Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices

Summary Very early embryonic mesoderm cells were taken from the primitive streak-stage chick embryo and cultured in a matrix of type I collagen in the presence of serum. Previous work has shown that under these conditions cells do not leave the explant and move in the collagen in the absence of supplemented avian transferrin. Cells explanted onto tissue culture plastic in the presence of serum do not require this transferrin supplement. These observations were investigated further by culturing cells in collagen in the presence of the lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), which can replace transferrin as an iron-delivery agent. Under conditions in which FePIH could effectively stimulate chick embryo myoblast growth, no such long-term stimulation was obtained with the early mesoderm cells in collagen. This suggested that for mesoderm cells, FePIH could not replace transferrin. Antibody to the transferrin receptor and to transferrin itself inhibited growth of myoblasts in collagen and on plastic, and of mesoderm cells in collagen. Mesoderm cells on plastic, however, were refractory to the presence of the antibody directed to the receptor and seemed to show a low dependency on transferrin-delivered iron under these conditions, inasmuch as antiserum to transferrin itself only caused a partial inhibition of outgrowth. The results suggest that mesoderm cells in collagen require transferrin for both iron uptake and for another unspecified function. It is consistent with the results to propose that transferrin binding might modulate the cells' attachment to collagen, thus influencing outgrowth. The distribution of the actin cytoskeleton in mesoderm cells actively migrating in collagen, such as in the presence of transferrin, suggests a stronger attachment to the collagen than nonmigrating cells. This work was supported by an operating grant from the Medical Research Council of Canada.     

The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor

Dissociated neurons from the trigeminal (V) region of the metencephalic basal plate or the ventral spinal cord from chick embryos of Day 4 (V basal plate) or Day 5 (spinal cord) were cultured on a laminin substratum either in the presence of nerve growth factor (NGF) or in control medium. Assessment was made of neuronal survival, the amount of neurite elaborated, and the percentage of neurons initiating neurites. The presence of motoneurons was verified by retrograde labeling with the fluorescent dye diI. NGF was found to significantly increase the quantity of neuritic processes produced by the spinal cord dissociates at both 24 and 48 hr in vitro. The percentage of neurons initiating neuritic processes was significantly increased by NGF in the trigeminal population at 48 hr in vitro. Neuronal survival was not enhanced by NGF in either group. Both trigeminal and spinal cord neurons were also found to specifically bind 125I-NGF in culture. These results provide direct evidence for an influence of NGF on process formation of early embryonic motoneurons in culture.     

Nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake

The human transferrin receptor is post-translationally modified by the addition of a fatty acyl moiety. In earlier studies, transient expression in Cos cells of human transferrin receptors in which Cys62 or Cys67 was altered to serine provided evidence that Cys62 is the major acylation site of the receptor (Jing, S., and Trowbridge, I. S. (1987) EMBO J. 6, 327-331). To determine whether acylation of the receptor is required for high efficiency endocytosis and iron uptake, wild type and mutant human transferrin receptors have been stably expressed in chick embryo fibroblasts using a helper-independent retroviral vector. In marked contrast to Cos cells, both Cys62 and Cys67 of the wild type human transferrin receptor were acylated in chick embryo fibroblasts. Moreover, their modification to serine did not abolish palmitate labeling, implying that one or both of these serine residues could serve as alternative lipid attachment sites in these cells. The relative labeling of mutant receptors with palmitate and the susceptibility of their lipid moieties to cleavage by hydroxylamine were consistent with Ser67 but not Ser62 serving as a lipid attachment site. Consequently, to obtain human transferrin receptors lacking covalently bound lipid in the chick embryo fibroblasts, it was necessary to alter Cys62 and Cys67 to alanine. Functional studies indicated that these non-acylated mutant receptors were internalized efficiently and mediated iron uptake from human transferrin at a similar rate to that of wild type receptors. We conclude, therefore, that acylation of the human transferrin receptor is not essential for endocytosis and recycling.     

Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures

We report here that cells present in embryonic chick retinal monolayer cultures express differentiated properties characteristic of chick cones developing in vivo. Cell suspensions from 8-d chick embryo retina (a stage when photoreceptor differentiation has not yet started) were cultured for up to 7 d in low density, glial-free monolayers. Under these conditions, monopolar cells represent approximately 40% of the total number of process-bearing neurons. After 6 d in vitro, most of these monopolar cells showed morphological features reminiscent of developing chick cones. These features could be detected with phase-contrast microscopy, lectin cytochemistry, and transmission and scanning electron microscopy. Characteristic cone traits expressed by cultured monopolar cells included the following: (a) a highly polarized organization; (b) a single, short, usually unbranched neurite; (c) the polarized position of the nucleus close to the origin of the neurite; (d) characteristic cone inner segment features such as abundant free ribosomes, a polarized Golgi apparatus, a cluster of mitochondria distal to the nucleus, a big, membrane-bound, pigment-containing vacuole reminiscent of the "lipid droplet" characteristic of chick cones, and at least in some cases, a well-developed paraboloid; (e) the presence of a complex of apical differentiations including abundant microvilli and in some cases also a cilium-like process; and (f) the staining of the apical region of the cell with peanut lectin, which has been shown to be selective for chick embryo cones (Blanks, J.C., and L.V. Johnson, 1983, J. Comp. Neurol., 221:31-41; and Blanks, J.C., and L.V. Johnson, 1984, Invest. Ophthalmol. Visual Sci., 25:546-557). This pattern of differentiation achieved by 8-d chick retina cells after 6 d in vitro is similar to that shown by 14-d-old chick embryo cones in vivo. Outer segments are not present at this stage of development either in vivo or in vitro. This experimental system is now being used to search for cellular and molecular signals controlling survival and differentiation of cone cells.     

Transferrin endocytosis and iron uptake in developing myogenic cells in culture: effects of microtubular and metabolic inhibitors, sulphydryl reagents and lysosomotrophic agents

The experiments described in this study were designed to investigate receptor-mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts (dividing and non-dividing) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor-mediated endocytosis of the protein.     

N-cadherin, NCAM, and integrins promote retinal neurite outgrowth on astrocytes in vitro

Retinal ganglion neurons extend axons that grow along astroglial cell surfaces in the developing optic pathway. To identify the molecules that may mediate axon extension in vivo, antibodies to neuronal cell surface proteins were tested for their effects on neurite outgrowth by embryonic chick retinal neurons cultured on astrocyte monolayers. Neurite outgrowth by retinal neurons from embryonic day 7 (E7) and E11 chick embryos depended on the function of a calcium-dependent cell adhesion molecule (N-cadherin) and beta 1-class integrin extracellular matrix receptors. The inhibitory effects of either antibody on process extension could not be accounted for by a reduction in the attachment of neurons to astrocytes. The role of a third cell adhesion molecule, NCAM, changed during development. Anti-NCAM had no detectable inhibitory effects on neurite outgrowth by E7 retinal neurons. In contrast, E11 retinal neurite outgrowth was strongly dependent on NCAM function. Thus, N-cadherin, integrins, and NCAM are likely to regulate axon extension in the optic pathway, and their relative importance varies with developmental age.     

Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.     

Purified proteins acting on cultured chick embryo ciliary ganglion neurons

Chick embryo ciliary ganglion neurons in dissociated monolayer culture have been used to examine molecular requirements for neuronal survival and neurite growth. These neurons will rapidly die in vitro unless supplied with an adequate level of ciliary neuronotrophic factor (CNTF), and even in the presence of CNTF they will not vigorously extend neurites on polyornithine substrata unless supplied with appropriate amounts of polyornithine-binding neurite-promoting factors (PNPFs). Recent work on the purification and partial characterization of embryonic chick eye CNTF and rat schwannoma PNPF is reviewed, and in vitro responses of ciliary ganglion neurons to other purified proteins such as laminin, fibronectin, insulin, and nerve growth factor are mentioned.     

Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development

The mechanism of iron uptake and the changes which occur during cellular development of muscle cells were investigated using primary cultures of chick embryo breast muscle. Replicating presumptive myoblasts were examined in exponential growth and after growth had plateaued. These were compared to the terminally differentiated cell type, the myotube. All cells, regardless of the state of growth or differentiation, had specific receptors for transferrin. Presumptive myoblasts in exponential growth had more transferrin receptors (3.78 +/- 0.24 X 10(10) receptors/micrograms DNA) than when division had ceased (1.70 +/- 0.14 X 10(10) receptors/micrograms DNA), while myotubes had 3.80 +/- 0.26 X 10(10) receptors/micrograms DNA. Iron uptake occurred by receptor-mediated endocytosis of transferrin. While iron was accumulated by the cells, apotransferrin was released in an undegraded form. There was a close correlation between the molar rates of endocytosis of transferrin and iron. Maximum rates of iron uptake were significantly higher in myotubes than in presumptive myoblasts in either exponential growth or after growth had plateaued. There were two rates of exocytosis of transferrin, implying the existence of two intracellular pathways for transferrin. These experiments demonstrate that iron uptake by muscle cells in culture occurs by receptor-mediated endocytosis of transferrin and that transferrin receptor numbers and the kinetics of transferrin and iron uptake vary with development of the cells.     

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.     

Analysis of ephrin-A2 in the chick retinotectal projection using a function-blocking monoclonal antibody

Eph receptor tyrosine kinases and their ligands have been shown to be involved in processes of cell migration and axon guidance during embryonic development. Here we describe the development of a function-blocking monoclonal antibody against chick ephrin-A2, and its effect on retinal ganglion cell axons studied both in vitro and in vivo. In the stripe assay, the blocking antibody completely abolished the repulsive effect of posterior tectal membranes. In vivo, in a loss-of-function approach, hybridoma cells secreting the antiephrin-A2 antibody were applied to chick embryos from embryonic day 3 (E3) on, and the retinotectal projection was subsequently analyzed at E16. DiI tracing analyses showed that although the projection of both temporal and nasal retinal ganglion axons in the tectum was, overall, normal, occasionally diffuse and extra termination zones were observed, in addition to axons over-shooting their termination zones. These data support the idea that ephrin-A2 contributes to the establishment of the chick retinotectal projection.     

Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.     

Differences between human and rabbit transferrins

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic.The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.     

Presence of nerve growth factor receptors and catecholamine uptake in subpopulations of chick sympathetic neurons: correlation with survival factor requirements in culture

The presence of nerve growth factor receptors and the imipramine-sensitive uptake of catecholamines in sympathetic neurons of chick embryos were investigated by autoradiography. Neurons were dissociated from paravertebral ganglia of different embryonic ages and receptors and catecholamine uptake were then determined both at the beginning of culture and after 2 days in culture, at which time the number of surviving neurons is determined by the survival factors present. It was found that while essentially all the neurons specifically bound 125I-NGF both after dissociation and at the end of the culture period, only 60% of the neurons take up [3H]norepinephrine after dissociation, and this proportion remained constant through the culture period under conditions where all the neurons survived. All of the neurons maintained by NGF in culture (35% of the total) displayed this uptake, and in contrast, only one-quarter of those maintained by heart cell-conditioned medium alone (60% of the total) took up catecholamines. The uptake was shown to be neither induced by NGF nor suppressed by heart cell-conditioned medium. These results support the hypothesis that chick sympathetic ganglia contain discrete subpopulations of neurons which may be selected in culture by virtue of their different requirements for survival factors.     

Differences between human and rabbit transferrins.

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic. The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.     

Neuronal precursor cells in chick dorsal root ganglia: differentiation and survival in vitro

Neuronal precursor cells present in dorsal root ganglia (DRG) during early development have been previously shown to differentiate in vitro to neurons, as characterized by morphology, cell surface antigens, and electrophysiological properties (H. Rohrer, S. Henke-Fahle, T. El-Sharkawy, H. D. Lux, and H. Thoenen, 1985, Embo J. 4, 1709-1714). In the present study the conditions necessary for the initial differentiation and long-term survival of these cells were established, and the neurotransmitter phenotype of the newly differentiated neurons was analyzed. Neuronal precursor cells isolated from chick DRG at Embryonic Day 6 (E6) were found to require the presence of a polyornithine substrate coated with either laminin or fibronectin for initial neurite production and long-term survival. Neurons were unable to develop on polyornithine alone or on polyornithine coated with BSA. The survival and neurite outgrowth from neuronal precursor cells was not affected by the presence of nerve growth factor (NGF) during the first 9 hr in culture. NGF also had no effect on the proportion of cells expressing the neuron-specific Q211 antigen. However, after this initial differentiation period the neurons did require the presence of a survival factor. The neurons could be maintained for at least 6 days in culture both in the presence of NGF and in the presence of brain-derived neurotrophic factor (BDNF). At saturating concentrations of both survival factors no additive effects could be observed, indicating a complete overlap of NGF- and BDNF-responsiveness. Although the same proportion of cells survived with either NGF or BDNF during the first 3 days in culture, survival decreased in the presence of BDNF but not in the presence of NGF during the following 3 days in culture. The loss of BDNF responsiveness in vitro was also observed in vivo. After 6 days in culture about 70% of the neurons expressed substance P immunoreactivity, and approximately the same proportion was positive for myelin-associated glycoprotein immunoreactivity. The neurons did not express properties of adrenergic neurons such as tyrosine hydroxylase immunoreactivity or norepinephrine uptake. These findings indicate that the neuronal precursor cells from E6 DRG acquire the same characteristics in vitro as in their normal in vivo environment.     

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.     

Culture conditions affect the cholinergic development of an isolated subpopulation of chick mesencephalic neural crest cells

Although neural crest cells are known to be very responsive to environmental cues during their development, recent evidence indicates that at least some subpopulations may be committed to a specific differentiation program prior to migration. Because the neural crest is composed of a heterogeneous mixture of cells that contributes to many vertebrate cell lineages, assessing the properties of specific subpopulations and the effect of the environment on their development has been difficult. To address this problem, we have isolated a pure subpopulation of chick mesencephalic neural crest cells by fluorescence no-flow cytometry after labeling them with monoclonal antibodies (Mabs) to a 75-kDa cell surface antigen that is associated with high affinity choline uptake. When cultures of chick mesencephalic neural crest cells are labeled with these Mabs and a fluorescent second step antibody, approximately 5% of the cells are antigen-positive (A+). After sorting, 100% of the resulting cultured mesencephalic neural crest cells are A+. The Mabs we used also label all of the neurons of the embryonic chick and quail ciliary ganglion in vivo and in vitro. We have compared the effect of various cell culture media on the isolated neural crest subpopulation and the heterogeneous chick mesencephalic neural crest from which it was derived. A+ cells were passaged and grown in a variety of media, each of which differently affected its characteristics and development. A+ cells proliferated in the presence of 15% fetal bovine serum (FBS) and high concentrations (10-15%) of chick embryo extract, but did not differentiate, although they retained basal levels of choline acetyltransferase (ChAT) activity. However, in chick serum and high (25 mM as opposed to 7 mM) K+, and heart-, iris-, or lung-conditioned medium, all of which are known to promote survival and/or cholinergic development of ciliary ganglion neurons, the cells ceased to proliferate and all of the cells in the culture became "neuron-like" within 10 days. No neuron-like cells were found in liver-, notocord-, or neural tube-conditioned media if FBS was used. When A+ cells were eliminated either by complement-mediated cytotoxicity or by laser-ablating A+ cells during no-flow cytometry, all ChAT activity was also eliminated, and no neuron-like cells or ChAT activity was found in cultures during a subsequent 3-week culture period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in the number of chick ciliary ganglion neuron processes with time in cell culture

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.     

The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis

The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis.