Fetal Brain Expression Research Articles

Cyclooxygenase‐1 and ‐2 inhibition alter hypothalamic‐pituitary‐adrenal axis (HPA) activity and COX expression in fetal brain.

In fetal sheep, reflex responses to hypotension are in part mediated by generation of prostaglandins (PG) in the CNS. We have previously demonstratd that inhibition of COX1 reduces the responsiveness of the fetal HPA to hypotension and increases expression of COX2 in specific regions of the fetal CNS. We hypothesized that generation of PG alters the transcription of COX2. The present study was designed to test this hypothesis. Chronically catheterized twin fetal sheep received cerebral ventricular infusions of inhibitor or vehicle. We used either the COX2 inhibitor nimesulide (NIM) at 1mg (HD) or 10 ug/day (LD), or the COX1 inhibitor resveratrol (RES) at 1 mg/day. Blood samples were collected to measure fetal blood gases, proopiomelanocortin (POMC), ACTH, and cortisol. After 5 days, fetal hypothalami and pituitaries were harvested, and COX1 and 2 mRNA were analyzed by real-time PCR. Arterial blood gases were unchanged by HD nimesulide or resveratrol. LD nimesulide significantly decreased pO2 of vehicle treated fetuses. HD NIM significantly reduced hypothalamic COX1 and 2 mRNA, but did not alter pituitary levels. COX1 and 2 were unchanged in the RES and LD NIM groups. Hypothalamic AVP, CRH, and POMC mRNA levels were unchanged in all groups. Plasma ACTH, POMC, and cortisol concentrations were unchanged in the HD NIM group, yet RES and LD NIM treatment significantly reduced plasma cortisol without changing POMC or ACTH. We conclude that blockade of COX2 with HD NIM alters the expression of COX1 and 2 mRNA; and 2) unstimulated fetal ACTH secretion is not altered by blockade of CNS COX isoforms, although adrenal sensitivity to ACTH appears to be influenced by both COX1 and 2 activity in the CNS. Supported by HD42135 and a Predoctoral Fellowship from the American Heart Association.

Effects of Maternal Campylobacter rectus Infection on Murine Placenta, Fetal and Neonatal Survival, and Brain Development.

Maternal periodontal infection has been associated with increased risk of prematurity and low birthweight. Infection and inflammatory pathways that mediate prematurity have also been implicated in neonatal developmental impairments. The objective of this study was to determine whether maternal Campylobacter rectus infection that induces fetal growth restriction in a mouse model also compromises neonatal pup survival, growth, and neurodevelopment. Timed pregnant mice were challenged with C. rectus on gestation day 7.5. One group of animals was sacrificed on embryonic day 16.5 for placental histology and measurement of fetal brain mRNA expression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Another group of animals was allowed to deliver to follow pup survival, growth, and brain structure at day 9. C. rectus challenge resulted in abnormal placental architecture with inflammation and a 2.8-fold increase in fetal brain expression of IFN-γ (P = 0.04). Pup birthweight was unaffected by C. rectus exposure, but lethality was 3.9-fold higher after 1 week. Ultrastructurally, the 9-day neonatal brain tissue displayed cellular and myelin alterations consistent with white matter damage. Maternal C. rectus infection induces placental inflammation and decidual hyperplasia as well as concomitant increase in fetal brain IFN-γ. Maternal infection increased pup mortality, and preliminary findings demonstrate ultrastructural changes in the hippocampal region of the neonatal brain, in a manner analogous to the effects of maternal infection on white matter damage seen in humans. Thus, the threat of maternal oral infectious exposure during pregnancy may not be limited to the duration of gestation, but may also potentially affect perinatal neurological growth and development.

Profiling of Alopecia Areata Autoantigens Based on Protein Microarray Technology

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.

Expression of T-type calcium channel splice variants in human glioma.

In humans, three isoforms of the T-type (Ca(v)3.1) calcium-channel alpha(1) subunit have been reported as a result of alternate splicing of exons 25 and 26 in the III-IV linker region (Ca(v)3.1a, Ca(v)3.1b or Ca(v)3.1bc). In the present study, we report that human glioma express Ca(v)3.1 channels in situ, that splicing of these exons is uniquely regulated and that there is expression of a glioma-specific novel T-type variant (Ca(v)3.1ac). Seven human glioma samples were collected at surgery, RNA was extracted, and cDNA was produced for RT-PCR analysis. In addition, three glioma cell lines (U87, U563, and U251N), primary cultures of human fetal astrocytes, as well as adult and fetal human brain cDNA were used. Previously described Ca(v)3.1 splice variants were present in glioma samples, cultured cells and whole brain. Consistent with the literature, our results reveal that in the normal adult brain, Ca(v)3.1a transcripts predominate, while Ca(v)3.1b is mostly fetal-specific. RT-PCR results on glioma and glioma cell lines showed that Ca(v)3.1 expression in tumor cells resemble fetal brain expression pattern as Ca(v)3.1bc is predominantly expressed. In addition, we identified a novel splice variant, Ca(v)3.1ac, expressed in three glioma biopsies and one glioma cell line, but not in normal brain or fetal astrocytes. Transient expression of this variant demonstrates that Ca(v)3.1ac displays similar current-voltage and steady-state inactivation properties compared with Ca(v)3.1b, but a slower recovery from inactivation. Taken together, our data suggest glioma-specific Ca(v)3.1 gene regulation, which could possibly contribute to tumor pathogenesis.

Conserved methylation imprints in the human and mouse GRB10 genes with divergent allelic expression suggests differential reading of the same mark.

Grb10/GRB10 encodes a cytoplasmic adapter protein which modulates coupling of a number of cell surface receptor tyrosine kinases with specific signalling pathways. Mouse Grb10 is an imprinted gene with maternal-specific expression. In contrast, human GRB10 is expressed biallelically in most tissues, except for maternal-specific expression of one isoform in muscle and paternal expression in fetal brain. Owing to its location in 7p11.2-p12, GRB10 has been considered a candidate gene for the imprinted growth disorder, the Silver-Russell syndrome (SRS), but its predominantly biallelic expression argues against involvement in the syndrome. To investigate the discrepant imprinting between mouse and human, we compared the sequence organization of their upstream regions, and examined their allelic methylation patterns and the splice variant organization of the mouse locus. Contrary to expectation, we detected both maternal and paternal expression of mouse Grb10. Expression of the paternal allele arises from a different promoter region than the maternal and, as in human, is restricted to the brain. The upstream regions are well conserved, especially the presence of two CpG islands. Surprisingly, both genes have a similar imprinted methylation pattern, the second CpG island is a differentially methylated region (DMR) with maternal methylation in both species. Analysis of 24 SRS patients did not reveal methylation anomalies in the DMR. In the mouse this DMR is a gametic methylation mark. Our results suggest that the difference in imprinted expression in mouse and human is not due to acquisition of an imprint mark but in differences in the reading of this mark.

Thyroid hormone regulates the expression of alpha-internexin in neurons in culture.

Maternal hypothyroidism in the rat compromises alpha-internexin (alpha-IN) expression in early fetal brain. We have therefore examined whether 3,5,3'-triiodothyronine (T3) regulates alpha-IN expression in fetal brain neurons in culture. Cells expressed transcripts encoding T3 nuclear receptor isoforms in a T3-independent manner. alpha-IN protein abundance was increased in cultures treated with 0.1 and 1 nM T3 for 20 h (177 and 185% control, respectively) and in cultures treated with 1 nM T3 for 40 h (131% control). alpha-IN transcript abundance was unaffected by T3 treatment. In conclusion, T3 at a physiological level, stimulates alpha-IN protein, but not mRNA, levels in early differentiating neurons in culture. This supports the hypothesis that maternal thyroid hormone directly regulates early neuronal differentiation.

Computational Identification, Cloning, and Characterization of IL-1R9, a Novel Interleukin-1 Receptor-like Gene Encoded over an Unusually Large Interval of Human Chromosome Xq22.2–q22.3

The Interleukin-1 receptor (IL-1R) and Toll signaling pathways share the evolutionarily conserved Toll homology domain (THD), which is a critical component in the signaling cascade of the host defense responses to infection and inflammation. Our initial genomic database searches uncovered a novel THD signature sequence between DNA markers DXS87 and DXS366. The feasibility of subsequently applying a coordinated computational approach, including various exon-finding programs, homology-based searches, and receptor profile searches, in revealing the exons encoding this novel IL-1R family member is described. IL-1R9 shows restricted expression in fetal brain and is highly homologous to IL1RAPL (A. Carrie et al., 1999 Nat. Genet. 23: 25–31), which is reportedly involved in nonsyndromic X-linked mental retardation. These genes are scattered over separate genomic intervals in excess of 1.0 Mb and encode receptors with extended C-terminal tails. In our functional NF-κB reporter assays, IL1RAPL, IL-1R9, or versions lacking the extended C-terminal sequences failed in responding either to IL-1 directly or to IL-18 when various permutations of IL-18R ectodomain chimeras were fused to their cytoplasmic domains. Evolutionary sequence analyses reinforce our conclusion that these novel orphan receptors probably form a functionally distinct subset of the IL-1R superfamily.

Maternal hypothyroxinemia influences glucose transporter expression in fetal brain and placenta.

The influence of maternal hypothyroxinemia on the expression of the glucose transporters, GLUT1 and GLUT3, in rat fetal brain and placenta was investigated. Fetal growth was retarded in hypothyroxinemic pregnancies, but only before the onset of fetal thyroid hormone synthesis. Placental weights were normal, but placental total protein concentration was reduced at 19 days gestation (dg). Immunoblotting revealed a decreased abundance of GLUT1 in placental microsomes at 16 dg, whereas GLUT3 was increased. Fetal serum glucose levels were reduced at 16 dg. In fetal brain, the concentration of microsomal protein was deficient at 16 dg and the abundance of parenchymal forms of GLUT1 was further depressed, whereas GLUT3 was unaffected. Northern hybridization analysis demonstrated normal GLUT1 mRNA levels in placenta and fetal brain. In conclusion, maternal hypothyroxinemia results in fetal growth retardation and impaired brain development before the onset of fetal thyroid function. Glucose uptake in fetal brain parenchyma may be compromised directly, due to deficient GLUT1 expression in this tissue, and indirectly, as a result of reduced placental GLUT1 expression. Though corrected by the onset of fetal thyroid hormone synthesis, these deficits are present during the critical period of neuroblast proliferation and may contribute to long term changes in brain development and function seen in this model and in the progeny of hypothyroxinemic women.

CAND3: A ubiquitously expressed gene immediately adjacent and in opposite transcriptional orientation to the ATM gene at 1lq23.1.

Using a magnetic beads-mediated cDNA selection procedure and a fetal brain expression library, we identified a transcriptional unit within a cosmid positive for the marker D11S384. Pursuit of its full-length cDNA led to the cloning of the third candidate gene (CAND3) we studied in our quest for the ataxiatelangiectasia (A-T) gene, ATM. CAND3 spans ~140 kb of genomic DNA and is located immediately centrimeric to ATM, with 544 bp of DNA separating the two genes. CAND3 encodes two ubiquitously expressed transcripts of ~5.8 kb and ~4.6 kb that are divergently transcribed from a promoter region common to ATM. Nucleotide sequence was determined for one of its alternately spliced transcripts. The predicted protein has 1175 amino acids and is novel in sequence, with only weak homologies to transcriptional factors, nucleoporin protein, and protein kinases, including members of the phosphatidylinositol 3-kinase (PI-3 kinase) family. Although neither homology to ATM nor any mutation of CAND3 in A-T patients has been found, the head-to-head arrangement of CAND3 and ATM, with expression of both housekeeping genes from a common stretch of 544 bp intergenic DNA, suggests a bi-directional promoter possibly for co-regulation of biologically related functions. YACs, BACs, cosmids, and STSs are defined to aid in the further study of this gene.

Glial fibrillary acidic protein expression in rat brain and in radial glia culture is delayed by prenatal ethanol exposure.

The alterations in astrocyte proliferation and differentiation induced by prenatal exposure to alcohol (PEA) suggest that ethanol exposure affects the radial glial cells, the main astrocytic precursors. We have investigated the effects of ethanol on the early stages of astrogliogenesis by analyzing the developmental pattern of vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity and their mRNA levels during embryonic/fetal brain development and in radial glia in primary culture. GFAP appeared late in gestation and at day 5 of culture of radial glia, whereas GFAP mRNA was first detected on fetal day 15 and increased in content on fetal day 21. In contrast, the levels of vimentin and its mRNA were high at fetal day 15 but decreased on day 21. Alcohol exposure delays the appearance of GFAP and its mRNA and significantly decreases the GFAP expression in fetal brain and in primary culture of radial glia. In addition, some morphological alterations were observed in PEA glial cells in culture. These results demonstrate that astroglial precursor cells are damaged by prenatal exposure to ethanol and suggest that abnormalities in the astrogliogenesis may underlie the disruption in neuronal migration and other CNS alterations observed after prenatal ethanol exposure.

CDNA Sequence, Genomic Organization, and Evolutionary Conservation of a Novel Gene from the WAGR Region

A new gene (239FB) with predominant and differential expression in fetal brain has recently been isolated from a chromosome 11p13–p14 boundary area near FSHB. The corresponding mRNA has an open reading frame of 294 amino acids, a 3′ untranslated region of 1247 nucleotides, and a highly GC-rich 5′ untranslated region. The coding and 3′ UT sequence is specified by 6 exons within nearly 87 kb of isolated genomic locus. The 5′ end region of the transcript maps adjacent to the only genomically defined CpG island in a chromosomal subregion that may be associated with part of the mental retardation of some WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome patients. In addition to nucleotide and amino acid similarity to an EST from a normalized infant brain cDNA library, the predicted protein has extensive similarity to twoCaenorhabditis eleganspolypeptides of, as yet, unknown function. The 239FB locus is, therefore, likely part of a family of genes with two members expressed in human brain. The extensive conservation of the predicted protein suggests a fundamental function of the gene product and will enable evaluation of the role of the 239FB gene in neurogenesis in model organisms.

The genes for human brain factor 1 and 2, members of the fork head gene family, are clustered on chromosome 14q

Brain factor-1 (BF-1) is a member of the fork head gene family which shows expression restricted to the neurons of the developing telencephalon in rodents and man. We have isolated a second human gene (HBF-2), which is also strongly expressed in embryonic brain and has very high homology to both the rat and human brain factor-1 genes and the retroviral oncogene qin. The HBF-2 cDNA was isolated from a human fetal brain expression library and contains a putative open reading frame of 479 amino acids. The HBF-2 gene is strongly expressed in fetal brain and also with lower levels of expression in several adult tissues. At the genomic level the gene for HBF-1 contains an 500 by intron situated between the DNA binding domain 11 and the fork head domain while that of HBF-2 is intronless. The two genes are clustered on human chromosome l4q11-13.

Prolactin Receptor mRNA Expression in Fetal Rat Brain.

One of the target tissues for prolactin (PRL) family members is the central nervous sytem (CNS) in adult animals. In this study, we tried to detect PRL receptor (PRL-R) mRNAs in fetal rat brains. mRNAs extracted from various tissues, including the brain, on embryonic days 12 (E12), 14, 16, 18 and 20 were subjected to reverse transcriptional polymerase chain reaction (RT-PCR) using two sets of primers specific to the short and long forms of the PRL-R. The short form cDNA band (330 bp) was detected in the fetal brain from E12 to 20, whereas the long band (420 bp) was not apparently observed, suggesting that the developing fetal brain is a target for PRL family members. Short form PRL-R mRNA was also expressed in the liver (E20), heart (E12 to 20), intestine (E16 to 20) and forelimb (E20), and long form PRL-R was observed in the heart on E20. Semi-quantitative PCR analysis revealed that short form PRL-R mRNA expression in the fetal brain was as biphasic: constant on E12 and 14, and increased progressively on E18 and 20. Thus, PRL-R gene expression is initiated very early in developing fetal tissues, including the CNS.

Cloning and characterization of a lambert‐eaton myasthenic syndrome antigen

Lambert-Eaton myasthenic syndrome is a paraneoplastic neuromuscular disorder in which an immune response directed against a small-cell lung tumor crossreacts with antigens in the neuromuscular junction. To isolate and characterize the antigens, we screened a human fetal brain expression library with a high-titer serum from a patient with Lambert-Eaton myasthenic syndrome. This screening resulted in the isolation of a complementary DNA clone encoding an antigen we call myasthenic syndrome antigen B (MysB). Approximately 43% (3 of 7) of Lambert-Eaton myasthenic syndrome sera specifically recognized MysB fusion protein, whereas none of 34 control sera did. The predicted amino acid sequence of this clone shows a high degree of homology to the beta subunit of calcium channel complexes. The MysB pre-messenger RNA is alternatively spliced to yield 3 forms of the protein differing in the domain between two highly conserved alpha-helical segments.

Monoclonal antibodies for studies on xenobiotic and endobiotic metabolism: Cytochromes P-450 as paradigm

Human exposure to bisphenol A (BPA) is mainly due to migration from plastic packaging into food and beverages. Studies reported BPA endocrine disruptions through interactions with different nuclear receptors, including the arylhydrocarbon receptor (AhR). AhR mediates xenobiotic responses and regulates expression of drug-metabolizing enzymes (DMEs), including many CYP450s. This study aimed to assess the effects of BPA maternal exposure on CYP450s expression in fetal brain. Sprague-Dawley dams were exposed to BPA concentrations of 0, 0.5, 5, and 50 mg/L in drinking water, individually, and with nicotine. Fetal brains were isolated at gestational days GD14 and GD19, and protein expression was assessed by Western blotting. Results showed a BPA-induced significant decrease in CYP1B1 expression levels at GD14 (p = 0.001), and CYP19A1 (aromatase) expression at both mid- and late-stage development (p < 0.001). In addition, nicotine individually decreased expression levels of all examined protein targets, significantly for CYP1B1 (p < 0.001), CYP19A1 (p = 0.010), AhRR (p = 0.042), and ARNT (p < 0.001), compared to control. When combined with BPA, nicotine suppressive effects were attenuated at both GD14 and GD19. In conclusion, BPA suppresses CYP1B1 and CYP19A1 expression in fetal brain, and attenuates the suppressive effects of nicotine. Observed effects may be mediated by AhR-ARNT independent mechanisms that need further examination.