During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.
- MeSH
- analýza jednotlivých buněk MeSH
- Caenorhabditis elegans cytologie genetika MeSH
- chromatin metabolismus MeSH
- genom genetika MeSH
- hybridizace in situ fluorescenční metody MeSH
- koncové značení zlomů DNA in situ metody MeSH
- lac operon genetika MeSH
- lac represory genetika MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIMS: The aim of this study was to characterize ventricular activation patterns in normal and connexin40-deficient mice in order to dissect the role of connexin40 in developing the conduction system. METHODS AND RESULTS: We performed optical mapping of epicardial activation between ED9.5-18.5 and analysed ventricular activation patterns and times of left ventricular activation. Mouse embryos deficient for connexin40 were compared with normal and heterozygous littermates. Morphology of the primary interventricular ring (PIR) was delineated with the help of T3-LacZ transgene. Four major types of ventricular activation patterns characterized by primary breakthrough in different parts of the heart were detected during development: PIR, left ventricular apex, right ventricular apex, and dual right and left ventricular apices. Activation through PIR was frequently present at the early stages until ED12.5. From ED14.5, the majority of hearts showed dual left and right apical breakthrough, suggesting functionality of both bundle branches. Connexin40-deficient embryos showed initially a delay in left bundle branch function, but the right bundle branch block, previously described in the adults, was not detected in ED14.5 embryos and appeared only gradually with 80% penetrance at ED18.5. CONCLUSION: The switch of function from the early PIR conduction pathway to the mature apex to base activation is dependent upon upregulation of connexin40 expression in the ventricular trabeculae. The early function of right bundle branch does not depend on connexin40. Quantitative analysis of normal mouse embryonic ventricular conduction patterns will be useful for interpretation of effects of mutations affecting the function of the cardiac conduction system.
- MeSH
- akční potenciály MeSH
- blokáda Tawarova raménka genetika metabolismus MeSH
- gestační stáří MeSH
- Hisův svazek embryologie metabolismus MeSH
- konexiny nedostatek genetika MeSH
- lac operon MeSH
- morfogeneze MeSH
- myši knockoutované MeSH
- myši transgenní MeSH
- myši MeSH
- penetrance MeSH
- převodní systém srdeční embryologie metabolismus MeSH
- srdeční komory embryologie metabolismus MeSH
- vývojová regulace genové exprese MeSH
- zobrazování pomocí barviva citlivého na potenciál MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To elucidate the physiological meaning of OmpR-dependent expression of invasin gene (inv) inhibition in Yersinia enterocolitica, the function of the EnvZ/OmpR regulatory pathway in osmoregulation of inv expression was analyzed in detail. The osmoregulation of inv expression was found to be a multifaceted process involving both OmpR-dependent and -independent mechanisms. Analysis of inv transcription in strains lacking OmpR or EnvZ proteins indicated that kinase EnvZ is not the only regulator of OmpR phosphorylation. Using the transcriptional inv::lacZ fusion in a heterologous system (Escherichia coli) we tried to clarify the role of OmpR in the inv regulatory circuit composed of negative (H-NS) and positive (RovA) regulators of inv gene transcription. We were able to show a significant increase in inv expression in E. coli ompR background under H-NS( Ecoli )-repressed condition. Moreover, H-NS-mediated inv repression was relieved when RovA of Y. enterocolitica was expressed from a plasmid. Furthermore, we showed that RovA may activate inv expression irrespective on the presence of H-NS protein. Using this strategy we showed that OmpR of Y. enterocolitica decrease RovA-mediated inv activation.
- MeSH
- bakteriální adheziny genetika MeSH
- bakteriální chromozomy genetika MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- fúze genů MeSH
- genetická transkripce MeSH
- lac operon genetika MeSH
- osmolární koncentrace MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u bakterií MeSH
- trans-aktivátory genetika metabolismus MeSH
- Yersinia enterocolitica genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this report, we describe the homing of hematopoietic stem cells (HSCs) to non-hematopoietic tissues in lethally irradiated (9Gy) hybrid mice transplanted intravenously with lin(-)/CD117(+) bone marrow cells from ROSA26 mice. The numbers of CFU-GM in spleen of irradiated transplanted mice were well above the levels found in non-irradiated group as early as day 8 after transplant. On 12th day regeneration of lymphocytes was observed, an increase in granulocytes was detected as late as on 33rd day. Transplanted cells containing lacZ gene were detected in recipient mice by histochemistry and their location in the thymus, liver, stomach and ileum was followed during 33days post-transplantation. On 8 and 33days post-transplantation, we found massive presence of donor (lacZ(+)) cells in the thymic cortex. Hematopoietic stem cell transplantation led not only to recovery of hematopoietic and lymphoid tissues but also facilitated recovery of the small intestinal mucosa, which was significantly damaged by ionizing radiation.
- MeSH
- časové faktory MeSH
- celotělové ozáření MeSH
- hematopoetické kmenové buňky fyziologie MeSH
- hematopoetický systém fyziologie MeSH
- lac operon MeSH
- lymfoidní tkáň fyziologie MeSH
- myši MeSH
- pohyb buněk MeSH
- protoonkogenní proteiny c-kit MeSH
- regenerace MeSH
- střevní sliznice fyziologie MeSH
- tenké střevo MeSH
- tkáňová distribuce MeSH
- transplantace hematopoetických kmenových buněk metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
ER81, a member of the ETS family of transcription factors, is involved in processes of specification of neuronal identity, control of sensory-motor connectivity, and differentiation of muscle spindles. Spindles either degenerate or are abnormal in mutant mice lacking ER81. We examined whether ER81 is required for the development of another class of mechanoreceptors, the Pacinian corpuscle. ER81 was expressed by the inner core cells of the corpuscles, as reflected by expression of the lacZ reporter gene in Er81(+/lacZ) mutants, thereby suggesting a role for ER81 in the corpuscle development. No Pacinian corpuscles or their afferent nerve fibers were present in the crus of Er81 null mice at birth. Legs of mutant embryos examined at E16.5 were also devoid of the corpuscles, but not of their afferents. Thus, Pacinian corpuscles do not form, and their afferents do not survive, in the absence of ER81. A deficiency of dorsal root ganglia neurons expressing calretinin, a marker for neurons subserving Pacinian corpuscles, correlated with the absence of corpuscles and their afferents in Er81 null mice. These observations indicate a requirement for ER81 in the assembly of Pacinian corpuscles and the survival of the sensory neurons that innervate them. (c) 2006 Wiley-Liss, Inc.
- MeSH
- biologické markery metabolismus MeSH
- delece genu MeSH
- DNA vazebné proteiny fyziologie genetika nedostatek MeSH
- financování organizované MeSH
- imunohistochemie MeSH
- lac operon MeSH
- myši knockoutované MeSH
- myši MeSH
- neurony aferentní cytologie metabolismus MeSH
- reportérové geny MeSH
- S100 kalcium vázající protein G metabolismus MeSH
- spinální ganglia cytologie MeSH
- transkripční faktory fyziologie genetika nedostatek MeSH
- Vater-Paciniho tělíska růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
