Page 159 - 2020_11-Haematologica-web

LETTERS TO THE EDITOR
NPAS4L is involved in avian hemangioblast
specification
Vertebrate primitive hematopoietic and vascular devel- opment is regulated by a conserved set of transcription factors. Their common precursors, the hemangioblasts, express Stem cell leukemia/T-cell acute lymphoblastic leukemia 1 (SCL/TAL1)1 and Lim only protein 2 (LMO2)2 in all vertebrate groups examined. Hemangioblast speci- fication from nascent mesoderm was reported to be less conserved, with Ets variant 2 (ETV2) and Neuronal PAS- domain containing protein 4-like (NPAS4L) identified as its master regulator in mammals3 and zebrafish,4 respec- tively. We show here that the ortholog of NPAS4L, but not of ETV2, is present in the avian genome. Chicken NPAS4L is expressed in hemangioblasts prior to SCL/TAL1 and LMO2. CRISPR-on mediated ectopic expression of endogenous NPAS4L leads to ectopic SCL/TAL1 and LMO2, as with ectopic expression of zebrafish NPAS4L. We propose that the ancestral amniote genome had both NPAS4L and ETV2 genes. The ETV2 gene was lost in the avian lineage without affecting
direct transcriptional regulation of SCL/TAL1 and LMO2 by NPAS4L.5,6 The NPAS4L gene was lost in the mam- malian lineage, with its roles partially replaced by ETV2.
Vertebrate primitive hematopoietic and vascular sys- tems are derived from the mesoderm germ layer.7,8 Lineage specification events taking place between gastru- lation and the onset of circulation are controlled by a set of evolutionarily-conserved transcription regulators.8,9 In birds,10-12 as in fish, amphibians and mammals,13-17 com- mon progenitors of blood and endothelial cells (the hemangioblasts) start to express transcription factors SCL/TAL1 and LMO2 at Hamburger and Hamilton stage 4+ (HH4+),18 soon after their exit from posterior primitive streak where ventral mesoderm cells originate. This is followed by FGFR-mediated segregation of blood and endothelial lineages and functional differentiation of blood cells starting from HH7,10 mediated by a conserved set of transcription factors including SCL/TAL1, LMO2, GATA-binding factor 2 (GATA2), LIM domain-binding protein 1 (LDB1) and transcription factor E2A (E2A).19 After the onset of circulation from HH12/13, the heman- gioblast markers SCL/TAL1 and LMO2 become restricted to the blood and endothelial lineages, respectively.
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haematologica | 2020; 105(11)
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Figure 1. The chicken genome has NPAS4L, but not ETV2 ortholog. (A) A simplified vertebrate phylogenetic tree. (B) Schematic view of blood and endothelial cell differentiation from mesoderm precursors in the streak. NPAS4L and ETV2 are proposed to function during hemangioblast specification in the ventral meso- derm. (C) The chicken genome has the NPAS4L orthologous gene flanked by KLHDC3 gene on one side and RRP36 and TMEM121L genes on the other. Similar syntenic organization is seen in lizard A. carolinensis and zebrafish D. rerio. These genes are missing in mammalian genomes. The chicken genome does not have ETV2 ortholog. The lizard genome has ETV2 and FLI1B as in the zebrafish genome. Mammals have ETV2, but not FLI1B. (It is to be noted that vertebrate genomes have three copies of such tandemly duplicated ETS family genes; not shown). In addition to the ETV2-FLI1B couplet which is the least conserved, the other two couplets (ETS1-FLI1 and ETS2-ERG) are well-conserved. (D) Summary of presence and absence of NPAS4L, ETV2, SCL/TAL1, LMO2 and NPAS4 genes in various vertebrate groups. The following protein sequences were used for comparison. For SCL/TAL1: NP_001274276.1 (human), NP_001274317.1 (mouse), XP_001374963.3 (opossum), DNA clone XX-200B24 (platypus), NP_990683.1 (chicken), XP_030427307.1 (desert turtle; sequence in Chinese soft-shell turtle is incomplete), XP_008114556.1 (lizard), NP_001081746.1 (Xenopus) and NP_998402.1 (zebrafish); for LMO2: AAH42426.1 (human), AAH57880.1 (mouse), XP_027693653.1 (opossum), XP_028917173.1 (platypus), AAL78036.1 (chicken), XP_030415938.1 (turtle), XP_003225211.1 (lizard), NP_001081112.1 (Xenopus) and AAH93136.1 (zebrafish); for ETV2: NP_055024.2 (human), NP_031985.2 (mouse), XP_007491908.1 (opossum), XP_028921116.1 (platypus), XP_008119144.1 (lizard), NP_001089600.1 (Xenopus) and NP_001032452.1 (zebrafish); for NPAS4L: EntrezID 101750093 (chicken), XP_008103134.1 (lizard), XP_008165306.1 (turtle) and NP_001316841.1 (zebrafish).