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

Effect of Hyperglycemia on Gene Expression during Early Organogenesis in Mice

BACKGROUND: Cardiovascular and neural malformations are common sequels of diabetic pregnancies, but the underlying molecular mechanisms remain unknown. We hypothesized that maternal hyperglycemia would affect the embryos most shortly after the glucose-sensitive time window at embryonic day (ED) 7.5 in mice. METHODS: Mice were made diabetic with streptozotocin, treated with slow-release insulin implants and mated. Pregnancy aggravated hyperglycemia. Gene expression profiles were determined in ED8.5 and ED9.5 embryos from diabetic and control mice using Serial Analysis of Gene Expression and deep sequencing. RESULTS: Maternal hyperglycemia induced differential regulation of 1,024 and 2,148 unique functional genes on ED8.5 and ED9.5, respectively, mostly in downward direction. Pathway analysis showed that ED8.5 embryos suffered mainly from impaired cell proliferation, and ED9.5 embryos from impaired cytoskeletal remodeling and oxidative phosphorylation (all P ≤ E-5). A query of the Mouse Genome Database showed that 20-25% of the differentially expressed genes were caused by cardiovascular and/or neural malformations, if deficient. Despite high glucose levels in embryos with maternal hyperglycemia and a ~150-fold higher rate of ATP production from glycolysis than from oxidative phosphorylation on ED9.5, ATP production from both glycolysis and oxidative phosphorylation was reduced to ~70% of controls, implying a shortage of energy production in hyperglycemic embryos. CONCLUSION: Maternal hyperglycemia suppressed cell proliferation during gastrulation and cytoskeletal remodeling during early organogenesis. 20-25% of the genes that were differentially regulated by hyperglycemia were associated with relevant congenital malformations. Unexpectedly, maternal hyperglycemia also endangered the energy supply of the embryo by suppressing its glycolytic capacity

Maternal diabetes causes developmental delay and death in early-somite mouse embryos

Maternal diabetes causes congenital malformations and delays embryonic growth in the offspring. We investigated effects of maternal diabetes on mouse embryos during gastrulation and early organogenesis (ED7.5-11.5). Female mice were made diabetic with streptozotocin, treated with controlled-release insulin implants, and mated. Maternal blood glucose concentrations increased up to embryonic day (ED) 8.5. Maternal hyperglycemia induced severe growth retardation (approx.1 day) in 53% of the embryos on ED8.5, death in most of these embryos on ED9.5, and the termination of pregnancy on ED10.5 in litters with >20% dead embryos. Due to this selection, developmental delays and reduction in litter size were no longer observed thereafter in diabetic pregnancies. Male and female embryos were equally sensitive. High-throughput mRNA sequencing and pathway analysis of differentially expressed genes showed that retarded embryos failed to mount the adaptive suppression of gene expression that characterized non-retarded embryos (cell proliferation, cytoskeletal remodeling, oxidative phosphorylation). We conclude that failure of perigastrulation embryos of diabetic mothers to grow and survive is associated with their failure to shut down pathways that are strongly down-regulated in otherwise similar non-retarded embryos. Embryos that survive the early and generalized adverse effect of maternal diabetes, therefore, appear the subset in which malformations become manifest

Cardiac septation: a late contribution of the embryonic primary myocardium to heart morphogenesis

Cardiac septation: a late contribution of the embryonic primary myocardium to heart morphogenesis. Lamers WH, Moorman AF. Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. [email protected] Heart morphogenesis comprises 2 major consecutive steps, viz. chamber formation followed by septation. Septation is the remodeling of the heart from a single-channel peristaltic pump to a dual-channel, synchronously contracting device with 1-way valves. In the human heart, septation occurs between 4 and 7 weeks of development. Cardiac looping and chamber formation bring the contributing structures into position to engage in septation. Cardiomyocytes that participate in chamber formation do not materially contribute to septation. The (re)discovery of the role of extracardiac mesenchymal tissue in atrioventricular septation, the appreciation that the formation of the right atrioventricular connection is more than a mere rightward expansion of the atrioventricular canal, the awareness that myocardium originating from the so-called anterior heart field regresses after its function as outflow-tract sphincter ceases, and the recent finding that the myocardialized proximal portion of the outflow-tract septum becomes the supraventricular crest have all significantly enhanced our understanding of the morphogenetic processes that contribute to septation. The bifurcation of the ventricular conduction system is the landmark that separates the contribution of the atrioventricular cushions and the outflow-tract ridges to septation and that divides the muscular ventricular septum in inlet, trabecular, and outlet portions