Encapsulating Pyrene in a Metal–Organic Zeolite for Optical Sensing of Molecular Oxygen

Pyrene is a good oxygen-sensitive probe with high fluorescence quantum yield, suitable sensitivity, and high photostability when dispersed in gas-permeable organic polymers, but it is a carcinogen and environmental pollutant and tends to aggregate and/or evaporate at high temperature/low pressure. In this work, we show that pyrene can be easily and firmly encapsulated in a metal–organic zeolite SOD-[Zn­(mim)₂] (Hmim = 2-methylimidazole, MAF-4 or ZIF-8) via an <i>in situ</i> loading strategy, giving fluorescence O₂-sensing materials not only with fast response, high photostability, and tunable sensitivity but also free of pyrene aggregation/leak and interference by other quenchers. Moreover, these host–guest inclusion crystals can be easily fabricated as thin film sensors and aerodynamic coatings

Mutational analysis of <i>dishevelled</i> genes in zebrafish reveals distinct functions in embryonic patterning and gastrulation cell movements

<div><p>Wnt signaling plays critical roles in dorsoventral fate specification and anteroposterior patterning, as well as in morphogenetic cell movements. Dishevelled proteins, or Dvls, mediate the activation of Wnt/ß-catenin and Wnt/planar cell polarity pathways. There are at least three highly conserved Dvl proteins in vertebrates, but the implication of each Dvl in key early developmental processes remains poorly understood. In this study, we use genome-editing approach to generate different combinations of maternal and zygotic <i>dvl</i> mutants in zebrafish, and examine their functions during early development. Maternal transcripts for <i>dvl2</i> and <i>dvl3a</i> are most abundantly expressed, whereas the transcript levels of other <i>dvl</i> genes are negligible. Phenotypic and molecular analyses show that early dorsal fate specification is not affected in maternal and zygotic <i>dvl2</i> and <i>dvl3a</i> double mutants, suggesting that the two proteins may be dispensable for the activation of maternal Wnt/ß-catenin signaling. Interestingly, convergence and extension movements and anteroposterior patterning require both maternal and the zygotic functions of Dvl2 and Dvl3a, but these processes are more sensitive to Dvl2 dosage. Zygotic <i>dvl2</i> and <i>dvl3a</i> double mutants display mild axis extension defect with correct anteroposterior patterning. However, maternal and zygotic double mutants exhibit most strongly impaired convergence and extension movements, severe trunk and posterior deficiencies, and frequent occurrence of cyclopia and craniofacial defects. Our results suggest that Dvl2 and Dvl3a products are required for the activation of zygotic Wnt/ß-catenin signaling and Wnt/planar cell polarity pathway, and regulate zygotic developmental processes in a dosage-dependent manner. This work provides insight into the mechanisms of Dvl-mediated Wnt signaling pathways during early vertebrate development.</p></div

Analysis of MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutants.

<p>(A) Schema illustrating the strategy to generate female mosaic m<i>dvl2</i>^+(-)/-;<i>dvl3a</i>^-/- adult fish with a new mutant allele (red) in <i>dvl2</i>. MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant embryos are present at varied proportions in the offspring from crosses between female m<i>dvl2</i>^{+(<i>-</i>)/-};<i>dvl3a</i>^-/- and male <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish, depending on the efficiency of germline mutations in the remaining <i>dvl2</i> WT allele. (B) An example of the sequencing chromatogram with both the original mutant allele and a new indel in <i>dvl2</i> locus. (C-F) Lateral (C, D) and dorsal (E, F) views of a representative WT embryo (C, E), and an MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant (D, F) at 11.5 hpf. Notice that the mutant embryo displays most severely impaired AP axis and convergence of paraxial mesoderm, with strongly widened somites (arrowheads). (G) A WT embryo at 30 hpf. (H) Lateral view of a representative MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant at 30 hpf shows deficiency of trunk and posterior regions, and cyclopia (arrowhead; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007551#pgen.1007551.s012" target="_blank">S12 Fig</a>). (I) The phenotype of a Z<i>dvl2</i>;MZ<i>dvl3a</i> mutant with characteristic caudal truncation at 30 hpf. (J) Genotyping of <i>dvl2</i> alleles in MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant embryos from 7 independent fish pairs. A novel indel (red) along with the original mutation (blue) are present in the mutants. The left and right TALEN targeting sites are indicated in green. Dots are introduced to optimize sequence alignment. (K) Quantitative analyses of the occurrence of MZ<i>dvl2</i>;MZ<i>dvl3a</i> and Z<i>dvl2</i>;MZ<i>dvl3a</i> mutants among offspring from 7 independent fish pairs. Each fish pair was crossed three times, and numbers on the top of each column indicate total embryos scored. Scale bar: (C-F) 400 μm; (G-I) 400 μm.</p

Zygotic Dvl2 and Dvl3a in axis extension.

<p>Mutant phenotypes were compared with WT embryos at indicated stages. Insets show eye phenotypes in ventral view, with bidirectional arrows indicating the distance of the two eyes. (A-C) WT embryos. (D-F) Double heterozygous <i>dvl2</i>^+/-;<i>dvl3a</i>^+/- mutants, from crosses between female <i>dvl2</i>^-/- fish and male <i>dvl3a</i>^-/- fish, show weakly delayed axis extension at 11.5 hpf, but are phenotypically normal at later stages. (G-I) Triallelic <i>dvl2</i>^+/-;Z<i>dvl3a</i> mutants,from crosses between <i>dvl2</i>^+/-;<i>dvl3a</i>^+/- carriers, display weak axis extension defect at 11.5 hpf and 30 hpf, and recover at 5 dpf. (J-L) Triallelic Z<i>dvl2</i>;<i>dvl3a</i>^+/- mutants, from crosses between <i>dvl2</i>^+/-;<i>dvl3a</i>^+/- carriers, exhibit more obvious axis extension defect at 11.5 hpf and 30 hpf, and develop shortened AP axis, compressed head, and reduced swim bladder at 5 dpf, which are referred as type I phenotype. (M-O) Z<i>dvl2</i>;Z<i>dvl3a</i> mutants, from crosses between female <i>dvl2</i>^+/-;<i>dvl3a</i>^+/- fish and male <i>dvl2</i>^+/-;Z<i>dvl3a</i> fish, show more strong axis extension defect at 11.5 hpf and 30 hpf, and present a severe type II phenotype, with shortened and wavy axis, craniofacial defects, and complete disappearance of swim bladder (sb) at 5 dpf. (P) Statistical analysis of the extent of axis extension delay, after genotyping of imaged embryos at 11.5 hpf. Capital letters of the abscissa correspond to the images at 11.5 hpf. Bars represent the mean ± s.d. from indicated numbers of embryos, and asterisks above the bars refer to comparison with WT embryos (***, <i>P</i><0.001; ****, <i>P</i><0.0001; NS, not significant). (Q) Quantitative analysis of type I and type II phenotypes at 5 dpf from three independent female <i>dvl2</i>^+/-;<i>dvl3a</i>^+/- and male <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish pairs. Control embryos were from crosses between female WT fish and male <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish. (R) Genotypes of type I and type II embryos. Numbers on the top of each column indicate total embryos analyzed. Scale bars: (A, D, G, J, M) 400 μm; (B, E, H, K, N) 400 μm; (C, F, I, L, O) 400 μm.</p

Analysis of <i>dvl2</i> and <i>dvl3a</i> mutant phenotypes.

<p>(A-C) WT embryos, the insets show the eyes in ventral view at 30 hpf and in dorsal view at 5 dpf. (D-F) Z<i>dvl2</i> mutants, obtained by crosses between heterozygous <i>dvl2</i>^+/- carriers, display weak axis extension defect at 11.5 hpf. They are normal at 30 hpf, and show reduced swim bladder (sb) at 5 hpf. (G-I) MZ<i>dvl2</i> mutants, obtained from crosses between female <i>dvl2</i>^-/- fish and male <i>dvl2</i>^+/- fish, display a reduced AP axis at 11.5 hpf and 30 hpf. They present fused eyes at 30 hpf (inset, ventral view; arrowhead indicates fused lenses), and develop craniofacial defects and cyclopia (inset, dorsal view), with pharyngeal cartilages protruding outward (arrow) at 5 dpf. (J-L) MZ<i>dvl3a</i> mutants from crosses between <i>dvl3a</i>^-/- carriers display weak axis extension defect, but are indistinguishable from WT embryos at 30 hpf and 5 dpf. (M) Statistical analysis of the extent of axis extension delay. The embryos were imaged at 11.5 hpf followed by genotyping. Those embryos with expected genotypes were used to measure the angle between the anterior end and posterior end, with vertex at the geometric center of the embryo (inset). Bars represent the mean ± s.d. from indicated numbers of embryos (****, <i>P</i><0.0001). (N) Alcian blue staining of head cartilages at 5 dpf. Cartilage structures of the basicranium are outlined in grey, showing the fusion of trabeculae and the absence of ethmoid plate (ep) in MZ<i>dvl2</i> mutants. (O) Quantitative analysis of MZ<i>dvl2</i> mutant phenotypes at 5 dpf in offspring from three independent female <i>dvl2</i>^-/- and male <i>dvl2</i>^+/- fish pairs. Control embryos were obtained from crosses between female WT fish and male <i>dvl2</i>^+/- fish. Numbers on the top of each column indicate total embryos analyzed. (P) Genotyping of <i>dvl2</i> mutants with normal and defective phenotypes. All embryos with a normal phenotype are <i>dvl2</i>^+/- mutants, whereas all defective embryos are MZ<i>dvl2</i> mutants. Numbers on the top of each column indicate total embryos genotyped from three independent fish pairs. Scale bars: (A, D, G, J) 400 μm; (B, E, H, K) 400 μm; (C, F, I, L) 400 μm; (N) 100 μm.</p

Knockdown of other <i>dvl</i> genes has no effect in MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutants.

<p>The embryos were injected with coMO (2.5 ng) or a mixture of equal amount of <i>dvl1a</i>/<i>1b</i>/<i>3b</i>MOs (6 ng in total) at 1-cell stage, except for immunostaining. All mutant embryos were imaged after different analyses, followed by genotyping. (A-D) In situ hybridization analysis of <i>goosecoid</i> (<i>gsc</i>) expression in indicated embryos at dome stage. Animal pole view with dorsal region on the right. (E) Endogenous ß-catenin nuclear accumulation (arrows) in dorsal marginal cells of a WT embryo at high stage. (F-I) In situ hybridization analysis of <i>chordin</i> expression in indicated embryos at dome stage. Animal pole view with dorsal region on the right. (J) Endogenous ß-catenin nuclear accumulation (arrows) in dorsal marginal cells of an MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant at high stage. (K-O) Phenotypes of indicated embryos at 11.5 hpf. Lateral view, with a <i>dvl1a</i>/<i>1b</i>/<i>3b</i>MOs-injected MZ<i>dvl2</i>;MZ<i>dvl3a</i> embryo also shown in dorsal view (O). (P-S) Phenotypes of indicated embryos at 30 hpf. Lateral view, note that injection of <i>dvl1a</i>/<i>1b</i>/<i>3b</i>MOs does not change the phenotype of WT and MZ<i>dvl2</i>;MZ<i>dvl3a</i> embryos. (T) Statistical analysis of the extent of axis extension delay in indicated embryos at 11.5 hpf. Bars represent the mean ± s.d. from indicated numbers of embryos (NS, not significant). Scale bars: (A-D, F-I) 400 μm; (E, J) 25 μm; (K-S) 400 μm.</p

The expression of organizer genes is not affected in MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutants.

<p>In situ hybridization analysis of the expression patterns of <i>goosecoid</i>, <i>chordin</i>, and <i>tbxta</i> at dome stage. Animal pole viewed embryos with dorsal region on the right. (A) Schematic representation for the analysis of MZ<i>dvl2</i>;MZ<i>dvl3a</i> embryos. (B-F) Representative images show similar expression patterns of <i>goosecoid</i> in WT embryos and in different mutants. (G) Knockdown of <i>ß-catenin2</i> strongly inhibits <i>goosecoid</i> expression. (H-L) Similar expression patterns of <i>chordin</i> in WT embryos and all indicated mutants. (M) Knockdown of <i>ß-catenin2</i> blocks <i>chordin</i> expression. (N-R) The expression patterns of <i>tbxta</i> are similar between WT embryos and different mutants. (S) Knockdown of <i>ß-catenin2</i> has no effect on <i>tbxta</i> expression. Scale bar: (B-J) 400 μm.</p

Dvl2 and Dvl3a dosages in axis extension and AP patterning.

<p>Both <i>dvl2</i>^+/-;MZ<i>dvl3a</i> and Z<i>dvl2;</i>MZ<i>dvl3a</i> mutants were from crosses between <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- carriers. Representative mutant embryos were imaged at indicated stages. Eye phenotypes are shown in the insets as ventral view. (A-C) WT embryos. (D-F) <i>dvl2</i>^+/-;MZ<i>dvl3a</i> mutants display moderate axis extension defect at 11.5 hpf and 30 hpf, and recover to a normal phenotype at 5 dpf. (G-I) Z<i>dvl2;</i>MZ<i>dvl3a</i> mutants show strong axis extension defect at different stages, and display caudal truncation, craniofacial defects, cardiac edema (arrow), and fused eyes or cyclopia (inset) at 30 hpf and at 5 dpf. (J) Quantitative analysis of the posterior truncation phenotype at 5 dpf in offspring derived from three independent <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- carriers. Control embryos were from crosses between female WT fish and male <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish. Posterior deficiency is present in the offspring from all three fish pairs with a proportion that follows the Mandel inheritance (about 25%). Numbers on the top of each column indicate total embryos analyzed. (K) Genotyping of normal and posteriorly truncated embryos. All defective embryos are <i>dvl2</i>^-/-;<i>dvl3a</i>^-/- mutants. Scale bars: (A, D, G) 400 μm; (B, E, H) 400 μm; (C, F, I) 400 μm.</p

The inducing-activity of Wnt8a is blocked in MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutants.

<p>In situ hybridization analysis of ectopic organizer gene expression at dome stage following Wnt8a overexpression in indicated embryos. Animal pole viewed embryos with dorsal region on the right. (A-F) <i>goosecoid</i> expression pattern in indicated embryos. (G-M) <i>chordin</i> expression pattern in indicated embryos. Notice that reduction of Dvl dosage progressively inhibits the inducing activity of Wnt8a, and MZ<i>dvl2</i>;MZ<i>dvl3a</i> embryos display a complete absence of ectopic <i>goosecoid</i> and <i>chordin</i> expression. All mutant embryos were derived from crosses between a female m<i>dvl2</i>^{+(<i>-</i>)/-};<i>dvl3a</i>^-/- and a male <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish, and were genotyped after in situ hybridization. Scale bar: 400 μm.</p

Dvl2 and Dvl3a dosages in CE movements and Wnt/PCP signaling activity.

<p>(A-H) Representative live images of WT and mutant embryos at 11.5 hpf. Lateral view, with anterior region on the top. (I-P) Dorsal view of indicated embryos simultaneously hybridized with <i>ctslb</i>, <i>dlx3</i>, and <i>tbxta</i> probes to reflect the position of prechordal plate mesoderm, neural plate borders, and notochord, respectively. MZ<i>dvl3a</i> mutants were from intercrosses between <i>dvl3a</i>^-/- carriers; MZ<i>dvl2</i> mutants were from crosses between female <i>dvl2</i>^-/- fish and male <i>dvl2</i>^+/- fish; Z<i>dvl2</i>;Z<i>dvl3a</i> mutants were from crosses between female <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish and male <i>dvl2</i>^+/-;<i>dvl3a</i>^+/- fish; Z<i>dvl2</i>;MZ<i>dvl3a</i> mutants were from intercrosses between <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- carriers; MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutants were from crosses between female m<i>dvl2</i>^+(-)/-;<i>dvl3a</i>^-/- fish and male <i>dvl2</i>^+/-;<i>dvl3a</i>^-/- fish. (Q) Statistical analysis shows that progressive reduction of Dvl2 and Dvl3a dosages increasingly aggravates axis extension defect. The embryos were imaged at 11.5 hpf, and genotyped before measuring the angle (inset). Bars represent the mean ± s.d. from indicated numbers of embryos collected from three independent experiments, and asterisks above the bars indicate significance with respect to WT embryos (***, <i>P</i><0.001; ****, <i>P</i><0.0001). (R) Reduced AP1 reporter activity in Z<i>dvl2</i>;MZ<i>dvl3a</i> and MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutants at 12 hpf. Bars represent the mean ± s.d. from three independent experiments (*, <i>P</i><0.05, ***, <i>P</i><0.001, ****, <i>P</i><0.0001). (S) Rescue of cell polarity of MZ<i>dvl2</i>;MZ<i>dvl3a</i> mutant cells by caJNK. Vertical bidirectional arrows indicate AP orientation. (T) Statistical analysis of the length (l) to width (w) ratio in indicated cells. Bars represent the mean ± s.d. from at least 10 cells in two representative images (*, <i>P</i><0.05, **, <i>P</i><0.01, ****, <i>P</i><0.0001). (U-X) Still frames from live time-lapse images show the dorsal convergence and movement behaviors of lateral cells in indicated embryos (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007551#pgen.1007551.s019" target="_blank">S1</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007551#pgen.1007551.s022" target="_blank">S4 Movies</a>). The trajectories of 10 randomly selected cells are traced. (Y) Statistical analysis of the ratio between the trajectory and the net mediolateral distance (as indicated by a horizontal arrow). Bars represent the mean ± s.d. from at least 15 cells in two or three representative images (***, <i>P</i><0.001; ****, <i>P</i><0.0001). Scale bars: (A-H) 400 μm; (I-P) 400 μm; (S) 20 μm; (U-X) 50 μm.</p