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Glucose-6-phosphatase activity has been determined in periportal and pericentral zones of the rat liver lobule using a quantitative histochemical method. The study was performed on unfixed cryostat sections of livers from fasted and fed female and male rats. Highest activity was found in periportal zones, and starvation caused a 2-3-fold increase of glucose-6-phosphatase activity in periportal and pericentral zones of both sexes. Unexpectedly, KM values were also significantly different in periportal and pericentral zones and were found to increase linearly with Vmax values, irrespective of sex and feeding condition. Because the cryofixation procedure was shown to permeabilize the biomembranes in the tissue sections, it can be concluded that the rise in KM and Vmax values has to be attributed to the catalytic unit of the glucose-6-phosphatase system. It is suggested that the enzyme exists in a high affinity configuration at low enzyme concentrations but that at high enzyme concentrations a hysteretic mechanism, as proposed by Berteloot et al. (Berteloot, A., Vidal, H., and Van de Werve, G. (1991) J. Biol. Chem. 266, 5497-5507), transforms the enzyme from a high to a low affinity configuration. The present study indicates that the concept of functional heterogeneity of liver parenchyma may be more complex than thus far assumed.

In the past decade we have made an inventory of the changing three-dimensional patterns of expression of a number of key proteins involved in contraction, energy metabolism and conduction in developing and adult chicken, rat, bovine and human hearts. These integrated morphological and immunohistochemical studies were complemented with electrophysiological studies in developing chicken hearts and have resulted in a preliminary model of heart development, that explains how the embryonic heart can function without valves and without an atrioventricular conduction system that is indispensable for the adult heart. Cardiomyocyte-specific proteins are first expressed in the cardiogenic plate when 6 somites have developed, while electrical activity becomes detectable only slightly later. Development proceeds as follows: 1. Upon its formation 'primary' myocardium is characterised by anteroposterior gradients in gene expression. Therefore cardiogenesis resembles many other developmental processes in the embryo. It serves as source for endocardial cells and cells specialized in mechanical contraction and in impulse generation/conduction supporting the view that a single population of cells (the 'primary' myocardium) serves as a precursor for these distinct cell types. 2. 'Primary' myocardium is characterized by the expression of alpha and beta myosin, acetylcholinesterase and the absence of fast sodium channels and of connexin 43. It has a peristaltoid contraction form due to a relatively slow propagation of the impulse. 3. In the looping stage, two cardiac segments appear due to the development of atrial and ventricular working myocardium, that is characterized by the expression of either alpha or beta myosin, connexin 43, fast sodium channels, the disappearance of acetylcholinesterase and by a relatively fast conduction.2+ sinuatrial and atrioventricular nodes.

The spatial distribution of alpha- and beta-myosin heavy chain isoforms (MHCs) was investigated immunohistochemically in the embryonic human heart between the 4th and the 8th week of development. The development of the overall MHC isoform expression pattern can be outlined as follows: (1) In all stages examined, beta-MHC is the predominant isoform in the ventricles and outflow tract (OFT), while alpha-MHC is the main isoform in the atria. In addition, alpha-MHC is also expressed in the ventricles at stage 14 and in the OFT from stage 14 to stage 19. This expression pattern is very reminiscent of that found in chicken and rat. (2) In the early embryonic stages the entire atrioventricular canal (AVC) wall expresses alpha-MHC whereas only the lower part expresses beta-MHC. The separation of atria and ventricles by the fibrous annulus takes place at the ventricular margin of the AVC wall. Hence, the beta-MHC expressing part of the AVC wall, including the right atrioventricular ring bundle, is eventually incorporated in the atria. (3) In the late embryonic stages (approx. 8 weeks of development) areas of alpha-MHC reappear in the ventricular myocardium, in particular in the subendocardial region at the top of the interventricular septum. These coexpressing cells are topographically related to the developing ventricular conduction system. (4) In the sinoatrial junction of all hearts examined alpha- and beta-MHC coexpressing cells are observed. In the older stages these cells are characteristically localized at the periphery of the SA node.

The developmental appearance and spatial distribution pattern of gap junctions were studied in prenatal and adult rat hearts. Gap junctions were visualized immunohistochemically with an antibody raised against a unique cytoplasmic epitope of connexin43, and the spatial distribution pattern was determined by three-dimensional reconstruction. The results demonstrate that from embryonic day 13 onward, connexin43 becomes detectable immunohistochemically in the myocardium of atria and ventricles. No expression is initially detectable in the myocardium of the sinus venosus, the sinoatrial node, the posterior wall of the atrium and pulmonary veins, the interatrial septum, the atrioventricular canal, including atrioventricular node and bundle, the interventricular septum, and the outflow tract. The developmental increase in the density of gap junctions in atria and ventricles of prenatal hearts correlates well with the reported developmental increase in conduction velocity. Whereas connexin43 becomes expressed in the derivatives of the sinus venosus (except for the sinoatrial node) and in the subepicardial layer of the ventricular free wall shortly before birth, it remains undetectable in the atrioventricular node and bundle and the proximal part of the ventricular conduction tissue, even in the adult heart. The apparent absence of an abundant expression of connexin43 at a location with a supposedly high conduction velocity (i.e., the atrioventricular bundle and bundle branches) is unexpected. These observations were confirmed in studies of the adult mouse heart, which showed, in addition, that connexin32 is not expressed in any part of the heart.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.

Using monoclonal antibodies against the M and B subunit isoforms of creatine kinase (CK) we have investigated their distribution in developing human skeletal and cardiac muscle immunohistochemically. It is demonstrated that in skeletal muscle, a switch from CK-B to CK-M takes place around the week 8 of development, whereas in the developing heart, CK-M is the predominant isoform from the earliest stage examined onward (i.e., 4 1/2 weeks of development). In all hearts examined, local differences in concentration of the CK isoforms are observed. The CK-M expression in the developing outflow tract (OFT) and conduction system is described in detail. Between the weeks 5 and 7 of development, the distal portion of the OFT is characterized by low CK-M expression, whereas around the week 8-10 of development the myocardium around the developing semilunar valves in the OFT expresses a very high level of CK-M. At all stages examined, a relatively low CK-M level is observed in those regions in which the "slow" components of the conduction system do develop (e.g., the sinoatrial junction and atrioventricular junction), whereas a relatively high concentration of CK-M is observed in those areas that are destined to become the "fast" components, i.e., the subendocardial myocardium of the ventricles. The high expression of CK-M in the developing "fast components" of the conduction system contrasts with the relatively low expression of CK-M in the force-producing myocardium of the interventricular septum and free ventricular wall.

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.

The effects of starvation, glucose refeeding, dibutyryl cAMP, and dexamethasone on expression of the gene for phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from rat liver cytosol was studied by using a cloned cDNA probe. The rate of transcription of the gene for phosphoenolpyruvate carboxykinase in hepatic nuclei isolated from starved rats decreased rapidly after refeeding with glucose. Administration of dibutyryl cAMP to glucose-refed animals increased the rate of phosphoenolpyruvate carboxykinase gene transcription seven-fold within 20 min. Phosphoenolpyruvate carboxykinase mRNA in the cytosol is 2.8 kilobases long whereas liver nuclei contain four precursor RNA species that are up to 6.5 kilobases long. Feeding glucose to starved rats rapidly decreased the sequence abundance of enzyme mRNA in both nuclei and cytosol. However, the decrease in cytosolic phosphoenolpyruvate carboxykinase mRNA was preceded by a transient increase in enzyme mRNA over the first 20 min after glucose refeeding. Administration of dibutyryl cAMP to glucose-refed starved animals increased the concentration of the nuclear RNA precursors of phosphoenolpyruvate carboxykinase five- to eight-fold within 30 min and induced the mRNA for the cytosolic enzyme over a period of 60 min. We conclude that cAMP induces phosphoenolpyruvate carboxykinase mRNA by increasing the rate of gene transcription.

In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and β-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of “primary” myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium.

The cellular distribution of histone H5 in embryonic and adult tissues of Xenopus laevis and chicken has been established with monoclonal antibodies to histone H5. Both in Xenopus and in chicken, the protein has presumably a more widespread cellular distribution than hitherto expected but is absent in most embryonic tissues. At least in Xenopus its presence seems not to be restricted to amitotic cells. Arguments will be put forward histone H5 in these animals should be considered as a H1(0) type of histone and that analogous to mammalian H1 degree this protein plays a role in differentiation.

Rat (Rattus norvegicus) and spiny mouse (Acomys cahirinus) are closely related species that mainly differ in the developmental timing of birth. A comparison between the developmental profiles of some characteristic enzymes of the small intestine (lactase and sucrase) and of the pancreas (amylase) of both species was carried out to elucidate the question to what extent these enzymic profiles and hence the maturation of these organs was related to the process of birth. It was found that these organ-specific enzymes become first detectable at the same developmental stage in both species. Likewise, the weaning phase of the enzymic profiles occurred at the same developmental time point in both species. It is argued that both the first appearance and the weaning increase in enzyme activity follow an inherent biological program that can only be modulated by hormones. In contrast, the perinatal phase of the enzymic profile is completely dependent on the developmental timing of birth, and therefore appears not to be anchored to a particular developmental time point but rather to be dependent on birth-associated (hormonal) adaptation. In accordance with this hypothesis it was found that the development of the microscopic anatomy of the small intestine proceeded independently of the functional adaptation of the intestine to the process of birth.

We prepared monoclonal antibodies against chicken histone H5. These antibodies could be divided into two classes, and we present the results obtained with one representative antibody of each class. One class reacted exclusively with chicken H5, whereas the other additionally cross-reacted with rat H1(0) and with material present in adult but not embryonic chicken liver. The cross-reacting material in adult liver was identified by Western blotting as representing a minor band in histone preparations. The protein was not present in histone extracts from chicken erythrocytes. It is likely that this newly identified protein is a chicken H1(0) histone.

The maintenance of the differentiated condition is supposed to be associated with the presence of a histone of the H1(0)/H5 subclass. If the H1(0)/H5 variant has an important role in differentiation distinct from that of H1, it should display differential expression in time and position during development. Here we report that this prediction is verified during Xenopus laevis development, in which tadpoles exhibit a very characteristic, developmentally regulated pattern of histone H1(0)/H5 expression that is different for the derivatives of each embryonic germ layer. However, the pattern of appearance of this variant during development does not reflect a simple correlation between its presence and the state of differentiation. Therefore, these results are pertinent to current ideas on differentiation and the involvement of lysine-rich histones in the repression of eukaryotic genes.

The hypothesis that amiodarone (AM) acts by inducing a local 'hypothyroid-like' state in thyroid hormone-responsive tissues was investigated in rat liver. Hypothyroid rats, pretreated orally for 8 consecutive days with AM (200 mg/kg) or water, were given a single i.p. injection of equimolar doses of T4, T3 or rT3 (1.00 to 1.20 mg/kg). Six hours later, the rats were killed and liver nuclear T3 receptor occupancy was determined. Simultaneously, the activity of two thyroid hormone-responsive enzymes was measured, together with the levels of their respective mRNAs by hybridization to specific cDNAs. The enzymes were phosphoenolpyruvate carboxykinase and glutamine synthetase. AM showed no effect on nuclear T3 receptor occupancy in rats injected with either vehicle, rT3, or T3, but it completely blocked the increase in receptor occupancy in rats injected with T4. With regard to postreceptor effects, T4 and T3 elicited an approximately two-fold increase in the levels of the mRNAs coding for the two enzymes, whereas rT3 had no effect. The increase of the two mRNAs was potentiated by AM, but this is probably secondary to an AM-induced state of anorexia. Remarkably, this potentiating effect of AM was not observed at the protein-level: enzyme activities were lower in rats pretreated with AM. AM-pretreatment thus results in lower enzyme activity to mRNA ratios for both enzymes, irrespective of hormonal treatment. Therefore, although no conclusions can be drawn about possible effects of AM at the transcriptional level, it is concluded that AM interferes with thyroid hormone responsive gene expression in rat liver at a post-transcriptional level. As a consequence, in the present experimental design the livers of AM-treated rats resemble the liver of hypothyroid rats with regard to specific enzyme activities, but not with regard to either nuclear T3 receptor occupancy or the levels of specific mRNAs.

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.