mouse anti fasciclin3 fasiii  (Developmental Studies Hybridoma Bank)

Journal: Development (Cambridge, England)

Article Title: DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm

doi: 10.1242/dev.199465

Figure Lengend Snippet: TaDa-identified Alk targets are enriched in the visceral mesoderm. (A) Schematic outline of embryonic scRNA-seq workflow. (B) UMAP plot displaying the cellular heterogeneity of whole embryo scRNA-seq as 13 cell clusters. (C) Dendrogram representing the relationship between the clusters. (D) Dot plot highlighting increased expression of factors involved in VM development, such as bin , bap , org-1 , Hand and Fas3 in the VM cell population cluster. (E) UMAP projection representing the five clusters of HandC-GFP -positive, FACS-sorted cells. (F) Correlation between the clusters across the population of the HandC-GFP dataset (Pearson's). (G) Heatmap indicating relative expression of TaDa-identified targets downstream of Alk in HandC-GFP -positive cells, highlighting low expression within the cardiac mesoderm population. (H) Dot plot representing the top canonical markers for the HandC-GFP scRNA-seq dataset, highlighting VM, cell cycle, muscle and cardiac markers. Expression levels are visualized as mean expression (red gradient, key below), as well as the fraction of cells in a group (dot size, key below).

Article Snippet: Primary antibodies used were: guinea pig anti-Alk (1:1000; ), guinea pig anti-Jeb (1:1000; ), rabbit anti-Alk (1:750; ), chicken anti-β-galactosidase (1:200; Abcam ab9361), mouse anti-Fasciclin3 (1:50; DSHB 7G10), rabbit anti-GFP (1:500; Abcam ab290), chicken anti-GFP (1:300; Abcam ab13970), mouse anti-Wg (1:50; DSHB 4D4), rat anti-OLLAS (1:200, pre-absorbed on w1118 embryos; Abnova), rabbit anti-Org-1 (1:1000; ), sheep anti-digoxygenin-AP fab fragment (1:4000, Roche), rabbit anti-phospho-Smad1/5 (41D10) (1:500; Cell Signaling Technologies 9516).

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm

doi: 10.1242/dev.199465

Figure Lengend Snippet: RNA-seq analysis identifies Kah target genes. (A) Schematic overview of Kah alleles: Kah Cterm.OLLAS , Kah ΔATG , Kah ΔZnF and Kah f06749 . Exon structure is depicted, highlighting protein coding regions (gray) and zinc-finger domains (red). (B) Dorsal views of stage 10–11 control [ Df(3L)Exel6085/TM3,Ubx-lacZ ] and Kah mutant [ Kah f06749 /Df(3L)Exel6085 , Kah ΔATG and Kah ΔZnF ] embryos stained with Alk (green), the FC-marker Org-1 (red), Fas3 (blue) and β-gal [blue, in control Df(3L)Exel6085/TM3,Ubx-lacZ ]. (C,D) Volcano plots of differential gene expression measured in RNA-seq from Kah ΔATG and Kah ΔZnF mutant embryos. See Table S2 for detailed results. Dashed lines indicate differential gene expression thresholds [FC≥1.5 and ≤−1.5 (log2FC≥0.59 and ≤−0.59)] for up- and downregulated genes respectively ( P adj≤0.05). Up- or downregulated genes are indicated in red or blue, respectively. A selection of differentially expressed genes are labeled. (E) Venn diagrams indicating the number of differentially expressed genes observed in Kah ΔATG and Kah ΔZnF mutants. Top panel, all significantly differentially expressed genes; lower left panel, significantly differentially expressed upregulated genes; lower right panel, significantly differentially expressed downregulated genes. (F) Correlation between the significantly differentially expressed genes (2524) observed in Kah ΔATG and Kah ΔZnf mutants. Thresholds used to determine differential expression are indicated by dashed lines [FC≥1.5 and ≤−1.5 (log2FC≥0.59 and ≤−0.59), and P adj≤0.05]. Pearson correlation coefficient is indicated in the lower right corner. (G) Heatmap detailing expression of genes in enriched pathways, such as Dpp, Toll, Notch and Hedgehog (Hh) in Kah ΔATG and Kah ΔZnf mutants, compared with controls (Ctrl). Color key indicates expression levels. Scale bar: 50 µm.

Article Snippet: Primary antibodies used were: guinea pig anti-Alk (1:1000; ), guinea pig anti-Jeb (1:1000; ), rabbit anti-Alk (1:750; ), chicken anti-β-galactosidase (1:200; Abcam ab9361), mouse anti-Fasciclin3 (1:50; DSHB 7G10), rabbit anti-GFP (1:500; Abcam ab290), chicken anti-GFP (1:300; Abcam ab13970), mouse anti-Wg (1:50; DSHB 4D4), rat anti-OLLAS (1:200, pre-absorbed on w1118 embryos; Abnova), rabbit anti-Org-1 (1:1000; ), sheep anti-digoxygenin-AP fab fragment (1:4000, Roche), rabbit anti-phospho-Smad1/5 (41D10) (1:500; Cell Signaling Technologies 9516).

Techniques: RNA Sequencing Assay, Mutagenesis, Staining, Marker, Expressing, Selection, Labeling

Journal: Development (Cambridge, England)

Article Title: DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm

doi: 10.1242/dev.199465

Figure Lengend Snippet: Kah mutants exhibit defects in midgut constriction. (A) Live imaging of control ( w 1118 ) embryos at stage 16 identifies three midgut constrictions, while Kah mutants ( Kah ΔATG and Kah ΔZnF ) fail to form the first midgut constriction (arrowheads indicate constrictions). Representative frames are shown (see Movies 5 - 7 ). (B) Midgut constriction defects in Kah ΔATG and Kah ΔZnF are not due to defective Mad signaling. Fas3 (white) highlights midgut structure at stage 16, while anti-pMAD (red) visualizes Mad signaling at stage 13/14; Alk identifies VM (green). Dorsal views. Asterisk indicates midgut constriction phenotype. (C) Quantification of the midgut constriction phenotype observed in Kah ΔATG ( n =89) and Kah ΔZnF ( n =109) mutants. (D) pnt Δ88 mutants display a midgut constriction phenotype similar to that observed in Kah mutants. Fas3 (white) highlights midgut structure; dorsal views. Asterisk indicates midgut constriction phenotype. (E) Kah mutants display abnormal midgut musculature organization, visualized with HandC-GFP (green). Lateral views. (F) Quantification of HandC-GFP -positive nuclei present in wild-type ( w 1118 , n =30) and Kah ΔATG /Kah ΔZnF ( n =30) mutants, P <0.001. (G) Representative images from live imaging of pnt Δ88 and Kah ΔATG pnt Δ88 mutant embryos (see Movies 8 and 9 ). (H) Quantification of pnt Δ88 ( n =22) and Kah ΔATG pnt Δ88 ( n =31) mutant midgut constriction phenotypes, indicating the increased severity midgut constriction phenotypes observed in Kah ΔATG pnt Δ88 double mutants. (I) Midgut morphology of representative stage 16 HandC-GFP control, HandC-GFP, bap3-Gal4; UAS-Alk.DN/+ and HandC-GFP; 2xPE-Gal4; UAS-jeb/+ embryos stained for Fas3 (red) and GFP (green). Transgene expression (blue) is revealed by Alk or Jeb antibody staining, as indicated. Scale bars: 50 µm.

Article Snippet: Primary antibodies used were: guinea pig anti-Alk (1:1000; ), guinea pig anti-Jeb (1:1000; ), rabbit anti-Alk (1:750; ), chicken anti-β-galactosidase (1:200; Abcam ab9361), mouse anti-Fasciclin3 (1:50; DSHB 7G10), rabbit anti-GFP (1:500; Abcam ab290), chicken anti-GFP (1:300; Abcam ab13970), mouse anti-Wg (1:50; DSHB 4D4), rat anti-OLLAS (1:200, pre-absorbed on w1118 embryos; Abnova), rabbit anti-Org-1 (1:1000; ), sheep anti-digoxygenin-AP fab fragment (1:4000, Roche), rabbit anti-phospho-Smad1/5 (41D10) (1:500; Cell Signaling Technologies 9516).

Techniques: Imaging, Mutagenesis, Staining, Expressing