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Hemoglobin switching in Klf1wt/Nan mice
observations were extended by the discovery that KLF1 haploinsufficiency caused hereditary persistence of fetal hemoglobin (HPFH) in a Maltese pedigree.14 To date, over 140 different KLF1 variants have been reported, and these have been linked to a broad range of hitherto unrelated human red cell disorders.1 In Sardinia, Thailand and southern China the frequency of KLF1 variants reaches endemic proportions, e.g., 1.25% in southern China.15 In these populations, cases with compound heterozygosity for KLF1 variants occur.16-18 In such cases, one allele invari- ably carries a missense variant which retains partial activity. KLF1 compound heterozygotes display more pronounced phenotypes, including HbF levels of >20%,16- 18 persistence of embryonic globins,18 microcytic hypochromic anemia,16,18 and pyruvate kinase deficien- cy.18 One case of a KLF1 null neonate was reported.19 This infant displayed severe non-spherocytic hemolytic ane- mia with elevated HbF levels (>70%). Thus, the vast majority of KLF1 variants displays classical autosomal recessive inheritance, and KLF1 haploinsufficiency is associated with mild erythroid phenotypes. The excep- tion is a KLF1 variant in which an ultra-conserved residue in the second zinc finger is affected. This KLF1 p.E325K variant causes congenital dyserythropoietic anemia type IV (CDA-IV; OMIM 613673).20-22 CDA-IV patients suffer from severe hemolytic anemia, splenomegaly, elevated HbF, iron overload, and dyserythropoiesis. Notably, the mouse neonatal anemia (Nan) phenotype is caused by a variant of the orthologous residue in mouse KLF1 p.E339D.23,24 Klf1Nan displays semidominant inheritance. Klf1wt/Nan animals suffer from hemolytic anemia while Klf1Nan/Nan embryos die around embryonic day 10.5 (E10.5) due to failure of pri-mitive erythropoiesis.23,24 This phenotype is more severe than that of Klf1ko/ko embryos which die around E14.5 due to failure of definitive erythropoiesis.6,7 During definitive erythropoiesis KLF1Nan is thought to exert a dominant-ne-gative effect on the function of wild-type KLF1.23,24 In primitive erythropoiesis, KLF1 and KLF2 have compensatory roles25 and the early lethality of Klf1Nan/Nan embryos could therefore be due to interference of KLF1Nan with normal KLF2 function. In addition, KLF1Nan leads to aberrant gene expression which exerts negative effects on erythropoiesis.26,27
In human HBB locus transgenic mice, the fetal HBG1/2 genes are expressed highly in primitive erythrocytes and early definitive erythrocytes. Switching to expression of the adult HBB gene occurs in the fetal liver between E12.5 and E14.5.28 Given the profound deregulation of embryo- nic and fetal globin genes in CDA-IV patients, we inves- tigated expression of the α-like and β-like globins in Klf1wt/Nan mice carrying a single-copy human HBB locus transgene29 at embryonic, fetal and adult stages of development.
Methods
Animals
All animal studies were approved by the Erasmus MC Animal Ethics Committee. Mouse strains used were Klf1wt/Nan (C3H101H-Klf1Nan/H30) crossed with PAC8.1 mice carrying a sin- gle-copy human HBB locus transgene (Tg(HBB)8.1Gvs29). For details see the Online Supplementary Materials and Methods.
Culture of mouse erythroid progenitors
E12.5 fetal livers were disrupted and single-cell suspensions were cultured as described.31 For details see the Online Supplementary Materials and Methods.
RNA isolation and RT-qPCR analyses
RNA was extracted using TRI reagent (Sigma-Aldrich). For details see the Online Supplementary Materials and Methods.
Flow cytometry analysis
Single-cell suspensions were washed twice with PBS and resus- pended in FACS buffer (PBS containing 1% [w/v] bovine serum albumin, 2 mM EDTA). Approximately 106 cells were incubated for 30 minutes at room temperature with the appropriate antibodies. Data were acquired on a Fortessa instrument (BD Biosciences), and analyzed with FlowJo software v10 (Tree Star). For details see the Online Supplementary Materials and Methods.
Cell morphology
Cell morphology was analyzed using cytospins stained with May Grünwald-Giemsa (Medion Diagnostics) and O-dianisidine (Sigma-Aldrich).32 Pictures were taken with a BX40 microscope (40x objective, NA 0.65) equipped with a DP50 CCD camera and Viewfinder Lite 1.0 acquisition software (all Olympus).
Statistical analysis
Statistical analysis of gene expression data was performed by using Mann-Whitney tests (GraphPad Prism). Excel 2010 was used to draw the graphs. Standard deviations and P-values <0.05 are displayed in the relevant figures (*).
Results
Expression patterns of the globin genes in Klf1wt/Nan mice
In order to assess the impact of KLF1Nan on developmen- tal regulation of globin gene expression, the Klf1wt/Nan strain30 was crossed with PAC8.1 mice carrying a single- copy human HBB locus transgene.29 RT-qPCR analysis was performed to determine α-like and β-like globin expression in Klf1wt/wt::HBB (control) and Klf1wt/Nan::HBB (Klf1wt/Nan) embryos at E11.5, E12.5, E13.5, E14.5 and E16.5. Primer pairs were designed to amplify the mouse and human embryonic, fetal and adult globin mRNA specifically, aiming to minimize inter-globin and inter-species cross- reactivity. Of note, the human HBG1 and HBG2 genes, encoding Aγ- and Gγ-globin respectively, arose by a recent duplication event and expression of these genes is assessed by a single primer pair. The same is true for the mouse Hba-a1/Hba-a2 genes, encoding α1- and α2-globin respectively, and Hbb-b1/Hbb-b2 genes, encoding major- and minor-globin respectively (see the Online Supplementary Information). Thus, we measured expression ofmouseα-likeglobins(zand α1/2),mouseβ-likeglobins (ey, βh1 and β major/minor) and human β-like globins (e, Aγ/Gγ, and β). For reasons of clarity we will refer to mouse α-like globins as mz and mα, mouse β-like globins as mey, mβh1 and mβ, and human β-like globins as he, hγ, and hβ in the remainder of this paper.
Globin gene expression patterns during development
The first erythroid cells are derived extra-embryonically from the blood islands in the yolk sac. They enter the cir-
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