IMAGE

Fig. S1

ID
ZDB-IMAGE-180827-17
Genes
Source
Figures for Ferrero et al., 2018
Image
Figure Caption

Fig. S1

The transcription factor tfec controls EMP-derived hematopoiesis in vivo, related to Figure 2

(A) Quantification of tfec-P1 (NM_001030105.2), tfec-P2 (XM_005164535.2) or tfec P3 (XM_009299965.1) expression in FACS sorted EMPs from lmo2:GFP/gata1:DsRed embryos (27-28hpf). Fold enrichment for each tfec splice variant was calculated from expression levels in whole embryos (27-28hpf) (n=3). Error bars represent mean ± S.E.M.

(B) Schematic diagram of translated proteins from tfec-P3 and a dominant-negative form of tfec (DN-tfec) mRNAs (bottom schematic). QR: glutamine rich domain, MAPK: consensus MAP kinase phosphorylation site, AD: transcriptional activation domain, bHLH: beta helix-loop-helix DNA binding domain, PKA: cAMP-dependant phosphorylation site, N: N terminal domain, C: C terminal domain, aa: amino acids. Schematics are not to scale.

(C) Experimental outline to assess the consequence of tfec modulation on EMP biology in vivo. tfec mRNA (tfec), or DN-tfec were injected in Tg(lmo2:GFP; gata1:DsRed) embryos at the onecell stage. Controls are non-injected (NI). Lmo2+ gata1+ EMPs were quantified by flow cytometry and isolated for gene expression analyses.

(D-D’) Representative FACS plots from NI controls (n=16), +tfec (n=13), +DN-tfec (n=12). Percentages in the top right hand corner represent mean ± S.E.M of EMPs within the gate. Comparative quantification is shown in the d’ panel.

(E) QPCR data showing gene expression in FACS sorted EMPs for NI (n=4 for all, except csf1ra where n=3), +tfec (n=4 for all, except csf1ra where n=3) or DN-tfec (n=3 for all) conditions, where n represents the average of biological triplicates performed. Our results show that tfec loss-of-function strongly affects the expression of myeloid genes in EMPs. Error bars represent mean ± S.E.M.

(F) Quantification of the number of mfap4+ cells (as determined by WISH) present within the tail region at 48hpf for NI (n=46), +tfec (n=28) or +DN-tfec (n=29) conditions. EMP-derived myelopoiesis is impaired in the loss of function condition. Error bars represent mean ± S.E.M.

(G-G’) mfap4 WISH on siblings or tfecnull embryos at 28hpf. (g’) Quantification of mfap4+ primitive macrophages per embryo (one side). Statistical analysis was completed using an unpaired Student’s t-test, comparing NI to either +tfec or DN tfec injected. Data represents mean ± S.E.M. NS: no significance, *: p<0.05. **: p<0.01, ***: p<0.001. NI: non-injected, +tfec: hlztfec injected, +DN-tfec: dominant negative tfec injected.

Figure Data
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Cell Rep.