Browsing by Subject "In Situ Hybridization"

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    BMP signaling in the development of the mouse esophagus and forestomach.
    (Development, 2010-12) Rodriguez, Pavel; Da Silva, Susana; Oxburgh, Leif; Wang, Fan; Hogan, Brigid LM; Que, Jianwen
    The stratification and differentiation of the epidermis are known to involve the precise control of multiple signaling pathways. By contrast, little is known about the development of the mouse esophagus and forestomach, which are composed of a stratified squamous epithelium. Based on prior work in the skin, we hypothesized that bone morphogenetic protein (BMP) signaling is a central player. To test this hypothesis, we first used a BMP reporter mouse line harboring a BRE-lacZ allele, along with in situ hybridization to localize transcripts for BMP signaling components, including various antagonists. We then exploited a Shh-Cre allele that drives recombination in the embryonic foregut epithelium to generate gain- or loss-of-function models for the Bmpr1a (Alk3) receptor. In gain-of-function (Shh-Cre;Rosa26(CAG-loxpstoploxp-caBmprIa)) embryos, high levels of ectopic BMP signaling stall the transition from simple columnar to multilayered undifferentiated epithelium in the esophagus and forestomach. In loss-of-function experiments, conditional deletion of the BMP receptor in Shh-Cre;Bmpr1a(flox/flox) embryos allows the formation of a multilayered squamous epithelium but this fails to differentiate, as shown by the absence of expression of the suprabasal markers loricrin and involucrin. Together, these findings suggest multiple roles for BMP signaling in the developing esophagus and forestomach.
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    Brain gene regulation by territorial singing behavior in freely ranging songbirds.
    (Neuroreport, 1997-05-27) Jarvis, ED; Schwabl, H; Ribeiro, S; Mello, CV
    To investigate the ecological relevance of brain gene regulation associated with singing behavior in songbirds, we challenged freely ranging song sparrows with conspecific song playbacks within their breeding territories. Males responded by approaching the speaker, searching for an intruder and actively singing. In situ hybridization of brain sections revealed significantly higher expression of the transcriptional regulator ZENK in challenged birds than in unstimulated controls in several auditory structures and song control nuclei. We conclude that singing behavior in the context of territorial defense is associated with gene regulation in brain centers that control song perception and production, and that behaviorally regulated gene expression can be used to investigate brain areas involved in the natural behaviors of freely ranging animals.
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    Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation.
    (J Clin Invest, 2010-05) Iskandar, Bermans J; Rizk, Elias; Meier, Brenton; Hariharan, Nithya; Bottiglieri, Teodoro; Finnell, Richard H; Jarrard, David F; Banerjee, Ruma V; Skene, JH Pate; Nelson, Aaron; Patel, Nirav; Gherasim, Carmen; Simon, Kathleen; Cook, Thomas D; Hogan, Kirk J
    The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.
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    FoxP2 expression in avian vocal learners and non-learners.
    (J Neurosci, 2004-03-31) Haesler, Sebastian; Wada, Kazuhiro; Nshdejan, A; Morrisey, Edward E; Lints, Thierry; Jarvis, Eric D; Scharff, Constance
    Most vertebrates communicate acoustically, but few, among them humans, dolphins and whales, bats, and three orders of birds, learn this trait. FOXP2 is the first gene linked to human speech and has been the target of positive selection during recent primate evolution. To test whether the expression pattern of FOXP2 is consistent with a role in learned vocal communication, we cloned zebra finch FoxP2 and its close relative FoxP1 and compared mRNA and protein distribution in developing and adult brains of a variety of avian vocal learners and non-learners, and a crocodile. We found that the protein sequence of zebra finch FoxP2 is 98% identical with mouse and human FOXP2. In the avian and crocodilian forebrain, FoxP2 was expressed predominantly in the striatum, a basal ganglia brain region affected in patients with FOXP2 mutations. Strikingly, in zebra finches, the striatal nucleus Area X, necessary for vocal learning, expressed more FoxP2 than the surrounding tissue at post-hatch days 35 and 50, when vocal learning occurs. In adult canaries, FoxP2 expression in Area X differed seasonally; more FoxP2 expression was associated with times when song becomes unstable. In adult chickadees, strawberry finches, song sparrows, and Bengalese finches, Area X expressed FoxP2 to different degrees. Non-telencephalic regions in both vocal learning and non-learning birds, and in crocodiles, were less variable in expression and comparable with regions that express FOXP2 in human and rodent brains. We conclude that differential expression of FoxP2 in avian vocal learners might be associated with vocal plasticity.
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    Inhibition of adaptive immune responses leads to a fatal clinical outcome in SIV-infected pigtailed macaques but not vervet African green monkeys.
    (PLoS Pathog, 2009-12) Schmitz, Jörn E; Zahn, Roland C; Brown, Charles R; Rett, Melisa D; Li, Ming; Tang, Haili; Pryputniewicz, Sarah; Byrum, Russell A; Kaur, Amitinder; Montefiori, David C; Allan, Jonathan S; Goldstein, Simoy; Hirsch, Vanessa M
    African green monkeys (AGM) and other natural hosts for simian immunodeficiency virus (SIV) do not develop an AIDS-like disease following SIV infection. To evaluate differences in the role of SIV-specific adaptive immune responses between natural and nonnatural hosts, we used SIV(agmVer90) to infect vervet AGM and pigtailed macaques (PTM). This infection results in robust viral replication in both vervet AGM and pigtailed macaques (PTM) but only induces AIDS in the latter species. We delayed the development of adaptive immune responses through combined administration of anti-CD8 and anti-CD20 lymphocyte-depleting antibodies during primary infection of PTM (n = 4) and AGM (n = 4), and compared these animals to historical controls infected with the same virus. Lymphocyte depletion resulted in a 1-log increase in primary viremia and a 4-log increase in post-acute viremia in PTM. Three of the four PTM had to be euthanized within 6 weeks of inoculation due to massive CMV reactivation and disease. In contrast, all four lymphocyte-depleted AGM remained healthy. The lymphocyte-depleted AGM showed only a trend toward a prolongation in peak viremia but the groups were indistinguishable during chronic infection. These data show that adaptive immune responses are critical for controlling disease progression in pathogenic SIV infection in PTM. However, the maintenance of a disease-free course of SIV infection in AGM likely depends on a number of mechanisms including non-adaptive immune mechanisms.
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    Lateralized activation of Cluster N in the brains of migratory songbirds.
    (Eur J Neurosci, 2007-02) Liedvogel, Miriam; Feenders, Gesa; Wada, Kazuhiro; Troje, Nikolaus F; Jarvis, Erich D; Mouritsen, Henrik
    Cluster N is a cluster of forebrain regions found in night-migratory songbirds that shows high activation of activity-dependent gene expression during night-time vision. We have suggested that Cluster N may function as a specialized night-vision area in night-migratory birds and that it may be involved in processing light-mediated magnetic compass information. Here, we investigated these ideas. We found a significant lateralized dominance of Cluster N activation in the right hemisphere of European robins (Erithacus rubecula). Activation predominantly originated from the contralateral (left) eye. Garden warblers (Sylvia borin) tested under different magnetic field conditions and under monochromatic red light did not show significant differences in Cluster N activation. In the fairly sedentary Sardinian warbler (Sylvia melanocephala), which belongs to the same phyolgenetic clade, Cluster N showed prominent activation levels, similar to that observed in garden warblers and European robins. Thus, it seems that Cluster N activation occurs at night in all species within predominantly migratory groups of birds, probably because such birds have the capability of switching between migratory and sedentary life styles. The activation studies suggest that although Cluster N is lateralized, as is the dependence on magnetic compass orientation, either Cluster N is not involved in magnetic processing or the magnetic modulations of the primary visual signal, forming the basis for the currently supported light-dependent magnetic compass mechanism, are relatively small such that activity-dependent gene expression changes are not sensitive enough to pick them up.
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    Neuron-specific Sumo1-3 knockdown in mice impairs episodic and fear memories.
    (Journal of psychiatry & neuroscience : JPN, 2014-07) Wang, Liangli; Rodriguiz, Ramona M; Wetsel, William C; Sheng, Huaxin; Zhao, Shengli; Liu, Xiaozhi; Paschen, Wulf; Yang, Wei
    BACKGROUND:Growing evidence suggests that small ubiquitin-like modifier (SUMO) conjugation plays a key role in brain plasticity by modulating activity-dependent synaptic transmission. However, these observations are based largely on cell culture experiments. We hypothesized that episodic and fear memories would be affected by silencing SUMO1-3 expression. METHODS:To investigate the role of SUMO conjugation in neuronal functioning in vivo, we generated a novel Sumo transgenic mouse model in which a Thy1 promoter drives expression of 3 distinct microRNAs to silence Sumo1-3 expression, specifically in neurons. Wild-type and Sumo1-3 knockdown mice were subjected to a battery of behavioural tests to elucidate whether Sumoylation is involved in episodic and emotional memory. RESULTS:Expression of Sumo1-3 microRNAs and the corresponding silencing of Sumo expression were particularly pronounced in hippocampal, amygdala and layer V cerebral cortex neurons. The Sumo knockdown mice displayed anxiety-like responses and were impaired in episodic memory processes, contextual and cued fear conditioning and fear-potentiated startle. LIMITATIONS:Since expression of Sumo1-3 was silenced in this mouse model, we need to verify in future studies which of the SUMO paralogues play the pivotal role in episodic and emotional memory. CONCLUSION:Our results indicate that a functional SUMO conjugation pathway is essential for emotionality and cognition. This novel Sumo knockdown mouse model and the technology used in generating this mutant may help to reveal novel mechanisms that underlie a variety of neuropsychiatric conditions associated with anxiety and impairment of episodic and emotional memory.
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    Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.
    (J Vis Exp, 2012-04-27) Chen, CC; Wada, K; Jarvis, Erich David
    Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)(1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.
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    Rapid behavioral and genomic responses to social opportunity.
    (PLoS Biol, 2005-11) Burmeister, SS; Jarvis, ED; Fernald, RD
    From primates to bees, social status regulates reproduction. In the cichlid fish Astatotilapia (Haplochromis) burtoni, subordinate males have reduced fertility and must become dominant to reproduce. This increase in sexual capacity is orchestrated by neurons in the preoptic area, which enlarge in response to dominance and increase expression of gonadotropin-releasing hormone 1 (GnRH1), a peptide critical for reproduction. Using a novel behavioral paradigm, we show for the first time that subordinate males can become dominant within minutes of an opportunity to do so, displaying dramatic changes in body coloration and behavior. We also found that social opportunity induced expression of the immediate-early gene egr-1 in the anterior preoptic area, peaking in regions with high densities of GnRH1 neurons, and not in brain regions that express the related peptides GnRH2 and GnRH3. This genomic response did not occur in stable subordinate or stable dominant males even though stable dominants, like ascending males, displayed dominance behaviors. Moreover, egr-1 in the optic tectum and the cerebellum was similarly induced in all experimental groups, showing that egr-1 induction in the anterior preoptic area of ascending males was specific to this brain region. Because egr-1 codes for a transcription factor important in neural plasticity, induction of egr-1 in the anterior preoptic area by social opportunity could be an early trigger in the molecular cascade that culminates in enhanced fertility and other long-term physiological changes associated with dominance.
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    Role of the midbrain dopaminergic system in modulation of vocal brain activation by social context.
    (Eur J Neurosci, 2007-06) Hara, Erina; Kubikova, Lubica; Hessler, Neal A; Jarvis, Erich D
    In a well-studied model of social behaviour, male zebra finches sing directed song to court females and undirected song, used possibly for practice or advertisement. Although the two song types are similar, the level of neural activity and expression of the immediate early gene egr-1 are higher during undirected than during directed singing in the lateral part of the basal ganglia song nucleus AreaX (LAreaX) and its efferent pallial song nuclei lateral magnocellular nucleus of the anterior nidopallium (LMAN) and the robust nucleus of the arcopallium (RA). As social interactions are dependent on brain motivation systems, here we test the hypothesis that the midbrain ventral tegmental area-substantia nigra pars compacta (VTA-SNc) complex, which provides a strong dopaminergic input to LAreaX, is a source of this modulation. Using egr-1 expression, we show that GABAergic interneurons in VTA-SNc are more active during directed courtship singing than during undirected singing. We also found that unilateral removal of VTA-SNc input reduced singing-dependent gene expression in ipsilateral LAreaX during both social contexts but it did not eliminate social context differences in LAreaX. In contrast, such lesions reduced and eliminated the social context differences in efferent nuclei LMAN and RA, respectively. These results suggest that VTA-SNc is not solely responsible for the social context gene regulation in LAreaX, but that VTA-SNc input to LAreaX enhances the singing-regulated gene expression in this nucleus and, either through LAreaX or through direct projections to LMAN and RA, VTA-SNc is necessary for context-dependent gene regulation in these efferent nuclei.
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    The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus.
    (Proc Natl Acad Sci U S A, 1999-02-16) Krebs, CJ; Jarvis, ED; Pfaff, DW
    Estrogen (E) and progesterone (P) orchestrate many cellular responses involved in female reproductive physiology, including reproductive behaviors. E- and P-binding neurons important for lordosis behavior have been located within the ventromedial hypothalamus (VMH), and several hormone-responsive genes have been observed there as well. In attempts to identify additional E- and P-responsive genes in the VMH that may contribute to sexual behaviors, we used the differential display mRNA screening technique. One of the genes identified encodes the 73-kDa heat shock cognate protein (Hsc73). Quantitative in situ hybridization analysis of brains from naturally cycling female rats revealed a significant increase in Hsc73 mRNA in the VMH and arcuate nucleus of animals during proestrus compared with those at diestrus-1. To confirm that these increases were steroid hormone dependent, we compared vehicle-treated ovariectomized females with ovariectomized females treated with estradiol benzoate and P. Northern analysis and in situ hybridizations showed that the Hsc73 gene is enhanced by E and P in the pituitary and subregions of the VMH. Incidentally, by examining the primary amino acid sequence of rat, human, and chicken progesterone receptors, we noticed that putative Hsc73 binding sites are conserved across species with similar sites existing in the androgen and glucocorticoid receptors. Together these findings suggest a possible mechanism through which E could influence the activities of progesterone, androgen, and glucocorticoid receptors, by enhancing the expression of Hsc73 in cells where these proteins colocalize.