Microinjecting recombinant rainbow trout Ea4-peptide of pro-IGF-I into zebrafish embryos causes abnormal development in heart, red blood cells, and vasculature

E-peptides and mature insulin-like growth factors (IGFs) are produced from pre-pro-IGFs during post-translational processing and co-secreted into the circulation. Previously, we reported that introduction of a transgene encoding the secreted form of rainbow trout (rt) Ea4-peptide or human (h) Eb-peptide into newly fertilized eggs of medaka (Oryzias latipes) and zebrafish (Danio rerio) resulted in developmental defects in heart, red blood cells and vasculature. In addition to vasculature and red blood cell developmental defects, multiple phenocopies of heart developmental defects categorized by developmental arrest at cardiomyocyte, heart tube and heart looping stages were also observed. These results raise a question of whether rtEa4- or hEb-peptide exerts pleiotropic inhibitory effects on heart, vasculature and red blood cell development in fish embryos. To answer this question, various amounts of recombinant rtEa4-peptide were microinjected into zebrafish eggs at 1.5, 2.5 and 5.5 h post-fertilization (hpf). Although a dose-dependent developmental defect in heart, vasculature and red blood cells was observed in embryos microinjected with rtEa4-peptide at 1.5 and 2.5 hpf, the heart development in all of the microinjected embryos was arrested at the cardiomyocyte stage. Furthermore, the mRNA levels of Nkx2.5, GATA5, VEGF, GATA1 and GATA2 genes in defective embryos were significantly reduced by rtEa4-peptide. These results confirm our previous findings that rtEa4- or hEb-peptide exhibits pleiotropic effects in inhibiting heart, vasculature and red blood cell development in zebrafish embryos.     

Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis

Blood vessels form either by the assembly and differentiation of mesodermal precursor cells (vasculogenesis) or by sprouting from preexisting vessels (angiogenesis). Endothelial-specific receptor tyrosine kinases and their ligands are known to be essential for these processes. Targeted disruption of vascular endothelial growth factor (VEGF) or its receptor kdr (flk1, VEGFR2) in mouse embryos results in a severe reduction of all blood vessels, while the complete loss of flt1 (VEGFR1) leads to an increased number of hemangioblasts and a disorganized vasculature. In a large-scale forward genetic screen, we identified two allelic zebrafish mutants in which the sprouting of blood vessels is specifically disrupted without affecting the assembly and differentiation of angioblasts. Molecular cloning revealed nonsense mutations in flk1. Analysis of mRNA expression in flk1 mutant embryos showed that flk1 expression was severely downregulated, while the expression of other genes (scl, gata1, and fli1) involved in vasculogenesis or hematopoiesis was unchanged. Overexpression of vegf(121+165) led to the formation of additional vessels only in sibling larvae, not in flk1 mutants. We demonstrate that flk1 is not required for proper vasculogenesis and hematopoiesis in zebrafish embryos. However, the disruption of flk1 impairs the formation or function of vessels generated by sprouting angiogenesis.     

BMP2 is required for early heart development during a distinct time period

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.     

Suppression of growth and cancer-induced angiogenesis of aggressive human breast cancer cells (MDA-MB-231) on the chorioallantoic membrane of developing chicken embryos by E-peptide of pro-IGF-I

E-peptide of the pro-Insulin-like growth factor-I (pro-IGF-I) is produced from pre-pro-IGF-I by proteolytic cleavage in the post-translational processing. Previous in vitro studies conducted in our laboratory showed that Ea4-peptide of rainbow trout (rt) pro-IGF-I or Eb-peptide of human (h) pro-IGF-I exhibited activities including induction of morphological differentiation, inhibition of anchorage-independent cell growth and suppression of invasion of several well established human cancer cell lines such as MDA-MB-231, HT-29, SK-N-F1, and HepG-2 (Chen et al. [2002] Gen Comp Endocrinol 126:342-351; Kuo and Chen [2002] Exp Cell Res 280:75-89). Seeding of aggressive human breast cancer cells, MDA-MB-231, on the chorioallantoic membrane (CAM) of 5 days old chicken embryos resulted in rapid growth and invasion of the cells and induction of blood vessel formation around the MDA-MB-231 cell mass in the chicken embryos. The invasion of MDA-MB-231 cells in the chicken embryos was further confirmed by immunocytochemistry. The rapid growth and invasion of MDA-MB-231 cells and the induction of blood vessel formation by MDA-MB-231 cells on chicken CAM are inhibited by treatment with a single or multiple doses of rtEa4- or hEb-peptide. Furthermore, a dose-dependent inhibition of angiogenesis by rtEa4- or hEb-peptide was also demonstrated by the chicken CAM assay. Results of microarray analysis of human gene chips (containing 9,500 unique cDNA clones) and confirmation by comparative real-time RT-PCR analysis showed that a group of genes related to cancer cell activities are up- or down-regulated in MDA-MB-231 cells transfected with a rtEa4-peptide gene. Together these results confirm the anti-tumor activity of rtEa4- and hEb-peptides, and further suggest that these peptides could be developed as therapeutics for treating human cancers.     

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.     

Mutations of the GATA4 and NKX2.5 genes in Chinese pediatric patients with non-familial congenital heart disease

A number of mutations in GATA4 and NKX2.5 have been identified to be causative for a subset of familial congenital heart defects (CHDs) and a small number of sporadic CHDs. In this study, we evaluated common GATA4 and NKX2.5 mutations in 135 Chinese pediatric patients with non-familial congenital heart defects. Two novel mutations in the coding region of GATA4 were identified, namely, 487C >T (Pro163Ser) in exon 1 in a child with tetralogy of Fallot and 1220C >A (Pro407Gln) in exon 6 in a pediatric patient with outlet membranous ventricular septal defect. We also found 848C >A (Pro283Gln) in exon 2 of the NKX2.5 gene in a pediatric patient with ventricular septal defect, patent ductus arteriosus and aortic isthmus stenosis. None of the mutations was detected in healthy control subjects (n = 114). This study suggests that GATA4 and NKX2.5 missense mutations may be associated with congenital heart defects in pediatric Chinese patients. Further clinical studies with large samples are warranted.     

Novel mutations in the TBX5 gene in patients with Holt-Oram Syndrome

The Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and cardiac malformations. Mutations in the TBX5 gene cause HOS and have also been associated with isolated heart and arm defects. Interactions between the TBX5, GATA4 and NKX2.5 proteins have been reported in humans. We screened the TBX5, GATA4, and NKX2.5 genes for mutations, by direct sequencing, in 32 unrelated patients presenting classical (8) or atypical HOS (1), isolated congenital heart defects (16) or isolated upper-limb malformations (7). Pathogenic mutations in the TBX5 gene were found in four HOS patients, including two new mutations (c.374delG; c.678G > T) in typical patients, and the hotspot mutation c.835C > T in two patients, one of them with an atypical HOS phenotype involving lower-limb malformations. Two new mutations in the GATA4 gene were found in association with isolated upper-limb malformations, but their clinical significance remains to be established. A previously described possibly pathogenic mutation in the NKX2.5 gene (c.73C > 7) was detected in a patient with isolated heart malformations and also in his clinically normal father.     

Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects

Smad5 has been implicated as a downstream signal mediator for several bone morphogenetic proteins (BMPs). To understand the in vivo function of Smad5, we generated mice deficient in Smad5 using embryonic stem (ES) cell technology. Homozygous mutant embryos die between E9.5 and E11.5, and display variable phenotypes. Morphological defects are first detected at E8.0 in the developing amnion, gut and heart (the latter defect being similar to BMP-2 knockout mice). At later stages, mutant embryos fail to undergo proper turning, have craniofacial and neural tube abnormalities, and are edematous. In addition, several extraembryonic lesions are observed. After E9.0, the yolk sacs of the mutants contain red blood cells but lack a well-organized vasculature, which is reminiscent of BMP-4, TGF-beta1 and TGF-beta type II receptor knockout mice. In addition, the allantois of many Smad5 mutants is fused to the chorion, but is not well-elongated. A unique feature of the Smad5 mutant embryos is that ectopic vasculogenesis and hematopoiesis is observed in the amnion, likely due to mislocation of allantois tissue. Despite the expression of Smad5 from gastrulation onwards, and in contrast to knockouts of Smad2 and Smad4, Smad5 only becomes essential later in extraembryonic and embryonic development.     

Antisense targeting of CXXC finger protein 1 inhibits genomic cytosine methylation and primitive hematopoiesis in zebrafish

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is approximately 70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.     

Cellular and molecular analyses of vascular tube and lumen formation in zebrafish

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.