Evidences of the PI5P Increasing the Expression of HBG and Gamma Globin Concentrations

Abstract Efforts have been made to identify modulators of increasing fetal hemoglobin (HbF - α2ɣ2) that may act as promising targets for hemoglobinopathies, such as in sickle cell disease and beta thalassemia. The inositide PI5P is a second messenger from the inositol pathway that comprehends in the main phosphorylation target of the enzyme phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIPKIIα), found in high concentrations in the erythrocyte. Some evidences have postulated a relation between this enzyme and the expression of the hemoglobin (Hb) genes (1,2). More recently, PI5P was found to stabilize UHRF1 protein (3), which has been reported as a possible repressor of HBG (from ɣ globin gene), in the switch from ɣ to β in the transition HbF- HbA (4). To evaluate the effects of the inositide PI5P (substrate of PIPKIIα) in globins, a preliminary time course experiment was performed. KU812 cells (ATCC CRL-2099), which express all three globin genes (HBA, HBB and HBG), were treated with 1μM of PI5P (Thermo Fischer Scientific, USA) in triplicate (4, 8 and 12 hours of treatment) - time 0h used as control. Gene expression and protein concentrations were determined by qPCR - SYBR Green Master Mix amplification and detection kit (Thermo Fischer Scientific, USA, in equipment StepOnePlus Real Time PCR System, Applied Biosystems - USA) using 2DDCT method with the ACTB and GAPDH genes as internal controls - and immunoblotting. Primers were design according to Zacariotto et al (2). No additional phosphate was imputed into the cell culture and the ATP consumption was monitored by ATP Determination Kit (Thermo Fisher Scientific, EUA). PI5P treatment resulted in significant increasement of HBG RNA transcripts (p= 0.001; 8 hours / treatment) and decrease of PIP4K2A (from PIPKIIα enzyme) (p=0.02; 12 hours / treatment), with no other significant changes into globin genes (HBA and HBB). At the protein level, there was a prominent increase in ɣ globin concentrations according to time course. No other consistent change in the concentrations of PIPKIIα or globin chains were observed. Due to protein similarities between the subfamilies of phosphatidylinositol-phosphate kinases (PIPKins) (5), the gene expression of these enzymes was also monitored by qPCR. There was a significant reduction of PIP5K1A (from PIPKIα enzyme) (p=0.01 - 4h; p=0.01 - 8h, and p=0.009 - 12h) as well as PIP4K2B (from PIPKIIβ enzyme) (p=0.03 - 4 hours/ treatment). No significant changes were observed in the other PIPKins (PIPKIβ, PIPKIɣ, PIPKIIɣ and PIPKIII). Considering that a significant consumption of intracellular ATP was observed in time course (p=0.006) it is possible to infer that the high concentrations of PI5P have shifted the inositol pathway resulting in the downregulation of its own PIPKin genes and culminating into the upregulation of HBG, reflected into the protein level. The mechanisms involving the inositide as second messenger, the role of PIPKins or other genes into the modulation of hemoglobin genes, particularly in HBG, should be further investigated. However, despite preliminary, these results reinforce the involvement of the PIPKins and its molecular targets (mainly PIPKIα and PI5P) in globin gene regulation and could represent a promising target for future therapeutic target for hemoglobinopathies. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp. 1.Wenning MR, Mello MP, Andrade TG, et al. PIP4KIIA and beta-globin: transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease. Eur J Haematol. 2009; 83: 490-3. 2.Zaccariotto TR, Lanaro C, Albuquerque DM, Santos MN, Bezerra MA, Cunha FG, et al. Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis. Genet Mol Res. 2012; 11: 3861-8. 3.Gelato KA, Tauber M, Ong MS, Winter S, et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol Cell. 2014;54(6):905-19. 4.Ruopeng Feng, Phillip A Doerfler, Yu Yao XT, et al. The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of H BG Promoter DNA Methylation. Blood. 2018;132:410. 5.Heck JN, Mellman DL, Ling K, et al. A conspicuous connection: structure defines function for the phosphatidylinositol-phosphate kinase family. Crit Rev Biochem Mol Biol. 2007; 42: 15-39. Disclosures Costa: Novartis: Consultancy.

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Formation of a Tissue-Specific Histone Acetylation Pattern by the Hematopoietic Transcription Factor GATA-1

Molecular and Cellular Biology ◽

10.1128/mcb.23.4.1334-1340.2003 ◽

2003 ◽

Vol 23 (4) ◽

pp. 1334-1340 ◽

Cited By ~ 108

Author(s):

Danielle L. Letting ◽

Carrie Rakowski ◽

Mitchell J. Weiss ◽

Gerd A. Blobel

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Histone Acetylation ◽

Time Course ◽

Globin Gene ◽

Erythroid Cell ◽

Chromatin Domain ◽

Globin Genes ◽

Tissue Specific ◽

Acetylation Pattern

ABSTRACT One function of lineage-restricted transcription factors may be to control the formation of tissue-specific chromatin domains. In erythroid cells, the β-globin gene cluster undergoes developmentally regulated hyperacetylation of histones at the active globin genes and the locus control region (LCR). However, it is unknown which transcription factor(s) governs the establishment of this erythroid-specific chromatin domain. We measured histone acetylation at the β-globin locus in the erythroid cell line G1E, which is deficient for the essential hematopoietic transcription factor GATA-1. Restoration of GATA-1 activity in G1E cells led to a substantial increase in acetylation of histones H3 and H4 at the β-globin promoter and the LCR. Time course experiments showed that histone acetylation occurred rapidly after GATA-1 activation and coincided with globin gene expression, indicating that the effects of GATA-1 are direct. Moreover, increases in histone acetylation correlated with occupancy of GATA-1 and the acetyltransferase CBP at the locus in vivo. Together, these results suggest that GATA-1 and its cofactor CBP are essential for the formation of an erythroid-specific acetylation pattern that is permissive for high levels of gene expression.

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Methyl Binding Domain Protein 2 Mediates gamma-Globin Silencing in Adult beta-YAC Transgenic Mice.

Blood ◽

10.1182/blood.v106.11.3626.3626 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3626-3626

Author(s):

Jeremy W. Rupon ◽

ShouZhen Wang ◽

Karin Gaensler ◽

Joyce Lloyd ◽

Gordon D. Ginder

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Quantitative Pcr ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Rnase Protection ◽

Gamma Globin ◽

Globin Gene Expression

Abstract The genes of the vertebrate beta-globin locus undergo a switch in expression during development whereby embryonic/fetal genes of the cluster are sequentially silenced and the adult genes are activated during erythroid development. DNA methylation has been shown to be associated with developmentally silenced globin genes, and compounds that inhibit cytosine methylation have been shown to activate transcription from developmentally silenced globin genes in several species, including humans. Previously, we have shown that the methyl domain binding protein 2 (MBD2) is involved in maintaining embryonic rho-globin gene silencing in adult avian erythroid cells. We describe here a role for MBD2 in the DNA methylation mediated silencing and maintenance of silencing of the human fetal gamma-globin gene in a transgenic mouse model. We confirmed the previously published report by Pace et al that the gamma-globin gene is reactivated, upon treatment with the DNA methyltransferase inhibitor, 5-azacytidine, of mice containing the entire beta-globin locus as a yeast artificial chromosome (BetaYAC) transgene. In order to elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2−/− mice were generated by breeding BetaYAC mice with MBD2−/− mice. Anemic adult betaYAC/MBD2−/− mice continue to express the gamma-globin gene at a level commensurate with animals treated with 5-azacytidine, which is10–20 fold over those treated with 1-acetyl-2-phenylhydrazine alone, as measured by both quantitative PCR and by RNase protection assays. In addition, the level of gamma-globin gene expression is consistently several fold higher in MBD2−/− compared to wild type BetaYAC mice in 14.5 and 16.5 dpc fetal liver erythroblasts. Furthermore, transcriptional activation of the gamma-globin gene in adult erythroblasts is associated with a modest decrease in DNA methylation around the gamma-globin promoters and a ~4-fold enrichment of histone H3 trimethylated at lysine 4 (TriK4), as measured by chromatin immunoprecipitation assay using quantitative PCR. Finally, treatment of MBD2 null mice with 5-azacytidine induces only a small, non-additive induction of gamma-globin expression indicating that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells. These results suggest that MBD2 is a potential target for therapeutic induction of gamma-globin gene expression in the settings of sickle cell anemia and beta-thalassemia.

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Analysis of DNA Methylation at the Human Alpha Globin Cluster during Hematopoiesis.

Blood ◽

10.1182/blood.v112.11.1861.1861 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1861-1861

Author(s):

Supachai Ekwattanakit ◽

Helena Ayyub ◽

Kevin Clark ◽

Fatima Marques-Kranc ◽

Sue Butler ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Globin Gene ◽

Regulatory Elements ◽

Human Cells ◽

Globin Genes ◽

Erythroid Cells ◽

Humanized Mouse ◽

Alpha Globin ◽

Globin Gene Expression

Abstract The analysis of globin gene regulation has elucidated many of the principles underlying mammalian gene expression. To understand how the epigenetic program that unfolds during hematopoiesis impacts on alpha (α)-globin gene expression, we analyzed DNA methylation at over 200 CpG dinucleotides in 130 kb around the human α-globin gene cluster focussing particularly on a region (12 kb) covering the α2 and α1-globin genes (HBA2 and HBA1), and the upstream regulatory sites (DNAse I hypersensitive sites (HS) -48, HS-40, HS-33, and HS-10); so-called multi-species conserved sequencesregulatory element1-4 (MCS-R1-4). Cultures of primary adult erythroid cells (at three stages: early, intermediate, and late) were analyzed together with non-erythroid human cells (neutrophils, peripheral blood mononuclear cells, and a human embryonic stem (ES) cell line). Using a recently published method involving bisulphite modification and MALDI-TOF Mass Spectrometry we quantified the level of DNA methylation across the alpha globin cluster. Most sequences outside of the CpG islands were methylated. However, the DNA methylation levels between erythroid and non-erythroid cells differed at regulatory elements. MCS-R1 contained only 1 CpG site and the average percentage of DNA methylation for erythroid and non-erythroid was 8% and 40%, respectively. While they were 8% vs. 42% (p=0.428), 11% vs. 99% (p=0.054), and 14% vs. 97% (p=0.001) in MCS-R2 to 4, respectively. To study changes in DNA methylation in a comprehensive, developmental and tissue-specific manner, we used a humanized-mouse model, in which the conserved α-globin syntenic region of mouse genomic sequence is replaced by that of human. This contains all cis-acting sequences required for fully regulated expression of the α-globin genes. Although the alpha globin cluster is expressed efficiently in this model, DNA methylation patterns differed in the 20 kb region encompassing the α-globin genes when comparing primary human cells and primary humanized cells. DNA in humanized cells were relatively hypomethylated both in erythroid and non-erythroid tissues. The methylation levels were higher in testis of the humanized mouse than in other tissues but still not as high as those in normal human non-erythroid cells. The cause of these differences is being investigated. Despite these differences, the humanized-mouse mimicked the patterns of methylation found in regulatory elements in erythroid and nonerythroid cells. This study shows that despite appropriate regulation of gene expression, epigenetic templating may differ between species (human and mouse). DNA methylation at the upstream regulatory elements might be involved in the regulation of α-globin gene expression during erythropoiesis, although it is equally possible that these changes in methylation are generated by passive demethylation secondary to transcription factor binding in erythroid cells. The mechanism(s) underlying demethylation of regulatory elements during differentiation remains to be clarified.

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Mi2β-mediated silencing of the fetal γ-globin gene in adult erythroid cells

Blood ◽

10.1182/blood-2012-11-466227 ◽

2013 ◽

Vol 121 (17) ◽

pp. 3493-3501 ◽

Cited By ~ 45

Author(s):

Maria Amaya ◽

Megha Desai ◽

Merlin Nithya Gnanapragasam ◽

Shou Zhen Wang ◽

Sheng Zu Zhu ◽

...

Keyword(s):

Gene Expression ◽

Major Part ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Cells ◽

Key Points ◽

Globin Gene Expression ◽

Partial Depletion

Key Points Mi2β exerts a major part of its silencing effect on embryonic and fetal globin genes by positively regulating the BCL11A and KLF1 genes. Partial depletion of Mi2β induces increased γ-globin gene expression in primary human erythroid cells without impairing differentiation.

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Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.398-402.1987 ◽

1987 ◽

Vol 7 (1) ◽

pp. 398-402

Author(s):

T Rutherford ◽

A W Nienhuis

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Gene Promoter ◽

Gene Promoters ◽

Globin Genes ◽

Specific Expression ◽

Tissue Specific ◽

Promoter Sequences ◽

Erythroleukemia Cells ◽

Murine Erythroleukemia

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.

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Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells

Blood ◽

10.1182/blood.v65.3.705.705 ◽

1985 ◽

Vol 65 (3) ◽

pp. 705-712 ◽

Cited By ~ 8

Author(s):

NP Anagnou ◽

TY Yuan ◽

E Lim ◽

J Helder ◽

S Wieder ◽

...

Keyword(s):

Gene Expression ◽

Human Chromosome ◽

Globin Gene ◽

Globin Genes ◽

Chromosome 16 ◽

Regulatory Factors ◽

Erythroleukemia Cells ◽

Erythroleukemia Cell ◽

Globin Gene Expression ◽

Mouse Erythroleukemia

Abstract In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

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Gene activation precedes DNA demethylation in response to infection in human dendritic cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814700116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 6938-6943 ◽

Cited By ~ 39

Author(s):

Alain Pacis ◽

Florence Mailhot-Léonard ◽

Ludovic Tailleux ◽

Haley E. Randolph ◽

Vania Yotova ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dendritic Cells ◽

Transcriptional Activation ◽

Time Course ◽

Gene Activation ◽

Dna Demethylation ◽

Epigenetic Mark ◽

Distal Enhancer ◽

Human Dendritic Cells

DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells withMycobacterium tuberculosis. We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.

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Ham-Wasserman Lecture

Hematology ◽

10.1182/asheducation-2004.1.1 ◽

2004 ◽

Vol 2004 (1) ◽

pp. 1-13 ◽

Cited By ~ 12

Author(s):

Douglas R. Higgs

Keyword(s):

Gene Expression ◽

Genetic Disease ◽

Globin Gene ◽

Hematopoietic Cells ◽

Current Understanding ◽

Globin Genes ◽

Human Genetic Disease ◽

The Past ◽

Inherited Mutations ◽

Globin Gene Expression

Abstract Over the past fifty years, many advances in our understanding of the general principles controlling gene expression during hematopoiesis have come from studying the synthesis of hemoglobin. Discovering how the α and β globin genes are normally regulated and documenting the effects of inherited mutations which cause thalassemia have played a major role in establishing our current understanding of how genes are switched on or off in hematopoietic cells. Previously, nearly all mutations causing thalassemia have been found in or around the globin loci, but rare inherited and acquired trans-acting mutations are being found with increasing frequency. Such mutations have demonstrated new mechanisms underlying human genetic disease. Furthermore, they are revealing new pathways in the regulation of globin gene expression which, in turn, may eventually open up new avenues for improving the management of patients with common types of thalassemia.

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Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation

Blood ◽

10.1182/blood-2007-01-068635 ◽

2007 ◽

Vol 110 (4) ◽

pp. 1343-1352 ◽

Cited By ~ 43

Author(s):

Rodwell Mabaera ◽

Christine A. Richardson ◽

Kristin Johnson ◽

Mei Hsu ◽

Steven Fiering ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Cells ◽

Promoter Dna Methylation ◽

Globin Promoter ◽

Promoter Dna

AbstractThe mechanisms underlying the human fetal-to-adult β-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human γ- and β-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at −162 of the γ promoter and −126 of the β promoter are hypomethylated in ABM and FL, respectively. We also studied γ-globin promoter methylation during in vitro differentiation of erythroid cells. The γ promoters are initially hypermethylated in CD34+ cells. The upstream γ promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient γ-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human γ- and β-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human β-globin locus gene switching.

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Endogenous Elevations of Short Chain Fatty Acids Up-Regulate Embryonic Globin Gene Expression during Primitive Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.1210.1210 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1210-1210

Author(s):

Lauren Sterner ◽

Toru Miyazaki ◽

Larry Swift ◽

Ann Dean ◽

Jane Little

Keyword(s):

Gene Expression ◽

Fatty Acids ◽

Fetal Liver ◽

Globin Gene ◽

Short Chain Fatty Acids ◽

Yolk Sac ◽

Globin Genes ◽

Wild Type ◽

Alpha Type ◽

Globin Gene Expression

Abstract We examined the effects of short chain fatty acids (SCFAs) on globin gene expression during development. We studied globin gene expression in transgenic mice that have endogenous elevations in the SCFA propionate due to a knockout (KO) of the gene for propionyl CoA carboxylase subunit A (PCCA, Miyazaki et al. JBC, 2001 Sep 21;276(38):35995–9). Serum propionate levels measured by gas chromatography were 2.5 to 3.6 mgms/ml in 2 adult PCCA KO mice and were undetectable in 2 wild type (wt) or heterozygous control adult mice. Embryonic PCCA KO offspring had propionate levels of 2.3 and 5.0 μgms/100 mgms of fetal liver, at day 16.5 (E16.5), while wt or heterozygotes at E14.5 had levels <1 μgm/100 mgms. Analysis of expression from alpha (α), beta major (βmaj), embryonic beta-type epsilon-y (εy), embryonic beta-type beta H1 (βH1) and embryonic alpha-type zeta (ζ) globin genes plus 18S ribosomal RNA as a control was undertaken using real-time PCR with gene-specific primers and taqman probes. cDNA was reverse-transcribed from the mRNA of yolk sac (YS) and fetal liver of PCCA KO and wt progeny of more than one litter from timed pregnancies. Individual PCCA embryos at E10 (n=10), E12 (n=9), and E14 (n=7) were analyzed for globin gene expression, normalized to18S expression and were compared to age-matched wt embryos (n>=4 for each time point). As expected, embryonic alpha- and beta-type globin gene expression (ζ and βH1 plus εy) predominated in E 10 YS, and definitive globin gene expression, α and βmaj, predominated in E12 or E14 fetal liver. Expression from embryonic alpha-type globin was calculated as normalized ζ/(ζ+α) and from embryonic beta-type globins as normalized (βH1+εy)/(βH1+εy+βmaj), see table. Embryonic globin gene expression was statistically significantly increased in PCCA KO E12 YS at 1.3 fold relative to wt ζ and in PCCA KO E14 YS at 1.8 fold and 2.1 fold relative to wt ζ or βH1 and εy respectively (p<.05). No increase in embryonic globin mRNA was seen in adult PCCA KO animals. We conclude that elevations of SCFAs during normal murine development causes a persistence of both embryonic alpha-type and embryonic beta-type globin gene expression during primitive, but not definitive, erythropoiesis, suggesting that SCFAs cannot reactivate silenced murine embryonic globin genes in the absence of erythroid stress. Embryonic Globin Gene Expression in Mice with Endogenous Elevations of SCFAs % Expression PCCA KO wild type p value, t test E10 ζ Yolk Sac 53+/− 2 nd E10 βH1 & ε y Yolk Sac 99 +/− 0.3 nd E12 ζ Yolk Sac 32 +/− 3 25 +/− 1 p < .05 E12 βH1 & ε y Yolk Sac 77 +/− 6 74 +/− 3 ns E14 ζ Yolk Sac 7 +/− 1.5 4 +/− 1.4 p < .05 E14 βH1 & ε y Yolk Sac 13 +/− 6 6 +/− 0.5 p < .05 E12 ζ Fetal Liver 11 +/− 4 9 +/− 2 ns E12 βH1 & ε y Fetal Liver 13 +/− 5 13+/− 3 ns E14 ζ Fetal Liver 1 +/− 0.4 0.7 +/− 0.2 ns E14 βH1 & εy Fetal Liver 6 +/− 1.8 4 +/− 1 ns

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