Red Blood Cell Precursors Research Articles

Cloning and expression pattern of the Xenopus erythropoietin receptor

Cytokine signaling plays an important role in the survival and differentiation of vertebrate hematopoietic cells. In red blood cells, erythropoietin is a key component of the differentiation program and maintains the homeostasis of the erythroid compartment. In the adult, anemia stimulates high levels of circulating erythropoietin that drives erythropoiesis to restore normal levels of red blood cells in circulation. Erythropoietin activates the erythropoietin receptor on immature red blood cell precursors to promote their survival and differentiation. Although extensively studied in mammalian systems, a complete understanding of the function of the erythropoietin receptor during primitive erythropoiesis has been lacking. To address this problem, we have cloned the Xenopus laevis erythropoietin receptor in order to further understand the development of primitive erythropoiesis. The amphibian erythropoietin receptor shares 33% amino acid sequence identity with the mammalian erythropoietin receptors and contains the conserved extracellular ligand binding and fibronectin domains, the WSXWS motif common to cytokine receptors, and several tyrosine phosphorylation sites located on the intracellular domain of the receptor. Expression of the erythropoietin receptor is first detected by in situ hybridization in the ventral blood island during tailbud stages.

The Role of Erythropoietin in Vascular Endothelial Cells and Thrombosis.

Erythropoietin (EPO) is an essential glycoprotein hormone that regulates erythrocyte production in hematopoietic tissues by stimulating growth, preventing apoptosis, and inducing the differentiation of red blood cell precursors. Clinically, EPO has been extensively used to increase levels of hemoglobin in the treatment of patients with anemia due to chronic renal failure, cancer chemotherapy, and HIV therapy. Recent studies suggest that the function of EPO and its cognate receptor, EPOR, are not strictly limited to erythroid lineages. EPOR expression has been identified in nonhematopoietic cells and tissues including endothelial, neuronal, and tumor cells. Epoetins have shown benefit in correcting anemia in chronic kidney disease and in chemotherapy-associated anemia in cancer patients. However, the incidence of thrombosis appears to be higher in patients administered EPO versus controls transfused to comparable hemoglobin levels. In particular, incidences seem higher in oncological patients treated with EPO and receiving combined radiation and chemotherapy than in those receiving chemotherapy alone. To address the potential interaction and responsiveness of the vasculature to EPO, we evaluated EPO using vascular endothelial cell assays. Biochemical analysis (eg, receptor expression, ligand-receptor binding, and activation of signaling cascades) as well as cellular analysis (eg, proliferation, migration, and differentiation) will be discussed. To evaluate the effects of EPO on thrombosis we used the in vivo pulmonary micoemboli (ME) model. In this fibrinolysis model, a preformed 125I-fibrin microemboli with a defined particle size is infused into mice intravenously. Microemboli lodge primarily in the lungs and the rate of radioactivity lost is measured over time. Defects in fibrinolysis have been identified in mice lacking either tPA or uPA. Mice were exposed acutely or chronically to EPO and the dissolution of ME was tested. Mice were injected subcutaneously with either a single dose of EPO (2500 IU/kg) and for the chronic studies EPO (1200 IU/kg) or control buffer three times a week for three weeks. The residual radioactivity in the lungs was measured after 10 and 90 minutes. Red blood cell count, hemoglobin, and hematocrit were analyzed weekly to confirm EPO activity. Our results indicate that neither a single (acute) dose of EPO nor chronic treatment using EPO affected the rate of pulmonary fibrinolysis. This suggests that EPO treatment does not significantly impair the function of the pulmonary fibrinolytic system proteins (eg, uPA, tPA, plasminogen, and PAI-1) using the ME model. We cannot exclude EPO's potential influence on other aspects of coagulation and fibrinolysis.

Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F(420)H(2):NADP(+) oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.

A deletion of 11 bp (CD 131–134) in exon 3 of the β-globin gene produces the phenotype of inclusion body β-thalassemia

Dominant inherited beta-thalassemias describe those beta-thalassemia variants that result in a thalassemia intermediate phenotype in individuals who have inherited only a single copy of the abnormal beta gene. This form of thalassemia is characterized by moderately severe anemia with jaundice and splenomegaly; it is also characterized by the presence of inclusion bodies in the red blood cell precursors and has, therefore, previously been referred to as inclusion body beta-thalassemia. We describe a case of inclusion body beta-thalassemia in a 51-year-old Spanish male caused by a deletion of 11 bp (CD 131-134) in exon 3 of the beta-globin gene. The deletion of 11 bp in exon 3 of the beta-globin chain is predicted to produce an anomalous chain of 134 amino acids instead of the normal 146 with an extremely altered amino acid sequence from residues 131-134. Although this shortened variant would lead to a missing H helix, which is involved in alpha1beta1 contact and alpha1beta2 subunit interactions, the variant chain can still be bound to the heme group and acquire a secondary structure that is not suitable for the formation of stable dimers or tetramers and also less susceptible to proteolytic degradation. This is the first report of such a beta-thalassemia mutation.

Characterization of cells in the developing human liver

Human hepatic progenitor cells (HPCs) have been shown to co-express the hematopoietic stem cell (HSC) markers, CD117 and CD34. These cells differentiate not only into hepatocytes and cholangiocytes but also into pancreatic ductal and acinar cells under certain conditions. The fetal liver (FL) is rich in precursor/stem cells; however, little is known about (i) the markers expressed by liver cells during fetal development and (ii) whether an equivalent to the adult liver stem-like progenitors exists in the FL. Here, (i) FL tissue obtained from human 5–18-week-old fetuses were evaluated by means of flow cytometry, immunocyto-, and histochemistry for the emergence of cells expressing and co-expressing known hematopoietic, hepatic, and pancreatic cell markers, and (ii) isolated putative HPCs were phenotypically and molecularly characterized. We report that (i) red blood and endothelial cell precursors were most abundant in early gestation. Cells expressing HSC and pancreatic markers were found in the first trimester, while cells expressing hepatic markers appeared in the second trimester. Very few committed cells were present in FLs obtained early in the first trimester. In addition, cells expressing pancreatic markers co-expressed the HSC marker CD117. (ii) Isolated CD117+/CD34+/CD90− cells in vitro expressed both the genes and proteins for the hepatic markers such as albumin, alpha feto protein (AFP), α1-antitrypsin, and cytokeratin 19 (CK19). Our study suggests that hepatoblast and ductal plate/bile duct development mainly occurs during the second trimester. FLs in gestation weeks 5–9 had the highest numbers of precursor cells and the least committed cells. Cells that differentiate into Alb+ or CK19+ can be isolated from early FLs and may be appropriate progenitors for establishing novel systems to investigate basic mechanisms for cell therapy.

Anemia of inflammation: the hepcidin link

The anemia of inflammation has been associated for nearly two decades with elevated cytokine levels, but the primary mediator of this condition was unknown. Recently hepcidin antimicrobial peptide has emerged as the hormone that links the type II acute phase response to iron handling and erythropoiesis. Hepcidin antimicrobial peptide likely modulates iron transport from macrophages and enterocytes to red blood cell precursors as a consequence of its interaction with SLC40A1/ferroportin, the only known transporter that facilitates iron egress. Insights into the regulation of hepcidin antimicrobial peptide expression by known iron metabolic proteins such as HFE, hemojuvelin, and transferrin receptor 2 are expanding the understanding of the genetic circuitry that controls iron absorption and utilization. Increasingly, experiments suggest the hepatocyte is not just the iron storage depot but is the 'command central' for the maintenance of iron homeostasis. It receives multiple signals related to iron balance and responds via transcriptional control of hepcidin antimicrobial peptide.

Improvement of Fc–erythropoietin structure and pharmacokinetics by modification at a disulfide bond

Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.

Transfer of Human α- to β-Hemoglobin via Its Chaperone Protein: EVIDENCE FOR A NEW STATE

The alpha-hemoglobin-stabilizing protein (AHSP), a small protein of 102 amino acids, is synthesized in red blood cell precursors. It binds specifically to alpha-hemoglobin (alpha-Hb) subunits acting as a chaperone protein, preventing the formation of alpha-hemoglobin-cytotoxic precipitates. We have engineered recombinant AHSP in a pGEX vector to study the functional consequence of interaction between AHSP and alpha-Hb. By in vitro binding assays, we have isolated the complexes glutathione S-transferase-AHSP.alpha-Hb and AHSP.alpha-Hb. The latter assembles as a heterodimer based on size-exclusion chromatography. These complexes exhibited monophasic CO binding kinetics, as observed for isolated alpha- and beta-subunits of hemoglobin. However, the rate of CO (or oxygen) binding to alpha-hemoglobin bound to its chaperone is three times slower than that observed for isolated alpha-hemoglobin, demonstrating a form that is intermediate to the R- and T-hemoglobin states. The physiologically relevant replacement of the chaperone by beta-hemoglobin chains could be detected by both ligand binding kinetics and tryptophan fluorescence quenching.

Expression of α-hemoglobin stabilizing protein gene during human erythropoiesis

α-Hemoglobin stabilizing protein (AHSP) is an abundant, erythroid-specific protein that forms a stable complex with free α-hemoglobin but not with β-hemoglobin or hemoglobin A. As such, AHSP is required for normal erythropoiesis, probably acting by blocking the deleterious effects of free α-hemoglobin precipitation. In order to study the levels of expression of the AHSP gene during the different phases of erythropoiesis, we carried out a two-phase liquid culture of erythroid cells and real-time quantitative polymerase chain reaction. Blood from control volunteers was cultured with erythropoietin to stimulate differentiation. The different stages of erythropoiesis were confirmed by morphologic and flow cytometric analysis. The results showed a progressive increase in AHSP gene expression following the expression of α- globin gene, during maturation of the red blood cell precursors, confirming the probable important function of this protein during normal erythropoiesis.

Persistence of human parvovirus B19 in human tissues

Human parvovirus B19 infection causes various clinical symptoms, such as rash, arthropathy, anemias and fetal death, but it can also remain asymptomatic. The arthropathies and anemias can become chronic for several years, not infrequently resembling autoimmune syndromes. B19 replicates only in red blood cell precursors of bone marrow or fetal liver, resulting in high-titred short-lived viremia, but viral DNA is detectable also in cells of several other types. Recently B19 DNA has been found, by very sensitive amplification tests, in certain tissues not only of symptomatic but also of healthy individuals for several years or decades after B19 infection. The mere presence of B19 DNA in these tissues of a symptomatic patient (e.g. joints in chronic arthritis or skin in dermatomyositis) thereby does not prove that the present disease is caused by B19. The diagnosis has to be verified by other innovative means. How and why viral DNA persists in the tissues of healthy individuals is under investigation.

Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency.

Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both alpha- and beta-globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron-deficient HRI(-/-) mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.

Fetal parvovirus B19 infection

Parvovirus B19 infection during pregnancy causes up to 27% cases of non-immune hydrops in anatomically normal fetuses. The virus is believed to cause arrest of maturation of red blood cell precursors at the late normoblast stage and also causes a decrease in the number of platelets. Fetal anemia is presently thought to be responsible for the development of skin edema and effusions. Myocarditis leading to heart failure may contribute to the development of fetal hydrops. We reviewed the literature regarding prevalence, transmission rates, clinical presentation, diagnostic techniques, current invasive vs. conservative management options, outcome and postmortem findings in a total of 82 studies involving 230 invasively and 435 conservatively managed pregnancies. In this non-selected population, the proportion of seronegative susceptible mothers ranged from 19 to 65%, seroconversion with an incubation time of up to 20 days occurred in 5.7-12.1%, and 188/230 (82%) who were transfused infected fetuses had a normal outcome as opposed to only 239/435 (55%) in the conservatively managed group. The average time from diagnosis to resolution in both groups was 6 weeks (range, 3-12 and 2-12 weeks, respectively). The most promising diagnostic techniques were PCR of amniotic fluid or fetal blood and electron microscopy. There are some reports of fetal abnormalities occurring (probably coincidentally) in cases of parvovirus, but the majority of postmortem findings were infection-related, in particular myocarditis and hepatic abnormalities. Although management guidelines cannot be derived from this study due to the variable degree of hydrops in the analyzed studies, the present data suggest a benefit of transfusion therapy over conservative management in infected fetuses. The only study which was corrected for severity of hydrops using ultrasound criteria showed a clear benefit of intrauterine transfusion.

Metallothionein isogene transcription in red blood cell precursors from human cord blood.

The in vitro transcription patterns for 10 functional metallothionein (MT) isogenes have been investigated in red blood cell (RBC) precursors from human cord blood. Active transcription status of the isogenes, MT-0, MT-1A, MT-1B, MT-1E, MT-1G, MT-1X, and MT-2A, was detected in both ex vivo expanded RBC precursors (burst-forming unit-erythroid) and glycophorin A(+) and CD71(+) cells separated by magnetic cell sorting. Transcription patterns of these isogenes were analyzed at different times of incubation with the addition of Zn supplement. In neither the ex vivo expanded precursors nor glycophorin A(+) and CD71(+) cells could MT-1F and MT-3 be detected. Transcripts of MT-4 were detected in glycophorin A(+) and CD71(+) cells. Erythropoietin-responsive constitutive transcription of MT-1X and possible interleukin-3-responsive downregulation of MT-2A in ex vivo expanded precursors reveal their effect on MT biosynthesis. Biosynthesis and induction of MT at the protein level in the RBC precursors was also demonstrated by immunoblotting.

A KOREAN FAMILY WITH A DOMINANTLY INHERITED β-THALASSEMIA DUE TO Hb DURHAM-N.C./BRESCIA [β114(G16)Leu→Pro

We describe the molecular and the hematological characteristics of a Korean family with a dominantly inherited β-thalassemia. Carriers were characterized by moderate anemia, hypochromia, microcytosis, elevated Hb A2 and Hb F levels, and splenomegaly. DNA analysis revealed a CTG (Leu) to CCG (Pro) substitution at codon 114 of the β-globin gene, that leads to a highly unstable hemoglobin variant, Hb Durham-N.C./Brescia, and this was linked to the β haplotype V, [+−−−−+−], and framework 2. RNA analysis showed that the proband had comparable levels of mutant and normal β-mRNA. Translation of the mutant mRNA would give rise to non-functional hyperunstable β-globin chains, and their degradation would, by placing an additional burden on the proteolytic process of the red blood cell precursors, result in a more severe phenotype.

Conditional deletion of the Bcl-x gene from erythroid cells results in hemolytic anemia and profound splenomegaly.

Bcl-x is a member of the Bcl2 family and has been suggested to be important for the survival and maturation of various cell types including the erythroid lineage. To define the consequences of Bcl-x loss in erythroid cells and other adult tissues, we have generated mice conditionally deficient in the Bcl-x gene using the Cre-loxP recombination system. The temporal and spatial excision of the floxed Bcl-x locus was achieved by expressing the Cre recombinase gene under control of the MMTV-LTR. By the age of five weeks, Bcl-x conditional mutant mice exhibited hyperproliferation of megakaryocytes and a decline in the number of circulating platelets. Three-month-old animals suffered from severe hemolytic anemia, hyperplasia of immature erythroid cells and profound enlargement of the spleen. We demonstrate that Bcl-x is only required for the survival of erythroid cells at the end of maturation, which includes enucleated reticulocytes in circulation. The extensive proliferation of immature erythroid cells in the spleen and bone marrow might be the result of a fast turnover of late red blood cell precursors and accelerated erythropoiesis in response to tissue hypoxia. The increase in cell death of late erythroid cells is independent from the proapoptotic factor Bax, as demonstrated in conditional double mutant mice for Bcl-x and Bax. Mice conditionally deficient in Bcl-x permitted us for the first time to study the effects of Bcl-x deficiency on cell proliferation, maturation and survival under physiological conditions in an adult animal.

EXPRESSION OF ANGIOTENSIN II TYPE I RECEPTOR ON ERYTHROID PROGENITORS OF PATIENTS WITH POST TRANSPLANT ERYTHROCYTOSIS1

The pathogenesis of posttransplant erythrocytosis (PTE) has been elusive. Angiotensin converting enzyme inhibitors (ACEI) are efficacious in lowering the hematocrit of patients with PTE and angiotensin II (AII) type I receptors (AT1R) were recently detected on red blood cell precursors, burst-forming unit-erythroid- (BFU-E) derived cells. The purpose of this study was to determine whether there is increased expression of the AT1R on BFU-E-derived cells of patients with PTE, which might contribute to the pathogenesis of PTE. Twelve healthy volunteers and 25 transplant recipients (13 patients with and 12 without PTE) were studied. BFU-E from peripheral blood were cultured in methylcellulose and BFU-E-derived colonies were harvested on day 10. Western blotting was used to detect AT1R and erythropoietin receptor (EpoR) expression. Intracellular free calcium in response to AII and erythropoietin (Epo) was measured with digital video imaging. There were no differences between transplant patients, with and without PTE, with respect to weight, age, sex, blood pressure, serum creatinine, circulating renin, angiotensin II, and Epo levels. Hematocrit, red blood cell number, BFU-E-derived colony number,and size were significantly increased in PTE compared with other two groups. AT1R expression was increased by 44% on the erythroid progenitors of PTE versus non posttransplant erythrocytosis patients and by 32% in PTE patients versus normal volunteers. AT1R expression correlated significantly with the hematocrit in PTE (Spearman r=0.68, P=0.01). In contrast, EpoR expression was equivalent in all groups. The AT1R was functional since a significant increase in [Ca(i)] was observed in Fura-2 loaded day 10 cells when stimulated with AII (182%, P<0.0001). An increase in AT1R density was observed in erythroid precursors of transplant patients with PTE compared to those without PTE and normal volunteers, and the level of AT1R expression in PTE correlated significantly with the hematocrit. In contrast, EpoR expression was not different in PTE compared with non posttransplant erythrocytosis or normal controls. This study supports a role for the AT1 receptor signaling pathway in the pathogenesis of PTE.

An in vitro system for efficiently evaluating gene therapy approaches to hemoglobinopathies.

A variety of gene therapy strategies are under development for the treatment of sickle cell anemia and other hemoglobinopathies. A number of alternative vectors have been developed to transfer and express the beta-globin gene and other therapeutic molecules, but none has resulted in efficient transduction and stable long-term expression in primary hematopoietic cells. One reason for this problem is that most vectors are initially evaluated in immortalized cell lines which may not faithfully recapitulate the biology of primary erythroid cells. In order to provide a more relevant system for efficiently evaluating alternative vector constructs for beta-globin disorders, we have developed (1) a simple method for generating primary human red blood cell (RBC) precursors in liquid culture established with mononuclear cells obtained from normal donors as well as patients with Hb SC disease; (2) a high titer retroviral vector which can be easily modified to optimize gene transfer and transgene expression; and (3) methods for transducing the RBC precursors at high efficiency. The development of simple and efficient methods and reagents for generating and transducing primary human RBC precursors provides a facile and effective means for screening alternative gene therapy strategies. Gene Therapy (2000) 7, 215-223.

Metallothionein Biosynthesis in Human RBC Precursors

The in vitro biosynthesis of metallothionein (MT) has been investigated in RBC precursors from human cord blood in order to support the hypothesis for the nucleated precursor origin of MT in human red blood cells (RBC). Human RBC precursors are obtained by (i) separating glycophorin A⁺ (gly A⁺) cells using a magnetic cell sorting (MACS) technique and by (ii) ex vivo expansion of precursors BFU-E (burst forming unit-erythroid) on methylcellulose semi-solid culture media from mononuclear cells of cord blood. Biosynthesis of MT is detected at the protein level, by immuno-histochemical staining using a mouse monoclonal antibody (E9) in ex vivo expanded RBC precursors obtained from BFU-E. Expression of MT is also detected at the mRNA level by MT specific reverse transcriptase polymerase chain reaction (RT-PCR) both in ex vivo expanded precursors from BFU-E and in MACS separated gly A⁺ cells. In addition, the expression of the fetal form of MT, MT-0 (also known as MT-1H) at the mRNA level in glycophorin A⁺ cells, is also confirmed by cDNA sequencing. With these observations, to our knowledge, MT biosynthesis in human erythroid precursors is reported for the first time. Moreover, the current findings of MT-0 expression at the mRNA level in gly A⁺ RBC precursors of hCB has added one more member in the list of cells/organs like fetal liver, human monocytes, non-neoplastic tissues of adenocarcinoma etc., in which the expression of the human fetal form of MT, i.e. MT-0, has also been reported.

Polycystin-2 expression is developmentally regulated.

PKD2 encodes a protein of unknown function that is mutated in 15% of autosomal dominant polycystic kidney disease (ADPKD) families. We used polyclonal antisera against PKD2 to examine the pattern of Pkd2 expression in staged mouse embryos. Staining for Pkd2 was documented as early as the 6th embryonic day (day E6) in the embryonic ectoderm and endoderm. Low-intensity staining is seen in metanephric ureteric bud at day E12.5. By day E15.5, the adult pattern of expression is established with low level staining in proximal tubules and high level, basolateral staining in distal tubules. Pkd2 expression is first detected in the medullary collecting ducts at postnatal day 14. Outside of the kidney, Pkd2 expression is widely distributed in utero and more restricted postnatally. The greatest intensity of staining is seen in the fetal but not adult adrenal cortex and in red blood cell precursors. Expression also is seen in multiple endocrine organs, in cardiac, skeletal, and smooth muscle, and in multiple mesenchymal tissues. The diffuse distribution and early expression of Pkd2 suggest a fundamental developmental role. The persistent strong expression in adult kidney is consistent with a more organ-specific function in the maintenance of the mature metanephric tubule.

Analysis of the pattern of QM expression during mouse development

QM, a novel gene that was originally identified as a putative tumor suppressor gene, has since been cloned from species encompassing members of the plant, animal, and fungal kingdoms. Sequence comparison indicates that QM has been highly conserved throughout eukaryotic evolution. QM is a member of a multigene family in both mouse and man, is expressed in a broad range of tissues, and is downregulated during adipocyte differentiation. Jif-1, a chicken homolog of QM, has been reported to interact with the protooncogene c-Jun, and to inhibit transactivation of AP-1 regulated promoters in vitro. Furthermore, disruption of the yeast QM homolog is lethal. Although these studies suggest that the QM gene product plays an important role within the normal cell, the precise role of QM has remained elusive. In this study, a thorough analysis of the pattern of QM expression during mouse development was undertaken, using the techniques of whole mount in situ hybridization and whole mount immunohistochemistry, in combination with conventional immunohistochemical analysis of tissue sections. QM is expressed in numerous embryonic tissues, and is differentially expressed throughout the embryo. The cytoplasmic localization of QM is consistent with its reported association with ribosomes, and inconsistent with its previously hypothesized function as a direct modulator of the nuclear protooncogene c-Jun. QM is expressed in the developing epidermis, and is particularly strong within developing limbs. Analysis of embryos of various stages of gestation indicate that QM is downregulated in the surface ectoderm of the embryo as development proceeds. QM protein is not detectable within either nucleated or enucleated red blood cell precursors. QM is strongly expressed within chondrocytes within the transition zone of developing limb cartilage, as well as within differentiated keratinocytes of the suprabasal regions of the epidermis. Furthermore, within both cartilage and skin, there is an inverse relationship between QM expression and proliferative capacity. This pattern of QM expression suggests that this novel gene product may be involved in processes such as posttranslational protein processing which are essential for differentiation of specific tissues during embryogenesis.