Alexander Pfeifer

Neuronal fate determinants of adult olfactory bulb neurogenesis.

Adult neurogenesis in mammals is restricted to two small regions, including the olfactory bulb, where GABAergic and dopaminergic interneurons are newly generated throughout the entire lifespan. However, the mechanisms directing them towards a specific neuronal phenotype are not yet understood. Here, we demonstrate the dual role of the transcription factor Pax6 in generating neuronal progenitors and also in directing them towards a dopaminergic periglomerular phenotype in adult mice. We present further evidence that dopaminergic periglomerular neurons originate in a distinct niche, the rostral migratory stream, and are fewer derived from precursors in the zone lining the ventricle. This regionalization of the adult precursor cells is further supported by the restricted expression of the transcription factor Olig2, which specifies transit-amplifying precursor fate and opposes the neurogenic role of Pax6. Together, these data explain both extrinsic and intrinsic mechanisms controlling neuronal identity in adult neurogenesis.

Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells in vitro (embryoid bodies) and in vivo (teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.

Defective smooth muscle regulation in cGMP kinase I‐deficient mice

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP‐dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross‐activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP‐dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI‐deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.

Cyld Inhibits Tumor Cell Proliferation by Blocking Bcl-3-Dependent NF-κB Signaling

Mutations in the CYLD gene cause tumors of hair-follicle keratinocytes. The CYLD gene encodes a deubiquitinase that removes lysine 63-linked ubiquitin chains from TRAF2 and inhibits p65/p50 NF-kappaB activation. Here we show that mice lacking Cyld are highly susceptible to chemically induced skin tumors. Cyld-/- tumors and keratinocytes treated with 12-O-tetradecanoylphorbol-13 acetate (TPA) or UV light are hyperproliferative and have elevated cyclin D1 levels. The cyclin D1 elevation is caused not by increased p65/p50 action but rather by increased nuclear activity of Bcl-3-associated NF-kappaB p50 and p52. In Cyld+/+ keratinocytes, TPA or UV light triggers the translocation of Cyld from the cytoplasm to the perinuclear region, where Cyld binds and deubiquitinates Bcl-3, thereby preventing nuclear accumulation of Bcl-3 and p50/Bcl-3- or p52/Bcl-3-dependent proliferation. These data indicate that, depending on the external signals, Cyld can negatively regulate different NF-kappaB pathways; inactivation of TRAF2 controls survival and inflammation, while inhibition of Bcl-3 controls proliferation and tumor growth.

Intestinal Secretory Defects and Dwarfism in Mice Lacking cGMP-Dependent Protein Kinase II

Cyclic guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinases (cGKs) mediate cellular signaling induced by nitric oxide and cGMP. Mice deficient in the type II cGK were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion. The cGKII-deficient mice also developed dwarfism that was caused by a severe defect in endochondral ossification at the growth plates. These results indicate that cGKII plays a central role in diverse physiological processes.

Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.

The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function

The vasodilator‐stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP‐ and cGMP‐dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, we inactivated the VASP gene in mice. Adult VASP‐deficient mice had normal agonist‐induced contraction, and normal cAMP‐ and cGMP‐dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP‐ and cGMP‐mediated inhibition of platelet aggregation was significantly reduced in the absence of VASP. Other cAMP‐ and cGMP‐dependent effects in platelets, such as inhibition of agonist‐induced increases in cytosolic calcium concentrations and granule secretion, were not dependent on the presence of VASP. Our data show that two different cyclic, nucleotide‐dependent mechanisms are operating during platelet activation: a VASP‐independent mechanism for inhibition of calcium mobilization and granule release and a VASP‐dependent mechanism for inhibition of platelet aggregation which may involve regulation of integrin function.

Efficient transgenesis in farm animals by lentiviral vectors.

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV‐PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV‐PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ‐line. Tissue‐specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV‐K14). LV‐K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV‐PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.

Functional Embryonic Cardiomyocytes after Disruption of the L-type α1C (Ca v 1.2) Calcium Channel Gene in the Mouse

The L-type α1C(Cav1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated theCa v 1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Cav1.2−/−) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/−), and homozygous (−/−) Cav1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 μm nisoldipine. In (+/+), (+/−), and (−/−) cardiomyocytes, an L-type Ba2+ inward current (I Ba) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of −80 mV, nisoldipine blocked I Ba of day 12.5 p.c. (+/+) and (+/−) cells with two IC50 values of ≈0.1 and ≈1 μm. Inhibition of I Ba of (−/−) cardiomyocytes was monophasic with an IC50 of ≈1 μm. The low affinity I Ba was also present in cardiomyocytes of homozygous α1D(Cav1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.