Frequenin--a novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system.

The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.

The opposite and antagonistic effects of the closely related POU family transcription factors Brn-3a and Brn-3b on the activity of a target promoter are dependent on differences in the POU domain.

The Brn-3a, Brn-3b, and Brn-3c POU family transcription factors are closely related to one another and are members of the group IV subfamily of POU factors. Here we show that despite this close relationship, the factors have different effects on the activity of a target promoter: Brn-3a and Brn-3c stimulate the promoter whereas Brn-3b represses it. Moreover, Brn-3b can antagonize the stimulatory effect of Brn-3a on promoter activity and can also inhibit promoter activation by the Oct-2.1 POU factor. The difference in the transactivation activities of Brn-3a and Brn-3b is dependent upon the C-terminal region containing the POU domain of the two proteins, since exchange of this domain between the two factors converts Brn-3a into a repressor and Brn-3b into an activator.

Expression pattern of Dkk-1 during mouse limb development.

dkk-1 has recently been identified as a secreted protein in Xenopus laevis which is sufficient and necessary to cause head induction by antagonizing Wnt signalling (Glinka et al., 1998, Nature 391, 357-362). Consistent with such a role dkk-1 is expressed in the Spemann organizer of the early frog gastrula. Later, expression can be observed in an endomesodermal domain corresponding to the prospective prechordal plate, in two longitudinal stripes flanking the anterior chordamesoderm and in the precursors of the liver. At late neurula stage expression occurs in the prechordal plate adjacent to the prospective forebrain and eyes and in a stripe corresponding to the forming somites. dkk-1 is part of a gene family with at least three family members which is conserved between species. Its mouse homologue, Dkk-1, is first expressed at embryonic day (E) 6.5 in mesodermal cells adjacent to the embryonic/extraembryonic junction. Starting at E7.5 transcripts can be detected in the head mesoderm and at E8.5 additionally in developing somites (Glinka et al., 1998, Nature 391, 357-362). In this study we focus on the highly dynamic pattern of Dkk-1 mRNA distribution during mouse limb development from E9.0-E14.5. The other currently known family members, Dkk-2 and -3, are not expressed in the limb bud before E11.5 (C. Niehrs, pers. commun.) while the limb pattern is established. We show that Dkk-1 expression starts with the first sign of forelimb budding, whereas in the presumptive hindlimb region transcription becomes already apparent before the limb starts to bud out. Expression then becomes confined to two mesenchymal domains at E10.5 and E11.5. Using double-whole mount in situ hybridization we show that the posterior Dkk-1 expression domain initially overlaps with that of Shh, one of the key signalling molecules in limb development. Later, the two expression domains become separated. At E12.5-E14.5 Dkk-1 transcripts are restricted to the interdigital mesenchyme.

Mouse Brn-3 family of POU transcription factors: a new aminoterminal domain is crucial for the oncogenic activity of Brn-3a.

The class IV POU domain genes Brn-3a, -b and -c are differentially expressed during neural development and at least Brn-3a also in neuroectodermal tumors. In contrast to Brn-3b and Brn-3c, Brn-3a encodes two protein variants: Brn-3a(l) and Brn-3a(s). Brn-3a(s) lacks 84 aminoterminal residues but is otherwise identical to Brn-3a(l). Outside the well conserved carboxyterminal POU domains all three Brn-3 proteins (-a, -b and -c) diverge until the aminoterminal end where a new domain of about 100 amino acids is identified. This domain is conserved only between Brn-3 proteins and other class IV POU factors. Brn-3a(l) that contains the complete domain but not Brn-3a(s) that lacks 84 amino acids of it is able to tumorigenically transform primary fibroblasts. Brn-3b that lacks 40 amino acids of the new domain does itself not transform, but abolishes the oncogenic potential of Brn-3a(l) when transfected together. This demonstrates not only that Brn3-a(l) is a proto-oncogene and may well be causally involved in the generation of neuroectodermal tumors but also suggests that the intactness of the new aminoterminal domain described here is crucial for oncogenic activity. In addition, our data indicate that Brn-3b acts as an inhibitor of Brn-3a(l) activity.

Implication of frequenin in the facilitation of transmitter release in Drosophila.

1. We have investigated the possible role of frequenin in the modulation of synaptic facilitation at the larval Drosophila neuromuscular junctions. Excitatory junctional currents (EJCs) and presynaptic nerve terminal currents were recorded by external electrodes in normal larvae and in transgenic larvae carrying an extra insertion of the frequenin cDNA. 2. Motor nerve stimulation by twin pulses or trains of stimuli provoked EJC facilitation which was about three times higher in transgenic larvae compared to controls. Unconditioned EJCs revealed, however, similar quantal content and Ca2+ sensitivity in both Drosophila strains. 3. Differences between normal and transgenic Drosophila in the quantal content of the facilitated EJC do not depend on differences in the duration of the repolarization phase of the presynaptic action potential. 4. Perfusion of tetrodotoxin or of low-Na+ solutions abolished the enhancement of the EJC facilitation observed in the transformants. These treatments only slightly affected the facilitation of normal junctions. 5. These results suggest that (i) internal Na+ accumulation can enhance facilitation of transmitter release in Drosophila neuromuscular junctions overexpressing frequenin, and (ii) this effect possibly depends on a modulation of the activity of the Na(+)-Ca2+ exchanger by frequenin.

Short isoform of POU factor Brn-3b can form a heterodimer with Brn-3a that is inactive for octamer motif binding.

The POU proteins Brn-3a and Brn-3b belong to a family of DNA binding transcription factors that share stretches of extensive homology. Both Brn-3a and Brn-3b are expressed as shorter and longer isoforms. The long form of Brn-3a is able to oncogenically transform primary fibroblasts. By contrast, the short form of Brn-3b (Brn-3b(s)) cannot transform fibroblasts but is able to specifically inhibit the transforming activity of Brn-3a(1). Moreover, Brn-3a(1) can act as a transcriptional transactivator, while Brn-3b(s) is not only unable to do so but in addition specifically inhibits the tranactivating activity of Brn-3a(1). Here, we show that the opposite and antagonistic activities of Brn-3a(1) and Brn-3b(s) proteins are due to their different DNA binding properties; Brn-3a(1) but not Brn-3b(s) can form stable complexes with several octamer-related target DNA sequences. The presence of Brn-3b(s) completely inhibits the binding of Brn-3a(1) to DNA by preventing the formation of Brn-3a(1)-DNA complexes as well as by disrupting preformed complexes. Experiments with GST fusion proteins and in vitro binding studies suggest that the inhibition of Brn-3a(1) activity by Brn-3b(s) occurs via direct interaction of the two transcription factors in solution. Therefore, we hypothesize that Brn-3b(s) can act as a direct antagonist of Brn-3a(1) by inhibiting its DNA binding through the formation of an inactive hetero-oligomeric complex.

Gli3 is required for Emx gene expression during dorsal telencephalon development.

Dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on its development, however, has been relatively poor. To date, only Emx genes were known to be required for dorsal telencephalon development. Here, we report on forebrain development in the extra toes (Xt(J)) mouse mutant which carries a null mutation of the Gli3 gene. This defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, Xt(J)/Xt(J) mice show absence of the hippocampus (Ammon's horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in Xt(J)/Xt(J) embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the Xt(J)/Xt(J) forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies Gli3 to be upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development.

Segmental expression of the EphA4 (Sek-1) receptor tyrosine kinase in the hindbrain is under direct transcriptional control of Krox-20.

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres (r) with distinct identities. Recent studies have uncovered regulatory links between transcription factors governing this process, but little is known of how these relate to molecules mediating cell-cell signalling. The Eph receptor tyrosine kinase gene EphA4 (Sek-1) is expressed in r3 and r5, and function-blocking experiments suggest that it is involved in restricting intermingling of cells between odd- and even-numbered rhombomeres. We have analysed the cis-acting regulatory sequences of the EphA4 gene in transgenic mice and identified a 470 bp enhancer element that drives specific expression in r3 and r5. Within this element, we have identified eight binding sites for the Krox-20 transcription factor that is also expressed in r3 and r5. Mutation of these binding sites abolishes r3/r5 enhancer activity and ectopic expression of Krox-20 leads to ectopic activation of the enhancer. These data indicate that Krox-20 is a direct transcriptional activator of EphA4. Together with evidence that Krox-20 regulates Hox gene expression, our findings reveal a mechanism by which the identity and movement of cells are coupled such that sharply restricted segmental domains are generated.

Chromosomal localization and sequences of the murine Brn-3 family of developmental control genes.

The POU proteins BRN-3a, -3b and -3c are transcription factors encoded by separate genes that are differentially expressed during neural development. Comparison of genomic and cDNA sequences revealed similar exon/intron structures for Brn-3a and -3c genes, whereas the Brn-3b gene appeared to contain an intronless coding region. Fluorescence in situ hybridization experiments with mouse chromosomes showed that Brn-3a maps to mouse chromosome 14E1-3, Brn-3b to XF1-5 and Brn-3c to 18B3-E1.

Activation of the herpes simplex virus immediate-early gene promoters by neuronally expressed POU family transcription factors.

Herpes simplex virus immediate-early (IE) promoters contain the TAATGARAT motif which acts as a target site for the cellular POU family transcription factors Oct-1 and Oct-2. Here we show that other members of the POU family that are expressed in sensory neurons can also affect IE promoter activity. In particular, two members of the Brn-3 family of POU proteins Brn-3a and Brn-3c can activate the IE-1 and IE-3 promoters when co-transfected into fibroblasts and neuronal cells whereas a third member Brn-3b represses IE promoter activity. In contrast, Brn-3 proteins cannot overcome the inhibitory effect of neuronal Oct-2 on IE promoter activity in co-transfections nor do they prevent transactivation of the IE promoters by the Oct-1-Vmw65 complex. The potential regulation of the IE promoters by several different neuronally expressed POU proteins during the initiation, maintenance and re-activation of latent infection in sensory neurons is discussed.

Gli genes and limb development.

During development of the limb Shh plays a key role as a mediator of zone of polarizing activity (ZPA). However, the molecular mechanisms by which Shh directs anterior/posterior patterning in the limb remain unknown. Members of the Gli gene family encode zinc-finger transcription factors and represent likely candidates for being regulators of Shh target genes. In this review we would like to summarize the current knowledge on expression and function of Gli genes in limb development.

The DNA target site for the Brn-3 POU family transcription factors can confer responsiveness to cyclic AMP and removal of serum in neuronal cells.

The POU factors Brn-3a and Brn-3b are closely related transcription factors which are expressed in neuronal cells. The levels of the transcripts encoding these factors are regulated in opposite directions in neuronal cells by specific cellular signalling pathways with dibutyryl cyclic AMP treatment and serum removal enhancing the level of Brn-3a and reducing the level of Brn-3b expression. This opposite expression pattern is paralleled by the ability of Brn-3a to specifically transactivate a target promoter bearing its DNA binding site whereas this promoter is repressed by Brn-3b. As predicted from these observations this target promoter is strongly activated by serum removal or addition of dibutyryl cyclic AMP. Therefore changes in Brn-3a and b expression can have a functional effect on promoter activity indicating that Brn-3a and Brn-3b can regulate gene expression via a specific binding site in response to the activation of specific cellular signalling pathways. The reasons for the differences in activity between these two related factors and their role in regulating gene activity in the nervous system are discussed.

PHF2, a novel PHD finger gene located on human chromosome 9q22.

We have isolated and characterized a novel PHD finger gene, PHF2, which maps to human Chromosome (Chr) 9q22 close to D9S196. Its mouse homolog was also characterized and mapped to the syntenic region on mouse Chr 13. The predicted human and mouse proteins are 98% identical and contain a PHD finger domain, eight possible nuclear localization signals, two potential PEST sequences, and a novel conserved hydrophobic domain. Northern analysis shows widespread expression of PHF2 in adult tissues, while in situ hybridization on mouse embryos reveals staining in the neural tube and dorsal root ganglia significantly above a ubiquitous low level expression signal. From its expression pattern and its chromosomal localization, PHF2 is a candidate gene for hereditary sensory neuropathy type I, HSN1.

Mouse Dac, a novel nuclear factor with homology to Drosophila dachshund shows a dynamic expression in the neural crest, the eye, the neocortex, and the limb bud.

Dac is a novel nuclear factor in mouse and humans that shares homology with Drosophila dachshund (dac). Alignment with available sequences defines a conserved box of 117 amino acids that shares weak homology with the proto-oncogene Ski and Sno. Dac expression is found in various neuroectodermal and mesenchymal tissues. At early developmental stages Dac is expressed in lateral mesoderm and in neural crest cells. In the neural plate/tube Dac expression is initially seen in the prosencephalon and gets gradually restricted to the presumptive neocortex and the distal portion of the outgrowing optic vesicle. Furthermore, Dac transcripts are detected in the mesenchyme underlying the Apical Ectodermal Ridge (AER) of the extending limb bud, the dorsal root ganglia and chain ganglia, and the mesenchyme of the growing genitalia. Dac expression in the Gli 3 mutant extra toes (Xt/Xt) shows little difference compared to the expression in wild-type limb buds. In contrast, a significant expansion of Dac expression are observed in the anterior mesenchyme of the limb buds of hemimelic extra toes (Hx/+) mice. FISH analysis reveals that human DAC maps to chromosome 13q22.3-23 and further fine-mapping defined a position of the DAC gene at 54cM or 13q21.1, a locus that associates with mental retardation and skeletal abnormalities.

A novel POU homeodomain gene specifically expressed in cells of the developing mammalian nervous system.

We report the isolation of a novel human POU domain encoding gene named RDC-1. The POU domain of the RDC-1 encoded protein is highly related to the POU domain potentially encoded by the rat brain-3 sequence and to that of the Drosophila I-POU protein; outside of the POU region, RDC-1 is unrelated to any previously characterized protein. The RDC-1 gene is expressed almost exclusively in normal tissues and transformed cells of neural origin. In the developing mouse and human fetus, RDC-1 is expressed in a spatially and temporally restricted pattern that suggests a critical role in the differentiation of neuronal tissues. In addition, RDC-1 is expressed in a unique subset of tumors of the peripheral nervous system including neuroepitheliomas and Ewing's sarcomas but not neuroblastomas. Based on its unique structural characteristics and expression pattern, we discuss potential functions for the RDC-1 protein.

Activation of the alpha-internexin promoter by the Brn-3a transcription factor is dependent on the N-terminal region of the protein.

The Brn-3a, Brn-3b, and Brn-3c proteins are closely related POU (Pit-Oct-Unc) family transcription factors which are expressed predominantly in neuronal cells. We have identified the alpha-internexin gene as the first reported, neuronally expressed, target gene whose promoter activity is modulated by these factors. Both the Brn-3a and Brn-3c factors can activate the alpha-internexin promoter while Brn-3b represses it and can prevent activation by Brn-3a. Using chimeric constructs containing different regions of Brn-3a or Brn-3b, we show that activation of the alpha-internexin promoter requires the N-terminal region of Brn-3a. In contrast the activation by Brn-3a but not Brn-3b of an artificial promoter containing a synthetic Brn-3 binding site can be shown using the same constructs to be dependent on the POU domain of Brn-3a. Moreover, the isolated POU domain of Brn-3a can activate this artificial promoter but not the alpha-internexin promoter. Hence Brn-3a contains two distinct transactivation domains, at the N terminus and within the POU domain, whose effect is dependent upon the target promoter. The relationship of gene transactivation by Brn-3a to its ability to transform primary cells which is also dependent on the N-terminal region of the protein is discussed.

Regulation of neurite outgrowth and SNAP-25 gene expression by the Brn-3a transcription factor.

SNAP-25 is a presynaptic nerve terminal protein which is also essential for the process of neurite outgrowth in vivo and in vitro. However the processes regulating its expression have not been characterized previously. We show that the gene encoding this protein, SNAP, is strongly activated by the Brn-3a POU (Pit-Oct-Unc) family transcription factor. Expression of both Brn-3a and SNAP-25 increases when ND7 neuronal cells are induced to extend neurite processes by serum removal. Inhibition of Brn-3a expression in these cells inhibits SNAP-25 expression and abolishes the neurite outgrowth that normally occurs in response to serum removal. These results identify Brn-3a as the first transcription factor having a role in process outgrowth in neuronal cells acting, at least in part, via the activation of SNAP-25 gene expression.