Dev Genes Evol (2009) 219:353–360 DOI 10.1007/s00427-009-0294-8
SHORT COMMUNICATION
Genomic organization of zebra finch alpha and beta globin genes and their expression in primitive and definitive blood in comparison with globins in chicken Cantas Alev & Kaori Shinmyozu & Brendan A. S. McIntyre & Guojun Sheng
Received: 30 March 2009 / Accepted: 16 June 2009 / Published online: 16 July 2009 # Springer-Verlag 2009
Abstract How alpha and beta globin genes are organized and expressed in amniotes is of interest to researchers in a wide variety of fields. Data regarding this from avian species have been scarce. Using genomic and proteomic approaches, we present here our analysis of alpha and beta globins of zebra finch, a passerine bird. We show that finch alpha globin gene cluster has three genes (alphas 1–3), each orthologous to its chicken counterpart. Finch beta globin gene cluster has three genes (betas 1–3), with an additional pseudogene at the 3′ end. Finch beta3 is orthologous to chicken betaA, but the orthology of beta1 and beta2 to chicken counterparts is less clear. All six finch globins are confirmed to encode functional proteins. Gene expression in both globin gene clusters is regulated developmentally. Communicated by A. Kispert Electronic supplementary material The online version of this article (doi:10.1007/s00427-009-0294-8) contains supplementary material, which is available to authorized users. C. Alev : B. A. S. McIntyre : G. Sheng (*) Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan e-mail:
[email protected] C. Alev Group for Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Japan K. Shinmyozu Mass Spectrometry Analysis Unit, RIKEN Center for Developmental Biology, Kobe, Japan B. A. S. McIntyre The Hospital for Sick Children, Toronto, ON, Canada
Adult finch blood has a globin profile similar to that of adult chicken, with high levels of beta3 and alpha3 and moderate levels of alpha2. Finch embryonic primitive blood exhibits a globin profile very different from that of equivalent stage chick embryos, with all six globins expressed at high levels. Overall, our data provide a valuable resource for future studies in avian globin gene evolution and globin switching during erythropoietic development. Keywords Globin . Hemoglobin . Alpha globin . Beta globin . Blood . Primitive . Definitive . Adult . Embryonic . Erythropoiesis . Chicken . Zebra finch . Bird . Avian . Gene duplication . Gene conversion
Introduction Globins are ancient proteins present in all three major kingdoms (eukaryotes, archaebacteria, and eubacteria; Vinogradov et al. 2006). Vertebrate hemoglobins, representing a small branch in the globin superfamily, are traditionally referred to as alpha or beta globins. Duplication of a single-copy hemoglobin gene gave rise to tandemly positioned proto-alpha and proto-beta globin genes in the ancestor of jawed vertebrates, and extant jawless vertebrates do not contain orthologous alpha or beta globin genes despite their having multiple hemoglobin genes (Aguileta et al. 2006; Fago et al. 2001; Hoffmann and Storz 2007; Patel et al. 2008; Qiu et al. 2000). Transchromosomal duplication of the proto-alpha-beta cluster is thought to have occurred during early amniote evolution, followed by gene loss and tandem duplication, resulting in distinct alpha and beta globin gene clusters in extant amniote lineages (Patel et al. 2008).
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In contrast to the wealth of molecular phylogenetic data in the literature of mammalian globin gene clusters (Aguileta et al. 2004; Cooper et al. 2006; Hoffmann et al. 2008; Hoffmann and Storz 2007; Opazo et al. 2008; Patel et al. 2008; Storz et al. 2007), the other two amniote lineages (reptiles and birds) have received little attention except for early work done in chicken (Dolan et al. 1981; Engel et al. 1983; Reitman et al. 1993). The split of ancestral mammals from other amniote lineages is thought to have taken place about 310 million years ago, whereas molecular and fossil data suggest that ancestral birds first appeared in the early cretaceous period (115–145 million years ago; Opazo et al. 2008; Patel et al. 2008). Comparative genomic analyses of hemoglobin genes in extant vertebrate species suggested that the common ancestor of all amniote vertebrates had three alpha-like globin genes and one beta-like globin gene. It is thus unclear how many beta globin genes may have been present in ancestral birds. Four chicken beta globin genes have been proposed to arise through three gene duplication and multiple gene conversion events (Reitman et al. 1993). Sequences of the major adult beta globin have been reported in many avian species, but those of major globins in embryonic primitive blood and of possible minor globins during development and in adult have been lacking except for one report of duckepsilon globin (NCBI no. X15740). Until recently, chicken (Gallus gallus) was the only non-mammalian amniote species with its genome sequenced and also the only avian species with genomic sequence of the globin gene clusters available. The completion of a draft assembly of zebra finch (Taeniopygia guttata) genome allowed us to carry out a comparative analysis of the globin gene clusters in these two avian species, representing two clades that have diverged early during avian evolution.
Materials and methods Genomic analysis Zebra finch alpha globin gene cluster, from the beginning of alpha1 exon 1 to the end of alpha3 exon3 and excluding enhancer regions, corresponds to approximately 2988K to 2979K (in the order of alpha1→ alpha3) of chromosome 14 using UCSC Genome Browser for July 2008 assembly of the zebra finch genome. Zebra finch beta globin gene cluster, from the beginning of beta1 exon 1 to the putative end of exon 3 of pseudo-beta4 and excluding enhancer regions, corresponds approximately to 886K to 872K (in the order of beta1→pseudo-beta4) of chromosome 1B. Three small regions in the beta globin gene cluster are without sequence information: two together covering approximately 200 base pairs within intron 2 (total of 2.3 kb) of beta2 and one covering the promoter, 5′ UTR,
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and first three amino acid residues of beta3. This last region, shown as 100 Ns in the genome assembly, was re-sequenced by us using primers for the flank regions. It contains 242 nucleotides. The actual sequence from our analysis is shown in the Electronic supplementary material. This and exons 2 and 3 of beta3 in the genome assembly match with known major zebra finch adult beta globin sequences. Two putative sequencing errors in exon2 of beta3 in the genome assembly (insertion of T at nucleotide positions 114 and 127 from the translation start site) were corrected based on comparison of beta3 genomic sequence with fourteen zebra finch adult beta globin cDNA/EST sequences available in public database. These corrections were also confirmed with our proteomic data. The final beta3 protein sequence used for comparison was the same as NCBI accession no. DQ215301. Molecular phylogeny In addition to zebra finch and chicken globins (see Electronic supplementary material), the following proteins were used for the phylogenetic analysis: turkey-alphaA with the NCBI accession number (p81023), turkey-alphaD (p81024), quail-alphaA (p24589), quail-alphaD (p30892), ostrich-alphaA (p01981), ostrichalphaD (p04242), duck-pi (p04243), human-alpha1 (np_000549), human-zeta (np_005323), wallaby-alpha (ay459589), wallaby-zeta (ay789121), tropicalis-alphal1 (nm_001005092), tropicalis-alpha1 (nm_203529), sphenodon-alphaD (p10062), sphenodon-alphaA (p10059), and zebrafish-alpha-a1 (nm_131257) for alpha globins; and duck-epsilon (x15740), duck-beta-major (x15739), turkeybeta-major (2qmb_b), flamingo-beta-major (p02121), pigeon-beta-major (p11342), ostrich-beta-major (p02123), macaw-beta-major (p02116), condor-beta-major (p07411), platypus-beta (ac192436), platypus-epsilon (ac192436), opossum-beta (j03643), opossum-epsilon (j03642), tropicalis-beta (nm_203528), tropicalis-epsilon1 (nm001016495), sphenodon-beta (p10061), land turtlebeta (gcu63145), alligator-beta (p02130), sea turtle-beta (q10733), lizard-beta (p18993), human-beta (nm_000518), human-gamma (bc130459), and human-epsilon (nm_005330) for beta globins. Phylogenetic trees were generated using the TreeTop cluster algorithm with a BLOSUM62 matrix (GeneBee server, Moscow State Univ.) with bootstrap values calculated from 100 replicates. Proteomic analysis Proteomic analysis was carried out as previously reported (Alev et al. 2008). Fertilized chicken eggs were purchased from Shiroyama Farm (Kanagawa, Japan), and adult chicken was purchased from MieHiyoko Co. (Mie, Japan). Adult zebra finch and zebra finch eggs were purchased from local pet shops. Four to 50 chicken embryos for each embryonic stage and one chicken for the adult stage were used to yield 20–50 μl
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of blood, although only a fraction of pooled blood was used for tryptic digestion and mass spectrometry runs. Data for each chicken stage are represented by three independent runs. Two zebra finch stage HH23 embryos and one adult zebra finch were used for obtaining primitive and adult blood samples, respectively. Two independently obtained samples from each finch embryo (total four independent samples) and four independently obtained samples from one adult finch were used for mass spectrometry runs. MASCOT searches were performed by using NCBI chicken protein database for chicken blood samples and by using a custom-made zebra finch globin “database” in combination with the NCBI chicken protein database for zebra finch blood samples. Mass spectrometry datasets from all eight zebra finch blood samples have been submitted to the proteomics identification database (PRIDE), with the accession numbers 9204–9211.
Results and discussion Genomic organization of zebra finch alpha globin genes To understand molecular evolution of globin genes in the avian lineage, we performed a comparative genomic analysis of zebra finch and chicken globin gene clusters. There are seven hemoglobin genes in chicken: three alpha (5′-pi-alphaD-alphaA-3′) and four beta (5′-rho-betaHbetaA-epsilon-3′) in alpha and beta gene clusters, respectively (Dolan et al. 1981; Engel et al. 1983; Reitman et al. 1993; Fig. 1a, b). Both chicken alpha and beta gene clusters are orthologous in their origin to eutherian counterparts (Patel et al. 2008). Pi is orthologous to human-zeta/pseudo-zeta globin, alphaD to human mu globin, and alphaA to human pseudo-alpha1/alpha2/alpha1/theta globins. Current annotation of the zebra finch genome is relatively poor. We therefore carried out manual annotation of these two genomic regions. Blast analyses with chicken globin genes using the UCSC Genome Browser interface indicated that zebra finch has alpha and beta globin gene clusters in chromosomes 14 and 1, respectively, same as in chicken. The alpha globin gene cluster contains three genes, hereby named alpha1, alpha2, and alpha3 based on their linear arrangement along the chromosome (Fig. 1a). The size of the gene cluster, all exon/intron boundaries of the constituent genes, and putative polypeptide lengths are conserved between zebra finch and chicken. Comparison of amino acid sequences indicated that the three zebra finch alpha globin genes are orthologous to the three chicken ones (alpha1 to pi, alpha2 to alphaD, and alpha3 to alphaA; Fig. 1c). This suggests that ancestral birds
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already had three alpha globin genes and supports the hypothesis based on comparative analysis between chicken and mammals that ancestral amniotes had three globin genes in the alpha globin gene cluster (Hoffmann and Storz 2007). Genomic organization of zebra finch beta globin genes Zebra finch beta globin gene cluster contains three putative beta globin genes and one putative pseudogene located at the 3′-end of the cluster. They will be called beta1, beta2, beta3, and pseudo-beta4 in this work (Fig. 1b). Taking the pseudo-beta4 into consideration, zebra finch beta globin gene cluster has an overall size similar to that in chicken. Beta1, beta2, and beta3 have conserved exon/intron boundaries for beta globins. Pseudo-beta4 has a recognizable exon 1 sequence with high homology to that of beta1. Its exon 2 and exon 3 are degenerate and contain insertions, although remnant sequences corresponding to the start regions of these two exons can be detected. Beta3 sequence is the same as the major beta globin sequence for adult zebra finch available in the NCBI database, indicating that beta3 has its gene location and expression profile similar to that of betaA, the chicken adult major beta globin, which is also the third gene in the beta globin gene cluster. Comparison of putative zebra finch beta1 and beta2 amino acid sequences with chicken beta globins, however, pointed to a more complex scenario (Fig. 1d). Zebra finch beta1 has high homology to chicken rho and epsilon, two primitive blood beta globins positioned as the first and fourth gene in the beta globin gene cluster, respectively. Epsilon and rho are thought to be formed after the first and second beta globin gene duplications, respectively, and to have undergone several gene conversion events between them in both directions (Reitman et al. 1993). Position wise, therefore, zebra finch beta1 is orthologous to chicken rho. Sequence wise, however, the orthology between beta1 and rho is not certain. Zebra finch beta2 does not show a clear orthology to any particular chicken beta globin, although phylogenetic analysis pointed to a slightly closer relationship with chicken betaH than with other chicken beta globin genes (Fig. 1d). Its exon 1 has an equal nucleotide homology to all four chicken beta globins and the highest amino acid homology to both chicken betaH and betaA. Its exon 2 has the highest nucleotide homology to betaH and the highest amino acid homology to epsilon, and its exon 3 has the highest homology to betaH. The size of intron 2 of beta2 is larger than all the other intron 2 sequences of beta globins in chicken and zebra finch. Zebra finch beta3, consistent with its location and expression, has the highest homology to chicken betaA. Overall, our analysis of zebra finch beta2 and beta3 supports the hypothesis (Reitman et al.
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Fig. 1 Genomic organization of zebra finch alpha and beta globin genes and proteomic confirmation of six functional globin proteins. First and third exons contain coding (red) and non-coding (gray) regions. Non-coding transcribed regions of zebra finch globins are inferred from sequence homology, but not confirmed experimentally. a Comparison of the alpha cluster genes in chicken (top) and zebra finch (bottom). E1, E2, and E3 refer to the first, second, and third exons, respectively. b Comparison of the beta cluster genes in chicken (top) and zebra finch (bottom). E1, E2, and E3 refer to the first, second, and third exons, respectively. The E1 of pseudo-beta4 is intact. E2 and E3 of pseudo-beta4 have recognizable remnant start and unrecognizable end sequences. c, d Comparison of six putative zebra finch globins (shown in e) with globins from chicken and other amniote lineages using fish and amphibian globins as outgroups. c Alpha globins; d Beta globins. Zebra finch and chicken globins are highlighted in red. Bootstrap values shown as percentage points at nodes, with values over 50% highlighted in red. e Mass spectrometry based confirmation of three alpha and three beta globins. Lys or Arg residues are highlighted in red. Blue underlines indicate regions covered by peptides from mass spectrometry runs
1993) that the second and third beta globin genes represent the third (and the last) gene duplication event in the avian beta globin gene cluster. Comparative analyses of beta globins in jawed vertebrates suggested that ancestral amniotes had one proto-beta globin and that tandem duplication of this gene took place independently in different amniote lineages (Patel et al. 2008). Eutherian mammals contain five paralogous beta globin genes (5′-epsilon-gamma-etadelta-beta-3′), while marsupials and monotremes have two (5′-epsilon-beta-3′; Opazo et al. 2008). Phylogenetic studies indicated that the marsupial epsilon gene is
orthologous to eutherian epsilon and gamma genes and marsupial beta orthologous to eutherian delta and beta. Monotreme epsilon and beta genes, however, are derived from an independent duplication, suggesting that ancestral mammals had only one proto-beta gene. Our analysis of the zebra finch beta globin gene cluster indicated that this species has three functional beta genes (beta1, beta2, and beta3) and one pseudogene (pseudo-beta4), suggesting that ancestral birds already had four beta genes. The high homology between exon 1 of beta1 and exon 1 of pseudo-beta4 in zebra finch may represent a recent gene conversion event similar to what have been documented
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for many mammalian beta globin genes and pseudogenes (Aguileta et al. 2004; Storz et al. 2007) and for chicken beta globin genes (Reitman et al. 1993). Our analysis, however, cannot establish a clear molecular orthology Fig. 2 Developmental changes of chicken alpha globin profiles and zebra finch alpha and beta globin profiles. a Semi-quantitative measurement of relative percentages of pi, alphaA, and alphaD in chicken blood samples from 15 different stages. Number represents embryonic day. P35 represents adult stage. Error bars represent standard deviations from three independent measurements for each stage. b Stage HH23 zebra finch embryo. Left HH23 zebra finch embryo with inset showing a comparison of zebra finch embryo (smaller one) with same stage chicken embryo. Right HH23 zebra finch embryo shown intact with extraembryonic tissues and the yolk. c Relative percentages of each zebra finch globin in blood samples from adult (red) and stage HH23 embryos (blue). Error bars represent standard deviations from four independent runs
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between zebra finch beta1 and chicken rho or between zebra finch beta2 and chicken betaH, clarification of which may require future comparison with a basal avian lineage such as ratites.
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Confirmation of zebra finch globin peptide sequences with mass spectrometry If each of the identified zebra finch globin genes is expressed, there are six distinct polypeptides that should be identifiable in erythrocytes from different stages of development. To confirm the existence of these putative proteins, we carried out peptide identification using a shotgun proteomics-based method. Globins are the most abundant proteins in erythrocytes and even minor globins are expressed at higher levels than most house-keeping genes (Alev et al. 2008; Nakazawa et al. 2009). Blood samples from adult and embryonic zebra finch were collected, digested with trypsin, and processed for mass spectrometry analysis using a 1-D μLC ESI-MS/MS setup. Lys and Arg residues, after which tryptic digestions occur, are shown in red in Fig. 1d. The majority of digested peptide sequences can be assigned unambiguously to individual alpha or beta globins. With a MASCOT cutoff score of 30, several hundred peptides specific for each globin were detected, covering 68% of alpha1, 93% of alpha2, 87% of alpha3, 94% of beta1, 92% of beta2, and 96% of beta3 (Fig. 1e, underlined regions). The regions that were not covered in our analysis represent small peptides, which are biased against in a normal mass spectrometry setting and in the case of alpha1 one long peptide. Overall, these data provide a validation for the genomic DNA sequence and the putative splicing and translation events of six globin genes in zebra finch. Furthermore, supporting our conclusion from the genomic analysis that beta4 is a pseudogene and does not encode a functional beta globin, we did not detect any putative peptide, which may correspond to a partial or truncated beta4 protein in our mass spectrometry analysis.
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understand globin heterogeneity in zebra finch embryonic blood, we first performed an analysis of developmental changes of chicken alpha globins for reference purpose, using the proteomics datasets obtained from our previous experiment (Alev et al. 2008). In summary, 16 embryonic stages (from embryonic day 3 to 17 with 1-day intervals) and one adult stage (post-hatching day 35) were used for the current analysis. Hypothetical tryptic digestions of three chicken alpha globins generate peptides, which can all be assigned unambiguously. For any given stage, relative percentage was calculated as the ratio between number of detected peptides for a given alpha globin and number of detected peptides for all alpha globins. As shown in Fig. 2a, E3, E4, and E5 blood has pi as the major alpha globin, with alphaA and alphaD also prominently expressed, but at lower levels. A shift in profiles takes place between E5 and E7, and after E7, alphaA becomes the major alpha globin, with alphaD being the second major one. Pi globin shows a gradual decrease throughout later embryonic development and becomes undetectable in adult. The difference between alphaA and alphaD remains stable during embryonic development and widens after hatching. The developmental changes of chicken alpha globins correlate in timing with those of chicken beta globins and with the switch from primitive to definitive blood during embryogenesis. Developmental regulation of zebra finch alpha and beta globins
Developmental regulation of chicken alpha globin genes
We then asked whether zebra finch globins show similar changes in their profiles between primitive and definitive blood. For this, we used adult finch for the profiling of definitive blood and, due to scarcity of zebra finch embryos, stage HH23 zebra finch embryos (equivalent to E4 in chicken; Fig. 2b) for the profiling of primitive blood. HH23 finch embryos are significantly smaller than same
Regulation of globin gene expression levels is important for proper red blood cell differentiation. Using a semiquantitative, shotgun proteomics-based measurement of globin protein heterogeneity, we have previously reported developmental changes of chicken beta globin proteins from establishment of embryonic circulation to adult (Alev et al. 2008). In that analysis and in our transcript-based studies (McIntyre et al. 2008; Nagai and Sheng 2008; Nakazawa et al. 2006), we showed that chicken primitive blood has high levels of epsilon and rho and low levels of betaA and undetectable betaH and that definitive blood during late period of embryogenesis has high levels of betaA, decreasing levels of epsilon and rho and low levels of betaH. In adult chicken, the predominant beta globin is betaA, with low levels of betaH and very low to undetectable levels of epsilon and rho. In order to
Fig. 3 Summary of the organization and expression of alpha and beta globins in zebra finch and chicken. Size of arrow represents relative robustness of protein expression. Arrowheads indicate barely detectable levels of expression
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stage chicken embryos (Fig. 2b, inset), but all main developmental landmarks are conserved. A detailed developmental staging system is currently unavailable for zebra finch. Embryonic development for one finch species, the society finch (Lonchura domestica), has been reported, with overall developmental features very similar to those in chicken (Yamasaki and Tonosaki 1988). With proteomic, transcriptomic, and in situ-based analyses, we have reported that stage HH23 chicken blood contains only primitive erythrocytes (Alev et al. 2008; McIntyre et al. 2008; Nagai and Sheng 2008). We therefore chose this stage for obtaining sufficient amount of zebra finch primitive blood and performed a similar semi-quantitative measurement of globin heterogeneity using mass spectrometry. We first analyzed globin heterogeneity in adult zebra finch blood. For alpha globins, zebra finch alpha3 is the major adult globin and alpha2 is the minor, but also highly expressed, one (Fig. 2c). The ratio between alpha3 and alpha2 is close to 3:1 (Fig. 2c). No zebra finch alpha1 could be detected in adult blood. This profile is identical to that of alpha globins in adult chicken blood. For beta globins, zebra finch beta3 is the predominant one, representing over 99% of all peptides (Fig. 2c). Trace levels of beta1 (0.2%) and beta2 (0.3%) were also detected in adult blood. This profile is also identical to that of beta globins in adult chicken blood. These profiles indicate a striking conservation of globin heterogeneity in adult blood between zebra finch and chicken. We next analyzed globin heterogeneity in embryonic zebra finch blood. Surprisingly, stage HH23 zebra finch blood has all six globins expressed at high levels. Comparatively, for alpha globins, the order in expression levels is alpha2 (55.2%)>alpha1 (28.5%)> alpha3 (16.3%) and for beta globins beta1 (43.8%)>beta2 (32.6%)>beta3 (23.6%). These profiles are very different from those in chicken embryos. For alpha globins, stage HH23 chicken blood has pi (first in the cluster)>alphaA (third in the cluster)>alphaD (second in the cluster) and alphaA levels overtake pi levels only during the mid-period of primitive to definitive blood switch (stage HH28, about E6; Fig. 2a). For beta globins, although zebra finch beta1 is the major primitive blood globin similar to chicken rho (first in the cluster) and epsilon (fourth in the cluster), zebra finch beta2 is very highly expressed, in contrast to chicken betaH (second in the cluster), which has no expression in primitive blood and very low expression in definitive blood. BetaH in chicken is considered to be a definitive blood specific beta globin, whereas our data in zebra finch suggest that beta2 is a primitive blood specific beta globin. Zebra finch beta3, the major definitive beta globin, is also expressed at quite high levels at stage HH23, suggesting that beta3 is not silent in primitive erythrocytes. This is in agreement with our recent observation that chicken betaA, although being the predominant beta globin in definitive
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blood, is also expressed during primitive erythropoiesis (Alev et al. 2008). In the case of zebra finch, beta3 is expressed even more robustly (Fig. 2c) in primitive blood than chicken betaA (Alev et al. 2008). In summary, using a combination of genomic and proteomic tools, we report here that there are three alpha and three beta globin genes in zebra finch alpha and beta globin gene clusters, respectively (Fig. 3). The beta cluster has an additional pseudogene, which is likely the remnant of the fourth beta gene in the cluster. All six globin genes are confirmed to encode functional proteins. Adult definitive blood in zebra finch has a similar globin composition to that in chicken, but embryonic primitive blood in zebra finch has a dramatically different globin profile from that of chicken primitive blood. Understanding the molecular and developmental basis of this difference will require similar studies in several representative avian species. Acknowledgments We thank the Genome Center at Washington University School of Medicine (St. Louis, MO, USA) and the International Zebra Finch Genome Analysis Consortium for making the zebra finch genome assembly available for our analysis, Dr. Scott Edwards (Harvard University, MA, USA) and Dr. Carlos Lois (Picower Institute, MA, USA) for helpful discussions and suggestions, Dr. Yukiko Nakaya and Ms. Kanako Ota (RIKEN, Kobe, Japan) for help in obtaining zebra finch eggs, and Dr. Wei Weng (RIKEN, Kobe, Japan) for comments on the manuscript. We apologize for many references that have not been included in this report due to space limitation. This work was supported by RIKEN. Conflict of interest statement conflict of interest.
The authors declare that they have no
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