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.

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.

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.

Sec14l3 potentiates VEGFR2 signaling to regulate zebrafish vasculogenesis.

Vascular endothelial growth factor (VEGF) regulates vasculogenesis by using its tyrosine kinase receptors. However, little is known about whether Sec14-like phosphatidylinositol transfer proteins (PTP) are involved in this process. Here, we show that zebrafish sec14l3, one of the family members, specifically participates in artery and vein formation via regulating angioblasts and subsequent venous progenitors' migration during vasculogenesis. Vascular defects caused by sec14l3 depletion are partially rescued by restoration of VEGFR2 signaling at the receptor or downstream effector level. Biochemical analyses show that Sec14l3/SEC14L2 physically bind to VEGFR2 and prevent it from dephosphorylation specifically at the Y1175 site by peri-membrane tyrosine phosphatase PTP1B, therefore potentiating VEGFR2 signaling activation. Meanwhile, Sec14l3 and SEC14L2 interact with RAB5A/4A and facilitate the formation of their GTP-bound states, which might be critical for VEGFR2 endocytic trafficking. Thus, we conclude that Sec14l3 controls vasculogenesis in zebrafish via the regulation of VEGFR2 activation.

A harlequin ichthyosis pig model with a novel ABCA12 mutation can be rescued by acitretin treatment.

Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 A>G in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies.

prpf4 is essential for cell survival and posterior lateral line primordium migration in zebrafish.

Prpf4 (pre-mRNA processing factor 4), a key component of spliceosome, plays critical roles in pre-mRNA splicing and its mutations result in retinitis pigmentosa due to photoreceptor defects. In this study, we characterized a zebrafish prpf4t243 mutant harboring a Tol2 transposon-based gene trap cassette in the third intron of the prpf4 gene. Cells in the brain and spinal cord gradually undergo p53-dependent apoptosis after 28 hpf in prpf4t243 mutants, suggesting that a widespread function of prpf4 in neural cell survival. In addition, prpf4 is essential for survival of posterior lateral line primordial (pLLP) cells. prpf4 deficiency perturbs Fgf, Wnt/ß-catenin and chemokine signaling pathways and impairs pLLP migration. RNA-Seq analysis suggests that prpf4 deficiency may impair spliceosome assembly, leading to compensatory upregulation of core spliceosomal genes and alteration of pre-mRNA splicing. Taken together, our studies uncover an essential role of prpf4 in pre-mRNA splicing, cell survival and pLLP migration.

Thyroid hormone regulates hematopoiesis via the TR-KLF9 axis.

Congenital hypothyroidism (CH) is one of the most prevalent endocrine diseases, for which the underlying mechanisms remain unknown; it is often accompanied by anemia and immunodeficiency in patients. Here, we created a severe CH model together with anemia and T lymphopenia to mimic the clinical features of hypothyroid patients by ethylnitrosourea (ENU) mutagenesis in Bama miniature pigs. A novel recessive c.1226A>G transition of the dual oxidase 2 (DUOX2) gene was identified as the causative mutation. This mutation hindered the production of hydrogen peroxide (H2O2) and thus contributed to thyroid hormone (TH) synthesis failure. Transcriptome sequencing analysis of the thymuses showed that Krüppel-like factor 9 (KLF9) was predominantly downregulated in hypothyroid mutants. KLF9 was verified to be directly regulated by TH in a TH receptor (TR)-dependent manner both in vivo and in vitro. Furthermore, knockdown of klf9 in zebrafish embryos impaired hematopoietic development including erythroid maturation and T lymphopoiesis. Our findings suggest that the TR-KLF9 axis is responsible for the hematopoietic dysfunction and might be exploited for the development of novel therapeutic interventions for thyroid diseases.

The signalling receptor MCAM coordinates apical-basal polarity and planar cell polarity during morphogenesis.

The apical-basal (AB) polarity and planar cell polarity (PCP) provide an animal cell population with different phenotypes during morphogenesis. However, how cells couple these two patterning systems remains unclear. Here we provide in vivo evidence that melanoma cell adhesion molecule (MCAM) coordinates AB polarity-driven lumenogenesis and c-Jun N-terminal kinase (JNK)/PCP-dependent ciliogenesis. We identify that MCAM is an independent receptor of fibroblast growth factor 4 (FGF4), a membrane anchor of phospholipase C-γ (PLC-γ), an immediate upstream receptor of nuclear factor of activated T-cells (NFAT) and a constitutive activator of JNK. We find that MCAM-mediated vesicular trafficking towards FGF4, while generating a priority-grade transcriptional response of NFAT determines lumenogenesis. We demonstrate that MCAM plays indispensable roles in ciliogenesis through activating JNK independently of FGF signals. Furthermore, mcam-deficient zebrafish and Xenopus exhibit a global defect in left-right (LR) asymmetric establishment as a result of morphogenetic failure of their LR organizers. Therefore, MCAM coordination of AB polarity and PCP provides insight into the general mechanisms of morphogenesis.

Embryonic hematopoiesis in vertebrate somites gives rise to definitive hematopoietic stem cells.

Hematopoietic stem cells (HSCs) replenish all types of blood cells. It is debating whether HSCs in adults solely originate from the aorta-gonad-mesonephros (AGM) region, more specifically, the dorsal aorta, during embryogenesis. Here, we report that somite hematopoiesis, a previously unwitnessed hematopoiesis, can generate definitive HSCs (dHSCs) in zebrafish. By transgenic lineage tracing, we found that a subset of cells within the forming somites emigrate ventromedially and mix with lateral plate mesoderm-derived primitive hematopoietic cells before the blood circulation starts. These somite-derived hematopoietic precursors and stem cells (sHPSCs) subsequently enter the circulation and colonize the kidney of larvae and adults. RNA-seq analysis reveals that sHPSCs express hematopoietic genes with sustained expression of many muscle/skeletal genes. Embryonic sHPSCs transplanted into wild-type embryos expand during growth and survive for life time with differentiation into various hematopoietic lineages, indicating self-renewal and multipotency features. Therefore, the embryonic origin of dHSCs in adults is not restricted to the AGM.

Eph/ephrin signaling maintains the boundary of dorsal forerunner cell cluster during morphogenesis of the zebrafish embryonic left-right organizer.

The Kupffer's vesicle (KV) is the so-called left-right organizer in teleost fishes. KV is formed from dorsal forerunner cells (DFCs) and generates asymmetrical signals for breaking symmetry of embryos. It is unclear how DFCs or KV cells are prevented from intermingling with adjacent cells. In this study, we show that the Eph receptor gene ephb4b is highly expressed in DFCs whereas ephrin ligand genes, including efnb2b, are expressed in cells next to the DFC cluster during zebrafish gastrulation. ephb4b knockdown or mutation and efnb2b knockdown cause dispersal of DFCs, a smaller KV and randomization of laterality organs. DFCs often dynamically form lamellipodium-like, bleb-like and filopodium-like membrane protrusions at the interface, which attempt to invade but are bounced back by adjacent non-DFC cells during gastrulation. Upon inhibition of Eph/ephrin signaling, however, the repulsion between DFCs and non-DFC cells is weakened or lost, allowing DFCs to migrate away. Ephb4b/Efnb2b signaling by activating RhoA activity mediates contact and repulsion between DFCs and neighboring cells during gastrulation, preventing intermingling of different cell populations. Therefore, our data uncover an important role of Eph/ephrin signaling in maintaining DFC cluster boundary and KV boundary for normal left-right asymmetrical development.

Coordinate involvement of Nodal-dependent inhibition and Wnt-dependent activation in the maintenance of organizer-specific bmp2b in zebrafish.

A vertebrate signaling center, known in zebrafish as the organizer, is essential for axis patterning and formation and is regulated by multiple cell signaling pathways, including Wnt, Nodal, and Bmp. Organizer-specific Bmp2b plays important roles in the maintenance of the Bmp activity gradient and dorsal-ventral patterning. However, it is unknown how transcription of bmp2b in the organizer is regulated. In this study, we generated a bmp2b transgenic line Tsg(-2.272bmp2b:gfp) that reproduced organizer-specific bmp2b expression. Dissection analysis revealed that a 0.273-kb minimal promoter was indispensable for bmp2b expression in the dorsal organizer. Reporter assays showed that organizer-specific bmp2b is negatively regulated by the Nodal signal and positively regulated by the Wnt signal in both embryos and cell lines. Promoter analysis and chromatin-immunoprecipitation (ChIP) indicated that one consensus Smad-binding element (SBE) (CAGAC) and one Lef/Tcf-binding element (LBE) (AGATAA) were present in the 0.273-kb promoter, and could be directly bound by Smad2 and ß-catenin proteins. Together, these results suggest that maintenance of organizer-specific bmp2b expression involves opposite and concerted regulation by Nodal and Wnt signaling.

The Kinase Activity-deficient Isoform of the Protein Araf Antagonizes Ras/Mitogen-activated Protein Kinase (Ras/MAPK) Signaling in the Zebrafish Embryo.

Raf kinases are important components of the Ras-Raf-Mek-Erk pathway and also cross-talk with other signaling pathways. Araf kinase has been demonstrated to inhibit TGF-ß/Smad2 signaling by directly phosphorylating and accelerating degradation of activated Smad2. In this study, we show that the araf gene expresses in zebrafish embryos to produce a shorter transcript variant, araf-tv2, in addition to the full-length variant araf-tv1. araf-tv2 is predicted to encode a C-terminally truncated peptide without the kinase activity domain. Araf-tv2 can physically associate with Araf-tv1 but does not antagonize the inhibitory effect of Araf-tv1 on TGF-ß/Smad2 signaling. Instead, Araf-tv2 interacts strongly with Kras and Nras, ultimately blocking MAPK activation by these Ras proteins. In zebrafish embryos, overexpression of araf-tv2 is sufficient to inhibit Fgf/Ras-promoted Erk activation, mesodermal induction, dorsal development, and neuroectodermal posteriorization. Therefore, different isoforms of Araf may participate in similar developmental processes but by regulating different signaling pathways.

The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression.

During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-ß signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-ß signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-ß/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization.

TGFß1a regulates zebrafish posterior lateral line formation via Smad5 mediated pathway.

The zebrafish sensory posterior lateral line (pLL) has become an attractive model for studying collective cell migration and cell morphogenesis. Recent studies have indicated that chemokine, Wnt/ß-catenin, Fgf, and Delta-Notch signaling pathways participate in regulating pLL development. However, it remains unclear whether TGFß signaling pathway is involved in pLL development. Here we report a critical role of TGFß1 in regulating morphogenesis of the pLL primordium (pLLP). The tgfß1a gene is abundantly expressed in the lateral line primordium. Knockdown or knockout of tgfß1a leads to a reduction of neuromast number, an increase of inter-neuromast distance, and a reduced number of hair cells. The aberrant morphogenesis in embryos depleted of tgfß1a correlates with the reduced expression of atoh1a, deltaA, and n-cadherin/cdh2, which are known important regulators of the pLLP morphogenesis. Like tgfß1a depletion, knockdown of smad5 that expresses in the pLLP, affects pLLP development whereas overexpression of a constitutive active Smad5 isoform rescues the defects in embryos depleted of tgfß1a, indicating that Smad5 mediates tgfß1a function in pLLP development. Therefore, TGFß/Smad5 signaling plays an important role in the zebrafish lateral line formation.

Destabilizing LSD1 by Jade-2 promotes neurogenesis: an antibraking system in neural development.

Histone H3K4 demethylase LSD1 plays an important role in stem cell biology, especially in the maintenance of the silencing of differentiation genes. However, how the function of LSD1 is regulated and the differentiation genes are derepressed are not understood. Here, we report that elimination of LSD1 promotes embryonic stem cell (ESC) differentiation toward neural lineage. We showed that the destabilization of LSD1 occurs posttranscriptionally via the ubiquitin-proteasome pathway by an E3 ubiquitin ligase, Jade-2. We demonstrated that Jade-2 is a major LSD1 negative regulator during neurogenesis in vitro and in vivo in both mouse developing cerebral cortices and zebra fish embryos. Apparently, Jade-2-mediated degradation of LSD1 acts as an antibraking system and serves as a quick adaptive mechanism for re-establishing epigenetic landscape without more laborious transcriptional regulations. As a potential anticancer strategy, Jade-2-mediated LSD1 degradation could potentially be used in neuroblastoma cells to induce differentiation toward postmitotic neurons.

Inhibition of endothelial ERK signalling by Smad1/5 is essential for haematopoietic stem cell emergence.

The earliest HSCs are derived from haemogenic endothelium via endothelial-to-haematopoietic transition during vertebrate embryogenesis; however, the underlying mechanism is largely unclear. Here we show that interplay of Smad1/5 and ERK signalling is essential for haemogenic endothelium-based HSC emergence. Smad1/5 directly inhibits erk expression through recruiting HDAC1 to and inducing de-acetylation of the erk promoter in endothelial cells. Over-activated ERK signalling conferred by inhibition of Smad1/5 promotes the arterial endothelial cell fate and constitutively strengthens the tight junction between endothelial cells, thereby repressing the specification of haemogenic endothelium and the following endothelial-to-haematopoietic transition process. These findings provide new insights into the in vitro generation of transplantable HSCs for potential clinical applications.

[The development of zebrafish research in China].

As one of the most important vertebrate model systems, the zebrafish plays increasingly important roles in the field of life sciences. At present, zebrafish is widely used in studies on developmental biology, molecular biology, cell biology, genetics, neurobiology, oncology, immunology, marine biology, pharmacology, toxicology, and environmental protection. The zebrafish-based studies have led to many important discoveries that contribute to the development of modern life sciences. The zebrafish model system was introduced into China in the late 1990s; since then, the Chinese zebrafish community has being expanded fast with more influencing research outcomes, promoting a wide range of disciplines. This review gives an overview of zebrafish-based studies and representative findings in the mainland and Hong Kang of China, aiming to promote wider utilization of zebrafish for high-level studies.

Protein phosphatase 4 cooperates with Smads to promote BMP signaling in dorsoventral patterning of zebrafish embryos.

BMP signals play pivotal roles in dorsoventral patterning of vertebrate embryos. The role of Ppp4c, the catalytic subunit of ubiquitous protein phosphatase 4, in vertebrate embryonic development and underlying mechanisms is poorly understood. Here, we demonstrate that knockdown of zebrafish ppp4cb and/or ppp4ca inhibits ventral development in embryos and also blocks ventralizing activity of ectopic Smad5. Biochemical analyses reveal that Ppp4c is a direct binding partner and transcriptional coactivator of Smad1/Smad5. In response to BMP, Ppp4c is recruited to the Smad1-occupied promoter, and its phosphatase activity is essential in inhibiting HDAC3 activity and, consequently, potentiating transcriptional activation. Consistently, genetic or chemical interference of Hdac3 expression or activity compromises the dorsalizing phenotype induced by ppp4cb knockdown. We conclude that Ppp4c is a critical positive regulator of BMP/Smad signaling during embryonic dorsoventral pattern formation in zebrafish.

The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response.

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. Here, we show that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. We observed similar roles for Fu during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism.

Interruption of cenph causes mitotic failure and embryonic death, and its haploinsufficiency suppresses cancer in zebrafish.

Kinetochore proteins associate with centromeric DNA and spindle microtubules and play essential roles in chromosome segregation during mitosis. In this study, we uncovered a zebrafish mutant, stagnant and curly (stac), that carries the Tol2 transposon element inserted at the kinetochore protein H (cenph) locus. Mutant embryos exhibit discernible cell death as early as 20 hours postfertilization, extensive apoptosis, and upward curly tail during the pharyngula period and deform around 5 days postfertilization. The stac mutant phenotype can be rescued by cenph mRNA overexpression and mimicked by cenph knockdown with antisense morpholinos, suggesting the responsibility of cenph deficiency for stac mutants. We demonstrate that the intrinsic apoptosis pathway is hyperactivated in stac mutants and that p53 knockdown partially blocks excess apoptosis in stac mutants. Mitotic cells in stac mutants show chromosome missegregation and are usually arrested in G(2)/M phase. Furthermore, compared with wild type siblings, heterozygous stac fish develop invasive tumors at a dramatically reduced rate, suggesting a reduced cancer risk. Taken together, our findings uncover an essential role of cenph in mitosis and embryonic development and its association with tumor development.