Cooperative non-cell and cell autonomous regulation of Nodal gene expression and signaling by Lefty/Antivin and Brachyury in Xenopus

AbstractDynamic spatiotemporal expression of the nodal gene and its orthologs is involved in the dose-dependent induction and patterning of mesendoderm during early vertebrate embryogenesis. We report loss-of-function studies that define a high degree of synergistic negative regulation on the Xenopus nodal-related genes (Xnrs) by extracellular Xenopus antivin/lefty (Xatv/Xlefty)-mediated functional antagonism and Brachyury-mediated transcriptional suppression. A strong knockdown of Xlefty/Xatv function was achieved by mixing translation- and splicing-blocking morpholino oligonucleotides that target both the A and B alloalleles of Xatv. Secreted and cell-autonomous inhibitors of Xnr signaling were used to provide evidence that Xnr-mediated induction was inherently long-range in this situation in the large amphibian embryo, essentially being capable of spreading over the entire animal hemisphere. There was a greater expansion of the Organizer and mesendoderm tissues associated with dorsal specification than noted in previous Xatv knockdown experiments in Xenopus, with consequent exogastrulation and long-term maintenance of expanded axial tissues. Xatv deficiency caused a modest animal-ward expansion of the marginal zone expression territory of the Xnr1 and Xnr2 genes. In contrast, introducing inhibitory Xbra-EnR fusion constructs into Xatv-deficient embryos caused a much larger increase in the level and spatial extent of Xnr expression. However, in both cases (Xatv/Xlefty-deficiency alone, or combined with Xbra interference), Xnr2 expression was constrained to the superficial cell layer, suggesting a fundamental tissue-specific competence in the ability to express Xnrs, an observation with direct implications regarding the induction of endodermal vs. mesodermal fates. Our experiments reveal a two-level suppressive mechanism for restricting the level, range, and duration of Xnr signaling via extracellular inhibition by Xatv/Xlefty coupled with potent indirect transcriptional repression by Xbra

Chemokine Signaling Directs Trunk Lymphatic Network Formation along the Preexisting Blood Vasculature

SummaryThe lymphatic system is crucial for fluid homeostasis, immune responses, and numerous pathological processes. However, the molecular mechanisms responsible for establishing the anatomical form of the lymphatic vascular network remain largely unknown. Here, we show that chemokine signaling provides critical guidance cues directing early trunk lymphatic network assembly and patterning. The chemokine receptors Cxcr4a and Cxcr4b are expressed in lymphatic endothelium, whereas chemokine ligands Cxcl12a and Cxcl12b are expressed in adjacent tissues along which the developing lymphatics align. Loss- and gain-of-function studies in zebrafish demonstrate that chemokine signaling orchestrates the stepwise assembly of the trunk lymphatic network. In addition to providing evidence for a lymphatic vascular guidance mechanism, these results also suggest a molecular basis for the anatomical coalignment of lymphatic and blood vessels

Effect of Exercise on Hepatic Gene Expression in an Obese Mouse Model Using cDNA Microarrays

To understand the molecular mechanisms involved in the effect of exercise training, we examined hepatic transcriptional profiles using cDNA microarrays in exercise-trained and untrained mice with diet-induced obesity. C57BL/6J male mice (n = 10/group) were fed with a normal diet, high-fat diet (HFD), or HFD with exercise training for 12 weeks. The expression level of 10,000 transcripts in liver tissues from each group was assessed using cDNA microarray analysis. Exercise training improved lipid profiles and hepatic steatosis and decreased body fat mass induced by the HFD. Seventy-three genes were differentially expressed in the HFD- and/or HFD with exercise training-treated groups, compared with the normal diet- and HFD-fed groups, respectively. Interestingly, the expression profiles involved in metabolism, such as elongation of very long chain fatty acids-like 2, lipin, and malic enzyme, were changed by exercise training. In addition, expression of genes altered by exercise training related to defense and stress response, including metallothionein 1 and 2 and heat shock protein, showed interesting findings. Our study showed beneficial effects of exercise training in preventing the development of obesity and metabolic disorders in mice with diet-induced obesity.This study was supported by the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (Grant 02-PJ10-PG4-PT02-0015) and by BK21 Program for Veterinary Science

Screening of <i>ROS1</i> Rearrangements in Lung Adenocarcinoma by Immunohistochemistry and Comparison with <i>ALK</i> Rearrangements

<div><p><i>ROS1</i> rearrangement is a predictive biomarker for response to the tyrosine kinase inhibitor, crizotinib. We investigated the usefulness of ROS1 immunohistochemistry (IHC) for the detection of patients who harbor ROS1 rearrangements in two separate cohorts. We also compared ROS1 IHC with ALK IHC in terms of diagnostic performance to predict each gene rearrangement. In a retrospective cohort, IHC was performed in 219 cases of lung adenocarcinoma with already known genetic alterations. In a prospective cohort, we performed IHC for 111 consecutive cases of lung adenocarcinoma and confirmed the results by subsequent FISH. In the retrospective cohort, all 8 <i>ROS1</i>-rearranged tumors were immunoreactive, and 14 of 211 <i>ROS1</i>-wild cases were immunoreactive (sensitivity 100% and specificity 93.4%). In the prospective cohort, all IHC-negative cases were FISH-negative, and 5 of 34 ROS1 immunoreactive cases were <i>ROS1</i>-rearranged (sensitivity 100% and specificity 72.6%). In <i>ROS1</i>-wild tumors, ROS1 protein was more expressed in the tumors of ever-smokers than in those of never-smokers (p = 0.003). ALK IHC showed 100% sensitivity and 98.1 to 100% specificity in both patient cohorts. In conclusion, ROS1 IHC is highly sensitive, but less specific compared with ALK IHC for detection of the corresponding rearrangement. ROS1 IHC-reactive tumors, especially when the tumor is stained with moderate to strong intensity or a diffuse pattern, are recommended to undergo FISH to confirm the gene rearrangement.</p></div

Proposed diagnostic algorithm using ROS1 and ALK IHC.

<p>When the tumor shows moderate to strong, or diffuse staining on IHC, FISH is recommend to confirm gene rearrangements. *The criteria should be determined based on each institutional method. According to this present study, subsequent FISH analysis is recommended for cases with an H-score ≥100, extent ≥75%, or the presence of intensity 2+ or 3+.</p

Immunohistochemical findings of <i>ROS1</i>-rearranged tumors.

<p>An <i>ROS1</i>-rearrangned tumor showed strong and diffuse cytoplasmic staining (a) (inset: <i>ROS1</i> FISH with split signals). Other tumors showed cytoplasmic staining with membrane accentuation (b), membranous staining with weaker cytoplasmic intensity (c), or cytoplasmic or paranuclear aggregates (d).</p

Comparison of the H-score in ROS1-non-rearranged tumors according to smoking history.

<p>The scatter dot plot shows that the ROS1 protein is more expressed in tumors of smokers' than in those of never-smokers (p = 0.003).</p

Heterogeneity of histology and immunohistochemical staining.

<p>One area of a <i>ROS1</i>-rearranged tumor shows a solid area with intense immunostaining for ROS1 (a and c). Another area shows a predominantly signet ring cell feature with weaker immunostaining (b and d). Both areas were ALK IHC-negative (e and f).</p