Journal: EMBO Reports
Article Title: A low-level Cdkn1c/p57 kip2 expression in spinal progenitors drives the transition from proliferative to neurogenic modes of division
doi: 10.1038/s44319-025-00653-9
Figure Lengend Snippet: ( A ) mRNA expression of Cdkn1c in chick embryonic neural tube after electroporation of each of the six shRNAs. In situ hybridization and GFP immunofluorescence on the same cryosection of thoracic region of chick embryo. Upper panel: visible downregulation of Cdkn1c mRNA was only observed with shRNA1 (sh1) and to a lesser extent shRNA4 (sh4) conditions, while comparable mRNA expression to the control condition (CTRL sh) was observed with the other shRNAs (sh2,3,5,6) (compare left versus right hemitubes). Lower panel: Corresponding level of electroporation for each embryo (GFP immunofluorescence). Scale bar: 50 µm. ( B ) Endogenous Cdkn1c-Myc protein expression pattern at E3,25 (30hae) using guide #1 to target the Cdkn1c locus upon downregulation of Cdkn1c via shRNA approach. Top row: Control sh, bottom row: Cdkn1c sh. Somatic knock-in of Myc tags at the Cdkn1c locus allows the visualization of Cdkn1c protein using an anti-Myc immunofluorescence (Myc, red) on E3,25 transverse vibratome sections. A H2B-GFP construct (green) was used as a control of electroporation. Cells with a knock-in event were visualized thanks to the coelectroporation of a UAS:H2B miRFP construct (magenta). Scale bar: 100 µm. The graduated scales in the third column illustrate the bins used for quantification in ( C ). ( C ) Quantification of Cdkn1c-Myc expression along the apico-basal axis upon downregulation of Cdkn1c via shRNA approach. Quantifications of the Cdkn1c-Myc signal intensity from knock-in insertions were performed in vibratome sections from embryos electroporated with a control shRNA or Cdkn1c shRNA1 (see representative examples in ( B )). Left graph: the Myc signal intensity measured in individual cells is plotted on the Y-axis towards the upper (CTRL sh) and lower (Cdkn1c sh1) parts of the graph as a function of their position along the apico-basal axis (X-axis). Right graph: bars represent the average Myc signal intensity in 25µm-wide bins along the apico-basal axis. CTRL sh: 347 cells from 5 embryos; Cdkn1c sh1: 233 cells from 5 embryos. Error bars show means ± SD; **** P < 0.0001 (Kolmogorov-Smirnov test). ( D ) Distribution of pRb-positive progenitors (red) in Cdkn1c shRNAs 1, 4, 5, 6 or control shRNA conditions at E4, 48 hae. (values for CTRL sh and Cdkn1c sh1 are identical to Fig. ). Error bars show means ± SD. CTRL vs sh1 and CTRL vs sh4, **** P < 0.0001; CTRL vs sh5 and CTRL vs sh6, * P = 0.0365; (unpaired Student’s t test relative to CTRL sh). Numbers of counted electroporated cells: sh4, 3111 cells from 8 embryos; sh5, 1552 from 3 embryos; sh6, 931 cells from 3 embryos were analyzed. ( E ) Distribution of the HuC/D-positive neurons (magenta) in shRNAs 1, 4, 5, 6 or control conditions at E4, 48 hae. (values for CTRL sh and Cdkn1c sh1 are identical to Fig. ). Error bars show means ± SD; CTRL vs sh1, **** P < 0.0001. All others: ns, P > 0.05; (unpaired Student’s t test relative to CTRL sh). Numbers of counted electroporated cells: sh4, 3111 cells from 8 embryos; sh5, 1552 from 3 embryos; sh6, 931 cells from 3 embryos were analyzed. ( F ) Cdkn1c knock-down did not induce cell death. Transverse vibratome sections of embryos 24 hae with a CTRL sh (left column) or Cdkn1c sh1 (right column). Immunostaining with an anti-Caspase3 antibody (red, 3rd row) show a signal in a few cells on the electroporated side (H2B-GFP, 2 nd row), which is similar in both conditions. Top row: DAPI staining. Scale bar: 100 µm. ( G , H ) Cdkn1c knock-down did not induce ectopic localization of progenitors in the mantle zone. Transverse vibratome sections of embryos 24 hae (E3, ( E )) or 48 hae (E4, ( F )) with a CTRL sh (left column) or Cdkn1c sh1 (right column). Electroporated cells are marked with H2B-GFP (green, 2nd row). Immunostaining with an anti-Sox2 antibody (magenta, 3rd row) was restricted to the ventricular zone, and no ectopic cells were observed in the mantle zone. Similarly, mitotic figures labeled with an anti-pH3 antibody (red, 4th row) were restricted to the apical surface. Scale bars: 100 µm. hae hours after electroporation, CTRL control, sh shRNA.
Article Snippet: Primary antibodies used are: chick anti-GFP (GFP-1020—1:2000) from Aves Labs; goat anti-Sox2 (clone Y-17—1:1000) from Santa Cruz; rabbit anti-pRb (Ser807/811— 1:1000) from Cell Signaling; mouse anti-c-myc tag (Clone 9E10—1:100) from Sigma-Aldrich; mouse anti Pax7 (monoclonal, 1:100) from DSHB; rabbit anti-phospho-Histone 3 (Polyclonal—1: 250) from Millipore; rabbit anti-active Caspase3 (Polyclonal—1:300) from R&D systems; mouse anti-HuC/D (clone 16A11—1:50) from Life Technologies.
Techniques: Expressing, Electroporation, In Situ Hybridization, Immunofluorescence, Control, shRNA, Knock-In, Construct, Knockdown, Immunostaining, Staining, Labeling
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