Irina Karavanova

Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development

Specification of cell lineages in mammals begins shortly after fertilization with formation of a blastocyst consisting of trophectoderm, which contributes exclusively to the placenta, and inner cell mass (ICM), from which the embryo develops. Here we report that ablation of the mouse Tead4 gene results in a preimplantation lethal phenotype, and TEAD4 is one of two highly homologous TEAD transcription factors that are expressed during zygotic gene activation in mouse 2-cell embryos. Tead4-/- embryos do not express trophectoderm-specific genes, such as Cdx2, but do express ICM-specific genes, such as Oct4 (also known as Pou5f1). Consequently, Tead4-/- morulae do not produce trophoblast stem cells, trophectoderm or blastocoel cavities, and therefore do not implant into the uterine endometrium. However, Tead4-/- embryos can produce embryonic stem cells, a derivative of ICM, and if the Tead4 allele is not disrupted until after implantation, then Tead4-/- embryos complete development. Thus, Tead4 is the earliest gene shown to be uniquely required for specification of the trophectoderm lineage.

Selective expression of ErbB4 in interneurons, but not pyramidal cells, of the rodent hippocampus.

NRG1 and ERBB4 have emerged as some of the most reproducible schizophrenia risk genes. Moreover, the Neuregulin (NRG)/ErbB4 signaling pathway has been implicated in dendritic spine morphogenesis, glutamatergic synaptic plasticity, and neural network control. However, despite much attention this pathway and its effects on pyramidal cells have received recently, the presence of ErbB4 in these cells is still controversial. As knowledge of the precise locus of receptor expression is crucial to delineating the mechanisms by which NRG signaling elicits its diverse physiological effects, we have undertaken a thorough analysis of ErbB4 distribution in the CA1 area of the rodent hippocampus using newly generated rabbit monoclonal antibodies and ErbB4-mutant mice as negative controls. We detected ErbB4 immunoreactivity in GABAergic interneurons but not in pyramidal neurons, a finding that was further corroborated by the lack of ErbB4 mRNA in electrophysiologically identified pyramidal neurons as determined by single-cell reverse transcription–PCR. Contrary to some previous reports, we also did not detect processed ErbB4 fragments or nuclear ErbB4 immunoreactivity. Ultrastructural analysis in CA1 interneurons using immunoelectron microscopy revealed abundant ErbB4 expression in the somatodendritic compartment in which it accumulates at, and adjacent to, glutamatergic postsynaptic sites. In contrast, we found no evidence for presynaptic expression in cultured GAD67-positive hippocampal interneurons and in CA1 basket cell terminals. Our findings identify ErbB4-expressing interneurons, but not pyramidal neurons, as a primary target of NRG signaling in the hippocampus and, furthermore, implicate ErbB4 as a selective marker for glutamatergic synapses on inhibitory interneurons.

Erbb transmembrane tyrosine kinase receptors are differentially expressed throughout the adult rat central nervous system

The neuregulin (NRG) family of growth and differentiation factors and their erbB receptors contribute importantly to the development of the nervous system, but their distribution and function in the adult brain are poorly understood. The present study showed that erbB2, erbB3, and erbB4 transcripts and protein are distributed throughout all areas of adult rat brain. These three receptors were differentially expressed in neurons and glia. Some neurons expressed only a subset of erbB kinases, whereas other neurons expressed all three erbB receptors but sequestered each of these polypeptides into distinct cellular compartments. In synapse‐rich regions, erbB immunoreactivity appeared as punctate‐, axon‐, and/or dendrite‐associated staining, suggesting that NRGs are involved in the formation and maintenance of synapses in adult brain. ErbB labeling also was present in neuronal soma, indicating that NRGs act at sites in addition to the synapse. Glia in adult brain also differentially expressed erbB3 and erbB4. Approximately half of the erbB3 labeling in white matter was associated with S100β+/glial fibrillary acidic protein negative macroglia (i.e., oligodendrocytes or glial fibrillary acidic protein negative astrocytes). In contrast, macroglia in gray matter did not express erbB3. The remaining erbB3 immunoreactivity in white matter and erbB4 glial staining seemed to be associated with microglia. These results showed that erbB receptors are expressed widely in adult rat brain and that each erbB receptor subtype has a distinct distribution. The differential distributions of erbB receptors in neurons and glia and the known functional differences between these kinases suggest that NRGs have distinct effects on these cells. The continued expression of NRGs and their erbB receptors in mature brain also implies that these molecules perform important functions in the brain throughout life. J. Comp. Neurol. 433:86–100, 2001.

THE IMPORTANCE OF THE NRG-1/ERBB4 PATHWAY FOR SYNAPTIC PLASTICITY AND BEHAVIORS ASSOCIATED WITH PSYCHIATRIC DISORDERS

Neuregulin 1 (NRG-1) and its receptor ErbB4 have emerged as biologically plausible schizophrenia risk factors, modulators of GABAergic and dopaminergic neurotransmission, and as potent regulators of glutamatergic synaptic plasticity. NRG-1 acutely depotentiates LTP in hippocampal slices, and blocking ErbB kinase activity inhibits LTP reversal by theta-pulse stimuli (TPS), an activity-dependent reversal paradigm. NRG-1/ErbB4 signaling in parvalbumin (PV) interneurons has been implicated in inhibitory transmission onto pyramidal neurons. However, the role of ErbB4, in particular in PV interneurons, for LTP reversal has not been investigated. Here we show that ErbB4-null (ErbB4−/−) and PV interneuron-restricted mutant (PV-Cre;ErbB4) mice, as well as NRG-1 hypomorphic mice, exhibit increased hippocampal LTP. Moreover, both ErbB4−/− and PV-Cre;ErbB4 mice lack TPS-mediated LTP reversal. A comparative behavioral analysis of full and conditional ErbB4 mutant mice revealed that both exhibit hyperactivity in a novel environment and deficits in prepulse inhibition of the startle response. Strikingly, however, only ErbB4−/− mice exhibit reduced anxiety-like behaviors in the elevated plus maze task and deficits in cued and contextual fear conditioning. These results suggest that aberrant NRG-1/ErbB4 signaling in PV interneurons accounts for some but not all behavioral abnormalities observed in ErbB4−/− mice. Consistent with the observation that PV-Cre;ErbB4 mice exhibit normal fear conditioning, we find that ErbB4 is broadly expressed in the amygdala, largely by cells negative for PV. These findings are important to better understand ErbB4s role in complex behaviors and warrant further analysis of ErbB4 mutant mice lacking the receptor in distinct neuron types.

Molecular Dissection of DNA Sequences and Factors Involved in Slow Muscle-Specific Transcription

ABSTRACT Transcription is a major regulatory mechanism for the generation of slow- and fast-twitch myofibers. We previously identified an upstream region of the slow TnI gene (slow upstream regulatory element [SURE]) and an intronic region of the fast TnI gene (fast intronic regulatory element [FIRE]) that are sufficient to direct fiber type-specific transcription in transgenic mice. Here we demonstrate that the downstream half of TnI SURE, containing E box, NFAT, MEF-2, and CACC motifs, is sufficient to confer pan-skeletal muscle-specific expression in transgenic mice. However, upstream regions of SURE and FIRE are required for slow and fast fiber type specificity, respectively. By adding back upstream SURE sequences to the pan-muscle-specific enhancer, we delineated a 15-bp region necessary for slow muscle specificity. Using this sequence in a yeast one-hybrid screen, we isolated cDNAs for general transcription factor 3 (GTF3)/muscle TFII-I repeat domain-containing protein 1 (MusTRD1). GTF3 is a multidomain nuclear protein related to initiator element-binding transcription factor TF II-I; the genes for both proteins are deleted in persons with Williams-Beuren syndrome, who often manifest muscle weakness. Gel retardation assays revealed that full-length GTF3, as well as its carboxy-terminal half, specifically bind the bicoid-like motif of SURE (GTTAATCCG). GTF3 expression is neither muscle nor fiber type specific. Its levels are highest during a period of fetal development that coincides with the emergence of specific fiber types and transiently increases in regenerating muscles damaged by bupivacaine. We further show that transcription from TnI SURE is repressed by GTF3 when overexpressed in electroporated adult soleus muscles. These results suggest a role for GTF3 as a regulator of slow TnI expression during early stages of muscle development and suggest how it could contribute to Williams-Beuren syndrome.

Neuregulin-2 is Developmentally Regulated and Targeted to Dendrites of Central Neurons

Neuregulin‐1 (NRG‐1) regulates numerous aspects of neural development and synaptic plasticity; the functions of NRG‐2 and NRG‐3 are presently unknown. As a first step toward understanding how NRGs contribute to distinct aspects of neural development and function, we characterized their regional and subcellular expression patterns in developing brain. The expression of NRG‐1–3 mRNAs was compared postnatally (P0, P7, adult) by using in situ hybridization. NRG‐1 expression is highest at birth, whereas NRG‐2 mRNA levels increase with development; expression of both genes is restricted to distinct brain regions. In contrast, NRG‐3 transcripts are abundant in most brain regions throughout development. NRG‐2 antibodies were generated to analyze protein processing, expression, and subcellular distribution. As with NRG‐1, the transmembrane NRG‐2 proprotein is proteolytically processed in transfected HEK 293 cells and in neural tissues, and its ectodomain is exposed and accumulates on the neuron surface. Despite the structural similarities between NRG‐1 and NRG‐2, we unexpectedly found that NRG‐2 colocalizes with MAP2 in proximal primary dendrites of hippocampal neurons in culture and in vivo, although it is not detectable in axons or in axon terminals. These findings were confirmed with NRG‐2 ectodomain antisera and epitope‐tagged recombinant protein. In cerebellum, NRG‐2 colocalizes with calbindin in proximal dendrites and soma of Purkinje cells. In contrast, NRG‐1 is highly expressed in axons of dissociated hippocampal neurons, as well as in somas and dendrites. The distinct temporal, regional, and subcellular expression of NRG‐2 suggests its unique and nonredundant role in neural function. J. Comp. Neurol. 472:156–172, 2004. Published 2004 Wiley‐Liss, Inc.

Neuregulin and dopamine modulation of hippocampal gamma oscillations is dependent on dopamine D4 receptors

The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal γ oscillations—a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal γ oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases γ oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for γ oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1–induced increase in γ oscillation power. This unique link between D4R and ErbB4 signaling on γ oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and γ oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits.

Cooperative effects of Sonic Hedgehog and NGF on basal forebrain cholinergic neurons.

In ventromedial cells of the developing CNS, Sonic hedgehog (Shh) has been shown to affect precursor proliferation, phenotype determination, and survival. Here we show that Shh and its receptor, Ptc-1, are expressed in the adult rat basal forebrain, and that Ptc-1 is expressed specifically by cholinergic neurons. In basal forebrain cultures, Shh was added alone and in combination with nerve growth factor (NGF), and the number of cholinergic neurons was determined by choline acetyltransferase (ChAT) immunocytochemistry. By 8 days in vitro, Shh and NGF show a synergistic effect: the number of ChAT-positive cells after treatment with both factors is increased over untreated cultures or cultures treated with either factor alone. While Shh increases the overall basal level of proliferation, double-labeling of dividing neuronal precursors with [(3)H]thymidine followed by ChAT immunocytochemistry after they mature, demonstrates that the specific increase in cholinergic neurons is not due to this proliferation enhancement. These experiments imply a role for Shh in the development of postmitotic cholinergic neurons and suggest a therapeutic value for Shh in neurodegenerative disease.

A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling

The neuregulin receptor ErbB4 is an important modulator of GABAergic interneurons and neural network synchronization. However, little is known about the endogenous ligands that engage ErbB4, the neural processes that activate them or their direct downstream targets. Here we demonstrate, in cultured neurons and in acute slices, that the NMDA receptor is both effector and target of neuregulin 2 (NRG2)/ErbB4 signalling in cortical interneurons. Interneurons co-express ErbB4 and NRG2, and pro-NRG2 accumulates on cell bodies atop subsurface cisternae. NMDA receptor activation rapidly triggers shedding of the signalling-competent NRG2 extracellular domain. In turn, NRG2 promotes ErbB4 association with GluN2B-containing NMDA receptors, followed by rapid internalization of surface receptors and potent downregulation of NMDA but not AMPA receptor currents. These effects occur selectively in ErbB4-positive interneurons and not in ErbB4-negative pyramidal neurons. Our findings reveal an intimate reciprocal relationship between ErbB4 and NMDA receptors with possible implications for the modulation of cortical microcircuits associated with cognitive deficits in psychiatric disorders.We have studied the impact of dust feedback on the survival and structure of vortices in protoplanetary discs using 2-D shearing box simulations with Lagrangian dust particles. We consider dust with a variety of sizes (stopping time ts = 10 −2Ω−1 – 102Ω−1), from fully coupled with the gas to the decoupling limit. We find that a vortex is destroyed by dust feedback when the total dust-to-gas mass ratio within the vortex is larger than 30-50%, independent of the dust size. The dust distribution can still be asymmetric in some cases after the vortex has been destroyed. With smaller amounts of dust, a vortex can survive for at least 100 orbits, and the maximum dust surface density within the vortex can be more than 100 times larger than the gas surface density, potentially facilitating planetesimal formation. On the other hand, in these stable vortices, small (ts < Ω −1) and large (ts & Ω−1) dust grains concentrate differently and affect the gas dynamics in different ways. The distribution of large dust is more elongated than that of small dust. Large dust (ts & Ω−1) concentrates in the centre of the vortex and feedback leads to turn-over in vorticity towards the centre, forming a quiescent region within an anticyclonic vortex. Such a turn-over is absent if the vortex is loaded with small grains. We demonstrate that, in protoplanetary discs where both large and small dust grains are present and under the right condition, the concentration of large dust towards the vortex centre can lead to a quiescent centre, repelling the small dust and forming a small dust ring around the vortex centre. Such anticorrelations between small and large dust within vortices may explain the discrepancy between ALMA and near-IR scattered light observations in the asymmetric region of transitional discs.

ErbB4 reduces synaptic GABAA currents independent of its receptor tyrosine kinase activity

Significance We identify a mode of Neuregulin signaling through ErbB4, requiring the receptor but not its canonical tyrosine kinase activity, that selectively decreases fast synaptic GABAA currents on hippocampal interneurons. Neuregulin promotes the clustering and association of ErbB4 with α1-containing GABA receptors, and results in the selective internalization of α1-containing receptors via a mechanism that requires PKC activity and clathrin-dependent endocytosis. These findings emphasize the diverse modes of Neuregulin signaling that can regulate interneuron network activity and which may contribute to the pathophysiology of neuropsychiatric disorders and epilepsy. ErbB4 signaling in the central nervous system is implicated in neuropsychiatric disorders and epilepsy. In cortical tissue, ErbB4 associates with excitatory synapses located on inhibitory interneurons. However, biochemical and histological data described herein demonstrate that the vast majority of ErbB4 is extrasynaptic and detergent-soluble. To explore the function of this receptor population, we used unbiased proteomics, in combination with electrophysiological, biochemical, and cell biological techniques, to identify a clinically relevant ErbB4-interacting protein, the GABAA receptor α1 subunit (GABAR α1). We show that ErbB4 and GABAR α1 are robustly coexpressed in hippocampal interneurons, and that ErbB4-null mice have diminished cortical GABAR α1 expression. Moreover, we characterize a Neuregulin-mediated ErbB4 signaling modality, independent of receptor tyrosine kinase activity, that couples ErbB4 to decreased postsynaptic GABAR currents on inhibitory interneurons. Consistent with an evolving understanding of GABAR trafficking, this pathway requires both clathrin-mediated endocytosis and protein kinase C to reduce GABAR inhibitory currents, surface GABAR α1 expression, and colocalization with the inhibitory postsynaptic protein gephyrin. Our results reveal a function of ErbB4, independent of its tyrosine kinase activity, that modulates postsynaptic inhibitory control of hippocampal interneurons and may provide a novel pharmacological target in the treatment of neuropsychiatric disorders and epilepsy.