ErbB4 signaling in dopaminergic axonal projections increases extracellular dopamine levels and regulates spatial/working memory behaviors.

Genetic variants of Neuregulin 1 (NRG1) and its neuronal tyrosine kinase receptor ErbB4 are associated with risk for schizophrenia, a neurodevelopmental disorder characterized by excitatory/inhibitory imbalance and dopamine (DA) dysfunction. To date, most ErbB4 studies have focused on GABAergic interneurons in the hippocampus and neocortex, particularly fast-spiking parvalbumin-positive (PV+) basket cells. However, NRG has also been shown to modulate DA levels, suggesting a role for ErbB4 signaling in dopaminergic neuron function. Here we report that ErbB4 in midbrain DAergic axonal projections regulates extracellular DA levels and relevant behaviors. Mice lacking ErbB4 in tyrosine hydroxylase-positive (TH+) neurons, but not in PV+ GABAergic interneurons, exhibit different regional imbalances of basal DA levels and fail to increase DA in response to local NRG1 infusion into the dorsal hippocampus, medial prefrontal cortex and dorsal striatum measured by reverse microdialysis. Using Lund Human Mesencephalic (LUHMES) cells, we show that NRG/ErbB signaling increases extracellular DA levels, at least in part, by reducing DA transporter (DAT)-dependent uptake. Interestingly, TH-Cre;ErbB4f/f mice manifest deficits in learning, spatial and working memory-related behaviors, but not in numerous other behaviors altered in PV-Cre;ErbB4f/f mice. Importantly, microinjection of a Cre-inducible ErbB4 virus (AAV-ErbB4.DIO) into the mesencephalon of TH-Cre;ErbB4f/f mice, which selectively restores ErbB4 expression in DAergic neurons, rescues DA dysfunction and ameliorates behavioral deficits. Our results indicate that direct NRG/ErbB4 signaling in DAergic axonal projections modulates DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors relevant to psychiatric disorders.

Neuregulin-2 ablation results in dopamine dysregulation and severe behavioral phenotypes relevant to psychiatric disorders.

Numerous genetic and functional studies implicate variants of Neuregulin-1 (NRG1) and its neuronal receptor ErbB4 in schizophrenia and many of its endophenotypes. Although the neurophysiological and behavioral phenotypes of NRG1 mutant mice have been investigated extensively, practically nothing is known about the function of NRG2, the closest NRG1 homolog. We found that NRG2 expression in the adult rodent brain does not overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC), and therefore generated NRG2 knockout mice (KO) to study its function. NRG2 KOs have higher extracellular dopamine levels in the dorsal striatum but lower levels in the mPFC; a pattern with similarities to dopamine dysbalance in schizophrenia. Like ErbB4 KO mice, NRG2 KOs performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. NRG2 KOs exhibit hyperactivity in a novelty-induced open field, deficits in prepulse inhibition, hypersensitivity to amphetamine, antisocial behaviors, reduced anxiety-like behavior in the elevated plus maze and deficits in the T-maze alteration reward test-a task dependent on hippocampal and mPFC function. Acute administration of clozapine rapidly increased extracellular dopamine levels in the mPFC and improved alternation T-maze performance. Similar to mice treated chronically with N-methyl-d-aspartate receptor (NMDAR) antagonists, we demonstrate that NMDAR synaptic currents in NRG2 KOs are augmented at hippocampal glutamatergic synapses and are more sensitive to ifenprodil, indicating an increased contribution of GluN2B-containing NMDARs. Our findings reveal a novel role for NRG2 in the modulation of behaviors with relevance to psychiatric disorders.

Sonic hedgehog signaling is essential for hair development.

BACKGROUND: The skin is responsible for forming a variety of epidermal structures that differ amongst vertebrates. In each case the specific structure (for example scale, feather or hair) arises from an epidermal placode as a result of epithelial-mesenchymal interactions with the underlying dermal mesenchyme. Expression of members of the Wnt, Hedgehog and bone morphogenetic protein families (Wnt10b, Sonic hedgehog (Shh) and Bmp2/Bmp4, respectively) in the epidermis correlates with the initiation of hair follicle formation. Further, their expression continues into either the epidermally derived hair matrix which forms the hair itself, or the dermal papilla which is responsible for induction of the hair matrix. To address the role of Shh in the hair follicle, we have examined Shh null mutant mice. RESULTS: We found that follicle development in the Shh mutant embryo arrested after the initial epidermal-dermal interactions that lead to the formation of a dermal papilla anlage and ingrowth of the epidermis. Wnt10b, Bmp2 and Bmp4 continued to be expressed at this time, however. When grafted to nude mice (which lack T cells), Shh mutant skin gave rise to large abnormal follicles containing a small dermal papilla. Although these follicles showed high rates of proliferation and some differentiation of hair matrix cells into hair-shaft-like material, no hair was formed. CONCLUSIONS: Shh signaling is not required for initiating hair follicle development. Shh signaling is essential, however, for controlling ingrowth and morphogenesis of the hair follicle.

Molecular dissection of DNA sequences and factors involved in slow muscle-specific transcription.

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.

Expression of an amphibian homolog of the Eph family of receptor tyrosine kinases is developmentally regulated.

In order to study the function of tyrosine kinase receptors during Xenopus development, we have isolated Xek (Xenopus Elk-like kinase), a tyrosine kinase receptor, which shows significant homology to rat Elk and chicken cek5, members of the Eph family. Xek exists as a maternally expressed mRNA which decreases in expression at the mid blastula transition and reappears at late neurulation in Xenopus. Xek mRNA is expressed at higher levels in the anterior and dorsal regions of embryonic stages 16, 24 and 37. In adult Xenopus tissues, Xek appears to be ubiquitously expressed with higher expression observed in brain and ovary. In situ hybridization analysis demonstrates localized mRNA expression in the brain, brachial arches, trigeminal facial ganglion, and the retina of the swimming tadpole stage of development. The similarities in sequence and expression pattern suggest that Xek is an amphibian member of the Eph family and may play a role in the development or function of the central nervous system.

Identification of XLerk, an Eph family ligand regulated during mesoderm induction and neurogenesis in Xenopus laevis.

We have isolated and characterized the first Xenopus transmembrane Eph ligand, XLerk (Xenopus Ligand for Eph Receptor Tyrosine Kinases). While this ligand has 72% identity with the closest mammalian family member, Lerk-2, it is the cytoplasmic domain of this molecule that is the most conserved domain with 95% identity. XLerk exists as a maternally expressed mRNA, however, expression of transcripts and protein increase during gastrulation and again in the late swimming tadpole stage. In the adult, XLerk is expressed at low levels in most adult tissues with increased levels observed in the kidney, oocytes, ovary and testis. While low levels of XLerk expression are observed in the adult brain, in situ hybridization analysis demonstrates prominent expression in the developing olfactory system, retina, hindbrain, cranial ganglia, and somites. Furthermore, we have shown that XLerk transcripts are significantly elevated during mesoderm induction caused by activin and FGF, but not during noggin-induced neuralization. These results suggest a role for XLerk in the developing mesenchymal and nervous tissue.

Transient and recurrent expression of the Egr-1 gene in epithelial and mesenchymal cells during tooth morphogenesis suggests involvement in tissue interactions and in determination of cell fate.

We have analyzed the expression of early growth response gene (Egr-1) by mRNA in situ hybridization during mouse embryonic tooth development and in experimental recombinations of dental epithelium and mesenchyme. Egr-1 was transiently and recurrently expressed both in epithelial and mesenchymal cells starting from day 13 of gestation and up to 4 days after birth. The expression correlated with developmental transition points of dental mesenchymal and epithelial cells suggesting a role for Egr-1 in sequential determination and differentiation of cells. In recombination cultures of early dental epithelium and mesenchyme Egr-1 RNA was localized at the epithelial-mesenchymal interface in mesenchymal cells, and in two cases also in epithelial cells. These data indicate that Egr-1 expression may be regulated by epithelial-mesenchymal interactions when they are specific enough to initiate differentiation. We have also analyzed by in situ hybridization whether Wilms' tumour-1 gene (wt-1) is expressed in the developing tooth as it was proposed on the bases of in vitro studies that it may inhibit Egr-1 expression. No wt-1 expression was detected at any stage of tooth development showing that wt-1 is not obligatory for regulation of Egr-1 expression.

Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene.

The main morphological features of the mammalian tooth crown are cusps, but the developmental mechanisms that cause the formation of cusps are unknown. Tooth cusp formation commences at cap-stage with the appearance of the enamel knot, which is a cluster of non-dividing epithelial cells. In this study, enamel knot was first seen in embryonic mice molar teeth at the onset of cap-stage. Later in tooth development, secondary enamel knot structures were observed at the cusp tips and their appearance corresponded to the formation of individual cusp morphology. Comparisons of the pattern of cell proliferation in embryonic mouse molars and the expression of fibroblast growth factor-4 (Fgf-4) gene revealed that expression of Fgf-4 mRNA is strictly localized to the non-dividing cells of the enamel knot. However, when FGF-4 protein was introduced onto isolated dental tissues in vitro, it stimulated the proliferation of both dental epithelial and mesenchymal cells. Based on these results, we suggest that the enamel knot may control tooth morphogenesis by concurrently stimulating cusp growth (via FGF-4 synthesis) and by directing folding of cusp slopes (by not proliferating itself).

Expression pattern of the rat Lim-1 homeobox gene suggests a dual role during kidney development.

This study describes an in situ hybridization and immunohistochemical analysis of Lim-1 homeobox gene expression during kidney development in the rat. Lim-1 is expressed at all stages of mesonephric and metanephric kidney development. In the metanephros, Lim-1 gene mRNA is first found at day 13 in the ureteric bud, but not in uninduced mesenchyme. Expression in the mesenchyme can be seen only after mesenchymal cells have condensed around the ureteric bud tips and primary vesicles have formed. Experiments with mesenchymal explants induced to differentiate in vitro by high levels of basic FGF in the absence of ureteric bud also indicate that Lim-1 expression is correlated with tubulogenesis and this experimental model faithfully reproduces its expression in vivo. During mesenchymal differentiation Lim-1 protein and mRNA were found in comma- and S-shaped bodies, proximal and distal tubules, and collecting ducts. Lim-1 mRNA and Lim-1 protein were seen transiently at early stages of glomerulus formation. In the fully differentiated kidney Lim-1 gene products disappear from mesenchymal derivatives but persist in the collecting ducts which are derived from the ureteric bud. These data suggest a dual role for the Lim-1 homeobox gene in the developing kidney, a transient developmental function in the mesenchyme and a maintenance function in the ureteric bud and its derivatives. Further we suggest that Lim-1 is not directly involved in mesenchymal induction but may participate in its epithelial transformation at later stages as its expression in mesenchyme begins only after the formation of primary vesicle.

The LIM homeodomain protein Lim-1 is widely expressed in neural, neural crest and mesoderm derivatives in vertebrate development.

Polyclonal antibodies to Xlim-1 homeodomain protein of Xenopus laevis were used to study the developmental expression pattern of this protein in Xenopus, rat and mouse. Western blotting of embryo extracts injected with different Xlim-1 constructs confirmed the specificity of the antibody. Beginning at the gastrula stage, Xlim-1 protein was detected in three cell lineages: (i) notochord, (ii) pronephros and (iii) certain regions of the central nervous system, in agreement with earlier studies of the expression of Xlim-1 RNA (Taira et al., Development 120: 1525-1536, 1994a). In addition, several new locations of Xlim-1 expression were found, including the olfactory organ, retina, otic vesicle, dorsal root ganglia and adrenal gland. Similar expression patterns were seen for the Lim-1 protein in frog and rodent tissues. These observations implicate the Xlim-1 gene in the specification of multiple cell lineages, particularly within the nervous system, and emphasize the conserved nature of the role of this gene in different vertebrate animals.

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 S100beta+/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.

[Specific protein markers in monolayer hepatocyte cultures from adult rats]. / Spetsificheskie belkovye markery v monosloinykh kul'turakh gepatotsitov vzroslykh krys.

By indirect immunofluorescence it has been shown that syntheses of protein A, ligandin, cytochrome P-450 PhB, and serum albumin persist in hepatocytes of adult rats during the first 2-3 days in culture. A surface protein--fibronectin--was also synthesized in cultured cells to be localized on the lower side of the free cell edge. On the 4-5th day of cultivation large regions of the lammelar cytoplasm appeared in hepatocytes accompanied by cell polarization. As a result, cells acquired a "fibroblast-like" form. During this period of cultivation, cells were characterized by the loss of cytochrome P-450 PhB, by a drastic decrease in protein A, and ligandin synthesis. At the same time, gamma-glutamyl transpeptidase, the protein characteristic of the embryonic stages, was revealed histochemically. Therefore, the impairment of tissue organization accompanying the transfer of hepatocytes into the vitro conditions results in gradual changes of their morphology, in a reduction or complete loss of some specific "adult" synthesis and activation of the "embryonic" synthesis.

[Differences in the expression of prekeratin and vimentin in organ and monolayer cultures of rat hepatocytes]. / Razlichiia ékspressii prekeratina i vimentina v organnykh i monosloinykh kul'turakh gepatotsitov krys.

Results obtained by the indirect immunofluorescence method employing specific monoclonal antibodies show that during the first 24 hours of cultivation in a monolayer there appears another protein of intermediate filaments--vimentin, which is a characteristic of most mesenchymal cells. At the same time, in the organ liver culture maintained in the same culture medium, no expression of vimentin was observed up to 5-7 days of cultivation. Vimentin was revealed only in cells that migrated from a tissuepiece to collagen. Besides the vimentin expression in these migrating cells and monolayer cultures of hepatocytes, a redistribution of prekeratin filaments took place: the cytoplasmic network appeared instead of thick fibers underlying membranes. The results of the present work suggest that the vimentin expression and the prekeratin filament redistribution in epithelial liver cells in vitro do not depend on the changes of natural humoral factors for the components of culture medium but are due to damages of the intact liver tissue structure.

[Role of microtubules and intermediate filaments in maintaining the shape of epithelial cells]. / Rol' mikrotrubochek i promezhutochnykh filamentov v podderzhanii formy épitelial'nykh kletok.

The role of microtubules and intermediate filaments in control of cell shape of cultured cells of hepatomas McA-RH-7777 and 27 was investigated. Indirect immunofluorescence with specific polyclonal antibodies against tubulin and monoclonal antibodies against prekeratin with molecular weight 49 kD and vimentin was used. Incubation of cells in colcemid, resulting in specific distribution of microtubules did not change either prekeratin or vimentin distribution in cells of both the hepatomas, but reversed polarization of elongated McA-RH-7777 cells. These data suggest that the effect of disruption of microtubular system on the cell shape is not mediated by alterations of intermediate filaments.

Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9.

To elucidate the roles of fibroblast growth factors (FGF) in tooth development, we have analyzed the expression patterns of fibroblast growth factor receptors (FGFR) in mouse teeth by in situ hybridization and studied the effects of FGF-2, -4, -8, and -9 on cell proliferation in vitro by local application with beads on isolated dental mesenchymes. mRNAs of FGFR-1, -2, and -3 were localized by probes specific for the alternative splice variants IIIb and IIIc. The expression patterns of FGFR1 -2, and -3 were completely different, and the two splicing variants of FGFR1 and 2 exhibited different expression domains. FGFR4 was not expressed in the developing teeth. The IIIb splice forms of FGFR1 and -2 were expressed in the dental epithelium during morphogenesis. The IIIc splice form of FGFR1 was expressed both in epithelium and mesenchyme whereas FGFR2 IIIc was confined to the mesenchymal cells of the dental follicle. Both splice forms of FGFR3 were expressed in dental papilla mesenchyme. None of the FGF-receptors was detected in the primary enamel knot, the putative signaling center regulating tooth morphogenesis. This may explain the fact that enamel knot cells do not proliferate, although they express intensely mitogenic FGFs. Beads releasing FGF-2, -4, -8, or -9 proteins stimulated cell proliferation in cultured dental mesenchymes. These data, together with our earlier data on FGF expression [Kettunen and Thesleff (1998): Dev Dyn 211:256-268] suggest that FGF-8 and -9 mediate epithelial-mesenchymal interactions during tooth initiation. During advancing morphogenesis FGF-3, -4, and -9 may act both on mesenchyme and epithelium. Finally, the intense expression of FGFR1 in odontoblasts and ameloblasts and FGFR2 IIIb in ameloblasts suggests that FGFs participate in regulation of their differentiation and/or secretory functions.

Basic fibroblast growth factor can mediate the early inductive events in renal development.

The earliest characterized events during induction of tubulogenesis in renal anlage include the condensation or compaction of metanephrogenic mesenchyme with the concurrent upregulation of WT1, the gene encoding the Wilms tumor transcriptional activator/suppressor. We report that basic fibroblast growth factor (FGF2) can mimic the early effects of an inductor tissue by promoting the condensation of mesenchyme and inhibiting the tissue degeneration associated with the absence of an inductor tissue. By in situ hybridization, FGF2 was also found to mediate the transcriptional activation of WT1 and of the hepatocyte growth factor receptor gene, c-met. Although FGF2 can induce these early events of renal tubulogenesis, it cannot promote the epithelial conversion associated with tubule formation in metanephrogenic mesenchyme. For this, an undefined factor(s) from pituitary extract in combination with FGF2 can cause tubule formation in uninduced mesenchyme. These findings support the concept that induction in kidney is a multiphasic process that is mediated by more than a single comprehensive inductive factor and that soluble molecules can mimic these inductive activities in isolated uninduced metanephrogenic mesenchyme.

Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development.

Growth factor-mediated signaling has been implicated in the regulation of epithelial-mesenchymal interactions during organogenesis. Bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta superfamily, is expressed in the presumptive dental epithelium at the initiation of tooth development. Subsequently, epithelial signaling leads to mesenchymal induction of BMP-4 expression. To address the role of this factor, BMP-4-releasing agarose beads were added to dental mesenchyme in culture. These beads induced a translucent mesenchymal zone similar to that induced by dental epithelium. Moreover, three transcription factors (Msx-1, Msx-2, and Egr-1) whose expression is governed by epithelial signaling were induced in response to BMP-4. In addition, BMP-4 induced its own mesenchymal expression. These findings support the hypothesis that BMP-4 mediates epithelial-mesenchymal interactions during early tooth development.

[Expression of vimentin and prekeratins in solid and ascites variants of Zajdela hepatoma]. / Ekspressiia vimentina i prekeratinov v solidnykh i astsitnykh variantakh gepatomy Zajdela.

Using indirect immunofluorescence with monoclonal antibodies against prekeratins and vimentin, the contents and intracellular distribution of these proteins have been investigated in Seidel hepatoma cells. In ascitic tumour, cells were organized in multicellular unilayer spheric or ellipsoid complexes with an inner cavity. Such complexes have been found to express intracellular vimentin and chaotically distributed prekeratin filaments. One of the constituents of the normal epithelial basal membrane--laminin was not found on the basal surface of cellular complexes but was localized in their inner lumens only. The expression of vimentin and prekeratin filaments was preserved in metastatic tumour cells found in paratracheal lymph nodes and in the majority of solid tumour cells induced by subcutaneous cell injections. In both cases tumour cells did not form regular morphological structures and laminin was visualized as extracellular granules and short fibrils. In several cases subcutaneous injections of Seidel hepatoma cells gave rise to adenocarcinomas. Prekeratin filaments in these tumours were localized predominantly under cellular membranes. Laminin "membranes" outlined the basal surface of adenomatous structures. Vimentin in these cellular structures was completely absent. It is suggested that vimentin expression in Seidel hepatoma cells was suppressed with morphological normalization of tumour structures manifested in the regular distribution of intercellular contacts and in basal membrane reconstitution.

Deficient expression of mRNA for the putative inductive factor bone morphogenetic protein-7 in chemically initiated rat nephroblastomas.

Wilms' tumor, or nephroblastoma, arises from metanephric blastema and caricatures renal organogenesis. An alteration in at least one of the genes involved in control of renal differentiation is therefore a likely event in tumorigenesis, and indeed some of the genes involved in renal development, for example, hepatocyte growth factor (HGF) and its receptor c-met, the transcription factor Wilms' tumor gene (WT1), and transforming growth factor-beta family member bone morphogenetic protein (BMP)-7, have also been implicated in various models of tumorigenesis. In a comparison of mRNA expression patterns for these genes in normal rat embryonic or fetal kidney and nephroblastoma, we found that the patterns for HGF, met, and WT1 detected by in situ hybridization or ribonuclease protection assay (RPA) in the nephroblastomas were similar to those of normal developing kidney. BMP-7 expression, on the other hand, was lower in most tumors examined both by in situ hybridization and RPA than in normal tissues. This deficiency in a defined inductive factor that has been shown to function in renal tubulogenesis may play a role in tumorigenesis by allowing the accumulation of blastemal populations typical of nephroblastomas.