During ontogeny, fetal liver (FL) acts as a major site for hematopoietic stem cell (HSC) maturation and expansion, whereas HSCs in the adult bone marrow (ABM) are largely quiescent. HSCs in the FL possess faster repopulation capacity as compared with ABM HSCs. However, the molecular mechanism regulating the greater self-renewal potential of FL HSCs has not yet extensively been assessed. Recently, we published RNA sequencing-based gene expression analysis on FL HSCs from 14.5-day mouse embryo (E14.5) in comparison to the ABM HSCs. We reanalyzed these data to identify key transcriptional regulators that play important roles in the expansion of HSCs during development. The comparison of FL E14.5 with ABM HSCs identified more than 1,400 differentially expressed genes. More than 200 genes were shortlisted based on the gene ontology (GO) annotation term “transcription.” By morpholino-based knockdown studies in zebrafish, we assessed the function of 18 of these regulators, previously not associated with HSC proliferation. Our studies identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish. In conclusion, we demonstrate that identification of genes involved in transcriptional regulation differentially expressed between expanding FL HSCs and quiescent ABM HSCs, uncovers novel regulators of HSC function.
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References
1.
MorrisonSJ, UchidaN and WeissmanIL. (1995). The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol, 11:35–71.
2.
EmaH and NakauchiH. (2000). Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood, 95:2284–2288.
3.
BowieMB, KentDG, DykstraB, McKnightKD, McCaffreyL, HoodlessPA and EavesCJ. (2007). Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties. Proc Natl Acad Sci U S A, 104:5878–5882.
4.
PietrasEM, WarrMR and PassegueE. (2011). Cell cycle regulation in hematopoietic stem cells. J Cell Biol, 195:709–720.
5.
IvanovaNB, DimosJT, SchanielC, HackneyJA, MooreKA and LemischkaIR. (2002). A stem cell molecular signature. Science, 298:601–604.
6.
McKinney-FreemanS, CahanP, LiH, LacadieSA, HuangHT, CurranM, LoewerS, NaveirasO, KathreinKL, et al. (2012). The transcriptional landscape of hematopoietic stem cell ontogeny. Cell Stem Cell, 11:701–714.
7.
MatsuokaS, EbiharaY, XuM, IshiiT, SugiyamaD, YoshinoH, UedaT, ManabeA, TanakaR, et al. (2001). CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages. Blood, 97:419–425.
8.
SatoT, LaverJH and OgawaM. (1999). Reversible expression of CD34 by murine hematopoietic stem cells. Blood, 94:2548–2554.
9.
CopleyMR, BabovicS, BenzC, KnappDJ, BeerPA, KentDG, WohrerS, TreloarDQ, DayC, et al. (2013). The Lin28b-let-7-Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells. Nat Cell Biol, 15:916–925.
10.
KimI, SaundersTL and MorrisonSJ. (2007). Sox17 dependence distinguishes the transcriptional regulation of fetal from adult hematopoietic stem cells. Cell, 130:470–483.
11.
HeS, KimI, LimMS and MorrisonSJ. (2011). Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors. Genes Dev, 25:1613–1627.
12.
ChenX, ZhangJ, ZhaoJ, LiuH, SunX, ZhaoM and LiuX. (2014). Lis1 is required for the expansion of hematopoietic stem cells in the fetal liver. Cell Res, 24:1013–1016.
13.
ZimdahlB, ItoT, BlevinsA, BajajJ, KonumaT, WeeksJ, KoechleinCS, KwonHY, AramiO, et al. (2014). Lis1 regulates asymmetric division in hematopoietic stem cells and in leukemia. Nat Genet, 46:245–252.
14.
BurnsCE, DeBlasioT, ZhouY, ZhangJ, ZonL and NimerSD. (2002). Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators. Exp Hematol, 30:1381–1389.
15.
ThompsonMA, RansomDG, PrattSJ, MacLennanH, KieranMW, DetrichHW3rd, VailB, HuberTL, PawB, et al. (1998). The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev Biol, 197:248–269.
16.
BurnsCE, TraverD, MayhallE, ShepardJL and ZonLI. (2005). Hematopoietic stem cell fate is established by the Notch-Runx pathway. Genes Dev, 19:2331–2342.
17.
LeeY, ManegoldJE, KimAD, PougetC, StachuraDL, ClementsWK and TraverD. (2014). FGF signalling specifies haematopoietic stem cells through its regulation of somitic Notch signalling. Nat Commun, 5:5583.
18.
HeC and ChenX. (2005). Transcription regulation of the vegf gene by the BMP/Smad pathway in the angioblast of zebrafish embryos. Biochem Biophys Res Commun, 329:324–330.
19.
GeringM and PatientR. (2005). Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos. Dev Cell, 8:389–400.
20.
Kalev-ZylinskaML, HorsfieldJA, FloresMV, PostlethwaitJH, VitasMR, BaasAM, CrosierPS and CrosierKE. (2002). Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis. Development, 129:2015–2030.
21.
BoissetJC, van CappellenW, Andrieu-SolerC, GaljartN, DzierzakE and RobinC. (2010). In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature, 464:116–120.
22.
KumanoK, ChibaS, KunisatoA, SataM, SaitoT, Nakagami-YamaguchiE, YamaguchiT, MasudaS, ShimizuK, et al. (2003). Notch1 but not Notch2 is essential for generating hematopoietic stem cells from endothelial cells. Immunity, 18:699–711.
23.
ManesiaJK, XuZ, BroekaertD, BoonR, van VlietA, EelenG, VanweldenT, StegenS, Van GastelN, et al. (2015). Highly proliferative primitive fetal liver hematopoietic stem cells are fueled by oxidative metabolic pathways. Stem Cell Res, 15:715–721.
24.
LoveMI, HuberW and AndersS. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol, 15:550.
25.
Tabas-MadridD, Nogales-CadenasR and Pascual-MontanoA. (2012). GeneCodis3: a non-redundant and modular enrichment analysis tool for functional genomics. Nucleic Acids Res, 40:W478–W483.
26.
KimmelCB, BallardWW, KimmelSR, UllmannB and SchillingTF. (1995). Stages of embryonic development of the zebrafish. Dev Dyn, 203:253–310.
27.
ThisseC and ThisseB. (2008). High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc, 3:59–69.
28.
AndersS and HuberW. (2010). Differential expression analysis for sequence count data. Genome Biol, 11:R106.
29.
AshburnerM, BallCA, BlakeJA, BotsteinD, ButlerH, CherryJM, DavisAP, DolinskiK, DwightSS, et al. (2000). Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet, 25:25–29.
30.
EckfeldtCE, MendenhallEM, FlynnCM, WangTF, PickartMA, GrindleSM, EkkerSC and VerfaillieCM. (2005). Functional analysis of human hematopoietic stem cell gene expression using zebrafish. PLoS Biol, 3:e254.
31.
MurayamaE, KissaK, ZapataA, MordeletE, BriolatV, LinHF, HandinRI and HerbomelP. (2006). Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development. Immunity, 25:963–975.
32.
JinH, XuJ and WenZ. (2007). Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development. Blood, 109:5208–5214.
33.
BeermanI, SeitaJ, InlayMA, WeissmanIL and RossiDJ. (2014). Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle. Cell Stem Cell, 15:37–50.
34.
RothM, BonevB, LindsayJ, LeaR, PanagiotakiN, HouartC and PapalopuluN. (2010). FoxG1 and TLE2 act cooperatively to regulate ventral telencephalon formation. Development, 137:1553–1562.
35.
SutherlandAP, ZhangH, ZhangY, MichaudM, XieZ, PattiME, GrusbyMJ and ZhangWJ. (2009). Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis. Mol Cell Biol, 29:2804–2815.
36.
XieZ, ZhangH, TsaiW, ZhangY, DuY, ZhongJ, SzpirerC, ZhuM, CaoX, et al. (2008). Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proc Natl Acad Sci U S A, 105:10859–10864.
37.
XieZ, MaX, JiW, ZhouG, LuY, XiangZ, WangYX, ZhangL, HuY, DingYQ and ZhangWJ. (2010). Zbtb20 is essential for the specification of CA1 field identity in the developing hippocampus. Proc Natl Acad Sci U S A, 107:6510–6515.
38.
KhaliliK, Del ValleL, MuralidharanV, GaultWJ, DarbinianN, OtteJ, MeierE, JohnsonEM, DanielDC, et al. (2003). Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse. Mol Cell Biol, 23:6857–6875.
39.
CortazarD, KunzC, SelfridgeJ, LettieriT, SaitoY, MacDougallE, WirzA, SchuermannD, JacobsAL, et al. (2011). Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature, 470:419–423.
40.
DrillerK, PagenstecherA, UhlM, OmranH, BerlisA, GrunderA and SippelAE. (2007). Nuclear factor I X deficiency causes brain malformation and severe skeletal defects. Mol Cell Biol, 27:3855–3867.
WicksSJ, HarosK, MaillardM, SongL, CohenRE, DijkePT and ChantryA. (2005). The deubiquitinating enzyme UCH37 interacts with Smads and regulates TGF-beta signalling. Oncogene, 24:8080–8084.
43.
Al-ShamiA, JhaverKG, VogelP, WilkinsC, HumphriesJ, DavisJJ, XuN, PotterDG, GerhardtB, et al. (2010). Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development. PLoS One, 5:e13654.
44.
WalshCA, QinL, TienJC, YoungLS and XuJ. (2012). The function of steroid receptor coactivator-1 in normal tissues and cancer. Int J Biol Sci, 8:470–485.
45.
NishiharaE, Yoshida-KomiyaH, ChanCS, LiaoL, DavisRL, O'MalleyBW and XuJ. (2003). SRC-1 null mice exhibit moderate motor dysfunction and delayed development of cerebellar Purkinje cells. J Neurosci, 23:213–222.
46.
TianY, ParamasivamM, GhosalG, ChenD, ShenX, HuangY, AkhterS, LegerskiR, ChenJ, et al. (2015). UHRF1 contributes to DNA damage repair as a lesion recognition factor and nuclease scaffold. Cell Rep, 10:1957–1966.
47.
LiangCC, ZhanB, YoshikawaY, HaasW, GygiSP and CohnMA. (2015). UHRF1 is a sensor for DNA interstrand crosslinks and recruits FANCD2 to initiate the Fanconi anemia pathway. Cell Rep, 10:1947–1956.
48.
WyattMD. (2013). Advances in understanding the coupling of DNA base modifying enzymes to processes involving base excision repair. Adv Cancer Res, 119:63–106.
49.
CortellinoS, XuJ, SannaiM, MooreR, CarettiE, CiglianoA, Le CozM, DevarajanK, WesselsA, et al (2011). Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair. Cell, 146:67–79.
50.
SharifJ, MutoM, TakebayashiS, SuetakeI, IwamatsuA, EndoTA, ShingaJ, Mizutani-KosekiY, ToyodaT, et al. (2007). The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature, 450:908–912.
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