Journal: BMC Genomics

Article Title: Genome-wide identification of AGO18b-bound miRNAs and phasiRNAs in maize by cRIP-seq

doi: 10.1186/s12864-019-6028-z

Figure Lengend Snippet: The AGO18b binding profile on the targets of miR166a for a negative regulation. a Volcano plot of the mRNAs specifically bound by AGO18b. The ratio of mRNA abundance in AGO18b RIP vs IgG RIP samples from ago18b::mum pre-meiotic tassels was normalized and plotted. Targets of mir159abfjk-3p, mir166a-3p and mir396cd were labeled with a different color, and targets of mir166a-3p were indicated with gene IDs. b Volcano plot of the AGO18b-regulated genes. The ratio of mRNA abundance in the RNA-seq samples from the ago18b::mum vs W22 pre-meiotic tassels was normalized and plotted. c A model to illustrate the AGO18b regulation of shoot apical meristem (SAM) and inflorescence meristem (IM) activity

Article Snippet: A portion of RNAs AGO18b-bound or IgG-bound RNAs was used to construct libraries using an mRNA library preparation kit (GnomeGen, Suzhou) according to the manufacture’s instruction.

Techniques: Binding Assay, Labeling, RNA Sequencing, Activity Assay

Journal: Nature Communications

Article Title: Cnidarian-bilaterian comparison reveals the ancestral regulatory logic of the β-catenin dependent axial patterning

doi: 10.1038/s41467-021-24346-8

Figure Lengend Snippet: a – c Scenarios of the direct correspondence of the cnidarian and bilaterian body axes. pb – polar bodies, aHox – anterior Hox gene, naHox – non-anterior Hox gene, asterisk denotes the mouth. Triangles with a β denote the direction of the β-catenin signaling gradient. d Putative topology of the gene regulatory network of the β-catenin-dependent O–A patterning in Nematostella . The GRN explains why the midbody domain does not expand into the oral and into the aboral domains, and why the aboral domain does not expand into the midbody. It does not explain, however, why the oral domain does not expand aborally. e Comparison of the early β-catenin-dependent patterning in sea urchin and Nematostella shows clear similarities. Unfertilized egg with maternal Fz5/8 and SoxB1 mRNA (future anterior/aboral markers) and maternal Dsh protein localized at the gastrulation pole , . Upon activation of β-catenin signaling in the embryo, first in the endomesodermal domain and then in the posterior/oral ectoderm the expression of Fz5/8 and SoxB1 is suppressed, and the anterior/aboral markers (including the zygotic genes Six3/6 and FoxQ2 ) become progressively confined to one side of the axis. The axis becomes patterned by mutually repressive transcription factors (T). Gray “T” in Nematostella indicate repressive interactions, for which candidate transcription factors are not known. Triangles with a β denote the direction of the β-catenin signaling gradient. β? indicates that in Nematostella , nuclear β-catenin could only be experimentally detected until midblastula stage , after which the presence of nuclear β-catenin gradient is deduced based on target gene response. After preendodermal plate is specified in Nematostella , β-catenin signaling becomes repressed there by an unknown mechanism , possibly involving ERG .

Article Snippet: Total RNA was extracted with TRIZOL (Life Technologies) or with GeneElute Mammalian Total RNA Miniprep Kit (Sigma) according to the manufacturer’s protocol; poly-A enriched mRNA library preparation (Lexogen), quality control, and multiplexed Illumina HiSeq2500 sequencing (50 bp, single-end) were performed at the Vienna BioCenter Core Facilities.

Techniques: Comparison, Activation Assay, Expressing

Journal: eLife

Article Title: Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator

doi: 10.7554/eLife.80803

Figure Lengend Snippet: ( A ) Maximum likelihood phylogeny shows that Nematostella ZSWIM4-6 clusters with ZSWIM4, ZSWIM5, and ZSWIM6 from zebrafish, frog, and mouse. ( B–F’ ) Nematostella zswim4-6 expression follows the dynamic BMP signaling domain (see for comparison). Double ISH shows zswim4-6 and chd transcripts localize to the opposite sides of the directive axis. ( G–I ) Mosaic expression of ZSWIM4-6-EGFP under the control of the ubiquitously active EF1α promoter in F0 transgenic animals demonstrates that ZSWIM4-6 is a nuclear protein. Full-length ZSWIM4-6-EGFP is translocated into the nuclei ( G ), while ZSWIM4-6ΔNLS-EGFP missing the predicted nuclear localization signal NLS remains cytoplasmic ( H ), similar to the EGFP control ( I ). Exposure time was the same in all images. ( J–L ) Microinjection of ZSWIM4-6-EGFP mRNA results in a weak EGFP signal detectable in the nuclei of the early blastula ( J ), which progressively disappears towards late gastrula ( K ). EGFP translated from EGFP mRNA remains readily detectable ( L ). To visualize the weak signal in ( J–K ), the exposure had to be increased in comparison to ( L ). Asterisks mark the oral side; scale bars 100 µm.

Article Snippet: Poly-A-enriched mRNA library preparation (Lexogen) and 50 bp SE Illumina HiSeq2500 sequencing was performed by the Vienna BioCenter Core Facilities.

Techniques: Expressing, Comparison, Control, Transgenic Assay, Microinjection

Journal: eLife

Article Title: Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator

doi: 10.7554/eLife.80803

Figure Lengend Snippet: Expression of BMP receptors ( A, A’ ) alk2, ( B, B’ ) alk3/6, and ( C, C’ ) bmprII is broadly detectable in endoderm with a slightly stronger expression on the ‘strong BMP signaling’ side of the directive axis, opposite to chordin (red). ( D ) qPCR quantification of knockdown (KD) efficiency for BMP receptor shRNAs in relation to shRNA against mOrange (sh_control, 100% expression). Black lines represent the median. ( E–I ) EGFP signal in late gastrula embryos injected with ( E ) EGFP mRNA and ( F–I ) mRNA of dominant-negative BMP receptor constructs in which Ser/Thr-kinase domain was replaced with the EGFP coding sequence. ( J ) Overview of the expected effect of the dominant negative BMP receptor overexpression and BMP receptor RNAi on BMP signaling. ( K–O ) zswim4-6 expression is weakly reduced upon overexpression of the dominant negative type I BMP receptors dnAlk2-EGFP and dn_Alk3/6-EGFP and strongly suppressed upon combined overexpression of dn_Alk2-EGFP and dn_Alk3/6-EGFP but remains unchanged upon overexpression of the dominant negative type II BMP receptor dn_BMPRII-EGFP. ( P–T ) Expression of zswim4-6 is weakly reduced upon Alk2 RNAi, the effect is stronger upon Alk3/6 KD, and zswim4-6 is practically abolished in a combined Alk2+Alk3/6 KD. zswim4-6 expression is unaffected by the BMPRII RNAi. Asterisks mark the oral side. Scale bars 100 µm. The numbers in the bottom-right corner indicate the ratio of embryos displaying the phenotype shown in the image to the total number of embryos treated and stained as indicated in the figure. In ( P–T ), KD efficiency is shown in the top-right corner of each image. Figure 5—figure supplement 1—source data 1. Source data for .

Article Snippet: Poly-A-enriched mRNA library preparation (Lexogen) and 50 bp SE Illumina HiSeq2500 sequencing was performed by the Vienna BioCenter Core Facilities.

Techniques: Expressing, Knockdown, shRNA, Control, Injection, Dominant Negative Mutation, Construct, Sequencing, Over Expression, Staining

Journal: eLife

Article Title: Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator

doi: 10.7554/eLife.80803

Figure Lengend Snippet: ( A ) Fluorescent signal in embryos injected with mRNA coding for the ZSWIM4/6 recognition sequence fused to the mCherry ( wt-zswim4/6-mCh ) coding sequence. ( B ) Co-injection with ZSWIM4/6 morpholino can suppress the translation of mRNAs containing the respective recognition sequence. ( C ) Translation of mRNA coding for the zswim4/6 recognition sequence carrying five mismatches and fused to mCherry ( mismatch-zswim4/6-mCh ) is no longer suppressed when co-injected with ZSWIM4/6 morpholino.

Article Snippet: Poly-A-enriched mRNA library preparation (Lexogen) and 50 bp SE Illumina HiSeq2500 sequencing was performed by the Vienna BioCenter Core Facilities.

Techniques: Injection, Sequencing

Journal: eLife

Article Title: Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator

doi: 10.7554/eLife.80803

Figure Lengend Snippet: ( A–B’’ ) Morpholino knockdown (KD) of zswim4-6 results in patterning defects. ( A ) and ( B ) show confocal sections across the pharyngeal region of 4d planulae stained with antiHoxE antibody and phalloidin. ( A’ ) and ( B’ ) show the areas boxed in ( A ) and ( B ). White dotted lines delineate mesenterial chambers. In the 4d planula, the HoxE-positive mesenterial chamber does not reach the pharynx, which leads to the fusion of neighboring chambers (compare A , A’ with B , B’ ). This results in the formation of three instead of four tentacles in the 9d polyp (compare A’’ with B’’ ). ( C–F’ ) Immunofluorescence and quantification of relative nuclear anti-pSMAD1/5 staining intensities in 2d ZSWIM4-6 morphants ( C–D’ ) and upon zswim4-6 mRNA overexpression in the late gastrula ( E–F’ ). Intensity measurements (arbitrary units, a.u.) are plotted as a function of the relative position of each nucleus in the endoderm or in the ectoderm along a 180° arc from 0 (high signaling side) to π (low signaling side). The measurements from Control MO embryos (n = 10) and ZSWIM4-6MO embryos (n = 10), as well as egfp mRNA embryos (n = 22 for the endodermal, and n = 24 for the ectodermal measurements) and zswim4-6 mRNA embryos (n = 8 for the endodermal, and n = 9 for the ectodermal measurements) are described by a LOESS smoothed curve (solid line) with a 99% confidence interval for the mean (shade). For visualization purposes, the intensity values were normalized to the upper quantile value among all replicates and conditions of each control-experiment pair. ( G–K’ ) Expression of zswim4-6 and BMP network components in the 2d planula upon morpholino KD of zswim4-6 ; All images except for ( H ) and ( H’ ) show oral views. ( L–P’ ) Expression of zswim4-6 and BMP network components in late gastrula (30 hr) upon zswim4-6 mRNA injection; oral views, purple dashed line marks the loss of a sharp boundary of chd expression. In ( A–P’ ), asterisks mark the oral side. In ( A-P’ ), scale bars 100 µm. ( Q ) ChIP with GFP-Trap detects ZSWIM4-6-EGFP fusion protein in the vicinity of the pSMAD1/5 binding site in the upstream regulatory region of chordin but not of gremlin . Experiment on biological quadruplicates. Mean enrichments and standard deviations are shown. Figure 6—source data 1. Source data for . Figure 6—source data 2. Source data for .

Article Snippet: Poly-A-enriched mRNA library preparation (Lexogen) and 50 bp SE Illumina HiSeq2500 sequencing was performed by the Vienna BioCenter Core Facilities.

Techniques: Knockdown, Staining, Immunofluorescence, Over Expression, Control, Expressing, Injection, Binding Assay

Journal: eLife

Article Title: Analysis of SMAD1/5 target genes in a sea anemone reveals ZSWIM4-6 as a novel BMP signaling modulator

doi: 10.7554/eLife.80803

Figure Lengend Snippet: ( A–F” ) Wild-type zebrafish embryos were left uninjected ( B ) or injected at the one-cell stage with 20, 40, 80, or 210 pg zzswim5 mRNA. ( A ) Phenotype quantification at 1 d post-fertilization (dpf) shows increasingly severe developmental defects at higher amounts of mRNA. A representative selection was imaged at 1 dpf ( B–F’’ ). Multiple embryos are shown for 80 ( E–E” ) and 210 ( F–F” ) pg to illustrate the variety of defects. Number of embryos – uninjected: 19, 20 pg: 20, 40 pg: 24, 80 pg: 21, 210 pg: 17. ( G–I ) Embryos were injected with 80 pg zzswim5 mRNA or left uninjected and fixed at 50% epiboly (early gastrulation). BMP signaling levels were assessed using pSmad1/5/9 immunostaining. Animal pole views are shown with ventral on the left. ( G ) Quantification of immunofluorescence (arbitrary units, a.u.) reveals lower amplitude BMP signaling gradients in zzswim5 -overexpressing embryos ( I ) compared to uninjected controls ( H ). The measurements from uninjected embryos (n = 8) and zzswim5 -injected embryos (n = 8) are described by a LOESS smoothed curve (solid line) with a 99% confidence interval for the mean (shade). Figure 6—figure supplement 2—source data 1. Source data for .

Article Snippet: Poly-A-enriched mRNA library preparation (Lexogen) and 50 bp SE Illumina HiSeq2500 sequencing was performed by the Vienna BioCenter Core Facilities.

Techniques: Injection, Selection, Immunostaining, Immunofluorescence