A. F. M. Moorman

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level.

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.

Heterogeneous expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase genes in the rat liver lobulus.

We investigated the lobular localization and molecular level of expression of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene transcription of cholesterol 7 alpha-hydroxylase were predominant in pericentral hepatocytes of control rats, being 7.9-, 9.9-, and 4.4-fold higher than in periportal hepatocytes, respectively. Similar localization was found for sterol 27-hydroxylase: 2.9-, 2.5-, and 1.7-fold higher enzyme activity, mRNA, and gene transcription, respectively, was found in pericentral hepatocytes. Interruption of the enterohepatic circulation with colestid resulted in upregulation of these parameters for both enzymes, as a consequence of stimulated gene expression mainly in the periportal zone. In contrast, mRNA levels and gene transcription of 3-hydroxy-3-methylglutaryl CoA reductase showed opposite lobular distribution. Selective periportal expression for the latter was enhanced, but remained local, after colestid treatment. In situ hybridization showed unambiguously that cholesterol 7 alpha-hydroxylase mRNA is localized exclusively in the pericentral zone and that sterol 27-hydroxylase mRNA is expressed preferentially in the pericentral region, though less pronounced. Administration of colestid led to expression of both genes within a larger area of the liver lobulus. In conclusion, we suggest that cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase are coordinately regulated by the bile acid gradient over the lobulus, resulting in predominant expression in the pericentral zone. Opposite lobular localization of cholesterol and bile acid synthesis provides an alternative view to interregulation of these metabolic pathways.

Arginine-metabolizing enzymes in the developing rat small intestine.

Before weaning, arginine biosynthesis from citrulline most likely takes place in the small intestine rather than in the kidney. We studied the expression of ornithine cycle enzymes in the rat small intestine during perinatal development. The spatiotemporal patterns of expression of ornithine aminotransferase, carbamoylphosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase mRNAs were studied by Northern blot analysis and in situ hybridization. In addition, the expression of carbamoylphosphate synthetase and argininosuccinate synthetase protein was studied by immunohistochemistry. Before birth, the developmentally more mature proximal loops of the intestine expressed the mRNAs at higher concentrations than the more distal loops. After birth, this difference was no longer obvious. The mRNAs of argininosuccinate synthetase and argininosuccinate lyase, the enzymes that metabolize citrulline to arginine, were detectable only in the upper part of the villi, whereas the other mRNAs were concentrated in the crypts. The distribution of argininosuccinate synthetase protein corresponded with that of the mRNA, whereas carbamoylphosphate synthetase protein was present in all enterocytes of the crypts and villi. Hepatic arginase mRNA could not be detected in the enterocytes. The spatial distribution of the respective mRNAs and proteins along the villus axis of the suckling small intestine indicates that the basal enterocytes synthesize citrulline, whereas the enterocytes in the upper half of the villus synthesize arginine.

Expression patterns of mRNAs for ammonia-metabolizing enzymes in the developing rat: the ontogenesis of hepatocyte heterogeneity

SummaryThe expression patterns of the mRNAs for the ammonia-metabolizing enzymes carbamoylphosphate synthetase (CPS), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in developing pre- and neonatal rat liver byin situ hybridization.In the period of 11 to 14 embryonic days (ED) the concentrations of GS and GDH mRNA increases rapidly in the liver, whereas a substantial rise of CPS mRNA in the liver does not occur until ED 18. Hepatocyte heterogeneity related to the vascular architecture can first be observed at ED 18 for GS mRNA, at ED 20 for GDH mRNA and three days after birth for CPS mRNA. The adult phenotype is gradually established during the second neonatal week, i.e. GS mRNA becomes confined to a pericentral compartment of one to two hepatocytes thickness, CPS mRNA to a large periportal compartment being no longer expressed in the pericentral compartment and GDH mRNA is expressed over the entire porto-central distance, decreasing in concentration going from central to portal. Comparison of the observed mRNA distribution patterns in the perinatal liver, with published data on the distribution of the respective proteins, points to the occurrence of posttranslational, in addition to pretranslational control mechanisms in the period of ontogenesis of hepatocyte heterogeneity.Interestingly, during development all three mRNAS are expressed outside the liver to a considerable extent and in a highly specific way, indicating that several organs are involved in the developmentally regulated expression of the mRNAs for the ammonia-metabolizing enzymes, that were hitherto not recognized as such.

Basic strategies for valid cytometry using image analysis

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetics in difficult imaging situations, such as low fluorescence signals and high absorbance signals.

Expression patterns of mRNAs for α-fetoprotein and albumin in the developing rat: the ontogenesis of hepatocyte heterogeneity

SummaryIn developing and normal adult rat liver the expression patterns of the mRNAs for α-fetoprotein (AFP) and albumin (ALB) were analysed byin situ hybridization using specific35S-labelled complementary DNA probes. In the developing liver AFP and ALB mRNA are found from embryonic day (ED) 11 and 12, respectively, onward. At ED 20 the first signs of a zonal distribution of these mRNAs across the liver lobule can be observed, AFP mRNA concentration being higher in the pericentral area and ALB mRNA concentration higher in the periportal area. This distribution pattern of reciprocal, overlapping gradients of mRNA can be clearly recognized in the neonatal period. In the adult liver AFP mRNA can no longer be detected and similar to the neonatal situation, ALB mRNA is expressed across the entire porto-central distance decreasing in concentration going from the portal to the central area.Transient extra-hepatic expression of AFP mRNA is found in the embryonic heart and in the epithelial lining of intestine and lung furthermore, AFP and ALB mRNA are found to be transiently expressed in the developing renal tubules. Similar expression patterns have been observed for other liver-characteristic mRNAs (Moormanet al., 1990), suggesting that common regulatory factors are operative during development.

Three-dimensional measurement and visualization of morphogenesis applied to cardiac embryology.

Volume growth and proliferation are key processes in heart morphogenesis, yet their regionalization during development of the heart has been described only anecdotally. To study the contribution of cardiomyocyte proliferation to heart development, a quantitative reconstruction method was designed, allowing the local mapping of this morphogenetic process. First, a morphological surface reconstruction is made of the heart, using sections stained specifically for cardiomyocytes. Then, by a comprehensive series of image processing steps, local three‐dimensional (3D) information of proliferation is obtained. These local quantitative data are then mapped onto the morphological surface reconstruction, resulting in a reconstruction that not only provides morphological information (qualitative), but also displays local information on proliferation rate (quantitative). The resulting 3D quantitative reconstructions revealed novel observations regarding the morphogenesis of the heart.

Developmental changes in the expression of the liver-enriched transcription factors LF-B1, C/EBP, DBP and LAP/LIP in relation to the expression of albumin, α-fetoprotein, carbamoylphosphate synthase and lactase mRNA

SummaryExpression of α-fetoprotein, carbamoylphosphate synthase and albumin, that are generally accepted markers for the hepatic phenotype, require a distinct set of transcription factors. We investigated by in situ hybridization whether this set of transcription factors, LF-B1, C/EBP, DBP and LAP/LIP, is expressed coordinately in the liver during embryonic development and to what extent they are also expressed elsewhere. Our results demonstrate that mRNA levels of all transcription factors tested are significantly above background in the whole embryo and are either reduced or enhanced in expression during subsequent development. Interestingly, cardiac mesoderm, which induces prehepatic endoderm to liver formation, is temporarily permissive to its own signals, showing enhanced expression of these transcription factors and, as a result, the hepatocyte-specific genes α-fetoprotein and carbamoylphosphate synthase. In addition, these transcription factors and many liver-specific structural genes rise concomitantly in intestine and kidney just before birth, suggesting the expression of hepatogenic factors in these tissues as well. Despite the extrahepatic expression of these transcription factors, expression of albumin remains confined to the liver at all developmental stages.

Detection of CAIII mRNA in rat skeletal muscle and liver by in situ hybridization.

We carried out a variety of in situ methods of hybridization on rat liver and rat skeletal muscle using 35S-labeled or biotin-labeled rat carbonic anhydrase III (CAIII) cDNA clone. The methods were compared and evaluated. Use of the biotin system produced defined but nonspecific results which were shown not to be due to the biotinylated cDNA probe binding to the mRNA in the muscle sections. This artifact was shown to persist despite various attempts to eliminate it. Alternatively, using 35S-labeled cDNA gave reproducible results which were shown to be consistent with probe binding specifically to mRNA in the muscle section.

Measure is treasure

See article by Ribadeau-Dumas et al. [1] (pages 426–436) in this issue. The paper of Ribadeau-Dumas et al. in this issue [1] deals with the interesting, but complex regulation of the SERCA2 gene. This gene encodes the sarcoplasmatic calcium pump, which is a crucial enzyme in the calcium handling of the cardiomyocyte. Insight into the regulation of this gene is therefore of paramount importance. A proper understanding of the regulation of the expression of a gene in adult, developing, experimental or diseased (cardiac) tissue requires precise knowledge of the control at each step from gene to the function of the encoded protein, comprising RNA and protein accumulation (being the product of synthesis and degradation) and biological activity of the studied protein. This list can of course be extended (Fig. 1). If we are to appreciate at which step in this pathway control is predominantly exerted, it is important to quantify as many of the parameters as possible. It is equally important to relate the disparate parameters with one another, to evaluate, for example, whether or not the increase in biological activity is proportional with the increase in the amount of protein, or whether the increase in protein follows a proportionate increase in the encoding mRNA. The vital role of quantification of the distinct parameters for our understanding of the regulation of (cardiac) gene expression is beyond any dispute. The lightheartedness concerning this topic is therefore surprising. Fig. 1 Potential steps at which control of gene expression can be exerted in the … * Corresponding author. Tel.: +31-20-5669111; fax: +31-20-6976177 m.j.vandenhoff{at}amc.uva.nl a.f.moorman{at}amc.uva.nl