Fig. 5: EC-derived Netrin-B controls EB protrusion formation and attracts their migration.
From: Frazzled/DCC directs spatial progenitor integration ensuring steady-state intestinal turnover
Midguts (R5) with esg>CD8::GFP (ISC/EB) in Net–/+,NetB–/+ heterozygous mutant background (Control, A) and NetA–/–,NetB–/+ (B) and NetA–/+,NetB–/– (C) mutant backgrounds. Scale bar is 20 µm. D Quantification of ISC/EB protrusion lengths (p = 0.1295 for NetA–/–,NetB+/+). E KluReDDMCas9 control (w1118) showing an old EC (white arrowhead, RFP-) positive for anti-NetB staining. Scale bar is 10 µm. Expression levels of fra, NetA and NetB measured by RT-qPCR in midgut-derived cDNA (F, H) after feeding of dioctyl sodium sulfosuccinate (DSS) (F, p = 0.8294;p = 0.0357;p = 0.0031), upon bacterial infection analyzed by RNA-Seq following FAC-sorting48 (G), and upon expression of >p35 in EC (mex > p35) (H, p = 0.0006). I Schematic illustration of EC-Rapport-tracing combining EC-specific mex> with independent lexA/Aop-driven esglexReDDM-tracing. esg» drives expression of »CD8::GFP with short half-life and »H2B::mCherry with longer half-life persisting also in newly differentiated cells. Both expression systems are temporally controlled by Gal80ts. Midguts (R5) after seven days (J–L), three days (M, N) and 24 h (O, P) of EC-Rapport-tracing in controls (w1118) (J, M, O), with expression of >p35 (K), >NetB-RNAi (L), >E-cad-RNAi (N), and >NetB (P). Quantification of protrusion lengths (Q) and numbers (R, p = 0.2202 for >NetB-RNAi;p = 0.0216 for >E-cad-RNAi;p = 0.0273 for >NetB). (S-T’) Midguts (R5) after three days of EC-Rapport-tracing in controls (w1118) (S-S’) and upon expression of >E-cad-RNAi (T-T’) with antibody staining targeting NetB and Dlg1. Scale bars are 20 µm. U Quantification of NetB intensities in EC (p = 0.0073). Midguts (R5) after 14 days of EC-Rapport-tracing in controls (w1118) (V) and upon knockdown of NetB (W). Scale bar is 20 µm. X Quantification of rEC renewal frequency (p = 0.0021). F, H, R, X Box plots show 25th and 75th percentiles and whiskers indicate the range of values. D, Q Bar plots show means and range indicated by whiskers. U Scatter dot plots show all data points with indication of means and standard deviations. All data points are shown by dots and means are indicated by light blues lines. ‘n’ are numbers of biological replicas and ‘N’ values describe numbers of technical replicas. Asterisks denote significances from multiple comparisons by Kruskal Wallis tests (D, F, Q, R) and two-sided Mann–Whitney tests (H, Q, R, U, X) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
