Increased Fos expression among midbrain dopaminergic cell groups during birdsong tutoring.

During avian vocal learning, birds memorize conspecific song patterns and then use auditory feedback to match their vocal output to this acquired template. Some models of song learning posit that during tutoring, conspecific visual, social and/or auditory cues activate neuromodulatory systems that encourage acquisition of the tutor's song and attach incentive value to that specific acoustic pattern. This hypothesis predicts that stimuli experienced during social tutoring activate cell populations capable of signaling reward. Using immunocytochemistry for the protein product of the immediate early gene c-Fos, we found that brief exposure of juvenile male zebra finches to a live familiar male tutor increased the density of Fos+ cells within two brain regions implicated in reward processing: the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). This activation of Fos appears to involve both dopaminergic and non-dopaminergic VTA/SNc neurons. Intriguingly, a familiar tutor was more effective than a novel tutor in stimulating Fos expression within these regions. In the periaqueductal gray, a dopamine-enriched cell population that has been implicated in emotional processing, Fos labeling also was increased after tutoring, with a familiar tutor again being more effective than a novel conspecific. As several neural regions implicated in song acquisition receive strong dopaminergic projections from these midbrain nuclei, their activation in conjunction with hearing the tutor's song could help to establish sensory representations that later guide motor sequence learning.

Human follistatin-related protein: a structural homologue of follistatin with nuclear localization.

Follistatin-related protein is a recently discovered glycoprotein that is highly homologous in both primary sequence and exon/intron domain structure to the activin-binding protein, follistatin. We explored their potential for functional redundancy by investigating the relative affinities and kinetics of their interactions with activin, bone morphogenic protein-6, and bone morphogenic protein-7 and by exploring their expression and distribution in human tissues and cells. Follistatin and follistatin-related protein mRNA were ubiquitous by Northern analyses, although their sites of peak distribution differed, with follistatin-related protein and follistatin predominating in the placenta and ovary, respectively. Follistatin-related protein, like follistatin, preferentially bound activin with high affinity and in an essentially irreversible fashion. Although follistatin-related protein, like follistatin, possesses a signal sequence and no known nuclear localization signals, its secretion was undetectable in most cell lines by RIA. Intriguingly, follistatin-related protein was identified as a nuclear protein in human granulosa cells and all human cell lines tested. Furthermore, Western analyses of CHO cells transfected with human follistatin-related protein revealed this protein to reside within the insoluble nuclear protein fraction. We conclude that despite its remarkably high level of similarity to follistatin with regard to structure and activin binding kinetics, follistatin-related protein is a nuclear as well as a secretory protein that may perform distinct intracellular actions.

Twisted gastrulation can function as a BMP antagonist.

Bone morphogenetic proteins (BMPs), including the fly homologue Decapentaplegic (DPP), are important regulators of early vertebrate and invertebrate dorsal-ventral development. An evolutionarily conserved BMP regulatory mechanism operates from fly to fish, frog and mouse to control the dorsal-ventral axis determination. Several secreted factors, including the BMP antagonist chordin/Short gastrulation (SOG), modulate the activity of BMPs. In Drosophila, Twisted gastrulation (TSG) is also involved in dorsal-ventral patterning, yet the mechanism of its function is unclear. Here we report the characterization of the vertebrate Tsg homologues. We show that Tsg can block BMP function in Xenopus embryonic explants and inhibits several ventral markers in whole-frog embryos. Tsg binds directly to BMPs and forms a ternary complex with chordin and BMPs. Coexpression of Tsg with chordin leads to a more efficient inhibition of the BMP activity in ectodermal explants. Unlike other known BMP antagonists, however, Tsg also reduces several anterior markers at late developmental stages. Our data suggest that Tsg can function as a BMP inhibitor in Xenopus; furthermore, Tsg may have additional functions during frog embryogenesis.

Cell dynamics in the embryonic and postnatal vomeronasal epithelium of snakes.

This review will discuss changes observed in the cell dynamics of the vomeronasal epithelium (VNE) of snakes during embryonic and postnatal growth. Recent work suggests that neuronal differentiation occurs early in VNE development. We have used an antibody to an evolutionarily conserved peptide sequence (the PSTAIRE region) in a family of cell cycle regulatory proteins, the cyclin-dependent kinases, to identify neuronal precursors in the embryonic and postnatal VNE. During prenatal development, the location of neuronal precursors changes in the VNE. Significant postnatal changes occur in cell proliferation in the VNE (as determined by 3H-thymidine autoradiography) and possibly in the larger complement of VNE receptor cell precursors (as determined by anti-PSTAIRE staining). A model is proposed for changes in cell proliferation and death during embryonic development and postnatal maintenance and senescence in VNE of snakes, which may be applicable to the VNE and olfactory epithelium of other vertebrates.

p110delta, a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes.

We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110delta. In addition, we have independently isolated p110delta from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110delta phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110delta is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system.

Preproenkephalin mRNA is regulated by an interaction between steroid hormones and nociceptive stimulation.

The expression of preproenkephalin (PPE) mRNA has previously been shown to be regulated by steroid hormones in the ventromedial nucleus of the hypothalamus (VMH) and to be regulated by noxious stimuli in the dorsal horn of the spinal cord (DH). The present in situ hybridization study in ovariectomized rats showed that PPE mRNA expression in both the VMH and the lumbar DH, responds to the interaction between a noxious peripheral stimulus and ovarian steroid hormones. In the VMH, either estradiol or estradiol + progesterone increased the mean PPE mRNA content per cell by 100% compared with vehicle-treated rats. Unilateral hindpaw injection of 5% formalin, as compared to saline, significantly increased mean PPE mRNA content per VMH cell in rats treated with vehicle or estradiol but not those treated with estradiol + progesterone. Regression analysis for mean PPE mRNA content per VMH cell as a function of intensity of hindpaw inflammation showed a significant positive correlation coefficient after vehicle and estradiol treatment (P < 0.02) but a strong trend towards a negative correlation coefficient after estradiol + progesterone treatment (P < 0.06). ANOVA for homogeneity of regression coefficients showed a significant difference across hormone groups (P < 0.01). In the lumbar DH, mean PPE mRNA content per cell was greater in rats injected with formalin than with saline and was greatest in rats given steroids + formalin. Mean PPE mRNA content per DH cell was greater ipsilateral than contralateral to the formalin injection in estradiol-treated rats, but no laterality difference was seen in the other hormone groups. No significant differences in mean PPE mRNA levels per DH cell were found among the rats treated with saline + hormone, saline + vehicle, formalin + vehicle, or uninjected rats. For all hormone groups combined, mean PPE mRNA per DH cell showed a significant positive regression on intensity of hindpaw inflammation (P < 0.05). Taken together these data are consistent with reports of increased pain threshold during pregnancy, descending control of antinociception from the basomedial hypothalamus and positive correlations between VMH levels of PPE mRNA and lordosis, a behavior evoked by somatosensory stimulation below nociceptive threshold.

The Schizosaccharomyces pombe rad3 checkpoint gene.

The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution.

The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes.

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia- and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are approximately 300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination.

Mapping actin surfaces required for functional interactions in vivo.

An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.

The yeast actin cytoskeleton.

Budding and fission yeast present significant advantages for studies of the actin cytoskeleton. The application of classical and molecular genetic techniques provides a facile route for the analysis of structure/function relationships, for the isolation of novel proteins involved in cytoskeletal function, and for deciphering the signals that regulate actin assembly in vivo. This review focuses on the budding yeast Saccharomyces cerevisiae and also identifies some recent advances from studies on the fission yeast Schizosaccharomyces pombe, for which studies on the actin cytoskeleton are still in their infancy.

Developmental changes in cytochrome oxidase histochemistry in the main and accessory olfactory bulbs of embryonic and neonatal garter snakes (Thamnophis sirtalis spp.).

Developmental studies examining the changes in oxidative metabolic activity are useful for understanding how and if the vomeronasal and olfactory systems respond to stimulation during embryogenesis. Garter snakes are good candidates for examining the potential functionality of the vomeronasal system in utero. In adult garter snakes, the vomeronasal system mediates many behaviors. Neonatal garter snakes exhibit these same behaviors, and the vomeronasal system has been shown to mediate feeding behavior in neonates. Using cytochrome oxidase histochemistry, we examined changes in the oxidative metabolic activity of main and accessory olfactory bulbs of embryonic and neonatal garter snakes (Thamnophis sirtalis sirtalis and T. s. parietalis). Cytochrome oxidase staining is greater in the accessory olfactory bulb than in the main olfactory bulb of embryonic garter snakes. However, neonates show no differences in the staining of the accessory and main olfactory bulbs, suggesting a change in the stimulation of the main olfactory bulb after birth. This is the first report of cytochrome oxidase histochemistry in reptiles and in the vomeronasal system of embryonic vertebrates.

Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae.

Abplp is a yeast cortical actin-binding protein that contains an SH3 domain similar to those found in signal transduction proteins that function at the membrane/cytoskeleton interface. Although no detectable phenotypes are associated with a disruption allele of ABP1, mutations that create a requirement for this protein have now been isolated in the previously identified gene SAC6 and in two new genes, SLA1 and SLA2. The SAC6 gene encodes yeast fimbrin, an actin filament-bundling protein. Null mutations in SLA1 and SLA2 cause temperature-sensitive growth defects. Sla1p contains three SH3 domains and is essential for the proper formation of the cortical actin cytoskeleton. The COOH terminus of Sla2p contains a 200 amino acid region with homology to the COOH terminus of talin, a membrane cytoskeletal protein which is a component of fibroblast focal adhesions. Sla2p is required for cellular morphogenesis and polarization of the cortical cytoskeleton. In addition, synthetic-lethal interactions were observed for double-mutants containing null alleles of SLA2 and SAC6. In total, the mutant phenotypes, sequences, and genetic interactions indicate that we have identified novel proteins that cooperate to control the dynamic cytoskeletal rearrangements that are required for the development of cell polarity in budding yeast.

The ontogeny of nasal chemical senses in garter snakes.

The development of the nasal chemical senses of reptiles is of particular interest since evidence suggests that neonatal reptiles use both their olfactory and vomeronasal systems. This review focuses on the embryonic development of these nasal chemosensory systems in garter snakes (Thamnophis spp.). Three approaches have been used to examine development: (1) Normal morphological studies, (2) 3H-thymidine autoradiographic studies, and (3) metabolic mapping of the accessory and main olfactory bulbs with cytochrome oxidase histochemistry. In addition, garter snake embryos have been grown in vitro to facilitate experimental procedures. The results of these studies show that reptiles are a rich and diverse group for investigating the developmental processes that form the neurobiological basis for naturally occurring chemosensory-mediated behaviors.

Axial muscle EMG responses evoked by cutaneous flank nerves in the female rat: effects of spinal transection, steroid hormones, and anesthesia.

In the lateral longissimus muscle (LL) of ovariectomized, female rats anesthetized with low surgical doses of urethane (1.0 g/kg), cutaneous reflexes with similar EMG and response patterns could be elicited from CNS-intact rats and from rats 24 h after complete thoracic spinal cord transection. The probability of eliciting a response to contralateral cutaneous nerve stimulation alone is much lower in rats with complete spinal transections compared to CNS-intact rats. For both CNS-intact and spinal-transected rats, responses to ipsilateral cutaneous nerve stimulation had a shorter latency and required significantly less current on average than responses to contralateral stimulation. The respective currents for eliciting threshold responses to ipsi- and contralateral stimulation are less for CNS-intact than spinal-transected rats. For both CNS-intact and spinal-transected rats, responses to bilateral cutaneous nerve stimulation were inconsistent in the same animal from run to run. With the variability of response at this anesthetic level, no consistent effects of progesterone (acute, i.v.) or estrogen (acute, i.v. and pretreatment, s.c.) were observed in spinal-transected rats. Intravenous progesterone reduced early, unilateral responses in CNS-intact rats anesthetized with 1.0 g of urethane/kg. For both CNS-intact and spinal-transected rats, additional anesthesia during EMG recording produced a gradual decline in response magnitude which could be recovered with a modest increase in stimulus intensity. However, spinal-transected rats appear to require less anesthesia to reduce comparable responses. The results suggest that supraspinal input is especially effective for facilitating contralateral cutaneous reflexes in back muscles, whereas it contributes more equally with afferent input and segmental circuitry to the efficacy of ipsilateral cutaneous reflexes.

Alternatively spliced murine lyn mRNAs encode distinct proteins.

Two lyn proteins of 56 and 53 kDa have been observed in immunoprecipitates from a variety of murine and human cell lines and tissues. We report the cloning and nucleotide sequence of two distinct murine lyn cDNAs isolated from an FDC-P1 cDNA library. One of the cDNAs, designated lyn11, encodes a protein of 56 kDa which shares 96% similarity with human lyn. The other cDNA, designated lyn12, encodes a protein of 53 kDa. The proteins differ in the presence or absence of a 21-amino-acid sequence located 24 amino acids C terminal of the translational initiation codon. Using RNase protection analysis, we have identified mRNAs corresponding to both cDNAs in murine cell lines and tissues. Sequence analysis of murine genomic clones suggests that the distinct mRNAs are alternatively spliced transcripts derived from a single gene. Expression of both cDNAs in COS cells leads to the production of lyn proteins with the same molecular weight as the two forms of lyn proteins immunoprecipitated from extracts of FDC-P1 cells and mouse spleen. Subcellular fractionation studies and Western immunoblotting analysis suggest that both isoforms of lyn are membrane associated. The association of both lyn isoforms with the membrane fraction supports the notion that lyn, like other src-related kinases, may interact with the intracellular domain of cell surface receptors.

Incorporation of 3H-thymidine in the embryonic vomeronasal and olfactory epithelial of garter snakes.

Previous studies have shown that the vomeronasal and olfactory epithelia of adult vertebrates provide good models for studying normal neuronal turnover and regeneration in response to axotomy. However, little is known about the cell dynamics in the embryonic vomeronasal and olfactory epithelia or the origins of different cell types in these structures. By using 3H-thymidine autoradiography, both in vivo and in vitro, the origins of receptor and supporting cells and the survival of labelled cells in the embryonic vomeronasal and olfactory epithelial of garter snakes were examined. The results of this study suggest that the receptor and supporting cells of both epithelial arise from separate stem cells and that two subpopulations of stem cells exist for receptor cells in the embryonic vomeronasal epithelium. One subpopulation generates cells that migrate through the receptor cell columns, while another subpopulation remains at the base of the epithelium for approximately 50 days. Although it is unclear how long receptor cells in the embryonic olfactory epithelium survive, the results of this study suggest that they survive at least 37 days and may survive over 56 days. In addition, the development of these sensory epithelia appears different in early versus late embryos, and regeneration in the vomeronasal and olfactory epithelia of adult garter snakes appears similar to development during late gestation. Cells in the developing receptor cell layer of the olfactory epithelium lose their ability to incorporate 3H-thymidine before those in the vomeronasal epithelium, suggesting that the onset of neuronal maturation occurs earlier in the olfactory epithelium than in the vomeronasal epithelium.

Incorporation of 3H-thymidine in telencephalic structures of the vomeronasal and olfactory systems of embryonic garter snakes.

Previous studies have shown that the vomeronasal and, possibly, olfactory systems are functional in newborn garter snakes. However, little is known about neurogenesis in these chemosensory pathways. In the companion paper, we describe the embryonic growth of the sensory epithelia for both the vomeronasal and olfactory systems. In the present study, we examine neurogenesis in the telencephalic structures of these chemosensory systems by using 3H-thymidine autoradiography (ARG). The majority of neurogenesis appears to occur before birth in the accessory and main olfactory bulbs and their principal projection sites, the nucleus sphericus and lateral cortex, respectively. The data suggest that some postnatal neurogenesis may occur in the accessory and main olfactory bulbs and in the nucleus sphericus. Although the neuronal constituents of the accessory and main olfactory bulbs appear to mature concurrently, those of the lateral cortex appear to mature before those of the nucleus sphericus. Along with previous findings, this latter result supports the hypothesis that the olfactory system develops before the vomeronasal system in garter snakes. There appears to be a rostral to caudal gradient of neurogenesis within the mural layer of the nucleus sphericus. However, an "outside to inside" gradient of neurogenesis was not observed in the mantle layer of the lateral cortex, as described for other reptiles. Similarities and differences observed by other investigators in other reptilian species and mammals are discussed.

Functional analysis and nucleotide sequence of the promoter region of the murine hck gene.

The structure and function of the promoter region and exon 1 of the murine hck gene have been characterized in detail. RNase protection analysis has established that hck transcripts initiate from heterogeneous start sites located within the hck gene. Fusion gene constructs containing hck 5'-flanking sequences and the bacterial Neor gene have been introduced into the hematopoietic cell lines FDC-P1 and WEHI-265 by using a self-inactivating retroviral vector. The transcriptional start sites of the fusion gene are essentially identical to those of the endogenous hck gene. Analysis of infected WEHI-265 cell lines treated with bacterial lipopolysaccharide (LPS) reveals a 3- to 5-fold elevation in the levels of endogenous hck mRNA and a 1.4- to 2.6-fold increase in the level of Neor fusion gene transcripts, indicating that hck 5'-flanking sequences are capable of conferring LPS responsiveness on the Neor gene. The 5'-flanking region of the hck gene contains sequences similar to an element which is thought to be involved in the LPS responsiveness of the class II major histocompatibility gene A alpha k. A subset of these sequences are also found in the 5'-flanking regions of other LPS-responsive genes. Moreover, this motif is related to the consensus binding sequence of NF-kappa B, a transcription factor which is known to be regulated by LPS.

Embryonic and neonatal development of the vomeronasal and olfactory systems in garter snakes (Thamnophis spp.).

Newborn, unfed garter snakes (Thamnophis spp.) respond preferentially to aqueous extracts of natural prey items, and these responses are mediated by the vomeronasal system (VNS). Since the VNS, and possibly the olfactory system (OS), are functional at birth, we examined the ontogeny of VNS and OS structures in four embryonic stages and two postnatal ages in garter snakes. The results of this study show 1) significant changes in thickness of the receptor epithelia for both systems; 2) temporal differences in the innervation of the telencephalon for each system; and 3) concurrent development of primary and secondary projection sites in both systems. Possible interactions between different cell populations and their significance for morphogenesis are discussed.

Synergistic activation by the glutamine-rich domains of human transcription factor Sp1.

We have examined the role of protein-protein interactions in modulating the activity of Sp1, a human transcription factor that utilizes glutamine-rich activation domains. These domains may represent a commonly used structural motif, since a nonhomologous glutamine-rich segment from the Drosophila Antennapedia protein is also a potent activator when fused to the Sp1 DNA binding domain. Sp1 is generally considered a proximal promoter factor that can only stimulate transcription when bound close to the initiation site. However, here we present evidence that distally and proximally bound Sp1 can stimulate transcription synergistically. In addition, a DNA binding-deficient mutant of Sp1 that retains glutamine-rich domains can interact with proximally bound Sp1 to superactivate transcription. Glutaraldehyde cross-linking provides direct evidence for an interaction between Sp1 monomers. Thus, Sp1-Sp1 interactions may play an important role in modulating promoter activity.