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.

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.

Molecular basis for bipartite recognition of histone H3 by the PZP domain of PHF14.

Histone recognition constitutes a key epigenetic mechanism in gene regulation and cell fate decision. PHF14 is a conserved multi-PHD finger protein that has been implicated in organ development, tissue homeostasis, and tumorigenesis. Here we show that PHF14 reads unmodified histone H3(1-34) through an integrated PHD1-ZnK-PHD2 cassette (PHF14PZP). Our binding, structural and HDX-MS analyses revealed a feature of bipartite recognition, in which PHF14PZP utilizes two distinct surfaces for concurrent yet separable engagement of segments H3-Nter (e.g. 1-15) and H3-middle (e.g. 14-34) of H3(1-34). Structural studies revealed a novel histone H3 binding mode by PHD1 of PHF14PZP, in which a PHF14-unique insertion loop but not the core ß-strands of a PHD finger dominates H3K4 readout. Binding studies showed that H3-PHF14PZP engagement is sensitive to modifications occurring to H3 R2, T3, K4, R8 and K23 but not K9 and K27, suggesting multiple layers of modification switch. Collectively, our work calls attention to PHF14 as a 'ground' state (unmodified) H3(1-34) reader that can be negatively regulated by active marks, thus providing molecular insights into a repressive function of PHF14 and its derepression.

Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype.

Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients.

Impairment of Cargo Transportation Caused by gbf1 Mutation Disrupts Vascular Integrity and Causes Hemorrhage in Zebrafish Embryos.

ADP-ribosylation factor GTPases are activated by guanine nucleotide exchange factors including Gbf1 (Golgi brefeldin A-resistant factor 1) and play important roles in regulating organelle structure and cargo-selective vesicle trafficking. However, the developmental role of Gbf1 in vertebrates remains elusive. In this study, we report the zebrafish mutant line tsu3994 that arises from N-ethyl-N-nitrosourea (ENU)-mediated mutagenesis and is characterized by prominent intracerebral and trunk hemorrhage. The mutant embryos develop hemorrhage accompanied by fewer pigments and shorter caudal fin at day 2 of development. The hemorrhage phenotype is caused by vascular breakage in a cell autonomous fashion. Positional cloning identifies a T → G nucleotide substitution in the 23rd exon of the gbf1 locus, resulting in a leucine → arginine substitution (L1246R) in the HDS2 domain. The mutant phenotype is mimicked by gbf1 knockouts and morphants, suggesting a nature of loss of function. Experimental results in mammalian cells show that the mutant form Gbf1(L1246R) is unable to be recruited to the Golgi apparatus and fails to activate Arf1 for recruiting COPI complex. The hemorrhage in tsu3994 mutants can be prevented partially and temporally by treating with the endoplasmic reticulum stress/apoptosis inhibitor tauroursodeoxycholic acid or by knocking down the proapoptotic gene baxb Therefore, endothelial endoplasmic reticulum stress and subsequent apoptosis induced by gbf1 deficiency may account for the vascular collapse and hemorrhage.

Uracil-DNA glycosylase is involved in DNA demethylation and required for embryonic development in the zebrafish embryo.

Uracil-DNA glycosylase (Ung) is a component of the base excision repair process and has the ability to remove uracil from U:G mispairs in DNA. However, its implications in development of vertebrate embryos are poorly understood. In this study, we found that zebrafish uracil-DNA glycosylase a (Unga) is maternally expressed at high levels and accumulated in nuclei during cleavage and blastulation periods. Knockdown of unga in zebrafish embryos causes an increase of the global DNA methylation level concomitantly with a reduction of overall transcriptional activity in the nucleus, ultimately resulting in embryonic lethality during segmentation period. Conversely, unga overexpression is sufficient to reduce the global DNA methylation level, to increase H3K4me3 and H3K27me3 marks, and to activate genome transcription. Furthermore, overexpression of unga(D132A) mRNA, encoding a mutant Unga without DNA glycosylase activity, does not affect global DNA methylation level, indicating that its involvement in DNA demethylation is dependent on its glycosylase activity. These results together suggest that Unga is implicated in postfertilization genomic DNA demethylation, zygotic gene transcription, and normal embryonic development in zebrafish.

SCFFBXL¹5 regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1.

Smad ubiquitination regulatory factor 1 (Smurf1), an homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligase, performs a crucial role in the regulation of the bone morphogenetic protein (BMP) signalling pathway in both embryonic development and bone remodelling. How the stability and activity of Smurf1 are negatively regulated remains largely unclear. Here, we report that F-box and LRR domain-containing protein 15 (FBXL15), an F-box protein of the FBXL family, forms an Skp1-Cullin1-F-box protein-Roc1 (SCF)(FBXL15) ubiquitin ligase complex and targets Smurf1 for ubiquitination and proteasomal degradation. FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of the Smurf1 HECT domain and promotes the ubiquitination of Smurf1 on K355 and K357 within the WW-HECT linker region. In this way, FBXL15 positively regulates BMP signalling in mammalian cells. Knockdown of fbxl15 expression in zebrafish embryos by specific antisense morpholinos causes embryonic dorsalization phenocoping BMP-deficient mutants. Injection of FBXL15 siRNAs into rat bone tissues leads to a significant loss of bone mass and decrease in bone mineral density. Collectively, our results demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signalling during embryonic development and adult bone formation.

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 zebrafish tufm mutant model for the COXPD4 syndrome of aberrant mitochondrial function.

Mitochondrial dysfunction is a critical factor leading to a wide range of clinically heterogeneous and often severe disorders due to its central role in generating cellular energy. Mutations in the TUFM gene are known to cause combined oxidative phosphorylation deficiency 4 (COXPD4), a rare mitochondrial disorder characterized by a comprehensive quantitative deficiency in mitochondrial respiratory chain (MRC) complexes. The development of a reliable animal model for COXPD4 is crucial for elucidating the roles and mechanisms of TUFM in disease pathogenesis and benefiting its medical management. In this study, we construct a zebrafish tufm-/- mutant that closely resembles the COXPD4 syndrome, exhibiting compromised mitochondrial protein translation, dysfunctional mitochondria with oxidative phosphorylation (OXPHOS) defects, and significant metabolic suppression of the tricarboxylic acid (TCA) cycle. Leveraging this COXPD4 zebrafish model, we comprehensively validate the clinical relevance of TUFM mutations and identify probucol as a promising therapeutic approach for managing COXPD4. Our data offer valuable insights for understanding mitochondrial diseases and developing effective treatments.

[Rbb4l enhances TGF-ß/Nodal signaling and promotes zebrafish embryonic dorsolization].

The TGF-ß/Nodal signaling pathway plays an important role in the zebrafish dorsoventral patterning process. To further explore the function and mechanism of this signaling pathway, we identified a set of Smad2/3a interacting proteins by the yeast two-hybrid screen. Rbb4l (Retinoblastoma binding protein 4, like) is one of the identified proteins. Human RBBP4 (Retinoblastoma binding protein 4), the homolog of zebrafish Rbb4l, has been shown to form complexes with other chromatin modifiers, but its roles in embryonic development remain unknown. In this study, we showed that Rbb4l directly interacted with Smad3a and enhances TGF-ß/Nodal signaling. In zebrafish embryos, rbb4l overexpression resulted in an expanded expression of dorsal markers with a reduction of ventral markers expression, suggesting a dorsalizing function. On the contrary, rbb4l knockdown caused ventralized phenotype of the embryos at 24 hours post-fertilization (hpf). Furthermore, a series of rescue experiments showed that rbb4l failed to cause embryonic dorsalization in the absence of Nodal signal. Together, our data suggested that Rbb4l acts as an enhancer of Nodal/Smad2/3 signaling during embryogene-sis, and depends on the existence of Nodal signaling.

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.

Global identification of SMAD2 target genes reveals a role for multiple co-regulatory factors in zebrafish early gastrulas.

Nodal and Smad2/3 signals play pivotal roles in mesendoderm induction and axis determination during late blastulation and early gastrulation in vertebrate embryos. However, Smad2/3 direct target genes during those critical developmental stages have not been systematically identified. Here, through ChIP-chip assay, we show that the promoter/enhancer regions of 679 genes are bound by Smad2 in the zebrafish early gastrulas. Expression analyses confirm that a significant proportion of Smad2 targets are indeed subjected to Nodal/Smad2 regulation at the onset of gastrulation. The co-existence of DNA-binding sites of other transcription factors in the Smad2-bound regions allows the identification of well known Smad2-binding partners, such as FoxH1 and Lef1/ß-catenin, as well as many previously unknown Smad2 partners, including Oct1 and Gata6, during embryogenesis. We demonstrate that Oct1 physically associates with and enhances the transcription and mesendodermal induction activity of Smad2, whereas Gata6 exerts an inhibitory role in Smad2 signaling and mesendodermal induction. Thus, our study systemically uncovers a large number of Smad2 targets in early gastrulas and suggests cooperative roles of Smad2 and other transcription factors in controlling target gene transcription, which will be valuable for studying regulatory cascades during germ layer formation and patterning of vertebrate embryos.

[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.

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.

Tob1 controls dorsal development of zebrafish embryos by antagonizing maternal beta-catenin transcriptional activity.

Maternal beta-catenin and Nodal signals are essential for the formation of the dorsal organizer, which, in turn, induces neural and other dorsal tissue development in vertebrate embryos. Tob (Transducer of ErbB2) proteins possess antiproliferative properties and are known to influence BMP signaling, but their relationship to other signaling pathways and to embryonic patterning in general was unclear. In this study, we demonstrate that zebrafish tob1a is required for correct dorsoventral patterning. Mechanistically, Tob1a inhibits beta-catenin transcriptional activity by physically associating with beta-catenin and preventing the formation of beta-catenin/LEF1 complexes. Although Tob1a can also inhibit the transcriptional activity of the Nodal effector Smad3, its role in limiting dorsal development is executed primarily by antagonizing the beta-catenin signal. We further demonstrate that Tob family members across species share similar biochemical properties and biological activities.

Mta3-NuRD complex is a master regulator for initiation of primitive hematopoiesis in vertebrate embryos.

Metastasis-associated antigens 1/2/3 (Mta1/2/3) are components of nucleosome remodeling and deacetylase (NuRD) complexes and have been found to play roles in embryonic development and homeostasis. However, their functions in primitive hematopoiesis are unknown. In this study, we demonstrate that knockdown of mta3 by antisense morpholinos abolishes primitive hematopoietic lineages and causes abnormal angiogenesis in zebrafish embryos. However, the expression of the pronephric duct and paraxial mesoderm markers is unaltered and the specification of angioblasts is unaffected in mta3 morphants. The results suggest that mta3 is specifically required for primitive hematopoiesis. Furthermore, inhibition of deacetylase activity with the inhibitors valproic acid (VPA) or trichostatin A (TSA) in zebrafish embryos completely blocks primitive hematopoiesis, resulting in hematopoietic defects almost identical to those seen in mta3 morphants. Importantly, overexpression of scl or scl and lmo2, 2 master genes for primitive hematopoiesis, is able to overturn effects of mta3 knockdown or VPA/TSA treatment; and overexpression of mta3, and human MBD3 or HDAC1, 2 other components of NuRD complex, enhances the expression of scl and lmo2 in the posterior lateral plate mesoderm during early primitive hematopoiesis. We conclude that Mta3-NuRD complex is essential for the initiation of primitive hematopoiesis. Thus, our findings provide new insight into the regulatory hierarchy of primitive hematopoiesis in vertebrates.

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.

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.

Smad2/3 activities are required for induction and patterning of the neuroectoderm in zebrafish.

Smad2 and Smad3, two essential nuclear effectors of transforming growth factor (Tgf)-beta signals, have been found to be implicated in mesoderm and endoderm development in vertebrate embryos. However, their roles in the induction and patterning of the neuroectoderm are not well established. In this study, we show that interference with Smad2/3 activities in zebrafish embryos, by injecting dnsmad3b mRNA encoding a dominant negative Smad3b mutant, inhibits the expression of the early neural markers sox2 and sox3 at the onset of gastrulation and results in reduction of the anterior neuroectodermal marker otx2 as well as the posterior neuroectodermal marker hoxb1b during late gastrulation, suggesting a role of Smad2/3 activities in neural induction. Conversely, excess Smad2/3 activities, caused by injecting smad3b mRNA, lead to an enhancement of sox2 and sox3 expression in the ventral domains but an inhibition of their expression in the dorsalmost region at early stages. Overexpression of smad3b also causes ventral expansion of the otx2 and hoxb1b expression domains accompanied with rostral shift of the hoxb1b domain at late gastrulation stages. Collectively, these data indicate that Smad2/3 activities are required for neural induction and neuroectodermal posteriorization in zebrafish. Knockdown of chordin partially inhibits effect of smad3b overexpression on neural induction, implying that Smad2/3 exert their effect on neural induction in part by regulating the expression of Bmp antagonists. Furthermore, down-regulation or up-regulation of Smad2/3 activities in MZoep mutant embryos, which lack the organizer and mesendodermal tissues due to deficiency of Nodal signaling, still affects induction and patterning of the neuroectoderm, suggesting that Smad2/3 activities are implicated in neural development in the absence of the organizer and mesendodermal tissues. We additionally demonstrate that Smad2/3 activities cooperate with Wnt and Fgf signals in neural development. Thus, Smad2/3 activities play important roles not only in mesendodermal development but also in neural development during early vertebrate embryogenesis.

High-throughput preparation of radioprotective polymers via Hantzsch's reaction for in vivo X-ray damage determination.

Radioprotectors for acute injuries caused by large doses of ionizing radiation are vital to national security, public health and future development of humankind. Here, we develop a strategy to explore safe and efficient radioprotectors by combining Hantzsch's reaction, high-throughput methods and polymer chemistry. A water-soluble polymer with low-cytotoxicity and an excellent anti-radiation capability has been achieved. In in vivo experiments, this polymer is even better than amifostine, which is the only approved radioprotector for clinical applications, in effectively protecting zebrafish embryos from fatally large doses of ionizing radiation (80 Gy X-ray). A mechanistic study also reveals that the radioprotective ability of this polymer originates from its ability to efficiently prevent DNA damage due to high doses of radiation. This is an initial attempt to explore polymer radioprotectors via a multi-component reaction. It allows exploiting functional polymers and provides the underlying insights to guide the design of radioprotective polymers.