Essential role of c-myb in definitive hematopoiesis isevolutionarily conservedCristian Soza-Ried1,2, Isabell Hess2, Nikolai Netuschil, Michael Schorpp, and Thomas Boehm3

Department of Developmental Immunology, Max-Planck Institute of Immunobiology, D-79108 Freiburg, Germany

Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved August 10, 2010 (received for review April 6, 2010)

The transcription factor c-myb has emerged as one of the keyregulators of vertebrate hematopoiesis. In mice, it is dispensablefor primitive stages of blood cell development but essentiallyrequired for definitive hematopoiesis. Using a conditional knock-out strategy, recent studies have indicated that c-myb is required forself-renewal of mouse hematopoietic stem cells. Here, we describeand characterize the c-mybmutant in a lower vertebrate, the zebra-fish Danio rerio. The recessive loss-of-function allele of c-myb (c-mybt25127) was identified in a collection of N-ethyl-N-nitrosourea(ENU)-induced mutants exhibiting a failure of thymopoiesis. The se-quence of the mutant allele predicts a missense mutation (I181N) inthe middle of the DNA recognition helix of repeat 3 of the highlyconserved DNA binding domain. In keeping with the findings in themouse, primitive hematopoiesis is not affected in the c-mybmutantfish. By contrast, definitive hematopoiesis fails, resulting in the lossofall blood cells byday20ofdevelopment. Thus, themutantfish lacklymphocytes and other white and red blood cells; nonetheless, theysurvive for 2–3 mo but show stunted growth. Because the mutantfish survive into early adulthood, it was possible to directly showthat their definitive hematopoiesis is permanently extinguished.Our results, therefore, suggest that thekey roleof c-myb indefinitivehematopoiesis is similar to that in mammals andmust have becomeestablished early in vertebrate evolution.

transcription factor | DNA binding domain | missense mutation | zebrafish

Research on the genetic hierarchy regulating the self-renewaland differentiation of hematopoietic progenitor cells pro-

vides critical information for diagnosing and treating a plethora ofhematological disorders, both benign andmalignant, and serves asa paradigm for stem-cell biology (1).One of the most important regulators of mammalian hemato-

poiesis is c-myb (2), an evolutionarily conserved transcriptionfactor (3). c-myb is part of a complex genetic network whosefunction is to specify and maintain hematopoietic progenitors andto regulate their differentiation (4). Among vertebrates, mostgenetic studies of c-myb function have been conducted in themouse model, primarily because the experimental armamentar-ium is well-developed but also because no c-myb mutations haveyet been described in other species.In zebrafish, primitive hematopoietic activity begins within the

first day after fertilization. In the first wave, the cephalic meso-derm gives rise to embryonic macrophages in the so-called rostralblood island (5); shortly thereafter, in a second wave, erythroidprecursors localized in the intermediate cell mass give rise toerythrocytes that exit into the circulation at the end of the first dayof embryonic development (6). After a brief transition period,which is characterized by the activity of erythromyeloid precursorsin the posterior blood island (7), primitive hematopoiesis endswith the formation of the first multipotent precursors that alsopossess lymphoid potential (8, 9). The latter exhibit self-renewalcapacity and are commonly referred to as hematopoietic stemcells (HSCs). Recent work has shown that these cells arise fromhemogenic endothelium lining the ventral wall of the dorsal aorta(10, 11), indicating that the cellular processes underlying thegeneration of these multipotent progenitors are similar in bothfish and mammals (12). Studies in the mouse model have sug-

gested that c-myb is required only for definitive hematopoiesis,during which it regulates HSC maintenance and differentiation(13–15). Given the evolutionary conservation of genes regulatingfunctional and temporo-spatial features of primitive hematopoi-esis, the question arises as to whether the same applies for de-finitive hematopoiesis in adult vertebrates. Here, we explore thispossibility by studying the phenotypic consequences of an ap-parent null mutation in the zebrafish gene encoding the c-mybtranscription factor.

ResultsIdentification of a Zebrafish c-myb Mutant. We have previouslyreported on a collection of mutant zebrafish lines identified inforward genetic screens for abnormalities in thymopoiesis (16).Here, we describe the positional cloning of the gene responsiblefor the recessive hematopoietic phenotype in line IP109 (t25217).Heterozygous fish are indistinguishable from homozygous wild-type animals. The mutant was originally recognized owing to itscomplete lack of ikaros- and rag1-expressing hematopoietic cellsin the thymus (Fig. 1A). Homozygous mutant fish grow slowerthan their wild-type and heterozygous littermates, and they aredistinguished by their severe anemia (Fig. 1B). Indeed, over thecourse of time, the number of blood cells diminishes, and by about20 d postfertilization (dpf), virtually no circulating red blood cellsare detectable in the vasculature of mutants (Movies S1, S2, S3,S4, S5, S6, S7, and S8). The absence of erythrocytes and otherblood cell types is also apparent in histological sections (Fig. 1C).The bloodless phenotype is accompanied by smaller body size,developmental retardation including incomplete ossification, andlack of sexual maturation (Fig. S1). Although they lack detectablered blood cells from about 3 wk of age onward (Fig. S1), mutantfish survive until 2–3 mo of age. We presume that the mutantsachieve residual oxygenation of their tissues through diffusion toan extent that is compatible with life into adulthood.The gene responsible for the phenotype in IP109 mutants was

mapped to chromosome 23; the marker closest to the mutation islocated on BAC BX927365, with a genetic distance of ≤0.1 cM.Interestingly, this BAC also contains the c-myb locus (Fig. 2A).Because c-myb has been implicated in the regulation of definitivehematopoiesis in mice (13–15), the gene was sequenced in itsentirety. This analysis revealed the presence of a thymidine toadenine (T > A) transversion (corresponding to nucleotide po-sition 220627 in accession number BX927365.7 and nucleotide

Author contributions: T.B. designed research; C.S.-R., I.H., N.N., and M.S. performed re-search; C.S.-R., I.H., N.N., M.S., and T.B. analyzed data; and C.S.-R., I.H., N.N., M.S., and T.B.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Commentary on page 17067.1Present address: Vertebrate Development Laboratory, Cancer Research United Kingdom,London Research Institute, London WC2A 3PX, United Kingdom.

2C.S.-R. and I.H. contributed equally to this work.3To whom correspondence should be addressed. E-mail: boehm@immunbio.mpg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1004640107/-/DCSupplemental.

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position 652 in accession number NM_131266). This causesa change from isoleucine to asparagine at amino acid residue 181,which is located in the middle of the DNA recognition helix of theR3 repeat of the evolutionarily conserved DNA binding domain(17) (Fig. 2B). Interestingly, amutation at residue 181 is among themany changes that occur in the avian myeloblastosis virus variantof chicken c-myb (18). The side chain of isoleucine 181 points awayfrom the DNA, and therefore, the replacement of a hydrophobicresidue with a polar dicarboxylic amino acid likely affects theoverall structure and/or stability of the R3 repeat (Fig. 2C and Fig.S2). The results of three additional experiments suggested that thephenotype in IP109 fish was indeed caused by the mutation inc-myb. First, all 257 mutant fish (identified by the absence of rag1expression in the thymus) were found to be homozygous for themutation, indicating close genetic linkage (<0.2 cM). Second,a c-myb–specific antisense morpholino resulted in a phenocopy (asdetermined by the lack of thymocytes) of the IP109 mutants(Fig. 2D). Third, thymopoietic abnormalities in 9 of 11 IP109mutants were rescued after injection of a BAC clone (Fig. 2A)encompassing a wild-type c-myb gene [P < 0.000111, Fisher exactprobability test (one-tailed)]. The missense mutation in the con-served DNA binding domain is an indication that the function ofc-myb as a sequence-specific transcription factor might be com-promised. To examine this possibility, wild-type and mutant formsof the DNA binding domains were expressed in Escherichia coli,purified, and used for electrophoretic mobility shift assays withan oligonucleotide containing a canonical c-myb recognition se-quence. As expected, the mutant protein lacks DNA binding ac-tivity in vitro (Fig. 2E). Although we cannot exclude the possibilitythat the mutant form of c-myb has residual activity in vivo, ourresults suggest that it corresponds to a null mutation.

Abnormal Hematopoiesis in c-myb Mutants. To examine the earlystages of hematopoiesis, whole-mount RNA in situ hybridizationswith several gene-specific probes were performed. In the zebrafish,thefirst hematopoietic precursors withmultilineage differentiationcapacity, the so-called erythromyeloid precursors (EMPs) are

found in the posterior blood island (PBI) and can be convenientlydetected by hybridization with a gata1 probe. At 24 h post-fertilization (hpf), the number of EMPs seems to be slightly re-duced in c-myb mutants, whereas the number of hematopoieticprogenitor cells expressing scl is similar inwild-type andmutantfish(Fig. 3A). However, in cells coexpressing scl and c-myb, the ex-pression levels of the latter are much higher in the mutants, whichare clearly indicated by double-fluorescent in situ hybridization(Fig. 3A and Fig. S3). Furthermore, although the expression levelsof ikaros are not affected in embryonic macrophages of mutants,ikaros expression is greatly diminished in the intermediate cellmass(ICM) and PBI regions, indicating a reduction in the number oflymphoid progenitors (Fig. 3A). Collectively, these data indicatethat even the early stages of definitive hematopoiesis in c-mybmutants are abnormal. Differences between wild-type and mutantfish are also detectable at 36 hpf (Fig. 3B) and later stages (Fig. 3C).The severe impairment of definitive hematopoiesis is also evidentin expression analyses using RT-PCR; the results confirm thetransient up-regulation of c-myb, which is also evident for spi1/pu.1,and show that, at 48 hpf, the expression levels of all hematopoieticgenes aremuch lower inmutants comparedwith controls (Fig. 3D).Thus, even in the absence of c-myb function, primitive hemato-

Fig. 1. Phenotype of IP109 mutants. (A) Whole-mount RNA in situ hybrid-ization of wild-type (+/+) and homozygous IP109 mutant (−/−) embryos withprobes for ikaros (lateral views; Left) and rag1 (dorsal views; Right) at 5 dpostfertilization (dpf). Note that ikaros is also expressed in neurons (arrows);this hybridization signal serves as an internal positive control for the hy-bridization process. In the hybridizations with rag1, a gh probe labelsgrowth hormone-producing cells in the hypophysis (arrows) and serves ascontrol for hybridization. The region of the thymus is encircled. No differ-ences were seen between +/+ and +/m

fish. Table S1 has probe details. (B)Macroscopic view of wild-type (Left) and mutant (Right) fish at 20 dpf. Notethe smaller size and pale appearance of mutants. (Scale bar: 1 mm.) (C)Histological sections through the regions of the heart (A, atrium; V, ventri-cle) of heterozygous (Left) and homozygous mutant (Right) fish at 8 wk ofage. Note the complete absence of erythrocytes in the mutant fish (Fig. S1).H&E staining. (Scale bars: 400 μm; Inset, 100 μm.)

Fig. 2. A deleterious missense mutation in the DNA binding domain ofzebrafish c-myb. (A) Genetic mapping of the IP109 mutation on chromosome23. The locations of informative markers are indicated; recombination fre-quencies are given in brackets below their names (details in Table S3). Theposition of BAC BX927365 encompassing the c-myb gene that was used forcomplementation analysis is shown. (B) Schematic indicating the location ofthe isoleucine (Ile) to asparagine (Asn) missense mutation relative to thethree repeat domains (R) of the DNA binding domain in the c-myb protein.(C) The side chain of isoleucine 181 in the third helix (h3) of repeat 3 (yellowarea on the carbon trace) points away from the DNA double helix (strands inblue and magenta) to the first (h1) and second (h2) helix of repeat 3. Thisfigure was rendered using Cn3D from PDB ID 1MSE (17). (D) Injection of anantisense c-myb morpholino oligonucleotide into wild-type fish transgenicfor an ikaros:eGFP reporter recapitulates the thymic homing defect ob-served in c-myb mutants. The thymic rudiment is encircled. Lateral views,4 dpf. (E) The mutant version of c-myb lacks in vitro DNA binding activity.The N-terminal one-half (amino acids 1–318) of the mouse c-myb protein,encompassing the highly conserved DNA binding domain, was expressed inE. coli; the I181N mutation was introduced by site-directed mutagenesis. Themutant protein (c-myb*) does not interact with the radioactively labeledDNA probe containing a c-myb consensus binding site (red box); excess un-labeled binding sites compete with the radiolabeled probe (two right-mostlanes). Equal amounts of wild-type and mutant proteins were used (Fig. S2).

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poiesis nevertheless gives rise to cells of the myeloid and erythroidlineages. However, in keeping with the progressive overall re-ductionof hematopoietic progenitor cells and the specific reductionof lymphocyte progenitors (Fig. 3A), the thymus is not colonized inmutant embryos (Fig. 4A and Fig. S4), although the thymic anlageas such is not affected (Fig. S4). As a consequence, thymopoiesis iscompletely absent in c-mybmutants, compatible with findings in themouse (19). For instance, in addition to the lack of ikaros and rag1expression (Fig. 1A), no expression of c-myb, gata3, ccr9b, and tcrbgenes is observed in the thymic anlage (Fig. 4B). Moreover, nofunctional tcrb transcripts could be found in mutant embryos at 5dpf (0/7 embryos), whereas they could be readily detected in het-erozygous fish [3/3; P < 0.0084, Fisher exact probability test (one-tailed)]. Time-lapse video recordings of fluorescent cells in trans-genic ikaros:eGFP reporter fish indicated that, although tissuemacrophage-like cells display their characteristic migratory be-havior, lymphocyte progenitors fail to develop and hence, do notappear in the thymic anlage of c-myb mutants (Movies S9 andS10). Collectively, these experiments suggest that T cell de-velopment is aborted at an early prethymic stage in c-mybmutants.

Failure of Definitive Hematopoiesis in c-myb Mutants. In contrast tomice with a null mutation in c-myb (14), a large fraction of c-mybmutant fish reaches adolescence. Hence, it was possible to directlyexamine the state of definitive hematopoiesis in these fish; ourresults indicate that it is severely compromised in the absence ofc-myb. Mutants lack hematopoietic tissue in the head kidney, asshown in histological sections (Fig. 5A) and by flow cytometry(Fig. 5B); in addition, expression of adult globin genes is not de-tectable (Fig. 5C). In keeping with these observations, an exten-sive survey of the expression of relevant genes by RNA in situhybridization indicated the complete absence of markers for im-mature cells of erythroid (gata1) and lymphoid (ikaros, rag1) lin-eages (Fig. 5D), compatible with the absence of red blood cells inthese fish (Fig. 1C and Fig. S1) and the lack of thymocytes (Fig. 4,Fig. S4, and Movies S9 and S10) and B cells (Fig. 5D). A few cellsexpressing markers associated with the myelo-monocytic lineage(spi1 and l-plastin) were detectable, whereas cells expressing thegranulocytic marker mpx were completely absent (Fig. 5B),compatible with the notion that short-lived progeny are no longerpresent after cessation of productive hematopoiesis. The formermight represent descendants of long-lived embryonic macro-phages, because they could also be observed in many tissues, in-

Fig. 3. Characterization of primitive hematopoiesis. (A) Whole-mount RNA in situ hybridizations (WISH) were performed at 24 hpf with probes specific for scl(merge composed of three pictures), gata1, and c-myb. Hybridizations using single probes are shown in the top three panels. A double-fluorescent in situ hy-bridization for c-myb (greenfluorescence) and scl (red fluorescence) is shown in the penultimate panel (merge of 22 optical slices at 5-μm thickness for wild-typeand 20 slices for mutant embryos; Fig. S3 has additional data). The expression of ikaroswas visualized using an ikaros:eGFP transgenic reporter (bottom panel).Note the normal number andfluorescence intensity of embryonicmacrophages located in the anterior part of the embryos (arrows) and the diminished signals inthe caudal hematopoietic tissue (CHT) and intermediate cell mass (ICM) of mutants (arrowheads); the embryonic macrophages are highly motile cells (Movies S9and S10). (B) WISH analyses at 36 hpf using the indicated probes. All photographs are composites of three pictures, with the exception of the gata1 hybridizationofmutant embryo, which is composed of four pictures. (C)WISH analyses for c-myb expression at the indicated time points. The photographs for the 48-hpf time-point are merged from three pictures. In the images taken at 72 hpf, the pharyngeal arches are marked with a black circle, and the thymic rudiment is markedwith a red circle (Middle); the hematopoietic tissue in the tail regions (Bottom) is indicated by lines. (D) Gene-expression analysis in c-myb mutants. RT-PCR wasperformed at the indicated time points for the indicated genes; elongation factor 1 α (ef1α) serves as a control for cDNA integrity and a standard.

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cluding the thymus (Fig. 4B). In wild-type fish, the expression ofscl, a marker associated with hematopoietic progenitor cells, wasdetected close to the nephric ducts and the intertubular space;some ventral renal tubules also express this gene. By contrast, sclexpression is lacking in the intertubular space in c-myb mutantfish, pointing to a dramatic reduction of hematopoietic progen-itor cells. Likewise, a large number of c-myb–expressing cells arepresent in wild-type fish, whereas only very few c-myb–expressingcells are found in the mutants. Using expression of cd41 asa marker for hematopoietic stem cells, it seems that such cells aregreatly diminished, if not absent, by 20 dpf (Fig. 5E); because cd41is also expressed in the thrombocyte lineage, its steadily declininglevels also argue against a transient increase of such cells, contraryto what was observed in hypomorphic mouse c-myb mutants (20,21). If hematopoietic progenitor cells survive until 2 mo of age inthe mutant, their subsequent differentiation must be severelycomprised. Collectively, the data show that the c-mybI181N mu-tation results in a state of anemia and immunodeficiency.

DiscussionFor about two decades, the potential role of c-myb in definitivehematopoiesis has been intensely scrutinized. Its pivotal functionin hematopoiesis was originally discovered in mice; nullizygousmice are severely anemic and die in midgestation, because de-finitive hematopoiesis does not occur (14). This phenotype is re-capitulated in themutant zebrafish.Thehematopoietic phenotypesobserved in animals carrying hypomorphic c-myb alleles (20, 21),chimeric animals established from mixtures of wild-type andc-myb–deficient cells (15, 19), and mice with lineage-specific in-activation of c-myb (13) suggested that this transcription factor isa pleiotropic regulator of hematopoiesis with nonredundant roles

in progenitor cells as well as in their differentiating progeny. Here,we show that, in general, the role of c-myb in the regulation ofself-renewal of hematopoietic progenitor cells and multilineagedifferentiation as originally defined in mice is evolutionarily con-served. As expected, not all aspects of the phenotypes observed inmice with hypomorphic c-myb alleles are seen in the fish mutant.For instance, thrombocytosis that occurs in some hypomorphs ofmouse c-myb (21, 22) is not observed in the mutant zebrafish.Whether this reflects functional differences of the c-myb tran-scription factor between mouse and zebrafish or is because of theparticular kind of missense mutation remains to be investigated.While this manuscript was under review, a report appeared de-scribing a c-mybmutation in the teleostOryzias latipes (23). Unlikethe situation in mouse and zebrafish, the loss of c-myb in medakaalready affects primitive hematopoiesis, supporting the notion ofspecies-specific differences in the requirement for c-myb in earlyand later stages of hematopoiesis.The unique biology of the fish has allowed us to study the

function of c-myb in adult animals, obviating the need for con-ditional lineage-specific gene inactivation or other experimentalmanipulations. Anemic zebrafish are able to survive for severalweeks (24), presumably owing to their ability to oxygenate tissuesthrough diffusion and to the minimal but nevertheless protectivefunction of long-lived innate immune cells established duringembryonic hematopoiesis. By contrast, fish with complete failureof erythropoiesis, for instance, because of mutations in gata1(25), are incapable of surviving into adulthood. This suggeststhat, after fish have developed beyond a critical threshold, theycan tolerate lack of oxygen much better than at earlier stages. Itseems that the critical transition between primitive and definitivehematopoiesis occurs between 15 dpf (when gata1 deficiencytakes its toll) and 20 dpf (when the function of primitive he-matopoiesis has lapsed).In conclusion, we have identified the first c-myb null allele in

lower vertebrates and have shown that the function of c-myb indefinitive hematopoiesis is evolutionarily conserved, contributingto the growing evidence that the genetic regulation of generalhematopoietic functions has deep evolutionary roots (26). Be-cause c-mybmutants survive into adulthood while lacking most oftheir hematopoietic cells, they could serve as a model to assess,through cell transplantation, the functional capacity of differenthematopoietic cell types without incurring damage to the hema-topoietic stromal compartments before cell transfer (27).

Materials and MethodsAnimals. Details of the forward genetic screens to identify genes regulatingthymopoiesis havebeendescribed (16); IP109mutants belong to the Tübingenarm of our screen. The ikaroseGFP line was described previously (28). The an-imal experiments reported here were approved by the regional government.

In Situ Hybridization Analysis. Procedures for RNA in situ hybridization weredescribedpreviously (16). Double in situhybridizationwas carriedour accordingto procedures described in ref. 29. Probes are listed in Table S1.

Gene-Expression Analysis. The primers for RT-PCR analysis of adult and em-bryonic gene expression have been described (30, 31); the primers for cd41were described in ref. 7. Other primers are listed in Tables S2 and S3.

Biochemistry. Details describing the expression, purification, and functionalanalysis of wild-type and mutant forms of the c-myb DNA binding domainscan be found in SI Materials and Methods.

Live Microscopy of Fish. The procedures for live imaging were describedpreviously (28).

Morpholino and BAC Injections. Injection of a c-myb–specific antisense mor-pholino was used to phenocopy the c-myb mutation; phenotypic rescue wasattempted by injection of a BAC clone encompassing a wild-type c-myb gene(details in SI Materials and Methods).

Fig. 4. Failure of thymopoiesis in c-myb mutants. (A) Lack of thymus colo-nization in c-mybmutants transgenic for ikaros:eGFP. Still photographs weretaken fromMovies S9 and S10 at the indicated time points of the observationperiod (t0 = 55 hpf), equivalent to ∼68 hpf. Note the cluster of green cells inthe thymus (encircled), whereas the thymus of mutants lacks such cells. Aprogenitor cell approaching the thymus is seen at the bottom right corner ofthe image of wild-type fish. (B) No evidence for thymopoiesis in early larvae.WISH was done with probes indicated at 5 dpf; the thymic area (encircled) isshown. Note the presence of small numbers of cells expressing l-plastin in thec-myb mutants, a marker associated with the myelo-monocytic lineage. Thismost likely represents embryonic macrophages situated in the thymus(Movies S9 and S10).

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ACKNOWLEDGMENTS. We thank the members of the Tübingen and Frei-burg screening group for help during the identification of the IP109mutant, Mike Bialecki, Dagmar Diekhoff, Fernando Mateos, Tanna Franz,

and Monika Held for help during various stages of the project, andthe Deutsche Forschungsgemeinschaft and the Max-Planck Society forfinancial support.

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31. Chan F-Y, et al. (1997) Characterization of adult alpha- and beta-globin genes in thezebrafish. Blood 89:688–700.

Fig. 5. Failure of adult hematopoiesis in c-mybmutants. (A) Histological sections of the head kidney at7 wk of age (Giemsa staining). Note the lack of hema-topoietic cells in the mutant tissue. (Scale bar: 10 μm.) (B)Flow cytometric analysis of whole kidney marrow (6 wkof age) according to side scatter (SSC) and forwardscatter (FSC) characteristics. Circles denote the positionsof various cell types detectable in wild-type fish: red,erythrocytes; blue, lymphocytes and thrombocytes; ma-genta, precursors; green, myelomonocytes. The residualcells obtained from disintegrated mutant kidney tissuelack these characteristic features. (C) Lack of adult he-moglobin gene expression in c-myb mutants. RT-PCR wasperformed at the indicated time points for adult (prefixa) and embryonic (prefix e) globin genes; ef1α serves asa control for cDNA integrity and as a standard. (D) RNAin situ hybridization of head kidney sections was per-formed with the indicated probes. The signals seen withspi1 and l-plastin presumably originate from long-livedembryonic macrophages; a single c-myb positive cell isindicated (arrow). In mutant tissue, no signals are ob-served for mpx, gata1, ikaros, rag1, and igh. (Scale bar:100 μm.) (E) Diminishing expression of cd41 in mutantembryos and larvae. RT-PCR was performed at the in-dicated time points; ef1α serves as a control for cDNAintegrity and as a standard.

17308 | www.pnas.org/cgi/doi/10.1073/pnas.1004640107 Soza-Ried et al.

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