Top PDF Kruppel-like factor 2 is required for normal mouse cardiac development.

Kruppel-like factor 2 is required for normal mouse cardiac development.

Kruppel-like factor 2 is required for normal mouse cardiac development.

In this work, we have demonstrated that KLF2 binds the promoters of, and positively regulates, the Tbx5 and Gata4 genes in the mouse E10.5 AV region. An endocardial specific Gata4 KO has multiple layers of endocardium in the AV canal, and hypocellular AV cushions at E10.5 [41], similar to FVB/N KLF2 2/2. Tbx5 KO embryos have hypoplastic endocardial cushions [42], like FVB/N KLF2 2/2. These phenotypes correlate with our observation that Gata4 and Tbx5 expression is reduced 3-fold in the absence of KLF2 in FVB/N AV canals. Interestingly, the Tbx5 and Gata4 proteins physically interact during cardiac development. A heterozygous mutation (mG295S) in the Gata4 gene disrupts these protein interactions, resulting in cardiac defects like atrial septal defects (ASD), AV septal defects (AVSD) and myocardial thinning beginning at E11.5 [35,36]. The AVSD and ASD in these mice are known to result from abnormal EMT and remodeling of endocardial cushions. Gata4 mG295S is Figure 7. Cardiovascular genes are dysregulated in FVB/N KLF22/2 AV region. Quantitative RT-PCR (qRT-PCR) of AV region RNA was used to test the amount of expression of genes important for AV cushion development (Tbx5 and Sox9) and atrial septation (Tbx5 and Gata4). Cyclophilin A mRNA was used as a normalization control. FVB/N WT and mix WT were designated as100%, and KLF22/2 were compared to WT. (A–C) In E10.5 FVB/N KLF22/2 AV region there is significantly decreased expression of (A) Tbx5 (p = 0.0175), (B) Gata4 (p = 0.0164) and (C) Sox9 (p = 0.019) mRNA compared to FVB/N WT, but Mix WT and Mix KLF22/2 hearts have no differences in expression of these genes. Error bars indicate standard deviation. n = 5. D) ChIP assays were performed on cells obtained from E10.5 WT AV regions. Approximately 8 to 10 WT AV regions were pooled to obtain cells for one replicate. Polyclonal anti-KLF2 and non-specific control pre-immune serum was used. The y-axis represents the relative fold-enrichment. The mean pre-immune enrichment was designated as ‘1.0’ and the enrichment with KLF2 antisera was scaled appropriately. The x-axis indicates the location of the primers used for qPCR; all were in gene promoters and are described in Table S2. Pr: Promoter. Primers specific for the b-actin gene were used as a negative control. n = 7 biological replicates.
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A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development.

A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development.

A challenging step in further understanding the MAPK pathway is the identification of downstream genes that are regulated by MAPK. Here, the analysis of 3 9 tag digital gene expression profiling using a PsSAK1 mutant obtained by PEG- mediated protoplast stable transformation led to the identification of a downstream gene of the MAPK PsSAK1 pathway, PsMYB1. PsMYB1 is transcriptionally suppressed after loss of PsSAK1, and silencing of PsMYB1 does not affect the transcriptional level of PsSAK1, which suggests partially that PsMYB1 is positively regulated by PsSAK1 (Figure 1C and Figure 2C). These data suggest that activation of the MAPK pathway leads directly or indirectly to increase production of PsMYB1 that in turn leads to zoosporogenesis and development of normal zoospores. Silencing of PsMYB1 results in undifferentiated sporangia that cannot form and release zoospores. Compared with PsSAK1-silenced lines which release less-active zoospores and contain obese protoplasmic balls, PsMYB1-silenced transformants display stronger malfunc- tions. However, loss of PsMYB1 has a weaker effect on virulence compared with the PsSAK1-silenced line. Thus, it is possible that the PsSAK1 MAPK pathway also regulates other genes which are important pathogenic factors in P. sojae. It is also possible that there is crosstalk between two pathways. Like in Cryptococcus neoformans, inhibition of calcineurin induces the phosphorylation of Mpk1 and Figure 5. Analysis of PsMYB1 gene expression during sporangial direct germination. The RNAs of sporulating hyphae were prepared by repeatedly washing 2-day-old hyphae incubated in 10% V8 broth with sterile distilled water and incubating the washed hyphae in the light at 25uC for 4–8 h until sporangia developed on most of the hyphae. "M+", washing the P6497 hyphae with sterile distilled water containing 0.8 M mannitol and induce the sporangia of P6497 to germinate directly. "CK", washing mycelia of the wild-type with sterile distilled water and induce the sporangia of P6497 to germinate indirectly. Evaluating expression of PsMYB1 in P6497 sporangia of germinated indirectly and that of germinated directly. Expression was measured by quantitative real time PCR and normalized to the level in the sporangia of germinated indirectly of wild-type P6497. Bars indicate standard errors from three independent replicates.
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Amyloid precursor protein is required for normal function of the rod and cone pathways in the mouse retina.

Amyloid precursor protein is required for normal function of the rod and cone pathways in the mouse retina.

Although APP may play a role in the function of these inner retinal neurons, it is not critical for their ongoing survival. Immunohistochemistry for specific retinal cell markers showed that the cell classes that usually express APP were present in both WT and APP-KO retinal tissue and that the morphology of these cells was qualitatively similar between the two genotypes. This suggests that although during early postnatal development there are changes in amacrine cell numbers in APP-KO compared to WT retina, by adulthood, with the exception of the AII amacrine cells, this difference is no longer observed [13]. This may, in part, be explained by the potential for other peptides, such as amyloid beta precursor-like proteins 1 and 2 (APLP1 and APLP2) to compensate for the role of APP during development and in the adult APP-KO mouse [39]. However, studies in the brain of APP- KO mice indicate APP is involved in neurite outgrowth [4] and modulation of synaptic activity [5,6,7,11], as well as being important in synaptic adhesion [12]. If APP plays a similar role in the retina then our findings indicate that APP may be important for synaptic fidelity between inner retinal neurons.
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Smyd3 is required for the development of cardiac and skeletal muscle in zebrafish.

Smyd3 is required for the development of cardiac and skeletal muscle in zebrafish.

Expression of cardiac markers in Smyd3 morphants To further disclose the mechanism(s) of heart defect in Smyd3 morphants, we studied the expression of seven markers; four anterior lateral plate mesoderm (ALPM) markers including GATA-binding protein 5 (gata5), stem cell leukemia protein (scl), NK2 transcription factor related 5 (nkx2.5), and heart and neural crest derivatives expressed transcript2 (hand2), and three cardiac chamber markers including ventricular myosin heavy chain (vmhc), atrial myosin heavy chain (amhc) and cardiac myosin light chain2 (cmlc2). The gata5, scl, nkx2.5 and hand2 are markers specific to ALPM, rostral ALPM, caudal ALPM and medial ALPM, respectively [10]. The three markers, vmhc, amhc and cmlc2 are specific to ventricle, atrium, and both chambers, respectively, and Figure 2. Effect of Smyd3 knockdown in zebrafish embryos by Smyd3-MO or Smyd3-SB-MO. (A, B, C, and D) Suppression of smyd3 was examined at 10 hpf in embryos injected with smyd3-EGFP mRNA alone (A), smyd3-EGFP mRNA and Smyd3-mis-MO (B), and smyd3-EGFP mRNA and Smyd3-MO (C). Signals of EGFP were examined in the fluorescent macroscope (lower panel). The frequencies of EGFP-positive embryos were (A) 78.7%62.3, (B) 83.7%61.5, and (C) 17.7%63.4. Data are shown as means6SEM. (D) Effect of Smyd3-SB-MO on smyd3 transcripts. The wild-type transcripts were detected as a band at 465 bp and the aberrant transcripts at 401 bp. The lower panel shows the expression of ef1a as a control. (E and F) Phenotype of embryos injected with Smyd3-MO (E) or Smyd3-mis-MO (F) at 72 hpf. The pericardial edema (arrow head) and curved trunk (arrow) were observed in Smyd3 morphants (E). (G, H, I, and J) Morphological classification of heart defect. The degree of cardiac defect in the morphants was classified into three grades at 48 hpf. Grade1: Heart shows abnormality with mild looping defect and pericardial edema (H); Grade2: Heart shows abnormality with moderate looping and defect and pericardial edema (I); Grade3: Heart shows abnormality with string-like heart with severe pericardial edema (J). Normal: Normal heart (G). Embryos are shown in lateral view.
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Cerebral Cortex Expression of Gli3 Is Required for Normal Development of the Lateral Olfactory Tract.

Cerebral Cortex Expression of Gli3 Is Required for Normal Development of the Lateral Olfactory Tract.

Correct formation of the lateral olfactory tract and specific innervation of the piriform cor- tex are prerequisites for the transmission of olfactory information [1,4,5]. Both of these pro- cesses are thought to depend on intrinsic properties of olfactory projection neurons that regulate axon outgrowth as well as environmental cues that control LOT axon navigation to the different structures of the olfactory cortex. This external control requires a number of axon guidance molecules, including EphrinA5 [6], Netrin1 [7] and Sema3B and F [8,9,10], as well as guidance by lot cells that are positioned on the telencephalic surface along the path followed by LOT axons [11]. In addition, LOT formation is controlled by several transcription factors [12,13,14], including the zinc finger transcription factor Gli3 which is expressed in progenitor cells of the dorsal and ventral telencephalon and in olfactory bulb progenitors but not in neu- rons of the piriform cortex or in mitral cells [15] (S1 Fig). Gli3 null (Gli3 Xt/Xt ) and Gli3 hypo- morphic (Gli3 Pdn/Pdn ) mouse mutants both show severe defects in the formation of the olfactory system [15,16,17]. Both mutants show no discernible olfactory bulb protrusion but form an olfactory bulb like (OB-like) structure containing mitral cells and OB interneurons in ectopic dorsal or lateral positions in the telencephalon [18]. In addition, Gli3 Pdn/Pdn mutants show apoptosis of precursor mitral cells in the OB-like structure [17] with residual surviving mitral cells creating a slender LOT [16,17]. Moreover, Gli3 Xt/Xt mutants show severe telence- phalic patterning defects resulting in the clustering of lot guidepost cells [19] and an expansion of the paleocortex [20]. Based upon these phenotypes, Gli3 could affect LOT development by controlling intrinsic OB development, the formation of environmental cues guiding LOT axons and/or the development of the LOT target area but the severity of the defects complicate the analysis of Gli3’s roles in LOT formation. To circumvent these difficulties, we made use of Emx1Cre;Gli3 fl/fl (Gli3 cKO ) conditional mutants [21] which we have previously shown to have an expanded piriform cortex [22]. These mutants formed an OB-like structure that did not protrude from the telencephalic surface but contained mitral cells and olfactory interneurons. Mitral cell axons formed a LOT which occupied a medially shifted position. LOT axons inner- vated an extended area of the piriform cortex and their collaterals penetrated deeper layers. No obvious defects were found in the expression of telencephalic guidance cues or in the formation of lot cells consistent with the formation of the LOT. However, time course analysis confirmed that the paleocortical primordium expanded from E13.5 onwards, coinciding with the arrival of the LOT axons. These findings suggest an important role for Gli3 in correctly positioning the LOT and controlling its innervation of the piriform cortex.
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Fibroblast growth factor 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis.

Fibroblast growth factor 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis.

We are not the first to observe a SPEM-like phenotype in association with FGF10 signaling. Spencer-Dene et al. demon- strated a disproportionately underdeveloped antrum with an enlarged simple unbranched gastric epithelium for both Fgf10 2/2 and Fgfr2b 2/2 embryos indicating a role of FGF10-FGFR2b mediated signaling in promoting antralization [13]. Furthermore, overexpression of Fgf10 during stomach development resulted in a SPEM-like phenotype with a shift in localization of TFF2 mRNA in the mouse and cSP mRNA in the chick, in addition to a reduction in the number of parietal cells in the mouse [11][23]. cSP is a marker of luminal epithelial cells in the chick, the analog to mucous neck cells in the mouse [8]. Nyeng et al. speculated that the antralization of the corpus in their model could be explained by increased FGF10 availability in the corpus as compared to the normal gradient of Fgf10 expression, which is higher in the antrum and lower in the corpus [11]. This could explain the SPEM-like phenotype seen during homeostasis as well, since Fgf10 was ubiquitously overexpressed in our model. Despite Fgf10 overex- pression resulting in a SPEM-like phenotype, well-established markers failed to confirm metaplasia during homeostasis, as has been similarly reported during stomach development [11]. Hence, we cannot exclude the possibility that FGF10-FGFR2b signaling may play a role in gastric metaplasia. In fact, ETV4, a downstream target of FGF10, is upregulated in human gastro- adenocarcinoma samples and is associated with decreased survival [46,47]. Since SPEM often results from parietal cell loss, and the average parietal cell lifespan is approximately 54 days [37], only 10 days of Fgf10 overexpression may not induce sufficient parietal cell loss and consequent metaplasia. Perhaps a higher Fgf10 expression level and/or a longer exposure time are required for
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Hypertension is a conditional factor for the development of cardiac hypertrophy in type 2 diabetic mice.

Hypertension is a conditional factor for the development of cardiac hypertrophy in type 2 diabetic mice.

Immediately after the dobutamine-stress test, the anesthetized mice were sacrificed by exsanguination via the abdominal aorta. Heart, lungs, liver, and kidneys were harvested, weighed and processed for further analysis. Tibia length was measured with a marking gauge. The isolated heart was divided into atria, right ventricle (RV) and LV. Part of LV was fixed in buffered 4% formaldehyde for 24 h and imbedded in paraffin for histological evaluation of cardiomyocyte cross-sectional surface area and interstitial fibrosis. For this, tissue sections of 5 m m were fixed at 56 uC overnight, deparaffinized, rehydrated and stained with hematoxylin and eosin (H&E) to determine cardiomyocyte cross- sectional surface area. To visualize interstitial fibrosis, the sections were stained with Picro-Sirius Red. The percentage of the LV wall consisting of interstitial collagen was calculated as the ratio of Picro-Sirius-Red positively stained area over total LV tissue area, excluding blood vessels. [7]. For CD45 staining slides were blocked in buffer containing normal Rabbit serum (Dako, Glostrup, Denmark), incubated with Anti-Mouse CD45 (BD Pharmingen, Franklin Lakes, NJ, USA) and then with biotin- conjugated secondary Rabbit anti-Rat antibody (Dako). Signal was amplified using TSA kit (PerkinElmer, Waltham, MA, USA) and visualized using DAB (Sigma, St. Louis, MO, USA).
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The activity of the Nodal antagonist Cerl-2 in the mouse node is required for correct L/R body axis

The activity of the Nodal antagonist Cerl-2 in the mouse node is required for correct L/R body axis

a Nodal probe revealed that although 40% of the em- bryos showed normal expression, 50% displayed bilat- eral expression, and 10% had an inverted pattern of ex- pression in the R-LPM (Fig. 3B–D). Interestingly, Nodal expression in the node remains unaffected in these three situations, always being stronger in the left side (red ar- rows in Fig. 3A–D). This evidence further supports pre- viously described work in which Nodal asymmetric ex- pression in the node is reported not to be necessary or linked to its later expression domain in the L-LPM (Bren- nan et al. 2002; Saijoh et al. 2003). At this same devel- opmental stage, Lefty1 is expressed along the ventral midline in the prospective floor plate of the wild-type embryo (Fig. 3E), whereas Lefty2 expression can be de- tected in the L-LPM (Fig. 3E; Meno et al. 1997). Both
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Jun is required in Isl1-expressing progenitor cells for cardiovascular development.

Jun is required in Isl1-expressing progenitor cells for cardiovascular development.

Several cell populations are important for normal OFT development and septation. The 100% incidence of PTA in Jun null embryos indicates that Jun is clearly required in one or more of these cell populations. An overview of Jun’s spatial and temporal expression pattern during embryonic development in the mouse is lacking in the literature, particularly prior to E14.5. In limited expression analyses by in situ hybridization and Northern blot, it has been reported that Jun mRNA is expressed in the developing heart, cartilage, gut, central nervous system, lung, kidney, adrenal gland and placenta of the developing mouse [16,17,18,19,20]. To determine the specific cell populations in which Jun might be functioning to regulate cardiac morphogenesis, we examined the expression of Jun by in situ hybridization and immunohistochem- istry at several stages of embryonic development between E8.5 and E15.5. Our Jun expression analysis revealed expression in multiple tissues important for heart development and aortic arch artery remodeling. At E8.5, Jun was expressed in the pharyngeal endoderm, dorsal aortae, common atrial chamber, endocardial cushions and in regions populated by SHF mesoderm (Fig. 1A). The anterior SHF expression was stronger than the posterior SHF (Fig. 1A). The expression of Jun in the SHF was also evident at E9.5 by whole mount in situ hybridization (Fig. 1B, C). This is consistent with our previous observation of Jun expression in SHF- derived OFT myocardium [21]. At E9.5, Jun was expressed in the otic vesicle, telencephalon, somites, and aortic arch arteries (Fig. 1B, C). The expression in the telencephalon, somites and pharyngeal arches is consistent with publically available in situ hybridization data at E11 (http://goo.gl/DoJro) [22]. At E10.5, Jun was highly expressed in the OFT endocardial cushions, AV endocardial cushions and cranial nerve IX (Fig. 1D). The high levels of Jun expression in the OFT endocardial cushions persists until E11.5 (Fig. 1E), where expression in pericardium (Fig. 1E) and dorsal root ganglia (data not shown) was also evident. At E15.5, Jun was broadly expressed in the myocardium and both the semilunar and AV valves (Fig. S1).
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Chemokine receptor CCR8 is required for lipopolysaccharide-triggered cytokine production in mouse peritoneal macrophages.

Chemokine receptor CCR8 is required for lipopolysaccharide-triggered cytokine production in mouse peritoneal macrophages.

reports of CCR8-positive T cells, CCR8-expressing macrophages also play significant roles in several pathological situations. For example, CCL1 and CCR8 mediate postoperative peritoneal adhesion development in mice [14], CCL1 is produced by mesothelial cells and macrophages in the peritoneal cavity and is a potent enhancer of CCR8 expression in peritoneal macrophages (PMQ) [14], and PMQ produce CCL1 upon inflammatory stimulation. The CCL1/CCR8 pathway activates itself through a positive autocrine/paracrine loop in the peritoneal cavity. In vitro stimulation of the PMQ with CCL1 on mesothelial cell layer leads to macrophage aggregation. In mice, such CCR8-positive macrophage aggregates are seen in vivo at the serosal sites of peritoneal adhesions induced by acute colitis or surgical manip- ulation of the peritoneal cavity. Adhesions are efficiently prevented by anti-CCL1 antibody or by CCR8 gene deficiency in mouse models [14]. Although CCL1 is not the primary chemokine secreted into the peritoneal cavity during laparotomy in humans [15], inflammatory macrophages in lung tissue from patients with chronic obstructive pulmonary disease (COPD) express high levels of CCR8. In COPD, potential interaction with Toll-like receptor (TLR)-4 was suggested because CCL1 induces superoxide and proinflammatory cytokine release from macrophages in the presence of lipopolysaccharide (LPS) [16]. A type 1 diabetes model demonstrated that CCL1 produced by diabetogenic CD4 +
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DNA methyltransferase 3b is dispensable for mouse neural crest development.

DNA methyltransferase 3b is dispensable for mouse neural crest development.

The minimal requirement for DNMT3b in neural crest cells is surprising given that animals fully mutant for DNMT3b have defects in neural crest derivatives [12,16]. While DNMT3a compensates for DNMT3b loss of function [12], and the DNMT3a/3b neural crest double mutant would likely be more severe, we were specifically interested in determining whether the known phenotypes of DNMT3b mutants reflected an essential role for DNMT3b in neural crest cells. Rather, our data suggest that DNMT3b must be required in cell types with which neural crest cells interact during their differentiation, such as the cardiac mesoderm or the branchial arch mesendoderm. It is also conceivable that DNMT3b is required to methylate particular targets during neural crest specification prior to the onset of Wnt1- driven cre expression [38], and the consequence of this methylation is not manifest until neural crest cells differentiate during organogenesis. Unfortunately we cannot formally evaluate this possibility as we are not aware of any neural crest cre lines expressed earlier than Wnt1-cre, and DNMT3b 2/2 embryos die after E13.5 ([16] and our unpublished observations). However, we find this interpretation unlikely for two reasons. First, DNA methylation is normally a late, irreversible event in gene silencing (see below and [1]). Second, neural crest migration was similarly affected in fully mutant DNMT3b 2/2 embryos and neural crest mutant Wnt1-cre; DNMT3b fl/ 2 embryos (Fig. 4). This suggests that DNMT3b is not essential prior to Wnt1-cre mediated recombi- nation. Thus, we favor the interpretation that DNMT3b is required in cell types that interact with neural crest cells during craniofacial and cardiac morphogenesis, and this leads to defects in neural crest derivatives in DNMT3b mutants [12,16].
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Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development.

Semaphorin-1a is required for Aedes aegypti embryonic nerve cord development.

The results of this investigation suggest that Sema1a plays a critical role during development of both the A. aegypti and D. melanogaster ventral nerve cords. Many similar defects, including thinning or loss of the commissural axons and breaks or fusion of the longitudinals, were observed when Sema1a function was Figure 3. A. aegypti sema1a knockdown embryonic CNS phenotypes. Anti-acetylated tubulin staining marks the axons of the ventral nerve cords at 56 hrs. of development post-injection of siRNA-control injected (A) and sema siRNA-injected (B–F) embryos. Control-injected embryos had a wild-type appearance (A; longitudinals are marked by black arrows, while the third (outermost) fascicle of the left longitudinal connective is marked by a white arrow; the anterior commissure is marked by a black arrowhead, and the posterior commissure is marked by a white arrowhead). sema siRNA-injected embryos (B–F) were injected with different siRNAs/combinations of siRNAs targeting sema1a. These included: siRNA 890 (B), siRNA 1198
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Ferritin Is Required in Multiple Tissues during Drosophila melanogaster Development.

Ferritin Is Required in Multiple Tissues during Drosophila melanogaster Development.

Following dechorionation with a commercial bleach solution, embryos from overnight collec- tions were devitellinized and fixed in a 1:1 mixture of heptane and 36% formaldehyde for 5 minutes and then washed in methanol. Embryos were then stored at -20 C or rehydrated, and used for staining. Primary antibodies used were: rat α-Elav 1:100, mouse α-BP102 1:100, mouse α-Eve 1:100 (Developmental Studies Hybridoma Bank, Indiana, USA); α-activated Cas- pase 3 1:100 (Cell Signaling, USA); and rat α-Deadpan 1:2, a gift from Cheng-Yu Lee. Second- ary antibodies used were: Alexa flour 546 α-rat 1:100 (Santa Cruz Biotechnology, USA), Cy5 α- mouse 1:1000, Cy3 α-mouse 1:1000, FITC α-rabbit 1:1000 (Zymax, USA). Signal from α-dead- pan staining was increased with the ABC kit from Vectastain (USA). A 510 Meta and 780 Duo confocal microscopes (Zeis, Germany) were used for fluorescent imaging, and images were processed with Zeiss software and ImageJ. Homozygous mutant embryos were selected by lack of TM3GFP of TM3LacZ. Accumulation of GFP::Fer1HCH in Sec23 mutants was evaluated using the fire LUT of ImageJ.
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A transcript cleavage factor of Mycobacterium tuberculosis important for its survival.

A transcript cleavage factor of Mycobacterium tuberculosis important for its survival.

the C-terminal globular domain of Gre factors (GreA, GreB of E. coli and MtbGre), which interacts with the RNAP, shows considerable variation, although certain specific residues in the hydrophobic patch are conserved in all these proteins. Importance of specific interactions between RNAP and Gre is suggested from the studies in T. aquaticus. GreA of T. aquaticus failed to induce transcript cleavage in EcRNAP elongation complexes [15] similar to the present observation with MtbGre. Thus it appears that the transcript cleavage activity requires species-specific interactions, although both partners viz RNAP and Gre have conserved characteristics across species. Gre may have a more important function in mycobacteria to compensate for the low intrinsic cleavage activity of mycobacterial RNAP compared to its E. coli and themophilic counterparts. This deficiency could affect the recovery from arrest of backtracked MtbRNAP in the absence of MtbGre. The similar mechanism has been recently proposed to explain growth inhibition of the yeast strains expressing the cleavage deficient mutant of the eukaryotic Gre homolog, TFIIS [43]. The results presented here and the data emerged till date from a number of studies with Gre factors of diverse group of organisms emphasize the biological importance of these secondary channel binding proteins. The deletion of greA led to hypersen- sitivity phenotype under various stress conditions in E. coli [39], Sinorhizobium meliloti [44] and Rhizobium tropici [45] implicating the importance of Gre factors in the survival of the organism in the restrictive environment. In contrast, the decrease in Gre levels Figure 4. Effect of Mtb Gre factor on promoter clearance and abortive transcription. (A) Promoter clearance assays were carried out in the absence (- N -) or presence (-D-) of 2 mM MtbGre. Transcripts were resolved on an 8% urea-PAGE and 109 nt long run-off transcripts were quantified
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Targeted Inactivation of Cerberus Like-2 Leads to Left Ventricular Cardiac Hyperplasia and Systolic Dysfunction in the Mouse

Targeted Inactivation of Cerberus Like-2 Leads to Left Ventricular Cardiac Hyperplasia and Systolic Dysfunction in the Mouse

E13.25, E15–E15.25 and P0. We analyzed the expression of transcription factors that are important for normal heart development such as Gata-4 and Nkx2.5 [3]. We also analyzed structural genes involved in contractility like a-Mhc and cTnT, which are activated by the transcription factors Gata-4 and Mef2 [48]. In addition, genes such as Anp, Bnp and Ankrd1, which are known to be involved in the hypertrophy program and cardiac stress [49,50] were evaluated. qRT-PCR analysis showed a dramatic reduction of Gata-4 expression and a slight decrease of c-TNT in Cer-2 2/2 hearts at E13 (Fig. 4A). In contrast, we detected an increase in Nkx2.5 expression levels, and at E15 we observed a decreased Nkx2.5 expression level. Since alterations in this gene are associated with conduction abnormalities [51], we speculate that Cerl2 2/2 may have impaired cardiac function already during fetal development. Concurrently, we also detected reduction of the encoding contractile genes a-Mhc and cTnT but not altered Gata-4 in Cerl2 2/2 (Fig. 4B) at this stage. According with other studies, the alterations of transcription factors such as Gata-4, Nkx2-5 and Mef2 and their target genes may compromise the cardiomyocyte differentiation program [48,52]. Therefore, we believed that the cardiac function of the null mutants might already be affected during embryogenesis. However, at E13 and E15 we did not detect any alteration of Anp and Bnp expression in Cerl2 2/2 mutants, which indicates that the blood pressure and blood volume regulating the hypertrophic response in embryonic stages are not de-regulated [49].
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Doxycycline inducible Kruppel-like factor 4 lentiviral vector mediates mesenchymal to epithelial transition in ovarian cancer cells.

Doxycycline inducible Kruppel-like factor 4 lentiviral vector mediates mesenchymal to epithelial transition in ovarian cancer cells.

Ovarian cancer presents therapeutic challenges due to its typically late detection, aggressive metastasis, and therapeutic resistance. The transcription factor Kru¨ppel-like factor 4 (KLF4) has been implicated in human cancers as a tumor suppressor or oncogene, although its role depends greatly on the cellular context. The role of KLF4 in ovarian cancer has not been elucidated in mechanistic detail. In this study, we investigated the role of KLF4 in ovarian cancer cells by transducing the ovarian cancer cell lines SKOV3 and OVCAR3 with a doxycycline-inducible KLF4 lentiviral vector. Overexpression of KLF4 reduced cell proliferation, migration, and invasion. The epithelial cell marker gene E-cadherin was significantly upregulated, whereas the mesenchymal cell marker genes vimentin, twist1and snail2 (slug) were downregulated in both KLF4-expressing SKOV3 and OVCAR3 cells. KLF4 inhibited the transforming growth factor b (TGFb)-induced epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Taken together, our data demonstrate that KLF4 functions as a tumor suppressor gene in ovarian cancer cells by inhibiting TGFb-induced EMT.
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E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

First, we applied qPCR analysis to quantify the expression levels of the E-cadherin transgene and the endogenous genes associated with pluripotency [9,10,11,12]. The results confirmed the in- ducible expression of E-cadherin in two of the transgenic mEpiSC lines EIN3 and EIN6 derived from PTmEpiSCs (Fig. 1A). Interestingly, we found that the expression levels of the endoge- nous E-cadherin in PTmEpiSCs were slightly higher than those of mESCs cultured with or without feeder cells, suggesting that the ability to integrate into the ICM is not simply correlated with the level of E-cadherin transcript. By activation of the E-cadherin transgene, Nanog expression was slightly upregulated in both lines but the other markers, such as Oct3/4, Klf4, Tbx3, and Esrrb, were unaffected. Klf4, Tbx3, and Esrrb, which are known to be specifically expressed in naı¨ve PSCs, showed much higher levels of expression in mESCs than in PTmEpiSCs, as shown in Fig. 1B. It has been reported that these naı¨ve marker genes are upregulated when mEpiSCs are reprogrammed to naı¨ve PSCs by either introduction of transgenes such as Nanog [13,14] or by long-term culture under conditions for naı¨ve PSCs [15]. Therefore, the maintenance of these markers at low levels after induction of the E-cadherin transgene in mEpiSCs indicated that upregulation of E- cadherin did not induce rapid promotion of reprogramming from the primed to the naı¨ve state. Essentially the same results were obtained with SvEIN3.4, SvEIN3.9, and SOmEpiSCs (data not shown).
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Inversin/Nephrocystin-2 is required for fibroblast polarity and directional cell migration.

Inversin/Nephrocystin-2 is required for fibroblast polarity and directional cell migration.

IFM analysis. In inv +/+ MEFs, both GTPases localized diffusely to the cytosol as well as to distinct punctae in cytoplasmic protrusions of migrating cells (Figure 5A,B, left panels). In comparison, inv 2/2 MEFs showed a markedly reduced Rac1 and RhoA localization at cell surfaces facing the wound (Figure 5A,B, right panels). Quantitative image analysis confirmed that localization of both Rho GTPases at the leading edge was significantly reduced in inv 2/2 MEFs (Figure 5C,D). These results show that while Rho GTPase activity was increased in inv 2/2 MEFs, the defective targeting of Rac1 and RhoA in these cells could compromise the organization of the cytoskeleton required for cell migration. Indeed, transcriptome analysis revealed a large extend of deregulated gene expression within pathways regulating focal adhesions, the actin cytoskeleton and adherens junctions in inv 2/2 MEFs (Figure 5E). In this regard, expression of Wasf1 (encoding Wave1) and Arp2/3, both of which are regulated by Rac1 and Cdc42 and essential for actin reorganization during lamellipodia formation [43], were significantly downregulated in inv 2/2 MEFs (Figure 5F). These findings demonstrate that Inversin regulates the activation and localization of Rho GTPases and the expression of a large number of key proteins in cell migration.
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The 3-hydroxy-2-butanone pathway is required for Pectobacterium carotovorum pathogenesis.

The 3-hydroxy-2-butanone pathway is required for Pectobacterium carotovorum pathogenesis.

The stability of a key P. carotovorum signal molecule, acyl- homoserine lactone (acyl-HSL), is also affected by pH and it is hydrolyzed into an inactive form above pH 6.8 [72]. As in many bacteria, acyl-HSL acts as a quorum sensing molecule in Pectobacterium and affects the regulation of approximately one quarter of Pectobacterium genes, including induction of both the budAB operon and plant cell wall degrading enzymes [60]. Since acyl-HSL hydrolyzes in alkaline pH, it should be less available once fermentation has started and the local pH has increased. Whether local concentrations of acyl-HSL are sufficient or other mechanisms, such as induction by plant cell wall fragments become more important in enzyme expression remains unknown. The butanediol pathway has been examined in plant growth promoting bacteria (PGPB), where it is thought to be responsible for the growth promoting effects of some PGPB [22,23]. A wide Figure 5. WPP14D budB is not impaired in growth in potato tubers. Bacterial suspensions containing 10 5 CFU were stab-inoculated into potato tubers (cv. ‘‘Superior’’) (n = 5). Tubers were placed into plastic bags and incubated at 28uC. Bacterial population was determined every 24 hrs after inoculation. Error bars indicate standard error. The graph shows data from one of two experiments, neither of which showed significant difference in bacterial growth.
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The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

This repression is mediated by a 90-nucleotide translational control element (TCE) in the 3 9UTR of nanos mRNA [14,15,16,17,18] to which Smaug (Smg) or Glorund (Glo) bind [19,20,21]. On the other hand, the localization in the germ plasm and subsequent translational activation of nanos mRNA is regulated by the Oskar (Osk) protein via 3 9UTR-dependent mechanisms [19,22,23]. In Danio rerio, maternal nanos1 mRNA is also present in a whole oocyte, but only a portion is localized to the germ plasm and translated specifically in the PGC. The translation of the bulk of nanos1 mRNA in somatic cells is then rapidly degraded during embryogenesis. The regulation of nanos1 both in the PGC and somatic cells depends on three elements within the nanos1-39UTR: (1) a site required for its localization to the germ plasm [24]; (2) two miR430 sites responsible for mRNA degradation in somatic cells; and (3) the binding site for the Dead end 1(Dnd1) protein that is expressed only in the PGC and protects mRNA from miR430-dependent degradation [2,25].
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