BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage- and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to down-regulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events.

Dorsal retinal fate is established early in eye development, via expression of spatially restricted dorsal-specific transcription factors in the optic vesicle; yet the events leading to initiation of dorsal fate are not clear. We hypothesized that induction of dorsal fate would require an extraocular signal arising from a neighboring tissue to pattern the prospective dorsal retina, however no such signal has been identified. We used the zebrafish embryo to determine the source, timing, and identity of the dorsal retina-inducing signal. Extensive cell movements occur during zebrafish optic vesicle morphogenesis, however the location of prospective dorsal cells within the early optic vesicle and their spatial relationship to early dorsal markers is currently unknown. Our mRNA expression and fate mapping analyses demonstrate that the dorsolateral optic vesicle is the earliest region to express dorsal specific markers, and cells from this domain contribute to the dorsal retinal pole at 24 hpf. We show that three bmp genes marking dorsal retina at 25 hpf are also expressed extraocularly before retinal patterning begins. We identified gdf6a as a dorsal initiation signal acting from the extraocular non-neural ectoderm during optic vesicle evagination. We find that bmp2b is involved in dorsal retina initiation, acting upstream of gdf6a. Together, this work has identified the nature and source of extraocular signals required to pattern the dorsal retina.

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

BACKGROUND

Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis.

RESULTS

Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1.

CONCLUSIONS

Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.

The bone morphogenetic proteins (BMPs), originally identified by their abilities to induce bone and/or cartilage formation, have been reported to be involved in various growth and differentiation processes, including reproduction. Although mammalian models are more frequently used to study the BMP system in reproduction, we have extended the study to the zebrafish, an excellent model for studying female reproduction in teleosts. Reverse transcription-PCR analysis revealed the expression of the Bmp ligands (bmp2a, bmp2b, bmp4, bmp6, and bmp7a) and the type II Bmp receptors (bmpr2a and bmpr2b) in various tissues, including the ovary. Spatiotemporal distribution of these Bmp ligands and receptors in the ovary was then investigated in this study. Reverse transcription-PCR on isolated follicle layers and denuded oocytes demonstrated that all Bmp ligands examined were exclusively or abundantly expressed in the oocyte, whereas the two receptors were expressed exclusively in the follicle layers, strongly suggesting a potential paracrine signaling from the oocyte towards the follicle layer by various Bmp ligands. This supports the current view that instead of being passively controlled and nurtured by the follicle layer for its growth and development, the oocyte may play an active role by releasing various growth differentiation factors to regulate follicle layer function. Quantitative analysis of temporal expression profiles during folliculogenesis revealed an increased expression of bmp2a, bmp2b, bmp4, and bmp6 from primary growth (stage I) to previtellogenic (stage II) stages, followed by steady declines toward the end of folliculogenesis when the follicles became fully grown. On the contrast, the BMP receptors (bmpr2a and bmpr2b) consistently showed an increase in expression during folliculogenesis, with the peak levels reached at the full-grown stage prior to final oocyte maturation. The spatiotemporal expression patterns of the Bmp family in the zebrafish follicles provide important insights into potential roles for Bmps during follicle development as oocyte-derived factors. Further experiments using recombinant zebrafish Bmp4 showed that Bmp4 had an inhibitory effect on spontaneous oocyte maturation in vitro, but not 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP)-induced oocyte maturation in vitro.

In vertebrates, extraembryonic tissues can act as signaling centers that impose a reproducible pattern of cell types upon the embryo. Here, we show that the zebrafish yolk syncytial layer (YSL) secretes a ventralizing signal during gastrulation. This activity is mediated by Bmp2b/Swirl (Swr) expressed under the control of Max's giant associated protein (MGA) and its binding partners, Max and Smad4. MGA coimmunoprecipitates with both Max and Smad4 in embryo extracts, and the three proteins form a complex in vitro. Furthermore, all three proteins bind to a DNA fragment upstream of the bmp2b transcription start site. Targeted depletion of MGA, its binding partners, or Bmp2b/Swr from the YSL reduces BMP signaling throughout the embryo, resulting in a mildly dorsalized phenotype. We conclude that MGA, Max, and Smad4 act in the extraembryonic YSL to initiate a positive feedback loop of Bmp signaling within the embryo.

Tetrabromobisphenol A (TBBPA), a brominated flame retardant, is detected commonly in aquatic environments, where it is thought to be highly toxic to the development of aquatic life. In this study, zebrafish embryos and larvae were used to investigate the protective effects of puerarin after exposure to TBBPA. Malformation, blood flow disorders, pericardial edema, and spawn coagulation rates increased, whereas survival decreased significantly after exposure to 0.5 and 1.0 mg L(-1) TBBPA. The measured indices of morphological toxicity improved after treatment with puerarin. TBBPA also induced reactive oxygen species (ROS) production in a dose-dependent manner. Acridine orange staining results revealed that TBBPA exposure caused cardiomyocyte apoptosis and induced the expression of three proapoptotic genes: P53, Bax, and Caspase9. In contrast, the expression of the antiapoptotic gene Bcl2 was down-regulated. When genes related to cardiac development were assessed, the expression of Tbx1, Raldh2, and Bmp2b changed after exposure to the combination of TBBPA and puerarin. These results suggest that TBBPA induces cardiomyocyte apoptosis and ROS production, resulting in cardiac developmental toxicity in zebrafish embryos or larvae. Therefore, puerarin regulates the expression of cardiac developmental genes, such as Tbx1, Bmp2b, and Raldh2 by inhibiting ROS production, and subsequently modulates cardiac development after the exposure of zebrafish larvae to TBBPA.

The zebrafish biklf gene encodes a novel Krüppel-like transcription factor containing three contiguous zinc fingers at the C-terminus. Expression of biklf is detected from the shield stage onward in the developing prechordal plate, and as a 'baseball seam'-like lateral stripe beginning at the end of gastrulation. The latter expression domain is suppressed in the swirl mutant in which bmp2b is disrupted. At the 5-somite stage the lateral expression domain separates into two distinct stripes, one in the ectoderm, the other in blood islands in the lateral plate mesoderm. Blood island staining of biklf continues through somitogenesis as the most prominent area of biklf expression.

Pharyngeal pouches, a series of outpocketings that bud from the foregut endoderm, are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus. However, whether pharyngeal pouch progenitors exist in the developing gut tube remains unknown. Here, taking advantage of cell lineage tracing and transgenic ablation technologies, we identified a population of nkx2.3+ pouch progenitors in zebrafish embryos and demonstrated an essential requirement of ectodermal BMP2b for their specification. At early somite stages, nkx2.3+ cells located at lateral region of pharyngeal endoderm give rise to the pouch epithelium except a subpopulation expressing pdgfαa rather than nkx2.3. A small-scale screen of chemical inhibitors reveals that BMP signaling is necessary to specify these progenitors. Loss-of-function analyses show that BMP2b, expressed in the pharyngeal ectoderm, actives Smad effectors in endodermal cells to induce nkx2.3+ progenitors. Collectively, our study provides in vivo evidence for the existence of pouch progenitors and highlights the importance of BMP2b signaling in progenitor specification.

BACKGROUND

Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH.

METHODS

We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH.

RESULTS

Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses.

CONCLUSIONS

Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Bone morphogenetic proteins (BMPs) are known to play roles in inner ear development of higher vertebrates. In zebrafish, there are several reports showing that members of the BMP family are expressed in the otic vesicle. We have isolated a novel zebrafish mutant gallery, which affects the development of the semicircular canal. Gallery merely forms the lateral and the immature anterior protrusion, and does not form posterior and ventral protrusions. We found that the expression of bmp2b and bmp4, both expressed in the normal optic vesicle at the protrusion stage, are extremely upregulated in the otic vesicle of gallery. To elucidate the role of BMPs in the development of the inner ear of zebrafish, we have applied excess BMP to the wild-type otic vesicle. The formation of protrusions was severely affected, and in some cases, they were completely lost in BMP4-treated embryos. Furthermore, the protrusions in gallery treated with Noggin were partially rescued. These data indicate that BMP4 plays an important role in the development of protrusions to form semicircular canals.

The Dapper/Frodo family of proteins are Dishevelled-interacting regulators of Wnt signaling. In this study, I characterize the regulation of the early expression patterns of dpr1 and dpr2. Although both dpr1 and dpr2 are expressed on the prospective dorsal side, I find that their pregastrula expression patterns have differences that have not been reported previously. Early dpr1 expression is much more dynamic than dpr2 expression. I use gain and loss of function experiments to identify dorsal organizer genes that regulate dpr1 and dpr2 expression. The dorsalizing factors beta-catenin, Bozozok (Boz), Noggin (Nog), and the mesendoderm-inducing factor Squint (Sqt) are all able to induce ectopic expression of dpr1 and dpr2. In reciprocal loss of function experiments, loss of maternal beta-catenin signaling leads to loss of early dorsal dpr1 and dpr2 expression, whereas loss of Boz and/or Nodal signaling does not. Ectopic expression of the ventralizing molecule Bmp2b leads to reduction of dpr1 and dpr2 expression. These results suggest that, in early zebrafish development, dpr1 and dpr2 are targets of beta-catenin and/or an unknown downstream effector. Their expression from 30% epiboly through shield is maintained by Nodal signaling and likely refined by the mutually antagonistic effects of Boz and bone morphogenetic protein signaling.

Zebrafish bmp2a and bmp2b mRNA expression in the developing median fins (caudal, anal, and dorsal) of late-stage larvae (>5 days post-fertilization) was analyzed by reverse transcriptase-PCR (RT-PCR) and in situ hybridization. bmp2a is expressed in developing fin rays, while bmp2b is expressed in developing fin rays, hypertrophic chondrocytes, and in the zone of segmentation (ZS) in developing anal and dorsal fin radials. This latter pattern of bmp2b expression in the ZS mirrors tetrapod bmp2 expression in developing joints. Additionally, both genes are expressed in neural and hemal arches and spines. bmp2a is strongly expressed in the lens; lens bmp2b expression is detected only weakly via RT-PCR.

Follicle-stimulating hormone receptor (fshr) and luteinizing hormone/choriogonadotropin receptor (lhcgr) exhibit differential temporal expression patterns during zebrafish folliculogenesis with fshr being dominant during vitellogenic growth and lhcgr increasing its expression dramatically before maturation. The dynamic and distinct expression patterns of fshr and lhcgr suggest that they are under tight regulatory control. However, the underlying mechanisms for the differential expression of the two receptors remain unknown. We have recently demonstrated that members of bone morphogenetic protein (BMP) family are largely expressed in the oocyte, while their receptors are exclusively localized on the follicle cells, suggesting a potential paracrine signaling from the oocyte to the follicle cells by BMPs. In this study, we investigated the effects of zebrafish BMP2b (zfBmp2b) and BMP4 (zfBmp4) on the expression of fshr and lhcgr using a novel co-culture approach. The recombinant zfBmp2b or zfBmp4-producing CHO cells were co-cultured with the zebrafish follicle cells followed by real-time qPCR analysis of fshr and lhcgr expression. Our results showed that zfBmp2b and zfBmp4 both down-regulated fshr, while up-regulated lhcgr expression at 24 h of co-culturing. This finding, together with the high expression level of BMP receptors in the follicle cells prior to oocyte maturation, strongly suggests a potential role for BMPs in the differential expression of fshr and lhcgr, especially in the full-grown follicles before maturation. As BMPs are largely expressed in the oocyte, this also implies an important role for the oocyte in orchestrating the differentiation and function of the follicle cells.

The enteric nervous system (ENS) is derived from neural crest cells (NCCs). Defects in ENS NCCs colonizing in the intestines lead to an absence of enteric ganglia in the colon and results in Hirschsprung's disease (HSCR). Bone morphogenetic proteins (BMPs) play diverse roles in the proliferation, migration and survival of ENS NCCs; however, whether BMPs are involved in HSCR and the underlying mechanism remains largely unknown. In this study, we found that BMP2 expression is significantly decreased in HSCR patients. Further experiments demonstrated that BMP2 is involved in the regulation of NCC proliferation, migration and differentiation. In a detailed analysis of the role of BMP2 in HSCR development in vivo, we demonstrated that BMP2b regulates the proliferation, migration and differentiation of vagal NCCs in zebrafish and that BMP2b is required for intestinal smooth muscle development. In addition, we showed that BMP2b is involved in regulating the expression of glial cell line-derived neurotrophic factor (GDNF) in the intestine, which mediates the regulation of ENS development by BMP2b in zebrafish. These results highlight a central role of the BMP-GDNF cascade in intestinal patterning and ENS development. Our results further demonstrate the key role of BMP2 in the etiology of HSCR in vitro and in vivo.

Wnt/β-catenin signaling controls various cell fates in metazoan development, and its dysregulation is often associated with cancer formation. However, regulations of this signaling pathway are not completely understood. Here, we report that Lzap, a tumor suppressor, controls nuclear translocation of β-catenin. In zebrafish embryos disruption of lzap increases the expression of chordin (chd), which encodes a bone morphogenetic protein (BMP) antagonist that is localized in prospective dorsal cells and promotes dorsal fates. Consistently, lzap-deficient embryos with attenuated BMP signaling are dorsalized, which can be rescued by overexpression of zebrafish lzap or bmp2b or human LZAP. The expansion of chd expression in embryos lacking lzap is due to the accumulation of nuclear β-catenin in ventral cells, in which β-catenin is usually degraded. Furthermore, the activity of GSK3, a master regulator of β-catenin degradation, is suppressed in lzap-deficient embryos via inhibitory phosphorylation. Finally, we also report that a similar regulatory axis is also likely to be present in a human tongue carcinoma cell line, SAS. Our results reveal that Lzap is a novel regulator of GSK3 for the maintenance of ventral cell properties and may prevent carcinogenesis via the regulation of β-catenin degradation.

The liver and pancreas originate from overlapping embryonic regions, and single-cell lineage tracing in zebrafish has shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of bipotential hepatopancreatic progenitors towards the liver or pancreas. Despite its pivotal role, the gene regulatory networks functioning downstream of Bmp2b signaling in this process are poorly understood. We have identified four and a half LIM domains 1b (fhl1b), which is primarily expressed in the prospective liver anlage, as a novel target of Bmp2b signaling. fhl1b depletion compromised liver specification and enhanced induction of pancreatic cells from endodermal progenitors. Conversely, overexpression of fhl1b favored liver specification and inhibited induction of pancreatic cells. By single-cell lineage tracing, we showed that fhl1b depletion led lateral endodermal cells, destined to become liver cells, to become pancreatic cells. Reversely, when fhl1b was overexpressed, medially located endodermal cells, fated to differentiate into pancreatic and intestinal cells, contributed to the liver by directly or indirectly modulating the discrete levels of pdx1 expression in endodermal progenitors. Moreover, loss of fhl1b increased the regenerative capacity of β-cells by increasing pdx1 and neurod expression in the hepatopancreatic ductal system. Altogether, these data reveal novel and critical functions of Fhl1b in the hepatic versus pancreatic fate decision and in β-cell regeneration.

In the genus Oryzias, the morphologies of the dorsal and anal fins are typical secondary sex characters. In the Japanese medaka (Oryzias latipes) and Thai medaka (Oryzias minutillus), androgen receptor (AR) expression levels in the dorsal, anal, and pectoral fins were higher in males than in females. Conversely, in both species estrogen receptor (ER) beta expression levels in the dorsal and anal fins were higher in females than in males. AR and ERbeta expression levels in the dorsal and anal fins of sex-undeterminable individuals of Thai medaka were intermediate between those in normal male and female Thai medaka. There was no difference in the bone morphogenic protein (Bmp) 2b expression level between male and female Japanese medaka. In contrast, the Bmp2b expression level in the dorsal fin of sex-undeterminable individuals was lower than in normal male and female Thai medaka. It is thus clear that androgen and estrogen regulate the sex-dependent characters of fin morphology in both Oryzias species. In sex-undeterminable individuals of Thai medaka, the low levels of Bmp2b expression in the dorsal fin are evidence that androgen and estrogen are necessary for adequate expression of Bmp2b in the normal development of at least the dorsal fin.

Iron is one of the essential elements of life. Iron metabolism is related to bone metabolism. Previous studies have confirmed that iron overload is a risk factor for osteoporosis. But the correlation between iron deficiency and bone metabolism remains unclear. Ferroportin 1 is identified as a cellular iron exporter and required for normal iron cycling. In zebrafish, the mutant of ferroportin 1 gene (fpn1), weh(tp85c) exhibited the defective iron transport, leading to developing severe hypochromic anemia. We used weh(tp85c) as a model for investigating iron deficiency and bone metabolism. In this study, we examined the morphology of the developing cartilage and vertebrae of the Weh(tp85) compared to the wild type siblings by staining the larvae with alcian blue for cartilage and alizarin red for the bone. In addition, we evaluated the expression patterns of the marker genes of bone development and cell signaling in bone formation. Our results showed that weh(tp85c) mutant larvae exhibited the defects in bone formation, revealing by decreases in the number of calcified vertebrae along with decreased expression of osteoblast novel genes: alpl, runx2a and col1a1a and BMPs signaling genes in osteoblast differentiation: bmp2a and bmp2b. Our data suggest that iron deficiency anemia affects bone formation, potentially through the BMPs signaling pathway in zebrafish.

Ovary development appears to be under polygenic control, and is influenced by multiple genetic factors that may vary from organism to organism. To gain a better insight into the molecular mechanisms of carp ovary development, Suppression Subtractive Hybridization (SSH) DNA libraries in two species of Yellow River carp were analyzed. Primordial gonads and stage II ovaries were used as testers, and adult ovaries as drivers. One hundred and fifty differentially-expressed candidate genes were examined by Southern blot microarray hybridization. We identified 41 differentially-expressed genes in the PG (Primordial gonad) library and 37 in the stage II ovary library. Gene Ontology Biological Pathway analysis showed the genes were involved in signal transduction, proteolysis process, cell differentiation, TGF-β signal and other biological responses. Twenty-two candidate genes were selected and further characterized using qRT-PCR. Pvalb, epd, and MYH were found specifically expressed in PG, while bmp2b, desmin and fp1 were specifically expressed in stage II ovary. Our results indicate that these genes could be used as biomarkers of the early development of carp ovary. This finding will provide a basis for further understanding of the complex gonad developmental molecular mechanisms in Yellow River carp.

Background: The epicardium is the outer mesothelial layer of the heart. It encloses the myocardium and plays key roles in heart development and regeneration. It derives from the proepicardium (PE), cell clusters that appear in the dorsal pericardium (DP) close to the atrioventricular canal and the venous pole of the heart, and are released into the pericardial cavity. PE cells are advected around the beating heart until they attach to the myocardium. Bmp and Notch signaling influence PE formation, but it is unclear how both signaling pathways interact during this process in the zebrafish.

Results: Here, we show that the developing PE is influenced by Notch signaling derived from the endothelium. Overexpression of the intracellular receptor of notch in the endothelium enhances bmp expression, increases the number of pSmad1/5 positive cells in the DP and PE, and enhances PE formation. On the contrary, pharmacological inhibition of Notch1 impairs PE formation. bmp2b overexpression can rescue loss of PE formation in the presence of a Notch1 inhibitor, but Notch gain-of-function could not recover PE formation in the absence of Bmp signaling.

Conclusions: Endothelial Notch signaling activates bmp expression in the heart tube, which in turn induces PE cluster formation from the DP layer.

Keywords: heart development; pericardium; zebrafish.

Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.

Intermuscular bones (IBs) only exist in the myosepta of lower teleosts and its molecular mechanism remains to be clarified. Bone morphogenetic proteins (BMPs) have been demonstrated to be involved in various physiological processes, including bone and cartilage formation. In this study, we firstly obtained and characterized nine bmp genes for Megalobrama amblycephala, which belongs to Cyprinidae and have a certain amount of IBs. Sequence alignment and phylogenetic analysis both documented that the mature proteins of M. amblycephala bmp genes were highly conserved with other corresponding homologs, respectively, indicating that the function of each bmp gene has been conserved throughout evolution. As a step to characterize potential involvement of bmp genes in IB formation and development, spatiotemporal expressions of nine bmp genes (bmp2a, bmp2b, bmp3, bmp4, bmp5, bmp7b, bmp8a, bmp14 and bmp16) were investigated during the key development stages of IBs. During the ossification process from stage I (the IBs haven't emerged) to stage IV (all of the IBs ossified in the tail with the mature morphology), the expression profiles revealed that bmp16 was the most abundant transcript while bmp4 had the lowest abundance. The mRNA levels of bmp3, bmp4, bmp5 and bmp8a increased significantly at stage II, suggesting their roles in stimulating IB formation. The expression of bmp7b reached the highest level at stage III (the rapid period of IB development), suggesting potential involvement of bmp7b in promoting osteoblast differentiation. With the exception of bmp7b and bmp16, most bmp genes appeared a significant increase at IB maturation phase (stage IV), which means that they may play important roles in maintenance of IB morphogenesis. Spatial tissue distribution of bmp genes showed that most bmp genes were observed at the highest level in developing IBs at one year old fish. Spatiotemporal expression patterns suggest the potential key roles of these bmp genes in IBs formation and maintenance in fish, being as possible promoters or inhibitors.

Pyrimethanil is a broad-spectrum fungicide commonly used in the prevention and treatment of Botrytis cinerea. However, little information is available in the literature to show the toxicity of Pyrimethanil to cardiac development. In this study, we used an experimental animal model to explore the developmental and cardiac toxicity of Pyrimethanil in aquatic vertebrates; we exposed zebrafish embryos to Pyrimethanil at concentrations of 2, 4, and 6 mg/L from 5.5 to 72 h post fertilisation. We found that Pyrimethanil caused a decrease in the hatching rate, heart rate, and survival rate of zebrafish embryos. Pyrimethanil exposure also resulted in pericardial and yolk sac edema, spinal deformity, and heart loop failure. Moreover, Pyrimethanil increased reactive oxygen stress levels and heightened the activity of superoxide dismutase and catalase. Alterations were induced in the transcription of apoptosis-related genes (p53, Bax, Bcl2, Casp 9, and Casp6l1) and heart development-related genes (Tbx2b, Gata4, Myh6, Vmhc, Nppa, Bmp2b, Bpm 4, and Bpm 10). Our data showed that the activation of Wnt signalling by BML-284 could partially rescue the malformed phenotype caused by Pyrimethanil. Our results provide new evidence for Pyrimethanil's toxicity and the danger of its residues in the environment and agricultural products.