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UXT potentiates angiogenesis by attenuating Notch signaling.

Zhou Y, Ge R, Wang R, Liu F, Huang Y, Liu H, Hao Y, Zhou Q, Wang C - Development (2015)

Bottom Line: This prevents RBP-Jκ/CSL from activation and thus inhibits the consequent gene inductions.Furthermore, blockade of Notch signaling rescues the angiogenesis defect caused by UXT knockdown both in vitro and in vivo.Taken together, the data presented in this study characterize UXT as a novel repressor of Notch signaling, shedding new light on the molecular regulation of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

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UXT is evolutionarily conserved and developmentally expressed. (A) A sequence alignment of human, mouse, rat and zebrafish UXT proteins by Clustal X. The histogram below the ruler indicates the degree of similarity. Peaks indicate positions of high similarity and valleys indicate low similarity. The asterisk indicates positions that have been fully conserved. The red rectangles indicate the conserved amino acid sites for UXT-2M mutation. (B) Whole-mount in situ hybridization of UXT in zebrafish embryos from 6 hpf to 72 hpf. The developmental time points are indicated in the panels. (C) Quantification of the UXT mRNA expression in zebrafish embryos from 6 hpf to 72 hpf by using RT-qPCR. Data show the mean±s.e.m. (at least three independent experiments). (D) Antibody staining of UXT in zebrafish embryos from 6 hpf to 72 hpf. (E) Quantification of UXT protein expression in zebrafish embryos with the UXT-specific antibody 4B4, from 0 hpf to 72 hpf, normalized to GAPDH. Densitometry was performed using ImageJ. Data show the mean±s.e.m. (at least three independent experiments). (F) Phenotypic analyses of zebrafish embryos at 24 hpf. Embryos were injected with 4 ng of control morpholino (Ctrl MO), 4 ng of UXT MO2 or 4 ng of UXT MO2 with 150 pg of UXT mRNA (UXT MO2+UXT mRNA). Additional embryos were injected with 4 ng of UXT MO2 plus 150 pg of UXT mRNA mutant (UXT MO2+UXT-2M mRNA). All embryos were analyzed at 24 hpf. The morphological defects are indicated by black arrowheads. (G) The knockdown efficiency of the morpholinos. Embryos were injected with 4 ng of control morpholino, 4 ng of UXT MO1, 4 ng of UXT MO2 or 4 ng of UXT MO3. The embryos were harvested at 24 hpf and the lysates were probed with the anti-zebrafish UXT-specific antibody 4B4. (H) Graphical representation of zebrafish phenotypic analyses; the number of embryos analyzed in each group is indicated above the bars.
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DEV112532F1: UXT is evolutionarily conserved and developmentally expressed. (A) A sequence alignment of human, mouse, rat and zebrafish UXT proteins by Clustal X. The histogram below the ruler indicates the degree of similarity. Peaks indicate positions of high similarity and valleys indicate low similarity. The asterisk indicates positions that have been fully conserved. The red rectangles indicate the conserved amino acid sites for UXT-2M mutation. (B) Whole-mount in situ hybridization of UXT in zebrafish embryos from 6 hpf to 72 hpf. The developmental time points are indicated in the panels. (C) Quantification of the UXT mRNA expression in zebrafish embryos from 6 hpf to 72 hpf by using RT-qPCR. Data show the mean±s.e.m. (at least three independent experiments). (D) Antibody staining of UXT in zebrafish embryos from 6 hpf to 72 hpf. (E) Quantification of UXT protein expression in zebrafish embryos with the UXT-specific antibody 4B4, from 0 hpf to 72 hpf, normalized to GAPDH. Densitometry was performed using ImageJ. Data show the mean±s.e.m. (at least three independent experiments). (F) Phenotypic analyses of zebrafish embryos at 24 hpf. Embryos were injected with 4 ng of control morpholino (Ctrl MO), 4 ng of UXT MO2 or 4 ng of UXT MO2 with 150 pg of UXT mRNA (UXT MO2+UXT mRNA). Additional embryos were injected with 4 ng of UXT MO2 plus 150 pg of UXT mRNA mutant (UXT MO2+UXT-2M mRNA). All embryos were analyzed at 24 hpf. The morphological defects are indicated by black arrowheads. (G) The knockdown efficiency of the morpholinos. Embryos were injected with 4 ng of control morpholino, 4 ng of UXT MO1, 4 ng of UXT MO2 or 4 ng of UXT MO3. The embryos were harvested at 24 hpf and the lysates were probed with the anti-zebrafish UXT-specific antibody 4B4. (H) Graphical representation of zebrafish phenotypic analyses; the number of embryos analyzed in each group is indicated above the bars.

Mentions: Our bioinformatics analysis reveals that UXT is highly conserved across vertebrate species (Fig. 1A). The zebrafish UXT displays 50.6% sequence homology and 87.8% sequence similarities to human sequences (Fig. 1A). Using the whole-mount in situ hybridization (WISH) technique, we observed a ubiquitous expression of uxt mRNA as early as 6 hours post fertilization (hpf) in zebrafish embryos, and later on we saw a relative enrichment in the head region and parts of the trunk (Fig. 1B). The real-time quantitative PCR (RT-qPCR) results further substantiated the expression of uxt mRNA during zebrafish embryo development (Fig. 1C). A mouse monoclonal antibody against the zebrafish UXT protein (4B4) was generated and its specificity was confirmed (ABmart, Shanghai, China). Consistently, the protein expression of UXT mirrors that of the mRNA (Fig. 1D), but with a relatively stable expression level (Fig. 1E; supplementary material Fig. S3). In addition, we screened out three anti-sense morpholino oligonucleotides (MO1, MO2 and MO3, listed in supplementary material Table S1) that effectively knocked down UXT expression (Fig. 1G). A scramble morpholino sequence was included as a negative control (Ctrl MO). We observed that MO2 was the most efficient, and therefore it was used in the following experiments (Fig. 1G; supplementary material Figs S1 and S2). MO1 and MO3 were also used in some experiments (supplementary material Figs S4, S5, S7 and S10).Fig. 1.


UXT potentiates angiogenesis by attenuating Notch signaling.

Zhou Y, Ge R, Wang R, Liu F, Huang Y, Liu H, Hao Y, Zhou Q, Wang C - Development (2015)

UXT is evolutionarily conserved and developmentally expressed. (A) A sequence alignment of human, mouse, rat and zebrafish UXT proteins by Clustal X. The histogram below the ruler indicates the degree of similarity. Peaks indicate positions of high similarity and valleys indicate low similarity. The asterisk indicates positions that have been fully conserved. The red rectangles indicate the conserved amino acid sites for UXT-2M mutation. (B) Whole-mount in situ hybridization of UXT in zebrafish embryos from 6 hpf to 72 hpf. The developmental time points are indicated in the panels. (C) Quantification of the UXT mRNA expression in zebrafish embryos from 6 hpf to 72 hpf by using RT-qPCR. Data show the mean±s.e.m. (at least three independent experiments). (D) Antibody staining of UXT in zebrafish embryos from 6 hpf to 72 hpf. (E) Quantification of UXT protein expression in zebrafish embryos with the UXT-specific antibody 4B4, from 0 hpf to 72 hpf, normalized to GAPDH. Densitometry was performed using ImageJ. Data show the mean±s.e.m. (at least three independent experiments). (F) Phenotypic analyses of zebrafish embryos at 24 hpf. Embryos were injected with 4 ng of control morpholino (Ctrl MO), 4 ng of UXT MO2 or 4 ng of UXT MO2 with 150 pg of UXT mRNA (UXT MO2+UXT mRNA). Additional embryos were injected with 4 ng of UXT MO2 plus 150 pg of UXT mRNA mutant (UXT MO2+UXT-2M mRNA). All embryos were analyzed at 24 hpf. The morphological defects are indicated by black arrowheads. (G) The knockdown efficiency of the morpholinos. Embryos were injected with 4 ng of control morpholino, 4 ng of UXT MO1, 4 ng of UXT MO2 or 4 ng of UXT MO3. The embryos were harvested at 24 hpf and the lysates were probed with the anti-zebrafish UXT-specific antibody 4B4. (H) Graphical representation of zebrafish phenotypic analyses; the number of embryos analyzed in each group is indicated above the bars.
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DEV112532F1: UXT is evolutionarily conserved and developmentally expressed. (A) A sequence alignment of human, mouse, rat and zebrafish UXT proteins by Clustal X. The histogram below the ruler indicates the degree of similarity. Peaks indicate positions of high similarity and valleys indicate low similarity. The asterisk indicates positions that have been fully conserved. The red rectangles indicate the conserved amino acid sites for UXT-2M mutation. (B) Whole-mount in situ hybridization of UXT in zebrafish embryos from 6 hpf to 72 hpf. The developmental time points are indicated in the panels. (C) Quantification of the UXT mRNA expression in zebrafish embryos from 6 hpf to 72 hpf by using RT-qPCR. Data show the mean±s.e.m. (at least three independent experiments). (D) Antibody staining of UXT in zebrafish embryos from 6 hpf to 72 hpf. (E) Quantification of UXT protein expression in zebrafish embryos with the UXT-specific antibody 4B4, from 0 hpf to 72 hpf, normalized to GAPDH. Densitometry was performed using ImageJ. Data show the mean±s.e.m. (at least three independent experiments). (F) Phenotypic analyses of zebrafish embryos at 24 hpf. Embryos were injected with 4 ng of control morpholino (Ctrl MO), 4 ng of UXT MO2 or 4 ng of UXT MO2 with 150 pg of UXT mRNA (UXT MO2+UXT mRNA). Additional embryos were injected with 4 ng of UXT MO2 plus 150 pg of UXT mRNA mutant (UXT MO2+UXT-2M mRNA). All embryos were analyzed at 24 hpf. The morphological defects are indicated by black arrowheads. (G) The knockdown efficiency of the morpholinos. Embryos were injected with 4 ng of control morpholino, 4 ng of UXT MO1, 4 ng of UXT MO2 or 4 ng of UXT MO3. The embryos were harvested at 24 hpf and the lysates were probed with the anti-zebrafish UXT-specific antibody 4B4. (H) Graphical representation of zebrafish phenotypic analyses; the number of embryos analyzed in each group is indicated above the bars.
Mentions: Our bioinformatics analysis reveals that UXT is highly conserved across vertebrate species (Fig. 1A). The zebrafish UXT displays 50.6% sequence homology and 87.8% sequence similarities to human sequences (Fig. 1A). Using the whole-mount in situ hybridization (WISH) technique, we observed a ubiquitous expression of uxt mRNA as early as 6 hours post fertilization (hpf) in zebrafish embryos, and later on we saw a relative enrichment in the head region and parts of the trunk (Fig. 1B). The real-time quantitative PCR (RT-qPCR) results further substantiated the expression of uxt mRNA during zebrafish embryo development (Fig. 1C). A mouse monoclonal antibody against the zebrafish UXT protein (4B4) was generated and its specificity was confirmed (ABmart, Shanghai, China). Consistently, the protein expression of UXT mirrors that of the mRNA (Fig. 1D), but with a relatively stable expression level (Fig. 1E; supplementary material Fig. S3). In addition, we screened out three anti-sense morpholino oligonucleotides (MO1, MO2 and MO3, listed in supplementary material Table S1) that effectively knocked down UXT expression (Fig. 1G). A scramble morpholino sequence was included as a negative control (Ctrl MO). We observed that MO2 was the most efficient, and therefore it was used in the following experiments (Fig. 1G; supplementary material Figs S1 and S2). MO1 and MO3 were also used in some experiments (supplementary material Figs S4, S5, S7 and S10).Fig. 1.

Bottom Line: This prevents RBP-Jκ/CSL from activation and thus inhibits the consequent gene inductions.Furthermore, blockade of Notch signaling rescues the angiogenesis defect caused by UXT knockdown both in vitro and in vivo.Taken together, the data presented in this study characterize UXT as a novel repressor of Notch signaling, shedding new light on the molecular regulation of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

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