pcdna3 egfp rab7a  (Addgene inc)

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-B Confocal images from a time-lapse of a tip cell from a transgenic Tg(kdrl:mCherry-CAAX) S916 embryo shown in magenta. B-B’’’ Inverted contrast of mCherry-CAAX from A shows CAAX dots which elongate along the apical membrane. Pink pseudo-color indicates the vascular lumen. C-E Confocal images of the tip cell of a double transgenic Tg(fli:eGFP-Rab7a) ubs48 ; Tg(kdrl:mCherry-CAAX) S916 embryo at 32hpf . D Inverted contrast image of the EGFP-Rab7 channel. E Inverted contrast image showing the membrane marker mCherry-CAAX in ECs. Arrows point to co-localisation of Rab7 and CAAX at dots. F Distribution of Pearson Correlation Coefficient (PCC) of EGFP-Rab7a ROIs in correlation to mCherry-CAAX, showing a correlation between EGFP and mCherry signal (n=8 tip cells, N>3 different embryos). F A tip cell from a transgenic Tg (kdrl:mCherry-CAAX) S916 embryo at 30hpf, transiently expressing fli:EGFP-Rab7a . F’-F’’’ Stills from the ROI from D showing EGFP-Rab7 and mCherry-CAAX dots that move along the expanding apical membrane. G-G’’’ EGFP-Rab7a signal alone. H-H’’’ mCherry-CAAX signal alone. Arrows point to the EGFP-Rab7a and mCherry-CAAX dots.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Transgenic Assay, Marker, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-B Confocal images of a tip cell from a transgenic TgBAC(Lamp2-RFP) pd1117 embryo at 30hpf, transiently expressing fli:GFP-Rab7a . A Z-projection of the tip cell. A’ Single z-slice of the ROI in A. Two vesicles are depicted with signal positive for GFP-Rab7a and Lamp2-RFP. The largest vesicle shows a reduced signal in the centre for both GFP-Rab7a and Lamp2-RFP, resembling late endosomal/lysosomal structures. B-B’ EGFP-Rab7a signal alone in endothelial cells. C-C’ Lamp2-RFP signal alone. D Schematic representation of the blood vessel (tip EC) undergoing transcellular lumen formation. Rab7, Lamp2 and CAAX co-localise at dots which migrate along the expanding apical membrane. D Tip cell of a double transgenic Tg(kdrl:GFP-CAAX) s916 ; TgBAC(Lamp2-RFP) pd1117 embryo at 34hpf. D’-D’’’ Timelapse from the ROI in E showing Lamp2-RFP dot-like structure elongating along the apical, invaginating membrane. E-E’’’ Lamp2 signal alone. F-F’’’ mCherry-CAAX signal alone. Arrows point to the EGFP-Rab7 and mCherry-CAAX dot-like structures

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Transgenic Assay, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A All three rab7 protein sequences and their predicted mutant sequence. Yellow boxes indicate an important prenylation site responsible for mediating post-translational prenylation of the C-terminal XCXC motif and membrane insertion, and the switch domains important for effector binding after GTP-activation and the hypervariable region responsible for proper membrane recognition. Green letters represent aa that are identical in all three Rab7 isoforms, while black letters represent different aa in Rab7 isoforms. In mutant protein sequences, blue letters are aa encoded out of frame and STOP marks the premature terminating codon. B-C Individual value scatter plots of relative protein expression of Rab7ba and the total Rab7 amount. Levels were measured in two different pooled samples of wild-type, rab7a mat-zyg, rab7ba mat-zyg and rab7bb mat-zyg homozygous embryos. Values were then normalized to total amount of protein measured per sample and to the amount of wild-type sample (n= 2 pools of 20 embryos). D-D’’ Sequencing results of PCR products of the respective rab7 loci from 3 months old homozygous fish. E Brightfield images of wild-type, rab7a, rab7ba and rab7bb homozygous mutant embryos at 30 hpf and 48 hpf. F Images of wild-type, rab7a, rab7ba and rab7bb adult fish at 5 months. G Percentage of rab7a, rab7ba and rab7bb mutations found in adult fish from a heterozygous incross of the respective mutant ( rab7a : N= 3 independent experiments, n= 100 fish; rab7b a: N= 3, n= 100; rab7bb N= 2, n= 70).

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Mutagenesis, Sequencing, Binding Assay, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A Phylogenetic tree constructed from the protein sequence derived from amphioxus (outgroup), human, mouse and zebrafish rab7 genes, scale: 0.9 amino acid substitutions. B Phylogenetic tree constructed from the protein sequences of rab7a, rab7b and zgc:100918 genes from zebrafish, other cyprinid family members and amphioxus (outgroup), scale: 0.3 amino acid substitutions. C Representation of the chromosomal region around the genes rab7b on chromosome 8 and zgc:100918 on chromosome 10. Indicated are the genes that are already annotated copies of each other. D Analysis of the expression of rab7 genes in endothelial and non-endothelial cells from zebrafish transcriptomics data (Lawson, Li et al. 2020). E Heatmap of expression of rab7 genes, a maternally contributed gene ( smarca2 ) and an endothelial-specific gene ( cdh5 ) during zebrafish development, based on data from an EMBL expression atlas (Papatheodorou et al. 2020 see Material and Methods). F Schematic representation of the gene structure of rab7 genes in zebrafish. 5’ and 3’ UTRs and ATG (start codon) are highlighted in dark blue, alternating coding exons are represented in light green and light blue. Sequence of the exons 2 of rab7a, rab7ba and rab7bb . Below each gene sequence appears the respective sequence of mutant alleles ubs51 , ubs52 and ubs53 . Deleted base pairs are underlined at the wild-type sequence, inserted base pairs are represented in light green and deletions are shown in red in mutant alleles. Red asterisk shows premature stop codon in the sequence of exon2.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Construct, Sequencing, Derivative Assay, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A All three rab7 amino acid sequences and their predicted mutant sequences. Peptides used for the Mass Spectrometry experiments are shown on top of each sequence. Peptides that recognise all three isoforms are shown in blue (PanRab7), isoform-specific peptides are shown in magenta and the common peptide for Rab7a and Rab7ba is shown in green. B Graph showing the contribution in % of the individual Rab7 isoforms to total Rab7 protein in wild-type (n= 2 pools of 20 embryos). C and D Individual value scatter plots of relative protein expression of the three different Rab7 isoforms. Levels were measured in two different pooled samples of wild-type, rab7a mat-zyg, rab7ba mat-zyg and rab7bb mat-zyg homozygous embryos. Values were then normalized to total amount of protein measured per sample and to the amount of wild-type sample (n= 2 pools of 20 embryos). G-J Bright field images of wild-type or maternal-zygotic rab7a; rab7bb double homozygous mutant embryos at 1-cell stage and 8-cell stage. K Bar graph showing the percentage of embryos with enlarged yolk granules in different rab7 mutant crosses (n= 289-515 embryos, N=3 different single crosses per condition). L Survival plot of clutches from different mutant crosses. Percentage of surviving embryos from wild-type and rab7a, rab7ba and rab7bb homozygous incrosses, as well as incrosses from rab7a homozygous , rab7bb heterozygous adults and rab7a; rab7bb double homozygous parents (n=499-1438 embryos N= 2-7 crosses per condition.). M Triple mutant survival from a cross of a rab7a -/- ; rab7ba -/- ; rab7bb +/- mother and a rab7a -/- ; rab7ba +/- ; rab7bb -/- father (n=16 embryos at 24hpf, 34 adults after 3 months).

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Mutagenesis, Mass Spectrometry, Sequencing, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-E Confocal still pictures from time-lapse movies from transgenic Tg(kdrl:EGFP-CAAX) s916 embryos at 34-44 hpf showing blood vessel lumenization in wild-type (A), maternal zygotic homozygous mutant for rab7a (B) , rab7ba (C) , maternal-zygotic double homozygous mutant for rab7a; rab7ba (D) and rab7a;rab7bb (E) . Black arrowheads show invaginating luminal front. The final image represents the fully lumenized state of the blood vessels around 44 hpf. F Schematic representation of how lumen diameter was measured. The diameter of the vessel was measured perpendicular to vessel axis at 3 positions. The membrane marker Tg(kdrl:mcherry-CAAX) s916 was used as reference to how far the lumen expanded. G Violin plot showing lumen diameter in rab7 mutants. Median is indicated by thick black line (wild-type: N= 8 fish, n= 24 blood vessels; rab7a : N= 4, n= 10 (maternal-zygotic homozygous); rab7ba : N= 8, n= 22 (maternal-zygotic); rab7a; rab7ba : N= 10, n= 31 (maternal-zygotic); rab7a; rab7bb : N=7, n= 19 (maternal-zygotic).

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Transgenic Assay, Mutagenesis, Marker

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-D Stills from high resolution confocal imaging of lumen fusion in the DLAV (anterior to the left) of double transgenic Tg(kdrl:mcherry-CAAX) s916 ; Tg(fli:Pecam1-EGFP) ncv27 wild-type ( A ), maternal-zygotic homozygous rab7a double ( B ), maternal-zygotic homozygous rab7ba ( C ) and maternal-zygotic double homozygous rab7a ubs51 ; rab7ba us521 ( D ) embryos. A’-D’ Isolated mcherry-CAAX signal labelling the apical membrane of ROIs from A-D. Arrows indicate the invaginating luminal front.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Imaging, Transgenic Assay, Isolation

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-B ’ Representation of measurement of vesicle size. Single z-stack of a non-lumenized tip-cell is chosen using the endothelial marker kdrl:EGFP-CAAX . B’ In a zoom-in window of the ROI in B, every Lamp2-RFP positive signal is measured (using as upper cut off 2 pixels). G Violin plots of measured vesicle sizes in rab7 mutants (wild-type N= 5, n= 283, rab7a;rab7ba heterozygous N= 4, n= 347, rab7a homozygous rab7ba heterozygous N= 5, n=356, rab7a heterozygous; rab7ba homozygous N= 7, n= 539, rab7a;rab7ba homozygous N= 6, n= 260, p***<0.001, red line indicates the median) and in rab7 morphants (standard-MO N= 8, n= 930, rab7a -MO N= 6, n= 129, rab7bb -MO N= 6, n=259, p****<0.0001, red line indicates the median). D-J Confocal images expressing an endothelial marker and the late endosome marker Lamp2 (double transgenic embryos Tg(kdrl:EGFP-CAAX);TgBAC(Lamp2-RFP) pd1117 or Tg(fli:Pecam1-EGFP) ncv27 ;TgBAC(Lamp2-RFP) pd1117 . D’-J’ Zoom-in areas indicated in D-J showing isolated Lamp2-RFP signal. D-F Images from wild-type or rab7 mutant embryos. G-J Images from embryos injected with a standard morpholino or rab7 morpholinos.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Marker, Expressing, Transgenic Assay, Isolation, Mutagenesis, Injection