Towards quantitative in situ hybridization

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissu

Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.1668F is a founder variant among Ashkenazi Jews (allele frequency of -.2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.Genetics of disease, diagnosis and treatmen

Trabeculated Right Ventricular Free Wall in the Chicken Heart Forms by Ventricularization of the Myocardium Initially Forming the Outflow Tract

Recent molecular lineage analyses in mouse have demonstrated that the right ventricle is recruited from anterior mesoderm in later stages of cardiac development. This is in contrast to current views of development in the chicken heart, which suggest that the initial heart tube contains a subset of right ventricular precursors. We investigated the fate of the outflow tract myocardium using immunofluorescent staining of the myocardium, and lineage tracer, as well as cell death experiments. These analyses showed that the outflow tract is initially myocardial in its entirety, increasing in length up to HH24. The outflow tract myocardium, subsequently, shortens as a result of ventricularization, contributing to the trabeculated free wall, as well as the infundibulum, of the right ventricle. During this shortening, the overall length of the outflow tract is maintained because of the formation of a nonmyocardial portion between the distal myocardial border and the pericardial reflections. Cell death and transdifferentiation were found to play a more limited contribution to the initial shortening than is generally appreciated, if they play any part at all. Cell death, nonetheless, plays an important role in the disappearance of the myocardial collar that continues to invest the aorta and pulmonary trunk around HH30, and in the separation of the intrapericardial arterial vessels. Taken together, we show, as opposed to some current beliefs, the development of the arterial pole is similar in mammals and bird

Foetal rise in hepatic enzymes follows decline in c-met and hepatocyte growth factor expression

BACKGROUND/AIMS: In the embryo, rapidly proliferating hepatocytes migrate from the liver primordium into the surrounding mesenchyme, whereas foetal hepatocytes are mitotically quiescent and accumulate hepatocyte-specific enzymes. We investigated the timing and topography of this behavioural switch. METHODS: The expression of the c-met receptor and its ligand, hepatocyte growth factor (HGF), was investigated in prenatal rat liver by in situ hybridization, immunohistochemistry and western-blot analysis. RESULTS: c-Met was expressed by hepatocytes and HGF by non-parenchymal liver cells. Their mRNA levels peaked during embryonic day (ED) 11-13. c-Met protein was weakly expressed in the entire liver during ED 11 and 12, but more abundantly at ED 13, when its expression withdrew to the hepatic periphery. Simultaneously, the periportal hepatocellular marker carbamoylphosphate synthetase began to accumulate in the centre of the liver. Although the definitive vascular architecture develops simultaneously, the downstream, pericentral hepatocytes began to express glutamine synthetase only 4 days later, suggesting a requirement for prior periportal hepatocyte maturation. Additionally, c-met protein appeared in the connective tissue surrounding the large veins. The c-met protein/mRNA ratio was substantially higher in non-epithelial cells (hepatic connective tissue, heart) than in endoderm-derived epithelia, including hepatocytes, indicating important post-transcriptional regulation. CONCLUSIONS: The decline in c-met expression reflects the end of the embryonic phase and heralds the onset of the fetal, maturational phase of liver developmen

Developmental changes in rat cardiac DNA, RNA and protein tissue base: implications for the interpretation of changes in gene expression

During cardiac development the expression levels of many genes change as determined by Northern blot, dot blot, RNase protection, quantitative RT-PCR. Western blot or immunoprecipitation analyses. It is not always realized that the total amount of RNA or protein per gram of heart, dubbed tissue base, may change significantly during development as well. If this would be the case, this has to be taken into account. So far, the (changing) tissue base has not been established during cardiac development. To this end developmental profiles of cardiac DNA, RNA and protein concentration were determined in rats ranging in age from embryonic day 13 until neonatal day 121. The profiles show significant development changes in each parameter, that closely match the distinct growth phases of the developing heart and provide the parameters that are essential for an adequate interpretation of changes in the amount of a distinct mRNA and/or protein. In a comparison between in situ hybridization and Northern blot analysis it is demonstrated that the same developmental profile leads to an almost opposite conclusion depending on whether or not the changing tissue base is taken into account. These findings are of great interest for studies aimed at unravelling the molecular mechanisms underlying the regulation of gene expression during cardiac developmen

Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation

Cell surface exposure of phosphatidylserine (PS) is shown to be part of normal physiology of skeletal muscle development and to mediate myotube formation. A transient exposure of PS was observed on mouse embryonic myotubes at E13, at a stage of development when primary myotubes are formed. The study of this process in cell cultures of differentiating C2C12 and H9C2 myoblasts also reveals a transient expression of PS at the cell surface. This exposure of PS locates mainly at cell-cell contact areas and takes place at a stage when the structural organization of the sarcomeric protein titin is initiated, prior to actual fusion of individual myoblast into multinucleated myotubes. Myotube formation in vitro can be inhibited by the PS binding protein annexin V, in contrast to its mutant M1234, which lacks the ability to bind to PS. Although apoptotic myoblasts also expose PS, differentiating muscle cells show neither loss of mitochondrial membrane potential nor detectable levels of active caspase-3 protein. Moreover, myotube formation and exposure of PS cannot be blocked by the caspase inhibitor zVAD(OMe)-fmk. Our findings indicate that different mechanisms regulate PS exposure during apoptosis and muscle cell differentiation, and that surface exposed PS plays a crucial role in the process of myotube formatio