Age-dependent amelioration of hypoplastic anemia in Ws/Ws rats with a small deletion at the kinase domain of c-kit

The white-spotting (Ws) locus of rats represents a 12-base deletion of the c-kit receptor tyrosine kinase. Homozygous Ws/Ws rats are deficient in melanocytes, mast cells, and erythrocytes. Although mice possessing two mutant alleles at the c-kit (W) locus, such as mice of W/Wv genotype, show severe anemia even in adult age, the anemia of Ws/Ws rats remarkably ameliorated with age. We investigated the mechanism of the age-dependent amelioration. Bone marrow cells of Ws/Ws rats did not form macroscopic colonies in the spleen of irradiated rats, and the concentration of burst-forming unit-erythroid in the marrow of Ws/Ws rats was comparable with that of +/+ rats. Therefore, the increase in morphologically identifiable erythroid precursors in the marrow of Ws/Ws rats was attributed to the increased concentration of colony- forming unit-erythroid (CFU-E). Furthermore, the increase in CFU-E appeared to result from the increased concentration of erythropoietin (EPO). Because injections of relatively low doses of EPO cured the slight anemia that remained in adult Ws/Ws rats, CFU-E and/or its immediate precursors of Ws/Ws rats appeared to be more sensitive to EPO than those of W/Wv mice, in which a huge dose of EPO was necessary to cure the anemia.     

Infection of Nippostrongylus brasiliensis induces normal increase of basophils in mast cell-deficient Ws/Ws rats with a small deletion at the kinase domain of c-kit

All basophils, mucosal-type mast cells (MMC) and connective tissue-type mast cells (CTMC) are derived from the multipotential hematopoietic stem cell. Mutations at the c-kit locus resulted in deficiency of MMC and CTMC in both mice and rats. To investigate the role of the c-kit receptor tyrosine kinase for production of basophils, we used white spotting/white spotting (Ws/Ws) mutant rats that have a small deletion at the tyrosine kinase domain of the c-kit gene. When Ws/Ws, nude athymic, and normal (+/+) rats were infected with Nippostrongylus brasiliensis (NB), the number of basophils increased greater than 50- fold in the peripheral blood of Ws/Ws and +/+ rats but did not increase in that of nude rats. Blood histamine concentration increased significantly in Ws/Ws and +/+ rats but did not increase in nude rats. Immature basophils increased greater than 10-fold in the bone marrow of Ws/Ws and +/+ rats but did not increase in that of nude rats. Mature and immature basophils that developed after the NB infection were identified by electron microscopy. The present result confirms that T- cell-derived cytokines are indispensable for the augmented production of basophils and suggests that stimulation via the c-kit receptor may not be necessary for the augmented production.     

Characterization of cultured mast cells derived from Ws/Ws mast cell- deficient rats with a small deletion at tyrosine kinase domain of c-kit

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP- I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I- /II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA- SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.     

Infection of Nippostrongylus brasiliensis induces development of mucosal-type but not connective tissue-type mast cells in genetically mast cell-deficient Ws/Ws rats

Ws/Ws rats have a small deletion at the tyrosine kinase domain of the c- kit gene and are deficient in both mucosal mast cells (MMC) and connective tissue-type mast cells (CTMC). The role of the c-kit receptor in the development of MMC and CTMC was investigated by infecting Ws/Ws and control +/+ rats with Nippostrongylus brasiliensis (NB), which induces T-cell-dependent mast cell proliferation. Although mast cells did not develop in the skin of Ws/Ws rats, a significant number of mast cells developed in the jejunum after NB infection. These mast cells had the MMC protease phenotype (rat mast cell protease [RMCP] I-/II+) and lacked heparin because they were not stained with berberine sulfate. Globule leukocytes were also detected in the mucosal epithelium of these rats. However, the number of MMC and the serum concentration of RMCP II in NB-infected Ws/Ws rats were only 13% and 7% of those of NB-infected +/+ rats, respectively. A small number of mast cells also developed in the lung, liver, and mesenteric lymph nodes of Ws/Ws rats after NB infection. Although mast cells in these tissues had the MMC phenotype throughout the observation period, the increased mast cells in the lung and liver of +/+ rats acquired a CTMC-like phenotype and were RMCP I+/II+, berberine sulfate+, and formalin resistant. These results indicate that the need for the stimulus through the c-kit receptor appears to be greater in the development of CTMC in the skin as well as for CTMC-like mast cells in the lung and liver than for the development of MMC.     

Analysis of the hematopoietic effects of new dominant spotting (W) mutations of the mouse. II. Effects on mast cell development

In addition to their anemia, sterility and lack of coat pigment (1,2), W/Wv mice are mast cell deficient (3,4). Our analysis of three recently described W alleles (5) confirms reports (3,6) that (a) W mutations alter skin mast cell number in parallel with their influence on red cell number (but not with pigmentation), (b) that mast cells arise from hematopoietic tissue (7) and (c) that injections of normal bone marrow cells, which cure the anemias of W/Wv recipients, also alleviate the deficiency of skin mast cells in these mice. Transplants of bone marrow cells from mice homozygous for two new anemia-causing W alleles, W39 and W41, fail to cure the anemias of W/Wv recipients (companion paper) or increase the number of mast cells in their skin. Marrow cell implants from non-anemic W44/W44 mice cure the anemia, but do not change the number of mast cells in the skin of W/Wv recipients. The fact that the bone marrows of all three new homozygotes have fewer than normal numbers of CFUs hematopoietic stem cells (see companion paper) and have reduced mast cell-regenerating capacities, supports Kitamura's contention (8) that mast cell precursors may be closely related to or identical with the CFUs.     

Mast cells may not play a crucial role in the pathogenesis of experimental closed duodenal loop-induced pancreatitis in rats

INTRODUCTION: Ws/Ws rats have a small deletion of the c-kit gene and are deficient in both mucosal-type mast cells and connective tissue-type mast cells. AIM: To investigate the role of pancreatic mast cells in the development of experimental closed duodenal loop (CDL)-induced pancreatitis using Ws/Ws rats. METHODOLOGY: Pancreatitis was induced by the CDL technique for 5 and 12 hours, and the subsequent ascites volume, wet pancreatic weight, pancreatic myeloperoxidase activities, and serum amylase levels were evaluated. The pancreatic tissue damage was also evaluated histologically. RESULTS: The CDL technique induced equally severe ascites, pancreatic edema and hyperemia, and hyperamylasemia in the Ws/Ws versus the control (+/+) rats. The microscopic mucosal damage score was also equivalent in the Ws/Ws and control (+/+) rats, and there were no significant differences in mucosal myeloperoxidase activity between the Ws/Ws and control (+/+) rats. CONCLUSION: These results indicate that mast cells may not be crucial for the development of CDL-induced pancreatitis.     

Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction in the rat

BACKGROUND AND AIMS: Stress may be an important factor in exacerbating inflammatory bowel disease but the underlying mechanism is unclear. Defective epithelial barrier function may allow uptake of luminal antigens that stimulate an immune/inflammatory response. Here, we examined the effect of chronic stress on colonic permeability and the participation of mast cells in this response. METHODS: Mast cell deficient Ws/Ws rats and +/+ littermate controls were submitted to water avoidance stress or sham stress (one hour/day) for five days. Colonic epithelial permeability to a model macromolecular antigen, horseradish peroxidase, was measured in Ussing chambers. Epithelial and mast cell morphology was studied by light and electron microscopy. RESULTS: Chronic stress significantly increased macromolecular flux and caused epithelial mitochondrial swelling in +/+ rats, but not in Ws/Ws rats, compared with non-stressed controls. Stress increased the number of mucosal mast cells and the proportion of cells showing signs of activation in +/+ rats. No mast cells or ultrastructural abnormalities of the epithelium were present in Ws/Ws rats. Increased permeability in +/+ rats persisted for 72 hours after stress cessation. CONCLUSIONS: Chronic stress causes an epithelial barrier defect and epithelial mitochondrial damage, in parallel with mucosal mast cell hyperplasia and activation. The study provides further support for an important role for mast cells in stress induced colonic mucosal pathophysiology.     

Infection with Nippostrongylus brasiliensis induces invasion of mast cell precursors from peripheral blood to small intestine

Precursors of mast cells were defined as cells that formed mast-cell colonies in methylcellulose culture (CFU-mast). Mononuclear cells (MNC) were obtained from the bone marrow, peripheral blood, and small intestine of Ws/Ws rats with a small deletion at the tyrosine kinase domain of c-kit and of control normal (+/+) rats. In the culture containing concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM) alone, the numbers of mast-cell colonies produced by Ws/Ws MNC were comparable with those of +/+ MNC. In the culture containing both ConA-SCM and stem cell factor (a ligand of c-kit), however, the numbers of mast-cell colonies produced by +/+ blood MNC were 107 times as great as that of Ws/Ws blood MNC. Using this culture condition, we investigated changes in concentration of CFU-mast in the marrow, blood, and intestine of +/+ rats after infection with Nippostrongylus brasiliensis (NB), which induced marked mast-cell accumulation in the small intestine. The concentration of CFU-mast in blood dropped to 21% of preinfection levels 1 week after the NB infection. In contrast, a sevenfold increase of CFU-mast occurred in the small intestine. The proportion of CFU-mast in S phase of the cell cycle remained at low levels in the marrow and blood after NB infection, but it increased significantly in the small intestine. The present result suggests that NB infection induces the invasion of CFU-mast into the intestine from blood and their subsequent proliferation in the tissue site.     

Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts

Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.     

Increased plasma transferrin, altered body iron distribution, and microcytic hypochromic anemia in ferrochelatase-deficient mice

Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, experience a painful type of skin photosensitivity called erythropoietic protoporphyria (EPP), which is caused by the excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial results have been reported regarding hematologic status and iron status of patients with EPP. We thoroughly explored these parameters in Fechm1Pas mutant mice of 3 different genetic backgrounds. FECH deficiency induced microcytic hypochromic anemia without ringed sideroblasts, little or no hemolysis, and no erythroid hyperplasia. Serum iron, ferritin, hepcidin mRNA, and Dcytb levels were normal. The homozygous Fechm1Pas mutant involved no tissue iron deficiency but showed a clear-cut redistribution of iron stores from peripheral tissues to the spleen, with a concomitant 2- to 3-fold increase in transferrin expression at the mRNA and the protein levels. Erythrocyte PPIX levels strongly correlated with serum transferrin levels. At all stages of differentiation in our study, transferrin receptor expression in bone marrow erythroid cells in Fech(m1Pas) was normal in mutant mice but not in patients with iron-deficiency anemia. Based on these observations, we suggest that oral iron therapy is not the therapy of choice for patients with EPP and that the PPIX-liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen, and the bone marrow.     

Evaluation of role of mast cells in the development of liver fibrosis using mast cell-deficient rats and mice.

BACKGROUND/AIMS: Several studies have suggested that mast cells participate in the development of liver fibrosis in rodent models. In this study mast cell-deficient mutant Ws/Ws rats and W/Wv mice were used to examine whether mast cells are involved in the development of liver fibrosis. METHODS: Liver fibrosis was induced in rats by bile duct resection (BDR), and by intraperitoneal injections of carbon tetrachloride (CCl4) or porcine serum, and in mice by intragastric administrations of CCl4, and BDR. The degree of fibrosis was evaluated by measuring the hydroxyproline content (microg/mg tissue) of the liver as an index of the collagen content. The density of mast cells (number/cm2 liver section) was determined by counting mast cells in liver sections stained with alcian blue. RESULTS: In the liver of control non-mutant (+/+) rats, mast cells were found principally in portal areas, and their average density was 200-300/cm2 liver section. BDR, and treatments with CCl4 and porcine serum increased the density of mast cells in the liver of +/+ rats several-fold, and induced liver fibrosis, increasing the liver hydroxyproline content markedly. BDR, and treatments with CCl4 and porcine serum also induced liver fibrosis in Ws/Ws rats, increasing the liver hydroxyproline content to a similar or higher level than that in +/+ rats. However, the average densities of mast cells in the liver of Ws/Ws rats after BDR and treatment with CCl4 and porcine serum were at most 10.2/cm2 liver section. The density of mast cells in the liver of control +/+ mice was extremely low (average, less than 2), and neither BDR nor treatment with CCl4 caused any significant increase in their density, whereas these treatments induced liver fibrosis and markedly increased the liver hydroxyproline content. Furthermore, treatment with CCl4 induced fibrosis in the liver of W/Wv mice similarly to that in +/+ mice, but the density of mast cells in the liver of W/Wv mice was very low (average, less than 1), and was not increased by treatment with CCl4. CONCLUSIONS: The present results indicate that mast cells play no role in the development of liver fibrosis in rats and mice.     

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts.

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.     

Heme oxygenase-1 deficiency alters erythroblastic island formation,steady-state erythropoiesis and red blood cell lifespan in mice

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119⁺-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119⁺-erythroid cells in the spleen, although α4β1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.     

Adventitial mast cells contribute to pathogenesis in the progression of abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is histologically characterized by medial degeneration and various degrees of chronic adventitial inflammation, although the mechanisms for progression of aneurysm are poorly understood. In the present study, we carried out histological study of AAA tissues of patients, and interventional animal and cell culture experiments to investigate a role of mast cells in the pathogenesis of AAA. The number of mast cells was found to increase in the outer media or adventitia of human AAA, showing a positive correlation between the cell number and the AAA diameter. Aneurysmal dilatation of the aorta was seen in the control (+/+) rats following periaortic application of calcium chloride (CaCl2) treatment but not in the mast cell-deficient mutant Ws/Ws rats. The AAA formation was accompanied by accumulation of mast cells, T lymphocytes and by activated matrix metalloproteinase 9, reduced elastin levels and augmented angiogenesis in the aortic tissue, but these changes were much less in the Ws/Ws rats than in the controls. Similarly, mast cells were accumulated and activated at the adventitia of aneurysmal aorta in the apolipoprotein E-deficient mice. The pharmacological intervention with the tranilast, an inhibitor of mast cell degranulation, attenuated AAA development in these rodent models. In the cell culture experiment, a mast cell directly augmented matrix metalloproteinase 9 activity produced by the monocyte/macrophage. Collectively, these data suggest that adventitial mast cells play a critical role in the progression of AAA.     

Delayed erythropoiesis in irradiated rats grafted with syngeneic marrow: effects of cytotoxic drugs and iron-deficiency anemia

Erythropoiesis in spleens of lethally irradiated Lewis rats grafted with 4-35 X 10(6) syngeneic marrow cells was inhibited or delayed during the test period of 5 days; this was in marked contrast to observation in irradiated mice. The mechanism of this inhibition was the subject of this study. Pretreatment of recipients 9 days prior to irradiation with the cytotoxic drugs cyclophosphamide (CY), busulfan (BUS), or dimethylmyleran (DMM), or the induction of iron deficiency with anemia in recipients reversed this delayed erythropoiesis. However, neither iron-deficiency anemia nor pretreatment with BUS or DMM affected the ability of irradiated recipients to reject 20 to 50 X 10(6) allogeneic marrow cells. The administration of commercial preparations of erythropoietin to hosts stimulated erythropoiesis moderately. However, proliferation of syngeneic marrow cells was not enhanced when infused into lethally irradiated Spontaneous Hypertensive (SH) inbred-strain rats which have high levels of endogenous erythropoietin. Finally, plasma from irradiated rats treated with phenylhydrazine to produce severe anemia was rich in erythropoietin but failed to stimulate erythropoiesis in the cell transfer system. Two hypotheses are considered: (1) Irradiation inhibits the secretion of a factor (not erythropoietin) responsible for initiating early stages in differentiation of transplanted stem cells; iron-deficiency anemia and cytotoxic drugs stimulate the secretion of this factor. (2) Normal rats secrete a factor which suppresses erythropoiesis; iron-deficiency anemia and cytotoxic drugs inhibit the production or function of this factor. Cellular rather than humoral factors may by involved.     

Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats

Mast cells have diverse roles throughout the body as evidenced by their heterogeneous nature. In the heart, cardiac mast cells have been implicated in left ventricular (LV) remodeling in response to elevated myocardial stress. Accordingly, the purpose of this study was to use mast cell deficient rats (Ws/Ws) to delineate the interaction between cardiac mast cell activation and LV remodeling. LV matrix metalloproteinase (MMP) activity, fibrillar collagen, TNF-α levels, and LV diameter were compared in Ws/Ws and wild type (WT) rats subjected to 5 d (n = 3/group) and 8 weeks (n = 4/group) of aortocaval fistula-induced volume overload. In contrast to attenuation of myocardial remodeling in the Ws/Ws group: 1) MMP-2 activity was significantly increased in the WT group at 5 days; 2) there was marked degradation of the extracellular collagen matrix in WT at 5 days and 8 weeks; 3) the percent increase in LV diameter from baseline was significantly greater in WT at 2, 4, 6, and 8 weeks post-fistula; and 4) myocardial TNF-α levels were markedly elevated in the WT group at 5 days post-fistula. These results underscore the importance of cardiac mast cells in mediating MMP activation, collagen degradation and LV dilatation and suggest that mast cell-derived TNF-α plays a role in early myocardial remodeling.     

Erythropoiesis in ha/ha and sph/sph mice, mutants which produce spectrin-deficient erythrocytes

In order to characterize chronically accelerated erythropoiesis, we studied the ultrastructure of bone marrow and spleen of ha/ha and sph/sph mice, two mutants with profound hemolytic anemia secondary to deficiency of the erythrocyte membrane protein spectrin. The marrows and spleens of both varieties were extremely erythropoietic and were without histological abnormalities directly related to spectrin deficiency. Erythropoiesis was consistently associated with distinctive, dark branched cells which constituted large proportions of the stroma of the mutant spleens and marrow. These dark cells were not present in untreated and acutely bled controls. Plasma clot assays for erythroid progenitors revealed that CFU-E concentrations in the mutant marrows were significantly increased over those in untreated controls while BFU-E concentrations were approximately half. In addition, mutant CFU-E often gave rise to abnormal appearing colonies. Spectrin, though crucial to erythrocyte function is probably not important to the process of erythroid differentiation and maturation. The status of erythroid precursors in the marrows of the spectrin deficient mice is similar to that of mice subjected to an acute bleed. The divergent changes in CFU-E and BFU-E may indicate that these two cells play different roles in accelerated erythropoiesis. The dark cells that we describe are similar to stromal cells observed in models of the early stages of erythropoiesis.