Progress 10/01/99 to 09/30/03
Outputs
We achieved our objectives for this research. To identify activities potentially dependent upon retinoids in the developing heart, we took three approaches. In the first we constructed subtractive hybridization cDNA libraries between normal and retinoic acid (RA)-treated chick embryos; from this we identified 32 cDNAs with dysregulated expression caused by RA. In the second approach, we used microarray filters to identify gene sets dysregulated in RA-deficient (RAD) embryos as compared with normal-fed controls. This identified 86 genes with increased or decreased expression in the RAD embryo. The list of these is being published (Flentke et al. 2004; in press); several of these are cardiac specific. We further tested a subset of these candidate genes, with priority assigned to those especially relevant to development. Eight of 9 of these showed disregulated expression in these same embryos using real-time PCR, thus confirming the microarray results. Further, these same
genes were also dysregulated in two separate models of gestational vitamin A deficiency, the retinol-binding protein (RBP) null-mutant mouse, and the retinaldehyde dehydrogenase (Raldh2) null-mutant mouse. The basis for the altered expression is currently being pursued. A third approach employed the RBP-deficient mouse. We ascertained that RBP is essential for normal heart development. Hearts of these mice exhibited defects in trabeculation and myocyte proliferation. Several of the genes identified above were also dysregulated in the RBP-null hearts. This work was recently published (Wendler et al. 2003). We are currently detailing the consequences of these changes to adult cardiac function. The results find that RBP is essential for normal function of the adult heart; and in its deficiency hearts are enlarged and have significant failures as assessed by echocardiography. We propose from these findings that adult heart requires proper vitamin A delivery to support its normal
function.
Impacts
Congenital heart malformation remains the leading birth defect in North American children. Implementation of programs to ensure adequate vitamin A status, especially during the early months of pregnancy, should help reduce the incidence of these problems.
Publications
- Wendler CC, Schmoldt A, Flentke GR, Case LC, QUadro L, Blaner WS, Lough J, Smith SM. 2003. Increased fibronectin deposition in embryonic hearts of retinol-binding protein null mice. Circ. Res. 92:920-928.
- Lin M, Zhang M, Abraham M, Smith SM, Napoli JL. 2003. Mouse RALDH4: molecular cloning, cellular expression, and activity of 9-cis-retinoic acid biosynthesis in intact cells. J. Biol. Chem. 278:9856-9861.
- Flentke GR, Baker MW, Docterman KE, Power S, Lough J, Smith SM. 2004. Microarray analysis of retinoid-dependent gene activity during rat embryogenesis: increased collagen fibril production in a model of retinoid insufficiency. Dev. Dynamics. In press.
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Progress 01/01/02 to 12/31/02
Outputs
We continue to make excellent progress on this project. Microarrays were performed that identified 100 genes with specific up or down regulation in retinoid deficient embryos. We used real time PCR to confirm the dysregulated expression of a subset of these, with emphasis upon those essential for normal cardiogenesis. In situ hybridizations to complete the descriptive analysis of their disregulation are in completion. A manuscript detailing these findings in the vitamin A deficient embryo is in preparation. A second manuscript detailing these findings as specifically applied to the vitamin A deficient heart is also in preparation. We applied the above microarray findings to a second model of retinoid insufficiency, a mouse line that contains a null mutation in the retinol binding protein (RBP), a protein that transports vitamin to target tissues. These mice have a cardiac phenotype. The manuscript detailing the changes within the embryonic heart has been submitted. In
brief, null mutant hearts experience an up regulation of fibronectin, a transient delay in trabeculation, and commensurate enhancements of cardiomyocyte proliferation and cushion mesenchymal content. Cardiac function and structure in the adult null mutants is underway, and a manuscript detailing those alterations will be prepared in the next several months.
Impacts
Vitamin A insufficiency is still observed in certain segments of the U.S. population and is not uncommon in locations with inadequate nutriture. Cardiac defects remain the most common birth defect in the U.S., affecting 0.7/1000 live births. It is critical to identify nutritional inadequacies that pose a developmental risk for the fetus.
Publications
- Wendler, C., S.M. Smith, W.S. Blaner, J. Lough. 2002. Heart development in mice that are null mutant for retinol binding protein. NIH/NHLBI Symposium on Phenotyping Mouse Cardiovascular Function and Development. (Abstract)
- Wendler C.C., A. Schmoldt, G.R. Flentke, L.C. Case, L. Quadro, W.S. Blaner, J. Lough, S.M. Smith. 2003. Increased fibronectin deposition in embryonic hearts of retinol-binding protein null mice. (Submitted).
- Lin, M., M. Zhang, M. Abraham, S.M. Smith, J.L. Napoli. 2002. Mouse RALDH4: molecular cloning, cellular expression, and activity in 9-cis-retinoic acid biosynthesis in intact cells. J. Biol. Chem. (in press).
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Progress 01/01/01 to 12/31/01
Outputs
We continue to make progress in this complex project. Two avenues are under investigation: 1. Gene arrays identified several signaling pathways that are critical for normal development and are dysregulated by retinoids. To test the validity of the array findings, we invested substantial time in models to test the hypothesis. The first model is of retinoid deficiency in whole embryo culture. Karen Downs provided us with training in how to culture e7.5 mouse embryos; this is technically challenging and required significant investment in time and equipment. We obtained retinoid antagonists which will be used to create a retinoid deficient environment in embryo culture. We are now developing protocols that will create retinoid-depleted rat serum, using uv irradiation of serum. Using these two approaches, we will incubate embryos in retinoid-normal and -deficient environments, to test whether signaling pathways identified in the arrays are truly retinoid responsive. We
also are testing the 20 most altered array targets for their normal distribution within the normal mouse embryo; most of these have not been characterized previously. 10 are known genes, 10 are ESTs. Half are increased and half decreased by retinoid deficiency. Candidates with normal distributions which potentially inform retinoid roles in morphogenesis will be investigated further using the above whole embryo culture system. 2) We acquired mice that are null mutant for the retinol binding protein. These embryos will be easy to make retinoid deficient, because of their absolute requirement for dietary retinoid. Once the colony is expanded and genotyping is mastered, we will examine relevant array targets from (1) in this model, to explore their retinoid dependence and participation in development. We also established that these null mutants display an early cardiac dysmorphology. We have identified a key matrix protein that is dysregulated in the hearts of these mice, and which may be
responsible for the disruption of myofibrillogenesis and trabeculation in their hearts. Interpretation of their hearts originated from the microarray results from (1) above. We are preparing a first manuscript that provides the initial characterization of their cardiac phenotype.
Impacts
Understanding the role of retinoids in embryo development and pathogenesis may provide therapeutic strategies for avoiding or treating human and animal diseases.
Publications
- Zhang, M., W. Chen, S.M. Smith, J.L. Napoli. 2001. Molecular characterization of mouse retinol dehydrogenase type 1 (RDH1): a rate-limiting enzyme of all-trans-retinoic acid synthesis in vivo. J. Biol. Chem. 276:44083-44090.
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Progress 01/01/00 to 12/31/00
Outputs
Work this year focused on the identification of target genes for retinoid action in the developing embryo. Two approaches were used. In the first approach, subtractive hybridization libraries were constructed to isolate genes whose expression is dysregulated by retinoid deficiency within the embryo. 42 genes were isolated in the first round of screening. Sequencing revealed that several of these clones were novel. Others were known genes not previously known to be targets of retinoid action. Their expression within the embryo is currently being characterized. Results from this work are not yet ready for disclosure. In the second approach, microarray filters were used to identify target genes of retinoid action within the embryo. A large number of candidates were identified. Current efforts focus on a fairly well-characterized signaling pathway that was not previously known to be retinoid responsive. Experiments are underway to characterize this interaction within
embryos further. Results are not ready for disclosure.
Impacts
Retinoids, the active form of vitamin A, are essential for normal health of humans and animals. It is suspected that many Americans are surprisingly retinoid deficient. Indeed, a recent Institute of Medicine study reveals that carotenoid intakes of Americans have been over-estimated and we have not been consuming as much of this vitamin A precursor as previously believed. We do not have good numbers to document the risk these deficiencies pose to human health. In particular, there are suggestions that retinoid deficiency poses a risk for birth defects in certain U.S. populations. To better understand this risk, we must have a clear grasp of how retinoids participate in gestation, so that we can identify birth defects that may be caused by retinoid deficiency. Having molecular markers for retinoid deficiency, such as those isolated here, are a critical part of reducing this risk.
Publications
- No publications reported this period
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