Site-1 protease is required for cartilage development in zebrafish

gonzo (goz) is a zebrafish mutant with defects in cartilage formation. The goz phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the goz phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (s1p). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in goz embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of goz are caused independently of the lipid defects.     

cpsf1 is required for definitive HSC survival in zebrafish

A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3' untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP(+) cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.     

VEGF-B-Neuropilin-1 signaling is spatiotemporally indispensable for vascular and neuronal development in zebrafish

Physiological functions of vascular endothelial growth factor (VEGF)-B remain an enigma, and deletion of the Vegfb gene in mice lacks an overt phenotype. Here we show that knockdown of Vegfba, but not Vegfbb, in zebrafish embryos by specific morpholinos produced a lethal phenotype owing to vascular and neuronal defects in the brain. Vegfba morpholinos also markedly prevented development of hyaloid vasculatures in the retina, but had little effects on peripheral vascular development. Consistent with phenotypic defects, Vegfba, but not Vegfaa, mRNA was primarily expressed in the brain of developing zebrafish embryos. Interestingly, in situ detection of Neuropilin1 (Nrp1) mRNA showed an overlapping expression pattern with Vegfba, and knockdown of Nrp1 produced a nearly identically lethal phenotype as Vegfba knockdown. Furthermore, zebrafish VEGF-Ba protein directly bound to NRP1. Importantly, gain-of-function by exogenous delivery of mRNAs coding for NRP1-binding ligands VEGF-B or VEGF-A to the zebrafish embryos rescued the lethal phenotype by normalizing vascular development. Similarly, exposure of zebrafish embryos to hypoxia also rescued the Vegfba morpholino-induced vascular defects in the brain by increasing VEGF-A expression. Independent evidence of VEGF-A gain-of-function was provided by using a functionally defective Vhl-mutant zebrafish strain, which again rescued the Vegfba morpholino-induced vascular defects. These findings show that VEGF-B is spatiotemporally required for vascular development in zebrafish embryos and that NRP1, but not VEGFR1, mediates the essential signaling.Angiogenesis is essential for embryonic development and contributes to the onset and development of many diseases (1). The angiogenic process is tightly regulated by angiogenic factors and inhibitors and involves cooperative and synchronized interactions between vascular endothelial cells and perivascular cells including pericytes and vascular smooth muscle cells. Among all known angiogenic factors, vascular endothelial growth factor (VEGF; also called VEGFA) is probably the best-characterized proangiogenic factor under physiological and pathological conditions (2, 3). There are five structurally and functionally related members in the VEGF family, which includes VEGF-A, -B, -C, and -D and placental growth factor (PlGF) (4). These factors bind primarily to three membrane tyrosine kinase receptors (TKRs), i.e., VEGFR1, VEGFR2, and VEGFR3, to display their biological functions (4). According to their receptor-binding patterns and biological functions, members of the VEGF family are divided into three subgroups: (i) VEGFA as the VEGFR1- and VEGFR2-binding ligand (5); (ii) VEGF-B and PlGF that exclusively bind to VEGFR1 (4, 6–8); and (iii) VEGF-C and VEGF-D as VEGFR3- and VEGFR2-binding ligands (9). Whereas VEGF-A potently stimulates angiogenesis, vascular permeability, and lymphangiogenesis, VEGF-C and VEGF-D primarily induce lymphangiogenesis although they also induce angiogenesis (9). VEGFR2 has been reported as the key receptor that transduces angiogenic and vascular permeability signals, and VEGFR3 is responsible mainly for lymphangiogenesis (10). In addition to TKRs, various heparin-binding isoforms of each member in the VEGF family have been reported to bind to neuropilins (NRPs), which is also crucial for angiogenesis, lymphangiogenesis, axon guidance, cell survival, migration, and invasion (11–14).VEGF-A is required for embryonic development in mammals, and deletion of only one allele of the Vegfa gene (haploinsufficiency) in mice results in a lethal embryonic phenotype, owing to inappropriate development of the vascular and hematopoietic systems (15, 16). Paradoxically, modest overexpression of VEGF-A in mice also causes embryonic lethality due to cardiovascular deficiency (17). These findings demonstrate that an optimal level of VEGF-A expression is needed for embryonic development. Unlike VEGF-A, deletion of the Vegfb gene in mice does not produce an overt phenotype, except slight cardiovascular impairments (18, 19). Recently, it has been found that VEGF-B–deficient animals exhibit defective lipid uptake in endothelial cells (20, 21). However, these findings could not be reproduced in another study (22). Based on these findings, VEGF-B is probably the least-characterized member in the VEGF-A family, and its physiological functions remain an enigmatic issue in mice (6). The key issue in VEGF-B research is what this factor does under physiological conditions. One of the main differences between developing mouse embryos and zebrafish embryos is the presence of tissue hypoxia during development. In mice and other mammals, embryonic tissues develop under a relatively hypoxic environment, and hypoxia is one of the key mechanisms behind up-regulation of VEGF-A expression (23). The increased VEGFA expression in various tissues would probably compensate the VEGF-B deletion-associated vascular and other defects. However, zebrafish embryos lack this hypoxia-related VEGF-A compensatory mechanism and allow us to study spatiotemporal functions of VEGF-B during embryonic development.To test this hypothesis, in the present study we have investigated the functions of VEGF-B in developing zebrafish embryos. Surprisingly, knockdown of the Vegfba gene in developing zebrafish embryos produced a lethal phenotype owing to vascular defects in the brain. The functional defects of VEGF-B–deficient zebrafish embryos impeccably correlate with the VEGF-B expression pattern in the developing brain in which VEGF-A expression is modestly low. Importantly, exposure of VEGF-B–defective zebrafish embryos to hypoxia rescues the VEGF-B deficiency-induced vascular defects by a VEGF-A–dependent mechanism. Our findings for the first time to our knowledge demonstrate the indispensable function of VEGF-B in vascular development in zebrafish embryos.     

Paired-related homeobox gene Prx1 is required for pulmonary vascular development

Herein, we show that the paired-related homeobox gene, Prx1, is required for lung vascularization. Initial studies revealed that Prx1 localizes to differentiating endothelial cells (ECs) within the fetal lung mesenchyme, and later within ECs forming vascular networks. To begin to determine whether Prx1 promotes EC differentiation, fetal lung mesodermal cells were transfected with full-length Prx1 cDNA, resulting in their morphological transformation to an endothelial-like phenotype. In addition, Prx1-transformed cells acquired the ability to form vascular networks on Matrigel. Thus, Prx1 might function by promoting pulmonary EC differentiation within the fetal lung mesoderm, as well as their subsequent incorporation into vascular networks. To understand how Prx1 participates in network formation, we focused on tenascin-C (TN-C), an extracellular matrix (ECM) protein induced by Prx1. Immunocytochemistry/histochemistry showed that a TN-C-rich ECM surrounds Prx1-positive pulmonary vascular networks both in vivo and in tissue culture. Furthermore, antibody-blocking studies showed that TN-C is required for Prx1-dependent vascular network formation on Matrigel. Finally, to determine whether these results were relevant in vivo, we examined newborn Prx1-wild-type (+/+) and Prx1-null (-/-) mice and showed that Prx1 is critical for expression of TN-C and lung vascularization. These studies provide a framework to understand how Prx1 controls EC differentiation and their subsequent incorporation into functional pulmonary vascular networks.     

Aquaporin 1 is required for hypoxia-inducible angiogenesis in human retinal vascular endothelial cells

Aquaporin 1 (AQP1) was first purified from red blood cell membranes and is now known to be an osmolarity-driven water transporter that is widely expressed in many epithelial and endothelial cells outside the brain. Several recent studies have shown strong expression of AQP1 in proliferating tumor microvessels, suggesting that AQP1 may have an important role in tumor angiogenesis. Hypoxia is thought to be a common precursor to neovascularization in many retinal diseases, including diabetic retinopathy, and therefore we analyzed the expression pattern and function of AQP1 in human retinal vascular endothelial cells cultured under hypoxic conditions. The levels of AQP1 mRNA and protein expression significantly increased under hypoxia, and inhibition of VEGF signaling did not affect AQP1 expression. To examine the effect of AQP1 on hypoxia-inducible angiogenesis, a tube formation assay was performed. Reduction of AQP1 expression using siRNA and inhibition of VEGF signaling both significantly inhibited tube formation, and these effects were additive. Therefore, our data suggest that AQP1 is involved in hypoxia-inducible angiogenesis in retinal vascular endothelial cells through a mechanism that is independent of the VEGF signaling pathway.     

Mnk1 is required for angiotensin II-induced protein synthesis in vascular smooth muscle cells

Angiotensin II (Ang II) stimulates protein synthesis in vascular smooth muscle cells (VSMCs), possibly secondary to regulatory changes at the initiation of mRNA translation. Mitogen-activated protein (MAP) kinase signal-integrating kinase-1 (Mnk1), a substrate of ERK and p38 MAP kinase, phosphorylates eukaryotic initiation factor 4E (eIF4E), an important factor in translation. The goal of the present study was to investigate the role of Mnk1 in Ang II-induced protein synthesis and to characterize the molecular mechanisms by which Mnk1 and eIF4E is activated in rat VSMCs. Ang II treatment resulted in increased Mnk1 activity and eIF4E phosphorylation. Expression of a dominant-negative Mnk1 mutant abolished Ang II-induced eIF4E phosphorylation. PD98059 or introduction of kinase-inactive MEK1/MKK1, but not SB202190 or kinase-inactive p38 MAP kinase, inhibited Ang II-induced Mnk1 activation and eIF4E phosphorylation, suggesting that ERK, but not p38 MAP kinase, is required for Ang II-induced Mnk1-eIF4E activation. Further, dominant-negative constructs for Ras, but not for Rho, Rac, or Cdc42, abolished Ang II-induced Mnk1 activation. Finally, treatment of VSMCs with CGP57380, a novel specific kinase inhibitor of Mnk1, resulted in dose-dependent decreases in Ang II-stimulated phosphorylation of eIF4E, protein synthesis, and VSMC hypertrophy. In summary, these data demonstrated that (1) Ang II-induced Mnk1 activation is mediated by the Ras-ERK cascade in VSMCs, and (2) Mnk1 is involved in Ang II-mediated protein synthesis and hypertrophy, presumably through the activation of translation-initiation. The Mnk1-eIF4E pathway may provide new insights into molecular mechanisms involved in vascular hypertrophy and other Ang II-mediated pathological states.     

Heat-shock protein 90alpha1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos

Heat-shock protein 90alpha (Hsp90alpha) is a member of the molecular chaperone family involved in protein folding and assembly. The role of Hsp90alpha in the developmental process, however, remains unclear. Here we report that zebrafish contains two Hsp90alpha genes, Hsp90alpha1, and Hsp90alpha2. Hsp90alpha1 is specifically expressed in developing somites and skeletal muscles of zebrafish embryos. We have demonstrated that Hsp90alpha1 is essential for myofibril organization in skeletal muscles of zebrafish embryos. Knockdown of Hsp90alpha1 resulted in paralyzed zebrafish embryos with poorly organized myofibrils in skeletal muscles. In contrast, knockdown of Hsp90alpha2 had no effect on muscle contraction and myofibril organization. The filament defects could be rescued in a cell autonomous manner by an ectopic expression of Hsp90alpha1. Biochemical analyses revealed that knockdown of Hsp90alpha1 resulted in significant myosin degradation and up-regulation of unc-45b gene expression. These results indicate that Hsp90alpha1 plays an important role in muscle development, likely through facilitating myosin folding and assembly into organized myofibril filaments.     

FGF-dependent left–right asymmetry patterning in zebrafish is mediated by Ier2 and Fibp1

Establishment of left–right asymmetry in vertebrates requires nodal, Wnt-PCP and FGF signaling and involves ciliogenesis in a laterality organ. Effector genes through which FGF signaling affects laterality have not been described. We isolated the zebrafish ier2 and fibp1 genes as FGF target genes and show that their protein products interact. Knock down of these factors interferes with establishment of organ laterality and causes defective cilia formation in Kupffer's Vesicle, the zebrafish laterality organ. Cilia are also lost after suppression of FGF8, but can be rescued by injection of ier2 and fibp1 mRNA. We conclude that Ier2 and Fibp1 mediate FGF signaling in ciliogenesis in Kupffer's Vesicle and in the establishment of laterality in the zebrafish embryo.     

SmyD1, a histone methyltransferase, is required for myofibril organization and muscle contraction in zebrafish embryos

Histone modification has emerged as a fundamental mechanism for control of gene expression and cell differentiation. Recent studies suggest that SmyD1, a novo SET domain-containing protein, may play a critical role in cardiac muscle differentiation. However, its role in skeletal muscle development and its mechanism of actions remains elusive. Here we report that SmyD1a and SmyD1b, generated by alternative splicing of SmyD1 gene, are histone methyltransferases that play a key role in skeletal and cardiac muscle contraction. SmyD1a and SmyD1b are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. Knockdown of SmyD1a and SmyD1b expression by morpholino antisense oligos resulted in malfunction of skeletal and cardiac muscles. The SmyD1 morphant embryos (embryos injected with morpholino oligos) could not swim and had no heartbeat. Myofibril organization in the morphant embryos was severely disrupted. The affected myofibers appeared as immature fibers with centrally located nuclei. Together, these data indicate that SmyD1a and SmyD1b are histone methyltransferases and play a critical role in myofibril organization during myofiber maturation.     

Piezo1, a mechanically activated ion channel,is required for vascular development in mice

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.Mechanotransduction, the conversion of physical forces into a biochemical response, is an essential signaling mechanism used by all organisms (1). Diverse physiological processes including hearing, touch, and blood pressure regulation require the ability of cells to sense and respond to mechanical stimuli (2–4). Impairment in mechanotransduction can lead to a wide range of pathologies including deafness and atherosclerosis (5, 6). The cellular response to mechanical force requires the coordinated action of multiple proteins; however, the identity of mechanotransducers in mammals remains poorly understood (7).Classical studies from auditory sensory cells first implicated ion channels to be inherently mechanically activated (8). Since then, stretch-activated cation channels have been recorded from various tissues (9), but their identity has remained unknown (10, 11). For an ion channel to be considered a physiologically relevant transducer of mechanical stimuli, several criteria must be met (12). The ion channel must be expressed in the appropriate mechanosensitive cells, its expression should be required for mechanotransduction in an in vivo setting, and ideally the expression of the channel in a naive cell must render that cell mechanosensitive.Progress toward identification of mechanically activated (MA) ion channels has been limited to a subset of ion channel families. In the invertebrates Caenorhabditis elegans and Drosophila melanogaster, members of the DEG/ENaC family and the TRP ion channel NOMPC have been conclusively shown to act as mechanotransduction ion channels (13–16). Mammals, however, do not have an ortholog of NOMPC, and the physiological relevance of the mammalian ENaC orthologs in mechanotransduction remains unclear. Many other TRP channels including TRPPs, TRPA1, and TRPV4 have been implicated in sensing mechanical stimuli; however, none has been shown to be required in vivo for sensing mechanical forces, nor to be necessary and sufficient for mechanotransduction (12). The two-pore potassium channels TREK-1, TREK-2, and TRAAK are mechanically activated in heterologous expression systems, and TREK-1 plays a role in modulating the mechanical sensitivity of dorsal root ganglia neurons (17). Overall, the identification of relatively few candidate genes has been a major impediment to studying mechanotransduction processes in vivo.We recently identified Piezo proteins as pore-forming subunits of an evolutionarily conserved MA cation channel family (18). Mouse Piezo1 forms a homo-multimeric complex of ∼1.2 mDa, with each monomer containing over 35 transmembrane domains (19). Piezo1 is necessary for mechanically activated currents in the Neuro2A cell line and is sufficient for conferring stretch-activated currents in heterologous cell expression. Purified mouse Piezo1 retains channel activity when reconstituted without accessory subunits into artificial lipid bilayers. The single Piezo member in Drosophila plays a role in sensing noxious mechanical stimuli (20), and mouse Piezo2 has been shown to be critically required for mechanotransduction in mammalian Merkel cells (21–23). Gain-of-function PIEZO1 mutations have recently been shown to cause hereditary xerocytosis in humans (24–26); however, it is still unclear whether the mammalian Piezo1 plays a role in mechanotransduction in vivo. Here we used a genetic strategy to ablate the function of Piezo1 in vivo and show that Piezo1 is a critical component of endothelial cell mechanotransduction.     

An antiapoptotic role of sorting nexin 7 is required for liver development in zebrafish

Sorting nexin (SNX) family proteins are best characterized for their abilities to regulate protein trafficking during processes such as endocytosis of membrane receptors, endosomal sorting, and protein degradation, but their in vivo functions remain largely unknown. We started to investigate the biological functions of SNXs using the zebrafish model. In this study, we demonstrated that SNX7 was essential for embryonic liver development. Hepatoblasts were specified normally, and the proliferation of these cells was not affected when SNX7 was knocked down by gene-specific morpholinos; however, they underwent massive apoptosis during the early budding stage. SNX7 mainly regulated the survival of cells in the embryonic liver and did not affect the viability of cells in other endoderm-derived organs. We further demonstrated that down-regulation of SNX7 by short interfering RNAs induced apoptosis in cell culture. At the molecular level, the cellular FLICE-like inhibitory protein (c-FLIP)/caspase 8 pathway was activated when SNX7 was down-regulated. Furthermore, overexpression of c-FLIP(S) was able to rescue the SNX7 knockdown-induced liver defect. CONCLUSION: SNX7 is a liver-enriched antiapoptotic protein that is indispensable for the survival of hepatoblasts during zebrafish early embryogenesis.     

Endothelial expression of the Notch ligand Jagged1 is required for vascular smooth muscle development

The Notch ligand Jagged1 (Jag1) is essential for vascular remodeling and has been linked to congenital heart disease in humans, but its precise role in various cell types of the cardiovascular system has not been extensively investigated. We show that endothelial-specific deletion of Jag1 results in embryonic lethality and cardiovascular defects, recapitulating the Jag1 null phenotype. These embryos show striking deficits in vascular smooth muscle, whereas endothelial Notch activation and arterial-venous differentiation appear normal. Endothelial Jag1 mutant embryos are phenotypically distinct from embryos in which Notch signaling is inhibited in endothelium. Together, these results imply that the primary role of endothelial Jag1 is to potentiate the development of neighboring vascular smooth muscle.     

PDGF signaling is required for epicardial function and blood vessel formation in regenerating zebrafish hearts

A zebrafish heart can fully regenerate after amputation of up to 20% of its ventricle. During this process, newly formed coronary blood vessels revascularize the regenerating tissue. The formation of coronary blood vessels during zebrafish heart regeneration likely recapitulates embryonic coronary vessel development, which involves the activation and proliferation of the epicardium, followed by an epithelial-to-mesenchymal transition. The molecular and cellular mechanisms underlying these processes are not well understood. We examined the role of PDGF signaling in explant-derived primary cultured epicardial cells in vitro and in regenerating zebrafish hearts in vivo. We observed that mural and mesenchymal cell markers, including pdgfrβ, are up-regulated in the regenerating hearts. Using a primary culture of epicardial cells derived from heart explants, we found that PDGF signaling is essential for epicardial cell proliferation. PDGF also induces stress fibers and loss of cell-cell contacts of epicardial cells in explant culture. This effect is mediated by Rho-associated protein kinase. Inhibition of PDGF signaling in vivo impairs epicardial cell proliferation, expression of mesenchymal and mural cell markers, and coronary blood vessel formation. Our data suggest that PDGF signaling plays important roles in epicardial function and coronary vessel formation during heart regeneration in zebrafish.     

Innervation is required for sense organ development in the lateral line system of adult zebrafish

Superficial mechanosensory organs (neuromasts) distributed over the head and body of fishes and amphibians form the “lateral line” system. During zebrafish adulthood, each neuromast of the body (posterior lateral line system, or PLL) produces “accessory” neuromasts that remain tightly clustered, thereby increasing the total number of PLL neuromasts by a factor of more than 10. This expansion is achieved by a budding process and is accompanied by branches of the afferent nerve that innervates the founder neuromast. Here we show that innervation is essential for the budding process, in complete contrast with the development of the embryonic PLL, where innervation is entirely dispensable. To obtain insight into the molecular mechanisms that underlie the budding process, we focused on the terminal system that develops at the posterior tip of the body and on the caudal fin. In this subset of PLL neuromasts, bud neuromasts form in a reproducible sequence over a few days, much faster than for other PLL neuromasts. We show that wingless/int (Wnt) signaling takes place during, and is required for, the budding process. We also show that the Wnt activator R-spondin is expressed by the axons that innervate budding neuromasts. We propose that the axon triggers Wnt signaling, which itself is involved in the proliferative phase that leads to bud formation. Finally, we show that innervation is required not only for budding, but also for long-term maintenance of all PLL neuromasts.     

The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb

The RNaseIII-containing enzyme Dicer is believed to be required for the processing of most, if not all, microRNAs (miRNAs) and for processing long dsRNA into small interfering RNAs. Because the complete loss of Dicer in both zebrafish and mice results in early embryonic lethality, it has been impossible to determine what role, if any, Dicer has in patterning later tissues in the developing vertebrate embryo. To bypass the early requirement of Dicer in development, we have created a conditional allele of this gene in mice. Using transgenes to drive Cre expression in discrete regions of the limb mesoderm, we find that removal of Dicer results in the loss of processed miRNAs. Phenotypically, developmental delays, in part due to massive cell death as well as disregulation of specific gene expression, lead to the formation of a much smaller limb. Thus, Dicer is required for the formation of normal mouse limbs. Strikingly, however, we did not detect defects in basic patterning or in tissue-specific differentiation of Dicer-deficient limb buds.