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Letters to the Editor
electroporated embryos were assessed for ectopic expres- sion of endogenous NPAS4L and of two hemangioblast markers SCL/TAL1 and LMO2. NPAS4L CRISPRa con- structs were able to ectopically activate endogenous NPAS4L (Figure 3D, oval areas) (11 of 12; 92%) in regions that are normally NPAS4L-negative (Figure 2A), as well as hemangioblast markers SCL/TAL1 (9 of 25; 36%) and LMO2 (6 of 21; 29%) (Figure 3E, oval areas in left two panels), albeit with reduced efficiency. Interestingly, sim- ilar inductive effect (5 of 13 for LMO2 and 4 of 9 for SCL/TAL1) was observed when we used zebrafish NPAS4L expression construct4 (cloned into the pCAGGS expression vector) (Figure 3E, oval area in right panel), supporting partial molecular conservation between the zebrafish and chicken NPAS4L genes.
In conclusion, we present evidence that during early chicken development, NPAS4L, instead of ETV2, is involved in hemangioblast formation. Data from our molecular phylogenetic analyses support the hypothesis that both the NPAS4L and ETV2 genes were present in the common reptilian ancestor and likely also in the com- mon amniote ancestor (Figure 3F). A conclusive confir- mation of their epistatic relationship, however, requires additional evidence from gain-of-function of ETV2 (e.g., using a reptilian ETV2 ortholog) and loss-of-function of NPAS4L (e.g., through CRISPR-mediated transcription inhibition) studies. In birds and other reptilian lineages which lack the ETV2 ortholog in their genome, it is pos- sible that other ETS family genes have been co-opted to play hemangioblast-specific roles of ETV2. Because ETV2 and NPAS4L are transcription factors with different DNA binding specificities and co-factor requirements, it remains to be shown how ETV2 took over molecular functions of NPAS4L during early mammalian evolution.
Wei Weng, Hiroki Nagai, Sofiane Hamidi and Guojun Sheng
International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan
Correspondence:
GUOJUN SHENG -sheng@kumamoto-u.ac.jp
doi:10.3324/haematol.2019.239434
References
1. PorcherC,ChagraouiH,KristiansenMS.SCL/TAL1:amultifaceted regulator from blood development to disease. Blood. 2017; 129(15):2051-2060.
2. Morishima T, Krahl AC, Nasri M, et al. LMO2 activation by deacety- lation is indispensable for hematopoiesis and T-ALL leukemogenesis. Blood. 2019;134(14):1159-1175.
3. Kataoka H, Hayashi M, Nakagawa R, et al. Etv2/ER71 induces vas- cular mesoderm from Flk1+PDGFRalpha+ primitive mesoderm. Blood. 2011;118(26):6975-6986.
4. Reischauer S, Stone OA, Villasenor A, et al. Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification. Nature. 2016;535(7611):294-298.
5. Liao EC, Paw BH, Oates AC, Pratt SJ, Postlethwait JH, Zon LI. SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes Dev. 1998;12(5):621-626.
6. Marass M, Beisaw A, Gerri C, et al. Genome-wide strategies reveal target genes of Npas4l associated with vascular development in zebrafish. Development. 2019;146(11).
7. Nagai H, Shin M, Weng W, et al. Early hematopoietic and vascular development in the chick. Int J Dev Biol. 2018;62(1-2-3):137-144.
8. Zon LI. Developmental biology of hematopoiesis. Blood. 1995;
86(8):2876-2891.
9. Shivdasani RA, Orkin SH. The transcriptional control of
hematopoiesis. Blood. 1996;87(10):4025-4039.
10. NakazawaF,NagaiH,ShinM,ShengG.Negativeregulationofprim-
itive hematopoiesis by the FGF signaling pathway. Blood. 2006;
108(10):3335-3343.
11. ShinM,NagaiH,ShengG.NotchmediatesWntandBMPsignalsin
the early separation of smooth muscle progenitors and blood/endothelial common progenitors. Development. 2009; 136(4):595-603.
12. MinkoK,BollerotK,DrevonC,HallaisMF,JaffredoT.Frommeso- derm to blood islands: patterns of key molecules during yolk sac ery- thropoiesis. Gene Expr Patterns. 2003;3(3):261-272.
13. Davidson AJ, Zon LI. The 'definitive' (and 'primitive') guide to zebrafish hematopoiesis. Oncogene. 2004;23(43):7233-7246.
14. Walmsley M, Cleaver D, Patient R. Fibroblast growth factor controls the timing of Scl, Lmo2, and Runx1 expression during embryonic blood development. Blood. 2008;111(3):1157-1166.
15. OrkinSH,ZonLI.Hematopoiesis:anevolvingparadigmforstemcell biology. Cell. 2008;132(4):631-644.
16. ManaiaA,LemarchandelV,KlaineM,etal.Lmo2andGATA-3asso- ciated expression in intraembryonic hemogenic sites. Development. 2000;127(3):643-653.
17. Drake CJ, Fleming PA. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood. 2000;95(5):1671-1679.
18. HamburgerV,HamiltonHL.Aseriesofnormalstagesinthedevel- opment of the chick embryo. J Morphol. 1951;88(1):49-92.
19. WengW,ShengG.FivetranscriptionfactorsandFGFpathwayinhi- bition efficiently induce erythroid differentiation in the epiblast. Stem Cell Reports. 2014;2(3):262-270.
20. LiuF,LiD,YuYY,etal.Inductionofhematopoieticandendothelial cell program orchestrated by ETS transcription factor ER71/ETV2. EMBO Rep. 2015;16(5):654-669.
21. Weng W, Sukowati EW, Sheng G. On hemangioblasts in chicken. PLoS One. 2007;2(11):e1228.
22. LizioM,DeviatiiarovR,NagaiH,etal.Systematicanalysisoftran- scription start sites in avian development. PLoS Biol. 2017;15(9):e2002887.
23. Cheng AW, Wang H, Yang H, et al. Multiplexed activation of endoge- nous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23(10):1163-1171.
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