Biosynthesis and processing of pro-opiomelanocortin-derived peptides during fetal pituitary development

We have investigated the molecular weight forms of pro-opiomelanocortin (POMC)-derived peptides present in rat pituitaries during fetal and early postnatal development (embryonic Day 14 to 3 day neonate). At all early ages examined, the major immunoreactive form of corticotropin (ACTH) was POMC. Only during late fetal and early postnatal stages did progressively larger amounts of 4.5K ACTH, a major POMC processing end product, appear. This form was found almost exclusively in isolated anterior lobes. In contrast, 3.5K size endorphin(s), another POMC derivative, were present in whole glands even at early stages (Day 14), and were the major POMC derivative(s) found in isolated intermediate-posterior lobes of older fetuses. Despite the early appearance of 3.5K endorphin(s), α-MSH did not appear until Day 19 and was detected only in isolated intermediate-posterior lobes. We have also cultured dispersed fetal pituitary cells in the presence of radioactive amino acids. After immunoprecipitation using affinity-purified antisera, followed by fractionation of the radiolabeled products, we found that POMC biosynthesis does occur in cultures of Day 14 embryonic pituitary cells, and that the major POMC-derived end product produced is 3.5K size endorphin(s). These findings demonstrate that POMC is synthesized at least by Day 14 of rat pituitary development and that lobe-specific processing characteristic of the corresponding adult lobe is apparent at the earliest stages that the lobes can be separated. The presence of 3.5K-sized endorphins at early ages is consistent with the possibility that POMC synthesis first occurs in the intermediate lobe. The noncoordinate appearance of α-MSH, 1–39 ACTH, and endorphins implies that the activities of certain cleavage enzymes and acetylation enzymes responsible for lobe-specific post-translational POMC processing may be expressed at different times during development.     

Expression of fetal and neonatal hepatic functions by mouse hepatoma-rat hepatoma hybrids

In order to analyze the mechanisms implicated in the expression of differentiated functions during development, we have studied ten hybrid clones arising from fusion of cells of a mouse hepatoma characterized by the expression of only fetal hepatic functions with those of a rat hepatoma which express, like adult hepatocytes, a set of neonatal as well as fetal hepatic functions. The cells of most hybrid clones contain one set of chromosomes of each parent and coexpress the hepatic functions common to both parents. Among the hepatic proteins characteristic of only one parental line, some continue to be expressed while others are extinguished. The three functions out of the eight examined which are subject to extinction are expressed uniquely by the rat parental cells and appear only near or at birth during normal liver development. These results suggest that regulatory mechanisms (whose final effect is negative) operate in fetal cells to inhibit the expression of differentiated functions limited to a later stage of development.     

Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development.

We have shown that DNA polymerase beta, the only nuclear DNA polymerase present in adult neurons, cannot discriminate between dTTP and dUTP, having the same Km for both substrates. This fact suggests that during reparative DNA synthesis, in adult neurons, dUMP residues can be incorporated into DNA. Since uracil DNA-glycosylase functions to prevent the mutagenic effects of uracil in DNA coming as a product of deamination of cytosine residues or as a result of dUMP incorporation by DNA polymerase, we have studied the perinatal activity of uracil DNA-glycosylase and of 2 enzymes (nucleoside diphosphokinase and dUTPase) involved in dUTP metabolism. Our data indicate that during neuronal development there is a rapid decrease in uracil DNA-glycosylase which could impair the removal of uracil present in DNA in adult neurons. However, misincorporation of dUMP into DNA might be kept to a low frequency by the action of dUTPase present at all developmental stages.     

Developmental changes in expression and subcellular localization of the DNA repair glycosylase, MYH, in the rat brain

Mammalian cells employ a network of DNA repair pathways. DNA repair is required during development to ensure accuracy of DNA replication in the rapidly dividing embryonic cells and to maintain genomic integrity in the mature organism. An enzyme involved in repair of replication errors generated on either normal or oxidatively damaged DNA templates, is the mammalian ortholog of the Escherichia coli MutY DNA glycosylase (MYH). We show that levels of MYH isoform, detected at the E14 embryonic stage, decrease during embryonic and neonatal rat development, while new isoforms appear and gradually increase in the neonate and adult brain. The temporally declining expression of embryonic MYH resembles the pattern of proliferating cell nuclear antigen (PCNA) decline during this period. Immunohistochemical analyses of the embryonic brain show that cells staining for MYH initially coincide with cells staining for PCNA. At later stages PCNA declines, while MYH is detected primarily outside the nucleus. MutY-like glycosylase activity for adenines misincorporated opposite oxidized guanines is detected in both, embryonic and adult brain extracts. Together, these findings suggest that in proliferating embryonic cells, MYH might be primarily involved in post replicative repair of nuclear DNA, whereas in post mitotic neurons, in the repair of mitochondrial DNA.     

Ghrelin in the gastrointestinal tract and blood circulation of perinatal low and normal weight piglets

Ghrelin, the ‘hunger’ hormone, is an endogenous growth hormone secretagogue that exerts a wide range of physiological functions. Its perinatal presence suggests that ghrelin might be involved in growth and metabolism processes during intrauterine and postnatal life. Intrauterine growth-restricted (IUGR) neonates have altered endocrine and metabolic pathways because of malnutrition during foetal development. These changes might include an altered gastrointestinal presence of ghrelin cells (GCs). As ghrelin is mainly secreted by the stomach, this altered presence might be reflected in its serum concentrations. Small-for-gestational age (SGA) pigs appear to be a natural occurring model for IUGR children. Therefore, the first aim of this study was to investigate the presence of gastrointestinal GCs expressing active ghrelin in normal weight (NW) foetal and postnatal piglets compared with their SGA littermates using immunohistochemical analysis in combination with stereological methods. Second, total ghrelin serum concentrations of these piglets were analysed with a porcine radioactive immunoassay. In addition, the growth of the gastric pars fundica in the NW and SGA piglets was analysed stereologically. Corresponding with humans and rats, it was shown that opened- and closed-type immunoreactive GCs are distributed along the entire gastrointestinal tract of the perinatal NW and SGA piglets. However, in contrast to the rat’s stomach, the porcine GCs do not disperse from the glandular base to the glandular neck during perinatal development. Furthermore, stereological analysis demonstrated that the NW neonates have a higher amount of gastric cells expressing active ghrelin compared with the SGA piglets that could result in higher milk consumption during the neonatal period. This finding is, however, not reflected in total serum ghrelin levels, which showed no difference between the NW and SGA piglets. Moreover, the stereological volume densities of the fundic layers demonstrate a similar growth pattern in the SGA and NW piglets.     

Transient biochemical compartmentalization of Purkinje cells during early cerebellar development

It has recently been observed that during early cerebellar development--from embryonic Day 17 to postnatal Day 3 in the rat--only certain discrete clusters of Purkinje cells (PCs) are immunoreactive to cyclic GMP-dependent protein kinase (cGK). In contrast, at later stages and in the adult, all the PCs are immunoreactive. These results obtained with cGK suggest a transitory intrinsic heterogeneity in the immature cerebellar cortex. It seemed therefore interesting to investigate the distribution of other PC markers during early development in the rat and in other species. The results presented here were obtained with two other antibodies--against vitamin D-dependent calcium binding protein and against Purkinje cell specific glycoprotein--which, like cGK, label all adult PCs. Each antibody gave a different and reproducible mosaic of positive and negative clusters of PCs in the perinatal cerebellum, thus indicating a transient biochemical compartmentalization resulting from the differential expression of parts of the same genotype by clusters of PCs. This compartmentalization in concomitant with the ingrowing of the cerebellar afferents. Once synaptogenesis starts, the biochemical heterogeneity of PCs disappears.     

Emergence of the mature myosin phenotype in the rat diaphragm muscle

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.     

Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.     

Changes in neuronal DNA content variation in the human brain during aging

The human brain has been proposed to represent a genetic mosaic, containing a small but constant number of neurons with an amount of DNA exceeding the diploid level that appear to be generated through various chromosome segregation defects initially. While a portion of these cells apparently die during development, neurons with abnormal chromosomal copy number have been identified in the mature brain. This genomic alteration might to lead to chromosomal instability affecting neuronal viability and could thus contribute to age-related mental disorders. Changes in the frequency of neurons with such structural genomic variation in the adult and aging brain, however, are unknown. Here, we quantified the frequency of neurons with a more than diploid DNA content in the cerebral cortex of normal human brain and analyzed its changes between the fourth and ninth decades of life. We applied a protocol of slide-based cytometry optimized for DNA quantification of single identified neurons, which allowed to analyze the DNA content of about 500 000 neurons for each brain. On average, 11.5% of cortical neurons showed DNA content above the diploid level. The frequency of neurons with this genomic alteration was highest at younger age and declined with age. Our results indicate that the genomic variation associated with DNA content exceeding the diploid level might compromise viability of these neurons in the aging brain and might thus contribute to susceptibilities for age-related CNS disorders. Alternatively, a potential selection bias of "healthy aging brains" needs to be considered, assuming that DNA content variation above a certain threshold associates with Alzheimer's disease.     

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.     

Evolution of odorant receptors expressed in mammalian testes

About 10% of mammalian odorant receptors are transcribed in testes, and odorant-receptor proteins have been detected on mature spermatozoa. Testis-expressed odorant receptors (TORs) are hypothesized to play roles in sperm chemotaxis, but they might also be ordinary nasal odorant receptors (NORs) that are expressed gratuitously in testes. Under the sperm-chemotaxis hypothesis, TORs should be subject to intense sexual selection and therefore should show higher rates of amino acid substitution than NORs, but under the gratuitous-expression hypothesis, TORs are misidentified NORs and therefore should evolve like other NORs. To test these predictions, we estimated synonymous and nonsynonymous divergences of orthologous NOR and TOR coding sequences from rat and mouse. Contrary to both hypotheses, TORs are on average more highly conserved than NORs, especially in certain domains of the OR protein. This pattern suggests that some TORs might perform internal nonolfactory functions in testes; for example, they might participate in the regulation of sperm development. However, the pattern is also consistent with a modified gratuitous-expression model in which NORs with specialized ligand specificities are both more highly conserved than typical NORs and more likely to be expressed in testes.     

Prenatal Stress Inhibits Hippocampal Neurogenesis but Spares Olfactory Bulb Neurogenesis

The dentate gyrus (DG) and the olfactory bulb (OB) are two regions of the adult brain in which new neurons are integrated daily in the existing networks. It is clearly established that these newborn neurons are implicated in specific functions sustained by these regions and that different factors can influence neurogenesis in both structures. Among these, life events, particularly occurring during early life, were shown to profoundly affect adult hippocampal neurogenesis and its associated functions like spatial learning, but data regarding their impact on adult bulbar neurogenesis are lacking. We hypothesized that prenatal stress could interfere with the development of the olfactory system, which takes place during the prenatal period, leading to alterations in adult bulbar neurogenesis and in olfactory capacities. To test this hypothesis we exposed pregnant C57Bl/6J mice to gestational restraint stress and evaluated behavioral and anatomic consequences in adult male offspring.We report that prenatal stress has no impact on adult bulbar neurogenesis, and does not alter olfactory functions in adult male mice. However, it decreases cell proliferation and neurogenesis in the DG of the hippocampus, thus confirming previous reports on rats. Altogether our data support a selective and cross-species long-term impact of prenatal stress on neurogenesis.     

Perinatal light imprinting of circadian clocks and systems (PLICCS): A signature of photoperiod around birth on circadian system stability and association with cancer

Recent findings from animal models suggest that plasticity of human circadian clocks and systems may be differentially affected by different paradigms of perinatal photoperiod exposure to the detriment of health in later life, including cancer development. Focusing on the example of cancer, we carry out a series of systematic literature reviews concerning perinatal light imprinting of circadian clocks and systems (PLICCS) in animal models, and concerning the risk of cancer development with the primary determinants of the perinatal photoperiod, namely season of birth or latitude of birth. The results from these systematic reviews provide supporting evidence of the PLICCS and cancer rationale and highlight that investigations of PLICCS in humans are warranted. Overall, we discuss findings from experimental research and insights from epidemiological studies. Considerations as to how to “test” PLICCS in epidemiological studies and as to the potential for non-invasive preventative measures during perinatal periods close our synthesis. If the PLICCS rationale holds true, it opens the exciting prospect for amenable, early-life, preventative measures against cancer development (and other disorders) in later life. Indeed, non-invasive anthropogenic light exposure may have enormous potential to alleviate the public health and economic burden of circadian-related diseases.     

Developmental mercury exposure elicits acute hippocampal cell death, reductions in neurogenesis, and severe learning deficits during puberty

Normal brain development requires coordinated regulation of several processes including proliferation, differentiation, and cell death. Multiple factors from endogenous and exogenous sources interact to elicit positive as well as negative regulation of these processes. In particular, the perinatal rat brain is highly vulnerable to specific developmental insults that produce later cognitive abnormalities. We used this model to examine the developmental effects of an exogenous factor of great concern, methylmercury (MeHg). Seven-day-old rats received a single injection of MeHg (5 μg/gbw). MeHg inhibited DNA synthesis by 44% and reduced levels of cyclins D1, D3, and E at 24 h in the hippocampus, but not the cerebellum. Toxicity was associated acutely with caspase-dependent programmed cell death. MeHg exposure led to reductions in hippocampal size (21%) and cell numbers 2 weeks later, especially in the granule cell layer (16%) and hilus (50%) of the dentate gyrus defined stereologically, suggesting that neurons might be particularly vulnerable. Consistent with this, perinatal exposure led to profound deficits in juvenile hippocampal-dependent learning during training on a spatial navigation task. In aggregate, these studies indicate that exposure to one dose of MeHg during the perinatal period acutely induces apoptotic cell death, which results in later deficits in hippocampal structure and function.     

In vivo study of the appearance and fluctuations of insulin binding sites in different tissues during rat development

The appearance and fluctuations of specific insulin binding sites in several tissues in vivo during rat development, have been determined. After intravenous administration of 125I-insulin to fetal, suckling and adult rats, changes on specific hormone uptake were observed depending on the tissues tested and on the age of animals. Thus, in liver, specific insulin uptake was much greater in 19 day-old fetuses and 10 day-old suckling animals than in adult rats. By contrast, brown fat and spleen insulin uptake was undetected in fetal animals but present in suckling rats, while lung insulin uptake was absent in the adults but present in fetal and suckling animals. Of interest were the specific insulin uptakes by three different muscle tissues. In fact, heart insulin uptake was much higher in younger animals than in adult rats, while in the diaphragm it was significantly smaller in all groups and in skeletal muscles hormone uptake was much smaller than in the other two muscle tissues and was even absent in the fetuses. In those tissues that had previously been shown to exhibit a specific insulin uptake, the iodinated hormone uptake decreased proportionally with simultaneous injection of increasing amounts of unlabelled insulin. These results indicate that insulin binding sites appear at different times and fluctuate in a different manner according to the tissues tested during rat development; this might be important in the stimulation of the functional activities of those tissues during perinatal age.     

Circadian regulation of the lark RNA‐binding protein within identifiable neurosecretory cells

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA‐binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)‐containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA‐binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly‐emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA‐binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 14–29, 2000     

Neural crest stem cells persist in the adult gut but undergo changes in self-renewal,neuronal subtype potential,and factor responsiveness

We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.     

Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.