mnope ecd  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: WFIKKN2 is a bifunctional axon guidance cue that signals through divergent DCC family receptors

doi: 10.1101/2023.06.15.544950

Figure Lengend Snippet: (A) Domain structure of murine (m)DCC family receptors and Netrin-1, drawn to scale. TM, transmembrane domain. (B) Brachial spinal cord transverse sections from E10.5, E11.5, and E12.5 mouse embryos were used for chromogenic in situ hybridization to detect Punc, Nope, and Prtg mRNA. All three genes show strong expression in DRGs (arrowheads) and the ventral horn (arrows) beginning at E11.5, with expression of Punc in ventral horn slightly delayed. Punc is also expressed in the progenitor zone (asterisk) at E10.5; all three receptors are expressed in the progenitor zone at E11.5 (C) An extracellular protein microarray was screened using Punc-ECD-Fc, Nope-ECD-Fc, and Prtg-ECD-His 6 as baits, revealing that all three receptors bind WFIKKN2. Black dots represent results for individual prey proteins from duplicate screening (arrays 1, 2). (D) Domain structure of mWFIKKN2. (E) Interactions of DCC family receptors with Netrin-1 and WFIKKN2 were examined in a COS-7-based binding assay. Netrin-1-AP specifically binds cells expressing DCC or Neo1, but not cells expressing Punc, Nope, or Prtg. Conversely, WFIKKN2-AP binds Punc-, Nope-, or Prtg-expressing cells, but not cells expressing DCC or Neo1. (F) WFIKKN2-Punc/Nope/Prtg binding was studied by SPR. Shown are SPR sensograms and Langmuir isotherms of steady-state responses fitted to a specific binding model for mWFIKKN2 as ligand on sensor chips with mPunc, mNope, or mPrtg ECDs as analytes at indicated concentrations. K D values are indicated, including standard error of the model fit. (G) Brachial-level transverse sections of E10.5, E11.5, and E12.5 embryos were used for RNAScope fluorescent in situ hybridization to detect WFIKKN2 mRNA. WFIKKN2 is expressed in tissue (yellow brackets) surrounding the spinal cord and DRG (red and orange dotted outlines, respectively) from E11.5 onwards. Asterisks indicate non-specific labeling from blood vessels. Scale bars, (B) 200 μm; (E) 50 μm; (G) 100 μm. Error bars indicate SEM.

Article Snippet: For the protein interaction screen, mPunc-ECD-Fc, a fusion of mNope ECD (G22-H953) to human Fc (Biotechne R&D Systems 1394-NP), and a fusion of mPrtg ECD (F36-A952) to a His 6 tag (Biotechne R&D Systems 6795-PR) were used as baits.

Techniques: Chromogenic In Situ Hybridization, Expressing, Microarray, Binding Assay, In Situ Hybridization, Labeling

Journal: bioRxiv

Article Title: WFIKKN2 is a bifunctional axon guidance cue that signals through divergent DCC family receptors

doi: 10.1101/2023.06.15.544950

Figure Lengend Snippet: (A) Domain structure of murine (m)DCC family receptors and Netrin-1, drawn to scale. TM, transmembrane domain. (B) Brachial spinal cord transverse sections from E10.5, E11.5, and E12.5 mouse embryos were used for chromogenic in situ hybridization to detect Punc, Nope, and Prtg mRNA. All three genes show strong expression in DRGs (arrowheads) and the ventral horn (arrows) beginning at E11.5, with expression of Punc in ventral horn slightly delayed. Punc is also expressed in the progenitor zone (asterisk) at E10.5; all three receptors are expressed in the progenitor zone at E11.5 (C) An extracellular protein microarray was screened using Punc-ECD-Fc, Nope-ECD-Fc, and Prtg-ECD-His 6 as baits, revealing that all three receptors bind WFIKKN2. Black dots represent results for individual prey proteins from duplicate screening (arrays 1, 2). (D) Domain structure of mWFIKKN2. (E) Interactions of DCC family receptors with Netrin-1 and WFIKKN2 were examined in a COS-7-based binding assay. Netrin-1-AP specifically binds cells expressing DCC or Neo1, but not cells expressing Punc, Nope, or Prtg. Conversely, WFIKKN2-AP binds Punc-, Nope-, or Prtg-expressing cells, but not cells expressing DCC or Neo1. (F) WFIKKN2-Punc/Nope/Prtg binding was studied by SPR. Shown are SPR sensograms and Langmuir isotherms of steady-state responses fitted to a specific binding model for mWFIKKN2 as ligand on sensor chips with mPunc, mNope, or mPrtg ECDs as analytes at indicated concentrations. K D values are indicated, including standard error of the model fit. (G) Brachial-level transverse sections of E10.5, E11.5, and E12.5 embryos were used for RNAScope fluorescent in situ hybridization to detect WFIKKN2 mRNA. WFIKKN2 is expressed in tissue (yellow brackets) surrounding the spinal cord and DRG (red and orange dotted outlines, respectively) from E11.5 onwards. Asterisks indicate non-specific labeling from blood vessels. Scale bars, (B) 200 μm; (E) 50 μm; (G) 100 μm. Error bars indicate SEM.

Article Snippet: Recombinant mWFIKKN2 (Biotechne/R&D Systems, 2070-GS) was applied (at indicated concentrations) to the Dunn chamber outer well, and ≍30-40 visual fields covering the bridge region of each chamber were imaged repeatedly over 2 h. For Nope-ECD-Fc blocking experiments, recombinant mNope-ECD-Fc protein (Biotechne/R&D Systems, 1294-NP) was added at 25 μg/ml (≍ 0.2 μM) to WFIKKN2-containing media (100 ng/ml ≍ 2 nM for sensory neurons, 1000 ng/ml ≍ 0.02 μM for motor neurons) immediately prior to Dunn Chamber assembly.

Techniques: Chromogenic In Situ Hybridization, Expressing, Microarray, Binding Assay, In Situ Hybridization, Labeling