Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-β superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.     

Distribution of endoglin in early human development reveals high levels on endocardial cushion tissue mesenchyme during valve formation

Endoglin is a component of the receptor complex for transforming growth factor (TGF)-β1 and TGF-β3. We analysed its expression by immunohistochemistry in human embryos at 4–8 weeks of gestation and in hearts ranging from 4–13 weeks old. We compared endoglin distribution with that of TGF-β receptors type I (TβR-I), type II (TβR-II) and betaglycan. Endoglin was found on endothelial cells in all tissues examined, consistent with its expression in adult blood vessels. TβR-I, TβR-II and betaglycan were observed on most cell types and had an overall similar pattern of distribution. Endoglin was detected on the endocardium as early as 4 weeks, but was absent from myocardium. It was present at high levels on the endocardial cushion tissue mesenchyme from 5–8 weeks’ gestation, during heart septation and valve formation, and subsequently decreased as the valves matured. Endoglin expression in heart extracts was confirmed by Western blot analysis. TβR-I, TβR-II and betaglycan were mostly found on cardiac myocytes, but were detectable at low levels on endocardium. They were expressed transiently on cushion mesenchyme, albeit at much lower levels than endoglin. All four components of the TGF-β receptor complex were detected by RT-PCR in embryonic heart. Thus transient up-regulation of the components of the TGF-β receptor complex, and particulartly of endoglin, is associated with heart septation and valve formation during early human development.     

Expression of a medaka (Oryzias latipes) Bar homologue in the differentiating central nervous system and retina

Endoglin is an auxiliary receptor for the transforming growth factor-β family of cytokines and is required for angiogenesis and heart development. Endoglin expression during mouse embryogenesis was analysed by monitoring β-galactosidase expression from a lacZ reporter cassette inserted downstream of the endoglin promoter. Expression was first detected at 6.5 days post-coitum (dpc) in the amniotic fold and developing allantois. Between 7.5 and 8.5 dpc, endoglin was expressed in endothelial cells of the yolk sac, dorsal aorta and primitive heart tube, and from 9.5 to 13.5 dpc in endothelial cells throughout the developing vasculature. Interestingly, this pattern of endoglin expression is almost identical to that reported for Alk1.     

Generation of new Notch2 mutant alleles

The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, and mutations in its components disrupt embryonic development in many organisms and cause inherited diseases in humans. We previously described construction and analysis of a hypomorphic allele of the Notch2 gene. Homozygosity for this allele leads to embryonic and perinatal lethality due to cardiovascular and kidney defects. We report here novel Notch2 mutant alleles generated by gene targeting in embryonic stem cells, including a conditional null allele in which exon 3 of the Notch2 gene is flanked by loxP sequences. These new Notch2 mutant alleles expand the set of tools available for studying the myriad roles of the Notch pathway during mammalian development and will enable analysis of Notch2 function at additional stages of embryogenesis and in adult mice.     

Generation of a conditional allele for the mouse endothelial nitric oxide synthase gene

Mice with endothelial nitric oxide synthase (eNOS) deletions have defined the crucial role of eNOS in vascular development, homeostasis, and pathology. However, cell specific eNOS function has not been determined, although an important role of eNOS has been suggested in multiple cell types. Here, we have generated a floxed eNOS allele in which exons 9–12, encoding the sites essential to eNOS activity, are flanked with loxP sites. Mice homozygous for the floxed allele showed normal eNOS protein levels and no overt phenotype. Conversely, homozygous mice with Cre‐deleted alleles displayed truncated eNOS protein, lack of vascular NO production, and exhibited similar phenotype to eNOS knockout mice, including hypertension, low heart rate, and focal renal scarring. These findings demonstrate that the floxed allele is normal and it can be converted to a non‐functional eNOS allele through Cre recombination. This mouse will allow time‐ and cell‐specific eNOS deletion. genesis 50:685–692, 2012. © 2012 Wiley Periodicals, Inc.     

Gene inactivation as a mechanism for the expression of recessive phenotypes.

A series of Chinese hamster ovary cell hybrids were constructed which were heterozygous at the emtB and chr loci. These loci encode two recessive drug-resistance genes (emetine resistance and chromate resistance, respectively) located on a structurally hemizygous region on the long arm of chromosome 2. These heterozygous hybrids therefore exhibit wild-type sensitivity to both emetine and chromate. Drug-resistant variants were then selected in medium containing either emetine or chromate, and the mechanism of reexpression of the recessive drug-resistant allele was determined by karyotypic analysis of the resultant colonies. In previous studies at these loci we have determined that segregation of the recessive phenotype occurs primarily by (1) the loss of the chromosome 2 carrying the wild-type, drug-sensitive, allele, (2) deletion of the long arm of chromosome 2, or (3) loss of one chromosome 2 followed by duplication of the remaining homologue. However, a small proportion of segregants have also been detected which may have arisen by the mechanisms of de novo gene inactivation or mutation. In this report, hybrids are described which were constructed to allow selection for the retention of the chromosome carrying the wild-type allele and which therefore optimize isolation of these rare segregants. We demonstrate by karyotypic analysis, mutation frequency analysis, and microcell-mediated chromosome transfer that these rare segregants occur primarily by gene inactivation. We also demonstrate a dramatic increase in the proportion of segregants occurring by gene inactivation in two of these hybrids as compared with those previously reported, indicating that this mechanism may be an important mode of phenotype segregation in diploid cells and, therefore, in the development of cancers--such as the childhood tumors retinoblastoma and Wilms tumor--resulting from recessive alleles     

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.     

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.     

Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor beta1 with higher affinity receptors and an activated Alk1 pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

New Insights into the Role of the Maize Ameiotic1 Locus

In maize the am1-1 mutant allele results in both the male and female meiocytes undergoing mitosis in place of the meiotic divisions. A second mutant allele am1-praI enables both the male and female meiocytes to proceed to the early zygotene stage of meiotic prophase I before being blocked. Here we report on three new alleles that allow all male meiocytes to undergo mitosis but in female meiocytes approximately one quarter (am1-2), one half (am1-485), or all (am1-489) of them are blocked at an abnormal interphase stage. Previous analysis has shown that am1-praI is dominant to am1-1 in male meiocytes. Cytological analysis of heteroallelic combinations in female meiocytes now indicates a dominance relationship of am1-praI > am1-1 > am1-2/am1-485 > am1-489. The evidence provided by the female phenotypes of the new mutant alleles suggest that, whereas the normal am1 allele is required for the meiocytes to proceed through meiosis, a partially functional allele may be required for their diversion into a mitotic division. The partial or complete blockage of mitosis in female meiocytes carrying the new am1 alleles rules out the possibility that the mitotic division of mutant meiocytes reflects a simple default pathway for cells that cannot initiate meiosis. This locus may have a dual function.     

Genetic analysis of mutations at the Glued locus and interacting loci in Drosophila melanogaster

A genetic analysis of the dominant mutation Glued that perturbs the development of the normal axonal architecture of the fly's visual system was undertaken. Ten new alleles at this locus were identified and characterized. Two complementation groups that were identified failed to complement the original allele, suggesting that it is a double mutant or that it resides at a complex locus. Several of the new alleles display visual-system abnormalities similar to those of the original mutation. Seven of the eight members of one complementation group are embryonic/early larval lethals, like the original mutation. The other allele in this group is temperature sensitive. Homozygous mutant adults exhibit a temperature-sensitive female sterile phenotype. Unsuccessful attempts to recover genetic mosaics carrying clones of cells homozygous for some of these mutations revealed that they are either essential for the viability of individual cells or that they affect some other fundamental cellular function, such as mitosis or the ability to participate in tissue level organization, which prevents them from being recovered in adult mosaics. This also indicates that these mutations do not specifically affect neural cells. A number of X-ray- and EMS-induced partial and complete phenotypic "revertants" of the original allele have also been isolated as material for a comparative analysis of visual system development. All "revertants" that alter the abnormal eye phenotype towards the wild type have similar impact on the organization of the optic lobe.     

Genetic and Developmental Analysis of the Locus vnd in DROSOPHILA MELANOGASTER

Genetic and developmental analysis of an X-linked vital locus vnd was undertaken. Embryos hemizygous for the original allele vnd did not hatch and exhibited a disorganized ventral nervous system (VNS). The mutation maps in the region 1B6-7 to 1B9-10, a subregion of an area previously shown to be essential to normal neural development. In this paper, we report isolation of five new alleles at the locus vnd. Genetic complementation analysis of all mutations at the vnd locus, with lethal alleles at adjacent loci, indicates that all lesions at the locus vnd affect only one vital gene function in the region. Four of the five alleles are embryonic lethal; one allele is subvital and behaves like an hypomorphic mutation. Hemizygous embryos for three of the four embryonic lethal alleles were inspected in histological sections; all exhibited disorganized VNS similar to the original allele. The developmental analysis in gynandromorphic genetic mosaics shows that (1) vnd+ gene function is not essential in most imaginal-disc cell derivatives, (2) only about 30% of the mosaic zygotes survive as adults, (3) mosaic zygotes with mutant tissue close to the head cuticle are least likely to survive, and (4) mutant tissue in the thoracic ganglion in the adult is not necessarily lethal. The mosaic data are consistent with the vnd+ gene function being necessary in neural cells derived from the anterioventral region of the blastoderm.     

Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.     

Endoglin is expressed in the chicken vasculature and is involved in angiogenesis.

Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex, highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant vascular disorder caused by a haploinsufficiency mechanism. Vascular lesions (telangiectasia and arteriovenous malformations) in HHT1 are associated with loss of the capillary network, suggesting the involvement of endoglin in vascular repair processes. Using the chick chorioallantoic membrane (CAM) as an angiogenic model, we have analyzed the expression and function of chicken endoglin. A pan-specific polyclonal antibody (pAb) recognized chicken endoglin as demonstrated by immunostaining and Western blot analysis. In ovo treatment of chicken embryos with this pAb resulted in a significantly increased area of CAM. This effect was likely mediated by modulation of the ligand binding to endoglin as this pAb was able to inhibit TGF-beta1 binding. These results support the involvement of endoglin in the angiogenic process.     

Phenotypic consequences and genetic interactions of a null mutation in the Drosophila Posterior Sex Combs gene.

The Posterior Sex Combs (Psc) gene of Drosophila is a member of the Polycomb (Pc) group of transregulatory genes. Previous analyses of the function of this gene in Drosophila embryogenesis have been hampered by the lack of a null mutation. We recently isolated a mutation that deletes the 5' end of the Psc gene. This allele appears to be a null mutation, and we have used it to determine the Psc zygotic null phenotype and to look at the interactions of a null allele of Psc with five other Pc group mutations. We find evidence for transformations along both the anterior-posterior and dorsal-ventral axes in embryos of a variety of genotypes that include a null mutation in Psc. The phenotypes of embryos that are doubly mutant for a null allele of Psc and a mutation in a second Pc group gene show dramatic synergistic effects, but in their specifics they are dependent on the identity of the second Pc group gene. This is different from the relatively uniform phenotypes seen among double mutants that contained the allele Psc1, which has both gain and loss of function properties. The differences in the phenotypes of the doubly mutant embryos allow us to eliminate one class of molecular models to explain the dramatic synergism seen with mutations in this group of genes.     

Association between a high-expressing interferon-gamma allele and a lower frequency of kidney angiomyolipomas in TSC2 patients

Tuberous sclerosis complex (TSC) is a familial hamartoma syndrome in which renal involvement is common and, at times, life threatening. We have investigated the potential effect of a non-TSC gene on renal disease in a cohort of 172 TSC patients with TSC2 mutations. Patients were genotyped for an interferon-gamma (IFN-gamma) microsatellite polymorphism, within intron 1, for which one common allele (allele 2, with 12 CA repeats) has been shown to have a higher expression of IFN-gamma. A chi(2) analysis was used to examine the association between IFN-gamma allele 2 and the development of kidney angiomyolipomas (KAMLs) in this TSC2 cohort. Because of the age-dependent development of KAMLs in TSC, we initially focused on the 127 patients who were >5 years old. Additional subgroup analyses were done to investigate the influence of age and gender. The transmission/disequilibrium test (TDT) was also performed in a subset of this cohort (46 probands) for whom parent and/or sibling samples were available for analysis. Both chi(2) analysis and TDT suggested an association between IFN-gamma allele 2 and the absence of KAMLs in patients who have known TSC2 mutations. Among the 127 patients who were >5 years old, KAMLs were present in 95 (75%) and were absent in 32 (25%). In the group with KAML present, the frequency of IFN-gamma allele 2 was 56%; in the group with KAML absent, the frequency of IFN-gamma allele 2 was significantly higher, at 78% (P=.02, by chi(2) analysis). The family-based TDT analysis gave similar results, with a TDT statistic (TDT chi2=5.45) corresponding to a P value of.02. Subgroup analyses show that both age and gender may influence the impact of this association. Although these results should be replicated in other populations with TSC, the present study suggests that modifier genes play a role in the variable expression of TSC and also suggests a potential therapy for KAMLs in patients with TSC.