Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Representative RFX family transcription factors are grouped according to functional domains. The first group consists of RFX1, RFX2, and RFX3, which have all the domains, including AD (activation domain), DBD (DNA binding domain), B (domain B), C (domain C), and DIM (dimerization domain). The second group consists of RFX4, RFX6, and RFX8, harboring the domains of DBD, B, C and DIM domains. The third group consists of RFX5 and RFX7, which only have the DBD. The green and red squares mark the start and stop codon positions, respectively. (B) Western blot with indicated antibodies in the whole cochlea at different developmental stages. Different developmental stages include E13.5, E15.5, E17.5, P0, P7, and adult (2M). (C,D,E) Single cell-level expression patterns of the RFX family TFs in three developmental stages in the mouse cochlea. IPhC: inner phalangeal cells/border cells, OC/OSC: Claudius cells/outer sulcus cells, L.PsC lateral prosensory cells, M.PsC medial prosensory cells, Oc90: Oc90+ cells, IPhc/IBC, inner phalangeal cell /inner border cell; ISC/OSC/CC: Claudius cells and inner and outer sulcus cells.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Functional Assay, Activation Assay, Binding Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A-C) Immunofluorescence detection of Rfx3 protein in organ of Corti at different developmental stages. All protein immunostaining signals are in red, nuclei were counterstained with DAPI (blue). Hereafter is the same. (A) Rfx3 expression on E17.5 cochlea section. (B) Rfx3 expression on P7 cochlea section. The white dashed boxed region was magnified and displayed below. Hereafter is the same. (C) Rfx3 expression on adult cochlea section. (D-F) Immunofluorescence detection of Rfx7 proteins in organ of Corti at different stages. (D) Rfx7 expression on E17.5 cochlea section. (E) Rfx7 expression on P7 cochlea section. (F) Rfx7 expression on adult cochlea section. (G-I) RNA in situ detection of Rfx3 gene in organ of Corti at different developmental stages. All the RNA expression signals were labeled in green. The cochlea sections from E17.5 (G), P7 (H), and adult (I) wild-type stages revealed a dynamic expression pattern of Rfx3 gene during the development. (J-L) RNA i n situ detection of Rfx7 gene on the cochlea sections. Rfx7 showed dynamic expression patterns from E17.5 (J), P7 (K), and adult stage (L). The white triangles indicate the position of hair cells. Representative images from n>3 experiments. Scale bars are indicated in the figure (µm).

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Immunofluorescence, Immunostaining, Expressing, In Situ, RNA Expression, Labeling

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Distance of Rfx3 ChIP-seq peaks to TSS positions. (B) Genomic distribution of Rfx3-enriched regions. (C) Localization of the Rfx3-motif within the peak sequence revealed a typical binding feature of transcriptional factor. (D) The most enriched gene ontology (GO) terms of the Rfx3-bound genes. (E) The top 5 most enriched motifs in the Rfx3-bound CREs, were revealed by the Homer Known motif analysis. (F) Rfx3 gene locus view revealed its regulatory features, including deposition of active marker H3K27ac, chromatin accessibility (ATAC-seq), and self-regulation by Rfx3. Two Rfx3-bound regions were indicated by red dashed box at the TSS and intron regions of Rfx3 gene locus. (G) Rfx7 gene locus view revealed its regulatory features, including H3K27ac deposition, chromatin accessibility, Rfx3 and Six1 binding.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: ChIP-sequencing, Sequencing, Binding Assay, Marker

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) The Venn diagram of 1543 common peaks between the E17.5 Rfx3 bound cis-regulatory elements (CREs) at E17.5 and E16.5 H3K27ac enriched regions. (B) There are 2979 common peaks between Rfx3 and hair cell specific ATAC-seq peaks. (C) Venn diagram showed 2501 common peaks between Rfx3 and spiral ganglion neuron (SGN) specific ATAC-seq peaks. (D) Comparisons between E17.5 Rfx3 ChIP-seq, P1 hair cell ATAC-seq, and P7 SGN ATAC-seq peaks together. Clustered heatmaps of Rfx3, H3K27ac, Hair cell ATAC-seq and SGN ATAC-seq peaks respectively within a -3 kb/+ 3 kb window. (E) Strong enrichments of Rfx3, Six1, and ATAC-seq signals in the promoter of Foxj1 genes. (F) Strong binding of Rfx3, however, no obvious deposits of H3K27ac, Six1 and Rfx3 in Dnah5 gene locus. (G) The large majority of 1543 common peaks (A) between Rfx3 and H3k27ac locate in or around the promoter region. (H) The Venn diagram shows that most of 2955 intergenic Rfx3 bound CREs do not have H3K27ac signals.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Single-cell level profiling of hair bundle related gene expresses in the adult cochlea. (B) RNA in situ detections of Triobp (upper panels) and Myo1h (lower panels) genes on cochlear sections in organ of Corti at P8. The Triobp and Myo1h probes were labelled in green, and nuclei were counterstained with DAPI in blue. The triangles indicate hair cells). (C) Genome browser visualization of Rfx3 peaks at the intron of Triobp gene. The RFX and SIX motifs were seen around the peak center. The red dashed box indicates Rfx3 bound regions. (D) Genome browser visualization of Rfx3 peaks at the TSS and intronic regions of Myo1h (E) Transient (G0) transgenic analysis of a 965-bp Rfx3-bound CRE (the red-dashed-boxed-region in ) in Triobp gene at P0 stage. This +40 kb intronic CRE ( Triobp +40000) drives specific expression of GFP reporter in hair cells (in 4/4 transgenic lines). Top panels, images of whole cochlea; middle panels, higher magnification of the partial areas in the top panel; bottom panels, images of cochlear sections showing GFP+ hair cells in the organ of Corti. (F) Transient (G0) transgenic analysis of an 847-bp Rfx3-bound CRE in the Myo1h intronic region (the red-dashed-boxed-region in , right) at P0 stage. The +70 kb intronic CRE ( Myo1h +70000) did not show expression activity in P0 cochlea (5/5, n=5 transgenic lines). Scale bars are indicated in the figure (µm).

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: In Situ, Transgenic Assay, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) The Venn diagram shows that there are 740 common CREs bound by Rfx3 (E17.5) and Six1 (E16.5). (B) SIX motif was one of the top 5 most enriched motifs in the 740 co-bound CREs, as revealed by the Homer Known motif analysis. (C) The Venn diagram shows 138 common peaks between Rfx3 and Foxp1 binding CREs in neural stem cells (NSC). (D) Homer motif analysis of 138 co-bound peaks identifies RFX as one of the most enriched motifs in Foxp1-bound CREs. (E) Single-cell level profiling of the Forkhead family TFs gene expressions in the adult cochlea. (F) The whole amount and section of immunofluorescence showing the expression pattern of Foxp1 in organ of Corti (P7). Top panels, whole cochlea duct. Bottom panels, images of cochlear sections. (G) Vangl2+22000 enhancer contains RFX, SIX and FOX-motifs. (H) Genome browser visualization of Rfx3 peaks at the TSS and intron of Vangl2 , as indicated by the red dashed boxes The tested CRE region is highly conserved as revealed by the conservation score track (black, bottom). (I) Genome browser visualization of Rfx3 peaks at the TSS and intron of Celsr1. Three Rfx3-bound regions were indicated by the red dashed boxes. Scale bars are indicated in the figure (µm).

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Binding Assay, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Rfx3 controls outer hair cell differentiation, maintenance, and hair bundle formation by regulating the expression of Insm1, Ikzf2 , and Triobp genes

doi: 10.1101/2024.09.24.614849

Figure Lengend Snippet: (A) Expression of RFX TFs, and hair cell differentiation and maintenance genes at E16, P7 and adult stages in inner ears. (B) Genome browser visualization of Rfx3 peaks at the Ikzf2 locus. Two Rfx3-bound regions were seen in the upstream intergenic and 3’ TTS regions. Left panels, higher magnification of the areas indicated by dashed lines at TSS; middle panels, whole Ikzf2 locus view; right panels, higher magnification of the upstream intergenic region. The Rfx3 bound CREs were indicated by the red dashed box3s. (C) Genome browser visualization of Rfx3 peaks at the CREs of Insm1 and intron of Tbx2 . (D) The schematic figure describing the dynamic expression pattern of Rfx3 and Rfx7 during inner ear development (left, E17.5; middle, P7; right, adult). The arrows indicate a shift from the transcriptional regulatory role towards a possible immune response function.

Article Snippet: The rabbit anti-RFX antibody from Sigma was raised against 270-405 aa of human RFX3 protein (HPA035689, Sigma) and another anti-RFX3 antibody against 1-413 aa of human RFX3 were both used (14784-1-AP, Proteintech).

Techniques: Expressing, Cell Differentiation

Journal: The Journal of Cell Biology

Article Title: Defective Ca 2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

doi: 10.1083/jcb.200703187

Figure Lengend Snippet: 8-CPT-cAMP treatment restores local Ca 2+ transients and N-type Ca 2+ channel (Ca v 2.2) immunoreactivity in axonal growth cones of Smn − / − ; SMN2 motoneurons. (A) Local Ca 2+ transients in growth cones of Smn- deficient motoneurons ( n = 30) that were cultured for 5 d on laminin-211/221 are restored by 8-CPT-cAMP in comparison with controls ( n = 30). (B) Quantitative analysis of immunoreactivity for Ca v 2.2 and β-actin in 5-d-old control (white bar; n = 30), Smn − / − ; SMN2 (gray bar; n = 30), and 8-CPT-cAMP–treated Smn − / − ; SMN2 motoneurons (checkered bar; n = 30) cultured on laminin-211/221. (C–E) In control motoneurons, Ca v 2.2 (C and D) and β-actin (E) are enriched in axonal growth cones. (F–H) Smn-deficient motoneurons exhibit reduced Ca v 2.2 and β-actin accumulation in comparison to controls. (B [checkered bar] and I–K) The reduction of signal intensity for Ca v 2.2 and β-actin is restored by 100 μM 8-CPT-cAMP. Results represent the mean ± SEM of pooled data from three independent experiments. n , number of motoneurons that were scored in total from control or Smn − / − ; SMN2 embryos. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against tau at 1 μg/ml (1:1,000; Sigma-Aldrich), an N-type Ca 2+ channel (1:200; Sigma-Aldrich), TRPC5 (1:200, Sigma- Aldrich), and TRPC6 (1:200, Chemicon), mouse monoclonal antibodies against 1 μg/ml β-actin (Abcam), 1 μg/ml microtubule-associated protein 2 (Sigma-Aldrich), and 2 μg/ml Smn (BD Biosciences).

Techniques: Cell Culture, Comparison, Control

Journal: The Journal of Cell Biology

Article Title: Defective Ca 2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

doi: 10.1083/jcb.200703187

Figure Lengend Snippet: 8-CPT-cAMP restores axon elongation, growth cone size, and responses to laminin-211/221 in Smn − / − ; SMN2 motoneurons. (A–E) The smaller growth cone size of 7-d-old Smn − / − ; SMN2 motoneurons (A and C) is rescued by 100 μM 8-CPT-cAMP (A [gray checkered bar] and D) and reaches the size of control motoneurons (A, B, and D), also in the presence of CTX (A and E; n = 30 for each genotype with and without 100 μM 8-CPT-cAMP). (F) Ratio of β-actin content in the most distal part of the axon and the most proximal part of the axon. Smn − / − ; SMN2 motor axons ( n = 82) show reduced distal β-actin signal intensity in comparison to control cells ( n = 78). 8-CPT-cAMP treatment of Smn − / − ; SMN2 restores distal β-actin levels (checkered gray bar; n = 90). (G–N) Analysis of actin mRNA levels in axonal growth cones by in situ hybridization in motoneurons from Smn − / − ; SMN2 (G, I, and L; n = 30), 8-CPT-cAMP–treated Smn − / − ; SMN2 (G, J, and M; n = 32), and control embryos (G, H, and K; n = 30). (G, J, and M) 8-CPT-cAMP restores the actin mRNA deficit in axon terminals of Smn − / − ; SMN2 motoneurons. (N) A sense actin probe was used as a negative control for the specificity of the actin probe. (O–R) 8-CPT-cAMP also restores the response of Smn − / − ; SMN2 motor axons to laminin-211/221. Axon elongation after 7 d on laminin-211/221 in control (P; n = 141), Smn − / − ; SMN2 (Q; n = 141) and 8-CPT-cAMP–treated Smn − / − ; SMN2 (R; n = 60) motoneurons. (O) Quantitative analysis of data. Results represent the mean ± SEM of pooled data from three independent experiments. n , number of motoneurons that were scored in total from control, Smn − / − ; SMN2 , or Smn − / − ; SMN2 embryos were treated with 100 μM 8-CPT-cAMP. *, P < 0.05; **, P < 0.001, tested by one-way ANOVA.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibodies against tau at 1 μg/ml (1:1,000; Sigma-Aldrich), an N-type Ca 2+ channel (1:200; Sigma-Aldrich), TRPC5 (1:200, Sigma- Aldrich), and TRPC6 (1:200, Chemicon), mouse monoclonal antibodies against 1 μg/ml β-actin (Abcam), 1 μg/ml microtubule-associated protein 2 (Sigma-Aldrich), and 2 μg/ml Smn (BD Biosciences).

Techniques: Control, Comparison, In Situ Hybridization, Negative Control