Differential adhesion leads to segregation and exclusion of N-cadherin-deficient cells in chimeric embryos.

Cadherin-mediated cell-cell interactions are thought to be critical in controlling cell sorting during embryogenesis. Here, we report that chimeric embryos generated with N-cadherin-deficient (N-cadherin(-/-)) embryonic stem cells develop further than embryos completely lacking N-cadherin only when the myocardium consists of N-cadherin-positive cells. Initially, the N-cadherin-negative and -positive cells mix together to form chimeric tissues; however, by embryonic day 9.5, the N-cadherin(-/-) cells segregate from the wild-type cells forming distinct aggregates. The chimeric embryos have large aggregates of N-cadherin(-/-) myocardial cells in the heart lumen, indicating that the cells are unable to maintain cell-cell contacts with N-cadherin-positive myocytes. This sorting-out phenomenon also is apparent in somites, neural tube, and developing brain where N-cadherin(-/-) cells form distinct lumenal structures. These studies demonstrate that N-cadherin-mediated adhesion is critical for maintaining cell-cell interactions in tissues undergoing active cellular rearrangements and increased mechanical stress associated with morphogenesis.

Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin.

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. We show that N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin was effective in rescuing the embryos. The rescued embryos exhibited an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development was severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos was corrected, demonstrating that this phenotype was secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos were more developed than null embryos, they were smaller than wild-type embryos, even though the integrity of the cardiovascular system appeared normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. Our data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. Our data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development.

Retinoid signaling is required to complete the vertebrate cardiac left/right asymmetry pathway.

Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window.

Retinoid signaling required for normal heart development regulates GATA-4 in a pathway distinct from cardiomyocyte differentiation.

Vitamin A is essential for normal embryonic cardiogenesis. The vitamin A-deficient phenotype in the avian embryo includes an abnormal heart tube closed at the sinus venosus and the absence of large vessels that normally connect the embryonic heart to the developing circulatory system. In vitamin A-deficient embryos the expression of cardiomyocyte differentiation genes, including atrial-specific myosin heavy chain, ventricular-specific myosin, and sarcomeric myosins as well as the putative cardiomyocyte specification gene Nkx-2.5, is not altered. However, the expression of transcription factor GATA-4 is severely decreased in the heart-forming regions of vitamin A-deficient stage 7-10 embryos. Significantly, GATA-4 transcripts are completely lacking in the lateral mesoderm posterior to the heart, in the area of the developing cardiac inflow tract that later displays prominent morphological defects, including a closed nonseptated heart lacking a sinus venosus. The administration of retinol to the vitamin A-deficient embryo restores GATA-4 expression and completely rescues the vitamin A-deficient phenotype. Our results indicate that GATA-4 is a component of the retinoid-mediated cardiogenic pathway unlinked to cardiomyocyte differentiation, but involved in the morphogenesis of the posterior heart tube and the development of the cardiac inflow tract.

Initial retinoid requirement for early avian development coincides with retinoid receptor coexpression in the precardiac fields and induction of normal cardiovascular development.

Vitamin A requirement for early embryonic development is clearly evident in the gross cardiovascular and central nervous system abnormalities and an early death of the vitamin A-deficient quail embryo. This retinoid knockout model system was used to examine the biological activity of various natural retinoids in early cardiovascular development. We demonstrate that all-trans-, 9-cis-, 4-oxo-, and didehydroretinoic acids, and didehydroretinol and all-trans-retinol induce and maintain normal cardiovascular development as well as induce expression of the retinoic acid receptor beta2 in the vitamin A-deficient quail embryo. The expression of RARbeta2 is at the same level and at the same sites where it is expressed in the normal embryo. Retinoids provided to the vitamin A-deficient embryo up to the 5-somite stage of development, but not later, completely rescue embryonic development, suggesting the 5-somite stage as a critical retinoid-sensitive time point during early avian embryogenesis. Retinoid receptors RARalpha, RARgamma, and RXRalpha are expressed in both the precardiac endoderm and mesoderm in the normal and the vitamin A-deficient quail embryo, while the expression of RXRgamma is restricted to precardiac endoderm. Vitamin A deficiency downregulates the expression of RARalpha and RARbeta. Our studies provide strong evidence for a narrow retinoid-requiring developmental window during early embryogenesis, in which the presence of bioactive retinoids and their receptors is essential for a subsequent normal embryonic development.

Vitamin A-deficient quail embryos have half a hindbrain and other neural defects.

BACKGROUND: Retinoic acid (RA) is a morphogenetically active signalling molecule thought to be involved in the development of severely embryonic systems (based on its effect when applied in excess and the fact that it can be detected endogenously in embryos). Here, we adopt a novel approach and use the vitamin A-deficient (A-) quail embryo to ask what defects these embryos show when they develop in the absence of RA, with particular reference to the nervous system. RESULTS: We have examined the anatomy, the expression domains of a variety of genes and the immunoreactivity to several antibodies in these A- embryos. In addition to the previously documented cardiovascular abnormalities, we find that the somites are smaller in A- embryos, otic vesicle development is abnormal and the somites continue up to and underneath the otic vesicle. In the central nervous system, we find that neural crest cells need RA for normal development and survival, and the neural tube fails to extend any neurites into the periphery. Using general hindbrain morphology and the expression patterns of Hoxa-2, Hoxb-1, Hoxb-4, Krox-20 and FGF-3 as markers, we conclude that segmentation in the myelencephalon (rhombomeres 4-8) is disrupted. In contrast, the dorsoventral axis of the neural tube using Shh, islet-1 and Pax-3 as markers is normal. CONCLUSIONS: These results demonstrate at least three roles for RA in central nervous system development: neural crest survival, neurite outgrowth and hindbrain patterning.

[A diagnostic analysis of a genetic mutation associated with a deficiency of leukocyte adhesion in cattle]. / Diagnosticheskii analiz geneticheskoi mutatsii, assotsiirovannoi s defitsitom adgezivnosti leikotsitov u krupnogo rogatogo skota.

Bovine leukocyte adhesion (BLAD) is a recessive autosomal disease in Holstein-Friesian cattle caused by point mutation in CD18 gene encoding neutrophil-surface glycoprotein. To determine BLAD carriers, the convenient primers were chosen to amplify the mutant region of gene with the following restriction analysis. A screening program for BLAD has been initiated. Among 190 animals from different Ukrainian farms 6 were heterozygous according to the tested trait, i.e., BLAD deficient. No homozygous BLAD carriers were detected.

Hensen's node from vitamin A-deficient quail embryo induces chick limb bud duplication and retains its normal asymmetric expression of Sonic hedgehog (Shh).

Both Hensen's node, the organizer center in chick embryo, and exogenous retinoic acid are known to induce limb duplication when grafted or applied to the host chick limb bud. Retinoic acid is known to be present in the node and has been proposed as the putative morphogen for chick limb development. Here, we report that Hensen's node from vitamin A-deficient quail embryo induces limb duplication in the host chick embryo similar to that induced by the node from vitamin A-sufficient control embryos. We also demonstrate that the expression of Sonic hedgehog (Shh), recently shown to be the mediator of polarizing activity in the chick limb bud, is not affected by the endogenous vitamin A status of the embryo. Furthermore, whole-mount in situ hybridization revealed asymmetry of Shh expression in the Hensen's node of both vitamin A-sufficient and -deficient quail embryos. Retinoids were not detectable in the eggs from which vitamin A-deficient embryos were obtained. Extracts from normal embryos induced a level of expression of reporter gene equivalent to the presence of 3.4 pg of active retinoids per embryo, while those from vitamin A-deficient embryos induced a baseline level of reporter gene expression similar to that of the controls. Our studies suggest that endogenous retinoic acid is not involved in Shh expression nor in regulating its asymmetry during normal early avian embryogenesis and support the current view that endogenous retinoic acid may not be a direct morphogen for limb bud duplication.

Vitamin A deficiency and the expression of retinoic acid receptors during early cardiogenesis in quail embryo.

The vitamin A deficiency-associated lethal syndrome observed in avian embryos may be linked to dysfunction of vitamin A-dependent genes. We tested this hypothesis in a quail embryo model by examining the expression of retinoic acid receptors (RARs) and cytosolic retinoic acid binding protein (CRABP) in normal and vitamin A-deficient embryos during early development. RARα and RARγ mRNA were expressed at the same level in normal and vitamin A-deficient embryos during all stages studied. Expression of CRABP I was low in normal and vitamin A-deficient quail embryos during early development, but increased rapidly at later stages. Two transcripts of RARß, 3.2 and 3.5 kb, were detected in quail embryos during developmental stages 6-12. In normal emryos the level of the 3.2-kb isoform increased as embryonic development advanced. The expression of the 3.5-kb transcript was significantly decreased in vitamin A-deficient embryos, while the 3.2-kb transcript was undetectable by northern analysis. In situ hybridization of stage 7-8 normal quail embryos using a chicken RARß2 riboprobe revealed that RARß2 expression was predominantly associated with the cell populations in heart-forming regions, somites, neural folds, notochord and the presumptive thyroid. In stark contrast, in the vitamin A-deficient quail embryo RARß2 was not expressed in any of the above cell populations. We conclude that the expressions of RARß and CRABP I are developmentally regulated. Additionally, the expression of RARß2 during early embryogenesis is regulated by vitamin A status. We propose that RARß2 plays an important role in the mechanism of action of retinoids in early avian development. The lack of expression of RARß2 may be linked to abnormalities of the cardiovascular system.

Comparative study of Msx-1 expression in early normal and vitamin A-deficient avian embryos.

Homeobox-containing genes may play an important role in establishing embryonic patterns during development of vertebrates. Retinoic acid is able to induce expression of Hox genes in cells in culture and to alter expression patterns in the developing vertebrate embryos. Using wholemount in situ hybridization, we have examined and compared the expression patterns of a homeobox-containing gene, Msx-1, in early normal and vitamin A-deficient quail embryos. At gastrulation stage, Msx-1 is primarily expressed in the posterior half of both normal and vitamin A-deficient embryos. However, the gene is expressed wider and stronger in the vitamin A-deficient embryos. At neurulation stages, Msx-1 is continuously expressed in the posterior region up to Hensen's node and in the edge of the neural fold in both normal and vitamin A-deficient embryos. Notably, in the vitamin A-deficient embryos, Msx-1 is expressed more strongly and is also expressed ectopically in the anterior and precardiac regions. These results provide evidence that endogenous retinoids are involved in the normal expression of Msx-1 in avian embryo and that the expression of Msx-1 is downregulated by endogenous and physiological retinoids in vivo during early avian embryogenesis.

[The mutagenic activity of the DNA of recombinant plasmids in a competent Bacillus subtilis culture]. / Mutagennaia aktivnost' DNK rekombinantnykh plazmid v kompetentnoi kul'ture Bacillus subtilis.

The method for testing foreign plasmid DNA mutagenicity on the competent culture of B. subtilis has been developed. High mutagenic effect of DNA of recombinant plasmids carrying a single human Alu-repeat or the same repeat in combination with human apoAi gene or human insulin gene was demonstrated. The vector plasmid pUC18 had no mutagenic activity. According to the data of dot-blotting some fragments of recombinant plasmid DNA of human origin can integrate in B. subtilis chromosome by means of illegitimate recombination. It is concluded that B. subtilis test system is suitable for detection of potential mutagenic polynucleotide sequences in recombinant plasmid constructions produced for gene therapy purposes.

[The induction of mutations in 6-mercaptopurine resistance in Chinese hamster cells under the action of the recombinant plasmid pAins containing the human insulin gene]. / Induktsiia mutatsii rezistentnosti k 6-merkaptopurinu v kletkakh kitaiskogo khomiachka pod deistviem rekombinantnoi plazmidy pAins, soderzhashchei gen insulina cheloveka.

The mutagenic activity of the pUC19 bacterial plasmid DNA and the pAins recombinant plasmid DNA carrying human insulin gene has been investigated. Both pUC19 and pAins plasmid DNAs have been shown to induce the gene mutations in hprt locus of Chinese hamster cell line. The high level of the gene mutations (similar to the indices of the gene mutations induced by the chemical mutagens) has been in the focus of attention. The conclusion has been made concerning impossibility to use pAins plasmid DNA in the diabetes mellitus gene therapy. It is necessary to test the mutagenic properties of the DNA molecules produced for gene therapy of human inherited diseases.

[The restoration by Escherichia coli RecA protein of the survival of gamma-irradiated HeLa cells reduced by the DNA repair inhibitors caffeine and 3-aminobenzamide]. / Vosstanovlenie RecA-belkom Escherichia coli vyzhivaemosti gamma-obluchennykh kletok HeLa, snizhennoi ingibitorami reparatsii DNK kofeinom i 3-aminobenzamidom.

It is confirmed that inhibitors of DNA repair caffeine and 3-aminobenzamide decrease the survival of gamma-irradiated HeLa cells. It is shown that the decreased survival of irradiated cells is reversed when Escherichia coli RecA protein is introduced into cell nucleases with the aid of liposomes. This effect is more expressed in caffeine-treated (before or after irradiation) than in 3-aminobenzamide-treated (before irradiation) cells. It is suggested that E. coli 38 kD RecA protein may compensate the function of HeLa RecA-like protein, inhibited by DNA repair inhibitors, which is necessary for the repair of single-strand breaks and double-strand breaks of DNA.