Enhanced hippocampal long-term potentiation in mice lacking heparin-binding growth-associated molecule.

Heparin-binding growth-associated molecule (HB-GAM) (pleiotrophin) is a highly conserved extracellular matrix-associated protein implicated in a diverse range of developmental processes, including the formation and plasticity of neuronal connections. Using gene targeting, we have in the present study created HB-GAM-deficient mice that are viable and fertile and show no gross anatomical abnormalities. The hippocampal structure as well as basal excitatory synaptic transmission in the area CA1 appear normal in the mice lacking HB-GAM. However, hippocampal slices from HB-GAM-deficient mice display a lowered threshold for induction of long-term potentiation (LTP), which reverts back to the wild-type level by application of HB-GAM. HB-GAM expression in hippocampus is activity-dependent and upregulated in several neuropathological conditions. Thus, we suggest that HB-GAM acts as an inducible signal to inhibit LTP in hippocampus.

CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.

Expression and functional analysis of Uch-L3 during mouse development.

Mice homozygous for the s(1Acrg) deletion at the Ednrb locus arrest at embryonic day 8.5. To determine the molecular basis of this defect, we initiated positional cloning of the s(1Acrg) minimal region. The mouse Uch-L3 (ubiquitin C-terminal hydrolase L3) gene was mapped within the s(1Acrg) minimal region. Because Uch-L3 transcripts were present in embryonic structures relevant to the s(1Acrg) phenotype, we created a targeted mutation in Uch-L3 to address its role during development and its possible contribution to the s(1Acrg) phenotype. Mice homozygous for the mutation Uch-L3(Delta3-7) were viable, with no obvious developmental or histological abnormalities. Although high levels of Uch-L3 RNA were detected in testes and thymus, Uch-L3(Delta3-7) homozygotes were fertile, and no defect in intrathymic T-cell differentiation was detected. We conclude that the s(1Acrg) phenotype is either complex and multigenic or due to the loss of another gene within the region. We propose that Uch-L3 may be functionally redundant with its homologue Uch-L1.

The temporal requirement for endothelin receptor-B signalling during neural crest development.

Endothelin receptor B (EDNRB) is a G-protein-coupled receptor with seven transmembrane domains which is required for the development of melanocytes and enteric neurons. Mice that are homozygous for a null mutation in the Ednrb gene are almost completely white and die as juveniles from megacolon. To determine when EDNRB signalling is required during embryogenesis, we have exploited the tetracycline-inducible system to generate strains of mice in which the endogenous Ednrb locus is under the control of the tetracycline-dependant transactivators tTa or rtTA. By using this system to express Ednrb at different stages of embryogenesis, we have determined that EDNRB is required during a restricted period of neural crest development between embryonic days 10 and 12.5. Moreover, our results imply that EDNRB is required for the migration of both melanoblasts and enteric neuroblasts.

Enhancer competition between H19 and Igf2 does not mediate their imprinting.

The linked H19 and Igf2 genes on mouse distal chromosome 7 are subject to genomic imprinting. Competition between the promoters of the genes for transcription from shared enhancers has been proposed as an explanation for the coordinate expression and reciprocal imprinting of these two genes. To test this model, we have used Cre-loxP technology to generate in mice a conditional deletion of the H19 promoter and structural gene that leaves no transcription unit in the locus. Contrary to the prediction of enhancer competition we find that transcriptional activity from the H19 promoter is not required for the imprinted silencing of the Igf2 gene.

Igf2 imprinting does not require its own DNA methylation or H19 RNA.

Three models have been proposed to explain the imprinting of the mouse Igf2 gene on the maternal chromosome. We ruled out the importance of DNA methylation at Igf2 by showing that silencing of Igf2 accompanying the loss of DNA methylation could be overcome by a mutation at the neighboring H19 gene that activates Igf2. By replacing the H19 structural gene with a protein-coding gene, we have ruled out a role for H19 RNA in the imprinting of Igf2. This replacement resulted in sporadic activation of the H19 promoter on the paternal chromosome without affecting the level of expression of Igf2, a finding that is inconsistent with strict promoter competition between the genes. We conclude that a transcriptional model involving access to a common set of enhancers shared between Igf2 and H19 is the most likely explanation for Igf2 imprinting.

Mutations in mouse Aristaless-like4 cause Strong's luxoid polydactyly.

Mutations that affect vertebrate limb development provide insight into pattern formation, evolutionary biology and human birth defects. Patterning of the limb axes depends on several interacting signaling centers; one of these, the zone of polarizing activity (ZPA), comprises a group of mesenchymal cells along the posterior aspect of the limb bud that express sonic hedgehog (Shh) and plays a key role in patterning the anterior-posterior (AP) axis. The mechanisms by which the ZPA and Shh expression are confined to the posterior aspect of the limb bud mesenchyme are not well understood. The polydactylous mouse mutant Strong's luxoid (lst) exhibits an ectopic anterior ZPA and expression of Shh that results in the formation of extra anterior digits. Here we describe a new chlorambucil-induced deletion allele, lstAlb, that uncovers the lst locus. Integration of the lst genetic and physical maps suggested the mouse Aristaless-like4 (Alx4) gene, which encodes a paired-type homeodomain protein that plays a role in limb patterning, as a strong molecular candidate for the Strong's luxoid gene. In genetic crosses, the three lst mutant alleles fail to complement an Alx4 gene-targeted allele. Molecular and biochemical characterization of the three lst alleles reveal mutations of the Alx4 gene that result in loss of function. Alx4 haploinsufficiency and the importance of strain-specific modifiers leading to polydactyly are indicative of a critical threshold requirement for Alx4 in a genetic program operating to restrict polarizing activity and Shh expression in the anterior mesenchyme of the limb bud, and suggest that mutations in Alx4 may also underlie human polydactyly.

Location of enhancers is essential for the imprinting of H19 and Igf2 genes.

Genomic imprinting is the process in mammals by which gamete-specific epigenetic modifications establish the differential expression of the two alleles of a gene. The tightly linked H19 and Igf2 genes are expressed in tissues of endodermal and mesodermal origin, with H19 expressed from the maternal chromosome and Igf2 expressed from the paternal chromosome. A model has been proposed to explain the reciprocal imprinting of these genes; in this model, expression of the genes is governed by competition between their promoters for a common set of enhancers. An extra set of enhancers might be predicted to relieve the competition, thereby eliminating imprinting. Here we tested this prediction by generating mice with a duplication of the endoderm-specific enhancers. The normally silent Igf2 gene on the maternal chromosome was expressed in liver, consistent with relief from competition. We then generated a maternal chromosome containing a single set of enhancers located equidistant from 1gf2 and H19; the direction of the imprint was reversed. Thus, the location of the enhancers determines the outcome of competition in liver, and the strength of the H19 promoter is not sufficient to silence Igf2.

Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene.

The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo.

Regulation of steroid production in ovarian stromal tissue from 5- to 8-week-old pullets and laying hens.

Studies were conducted to evaluate the regulation of steroid production in dispersed cells from ovarian stromal tissue from 5- to 8-week-old-pullets (IM cells) and laying hens (MAT cells). Short-term incubation of IM and MAT cells with ovine (o) LH resulted in a dose-dependent increase in progesterone, androstenedione and oestradiol production; progesterone production was greater in MAT cells than in IM cells (P < 0.05) in response to 2-200 ng oLH ml-1, whereas androstenedione and oestradiol production was greater in MAT cells following treatment with 20 and 200 ng oLH ml-1 (P < 0.05). In both cell populations the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (1 and 10 mmol l-1) stimulated progesterone and androstenedione production, whereas oLH (200 ng ml-1) and forskolin (1-10 mumol l-1) promoted cAMP accumulation (P < 0.05 compared with basal values). However, treatment with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), did not alter basal or oLH-stimulated cAMP accumulation or progesterone production in either IM or MAT cells (P > 0.10). PMA did, however, inhibit agonist-induced androstenedione production (P < 0.05); co-treatment with the calcium ionophore A23187 potentiated this inhibitory effect. Finally, treatment with transforming growth factor-alpha (TGF-alpha; 1.8-18 pmol l-1) did not affect basal or oLH-stimulated progesterone or androstenedione production by IM cells, MAT cells, theca cells from 6-8 mm follicles or theca cells from the second largest (F2) follicle (P > 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)

Plasminogen activator activity and thymidine incorporation in avian granulosa cells during follicular development and the periovulatory period.

The hormonal and second messenger regulation of plasminogen activator (PA) activities in avian granulosa and theca cells has been documented. However, the physiological role(s) of PAs in the avian ovary remains poorly understood. The present studies were designed to evaluate PA activity in hen granulosa cells collected from the most mature (F1) preovulatory follicle at three discrete time points relative to a spontaneous ovulation and from follicles collected at various stages of follicular development. Levels of PA activity in the granulosa layer of the F1 follicle declined by greater than 90% as follicles were collected closer to their anticipated time of ovulation (e.g., from 17-16 h to 0.75-0.15 h; p less than 0.05). Timing of tissue collection was confirmed by evaluation of serum progesterone levels, which peaked as expected at the 6-5-h time point. During follicular development, PA activity was several times greater in rapidly growing follicles (6-12 mm, 1-3 wk prior to ovulation) than in slowly growing (1-5 mm) or preovulatory (F3 and F1) follicles (p less than 0.05). Granulosa cells of these rapidly growing follicles also incorporated significantly higher levels of 3H-thymidine than did granulosa cells of mature follicles (p less than 0.05), suggesting a higher level of DNA synthesis. Similarly, granulosa cells of the mitotically active germinal disc region of the F1 granulosa layer were found to possess at least 3-fold higher (p less than 0.05) levels of PA activity and a 2-fold greater level of 3H-thymidine incorporation than the more mature granulosa cells isolated from the remaining F1 granulosa layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of calcium in the regulation of theca cell androstenedione production in the domestic hen.

Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100 microM) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion. The calmodulin inhibitors trifluoperazine (100 microM) and R24571 (50 microM) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.

Possible role for arachidonic acid in the control of steroidogenesis in hen theca.

Studies were conducted to evaluate if arachidonic acid (C20:4) could function as a second messenger within theca cells from the second largest preovulatory (F2) follicle from the ovary of the domestic hen. Arachidonic acid stimulated basal progesterone and androstenedione production, but inhibited LH-induced androstenedione production. The stimulatory effects of arachidonic acid were not altered by either cyclooxygenase or lipoxygenase pathway inhibitors (indomethacin and nordihydroguaiaretic acid, respectively), but were blocked by agents that prevented mobilization and/or efflux of calcium (TMB-8 and verapamil). The inhibitory effects of arachidonic acid on LH-stimulated steroidogenesis were determined to occur both prior and subsequent to cAMP formation. Fifty and 100 microM arachidonic acid attenuated LH- (10 ng) and forskolin- (0.2 microM) induced cAMP levels, and decreased androstenedione and estradiol production following treatment with 8-bromo-cAMP. Phospholipase A2 (PLA2) and the calcium ionophore, A23187, stimulated the release of 3H from theca cells prelabeled with [3H]arachidonic acid, and both PLA2 and the closely related fatty acid, eicosatrienoic acid (C20:3), could replicate the inhibitory effects of arachidonic acid on LH-stimulated androstenedione production. Finally, neither indomethacin nor nordihydroguaiaretic acid blocked the inhibitory effects of arachidonic acid on LH-promoted androstenedione production. We conclude that arachidonic acid can be released within theca cells in response to physiologic (PLA2) and pharmacologic agents (A23187), and accordingly, that it may act directly as a second messenger to modulate both basal and LH-stimulated steroid production.