Calibration of Densitometry in Radio-Isotopic in Situ Hybridization

Molecular imaging, which is the three-dimensional (3D) visualization of gene expression patterns, is indispensable for the study of the function of genes in cardiac development. The instrumentation, as well as the development of specific contrast agents for molecular imaging, has shown spectacular advances in the last decade. In this review, the spatial resolutions, contrast agents, and applications of these imaging methods in the field of cardiac embryology are discussed. Apart from 3D reconstructions from histological sections, not many of these methods have been applied in embryological research. This review shows that, for most methods, neither the spatial resolutions nor the specificity and applicability of the contrast agents are adequate for the reliable imaging of specific gene expression at the microscopic resolution required for embryological studies of small organs like the developing heart. Although a 3D reconstruction from sections will always suffer from imperfections, the resulting reconstructions meet the aim of most biological studies, especially since the original microscopic images are linked. With respect to imaging of gene expression, only histological sections and laser scanning microscopy provide the required resolution and specificity at the tissue and cellular level. Episcopic fluorescence image capturing and optical projection tomography are being used for microscopic phenotyping and lineage analysis, and both show potential for detailed molecular imaging. Other methods can be used very efficiently in rapid evaluation of biological experiments and high-throughput screens of large-scale gene expression profiling efforts when high spatial resolution is not required.

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Molecular imaging of the embryonic heart: Fables and facts on 3D imaging of gene expression patterns

Soufan

Hagoort

Moorman

2004

Birth Defects Research Pt C

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Prenatal exposure to thyroid hormone is necessary for normal postnatal development of murine heart and lungs

Tuyl

Blommaart

Boer

et al. 2004

Developmental Biology

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Maternal hypothyroxinemia during early pregnancy poses an increased risk for poor neuropsychological development of the fetus. We tested the hypothesis that maternal hypothyroidism before the onset of fetal thyroid function also affects postnatal development of heart and lungs. This question was addressed in transgenic mice that express herpes simplex virus thymidine kinase in their thyroidal follicle cells. Treatment with ganciclovir rendered these mice severely hypothyroid because viral thymidine kinase converts ganciclovir into a cytotoxic nucleoside analog. Since ganciclovir crosses the placenta, it also destroyed the thyroid of transgenic embryos while leaving the thyroids of nontransgenic littermates unaffected. Hypothyroidism of both mother and fetus did not affect prenatal heart and lung development. However, the postnatal switch from beta- to alpha-myosin heavy chain (beta- and alpha-MHC, respectively) gene expression and the increase of SERCA-2a mRNA expression did not occur in the ventricular myocardium of either the transgenic (thyroid destroyed) or nontransgenic (intact thyroid) offspring of hypothyroid mothers. Similarly, postnatal animals of the latter two groups retained elevated surfactant protein (SP) A, B, and C mRNA levels in their alveolar epithelium. In hypothyroid pups from hypothyroid mothers, these changes were accompanied by decreased alveolar septation. Our study shows that these effects of maternal hypothyroidism become manifest after birth and are aggravated by the concomitant existence of neonatal hypothyroidism.

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Three-dimensional reconstruction of gene expression patterns during cardiac development

Soufan

Ruijter

Hoff

et al. 2003

Physiological Genomics

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The study of the genetic regulation of embryonic development requires the three-dimensional (3D) mapping of gene expression at the microscopic level. Despite the recent burst in the number of methods focusing on 3D reconstruction of embryonic specimens, an adequate and accessible 3D reconstruction protocol for the visualization of patterns of gene expression is lacking. In this communication we describe a protocol that was developed for the 3D visualization of patterns of gene expression determined by in situ hybridization (ISH) on serial sections. The method still requires tissue sectioning, due to penetration limits of the specific staining agents into whole embryo preparations. With regard to expenditure of resources, i.e., hardware, software, and time, the protocol is relatively undemanding. Because the variation between specimens requires the visualization of multiple specimens per stage, it was decided to "do more, less well." The current protocol, therefore, results in reconstructions of sufficient, but not the highest, quality. The use of the protocol is demonstrated on a series of serially sectioned mouse hearts, ranging from embryonic day 8.5 to 14.5. The myocardium of the hearts was identified by ISH using a mixture of specific mRNA probes and reconstructed.

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Calibration of Densitometry in Radio-Isotopic in Situ Hybridization

Cited by 4 publications

References 14 publications

Molecular imaging of the embryonic heart: Fables and facts on 3D imaging of gene expression patterns

Molecular imaging of the embryonic heart: Fables and facts on 3D imaging of gene expression patterns

Prenatal exposure to thyroid hormone is necessary for normal postnatal development of murine heart and lungs

Three-dimensional reconstruction of gene expression patterns during cardiac development

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