Bcl-2 is required for cranial sensory neuron survival at defined stages of embryonic development

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4173-4178 ◽  
Author(s):  
L.G. Pinon ◽  
G. Middleton ◽  
A.M. Davies
Keyword(s):  
Embryonic Development ◽  
Sensory Neurons ◽  
Neuronal Death ◽  
Null Mouse ◽  
Wild Type ◽  
Naturally Occurring ◽  
Age Related ◽  
Trigeminal Neurons ◽  
Ganglion Neurons

To ascertain the role of endogenous Bcl-2 in maintaining the survival of developing neurons and modulating their responses to neurotrophins, we compared the in vitro and in vivo survival of cranial sensory neurons of wild-type and bcl-2 null mouse embryos. At the peak of naturally occurring neuronal death in the trigeminal ganglion at E14, trigeminal neurons from bcl-2(−/−) embryos initially survived in culture in response to NGF but were not sustained as well as neurons from wild-type embryos. At the end of the period of naturally occurring neuronal death at E18, Bcl-2-deficient trigeminal neurons survived with NGF as well as wild-type neurons. At E14 in vivo, the number of trigeminal neurons undergoing apoptosis was significantly greater in bcl-2(−/−) embryos, and there were significantly fewer neurons in the trigeminal ganglia of bcl-2(−/−) embryos at E16 and E18. Similar age-related changes in the responses of nodose ganglion neurons to BDNF were observed in cultures established from bcl-2(−/−) and wild-type embryos between E14 and E18. These results suggest that endogenous Bcl-2 is required for the sustained survival response of a subset of cranial sensory neurons to neurotrophins at particular stages of embryonic development and show that its absence leads to reduced numbers of these neurons in vivo.

scholarly journals MRG15 Regulates Embryonic Development and Cell Proliferation

2005 ◽  
Vol 25 (8) ◽  
pp. 2924-2937 ◽  
Author(s):  
Kaoru Tominaga ◽  
Bhakti Kirtane ◽  
James G. Jackson ◽  
Yuji Ikeno ◽  
Takayoshi Ikeda ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Embryonic Development ◽  
Chromatin Remodeling ◽  
Histone Deacetylases ◽  
Globin Gene ◽  
Immunohistochemical Analysis ◽  
Wild Type ◽  
Proliferating Cells ◽  
Globin Promoter

ABSTRACT MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15 − / − embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15 − / − mouse embryonic fibroblasts. The hearts of the Mrg15 − / − embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15 − / − embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.

scholarly journals Mutant tryptophan aporepressors with altered specificities of corepressor recognition.

Genetics ◽  
1991 ◽  
Vol 128 (1) ◽  
pp. 29-35
Author(s):  
D N Arvidson ◽  
M Shapiro ◽  
P Youderian
Keyword(s):  
Dna Binding ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Mutant Proteins ◽  
Naturally Occurring ◽  
Repressor Complex ◽  
Genes Encoding ◽  
Active Repressor

Abstract The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex. The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket. Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made. Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor. Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo. These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition.

Abstract 123: Age-Related Endothelial Senescence is Driven by Thrombospondin-1-Activated NADPH Oxidase Nox1

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Daniel N Meijles ◽  
Imad Al Ghouleh ◽  
Sanghamitra Sahoo ◽  
Jefferson H Amaral ◽  
Heather Knupp ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nadph Oxidase ◽  
Vascular Diseases ◽  
Dependent Manner ◽  
Wild Type ◽  
Molecular Control ◽  
Redox Biology ◽  
Age Related ◽  
P53 Activation

Organismal aging represents an independent risk factor underlying many vascular diseases, including systemic and pulmonary hypertension, and atherosclerosis. While the mechanisms driving aging are largely elusive, a steady persistent increase in tissue oxidative stress has been associated with senescence. Previously we showed TSP1 elicits NADPH oxidase (Nox)-dependent vascular smooth muscle cell oxidative stress. However mechanisms by which TSP1 affects endothelial redox biology are unknown. Here, we tested the hypothesis that TSP1 induces endothelial oxidative stress-linked senescence in aging. Using rapid autopsy disease-free human pulmonary (PA) artery, we identified a significant positive correlation between age, protein levels of TSP1, Nox1 and the cell-cycle repressor p21cip (p<0.05). Age also positively associated with increased Amplex Red-detected PA hydrogen peroxide levels (p<0.05). Moreover, treatment of human PA endothelial cells (HPAEC) with TSP1 (2.2nM; 24h) increased expression (~1.9 fold; p<0.05) and activation of Nox1 (~1.7 fold; p<0.05) compared to control, as assessed by Western blot and SOD-inhibitable cytochrome c reduction. Western blotting and immunofluorescence showed a TSP1-mediated increase in p53 activation, indicative of the DNA damage response. Moreover, TSP1 significantly increased HPAEC senescence in a p53/p21cip/Rb-dependent manner, as assessed by immunofluorescent detection of subcellular localization and senescence-associated β-galactosidase staining. To explore this pathway in vivo, middle-aged (8-10 month) wild-type and TSP1-null mice were utilized. In the TSP1-null, reduced lung senescence, oxidative stress, Nox1 levels and p21cip expression were observed compared to wild-type supporting findings in human samples and cell experiments. Finally, prophylactic treatment with specific Nox1 inhibitor NoxA1ds (10μM) attenuated TSP1-induced HPAEC ROS, p53 activation, p21cip expression and senescence. Taken together, our results provide molecular insight into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence, with implications for molecular control of the aging process.

scholarly journals Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 596-604 ◽  
Author(s):  
Ian S. Hitchcock ◽  
Norma E. Fox ◽  
Nicolas Prévost ◽  
Katherine Sear ◽  
Sanford J. Shattil ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Platelet Function ◽  
Therapeutic Benefit ◽  
Negative Regulator ◽  
Null Mouse ◽  
Wild Type ◽  
Platelet Recovery ◽  
Lyn Kinase

Focal adhesion kinase (FAK) plays a key role in mediating signaling downstream of integrins and growth factor receptors. In this study, we determined the roles of FAK in vivo by generating a megakaryocyte lineage–specific FAK-null mouse (Pf4-Cre/FAK-floxed). Megakaryocyte and platelet FAK expression was ablated in Pf4-Cre/FAK-floxed mice without affecting expression of the FAK homologue PYK2, although PYK2 phosphorylation was increased in FAK−/− megakaryocytes in response to fibrinogen. Megakaryopoiesis is greatly enhanced in Pf4-Cre/FAK-floxed mice, with significant increases in megakaryocytic progenitors (CFU-MK), mature megakaryocytes, megakaryocyte ploidy, and moderate increases in resting platelet number and platelet recovery following a thrombocytopenic stress. Thrombopoietin (Tpo)–mediated activation of Lyn kinase, a negative regulator of megakaryopoiesis, is severely attenuated in FAK-null megakaryocytes compared with wild-type controls. In contrast, Tpo-mediated activation of positive megakaryopoiesis regulators such as ERK1/2 and AKT is increased in FAK-null megakaryocytes, providing a plausible explanation for the observed increases in megakaryopoiesis in these mice. In Pf4-Cre/FAK-floxed mice, rebleeding times are significantly increased, and FAK-null platelets exhibit diminished spreading on immobilized fibrinogen. These studies establish clear roles for FAK in megakaryocyte growth and platelet function, setting the stage for manipulation of this component of the Tpo signaling apparatus for therapeutic benefit.

scholarly journals Blocking TLR2 in vivo Protects against Accumulation of Inflammatory Cells and Neuronal Injury in Experimental Stroke

2010 ◽  
Vol 31 (2) ◽  
pp. 757-766 ◽  
Author(s):  
Gina Ziegler ◽  
Dorette Freyer ◽  
Denise Harhausen ◽  
Uldus Khojasteh ◽  
Wilfried Nietfeld ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Neuronal Death ◽  
Focal Cerebral Ischemia ◽  
Neuronal Survival ◽  
Inflammatory Cells ◽  
Experimental Stroke ◽  
Wild Type ◽  
Antibody Treatment ◽  
Cell Counts

Reduced infarct volume in TLR2-knockout mice compared with C57Bl/6 wild-type mice has recently been shown in experimental stroke and confirmed in this study. We now also show a significant decrease of CD11b-positive cell counts and decreased neuronal death in the ischemic hemispheres of TLR2-deficient mice compared with C57Bl/6wt mice 2 days after transient focal cerebral ischemia. To examine the potential benefit of intravascular TLR2 inhibition, C57Bl/6wt mice were treated intraarterially with TLR2-blocking anti-TLR2 antibody (clone T2.5) after 45 minutes of cerebral ischemia and compared with control antibody (isotype) treated wild-type mice. Whereas T2.5-treated mice had no reduction in infarct volumes at 48 hours after reperfusion, they did have decreased numbers of CD11b-positive inflammatory cells and decreased neuronal death compared with isotype-treated control mice. Comparison of the isotype antibody treatment to control (saline) treatment showed no effects on infarct volumes or neuronal survival. However, mice treated with the control isotype antibody had increased numbers of CD11b-positive inflammatory cells compared with saline-treated animals. Thus, antibody treatment itself (i.e., control isotype antibody, but potentially of any antibody) may have adverse effects and limit therapeutic benefit of anti-TLR2-antibody therapy. We conclude that TLR2 mediates leukocyte and microglial infiltration and neuronal death, which can be attenuated by TLR2 inhibition. The TLR2 inhibition in vivo improves neuronal survival and may represent a future stroke therapy.

scholarly journals Miz1 Is Required for Early Embryonic Development during Gastrulation

2003 ◽  
Vol 23 (21) ◽  
pp. 7648-7657 ◽  
Author(s):  
Sovana Adhikary ◽  
Karen Peukert ◽  
Holger Karsunky ◽  
Vincent Beuger ◽  
Werner Lutz ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Binding Sites ◽  
Target Gene ◽  
Physiological Function ◽  
Core Promoter ◽  
Early Embryonic Development ◽  
Wild Type ◽  
Transcription Factor Family ◽  
Massive Apoptosis

ABSTRACT Miz1 is a member of the POZ domain/zinc finger transcription factor family. In vivo, Miz1 forms a complex with the Myc oncoprotein and recruits Myc to core promoter elements. Myc represses transcription through Miz1 binding sites. We now show that the Miz1 gene is ubiquitously expressed during mouse embryogenesis. In order to elucidate the physiological function of Miz1, we have deleted the mouse Miz1 gene by homologous recombination. Miz1+/− mice are indistinguishable from wild-type animals; in contrast, Miz1−/− embryos are not viable. They are severely retarded in early embryonic development and do not undergo normal gastrulation. Expression of Goosecoid and Brachyury is detectable in Miz1−/− embryos, suggesting that Miz1 is not required for signal transduction by Nodal. Expression of p21Cip1, a target gene of Miz1 is unaltered; in contrast, expression of p57Kip2, another target gene of Miz1 is absent in Miz1−/− embryos. Miz1−/− embryos succumb to massive apoptosis of ectodermal cells around day 7.5 of embryonic development. Our results show that Miz1 is required for early embryonic development during gastrulation.

scholarly journals Timing of neuronal death in trkA, trkB and trkC mutant embryos reveals developmental changes in sensory neuron dependence on Trk signalling

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3255-3261 ◽  
Author(s):  
L.G. Pinon ◽  
L. Minichiello ◽  
R. Klein ◽  
A.M. Davies
Keyword(s):  
Neuronal Death ◽  
Tyrosine Kinases ◽  
Marked Decrease ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Embryonic Mouse ◽  
Target Field ◽  
Significant Difference

The sensory neurons of the embryonic mouse trigeminal ganglion are supported in culture by different neurotrophins at successive stages of development. Initially the neurons survive in response to BDNF and NT3 and later switch to becoming NGF-dependent (Buchman, V. I. and Davies, A. M. (1993), Development 118, 989–1001). To determine if this in vitro switch in neurotrophin responsiveness is physiologically relevant, we studied the timing of neuronal death in the trigeminal ganglia of embryos that are homozygous for null mutations in the trkA, trkB and trkC genes, which encode receptor tyrosine kinases for NGF, BDNF and NT3, respectively. In wild-type embryos, the number of pyknotic nuclei increased from E11 to peak between E13 and E14, and decreased gradually at later ages, becoming negligible by birth. Neuronal death in the trigeminal ganglia of trkA−/− embryos also peaked between E13 and E14, but was almost threefold greater than in wild-type embryos at this stage. Whereas there was no significant difference between the number of pyknotic nuclei in trkA−/− and wild-type embryos at E11 and E12, there was a substantial increase in the number of pyknotic nuclei in the trigeminal ganglia of trkB−/− at these earlier stages. Counts of the total number of neurons in E13 trigeminal ganglia revealed a marked decrease in trkB−/− but not trkA−/− or trkC−/− embryos. Consistent with the later onset of excessive neuronal death in trkA−/− embryos, there was a marked decrease in the neuronal complement of the trigeminal ganglia of trkA−/− embryos at E15. These results demonstrate that TrkB signalling is required for the in vivo survival of many trigeminal neurons during the early stages of target field innervation before they become NGF-dependent.

scholarly journals The Anatomical Distribution of Mechanoreceptors in Mouse Hind Paw Skin and the Influence of Integrin α1β1 on Meissner-Like Corpuscle Density in the Footpads

2021 ◽  
Vol 15 ◽  
Author(s):  
Valerie Wai ◽  
Lauren Roberts ◽  
Jana Michaud ◽  
Leah R. Bent ◽  
Andrea L. Clark
Keyword(s):  
Sensory Feedback ◽  
Receptive Fields ◽  
Glabrous Skin ◽  
Null Mouse ◽  
Merkel Cells ◽  
Wild Type ◽  
Anatomical Distribution ◽  
Footfall Patterns

Afferent neurons and their mechanoreceptors provide critical sensory feedback for gait. The anatomical distribution and density of afferents and mechanoreceptors influence sensory feedback, as does mechanoreceptor function. Electrophysiological studies of hind paw skin reveal the different types of afferent responses and their receptive fields, however, the anatomical distribution of mechanoreceptor endings is unknown. Also, the role of integrin α1β1 in mechanoreceptor function is unclear, though it is expressed by keratinocytes in the stratum basale where it is likely involved in a variety of mechanotransduction pathways and ion channel functionalities. For example, it has been shown that integrin α1β1 is necessary for the function of TRPV4 that is highly expressed by afferent units. The purpose of this study, therefore, was to determine and compare the distribution of mechanoreceptors across the hind paw skin and the footfall patterns of itga1-null and wild type mice. The itga1-null mouse is lacking the integrin α1 subunit, which binds exclusively to the β1 subunit, thus rendering integrin α1β1 nonfunctional while leaving the numerous other pairings of the β1 subunit undisturbed. Intact hind paws were processed, serially sectioned, and stained to visualize mechanoreceptors. Footfall patterns were analyzed as a first step in correlating mechanoreceptor distribution and functionality. Merkel cells and Meissner-like corpuscles were present, however, Ruffini endings and Pacinian corpuscles were not observed. Meissner-like corpuscles were located exclusively in the glabrous skin of the footpads and digit tips, however, Merkel cells were found throughout hairy and glabrous skin. The increased density of Merkel cells and Meissner-like corpuscles in footpads 1 and 3 and Meissner-like corpuscles in footpad 4 suggests their role in anteroposterior balance, while Meissner-like corpuscle concentrations in digits 2 and 5 support their role in mediolateral balance. Finally, a larger density of Meissner-like corpuscles in footpads 3 and 4 in male itga1-null mice compared to wild type controls paves the way for future site-specific single fiber in vivo recordings to provide insight into the role of integrin α1β1 in tactile mechanotransduction.

scholarly journals Polo-Like Kinase 1 Is Essential for Early Embryonic Development and Tumor Suppression

2008 ◽  
Vol 28 (22) ◽  
pp. 6870-6876 ◽  
Author(s):  
Lin-Yu Lu ◽  
Jamie L. Wood ◽  
Katherine Minter-Dykhouse ◽  
Lin Ye ◽  
Thomas L. Saunders ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Cell Cycle Progression ◽  
Physiological Function ◽  
S Phase ◽  
Tumor Formation ◽  
Early Embryonic Development ◽  
Wild Type ◽  
Cell Stage ◽  
Cycle Progression

ABSTRACT Polo-like kinases (Plks) are serine/threonine kinases that are highly conserved in organisms from yeasts to humans. Previous reports have shown that Plk1 is critical for all stages of mitosis and may play a role in DNA replication during S phase. While much work has focused on Plk1, little is known about the physiological function of Plk1 in vivo. To address this question, we generated Plk1 knockout mice. Plk1 homozygous null mice were embryonic lethal, and early Plk1−/− embryos failed to survive after the eight-cell stage. Immunocytochemistry studies revealed that Plk1-null embryos were arrested outside the mitotic phase, suggesting that Plk1 is important for proper cell cycle progression. It has been postulated that Plk1 is a potential oncogene, due to its overexpression in a variety of tumors and tumor cell lines. While the Plk1 heterozygotes were healthy at birth, the incidence of tumors in these animals was threefold greater than that in their wild-type counterparts, demonstrating that the loss of one Plk1 allele accelerates tumor formation. Collectively, our data support that Plk1 is important for early embryonic development and may function as a haploinsufficient tumor suppressor.

scholarly journals Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Szu-Yao Wu ◽  
Ya-Shu Chang ◽  
Tien-Hua Chu ◽  
Chiaho Shih
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Mouse Model ◽  
Core Protein ◽  
Wild Type ◽  
Viral Dna ◽  
Naturally Occurring ◽  
B Virus

ABSTRACT Hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations in natural infection. Wild-type HBV is known to secrete predominantly virions containing mature DNA genome. However, a frequent naturally occurring HBc variant, I97L, changing from an isoleucine to a leucine at amino acid 97, exhibited an immature secretion phenotype in culture, which preferentially secretes virions containing immature genomes. In contrast, mutant P130T, changing from a proline to a threonine at amino acid 130, exhibited a hypermaturation phenotype by accumulating an excessive amount of intracellular fully mature DNA genome. Using a hydrodynamic delivery mouse model, we studied the in vivo behaviors of these two mutants, I97L and P130T. We detected no naked core particles in all hydrodynamically injected mice. Mutant I97L in mice exhibited pleiotropic phenotypes: (i) excessive numbers of serum HBV virions containing immature genomes, (ii) significantly reduced numbers of intracellular relaxed-circle and single-stranded DNAs, and (iii) less persistent intrahepatic and secreted HBV DNAs than wild-type HBV. These pleiotropic phenotypes were observed in both immunocompetent and immunodeficient mice. Although mutant P130T also displayed a hypermaturation phenotype in vivo, it cannot efficiently rescue the immature virion secretion of mutant I97L. Unexpectedly, the single mutant P130T exhibited in vivo a novel phenotype in prolonging the persistence of HBV genome in hepatocytes. Taken together, our studies provide a plausible rationale for HBV to regulate envelopment morphogenesis and virion secretion via genome maturity, which is likely to play an important role in the persistence of viral DNA in this mouse model. IMPORTANCE Chronic infection with human hepatitis B virus (HBV) could lead to cirrhosis and hepatoma. At present, there is no effective treatment to eradicate the virus from patients. HBV in chronic carriers does not exist as a single homogeneous population. The most frequent naturally occurring mutation in HBV core protein occurs at amino acid 97, changing an isoleucine to leucine (I97L). One dogma in the field is that only virions containing a mature genome are preferentially secreted into the medium. Here, we demonstrated that mutant I97L can secrete immature genome in mice. Although viral DNA of mutant I97L with immature genome is less persistent than wild-type HBV in time course experiments, viral DNA of mutant P130T with genome hypermaturation, surprisingly, is more persistent. Therefore, virion secretion regulated by genome maturity could influence viral persistence. It remains an open issue whether virion secretion could be a drug target for HBV therapy.

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