Altered gene expression and spongiotrophoblast differentiation in placenta from a mouse model of diabetes in pregnancy.

AIMS/HYPOTHESIS: Pregnancies complicated by diabetes have a higher risk of adverse outcomes for mothers and children, including predisposition to disease later in life, e.g. metabolic syndrome and hypertension. We hypothesised that adverse outcomes from diabetic pregnancies may be linked to compromised placental function, and sought to identify cellular and molecular abnormalities in diabetic placenta. METHODS: Using a mouse model of diabetic pregnancy, placental gene expression was assayed at mid-gestation and cellular composition analysed at various stages. Genome-wide expression profiling was validated by quantitative PCR and tissue localisation studies were performed to identify cellular correlates of altered gene expression in diabetic placenta. RESULTS: We detected significantly altered gene expression in diabetic placenta for genes expressed in the maternal and those expressed in the embryonic compartments. We also found altered cellular composition of the decidual compartment. In addition, the junctional and labyrinth layers were reduced in diabetic placenta, accompanied by aberrant differentiation of spongiotrophoblast cells. CONCLUSIONS/INTERPRETATION: Diabetes during pregnancy alters transcriptional profiles in the murine placenta, affecting cells of embryonic and maternal origin, and involving several genes not previously implicated in diabetic pregnancies. The molecular changes and abnormal differentiation of multiple cell types precede impaired growth of junctional zone and labyrinth, and of placenta overall. Regardless of whether these changes represent direct responses to hyperglycaemia or are physiological adaptations, they are likely to play a role in pregnancy complications and outcomes, and to have implications for developmental origins of adult disease.

09/15: Comparative genomics of a conserved chromosomal region associated with a complex human phenotype.

Three genes that encode related immunoglobulin superfamily molecules have recently been mapped to human chromosome 15 in the region q22.3-q23 and to the syntenic region on mouse chromosome 9. These genes presumably derived from gene duplications, and they are highly similar to Deleted in Colorectal Cancer (DCC), which functions as an axon guidance molecule during development of the nervous system. To find out whether additional genes of this class were present in a chromosomal cluster, we produced a comparative physical map within the region of synteny between mouse chromosome 9 and human chromosome 15. This interval overlaps the critical region for the fourth genetic locus for Bardet-Biedl syndrome (BBS4) in humans. Bardet-Biedl syndrome (OMIM 600374) is characterized by poly/syn/brachydactyly, retinal degeneration, hypogonadism, mental retardation, obesity, diabetes, and kidney abnormalities. A detailed map of this locus will help to identify candidate genes for this disorder.

Cloning and expression of nope, a new mouse gene of the immunoglobulin superfamily related to guidance receptors.

The novel mouse gene Nope was identified due to its proximity to the Punc gene on chromosome 9. With a domain structure of four immunoglobulin domains, five fibronectin type III repeats, a single transmembrane domain, and a cytoplasmic domain, Nope encodes a new member of the immunoglobulin superfamily of cell surface proteins. It displays a high level of similarity to Punc, as well as to guidance receptors such as the Deleted in Colorectal Cancer protein and Neogenin. Nope is expressed during embryonic development in the notochord, in developing skeletal muscles, and later in the ventricular zone of the nervous system. In the adult brain, Nope can be detected in the hippocampus. Radiation hybrid mapping of Nope, Punc, and Neogenin placed all three genes in close vicinity on mouse chromosome 9.

Genomic structure and chromosomal localization of the mouse gene Punc.

The mouse gene Punc encodes a member of the immunoglobulin superfamily of cell surface proteins. It is highly expressed in the developing embryo in nervous system and limb buds. At mid-gestation, however, expression levels of Punc decrease sharply. To allow investigation of such a regulatory mechanism, the genomic locus encompassing the Punc gene was cloned, characterized, and mapped. Fluorescent in situ hybridization was used to determine the chromosomal location of the Punc gene of mouse and human. Mouse Punc maps to Chromosome (Chr) 9 in the region D-E1, whereas the human PUNC gene is localized to Chr 15 at 15q22.3-23, a region known to be syntenic to mouse 9D-E1. The human PUNC gene therefore maps close to a genetic locus that is linked to Bardet-Biedl Syndrome, an autosomal recessive human disorder. Confirmation for the location of human PUNC was obtained through sequence relationships between mouse Punc cDNA, human PUNC cDNA, genomic sequence upstream of the murine Punc gene, and human STS markers that had been previously mapped on Chr 15. The STS sequence WI-14920 is in fact derived from the 3'-untranslated region of the human PUNC gene. WI-14920 had been placed at 228cR from the top of the Chr 15 linkage group, which provided positional information for the human PUNC gene at high resolution. Thus, this study identifies PUNC as the gene corresponding to a previously anonymous marker and serves as a basis to investigate its role in genetic disorders.

Mice transgenic for a human amyloid precursor protein promoter-lacZ reporter construct.

Transgenic mouse lines were generated that expressed a 2-kb amyloid precursor protein (APP) promoter/beta-galactosidase reporter gene construction. In brain, hippocampal pyramidal neurons, neurons in the deeper layers of cerebral cortex, and neurons in several thalamic nuclei were heavily labeled by beta-galactosidase histochemistry. In general, molecular layers and white matter regions did not express the reporter gene. When compared with in situ hybridization for endogenous murine APP RNA, the striatum and outer layers of cerebral cortex had little reporter expression. Thus, the match between reporter expression and endogenous APP expression in brain was not perfect. A similar mismatch between the relative expression of the reporter gene and endogenous APP RNA distribution was found in homogenates from several organs. Although prior work in transgenic mice found similar mismatches in reporter gene distribution, none had tested the APP promoter construct in response to neuronal injury. Kainic acid injections successfully increased murine APP expression in the transgenic mice, but had no effect on the reporter gene expression. Based on these data and those collected by others, we conclude that the 2-kb region upstream of the APP transcription initiation site contains some elements responsible for the tissue-specific expression of this gene, but does not contain all the cis-acting elements sufficient for either the differential tissue distribution of this gene or the regulation of this gene subsequent to neural damage.

Punc, a novel mouse gene of the immunoglobulin superfamily, is expressed predominantly in the developing nervous system.

A search for genes regulated by the LlM/homeodomain transcription factor islet-1 during development identified the novel mouse gene punc (putative neuronal cell adhesion molecule). Punc is a member of the immunoglobulin superfamily, exhibits a novel configuration of four immunoglobulin domains and two fibronectin-type III repeats, and resembles proteins involved in axon guidance. Punc expression in the embryo occurs in restricted patterns in both the nervous system and limb mesoderm. A sharp decrease in punc expression after embryonic day 11.5 suggests a function for punc early in mouse embryogenesis.

Evolutionary conservation of the immediate-early gene ZENK.

Immediate-early genes are part of a cellular response mechanism that reacts to biochemical, electrical, pharmacological, and physiological stimuli as well as changes in behavioral state. In the brain, immediate-early genes-such as egr-1 have been used as markers for neuronal activity. These markers could be invaluable in studies that utilize the chick-quail chimaera system to investigate neural components of behavior. Therefore, we decided to clone avian homologs of immediate-early genes to allow an expression analysis in behavioral paradigms and to determine the degree of conservation among diverse species. We report in this study the cloning of the ZENK gene, an egr-1 homolog, from chicken, quail, zebrafinch, and canary. We show that the coding region of this gene is highly conserved and follows established phylogenetic relationships. In situ hybridization demonstrates that the expression pattern is also conserved among species. We further demonstrate that there are regions in the 3' untranslated area of the ZENK gene that are as highly conserved as the protein-coding region and that may play a role in postranscriptional regulatory mechanisms of ZENK gene expression.

Embryonic expression pattern of amyloid protein precursor suggests a role in differentiation of specific subsets of neurons.

Immunohistochemical analysis revealed the temporal and spatial expression pattern of the amyloid protein precursor (APP) during the development of the mouse embryo. APP was first detected at day 9.5 of gestation in motor neurons of the hind brain and the spinal cord. APP proteins were also evident in cells of the floor plate, and in neurons of the cranial, dorsal root, and sympathetic ganglia shortly after their formation. Except for floor plate cells, APP expression was restricted to differentiated neurons. Comparison with the expression of microtubule-associated protein 2 (MAP-2), a marker for neurodifferentiation, showed that APP is expressed on a subset of differentiated neurons. APP can also serve as an early marker for the developing nuclei of the hind brain. The onset of APP expression in neurons appeared to be correlated with axonal outgrowth, whereas later expression of APP may be associated with functional specialization in the developing nervous system.

Repression of the beta-amyloid gene in a Hox-3.1-producing cell line.

Mammalian homeobox genes are widely expressed in the developing central nervous system and are postulated to control developmental processes by regulating gene expression at the transcriptional level. In vitro studies have identified consensus DNA sequences that contain an ATTA core as sites for interaction with homeodomain proteins. Such elements have been found in the upstream regulatory region of the gene encoding beta-amyloid precursor protein, which is associated with the neurological disorder Alzheimer disease. As the beta-amyloid precursor protein gene is also expressed in the developing central nervous system and appears to play a role in cellular regulatory processes, we have examined the possibility that a homeobox gene product can regulate its transcription. We demonstrate by Northern blot analyses and transfection experiments that the expression of the beta-amyloid precursor protein gene is decreased in cultured cells expressing the mouse homeobox gene Hox-3.1.

Alternative splicing of the beta A4 amyloid gene of Alzheimer's disease in cortex of control and Alzheimer's disease patients.

An S1 nuclease protection assay was designed to study the splicing pattern of the alternatively spliced beta A4 amyloid gene (APP gene) of Alzheimer's disease (AD). We determined the splicing pattern of the APP gene in fetal, adult, aged adult and AD human cortex. The results suggest that alternative splicing of the APP gene in AD is not significantly different from age-matched controls, but distinct from the developing fetal brain.

Structure and expression of a single actin gene in Volvox carteri.

Southern blot analysis of Volvox carteri DNA indicated the presence of a single actin gene; the nucleotide sequence of that gene is reported here. In comparison with plant animal and fungal actins, the derived primary structure of 377 amino acids is highly conserved yielding similarity values of 79% to 94% (including non-identical conservative exchanges). In contrast, the intron structure of the gene is highly unusual: in addition to one intron in the 5' untranslated region (ten nucleotides upstream of the initiator ATG), it has eight introns in the coding region, only three of which are in locations where introns have previously been reported. Transcription starts 26 nucleotides downstream of the putative TATA box and 70 nucleotides downstream of a conspicuous CCAAT motif. A potential polyadenylation signal, TGTAA, is located 366 nucleotides downstream of the terminator TAA. Northern hybridization indicates that the actin gene is transcribed throughout the Volvox life cycle with only a slight depression during the release of juveniles from mother spheroids. This pattern of gene expression suggests that actin may assume various functional roles in the differentiation and growth of Volvox.

Study of the Alzheimer's A4 precursor gene promoter region by genomic sequencing using Taq polymerase.

The beta-amyloid protein has been identified as the prominent component of the fibrillary aggregates of the neuritic plaques found in Alzheimer's disease (AD). In this paper, the DNA methylation pattern of the promoter region of the Alzheimer's disease amyloid precursor gene (PAD) was assessed using the recently developed genomic sequencing technique with Taq polymerase. We analyzed seven potential methylation sites between position -460 and -275 of the PAD promoter. Three of the CpG dinucleotides we analyzed are located in the flanking regions of the AP-1 binding site and heat-shock response element consensus sequences. Of the seven CpG dinucleotides present in this region, we found none to be methylated. This finding indicates that, in healthy brain tissue, cytosine methylation of this binding motif seems not to affect protein/DNA interaction. However, it remains to be determined whether methylation of these sites is significant in AD patients.

Two alleles of a neural protein gene linked to scrapie in sheep.

Sheep are the natural hosts of the pathogens that cause scrapie, an infectious degenerative disease of the central nervous system. Scrapie-associated fibrils [and their major protein, prion protein (PrP)] accumulate in the brains of all species affected by scrapie and related diseases. PrP is encoded by a single gene that is linked to (and may be) the major gene controlling the incubation period of the various strains of scrapie pathogens. To investigate the role of PrP in natural scrapie, we have determined its gene structure and expression in the natural host. We have isolated two sheep genomic DNA clones that encode proteins of 256 amino acids with high homology to the PrPs of other species. Sheep PrPs have an arginine/glutamine polymorphism at position 171 that may be related to the alleles of the scrapie incubation-control gene in this species.

Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein.

To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.

PreA4 mRNA distribution in brain areas.

The A4 amyloid precursor gene of Alzheimer's Disease (PreA4 gene, App gene, PAD gene) gives rise to three different transcripts, which are generated by alternative splicing. The three transcripts are PreA4695, PreA4751 and PreA4770, according to the number of amino-acids in the primary translation product. Previous expression studies did not discriminate between PreA4751 and PreA4770. We have analyzed the distribution of PreA4 transcripts in four cortical brain areas in a way that allows us the selective identification of each transcript. We used a sensitive S1 nuclease protection assay with a probe derived from PreA4770 cDNA. This radiolabeled probe gives rise to three specifically protected DNA fragments, each corresponding to one of the three PreA4 transcripts. These fragments could readily be resolved in a single lane of a denaturing polyacrylamide gel. PreA4 transcripts were quantified by scanning autoradiograms. In younger individuals PreA4695 mRNA is the dominant transcript with 55% (mean values). PreA4770 mRNA is the minor PreA4 transcript with less than 5% and PreA4751 ranges at 40%. The exception is observed in occipital cortex, where PreA4751 (50%) is higher than PreA695 (45%). A different situation was observed in older individuals. In the latter PreA4(695) transcripts are significantly reduced, with the exception of frontal cortex, whereas PreA4770 transcripts show an increase. PreA4751 has a constant level of 45% and becomes the dominant transcript.

The promoter of Alzheimer's disease amyloid A4 precursor gene.

The promoter of the gene for the precursor of Alzheimer's Disease A4 amyloid protein (PAD gene) resembles promoters of housekeeping genes. A typical TATA box is missing, and transcription initiates at multiple sites. It shows a high GC content of 72% in a DNA region that confers promoter activity to a reporter gene in vivo. Upstream of the RNA start sites we found sequences homologous to the consensus binding sites of transcription factor AP-1 and the heat shock control element binding protein. Six copies of a 9bp long GC-rich element are located between positions -100 and -200 of the sequence. A protein-DNA interaction could be mapped to this element. The ratio of the dinucleotide CpG, the target for DNA methylation, versus GpC is about 1:1 around the RNA start site, in contrast to the normal ratio of 1:5 in eucaryotic DNA. These findings suggest that four mechanisms may participate in the regulation of the PAD gene: the stress-related heat shock; the AP-1/Fos binding; the GC-rich element, and the possible methylation of the CpG region. PAD gene regulation could be of relevance for the progression of amyloid deposition in Alzheimer's Disease.

The PreA4(695) precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons.

Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4(695] of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.

The promoter of Alzheimer's disease amyloid A4 precursor gene.

The promoter of the gene for the human precursor of Alzheimer's disease A4 amyloid protein (PAD gene) resembles promoters of housekeeping genes. It lacks a typical TATA box and shows a high GC content of 72% in a DNA region that confers promoter activity to a reporter gene in an in vivo assay. Transcription initiates at multiple sites. Sequences homologous to the consensus binding sites of transcription factor AP-1 and the heat shock control element binding protein were found upstream of the RNA start sites. Six copies of a 9-bp-long GC-rich element are located between positions -200 and -100. A protein--DNA interaction could be mapped to this element. The 3.8 kb of the 5' region of the PAD gene include two Alu-type repetitive sequences. These findings suggest that four mechanisms may participate in the regulation of the PAD gene and could be of relevance for the progression of amyloid deposition in Alzheimer's disease.

Organization and structure of Volvox alpha-tubulin genes.

Southern analysis of Volvox genomic DNA revealed two genes homologous to Chlamydomonas reinhardtii alpha-tubulin cDNA. Restriction fragment length polymorphism analysis indicated that the two genes are not genetically linked. Clones representing one of the alpha-tubulin genes have been isolated from a genomic library of Volvox carteri f. nagariensis. A 3153 bp BamHI fragment containing the entire alpha-tubulin gene (1802 bp) plus 707 bp of the 5'- and 644 bp of the 3'-untranslated regions has been sequenced, revealing the following features: (1) the derived alpha-tubulin primary structure of 451 amino acids is highly conserved, differing in two residues from the alpha 1- and in two additional residues from the alpha 2-tubulin of C. reinhardtii; (2) in comparison to the C. reinhardtii genes, the Volvox alpha-tubulin gene contains a third intron; positions of the other two introns are precisely conserved; (3) codon usages are biased towards G or C, and against A, in the third position; 19 codons are absent from the alpha-tubulin coding sequence, and 5 of these are not used in any of 7 compiled Volvox genes; (4) transcription begins with an A, 30 bp downstream of the putative TATA box; upstream of the TATA box is a 14 bp sequence similar to consensus sequences found in all 4 C. reinhardtii tubulin genes and believed to regulate promoter function.

Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post-translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.