Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.

Abstract Vertebrate heart development requires precise regulation of morphogenetic movements and dysregulation causes congenital heart defects. However, cell polarity remodeling during early heart tube formation remains underexplored. Here, we show that zebrafish ventricular cardiomyocyte precursors (vCPCs) undergo striking polarity reorientation as they transition from a single-layered epithelium to a transient double-layered configuration by involution. During this process, vCPCs remove inner adherens components and re-establish polarity toward the ECM-attached outer surface, enabling proper directional extension to form the ventricle. We identify the junctional scaffold protein Afadin a (Afdna) as a critical regulator. Afdna localizes to the outer and lateral membrane to restrict Rap1 activity, facilitating Podocalyxin translocation and adhesion disassembly at the inner interface between vCPC double layers. afdna mutants exhibit a multilayered ventricle with impaired blood flow, phenocopying human congenital ventricular obliteration. Our findings uncover a polarity-based mechanism ensuring ventricular chamber formation and provide insight into ventricular obliteration.

During vertebrate embryogenesis, hematopoietic stem cells (HSCs) are believed to almost exclusively arise from hemogenic endothelial cells (ECs) of the dorsal aorta through endothelial-to-hematopoietic transition (EHT). It remains elusive whether HSCs can be generated by other tissues. Sclerotomal cells (SCs) in embryonic somites predominantly form vertebrae and ribs, and some SCs may also insert into aortic endothelia. Here we show that a subset of SCs directly enter the vascular lumen to become hematopoietic stem and progenitor cells (HSPCs) in an EHT-independent way in both zebrafish and mouse embryos. The sclerotome-derived HSPCs (scHSPCs) contribute to various blood cells throughout the lifetime. The proportion of scHSPCs increases dramatically when endothelium hematopoiesis is defective. Thus, the sclerotomal hematopoiesis is evolutionarily conserved and would ensure the robustness of hematopoiesis.

Sex determination systems display striking evolutionary flexibility, yet the mechanisms underlying their transitions remain poorly understood. Using newly generated genome assemblies, we investigated the evolving sex-determining system in the amphioxus Branchiostoma belcheri. We identified two female-specific sex-determining regions (SDRs) on chromosome 13, both derived from independent transpositions of the autosomal gene tesD, which shows testis-specific expression in amphioxus species. CRISPR/Cas9 knockout experiments in Branchiostoma floridae confirmed that tesD functions as a male-determination gene, with loss of function producing an all-female phenotype. In B. belcheri, the older SDR (tesDwa) inserted into the coding region of twai, while the younger SDR (tesDwb), flanked by active Zator-1 transposons, inserted into the 3' UTR of vps9c and later translocated to autosomes in ~10% of individuals. Transcriptomic analyses revealed that W-linked tesDwa and tesDwb produce antisense long non-coding RNAs that likely suppress tesD transcription in females, whereas autosomal tesDwb is not expressed and appears non-functional. The insertion sites and co-transcription with host genes suggest promoter hijacking. Together, these findings demonstrate that recurrent transpositions can generate new functional SDRs that coexist with older ones, driving dynamic turnover of sex determination in B. belcheri.

The germ cell fate in zebrafish is determined by germ plasm, whereas mammalian germ cell fate is induced by bone morphogenetic protein (BMP) signaling. It remains elusive whether BMP signaling is implicated in zebrafish germ cell development. Here, we demonstrate that BMP-Smad1/9 signaling plays a critical role in zebrafish primordial germ cell (PGC) maintenance rather than fate determination. BMP inhibition or smad1/9 knockdown reduces PGC numbers. Furthermore, we generated PGC-specific smad1/9 knockouts using a transgenic approach with PGC-specifically expressed Cas9 and ubiquitously expressed guide RNAs. Smad1/9 deficiency in PGCs leads to impaired PGC proliferation and increased apoptosis, consequently reducing PGC numbers. Transcriptome analysis revealed unchanged PGC-specific gene expression, but a marked upregulation of DNA damage response-related genes, which is validated by ectopic ATR-pChk1 activation in PGCs and PGC restoring by ATR inhibition. Collectively, these findings underscore conserved but functionally distinct roles of BMP signaling in vertebrate PGC development.

The dorsal organizer, essential for vertebrate embryonic axis formation, is induced by microtubule-mediated transport of maternal determinants. Maternal Huluwa (Hwa) has been identified as an essential organizer inducer in zebrafish and frogs, functioning at midblastula stages to activate β-catenin signaling in the preorganizer. It remains unknown if maternal Hwa functions at or before fertilization. Here, we report that maternal Hwa protein is critical for organizing the vegetal parallel microtubule array immediately after fertilization in zebrafish. Hwa protein and mRNA are enriched at the vegetal pole and facilitate microtubule network formation, enabling asymmetrical transport of dorsal determinants. Loss of maternal Hwa disrupts this microtubule architecture and abrogates mRNA transport, revealing a self-reinforcing mechanism where Hwa regulates its own asymmetrical distribution. Hwa may regulate the vegetal microtubule network through other signaling pathways and microtubule-associated proteins. Our findings establish a dual-phase model of dorsal specification: Hwa initially governs symmetry breaking through postfertilization microtubule organization and later on activates β-catenin signaling at blastula stages. This work provides fundamental insights into how the key maternal factor regulates the organizer and body axis formation at different developmental stages. Here they show that maternal Huluwa protein localizes at the zebrafish egg’s vegetal pole, organizing postfertilization microtubules to transport dorsal determinants and establishing the body axis through a dual-phase mechanism.

Proteins without transmembrane domains could be anchored to the cell surface for regulating various biological processes when covalently linked to glycosylphosphatidylinositol (GPI) molecules by the GPI transamidase (GPIT) complex. However, it remains poorly understood whether and how the GPIT complex affects primordial germ cell (PGC) development. In this study, we report the important roles of the GPIT complex in PGC migration and development in zebrafish embryos. Mutation of pigu or pigk, both encoding essential GPIT complex subunits, resulted in defective PGC migration with ectopically located PGCs and reduction of PGC counts. Notably, a detailed analysis of filopodia in PGCs revealed the attenuated polarity of filopodia distribution along the migration direction in mutant embryos. PGC transplantation and PGC-specific rescue experiments demonstrated that both PGC and somatic cell-expressed Pigu are required for PGC migration. Furthermore, expression levels of PGC-specific genes decreased in pigu mutant PGCs with the derepression of somatic cell genes. Hence, we propose that the GPIT complex plays a critical role during PGC migration and development.

The specification and maintenance of germ cell lineage are vital for reproductive development and heredity. The germ cell fate in some species, such as zebrafish and Xenopus , is determined by maternally supplied germ plasm, whereas in mammals it is induced by bone morphogenetic protein (BMP) signaling after implantation. It remains elusive whether BMP signaling is implicated in zebrafish germ cell development. Here, we demonstrate that, in zebrafish, BMP–Smad1/9 signaling is required for primordial germ cell (PGC) maintenance but not for PGC fate determination or migration. BMP inhibitor treatment or whole-embryo smad1/9 knockdown reduces the number of PGCs. To investigate cell-autonomous roles of BMP–Smad1/9 in PGCs, we generated PGC-specific smad1 / 9 knockouts using a double-transgenic approach with PGC-specific expression of Cas9 and ubiquitous expression of guide RNAs (gRNAs). Loss of Smad1/9 in PGCs leads to impaired PGC proliferation and increased apoptosis, resulting in reduced PGC numbers and adult sex bias. Moreover, transcriptome analysis of smad1 -deficient PGCs revealed no significant changes in the expression of PGC-specific genes, but a marked upregulation of genes involved in cell cycle checkpoints and DNA damage repair. Notably, ectopic ATR–pChk1 activation in smad1 -cKO PGCs validates cell cycle defects and DNA replication stress. ATR inhibition restores PGC numbers in mutant embryos. Collectively, these findings underscore a critical role of BMP–Smad1/9 signaling in zebrafish PGC population maintenance, while it is dispensable for PGC fate determination and migration.

A central goal of evolutionary developmental biology is to decipher the evolutionary pattern of gene regulatory networks (GRNs) that control embryonic development, and the mechanism underlying GRNs evolution. The Nodal signaling that governs the body axes of deuterostomes exhibits a conserved GRN orchestrated principally by Nodal, Gdf1/3, and Lefty. Here we show that this GRN has been rewired in cephalochordate amphioxus. We found that while the amphioxus Gdf1/3 ortholog exhibited nearly no embryonic expression, its duplicate Gdf1/3-like, linked to Lefty, was zygotically expressed in a similar pattern as Lefty. Consistent with this, while Gdf1/3-like mutants showed defects in axial development, Gdf1/3 mutants did not. Further transgenic analyses showed that the intergenic region between Gdf1/3-like and Lefty could drive reporter gene expression as that of the two genes. These results indicated that Gdf1/3-like has taken over the axial development role of Gdf1/3 in amphioxus, possibly through hijacking Lefty enhancers. We finally demonstrated that, to compensate for the loss of maternal Gdf1/3 expression, Nodal has become an indispensable maternal factor in amphioxus and its maternal mutants caused axial defects as Gdf1/3-like mutants. We therefore demonstrated a case that the evolution of GRNs could be triggered by enhancer hijacking events. This pivotal event has allowed the emergence of a new GRN in extant amphioxus, presumably through a stepwise process. In addition, the co-expression of Gdf1/3-like and Lefty achieved by a shared regulatory region may have provided robustness during body axis formation, which provides a selection-based hypothesis for the phenomena called developmental system drift.

Despite their pivotal role, the evolutionary origins of vertebrate digestive systems remain enigmatic. We explored the cellular characteristics of the amphioxus ( Branchiostoma floridae ) digestive tract, a model for the presumed primitive chordate digestive system, using bulk tissue companioned with single-cell RNA sequencing. Our findings reveal segmentation and a rich diversity of cell clusters, and we highlight the presence of epithelial-like, ciliated cells in the amphioxus midgut and describe three types of endocrine-like cells that secrete insulin-like, glucagon-like, and somatostatin-like peptides. Furthermore, Pdx , Ilp1 , Ilp2 , and Ilpr knockout amphioxus lines revealed that, in amphioxus, Pdx does not influence Ilp expression. We also unravel similarity between amphioxus Ilp1 and vertebrate insulin-like growth factor 1 (Igf1) in terms of predicted structure, effects on body growth and amino acid metabolism, and interactions with Igf-binding proteins. These findings indicate that the evolutionary alterations involving the regulatory influence of Pdx over insulin gene expression could have been instrumental in the development of the vertebrate digestive system.

Dynamic epigenomic reprogramming occurs during mammalian oocyte maturation and early development. However, the underlying transcription circuitry remains poorly characterized. By mapping cis-regulatory elements using H3K27ac, we identified putative enhancers in mouse oocytes and early embryos distinct from those in adult tissues, enabling global transitions of regulatory landscapes around fertilization and implantation. Gene deserts harbour prevalent putative enhancers in fully grown oocytes linked to oocyte-specific genes and repeat activation. Embryo-specific enhancers are primed before zygotic genome activation and are restricted by oocyte-inherited H3K27me3. Putative enhancers in oocytes often manifest H3K4me3, bidirectional transcription, Pol II binding and can drive transcription in STARR-seq and a reporter assay. Finally, motif analysis of these elements identified crucial regulators of oogenesis, TCF3 and TCF12, the deficiency of which impairs activation of key oocyte genes and folliculogenesis. These data reveal distinctive regulatory landscapes and their interacting transcription factors that underpin the development of mammalian oocytes and early embryos. Liu, Xie and colleagues profile putative cis-regulatory elements in mouse oocytes and pre-implantation embryos. They further validate putative enhancers in oocytes and identify the transcription factors TCF3 and TCF12 as key regulators of folliculogenesis.

The cell fate of primordial germ cell (PGC) in zebrafish is pre-determined by maternally deposited germ plasm, which is packaged into ribonucleoprotein complex in oocytes and inherited into PGC-fated cells in embryos. However, the maternal factors regulating the assembly of germ plasm and PGC development remain poorly understood. In this study, we report that the maternal transcription factor Znf706 regulates the assembly of germ plasm factors into a granule-like structure localized perinuclearly in PGC during migration. Maternal and zygotic mutants of znf706 exhibit deficient germ plasm scattering at the early embryonic stage, decreased PGC numbers with some mislocation during PGC migration, and a lower female ratio in adulthood. Notably, the implementation of Znf706 CUT&Tag and RNA-seq on immature oocytes uncovers that Znf706 in stage I oocytes may promote transcription of several mitochondrial genes in addition to other functions. Hence, we propose that Znf706 is implicated in germ plasm assembly and PGC development in zebrafish.

Histone modification signatures mark sites of transcriptional regulatory elements and regions of gene activation and repression. These sites vary among cell types and undergo dynamic changes during development and in diseases. Oocytes produce numerous maternal factors essential for early embryonic development, which are significantly influenced by epigenetic modifications. The profiling of epigenetic modifications during oogenesis remains uniquely challenging due to the presence of numerous tightly wrapped granulosa cells. Here, we successfully established a low-input CUT&Tag (Cleavage Under Targets and Tagmentation) method tailored for zebrafish stage I oocytes. This advanced technique enables high-resolution profiling of histone modifications and DNA-binding proteins, critical for understanding chromatin dynamics in developing oocytes. In this study, we detailed the workflow for this technique, including the isolation of pure stage I oocytes without somatic cells, library construction and quality monitoring. Our results demonstrate the method’s efficacy by identifying distinct histone modification patterns and analyzing differentially expressed genes in oocytes with and without granulosa cells. We also successfully profiled divergent histone modifications in oocytes derived from wild-type and huluwa mutants. These advancements overcome technical challenges in epigenetic research on zebrafish oocytes and establish a solid foundation for exploring the epigenetic regulatory mechanisms of maternal contribution.

Spatiotemporal regulation of cell type-specific gene expression is essential to convert a zygote into a complex organism that contains hundreds of distinct cell types. A class of cis-regulatory elements called enhancers, which have the potential to enhance target gene transcription, are crucial for precise gene ex-pression programs during development. Following decades of research, many enhancers have been discovered and how enhancers become activated has been extensively studied. However, the mechanisms underlying enhancer si-lencing are less well understood. We review current understanding of enhancer decommissioning and dememorization, both of which enable enhancer silenc-ing. We highlight recent progress from genome-wide perspectives that have re-vealed the life cycle of enhancers and how its dynamic regulation underlies cell fate transition, development, cell regeneration, and epigenetic reprogramming.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

The gene family protein phosphatase 2C (PP2C) is related to developmental processes and stress responses in plants. Barley (Hordeum vulgare L.) is a popular cereal crop that is primarily utilized for human consumption and nutrition. However, there is little knowledge regarding the PP2C gene family in barley. In this study, a total of 1635 PP2C genes were identified in 20 barley pan-genome accessions. Then, chromosome localization, physical and chemical feature predictions and subcellular localization were systematically analyzed. One wild barley accession (B1K-04-12) and one cultivated barley (Morex) were chosen as representatives to further analyze and compare the differences in HvPP2Cs between wild and cultivated barley. Phylogenetic analysis showed that these HvPP2Cs were divided into 12 subgroups. Additionally, gene structure, conserved domain and motif, gene duplication event detection, interaction networks and gene expression profiles were analyzed in accessions Morex and B1K-04-12. In addition, qRT-PCR experiments in Morex indicated that seven HvMorexPP2C genes were involved in the response to aluminum and low pH stresses. Finally, a series of positively selected homologous genes were identified between wild accession B1K-04-12 and another 14 cultivated materials, indicating that these genes are important during barley domestication. This work provides a global overview of the putative physiological and biological functions of PP2C genes in barley. We provide a broad framework for understanding the domestication- and evolutionary-induced changes in PP2C genes between wild and cultivated barley.

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.

Amphioxus is a promising model organism for understanding the origin and evolution of vertebrates due to its basal phylogenetic position among chordates. We here compared the mutation efficacy and mutation type of tail tips and gametes of amphioxus founders injected with Cas9 protein and six different sgRNAs targeting five distinct genes, and revealed a strong correlation for mutation efficacy and a mild correlation for mutation type among the two tissues. In addition, we also observed a positive relationship between gene insertions observed in tail tips and gametes of amphioxus founders injected with Tol2 transposase and two different transgenic constructs. Finally, we showed that amphioxus larvae which had their tail tips cut at the 3–4 gill-slit stage were able to recover within 6 days and developed a normal number of gonads at the adult stage, and that F0 larvae carry similar mutation efficacy and type in the posterior end to that in the tail tips after their metamorphosis. Together, these findings suggest a great potential for obtaining valid amphioxus founders with desired mutations and transgenes at as early as the early larval stage, which will certainly speed up the generation of amphioxus mutants and transgenes and make it more cost- and labor-effective.

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 tissue–specific 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.