Journal: PLoS Genetics

Article Title: The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

doi: 10.1371/journal.pgen.1009765

Figure Lengend Snippet: ( A ) Gross phenotype of Tmem2 CKO embryos at E10.5. Whole-mount images of unstained embryos ( left panels ) and fluorescence microscopic images of DAPI-stained embryos ( center and right panels ) are shown. Hypoplasia of the frontonasal process, maxillary process, and mandibular process is observed in Tmem2 CKO ( open arrowheads ). Arrow indicates edema in the facial region. Asterisk indicates a partial tear of the right forelimb bud inadvertently caused during dissection. ( B ) Gross phenotype of Tmem2 CKO embryos at E12.5. Whole-mount images reveal severe developmental malformations of the craniofacial region of Tmem2 CKO embryos, with marked hypoplasia of the facial structures ( white arrows ) and lack of fusion of the frontonasal processes ( black open arrowheads ; see Fig 1C for more detailed images). White open arrowhead indicates mild exencephaly observed in some of the Tmem2 CKO embryos. Hemorrhagic lesions are also frequently observed in Tmem2 CKO embryos. ( C, D ) Phenotypes in the craniofacial region of E12.5 Tmem2 CKO embryos. ( C ) Fluorescence microscopic images of the craniofacial region of DAPI-stained embryos. Tmem2 CKO embryos show lack of fusion of the bilateral medial nasal processes ( filled arrowhead ) and bilateral mandible processes at the midline, lack of fusion between frontonasal process and maxillary process ( open arrowheads ), and wide opening of the nasal cavity. Broken lines (i, ii, iii and iv) indicate the orientations of sections shown in D . ( D ) H&E staining of transverse sections through the forebrain (i, ii) and the maxillary region (iii, iv). (v, vi) High magnification images corresponding to the area of NCC-derived peripheral nervous tissues, including trigeminal ( tg ), vestibular ( vg ), and facial ( fg ) ganglia. Note that the size of these NCC-derived ganglia is reduced in Tmem2 CKO embryos. Tmem2 CKO embryos also exhibit blister-like epithelial detachment, often accompanied with hemorrhaging, in the lateral portion of the frontonasal process and at the midline of the mandibular arches ( arrows ). ( E ) Accumulation of HA in craniofacial tissues of E12.5 Tmem2 CKO embryos. Transverse sections of the facial processes were double-labeled with bHABP and/or DAPI. Increased HA staining is observed in the facial processes of Tmem2 CKO embryos ( HA ). Bar graph shows the quantification of HA in the facial tissue of E12.5 embryos (ng/μg tissue protein). Data represent means ± SD ( n = 5). ** p < 0.01 by Student’s t -test. ( F ) H&E staining of the frontonasal tissue demonstrates expanded extracellular space in E12.5 Tmem2 CKO and control embryos. Bar graph shows the quantification of the size of the extracellular space in the frontonasal tissue. Data represent means ± SD ( n = 5). *** p < 0.001 by Student’s t -test. ba , branchial arch; fnp , frontonasal process; max , maxillary process; man , mandibular process; fb , forebrain; mb , midbrain; hb , hindbrain; nc , nasal cavity; mnp , medial nasal process; lnp , lateral nasal process; t . tongue; phv , primary head vein; tg , trigeminal ganglion; v , fourth ventricle; vg , vestibular ganglion; fg , facial ganglion. Scale bars, 500 μm in A - D ; 250 μm in E; 25 μm in F .

Article Snippet: A conditional Tmem2 null allele ( Tmem2 flox ) was created by Cyagen Biosciences (Santa Clara, CA) using TurboKnockout gene targeting methods.

Techniques: Fluorescence, Staining, Dissection, Derivative Assay, Labeling, Control

Journal: PLoS Genetics

Article Title: The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

doi: 10.1371/journal.pgen.1009765

Figure Lengend Snippet: ( A ) Sagittal sections of Tmem2-FLAG KI reporter embryos at E11.0 were double-labeled with anti-FLAG antibody and bHABP. Nuclei were counterstained with DAPI. fb , forebrain; mb , midbrain; hb , hindbrain; ht , heart; drg , dorsal root ganglia. ( B ) High magnification images of the facial prominence, heart, branchial arch, and dorsal root ganglion in Tmem2-FLAG KI embryos at E11.0 double-labeled for TMEM2-FLAG protein and HA. Areas indicated by boxes are enlarged in lower panels. fb , forebrain; fp , facial prominence; ba , branchial arch; ht , heart; ec , endocardial cushion; drg , dorsal root ganglia. ( C ) Transverse sections of the neural tube of Tmem2-FLAG KI embryos at E9.0. Sections at the cranial and trunk levels of the neural tube were double-labeled for TMEM2-FLAG protein and HA as in A . TMEM2 expression is observed in the neural plate and the border region of the neural tube ( filled arrowheads ), whereas these sites are devoid of HA staining ( open arrowheads ). ( D ) Double-labeling of neural crest cells for TMEM2-FLAG and Sox9. Transverse sections of E9.0 neural tube were stained for TMEM2-FLAG and Sox9. Sox9-positive pre-migratory and emigrating NCCs at the edge of the neural tube highly express TMEM2. nt , neural tube. Scale bars, 250 μm in A ; 50 μm in B ; 300 μm in C ; 100 μm in D .

Article Snippet: A conditional Tmem2 null allele ( Tmem2 flox ) was created by Cyagen Biosciences (Santa Clara, CA) using TurboKnockout gene targeting methods.

Techniques: Labeling, Expressing, Staining

Journal: PLoS Genetics

Article Title: The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

doi: 10.1371/journal.pgen.1009765

Figure Lengend Snippet: ( A ) Transverse sections at three different levels of the neural tube of Tmem2 CK O and control embryos at E9.0 were triple-stained for Sox9 ( green ), HA ( red ), and nuclei ( blue ). Broken lines indicate the external contour of the neural tube. Note that emigration of Sox9-positive cells out of the neural tube is reduced in Tmem2 CK O embryos. Also, in Tmem2 CK O embryos, the dorsal surface of the neural tube exhibits HA staining ( filled arrowheads ), whereas little HA staining is detectable in the corresponding area of control embryos ( open arrowheads ). ( B ) Transverse sections of Tmem2 CK O and control embryos at E9.0 were double-stained for Sox10 ( red ) and nuclei ( blue ). In Tmem2 CKO embryos, the number of Sox10-positive cells outside of the neural tube is significantly decreased. The image in the caudal neural tube reveals that some Sox10-positive cells persist within the neural tube in Tmem2 CKO embryos. ( C ) Quantification of pre-migratory and migrating NCCs in Tmem2 CKO and control embryos. The numbers of Sox9-positive cells within the neural tube and Sox10-positive cells outside of the neural tube were counted as pre-migratory and migrating NCCs, respectively ( top and middle bar graphs ). The ratio of migratory NCCs relative to the total number of NCCs was also compared between Tmem2 CKO and control embryos ( bottom graph ). Data represent means ± SD ( n = 5). *** p < 0.001 by Student’s t -test. Scale bars, 300 μm in A and B .

Article Snippet: A conditional Tmem2 null allele ( Tmem2 flox ) was created by Cyagen Biosciences (Santa Clara, CA) using TurboKnockout gene targeting methods.

Techniques: Control, Staining

Journal: PLoS Genetics

Article Title: The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

doi: 10.1371/journal.pgen.1009765

Figure Lengend Snippet: ( A ) Whole-mount fluorescence images of Tmem2 CKO ;ZsGreen and control embryos at E9.5. In Wnt1 - Tmem2 CKO ;ZsGreen embryos, domains occupied by ZsGreen-labeled NCCs are smaller and the intensity of ZsGreen signals is reduced in the facial prominence and in the first and second branchial arches ( open arrowheads ). Aberrant accumulation of ZsGreen -positive cells is observed in the dorsal midbrain and hindbrain of Tmem2 CKO ;ZsGreen embryos ( filled arrowheads ). ( B ) Transverse sections of Tmem2 CKO ;ZsGreen and control embryos at E10.5 stained for Sox10. Arrowheads indicated trigeminal ganglia ( tg ). The number of Sox10-positive NCCs arrived at the trigeminal ganglia is significantly reduced in Tmem2 CKO ;ZsGreen embryos. Bracket indicates abnormal folding of the hindbrain neuroepithelium. ( C ) Transverse sections through the caudal neural tube of Tmem2 CKO ;ZsGreen and control embryos at E10.5 stained for Sox10. In Tmem2 CKO ;ZsGreen embryos, the number of NCCs migrated to the dorsal root ganglia ( arrowheads ) is decreased, while the number remaining in the neural tube is increased ( brackets ). Bar graph shows the quantification of Sox10-positive cells in the dorsal root ganglia at E10.5. Data represent mean ± SD ( n = 5). *** p < 0.001 by unpaired Student’s t -test. Scale bars, 500 μm in A - C . fp , facial prominence; ba1 , the first branchial arch; ba2 , the second branchial arch; lnp , lateral nasal process; max , maxillary process; mnp , medial nasal process; tg , trigeminal ganglion; drg , dorsal root ganglia.

Article Snippet: A conditional Tmem2 null allele ( Tmem2 flox ) was created by Cyagen Biosciences (Santa Clara, CA) using TurboKnockout gene targeting methods.

Techniques: Fluorescence, Control, Labeling, Staining

Journal: PLoS Genetics

Article Title: The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

doi: 10.1371/journal.pgen.1009765

Figure Lengend Snippet: ( A ) Cell-based hyaluronidase assay. Tmem2 -depleted and control O9-1 cells were cultured for 48 h on glass coverslips coated with fluoresceinated HA (FA-HA). HA degrading activity is revealed as dark areas in the fluorescent background. The level of HA degradation was also quantitatively compared between Tmem2 -depleted and control O9-1 cells as described in Materials and Methods ( bar graph ). Data represent mean ± SD of the fluorescence intensity underneath a cell relative to that in cell-free area (n > 50 cells per condition pooled from three independent experiments). *** p < 0.001 by unpaired Student’s t -test. ( B ) O9-1 cells degrade substrate-bound HA at FAs. Cell-based hyaluronidase assays were performed for 16 h and cells were stained for vinculin. In control O9-1 cells, HA degradation occurs coincident with vinculin-positive FAs. In Tmem2 -depleted O9-1 cells, HA degradation and FA formation are greatly diminished. The number of FAs per cell was quantitatively compared between Tmem2 -depleted and control O9-1 cells ( bar graph ). Data represent mean ± SD (n >30 cells per condition pooled from three independent experiments). *** p < 0.001 by unpaired Student’s t -test. ( C ) Representative images of the migration of Tmem2 -depleted and control O9-1 cells into a cell-free gap on Col1/HA mixed substrates. Top panels show images of gaps immediately after removal of the ibidi 2-well Culture-Insert. Other panels show images of gaps after a 24 h or 48 h incubation. Data are representative of three independent experiments. Bar graph shows the quantitative analysis of cell migration. Data represent the mean ± SD of the gap area covered by migratory cells relative to the area of the original gap (n = 5 per condition). *** p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparison test. ( D, E ) Transverse sections of the neural tube in the caudal and cranial regions of E9.5 Tmem2 CKO ;ZsGreen and control embryos were stained for vinculin ( D ) or N-cadherin ( E ). ( D ) Vinculin accumulation in the cellular cortex is reduced in NCCs in Tmem2 CKO ;ZsGreen embryos. Insets show enlarged images of migrating NCCs in the boxed areas. ( E ) Cell surface expression of N-cadherin is reduced in NCCs in Tmem2 CKO ;ZsGreen embryos. Lower panels show enlarge images of the boxed areas. nt , neural tube. Scale bars, 25 μm in A ; 2.5 μm in B ; 200 μm in C; 150 μm in D and E .

Article Snippet: A conditional Tmem2 null allele ( Tmem2 flox ) was created by Cyagen Biosciences (Santa Clara, CA) using TurboKnockout gene targeting methods.

Techniques: Control, Cell Culture, Activity Assay, Fluorescence, Staining, Migration, Incubation, Comparison, Expressing

Journal: PLoS Genetics

Article Title: The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

doi: 10.1371/journal.pgen.1009765

Figure Lengend Snippet: ( A ) Analysis of cell death in the facial processes of E12.5 embryos. Transverse sections through the facial processes were analyzed by TUNEL assays. Bar graphs on the right represent the number of TUNEL-positive cells per unit area of the facial processes ( top ) and the first branchial arch ( bottom ). Means ± SD ( n = 3) are shown as horizontal bars. ** p < 0.01, *** p < 0.001 by unpaired Student’s t -test. ( B ) Analysis of cell proliferation in the facial processes of E12.5 embryos. Transverse sections through the facial processes were stained with anti-PHH3 antibody. Bar graphs on the right represent the number of PHH3-positive cells per unit area of the facial processes ( top ) and the first branchial arch ( bottom ). mnp , medial process; lnp , lateral nasal process; nc , nasal cavity. Means ± SD ( n = 3) are shown as horizontal bars. n . s ., not significant by unpaired Student’s t -test. ( C, D ) Analysis of apoptosis of Tmem2 -depleted and control O9-1 cells cultured on HA-free and HA-containing substrates. Tmem2 -depleted and control O9-1 cells were cultured for 48 h on Col1 alone ( C ) and on Col1/HA mixed ( D ) substrates. Cells were then stained with propidium iodide (PI) and anti-annexin V antibody, followed by the analysis of apoptosis by flow cytometry. Tables show the quantification of live (lower left quadrant in the charts), early apoptotic (lower right quadrant), and late apoptotic (upper right quadrant) cells. *** p < 0.001; n . s ., not significant by unpaired Student’s t -test. Scale bars, 250 μm in A and B .

Article Snippet: A conditional Tmem2 null allele ( Tmem2 flox ) was created by Cyagen Biosciences (Santa Clara, CA) using TurboKnockout gene targeting methods.

Techniques: TUNEL Assay, Staining, Control, Cell Culture, Flow Cytometry

Journal: bioRxiv

Article Title: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

doi: 10.1101/2021.02.24.432780

Figure Lengend Snippet: A. Genomic region encompassing the 3′-most terminal exons of the Srrm234 gene and the corresponding protein domains encoded therein. Arg/Ser: Arginine/Serine-rich, eMIC: enhancer of microexons, 3′UTR: 3′ untranslated region. Putative AS and poly-adenylation (pA) sites are indicated together with their associated reference transcripts (A, C, F, G). B. Genomic architecture of the 3′ end of the Srrm234 locus in different insect species. Reference isoforms encoding the eMIC domain are indicated. mya: million years ago. C. Sashimi plots of RNA-seq data from different tissues at Srrm234 3′ end region. Average numbers of reads spanning each splice junction between male and female samples are indicated. Y-axis represents absolute number of mapping reads (without normalization for library size). Bottom: main transcript isoforms annotated for the Srrm234 gene (FlyBase annotation). Pie charts depict isoform usage quantified based on junction reads only. Data from FlyAtlas 2 . D. Protein domains of Drosophila (d) Srrm234 isoforms and human (h) SRRM4. IR: intron retention, aa: amino acids, K: lysine, Srrm2/4-N: conserved regions at Srrm2/4 N-termini. E. RT-PCR assays for alternatively spliced exons in SL2 cells overexpressing different Srrm234 isoforms. F. Representative pictures of fly wings overexpressing either UAS-Srrm234-C or -A under the control of spalt (Sal E|PV -GAL4) driver line, active in the centre of the wing blade .

Article Snippet: The eMIC-null allele for Srrm234 ( CG7971 ) was generated by GenetiVision CRISPR gene targeting services.

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

doi: 10.1101/2021.02.24.432780

Figure Lengend Snippet: A. Transcriptomic data mapping at the Srrm234 gene. Top, 3′ seq data from . Middle, CAGE-seq (cap analysis gene expression) data from . Bottom, RNA-seq data from Fly Atlas 2 .

Article Snippet: The eMIC-null allele for Srrm234 ( CG7971 ) was generated by GenetiVision CRISPR gene targeting services.

Techniques: Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

doi: 10.1101/2021.02.24.432780

Figure Lengend Snippet: A. Expression levels as quantified by qPCR of heterologous expressed Srrm234 constructs ( Drosophila isoforms A, C, I, E and hSRRM4 ) in SL2 cells, relative to the housekeeping Gapdh2 gene. B. Representative wings (white arrows) and halteres (yellow arrows) of flies expressing Srrm234 -derived transgenes under the control of nub-GAL4 ( nubbin ) driver lines. C. Integration cassette introduced at the 3′ end of Srrm234 replacing the endogenous sequence that encodes for the eMIC domain (region delimited by guide RNAs in ). D. Expression levels of are neuronally secreted insulin-like peptides (Ilps) in Drosophila , in control and eMIC-L3 CNS and adult brain. E. RT-PCRs of alternatively spliced exons in fly heads for control (w 1118 ) and eMIC-flies. In brackets, exon lengths. F. Sleep patterns of 21 day-old flies in 12h light – 12h dark cycles. Left, average time flies spend sleeping (inactive for >= 5min) at different times of the light:dark cycle. ZT: Zeitbeger Time (switch from light to dark conditions), vertical lines: standard error of the mean. Top right, maximum sleep episode during the night. Bottom right, total number of activity counts per hour during the night. P-values from Mann-Whitney U tests comparing with w 1118 controls. G. Top, mating scheme for the determination of relative fitness values presented in . Sb: stubbl e. Middle, total number of flies quantified per experiment. Bottom, allele frequencies in the F1 generation. H. Deleterious effects of overexpressing dSrrm234-I pan-neuronally in the eMIC-background: failed wing expansion (1) and open positioning of wings (2) and legs (3). I. RT-PCRs from female fly heads of candidate eMIC-sensitive exons. dI: Drosophila Srrm234-I, h4: human SRRM4, dA: Drosophila Srrm234-A. J. Expression levels as quantified by qPCR of Srrm234 gene, each of the two alleles (eMIC+ and eMIC-), and UAS-transgenes in female fly heads, relative to the housekeeping Sply gene.

Article Snippet: The eMIC-null allele for Srrm234 ( CG7971 ) was generated by GenetiVision CRISPR gene targeting services.

Techniques: Expressing, Construct, Derivative Assay, Sequencing, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

doi: 10.1101/2021.02.24.432780

Figure Lengend Snippet: A. CRISPR strategy to generate mutant flies with eMIC-specific deletion at the Srrm234 locus. The deleted part of the ‘enhancer of microexons’ (eMIC) protein domain is encoded in the alternative last exon and is essential for its function. The dotted red line spans the deleted genomic region, which was replaced by an integration cassette . Arg/Ser: Arginine/Serine-rich, eMIC: enhancer of microexons, 3′UTR: 3′ untranslated region, gRNA: guide RNA, pA: poly-adenylation site. B. Left: eMIC+/− male 1 day-old fly from the cross with w 1118 . Right: eMIC-male. C. Average weight of w 1118 controls, eMIC-/+ heterozygous flies and two independent eMIC-clones (#1, #2) from the CRISPR targeting, less than 24 hours after hatching. White numbers indicate mean values. P-values from two-sided t-tests comparing with w 1118 controls, n.s.: non-significant or p > 0.05. D. Longevity assay, n=100 flies per sex and genotype. P-value from log-rank tests of eMIC-flies compared to controls for each sex. E. Kymographs displaying Drosophila negative geotaxis behaviour. Coloured lines indicate average height at each timepoint. F. Sensitivity of adult flies to mechanical stimulation (10s vortex). Left: probability of recovering from mechanical-induced paralysis over time. Top right: number of flies tested and percentage of them that are sensitive to mechanical stress. Bottom right: time spent in recovering from paralysis until the fly is in upright position. P-values from Mann-Whitney U-tests. G. Sleep patterns of 3 day-old flies in 12h light – 12h dark cycles. Left: average time flies spend sleeping (inactive for >= 5min) at different times of the light:dark cycle. ZT: Zeitbeger Time (switch from light to dark conditions), vertical lines: standard error of the mean. Top right: maximum sleep episode during the night. Bottom right: total number of activity counts per hour during the night. P-value from Mann-Whitney U-tests comparing eMIC-with control flies, n.s.: p > 0.05. H. Relative fitness of flies with varying number of eMIC+ alleles in the F1 generation. See Supplementary Figure 2F for a detailed mating scheme. P-values from χ 2 -tests on the observed frequencies of genotypes per cross, compared with crosses marked with a diamond. Transgenic construct expression is induced pan-neuronally using an elav-GAL4 driver. I. Representative pictures of eyes from young flies expressing dSrrm234 isoforms under an elav-GAL4 driver in the eMIC-background. J. RT-PCR assays of short neural exons from female fly heads. Numbers indicate mean and standard deviation values from three replicates. K-L. Kymographs displaying the negative geotaxis behaviour of Drosophila eMIC-flies upon expression of UAS- hSRRM4 pan-neuronally using an elav-GAL4 driver line (K-L) or in glutamatergic neurons (including motoneurons) under a vGlut-GAL4 line (L). Coloured lines indicate average height at each timepoint and ribbons, 95% non-parametric bootstrap confidence intervals.

Article Snippet: The eMIC-null allele for Srrm234 ( CG7971 ) was generated by GenetiVision CRISPR gene targeting services.

Techniques: CRISPR, Mutagenesis, Clone Assay, MANN-WHITNEY, Activity Assay, Transgenic Assay, Construct, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: bioRxiv

Article Title: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

doi: 10.1101/2021.02.24.432780

Figure Lengend Snippet: A. Length of the exon and neighbouring introns for six exon groups, from top to bottom: eMIC-dependent, eMIC-sensitive, Neural, Other AS exons (ASE), cryptic and constitutive exons (Table S2). Box limits represent interquartile ranges; central lines, median values (also indicated with numbers). P-values from Mann-Whitney U tests are shown for the comparison of each class against eMIC-dependent exons. Red font indicates the difference goes in the opposite direction. Number of exons per group is indicated in parentheses. B. Maximum entropy scores for the 5′ splice site and AG region, relative to constitutive (High PSI) exons. P-values from Mann-Whitney U tests are shown for the comparison with the eMIC-dependent group: * < 0.01, ** < 0.001, *** < 10 −4 . C. RNA maps for motifs enriched or depleted in the intronic regions surrounding eMIC-dependent exons. From left to right: distribution of AG motifs in the 75 nt upstream of the alternative exon start (3′ ss), UGC motif distribution close to the 3′ ss, Polypyrimidine tract profiles for YYYY tetramers (pY) or CU-rich (UCUC/CUCU) tetramers, and profile of the Drosophila branch point consensus sequence CUAA[U/C], in both upstream and downstream introns. Length of sliding window: 15 nt for AG and UGC and 27 nt for the others. Regions with a significant difference in the motif coverage (FDR < 0.05) compared to ASE group are marked with a coloured rectangle underneath. D. Regulation of eMIC-dependent exons by PTB proteins, quantified as the difference in PSI (ΔPSI) relative to control. Left: heph ( Drosophila PTBP1/2/3 ortholog) knockdown in SL2 cells, data from modENCODE. Right: double knockdown of PTBP1 and PTBP2 in HEK293 cells, data from . P-values from Mann-Whitney U tests. E. Regulation of eMIC-dependent exons by U2af1. Left: effect of the knockdown of U2af38 ( Drosophila U2AF1 ortholog) in SL2 cells, data from . Right: effect of U2AF1 KD in HEK293 cells, data from . P-values from Mann-Whitney U tests. F. Cross-regulation of eMIC-dependent exons by other RNA binding proteins (RBPs) in fly brains, data from . In red/blue, number of exons regulated in the same/opposite direction between each RBP and the eMIC domain at two ΔPSI levels. G. Effect of the double knockout of elav and fne on exon inclusion (PSI) genome-wide in the first instar larval (L1) CNS. Data from . Exons are grouped based on their response to eMIC insufficiency (see Methods). PSI frequency distributions of eMIC-dependent exons in control and mutant CNS are depicted in green. H. Overlap between eMIC-dependent exons, neural-enriched exons and Elav/Fne up-regulated exons. The stage of the samples used for the definition of each exon group is indicated. I. Sashimi plot of RNA-seq data from L1 CNS upon knockdown of elav and fne . Pink arrows indicate putative binding motifs of Elav. Data from . J,K. RT-PCR assays of Srrm234 terminal exons from wild-type (wt) and elav -hypomorphic ( elav edr ) larval eye imaginal discs (J) and from SL2 cells upon overexpression of Elav (K). Blue triangles mark primer positions. The main splicing products corresponding to annotated isoforms from the Srrm234 gene (A, C/F, G) are labelled. Non-labelled bands correspond to intermediate splicing products or unspecific amplification that do not differ substantially between samples. wt: wild-type.

Article Snippet: The eMIC-null allele for Srrm234 ( CG7971 ) was generated by GenetiVision CRISPR gene targeting services.

Techniques: MANN-WHITNEY, Sequencing, RNA Binding Assay, Double Knockout, Genome Wide, Mutagenesis, RNA Sequencing Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Over Expression, Amplification

Journal: bioRxiv

Article Title: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

doi: 10.1101/2021.02.24.432780

Figure Lengend Snippet: A. Inclusion of eMIC-dependent exons in PSI (percent spliced in) upon perturbation of heph (Ptbp1/2/3 Drosophila ortholog) expression levels. Left, heph knockdown (KD) and control Drosophila embryos from . Right, Ptbp1/2 double knockdown (DKD) and control mouse embryonic stem cells (ESCs) from . B,C. Expression of RNA binding proteins (C) and change in inclusion levels (dPSI) for different types of exons upon knockdown of an array of RBPs in Drosophila SL2 cells (D). Data from modENCODE or (the latter are marked with an asterisk). FC: fold-change relative to control KD samples, eMICdep: eMIC-dependent exons, eMICsens: eMIC-sensitive exons, ASE: other alternatively spliced exons. In boxplots, centre of the box marks median values, box limits mark interquartile ranges (IQR) and whiskers 1.5 IQR. D,E . Expression of RBPs (E) and AS at the 3′ end of Srrm234 (E) upon KD of several RBPs in Drosophila adult brains. Data from . F. 3′ seq reads at the Srrm234 locus upon perturbation of elav/fne levels: overexpression in SL2 cells or knockout in L1 larva central neural system (CNS). pA: poly-adenylation site, ctrl: control, rep: replicate. Data from . G. Elav expression in SL2 cells detected by western blot. Ponceau staining was used as loading control. H. Elav iCLIP tags at the Srrm234 3’end region from adult heads. Data from . Bottom, annotated Srrm234 isoforms based on AS and poly-adenylation at this region.

Article Snippet: The eMIC-null allele for Srrm234 ( CG7971 ) was generated by GenetiVision CRISPR gene targeting services.

Techniques: Expressing, RNA Binding Assay, Over Expression, Knock-Out, Western Blot, Staining

Journal: bioRxiv

Article Title: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals

doi: 10.1101/2021.02.24.432780

Figure Lengend Snippet: A. Heatmap for the inclusion levels of eMIC-dependent exons in L1 and L3 CNS and adult brains for control and mutant flies. PSI: Percent Spliced In. B. Comparison of the PSI values of eMIC-dependent exons in larval central neural system (CNS) and adult brains. Error bar ends indicate the difference between male and female samples. Data generated in this study. C. PSI values of eMIC exons in L1 and L3 larva central neural system (CNS). L1 data from , error bars representing the PSI range across replicates. L3 data, from FlyAtlas 2 and this study, error bar ends indicating PSI values in each source. D. eMIC-dependent exon inclusion across cell types in the Drosophila adult optic lobe. Data from . PSI: percent spliced in. NC: no sufficient read coverage. F. Alluvial plot depicting the overlap between groups of eMIC-dependent exons based on their inclusion profile across tissues (left) or neural cell types (right). E. Inclusion levels (scaled for each exon) of all alternatively spliced (AS) exons among different cell types in the fly optic lobe. Sample sources are listed in Table S1. G. Sample to sample correlation (corr.) distance matrix for different cell types in the fly optic lobe using either all AS exons (left) or eMIC-dependent exons only (right). PR: photoreceptors, KC: Kenyon cells, OCT: octopaminergic neurons, nSyb: all neurons. H. Alternative last exon usage and gene expression levels of Srrm234 (grey line) across cell types in the optic lobe. Data from . RPKM: corrected reads per kilobase per million mapped reads.

Article Snippet: The eMIC-null allele for Srrm234 ( CG7971 ) was generated by GenetiVision CRISPR gene targeting services.

Techniques: Mutagenesis, Generated, Expressing

Journal: Biochemical and Biophysical Research Communications

Article Title: Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

doi: 10.1016/j.bbrc.2007.10.026

Figure Lengend Snippet: ES-MS and -MS/MS analysis of authentic acetyl- p ABAglu. (A) Negative ion ES-MS spectrum of authentic acetyl- p ABAglu, giving a [M-H] − ion of m / z 307.06. Inset; chemical structure of the [M-H] − ion of acetyl- p ABAglu showing exact mass. (B) MS/MS of the ion of [M-H] − ion ( m / z 307) from authentic acetyl- p ABAglu. (C) Negative ion ES-MS spectrum of urine from Nat2 +/+ mice given dietary folate supplement. (D) MS/MS of the ion of [M-H] − ion ( m / z 307) from urine of Nat2 +/+ mice given dietary folate supplement. See for interpretation of fragment ions.

Article Snippet: The Nat2 ∗null allele was bred from a129/Ola background onto two different genetic backgrounds (A/J and C57Bl/6, supplied by Harlan, UK) by backcrossing over ten generations.

Techniques: Tandem Mass Spectroscopy

Journal: Biochemical and Biophysical Research Communications

Article Title: Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

doi: 10.1016/j.bbrc.2007.10.026

Figure Lengend Snippet: MS/MS analysis of the ion of m/z 307 in the formic acid fraction from the anion-exchange column from urine of Nat2 +/+ and Nat2 −/− mice

Article Snippet: The Nat2 ∗null allele was bred from a129/Ola background onto two different genetic backgrounds (A/J and C57Bl/6, supplied by Harlan, UK) by backcrossing over ten generations.

Techniques:

Journal: Biochemical and Biophysical Research Communications

Article Title: Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

doi: 10.1016/j.bbrc.2007.10.026

Figure Lengend Snippet: Wholemount views of embryos showing neural tube defects. Embryos derived by crossing C57Bl/6 Nat2 +/+ and Nat2 −/− mice, and harvested at e10.5–e11.5. (A) Normal embryo (e11.5) viewed from right hand side. (B) Embryo (e11.5) with neural tube defect, showing incomplete closure of the anterior neuropore, viewed from right hand side in main panel; inset, dorsal view. (C) Embryo (e10.5) with neural tube defect at cervical level, viewed from right in main panel; inset, dorsal view. Arrowheads point to regions of incomplete closure of the neural tube. The orientation of the embryos is indicated in dorsal views; C, caudal; R, rostral.

Article Snippet: The Nat2 ∗null allele was bred from a129/Ola background onto two different genetic backgrounds (A/J and C57Bl/6, supplied by Harlan, UK) by backcrossing over ten generations.

Techniques: Derivative Assay