Fig. 8: Atg5 regulates endothelial to hematopoietic transition through Ncl pathway.

a Comparison analysis of the expression/interactions (Ptn-Ncl and Mdk-Ncl) between ligands from niche cells (excluding EC, pre-HSCs and Mk) and receptors from HEC, pre-HSC I and pre-HSC II in the control and KO group, showing the enhanced Ptn-Ncl and Mdk-Ncl interplays in these three fractions of KO group compared to control cells. b Representative immunostaining data of Ncl in the EC, pre-HSC I (I Pre) and pre-HSC II (II Pre) sorted from E10.5 control (Ctr) and KO AGM regions. Red = Ncl, Blue = Hoe (Hoechst). c–e Fluorescence intensity (FI) in each cell fraction was calculated by image J and hubs (maximum length of each hub over one micrometer) of Ncl signal in the detected cells. Error bars represent mean ± SEM. Each dot represents a cell. EC (n = 32 for control, n = 27 for KO), I Pre (n = 17 for control, n = 29 for KO) and II Pre (n = 26 for control, n = 39 for KO) were examined over 3 independent experiments). **p < 0.001, ***p = 0.0005, ****p < 0.0001. f Experimental set-up for 3 days explant cultures. Adobe Illustrator software was used for drawing this figure. g–i 3 days E10.5 AGM explant cultures showing the rescue function in the percentages of HEC (g) after 3-MA and AS1411 treatment, but not in the pre-HSC I (h) and pre-HSC II (i) fractions. AS = AS1411, the aptamer of Ncl (also known as AGRO100). 3 MA = 3-MA, the inhibitor of autophagy. A + 3 = AS1411 + 3 MA. Error bars represent mean ± SEM. n = 11 embryos over 11 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001. j Total CFU-C number per AGM explant and number of each hematopoietic colony type (indicated by color bars) in the existence of AS1411 and 3-MA. Error bars represent mean ± SEM. n = 7 embryos examined over 7 independent experiments,**p < 0.01. k–m The number of BFU-E per AGM explant was increased in the AS1411 + 3-MA group compared to 3-MA group, but not in the CFU-GM and CFU-GEMM. Error bars represent mean ± SEM. n = 7 embryos over 7 independent experiments, *p < 0.05, **p < 0.01. n Experimental set-up for 1 + 2 days explant cultures. Adobe Illustrator software was used for drawing this figure. o–q The number of PK44 (CD31⁺CD201⁺cKit⁺CD44⁺CD45⁻CD41⁻) and pre-HSC I^plus was rescued by AS1411 in the inhibition of 3-MA, but not in pre-HSC II^plus. n = 3 embryos over 3 independent experiments, *p < 0.05. Statistical significance was determined by one side Student’s t-test.