Journal: Brain

Article Title: A novel disorder reveals clathrin heavy chain-22 is essential for human pain and touch development

doi: 10.1093/brain/awv149

Figure Lengend Snippet: Discovery of a CLTCL1 mutation in individuals with lack of pain or touch sensing and severe intellectual disability. ( A ) Image of an affected child aged 5 years, noting corneal opacity secondary to lack of a corneal reflex. ( B ) Family pedigree with parents being double first cousins and the filled in symbols indicating their affected children (two female and one male). ( C ) The affected individuals were concordant and homozygous for consecutive variants in five autosomal regions. The candidate mutation was localized to one of these regions on chromosome 22. This contained the gene CLTCL1 for which a cartoon of the exon/intron structure is shown. In exon 7 we identified the c.988G>A mutation in CLTCL1 , for which electrophoretograms of the Sanger sequencing results are shown for an affected child homozygous for the mutant A allele, an unaffected carrier parent heterozygous for the wild-type G and mutant A alleles, and a typical control wild-type G allele. The gene mutation resulted in the alteration of an invariant negatively charged glutamic acid 330 to a basic charged lysine in the encoded protein CHC22. The amino acid at position 330 in CHC22 is the first of an invariant pair of acidic amino acids present in all clathrin heavy chain proteins, which is evolutionarily conserved through vertebrates (with two clathrin heavy chains, CHC17 and CHC22, termed 1 and 2, respectively in this figure), insects and amoeba (with a single CHC17 like clathrin heavy chain). Protein sequence data are for human as a representative mammal (CHC22 is NP_009029.3; CHC17 is NP_004850.1), Xenopus tropicalis for amphibians (XP_002931934.2; NP_001011039.1), Danio rerio for fish (XP_005172491.1; XP_005157480.1), Gallus gallus for birds (Human Genome Browser, BLASTp and ACA13364.1; NP_001073586.1), Drosophila melanogaster for flies (NP_477042.1), Caenorhabditis elegans as a nematode (NP_499260.1), and Dictyostelium discoideum , a slime mould, as an amoeba (XP_642717.1). Upper case amino acid nomenclature is for invariance within a class, lowercase for amino acids that vary between species of the class.

Article Snippet: Primary antibodies used include Human Clathrin Heavy Chain 2/CHC22 Polyclonal Sheep IgG Antibody (R&D Systems), Human Clathrin Heavy Chain 1/CHC17 Affinity Purified PAb (R&D Systems) (antibodies were proven not to cross-react with either endogenous or over-expressed protein; see Supplementary Fig. 2 ), Trk (pan) C17F1 Rabbit mAb 4609 (Cell Signalling Technology), anti-TrkB Mouse mAb 610 101 (BD Transduction Laboratories), GFP (D5.1) XP Rabbit mAb #2956 (Cell Signalling Technology), anti-beta Actin (AC-15) antibody (ab6276) (Abcam).

Techniques: Mutagenesis, Sequencing, Control

Journal: Brain

Article Title: A novel disorder reveals clathrin heavy chain-22 is essential for human pain and touch development

doi: 10.1093/brain/awv149

Figure Lengend Snippet: CLTCL1 is highly expressed in the developing human cortex. Heat maps demonstrating the expression profiles of CLTCL1 ( A ) and CLTC ( B ), respectively, throughout development of various regions of the human brain. Note the increased expression of CLTCL1 during early prenatal development and a drop after birth contrasting with the more linear expression profile for CLTC . The colours of the heat map are expression values, transformed to a log2 scale and calculated as a percentage of the highest expression time point for each brain region, respectively. The colour scale ranges from dark blue representing low expression through yellow to dark red representing high expression. The key below demonstrates the colours assigned to each percentage expression. Blank boxes represent where no data were available. ( B ) Graphs comparing the percentage of clathrin heavy chain expression ( CLTC and CLTCL1 ) represented by CLTCL1 in various regions of the cortex. All regions show a significant downregulation in the proportion of clathrin expression attributed to CLTCL1 after birth. On average CLTCL1 was between and 6% of total clathrin before birth and ∼3% after birth. Horizontal bars represent the mean and error bars, the standard error. Significance tests were Student’s t -test. Three stars represent P < 0.0001.

Article Snippet: Primary antibodies used include Human Clathrin Heavy Chain 2/CHC22 Polyclonal Sheep IgG Antibody (R&D Systems), Human Clathrin Heavy Chain 1/CHC17 Affinity Purified PAb (R&D Systems) (antibodies were proven not to cross-react with either endogenous or over-expressed protein; see Supplementary Fig. 2 ), Trk (pan) C17F1 Rabbit mAb 4609 (Cell Signalling Technology), anti-TrkB Mouse mAb 610 101 (BD Transduction Laboratories), GFP (D5.1) XP Rabbit mAb #2956 (Cell Signalling Technology), anti-beta Actin (AC-15) antibody (ab6276) (Abcam).

Techniques: Expressing, Transformation Assay

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) Schematic of cardiac progenitors and their contributions to linear heart tube development from cardiac crescent (E7.75) to looped heart tube (E9) stage. B) Immunofluorescent staining of MEF2C (cyan) and cardiac Troponin T (cTnT, magenta) in E7.75, E8.5, and E9 WT embryos. C) Representative images of WT and MEF2C KO embryos at E7.75, E8.5 and E9. Cardiac progenitors are marked by the Smarcd3 -F6-eGFP reporter transgene (green). BF, brightfield. D) Schematic of the methodology and biological insights presented in the current study. Elements of this panel were created in BioRender. B, B. (2024) https://BioRender.com/i72e213 . Scale bars = 200 μm. FHF, first heart field; aSHF, anterior second heart field; pSHF, posterior second heart field; LV, left ventricle; RV, right ventricle; V, ventricle; IFT, inflow tract; OFT, outflow tract.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Staining

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A-C) UMAPs of snRNA-seq data for cardiac progenitors, cardiomyocytes, and related mesoderm subtypes from E7.75 (A), E8.5 (B), and E9 (C) embryos labeled by cell type (left) and genotype/sample ID (right). D-F) Bar plots displaying the number of up-regulated and down-regulated genes (MEF2C KO-vs-WT) for cell types of interest at E7.75 (D), E8.5 (E), and E9 (F). G-I) Dot plots displaying gene expression of key CM genes and anterior/posterior (A/P) markers at E7.75 (G), E8.5 (H), and E9 (I). J) Fluorescence in situ hybridization of key CM genes and A/P markers in E8-E8.5 WT and MEF2C KO embryos. Scale bars = 200 μm. CMs, cardiomyocytes; FHF, first heart field; SHF, second heart field; JCF, juxtacardiac field; CrM, cranial mesoderm; PrxM, paraxial mesoderm; LPM, lateral plate mesoderm; SoM, somitic mesoderm; NMPs, neuromesodermal progenitors; KPs, kidney progenitors; ExM, extraembryonic mesoderm; HSCs, hematopoietic stem cells; V-CMs, ventricular cardiomyocytes; IFT-CMs, inflow tract cardiomyocytes; OFT-CMs, outflow tract cardiomyocytes; aSHF, anterior second heart field; pSHF, posterior second heart field; PostM, posterior mesoderm; PhM, pharyngeal mesoderm; MixM, mixed mesoderm; A-CMs, atrial cardiomyocytes; AVC-CMs, atrioventricular canal cardiomyocytes; Pe, proepicardium; VP, venous pole; *, Genes known to be associated with CHDs ; #, Direct targets of MEF2C based on MEF2C ChIP-seq data .

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Labeling, Gene Expression, Fluorescence, In Situ Hybridization, ChIP-sequencing

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) UMAP of integrated snRNA-seq and snATAC-seq data for cardiac progenitors, cardiomyocytes, and related mesoderm subtypes at E8.5 labeled by cell types determined from snRNA-seq clustering (left) and genotype/sample ID (right). B) Bar plots displaying the number of gained and lost DARs (MEF2C KO-vs-WT) for cell types of interest at E8.5. C) Venn diagrams displaying unique and overlapping DARs in the three heart tube segments. D) Scatter plots displaying the relationship between the Log2 fold change (Log2FC) values of the DEG and DAR analyses for all identified Peak2Gene (P2G) links in the three heart tube segments. Dots are colored by the P2G correlation score. E-F) Genome browser tracks displaying snATAC-seq accessibility profiles at the Myh6 / Myh7 (E) and Wnt2 (F) loci for the indicated pseudobulked cell types. DARs are highlighted and the peaks are indicated by red bars (MEF2C KO-vs-WT IFT-CMs). Loops indicate P2G links, colored by the P2G correlation score. V-CMs, ventricular cardiomyocytes; IFT-CMs, inflow tract cardiomyocytes; OFT-CMs, outflow tract cardiomyocytes; aSHF, anterior second heart field; pSHF, posterior second heart field; LPM, lateral plate mesoderm; PostM, posterior mesoderm; PhM, pharyngeal mesoderm; NA, cells not available in the snRNA-seq dataset.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Labeling

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) Schematic of selection process to identify candidate MEFC-dependent enhancers from integrated snRNA-seq and snATAC-seq data. B) Genome browser tracks displaying snATAC-seq accessibility profiles, MEF2C ChIP-seq occupancy profiles , and H3K27ac ChIP-seq occupancy profiles at example loci containing IFT-specific (left), V-specific (middle), and OFT-specific (right) candidate enhancers (yellow highlights). C) Genome browser tracks displaying snATAC-seq accessibility profiles, MEF2C ChIP-seq occupancy profiles , and H3K27ac ChIP-seq occupancy profiles at the Myh6 / Myh7 locus, which contains two candidate enhancers (yellow highlights, MVEB1 and MVEB2) with IFT-specific altered accessibility that overlap with regions found in the VISTA Enhancer Browser database . D) Images of E11.5 mouse embryos from the Vista Enhancer Browser database demonstrating positive enhancer activity of regions that overlap with candidates MVEB1 and MVEB2. E) Schematic of the Tol2 Transgenesis assay used to screen candidate enhancers in zebrafish. Elements of this panel were created in BioRender. B, B. (2024) https://BioRender.com/h83r503 . F) Representative ventral view images of zebrafish embryos at 72 hpf injected with candidate enhancers that demonstrated positive activity in the heart. Boxed area in the representative brightfield image (left) indicates the anatomical region of interest captured in the fluorescent images. G) Schematic representation of the observed onset of enhancer activity for candidate enhancers that demonstrated positive activity in the heart. H) Representative ventral view images of zebrafish embryos at 24 and 72 hpf injected with MVEB2:eGFP or MVEB6:eGFP reporter constructs. Scale bars = 100 μm.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Selection, ChIP-sequencing, Activity Assay, Injection, Construct

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) TF binding motif enrichment analysis for gained DARs (MEF2C KO-vs-WT) in IFT-CMs at E8.5. B) Pie charts showing the proportion of gained or lost DARs (MEF2C KO-vs-WT) containing NR and MEF2C motifs or only NR motifs (top), and those containing GATA and MEF2C motifs or only GATA motifs (bottom). C) Odds ratio analysis for NR2F2 target genes (left) and GATA4 target genes (right) amongst DEGs up-regulated in MEF2C KO IFT-CMs. p-values were calculated using Fisher’s exact test. D) Ridge plots displaying module scores for up-regulated NR2F2 targets (left) and GATA4 targets (right) in MEF2C KO and WT heart tube segments. E) Inferred GRNs constructed for MEF2C KO and WT IFT-CMs at E8.5 and E9. F) Schematic of GRN validation by predicting gene expression with Mef2c knockdown in the E8.5 WT network and comparing to measured gene expression changes at E9. G) Results of GRN validation displaying predicted and measured gene expression changes. H) Visualizations of cardiac subnetworks within the WT and MEF2C KO E8.5 IFT-CM GRNs. I-J) Visualizations of direct NR2F2 (I) and GATA4 (J) interactions in the WT and MEF2C KO E8.5 IFT-CM GRNs. Direct interactions that occur upon MEF2C KO are highlighted. #, up-regulated DEG in MEF2C KO IFT-CMs at E8.5; *, Direct target of NR2F2 or GATA4 ( , ) . K-L) Immunofluorescent staining of cardiac Troponin T (cTnT, green) in representative E9.5 embryos (18-24 somites) collected from Mef2c del/+ ; Nr2f2 del/+ to Mef2c del/+ crosses. Boxed regions in (K) are shown at higher magnification in (L). Arrows indicate more expanded ventricle phenotype in Mef2c del/del ; Nr2f2 del/+ embryos. Asterisks indicate better developed and looping atria in Mef2c del/del ; Nr2f2 del/+ embryos. n=5 Mef2c del/del and n=4 Mef2c del/del ; Nr2f2 del/+ embryos from 5 independent litters. Scale bars = 200 μm.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Binding Assay, Construct, Biomarker Discovery, Gene Expression, Knockdown, Staining

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or NLRP1 −/− HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Infection, Knockdown, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) Different NLRP1 promoter regions were cloned into a luciferase reporter vector. At 24 hours after transfection, HeLa cells were treated with BFA for 20 hours before measuring luciferase activity. (B) HeLa cells were transfected with wild-type NLRP1 promoter or a version carrying mutations in the ATF4-binding motif. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (C) The indicated NLRP1 promoter-luciferase vectors were transfected into either wild-type or ATF4 −/− HeLa cells. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (D) ChIP analysis of un-stimulated (DMSO) or BFA-stimulated HeLa cells, followed by qPCR analysis of ATF4 occupancy at the NLRP1 and ATF3 promoter. ATF3 was used as positive control. Each panel is representative of at least three independent experiments.

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Binding Assay, Positive Control