Developmental Changes In Expression Research Articles

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.

Developmental Programming: Impact of Prenatal Testosterone Excess on Ovarian Cell Proliferation and Apoptotic Factors in Sheep1

Prenatal testosterone (T) excess leads to reproductive dysfunctions in sheep, which include increased ovarian follicular recruitment and persistence. To test the hypothesis that follicular disruptions in T sheep stem from changes in the developmental ontogeny of ovarian proliferation and apoptotic factors, pregnant Suffolk sheep were injected twice weekly with T propionate or dihydrotestosterone propionate (DHT; a nonaromatizable androgen) from Days 30 to 90 of gestation. Changes in developmental expression of proliferating cell nuclear antigen (PCNA), BCL2, BAX, activated CASP3, and FAS/FASLG were determined at Fetal Days 90 and 140, 22 wk, 10 mo, and 21 mo of age by immunocytochemisty. Prenatal T treatment induced changes in expression of proliferative and apoptotic markers in a follicle-, age-, and steroid-specific manner. Changes in BAX were evident only during fetal life and PCNA, BCL2, and CASP3 only postnatally. Prenatal T and not DHT increased PCNA and decreased BCL2 in granulosa/theca cells of antral follicles at 10 and 21 mo but decreased CASP3 in granulosa/theca cells of antral follicles at 22 wk (prepubertal) and 10 and 21 mo. Both treatments decreased BAX immunostaining in granulosa cells of Fetal Day 90 primordial/primary follicles. Neither treatment affected FAS expression at any developmental time point in any follicular compartment. Effects on BAX appear to be programmed by androgenic actions and PCNA, BCL2, and CASP3 by estrogenic actions of T. Overall, the findings demonstrate that fetal exposure to excess T disrupts the ovarian proliferation/apoptosis balance, thus providing a basis for the follicular disruptions evidenced in these females.

Developmental changes in expression, subcellular distribution, and function of Drosophila N-cadherin, guided by a cell-intrinsic program during neuronal differentiation

Cell adhesion molecules (CAMs) perform numerous functions during neural development. An individual CAM can play different roles during each stage of neuronal differentiation; however, little is known about how such functional switching is accomplished. Here we show that Drosophila N-cadherin (CadN) is required at multiple developmental stages within the same neuronal population and that its sub-cellular expression pattern changes between the different stages. During development of mushroom body neurons and motoneurons, CadN is expressed at high levels on growing axons, whereas expression becomes downregulated and restricted to synaptic sites in mature neurons. Phenotypic analysis of CadN mutants reveals that developing axons require CadN for axon guidance and fasciculation, whereas mature neurons for terminal growth and receptor clustering. Furthermore, we demonstrate that CadN downregulation can be achieved in cultured neurons without synaptic contact with other cells. Neuronal silencing experiments using Kir2.1 indicate that neuronal excitability is also dispensable for CadN downregulation in vivo. Interestingly, downregulation of CadN can be prematurely induced by ectopic expression of a nonselective cation channel, dTRPA1, in developing neurons. Together, we suggest that switching of CadN expression during neuronal differentiation involves regulated cation influx within neurons.

Immunolocalisation of 11β‐HSD‐1 and ‐2, glucocorticoid receptor, mineralocorticoid receptor and Na+K+‐ATPase during the postnatal development of the rat epididymis

Glucocorticoids have been implicated in male reproductive function and 11β-HSD-1 and -2, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), all of which are known to modulate glucocorticoid action, have been localised in the adult rat epididymis, but their developmental expression has not been investigated. Na(+)K(+)-ATPase activity, responsible for sodium transport, is induced by both mineralocorticoids and glucocorticoids in the kidney and colon, and has been localised in epididymal epithelium. This study examined the immunolocalisation of 11β-HSD-1 and -2, GR, MR and Na(+)K(+)-ATPase in rat epididymal epithelium (n = 5) at postnatal days (pnd) 1, 7, 15, 28, 40, 60, 75 and 104, and relative mRNA expression of 11β-HSD-1 and -2, and GR at pre-puberty (pnd 28) and post-puberty (pnd 75). 11β-HSD-1, GR and MR were localised in the epididymal epithelium from pnd 1, and 11β-HSD-2 and Na(+)K(+)-ATPase reactivity from pnd 15. At pnd 28 there was maximal immunoreactivity for both the GR and MR and 11β-HSD-1 and -2. 11β-HSD-1 mRNA expression in the caput increased from pre- to post-puberty, whereas 11β-HSD-2 mRNA expression fell over the same period (P < 0.01). GR mRNA expression was similar at pre- and post-puberty in both caput and cauda. Developmental changes in expression of 11β-HSD-1 and -2 suggest that overall exposure of the epididymis to glucocorticoids increases post-puberty, but cell-specific expression of the 11β-HSD enzymes still provides a capacity for intricate local control of glucocorticoid exposure.

Quantitative Membrane Protein Expression at the Blood–Brain Barrier of Adult and Younger Cynomolgus Monkeys

Cynomolgus monkey has been used as a model for the prediction of drug disposition in human brain. The purpose of this study was to clarify protein expression levels of membrane proteins affecting drug distribution to brain, such as transporters, receptors, and junctional proteins, in cynomolgus monkey brain microvessels by using liquid chromatography tandem mass spectrometory. In adult monkeys, three ATP-binding cassette transporters (multidrug resistance 1 (MDR1), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4)), six solute carrier transporters (glucose transporter 1 (GLUT1), GLUT3/14, monocarboxylate transporter 1 (MCT1), MCT8, organic anion transporting polypeptide 1A2, and equilibrative nucleoside tranporter 1), two junctional proteins (claudin-5 and vascular endothelial cadherin), and two receptors (insulin receptor and low-density lipoprotein receptor-related protein 1) were detected. Comparison of the expression levels with those in mouse, which we reported previously, revealed a pronounced species difference. BCRP expression in monkey was greater by 3.52-fold than that in mouse, whereas MDR1 and MRP4 expression levels in monkey were lower by 0.304- and 0.180-fold, respectively, than that in mouse. This study also investigated the developmental changes in expression of membrane proteins in neonate and child monkeys. Expression of MDR1 was similar in neonate and adult monkeys, whereas in rat, P-glycoprotein expression was reported to be significantly lower in brain microvessels of neonate as compared with adult rat. These results will be helpful to understand and predict brain concentrations of drugs in different species and at different ages of primates. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3939–3950, 2011

Serotonin receptor expression in human prefrontal cortex: balancing excitation and inhibition across postnatal development.

Serotonin and its receptors (HTRs) play critical roles in brain development and in the regulation of cognition, mood, and anxiety. HTRs are highly expressed in human prefrontal cortex and exert control over prefrontal excitability. The serotonin system is a key treatment target for several psychiatric disorders; however, the effectiveness of these drugs varies according to age. Despite strong evidence for developmental changes in prefrontal Htrs of rodents, the developmental regulation of HTR expression in human prefrontal cortex has not been examined. Using postmortem human prefrontal brain tissue from across postnatal life, we investigated the expression of key serotonin receptors with distinct inhibitory (HTR1A, HTR5A) and excitatory (HTR2A, HTR2C, HTR4, HTR6) effects on cortical neurons, including two receptors which appear to be expressed to a greater degree in inhibitory interneurons of cerebral cortex (HTR2C, HTR6). We found distinct developmental patterns of expression for each of these six HTRs, with profound changes in expression occurring early in postnatal development and also into adulthood. However, a collective look at these HTRs in terms of their likely neurophysiological effects and major cellular localization leads to a model that suggests developmental changes in expression of these individual HTRs may not perturb an overall balance between inhibitory and excitatory effects. Examining and understanding the healthy balance is critical to appreciate how abnormal expression of an individual HTR may create a window of vulnerability for the emergence of psychiatric illness.

RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

BackgroundPCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.ResultsJuvenile and adult (male and female) guppies express 10 visual opsins at varying levels in the eye. Two opsin genes in juveniles, SWS2B and RH2-2, accounted for >85% of all visual-opsin transcripts in the eye, excluding RH1. This relative abundance (RA) value dropped to about 65% in adults, as LWS-A180 expression increased from approximately 3% to 20% RA. The juvenile-to-female transition also showed LWS-S180 upregulation from about 1.5% to 7% RA. Finally, we found that expression in guppies' SWS2-LWS gene cluster is negatively correlated with distance from a candidate locus control region (LCR).ConclusionsSelective pressures influencing visual-opsin gene expression appear to differ among age and sex. LWS upregulation in females is implicated in augmenting spectral discrimination of male coloration and courtship displays. In males, enhanced discrimination of carotenoid-rich food and possibly rival males are strong candidate selective pressures driving LWS upregulation. These developmental changes in expression suggest that adults possess better wavelength discrimination than juveniles. Opsin expression within the SWS2-LWS gene cluster appears to be regulated, in part, by a common LCR. Finally, by comparing our RT-qPCR data to MSP data, we were able to propose the first opsin-to-λmax assignments for all photoreceptor types in the cone mosaic.

Developmental expression changes of the genes involved in IGFI signaling pathway in longissimus dorsi muscle of Tongcheng and Yorkshire pigs during postnatal growth

IGFI signaling pathway is sufficient to regulate myofibre hypertrophy postnatally, which is associated with muscle mass in economically livestock. In the present study, we drafted the developmental expression pattern of eight genes implicated in IGFI system across six stages of postnatal myofibre growth in Yorkshire and Tongcheng pigs. The results indicated that GRB2 may contribute to increased DNA content in postnatal myofibre hypertrophy via GRB2-Ras-Raf-MEK-ERK sub-pathway; INSR, PDK1, IRS1 and eIF4E may contribute to high growth rate via stimulating the rate of protein synthesis and inhibiting the rate of protein degradation. In addition, the results suggested 60 days maybe a very important stage in postnatal myofibre growth. Moreover, higher mRNA level of IRS1 and GLUT4 maybe associated with inferior meat quality in Yorkshire compared to Tongcheng pig. Therefore, IGFI signaling pathway regulates myofibre hypertrophy postnatally via complicated signal effectors, which may have negative impact on meat quality simultaneously.

Developmental Programming: Prenatal Testosterone Excess Disrupts Expression of Oocyte and Granulosa Cell Growth Factors in Sheep.

Prenatal testosterone (T) excess in sheep leads to ovarian dysfunction including increased ovarian follicular recruitment and persistence culminating in an adult Polycystic Ovary Syndrome (PCOS)-like reproductive phenotype. Growth factors expressed in granulosa cells such as antimullerian hormone (AMH) and c-kit ligand (KL) and the oocyte growth differentiation factor-9 (GDF-9) have been implicated in follicular differentiation. We hypothesized that the prenatal T excess would alter the relative expression of these growth factors thus contributing to aberrant follicular differentiation. Pregnant Suffolk sheep were given T propionate (100 mg, i.m. twice weekly) in cottonseed oil from days 30-90 of gestation (term is ~147 d). Controls (C) received vehicle. Changes in developmental expression of AMH and KL were determined in two ovarian sections (5μm thickness) <5mm apart from each of 6 animals/age/treatment group at fetal (days 90 and 140), postpubertal (10 months), and adult (21 months) ages by immunohistochemistry (IHC). Changes in oocyte GDF-9 expression were evaluated only during postpubertal and adult ages. Consecutive sections were used to relate changes in AMH and KL expression within the same follicle. Follicles were classified as primordial, transitional, primary, small preantral (SPF), large preantral (LPF), small antral (SAF) and large antral (LAF). Degree of expression was scored in a 0 to 5 scale and analyzed using T-test and linear regression. Fetal day 90 ovaries had only primordial to primary follicles while day 140 ovaries had few SPF (range/animal: 2-13), LPF (1-15), SAF (0-9), and LAF (0-1) follicles. All follicles at both ages expressed KL with AMH expressed only in the preantral and antral follicles. Number of preantral follicles studied ranged from 10-45/animal in 10 mo-old and 15-79 in 20 mo-old animals. Consistent with increased follicular recruitment, number of follicles beyond the SPF stage was higher in T females compared to C (P<0.05). Prenatal T treatment did not affect KL expression at any follicular stage. AMH expression increased with follicular stage from SPF to SAF in both groups of females. There was an age effect in AMH expression with it being higher at 20 mo compared to 10 mo in LPF and LAF in C and SPF, LPF and LAF in T females (P<0.05). AMH expression decreased between SAF and LAF (P<0.05) in C but not T females at 20 mo consistent with the altered follicular growth seen in T females. As a result, AMH expression was relatively low in LPF and high in LAF in T females compared to C (P<0.01). The percentage of LPF that had lower AMH expression (scale 0-1) was 1.8 times higher in the T group (C: 15.8 vs. T: 28.4%; P<0.05) while percentage of LAF that had lower AMH expression (0-1) was 1.4 times higher in the C group (C: 52.5 vs. 37.1%; P=0.057). Because AMH is known to reduce follicular sensitivity to FSH, the increased AMH expression in LAF in T females may contribute to the follicular persistence seen in T females. Changes in GDF-9 were evident only at 10 mo with GDF-9 expression being lower in LPF of T compared to the C group (P<0.01) suggestive of altered oocyte quality. The small sample size precluded statistical comparison of GDF-9 expression in LAF. Overall, the imbalance in paracrine environment stemming from altered AMH and GDF-9 expression in T females may underlie the follicular disruptions namely, recruitment and persistence, seen in the T females. Supported by P01 HD44232. (platform)

Developmental Changes in Expression of Genes Involved in Regulation of Apoptosis in the Bovine Preimplantation Embryo1

The early bovine preimplantation embryo is resistant to proapoptotic signals until around the 8- to 16-cell stage. We hypothesized that 2-cell embryos have higher amounts of antiapoptotic proteins and lower amounts of proapoptotic proteins when compared to embryos ≥16 cells. Steady-state concentrations of mRNA for the antiapoptotic genes BCL2 and HSPA1A were higher for MII oocytes, 2-cell embryos, and 2-cell embryos treated with alpha-amanitin as compared to ≥16-cell embryos. Steady-state concentrations of mRNA for the proapoptotic gene BAD increased in embryos ≥16 cells. There was no significant effect of stage of development on steady-state mRNA concentrations of BCL2L1, DFFA, or BAX. Using immunohistochemistry, it was found that BCL2 was present in greater relative concentrations for 2-cell embryos than for embryos ≥16 cells. These results were confirmed by Western blotting. Relative amounts of immunoreactive BAX detected by immunofluorescence were lower for 2-cell embryos than for embryos ≥16 cells. Using Western blotting, a high molecular weight (46 kDa) form of BAX was highest in ≥16-cell embryos, intermediate in 2-cell embryos, and lowest in MII oocytes. There were no effects of stage of development on relative amounts of immunoreactive BCL2L1, HSPA1A, or BAD, as determined by immunofluorescence. Treatment of embryos with alpha-amanitin from Day 0 to Day 5 or Day 4 to Day 5 after insemination reduced activation of group II caspases and terminal deoxynucleotidyl transferase dUTP nick end labeling after treatment with the proapoptotic signal C(2) ceramide at Day 5 after fertilization. Thus, transcription of BAX or other proteins is required for acquisition of the capacity for apoptosis. Results support the idea that changes in amounts of BCL2 family members are important for the inhibition of apoptosis in the 2-cell embryo and in the establishment of the capacity for apoptosis later in development.

Developmental expression changes of insulin-like growth factors (IGFs) system genes in &lt;I&gt;longissimus dorsi&lt;/I&gt; muscle of two pig breeds

Real-time fluorescence quantitative PCR was applied to investigate the developmental expression patterns of insulin like growth factor-1 and -2 (IGF-1 and -2), insulin like growth factor receptor -1 and -2 (IGF-1R and -2R), insulin like growth factor binding protein-3 and -5 (IGFBP-3 and -5) genes at the age of birth (0), 1, 2, 3, 4, and 5 months in longissimus dorsi muscle of Landrace and Meishan pigs. The expression level of IGF-1 exhibited up-regulation, in contrast, the expression level of IGF-2 showed down-regulation after birth in the two pig breeds. These findings are consistent with the previous knowledge that IGF-2 is mainly expressed during the embryonic phase, IGF-1 is mainly expressed during the postnatal period, which could promote cell proliferation and ontogenesis. The expression patterns of IGFRs and IGFs were dissimilar from 0 to 5 months in the two pig breeds, which suggest that the expression changes of IGFRs may not be subjected to the regulation by local secretion of IGFs. Strikingly, at the age of 2 months, the expression levels of IGF-1R, IGF-2R, and IGFBP-3 reached their maximum peak values in Landrace pigs, which suggest that the 2 months after birth may be the most important growth stage for genes of insulin-like growth factors (IGFs) system to exert their biological functions from birth to 5 months. These results indicate that the mRNAs of IGFs system genes exhibit specific developmental expression changes between Landrace and Meishan pigs, and provides some data for further study on the mutual regulation mechanism of genes in IGFs system.

Kindlin-1 Is Required for RhoGTPase-Mediated Lamellipodia Formation in Keratinocytes

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

Sept8 controls the binding of vesicle‐associated membrane protein 2 to synaptophysin

Septins, a conserved family of GTP/GDP-binding proteins, are present in organisms as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, Sept8, Sept9 and Sept11, in various rat tissues by western blot analyses and found all septins to be expressed in brain. We also examined the developmental changes of expression of these septins in the rat brain and found that the level of Sept8 increased during post-natal development. Morphological analyses revealed that Sept8 is enriched at pre-synapses. Using yeast two-hybrid screening, we identified vesicle-associated membrane protein 2 (VAMP2), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), as an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 to synaptic vesicles and dissociate prior to forming the SNARE complex consisting of VAMP2, syntaxin and synaptosome-associated protein of 25 kDa. We showed that Sept8 suppresses the interaction between VAMP2 and synaptophysin through binding to VAMP2. In addition, we found that Sept8 forms a complex with syntaxin1A, and the Sept8-VAMP2 interaction is disrupted by synaptosome-associated protein of 25 kDa. These results suggest that Sept8 may participate in the process of the SNARE complex formation and subsequent neurotransmitter release.

Nonhuman primates as models for human adrenal androgen production: Function and dysfunction

The origin of circulating DHEA and adrenal-derived androgens in humans and nonhuman primates is largely distinct from other mammalian species. In humans and many Old world primates, the fetal adrenal gland and adult zona reticularis (ZR) are known to be the source for production of DHEA (and DHEAS) in mg quantities. In spite of similarities there are also some differences. Herein, we take a comparative endocrine approach to the diversity of adrenal androgen biosynthesis and its developmental timing in three primate species to illustrate how understanding such differences may provide unique insight into mechanisms underlying adrenal androgen regulation and its pathophysiology in humans. We contrast the conventional developmental onset of adrenal DHEA biosynthesis at adrenarche in humans with (1) an earlier, peri-partutrition onset of adrenal DHEA synthesis in rhesus macaques (Old World primate) and (2) a more dynamic and reversible onset of adrenal DHEA biosynthesis in female marmosets (New World primate), and further consider these events in terms of the corresponding developmental changes in expression of CYP17, HSD3B2 and CYB5 in the ZR. We also integrate these observations with recently described biochemical characterization of CYP17 cDNA cloned from each of these nonhuman primate species and the corresponding effects of phosphorylation versus CYB5 coexpression on 17,20 lyase versus 17-hydroxylase activity in each case. In addition, female rhesus macaques exposed in utero to exogenous androgen excess, exhibit symptoms of adrenal hyperandrogenism in adult females in a manner reminiscent of that seen in the human condition of PCOS. The possible mechanisms underlying such adrenal hyperandrogenism are further considered in terms of the effects of altered relative expression of CYP17, HSD3B2 and CYB5 as well as the altered signaling responses of various kinases including protein kinase A, or the insulin sensitive PI3-kinase/AKT signaling pathway which may impact on 17,20 lyase activity. We conclude that while the triggers for the onset of ZR function in all three species show clear differences (age, stage of development, social status, gender), there are still common mechanisms driving an increase in DHEA biosynthesis in each case. A full understanding of the mechanisms that control 17,20 lyase function and dysfunction in humans may best be achieved by comparative studies of the endocrine mechanisms controlling adrenal ZR function and dysfunction in these nonhuman primate species.

Developmental expression changes of &lt;I&gt;IGF2&lt;/I&gt; and &lt;I&gt;IGFBP3&lt;/I&gt; genes in adipose tissue of two pig breeds

To investigate the developmental expression patterns of Insulin-like Growth Factor 2 (IGF2) and Insulin-like Growth Factor Binding protein 3 (IGFBP3) genes at the age of 30, 60, 90, 120 and 150 days in back subcutaneous fat tissue of Landrace and Taihu pigs, real-time fluorescence quantitative PCR was applied. Results showed that the expression level of IGF2 mRNA in Landrace pigs was significantly higher at 30 days than other days (P<0.01), then gradually declined and lowest at 120 days, obviously increased at 150 days. In Taihu pigs, it was relatively higher at 30~60 days, lowest at 90 days, increased rapidly at 120 days, and then decreased. Developmental expression patterns of IGFBP3 mRNA in the two pig breeds were similar, which were highest at 30 days, declined significantly at 60 days (P<0.05) then maintained steady, but a little fluctuant between ages in each breed. In addition, the expression level of IGF2 mRNA in Taihu pigs was significantly higher than that of Landrace pigs at the age of 120 days (P<0.01), and the expression level of IGFBP3 mRNA in Taihu pigs was significantly lower than that of Landrace at the age of 150 days (P<0.01), with no significant difference between the two pig breeds at other ages (P>0.05). The results suggest that the expression of IGF2 and IGFBP3 mRNA in the adipose tissue of pigs exhibits specific developmental changes and different patterns between the two pig breeds. The expression decrease of IGF2 may be related to adipocyte hyperplasia.

Developmental changes in expression and self-editing of adenosine deaminase type 2 pre-mRNA and mRNA in rat brain and cultured cortical neurons

Adenosine deaminase-1 and -2 (ADAR-1 and -2) are double-stranded RNA-specific enzymes involved in the editing of genes including serotonin 2C receptor (5-HT2CR) mRNA and ADAR-2 pre-mRNA. We reported that the editing efficacy of 5-HT2CR mRNA altered during brain development in rats. The present study aimed to clarify if changes in the expression of ADAR genes and the editing of ADAR-2 pre-mRNA occur during development. The expression level of ADAR-1 mRNA was constant during development, whereas the expression levels of ADAR-2 mRNA and ADAR-2 pre-mRNA markedly increased during development. ADAR-2 pre-mRNA possesses six editing sites. Editing of these sites did not occur during the embryonic period; however, the number of edited sites and the editing frequency at these sites increased after birth and cultivation period. These results suggest that the increases in ADAR-2 pre-mRNA editing and mRNA expression of the enzyme may play a role in development. We also discuss the relationship between 5-HT2CR mRNA editing and the expression/RNA editing of ADAR-1 and ADAR-2 mRNA.

Changes in expression of some two-pore domain potassium channel genes ( KCNK) in selected brain regions of developing mice

Two P loop domain potassium (K2P or KCNK) channels produce transmitter-modulated K + currents that could influence brain development. We mapped by in situ hybridization the expression of the K2P gene family in the developing mouse brain. All the K2P genes had different expression patterns, and it is likely that many neuronal types change their K2P channel subunit composition during development. Fitting with a possible role in the control of cell division, three K2P genes ( tandem of P domains in a weak inwardly-rectifying K + channel– related K + channel (TREK) -1, TREK-2 and weak inwardly-rectifying K + channel–related acid- sensitive K + channel (TASK) -2) had high expression in the embryonic subventricular and ventricular zones, and the tandem of P domains in a weak inwardly-rectifying K + channel (TWIK) -1, TREK-1, TREK-2 and TASK-3 genes were significantly expressed in the external cerebellar granule cell layer. There were also some clear changes in developmental expression of the K2P genes: for example, TREK-1 goes from high to low expression in post-migratory cerebellar granule cells; TREK-2 has one of the highest expressions in the embryonic and early postnatal brain of any K2P gene, but transcript levels fall strongly in the postnatal periods, except for cerebellar granule cells. TASK-1 and tandem pore domain halothane- inhibited K + channel (THIK) -2 genes both turn on specifically in post-migratory cerebellar granule cells, whereas the TASK-3 gene, for example, is strongly expressed in pre-migratory cells as well as post-migratory cells. On the other hand, young postnatal dentate granule cells express TWIK-1, TREK-1 and TREK-2 before P7, but TASK-3 expression only begins to become clear in these cells in the second postnatal week. THIK-2 mRNA was up-regulated with TASK-1 and TASK-3 transcripts in cerebella of GABA A receptor α6 subunit knockout mice, possibly implying a functional association of THIK-2, TASK-1 and TASK-3.

Developmental Programming: Impact of Prenatal Testosterone Excess on Pre- and Postnatal Gonadotropin Regulation in Sheep1

The goal of this study was to explore mechanisms that mediate hypersecretion of LH and progressive loss of cyclicity in female sheep exposed during fetal life to excess testosterone. Our working hypothesis was that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH (but not FSH) secretion and, thus, hypersecretion of LH in adulthood, and that this results from altered developmental gene expression of GnRH and estradiol (E2) receptors, gonadotropin subunits, and paracrine factors that differentially regulate LH and FSH synthesis. We observed that, relative to controls, females exposed during fetal life to excess testosterone, as well as the nor-aromatizable androgen dihydrotestosterone, exhibited enhanced LH but not FSH responses to intermittent delivery of GnRH boluses under conditions in which endogenous LH (GnRH) pulses were suppressed. Luteinizing hormone hypersecretion was more evident in adults than in prepubertal females, and it was associated with development of acyclicity. Measurement of pituitary mRNA concentrations revealed that prenatal testosterone excess induced developmental changes in gene expression of pituitary GnRH and E2 receptors and paracrine modulators of LH and FSH synthesis in a manner consistent with subsequent amplification of LH release. Together, this series of studies suggests that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH response, leading to LH hypersecretion and acyclicity in adulthood, and that this programming involves developmental changes in expression of pituitary genes involved in LH and FSH release.

Growth factors differentially regulate neuronal Ca v channels via ERK-dependent signalling

Voltage-gated calcium channels (Ca v) are tonically up-regulated via Ras/extracellular signal-regulated kinase (ERK) signalling in sensory neurones. However, the mechanisms underlying the specificity of cellular response to this pathway remain unclear. Neurotrophic factors are attractive candidates to be involved in this process as they are key regulators of ERK signalling and have important roles in neuronal survival, development and plasticity. Here, we report that in rat dorsal root ganglion neurones, endogenous nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) and epidermal growth factor (EGF) are all involved in tonic ERK-dependent up-regulation of Ca v channels. Chronic (overnight) deprivation of growth factors inhibits total Ca v current according to developmental changes in expression of the cell surface receptors for NGF, GDNF and EGF. Whilst EGF specifically regulates transcriptional expression of Ca vs, NGF and GDNF also acutely modulate Ca v channels within a rapid (∼10 min) time-frame. These acute effects likely involve changes in the biophysical properties of Ca vs, including altered channel gating rather than changes in surface expression. Furthermore, NGF, GDNF and EGF differentially regulate specific populations of Ca vs. Thus, ERK-dependent regulation of Ca v activity provides an elegant and extremely flexible system with which to tailor calcium influx to discrete functional demands.

Control of beta globin genes

The developmental changes in expression of the beta like genes from embryonic to adult stages of human life are controlled at least partially at the level of the promoter sequences of these genes and their binding factors, and competition for promoter specific interactions with the locus control region (LCR). In recent years, the control of beta globin genes has also been investigated at the level of chromatin structure involving the chemical modification of histones and their remodelling by DNA dependent ATPases (SMARCA) containing protein complexes. The role of intergenic RNA is also being investigated with renewed interest. Although a wealth of information on the structure/function relationship of the LCR and globin promoters has been gathered over more than two decades, the fundamental nature of the control of these genes at the molecular level is still not completely understood. In the following pages, we intend to briefly describe the progress made in the field and discuss future directions.