasis, numerous TGFß family members are expressed in various cell types with temporally changing levels, playing diverse roles in embryonic development, adult tissue homeostasis and human diseases by regulating cell proliferation, differentiation, adhesion, migration and apoptosis. Here, we discuss the molecular mechanisms underlying signal transduction and regulation of the TGFß subfamily pathways, and then highlight their key functions in mesendoderm induction, dorsoventral patterning and laterality development, as well as in the formation of several representative tissues/organs.

Mini-III RNase-based dual-color system for in vivo mRNA tracking.

Mini-III RNase (mR3), a member of RNase III endonuclease family, can bind to and cleave double-stranded RNAs (dsRNAs). Inactive mR3 protein without the α5ß-α6 loop loses the dsRNA cleavage activity, but retains dsRNA binding activity. Here, we establish an inactive mR3-based non-engineered mR3/dsRNA system for RNA tracking in zebrafish embryos. In vitro binding experiments show that inactive Staphylococcus epidermidis mR3 (dSmR3) protein possesses the highest binding affinity with dsRNAs among mR3s from other related species, and its binding property is retained in zebrafish embryos. Combined with a fluorescein-labeled antisense RNA probe recognizing the target mRNAs, dSmR3 tagged with a nuclear localization sequence and a fluorescent protein could allow visualization of the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach for tracking non-engineered RNAs in real-time, which will help understand how endogenous RNAs dynamically move during embryonic development.

Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species.

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.