Journal: bioRxiv

Article Title: The temporal refinement of Dach1 is a key step in the functional maturation of primary somatosensory neurons

doi: 10.1101/2024.07.18.604081

Figure Lengend Snippet: (A) Triple immunostaining of Dach1, Prdm12 and TrkA performed on E12.5 and E16.5 DRG transverse sections of control embryos. (B) Left, double immunostaining of Dach1 and Islet1 performed on E18.5 DRG transverse sections of control and NGF -/- ; Bax -/- embryos. Note the maintenance of Dach1 restricted expression pattern in absence of NGF . Right, Immunostaining targeting Ret performed as a positive control showing the proper inactivation of the NGF signalling pathway in NGF -/- ; Bax -/- E18.5 DRG. (C) Immunostaining targeting Prdm12 performed on E18.5 DRG transverse sections of control and NGF -/- ; Bax -/- embryos. Note the maintained expression of Prdm12 in absence of NGF. (D) Double immunostaining of Dach1 and Islet1 performed on E18.5 DRG transverse sections of control and Scn10a Cre/+ ; Prdm12 flox/flox embryos. Note the broadening of Dach1 + cells in absence of Prdm12. (E) Quantification analysis of the mean percentage of Islet1 + neurons expressing Dach1 in DRG of E18.5 control and Scn10a Cre/+ ; Prdm12 flox/flox (CKO) embryos. Mann-Whitney tests. (F) Schematic representation of the CRISPR-Cas9-based strategy used to generate the Prdm12 KI allele. (G) Left, double immunostaining of Prdm12 and V5 performed on E12.5 spinal cord transverse sections of Prdm12 +/+ and Prdm12 KI/+ embryos. Right, quantification analysis of the mean percentage of V5 + nuclei expressing Prdm12 in E12.5 spinal cords of Prdm12 +/+ and Prdm12 KI/+ embryos. (H) Left, double immunostaining of Prdm12 or TrkB with V5 performed on E12.5 DRG transverse sections of Prdm12 +/+ and Prdm12 KI/+ embryos or Prdm12 KI/+ embryos alone. Top right, quantification analysis of the mean percentage of V5 + nuclei expressing Prdm12 in E12.5 DRG of Prdm12 +/+ and Prdm12 KI/+ embryos. Bottom right, quantification analysis of the mean percentage of V5 + nuclei found in TrkB + neurons in E12.5 DRG of Prdm12 KI/+ embryos. Bottom, colocalization analysis of Prdm12 and V5 in a E12.5 DRG cell counterstained with DAPI (scale bar, 5µm). (I) Mouse genomic region surrounding Dbx1 (top) or Dach1 (middle) showing sequencing data for input DNA recovered from Prdm12 +/+ and Prdm12 KI/+ and V5 ChIP-enriched DNA recovered from Prdm12 +/+ and Prdm12 KI/+ E12.5 spinal cord with attached DRG. Bottom trace view shows that the peak observed at E12.5 is still found following V5 targeted ChIP-seq performed on isolated E13.5 DRG. Peaks of interest are highlighted by red arrowheads. The homologous Prdm12 bound region near Dbx1 previously characterized in Xenopus laevis is highlighted by a yellow band. (J) ChIP-qPCR validation of the peak identified in the vicinity of the Dbx1 locus performed on dissected spinal cord and DRG of E12.5 embryos of indicated genotypes. Graphical data in this panel and in the subsequent ones are presented as mean ± SEM. One way ANOVA test with Dunnett’s post hoc multiple comparison. (K) ChIP-qPCR validation of the peak identified in the fourth intron of the Dach1 gene performed on dissected spinal cord and DRG of E12.5 embryos of indicated genotypes. One way ANOVA test with Dunnett’s post hoc multiple comparison. DRG in pictures are delineated by white dashed lines. Scale bars, 100 µm.

Article Snippet: 2.4 pmol/μl of annealed crRNA (10nM, Integrated DNA Technologies, 5’-GGCGCTCACAGCACCATGGC-3’) and the tracrRNA were injected with 200 ng/μl of recombinant Cas9 protein (Integrated DNA Technologies) and 10 ng/μl of a single-stranded DNA template (Megamer® single-stranded DNA fragment, GenScript) into the pronucleus of B6D2F2 zygotes which were subsequently transferred to pseudopregnant CD1 mice.

Techniques: Triple Immunostaining, Control, Double Immunostaining, Expressing, Immunostaining, Positive Control, MANN-WHITNEY, CRISPR, Sequencing, ChIP-sequencing, Isolation, ChIP-qPCR, Biomarker Discovery, Comparison