Vimentin downregulation is an inherent feature of murine ...Vimentin expression in erythropoiesis 87 described by Cudennec et al. (1981). Murine fibroblasts were isolated from the

Development 110, 85-96 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

85

Vimentin downregulation is an inherent feature of murine erythropoiesis and

occurs independently of lineage

FRANK SANGIORGI, CATHERINE M. WOODS and ELIAS LAZARIDES

Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125, USA

Summary

In mammalian erythropoiesis, the mature cells of theprimitive lineage remain nucleated while those of thedefinitive lineage are anuclear. One of the molecular andstructural changes that precedes enucleation in cells ofthe definitive lineage is the cessation in the expression ofthe gene for the intermediate filament (IF) proteinvimentin and the removal of all vimentin filaments fromthe cytoplasm. We show here that in immature primitivecells vimentin is synthesized and forms a cytoplasmicnetwork of IFs. As differentiation proceeds in vivo,vimentin gene expression is downregulated in these cells;this is accompanied by the loss of vimentin filamentsfrom the cytoplasm. This loss temporally coincides with

the nucleus becoming freely mobile within the cyto-plasm, suggesting that, while IT removal is not directlylinked to the physical process of enucleation, it may be aprerequisite for the initiation of nuclear mobility in bothlineages. These changes are also observed in earlyprimitive cells cultured in vitro, suggesting that theyconstitute an intrinsic part of the murine erythroiddifferentiation program independent of lineage andhematopoietic microenvironment.

Key words: murine erythropoiesis, intermediate filaments,primitive erythrocytes, vimentin, mouse.

Introduction

During vertebrate development, erythropoiesis occursin a succession of distinct loci, with the sequentialappearance of two erythroid lineages each possessingspecific morphological and physiological characteristics(Sabin, 1920; Kovach et al. 1967; Bruns and Ingram,1973). In both avian and mammalian species, the firstlineage to be produced, the primitive lineage, originatesin the blood islands of the yolk sac. The cells of thislineage are large (12-13 /zm in diameter), amoeboid andnucleated, expressing characteristic embryonic hemo-globins. These cells differentiate as a cohort, beingreleased into the circulation while still mitotically active(around 9 days of embryonic development for themouse). They are short-lived, being progressively re-placed by the definitive lineage of erythroid cells (from12 to 13 days in the mouse) (Bruns and Ingram, 1973;Fantoni et al. 1967, 1969a).

The second lineage, the definitive lineage, arises inspecific interim sites before the bone marrow becomesthe ultimate erythropoietic site just prior to hatching orbirth (Sabin, 1920; Bruns and Ingram, 1973; Dieterlen-Lievre and Martin, 1981; Russell and Bernstein, 1966;Fantoni et al. 1967, 19696). In mammals, the first waveof definitive erythropoiesis occurs specifically in thefetal liver (Fantoni et al. 1969a,b). In mice the first

hematoblasts can be distinguished at 10 days of ges-tation, developing within the liver before being releasedinto the circulation as intermediate-sized (8/zm diam-eter) erythrocytes but with identical anuclear, bicon-vex, disc-shaped morphology to the smaller (6/zm)spleen- and bone-marrow-derived erythrocytes found incirculation after birth (Russell and Bernstein, 1966;Fantoni et al. 1967, 1969a,b). These mammalian defini-tive erythrocytes differ markedly from their nonmam-malian counterparts which remain nucleated andexhibit a biconcave disc morphology (Bruns andIngram, 1973; Lazarides, 1987). Thus, despite thesimilar differentiation pathway from early BFU-e andCFU-e precursor to erythroblast cell stages (Till andMcCulloch, 1980; Samarut and Gazzolo, 1982), mam-mals have an additional differentiation stage where thenucleus is actively extruded to give rise to an anuclearreticulocyte. This reticulocyte then matures into thecharacteristic biconcave disc-shaped erythrocyte.

Morphological studies have shown that enucleation isa complex process that involves an asymmetric position-ing of the nucleus and a concentration of the erythroidmembrane skeleton in the incipient reticulocyte prior toenucleation, a process resembling asymmetric cytokin-esis (Geidushek and Singer, 1979). This step occursonly within the liver, spleen and bone marrow, andappears to involve the association of the nucleated

86 F. Sangiorgi and others

erythroblasts with stromal cells, possibly a distinctsubset of macrophages (Crocker et al. 1988). Theextruded nuclei are then phagocytosed by the suppor-tive macrophages. The exact role of cell-cell contact orextracellular factors in this morphogenetic process re-mains to be determined since murine erythroblaststransformed in vivo with the Friend murine leukemiaviral complex will reliably differentiate and enucleate invitro in the absence of supportive stromal cells orextracellular matrix (Koury et al. 1982,1984). However,it is clear that, for enucleation to occur, multiplemorphogenetic changes must have taken place duringthe divergence of nucleated and anuclear red bloodcells.

One such change occurs in the structure of thecytoplasm that allows the nucleus to rotate and movefreely, a prerequisite for its asymmetric positioning inthe cytoplasm prior to extrusion. Normally, the nucleusis anchored to the plasma membrane and maintained inan invariant position. In avian red blood cells this hasbeen shown to be effected by a network of vimentinintermediate filaments (IFs) (Granger and Lazarides,1982; Virtanen et al. 1979). This vimentin network isaugmented during the final stages of avian erythroidterminal differentiation in both primitive and definitivelineages resulting from the upregulation of vimentinexpression at the transcriptional and translational levels(Blikstadt and Lazarides, 1983; Capetanaki etal. 1983).In contrast, in murine erythroleukemia (MEL) cells,used as a model of murine definitive erythroid differen-tiation, a marked and rapid downregulation in vimentinexpression takes place when they are chemicallyinduced to differentiate, with complete disappearanceof vimentin IFs within 96 h (Ngai et al. 1984). Similarremoval of vimentin filaments occurs in human eryth-roblasts grown in vitro (Dellagi et al. 1983). Thisdivergence in vimentin expression appears to be due tochanges in regulatory sequences in the chicken andmammalian vimentin genes and is effected at the lateCFU-e stage (Ngai etal. 1987; Koury etal. 1989). Theseobservations have led to the hypothesis that the re-moval of the vimentin IF network is a necessary earlyprerequisite for enucleation to proceed and one of thefundamental changes that has occurred in the diver-gence of the morphogenetic pathways of definitiveavian and mammalian erythroid cells (Ngai et al. 1987).

In this report, we analyze the expression of vimentinduring the differentiation of murine primitive erythro-cytes and compare it with that in hepatic and adultdefinitive erythrocytes. We demonstrate that a vimentinIF network exists in primitive cells early in develop-ment. However, as differentiation proceeds, vimentinexpression is markedly downregulated and is ac-companied by the loss of the vimentin IF network.These changes occur at the same developmental stagewhen the primitive erythroblasts are cultured in vitro.The loss of IFs correlates with the nucleus becomingfreely mobile within the cell. These results are consist-ent with the hypothesis that the downregulation andloss of vimentin IFs represent an intrinsic part of theerythroid differentiation program in both the primitive

and definitive lineages and that these events are inde-pendent of both hematopoietic microenvironment andthe physical process of enucleation.

Materials and methods

AnimalsAll mice were of the outbred MTS strain, bred and main-tained at Harlan Sprague Dawley Laboratories (San Diego,CA). The dating of fetal development was based on timedmatings of both hormonally primed and non-primed females,with day 0 of gestation commencing on the morning aftermating.

CellsPrimitive erythroid cells were isolated by a modification of theprocedure described by Kovach et al. (1967). At selectedgestational ages (8 to 14 days), pregnant mice were sacrificedby cervical dislocation and their uteri removed and placed incalcium-/magnesium-free phosphate-buffered saline (PBS)containing 35Uml-1 heparin. The embryos were then dis-sected out either within the decidua in the case of 8 and 9 dayembryos or within the amniotic sac in the case of olderembryos. After removal of extraembryonic tissues, erythroidcells were released by mechanical disruption of the yolk sacblood islands (8 to 10 days) or by transection of the dorsalaorta or umbilical vessels (older embryos). From 13 days ofgestation onward, circulating cells within the embryo alsoincluded hepatic erythrocytes. The older the embryo, thecleaner the dissection from maternal tissues, including theplacenta, with greatly reduced contamination by maternalerythrocytes. The cells were collected in Dulbecco's modifiedEagle's medium (DMEM) with high glucose supplementedwith 1% bovine serum albumin (BSA) and 3 5 U m r 'heparin. Residual tissue fragments and contaminating ma-ternal leucocytes were removed by filtering the cell suspen-sion through a combined cheese cloth-Leuko-Pak filter (Fen-wal Laboratories, IL). The cells were subsequently collectedby centrifugation and resuspended in DMEM. The cellsuspension was further enriched for primitive or hepaticerythroid cells by centrifugation through a discontinuous stepgradient of Percoll (Pharmacia LKB Biotechnology, NJ) in0.15 M NaCl. The gradient consisted of five 2 ml steps ofincreasing Percoll densities ranging from 1.065 to l.lOgml"1,the exact composition being modified for each time point tocompensate for the increasing density of the primitive eryth-rocytes with gestational age due to accumulation of hemoglo-bin and their shrinking size. Typically, the primitive cellssedimented in the 1.075 to l.OSgml"1 fraction at 9 days ofgestation but by 14 days they cosedimented with maturedefinitive cells in the 1.1 gml"1 fraction. Hepatic erythroidcells were typically found in the 1.09 to 1.1 g ml"1 fraction at14 days of gestation. The cells were layered onto the gradientand centrifuged at 10000 revs min"1 for lOmin at 4°C in anSS34 rotor. 2 ml fractions of the gradient were collected,diluted in DMEM and cells collected by centrifugation. Thosefractions containing primitive or hepatic erythroid cells wereidentified by microscopy during cell counts. This identifi-cation was subsequently confirmed by nuclear staining (Paul,1975) and benzidine staining for hemoglobin (Friedman andSchildkraut, 1977). Contamination of the primitive cell frac-tion with embryo-derived myeloid cells was insignificant asmyelopoiesis does not commence until the 15th or 16th day ofgestation (Russell, 1979).

Culture of isolated yolk sac cells was carried out exactly as

Vimentin expression in erythropoiesis 87

described by Cudennec et al. (1981). Murine fibroblasts wereisolated from the ear shell of an MTS mouse and maintainedin DMEM supplemented with 10% fetal calf serum (FCS),"lOUmr1 penicillin and 40figmT1 streptomycin. Avianerythrocytes were isolated from 15 day embryos as describedby Blikstadt and Lazarides (1983).

Electrophoresis and immunoblottingCells were lysed in sodium dodecyl sulfate (SDS) samplebuffer, and lxlO6 cells were electrophoresed on a 7.5%polyacrylamide-SDS gel and transferred to nitrocellulose aspreviously described (Granger and Lazarides, 1984). Afterblocking at 37 °C in Tris-buffered saline (TBS) containing0.25 % gelatin and 0.1 % Tween-20 (TBS-GT) the filters wereincubated overnight at room temperature in TBS-GT with a1/1000 dilution of either a rabbit anti-chicken vimentin(Granger and Lazarides, 1979) or a goat anti-mouse vimentin(ICN, Costa Mesa, CA) antiserum. After washing, the filterswere incubated in TBS-GT containing 125I-Protein A for30min at room temperature, washed extensively, dried andexposed to Kodak XAR-5 X-ray film at -80°C. Alternatively,immunoblotting was performed on two-dimensional gels. Forthis the cells were either lysed directly in 9 M urea orfractionated into Triton X-100 soluble and insoluble fractionsas described by Blikstadt and Lazarides (1983). The insolublefraction was resuspended in Triton-saline lysis buffer (1 %Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH7.5, 5mMMgCl2, 2mM EGTA, 6mM /3-mercaptoethanol, lmM phenyl-methylsulphonyl fluoride, SOUml"1 aprotinin), sonicatedwith two 15 s pulses at 100 watts and centrifuged for 5 min at12 000 revs min"1 in an Eppendorf centrifuge to give a cyto-skeletal supernatant fraction free of nuclei. The soluble andcytoskeletal fractions were then denatured in 9 M urea, 5mM/3-mercaptoethanol. Two-dimensional electrophoresis wasperformed by a modification of the method of O'Farrell(1975) as described by Hubbard and Lazarides (1979), using a12.5 % polyacrylamide-SDS gel in the second dimension. Forthe immunoblotting of two-dimensional gels, the vimentinantiserum was visualized using the Protoblot alkaline phopha-tase-conjugated goat anti-rabbit IgG system (Promega Corp.,Madison, WI).

Metabolic labelling and autoradiographyPrimitive erythrocytes (6X106) from 10- to 14-day embryoswere washed with methionine-free minimum essential me-dium (MEM) supplemented with 10% dialysed FCS andincubated in 0.5 ml of this medium for 15 min prior to additionof 400^Ci [35S]methionine (1100 to 1200Cimmor'; Amer-sham Corp., IL). After 1 h at 37°C the cells were washed withDMEM and lysed in Triton-saline lysis solution. Cytoskeletaland soluble fractions were prepared as described above,denatured in 9 M urea, 6mM /3-mercaptoethanol and subjectedto two-dimensional electrophoresis. The resultant gel wasprocessed for autoradiography by impregnating with 2,5-diphenyloxazole (PPO), dried and exposed to XAR-5 X-rayfilm as previously described (Ngai et al. 1987). Incorporationof [35S]methionine into protein in total lysates or fractionatedsamples was determined as trichloroacetic acid (TCA)-pre-cipitable counts.

ImmunofluorescenceVimentin filaments in murine primitive cells were visualizedby immunofluorescence essentially as described by Grangerand Lazarides (1982), with the following modifications. Primi-tive red cells from 9 to 13 days of gestation or culturedprimitive cells were allowed to settle onto coverslips at roomtemperature. Adherent cells were fixed in 2% formaldehyde

in TBS for 2 min, rinsed and permeabilized in TBS containing1 % Triton X-100. Polyclonal anti-chicken vimentin (Grangerand Lazarides, 1979), anti-chick protein 4.1 (Granger andLazarides, 1984), anti-chicken /S-spectrin (Nelson and Lazar-ides, 1983) or anti-mouse tubulin (kindly provided by Dr J.Olmsted) all at 1/40 dilution were added to the coverslips.Cells were subsequently incubated in fluorescein isothiocyan-ate-conjugated goat anti-rabbit IgG diluted 1/150 (Miles-Yeda Ltd., Rehovot, Israel). Coverslips were mounted inElvanol and viewed with a Leitz phase-epifluorescence micro-scope using a 63x lens.

RNase protection analysisSteady-state mRNA levels of murine vimentin and y-actinwere assayed by protection from RNase digestion of 32P-labelled RNA probes synthesized in vitro (Melton et al. 1984;Zinn et al. 1983). For detection of vimentin sequences a 315 bpEcoRI-ffiwcII fragment from a 1.4 kb murine vimentincDNA was subcloned into the pGEM-4 vector (Promega Co.,Madison, WI). This fragment is derived from a cDNAisolated from a mouse spleen library and corresponds to thesequence towards the 5' end of vimentin mRNA extendingfrom nucleotides 324 to 630 (Wood et al. 1989). Afterlinearizing the recombinant plasmid with PvuU, a 383 nucleo-tide ^P-labelled RNA probe was generated with SP6 polym-erase as described (Ngai et al. 1987). Murine y-actin mRNAwas detected with a 145 nucleotide 32P-labelled RNA probesynthesized from a HinQ linearized template containing the 3'non-coding region of the human y-actin cDN A (Gunning et al.1983) cloned into pSP64 (Enoch et al. 1986), as previouslydescribed (Ngai et al. 1987).

Cell lysates were hybridized with probe using the protocolsof Firestein et al. (1987) and Thompson and Gillespie (1987),with the following modifications. Primitive erythroid cellsisolated from 9 to 14 days of gestation and cultured mousefibroblasts were washed with DMEM, counted and pelleted.The cell pellets were dissolved in 4 M guanidinium thiocya-nate, 25 mM sodium citrate, pH7.0, 100 mM /3-mercapto-ethanol to give a suspension of 108cells/ml. Aliquots of thelysates were stored at -70°C until used.

The hybridization mixture typically contained 1 to2xl05ctsmin~1, corresponding to 1 to 2ng of lyophilizedprobe and lysate equivalent to 1 to 2X106 primitive erythroidcells or 5X104 fibroblasts in a final volume of 75 ul in 4 Mguanidinium thiocyanate, 25mM sodium citrate, pH7.0,100 mM /J-mercaptoethanol. The mixture was heated to 65 °Cfor 5 min and incubated at 22°C for 17h. The hybridizationreaction was diluted with 500^1 of lOmM Tris-HCl, pH7.5,5mM EDTA, 0.3M NaCl, 900Uml-1 RNase Tl and500/igmT1 RNaseA. After lh at 30°C, SDS and proteinaseK were added to give a final concentration of 0.6% and0.3 ing ml"1, respectively, and the incubation continued at37°C for 30min. The digests were extracted with phenol-chloroform-isoamyl alcohol and coprecipitated with 10 fig ofyeast tRNA in isopropanol and lyophilized. The pellets wereresuspended in 90% formamide, lmM EDTA, 0.1% (w/v)xylene cyanole and the fragments were resolved on a 5%polyacrylamide-8 M urea gel. The gel was exposed to KodakXAR-5 film with an intensifying screen. Protection of the invitro synthesized vimentin probe by cellular vimentin mRNAshould yield a 307 nucleotide fragment while protection of they-actin probe should yield an 80 nucleotide fragment.

Flow cytometryAcridine orange (AO) cell cycle analysis as developed byDarzynkiewicz et al. (1980b), was performed on 10 to 14 dayprimitive cells that had been previously fixed and stored in


50% ethanol at 4°C. Cells were resuspended in physiologicalsaline at a concentration of 106ml~1 immediately before use.To an aliquot of 2X105 cells, 0.4 ml of 80 mM HC1, 0.2%Triton X-100 in saline was added. After 30 s of permeation thecells were stained by adding 1.2 ml of 126mMNaHPO4, 37 mMacetic acid, lmM EDTA, 150 mM NaCl and 10/igmP1 of AO.To confirm the specificity of AO staining for RNA, 10 dayprimitive cells were permeabilized and treated with1000 U ml"1 of RNase A in 150 mM sucrose, 5mM MgCl2,20mM Tris-HCl, pH6.5 at 37°C for 30min prior to stainingwith AO. Samples were filtered through a 50 /xm mesh prior tofluorescent-activated cell sorting (FACS) analysis. Analysis ofAO-stained samples was performed on an Ortho Cytofluoro-graph 50H using an argon laser excitation beam of 250 mW at488 nm as described (Boyer et al. 1989).

Results

Vimentin IFs expressed in early murine primitiveerythroblasts are lost during differentiationTo establish whether murine nucleated primitive eryth-roblasts express a vimentin-based IF network at anystage during their development, purified primitive cellsfrom 9 to 14 days of gestation were fixed and processedfor immunofluorescence using a polyclonal anti-chickenvimentin antiserum which is known to react with bothavian and murine vimentin with high affinity (Grangerand Lazarides, 1979; Ngai et al. 1987). In primitive redcells from 9 and 10 days of gestation this antiserumdetects a distinct IF network extending from the nu-cleus to the cell membrane (Fig. 1A and B). However,as these cells mature, they shrink in size and thenetwork becomes reduced exhibiting a diffuse distri-bution between day 11 and 12 until by day 13 it hasdisappeared completely from the cell (Fig. 1C to E).One striking correlation with this loss of vimentin IFswas readily seen by microscopy when living cells wereflushed under the coverslip. From 13 days onwards, thenuclei of the primitive cells were completely mobile,tumbling within the cell as they floated across the fieldof view. Hepatic erythrocytes from the circulation of 13or 14 day embryos, readily distinguishable from theirbone-marrow-derived counterparts on the basis of size(8/zm compared to 6/im in diameter), were completelynegative for vimentin.

For comparison, immunofluorescence was performedon these primitive cells using anti-/S-spectrin, protein4.1 and tubulin antibodies. Spectrin and protein 4.1 arecomponents of the erythroid membrane skeleton andwould be expected to accumulate under the membraneduring terminal differentiation. Tubulin is the mainstructural component of the characteristic marginalband found encircling the equator of nucleated defini-tive cells in nonmammalian vertebrates (Goniakowska-Witalinska and Witalinski, 1976). It was therefore ofinterest to determine whether these structural domainswere also present in primitive murine red cells. Adiffuse uniform fluorescence, localized under theplasma membrane, was observed with anti-/5-spectrinand protein 4.1 antibodies. This fluorescence becamemore intense as these cells matured (data not shown).Immunofluorescence with anti-tubulin revealed the

presence of a loosely organized band of microtubulesaround the cell periphery in non-mitotic cells around 10to 12 days as reported by Koury et al. (1987). However,these microtubules subsequently vanished from thecytoplasm from 13 days of gestation onwards (data notshown).

To confirm that the putative IF network was indeedvimentin, immunoblot analysis was performed onsamples prepared from erythroid cells isolated between8 and 14 days of gestation. It should be noted that with 8day embryos it was difficult to obtain primitive cellscompletely free of the contaminating maternal erythro-cytes. For this time point the loading was based on thenumber of primitive cells in the sample. Since adulterythrocytes do not express vimentin, any signal mustarise solely from the primitive cells. As seen in Fig. 2,the anti-chicken vimentin antibody readily detects asingle band of MT 57 000 of equal intensity on a per cellbasis in primitive cells from 8 and 9 day embryos, whichcomigrates with the band detected in murine lympho-cytes, cells known to express vimentin (Lilienbaum etal. 1986; Dellagi et al. 1983), and has a slightly slowerelectrophoretic mobility than chicken vimentin(Granger and Lazarides, 1979). The signal then rapidlydeclines from 10 days onwards, such that after day 12 itcan no longer be detected even after overexposure ofthe immunoblot (Fig. 2, lower panel). Since hepaticerythrocytes are released into the circulation around 12to 13 days of gestation and can be separated readilyfrom the primitive cell population, these cells were alsoanalyzed for the presence of vimentin. As seen inFig. 2, fetal hepatic red cells are also negative forvimentin. Immunoblotting of two-dimensional gels of10 day primitive cells confirmed that this immuno-reactive protein comigrates as murine vimentin(Fig. 3A and B), migrating adjacent to a- and/S-tubulins.

Cessation of vimentin expression during primitiveerythroblast differentiationTo assess the relative changes in vimentin synthesisduring primitive erythroid development, cells werepulse labeled with [ Sjmethionine and the amount ofvimentin synthesized and assembled was monitored bytwo-dimensional gel electrophoresis of both the solubleand cytoskeletal cell fractions followed by autoradi-ography. Newly synthesized vimentin can readily bevisualized by such analysis (Blikstadt and Lazarides,1983; Ngai et al. 1987). Fig. 3 depicts the area of theautoradiographs that include vimentin, the tubulins andactin in the cytoskeletal fractions. Newly synthesizedvimentin was not evident in the soluble fraction (datanot shown), consistent with the previous observationthat any vimentin synthesized is rapidly assembled intofilaments (Blikstadt and Lazarides, 1983). Newly syn-thesized, cytoskeletal vimentin is readily visualized inautoradiograms of 10 day primitive cell cytoskeletalfractions but the amount of vimentin synthesizedrapidly declines thereafter needing much longer ex-posure times to be visualized for 11 and 12 day cells,being undetectable in 13 and 14 day cells (Fig. 3B and


9 day

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13 day

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C). This decline is much more precipitous than theoverall decline in total protein synthesis occurringduring the terminal differentiation of these cells asdetermined by the incorporation of [35S]methionineinto TCA-precipitable counts (data not shown). RNaseprotection analysis of RNA was then performed inorder to correlate these changes in protein synthesiswith changes in mRNA levels. As seen in Fig. 4, adecline in vimentin mRNA levels exactly correlateswith changes in synthesis of the protein. As illustratedin Fig. 4A, a 307 nucleotide fragment is protected bycellular RNA from 9 day cells but not by RNA from 12,13 or 14 day cells. By using twice the lysate (2xlO6 cell

Fig. 1. Immunofluorescencelocalization of vimentin duringmurine erythropoiesis. Murineprimitive erythroblasts wereisolated from embryos at 9 (A,B),11 (C) and 13 (D,E) days ofgestation, left panels (in vivo); orisolated from 9 day embryos andgrown for 2 (F) and 4 (G,H) daysin culture (i.e. they correspond to11 and 13 days of in vivodevelopment, respectively), rightpanels (in vitro). Cells wereprocessed for immunofluorescenceusing a vimentin-specific antibodyand viewed with epifluorescence(A,C,D,F,G) or phase contrast(B,E,H) optics. Note the loss ofvimentin from day 11 to 13whether grown in vivo or culturedin vitro. Bar,in vitro

equivalents) vimentin mRNA can be reliably detectedin 10 day cells but appears to be absent from 12 day cells(Fig. 4B).

Among other cytoskeletal proteins readily identifiedin the two-dimensional gel autoradiograms, the syn-thesis of tubulins also declines precipitously around day12 (Fig. 3B and C), an event similar to the downregu-lation seen during MEL cell-induced differentiation(Ngai et al. 1987). This correlates with the loss ofperipheral microtubules seen by immunofluorescencedescribed above. In contrast, actin synthesis declines inparallel with the general decline in protein synthesis,with newly synthesized actin being readily visible up to


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Fig. 2. Steady-state levels of vimentin during primitiveerythroid cell differentiation. Total cell lysates fromdefinitive chicken erythrocytes, murine primitive red cellsand murine lymphocytes were solubilized in SDS buffer andsubjected to immunoblot analysis. CRBC 1.5 and 0.5 referto loadings of a chicken erythrocyte lysate equivalent to 1.5and 0.5 xlO6 cells, respectively. Murine primitive cells(MPrRBC) from 8, 9, 10, 12, 14 and 15 days were 1.5xlO6

cell equivalents per lane. Definitive hepatic erythroid cells(isolated from embryonic circulation on day 14 of gestation)and lymphocytes (purified from adult mouse blood) wereboth 1.5x10° cell equivalents. The upper, larger panelrepresents an exposure of 3h with intensifying screen. Thelower panel presents a 3-day exposure without screen of thecorresponding lanes immediately above. V indicatesvimentin. Note the difference in MT between avian andmouse vimentin with the gel system employed here (Ngai etal. 1987).

14 days (Fig. 3B and C). Furthermore, the steady-statelevel of y-actin mRNA (Fig. 4C) parallels the continuedsynthesis of actin through the later days of differen-tiation (Fig. 3B and C).

Downregulation of the vimentin IF network inprimitive cells grown in cultureTo determine whether this pattern of vimentin regu-lation in murine primitive cells is an intrinsic property ofthese cells' differentiation program or is due to externalfactors provided by the yolk sac, purified 9 day primi-tive cells were grown in culture for up to 4 days and thepresence of a vimentin network was then assayed byimmunofluorescence after different times of culture.

We chose to use 9 day cells for this experiment becauseof the difficulty of obtaining sufficient numbers ofpurified 8 day cells. Given that the steady-state level ofvimentin was constant up to the 9 day stage, it seemedreasonable to suppose that those events leading tovimentin downregulation had not yet occurred. Asshown in Fig. IF a diffuse vimentin network wasevident for up to two days in culture, but after 4 days ofculture this network had disappeared (Fig. 1G and H).Immunoblot analysis confirmed that this disappearanceof vimentin filaments correlated with the loss of vimen-tin protein from these cells (data not shown). Hence,the disappearance of the vimentin network follows thesame time course in vitro as in in vivo.

Changes in mitotic state and RNA content of primitiveerythrocytes during differentiationFACS cell cycle analysis of primitive erythrocytesisolated on different days of gestation was performed toexamine how the pattern of vimentin downregulationcompares with changes in the mitotic state duringdifferentiation. The cytogram (RNA versus DNA con-tent) of 10 day primitive cells (Fig. 5) is characteristic ofa proliferating cell population and is comparable to thatof an MEL cell population in exponential growth(Darzynkiewicz ef a/. 1980a;Traganoseia/. 1979). Fromthe eleventh day onward, however, the RNA/DNAprofile of this population changes. The relative contentof RNA decreases in conjunction with a shift in DNAcontent from 4C to 2C (where C is the DNA content perhaploid nucleus) so as to compress the cytogram patterntowards the Go compartment of the cell cycle. Thetwelfth day therefore represents the point when primi-tive cells begin to cease proliferation and becomequiescent such that by day 13 and 14 the vast majority ofcells are quiescent. The withdrawal of these cells fromthe cell cycle and the appearance of the terminallydifferentiated state would tentatively place these cells inthe G1D compartment (Darzynkiewicz, 1987). How-ever, the altered accessibility of chromosomal DNA tosmall intercalating agents such as ethidium bromide orAO, and the degree to which the chromatin is con-densed, both characteristic of Glp cells (Darzynkiewiczet al. 1980), remain to be determined for these primitivecells.

Treatment of 10 day cells with RNase A at 37 °C for30 min abolishes the y-axis component of the cytogramwhile leaving the histogram of DNA content intact(Fig. 5). This confirms the specificity of AO staining forRNA and reveals the contribution of RNA to thepattern of the cytogram. By comparing the RNA datafrom the cytograms in Fig. 5, 10 day cells are seen tohave a broad range of RNA content. As these cellsmature, progressively more cells exhibit a lower RNAcontent with this shift in relative values becoming quitemarked by 12 days of gestation and being most pro-nounced by day 13.

Discussion

Enucleation is a process unique to mammalian defini-


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Fig. 3. Relative synthetic rate of vimentin, tubulins and actins during primitive erythroid cell differentiation. All panelsrepresent two-dimensional electrophoretic patterns of [35S]methionine-labelled cytoskeletal fraction of primitive red cellsisolated at the designated days of gestation. Only a small area of the total two-dimensional gel, corresponding to the regioncontaining tubulin, actin and vimentin, is shown. (A) Autoradiograph of a Western blot of the cytoskeletal fraction of 3xlO6

10 day primitive cells labelled for 1 h with [35S]methionine (left panel) and the same filter incubated with an anti-vimentinantibody and developed with alkaline phosphatase to detect vimentin (right panel). Exposure of the autoradiogram was for 7days. (B) Fluorograms of two-dimensional gels of cytoskeletal fractions equivalent to 106 primitive cells labeled at differentdays of development. FluorogTams were exposed for 2 days. Symbols: at, a^tubulin; fit, /3-tubulin; a, actin; v, vimentin.(C) Extended exposure (3 weeks) of the same 11 to 14 day gels presented immediately above. Note the loss of vimentin byday 12.

tive erythropoiesis. Since erythrocytes of the primitivelineage remain nucleated, a comparative analysis of thedefinitive and primitive lineages should delineate thosechanges that have occurred independently of the physi-cal process of enucleation and those molecular changesthat are lineage specific and intimately coupled to thisprocess. In nonmammalian nucleated erythrocytes, twomajor structural domains exist that are absent in anuc-lear mammalian definitive red cells. The first is amarginal band of microtubules that forms around theequatorial plane of the cell periphery during terminaldifferentiation (Behnke, 1970; Barrett and Dawson,

1974). The second is the vimentin-based IF networkthat emanates from the nucleus and anchors it to theplasma membrane at a distinct and separate site fromthe marginal band attachment sites (Granger andLazarides, 1982; Virtanen et al. 1979). Given theapparent structural linkage of the IF network and thenucleus, the hypothesis has been advanced that thedissolution of this network is a necessary prerequisitefor enucleation to proceed (Ngai et al. 1987). A com-parison of vimentin expression in avian definitive andmurine primitive and definitive erythropoiesis thereforeprovides an opportunity to examine how the expression


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Probe (383 nucleotides)

Protected fragment (307 nucleotides)

of this gene relates to the process of enucleation. Wehave shown here that murine primitive cells contain anIF network early in their development which is similarto that found in avian erythroblasts and early mam-malian definitive erythroblasts. However, during thesubsequent stages of terminal differentiation vimentinexpression is downregulated and the IF network isremoved as occurs during terminal differentiation of themurine definitive lineage (Ngai etal. 1984,1987). Otherworkers have analyzed the cytoskeleton of 12 daymurine primitive erythroid cells, demonstrating the

presence of an IF network and peripheral microtubules,and concluded that murine primitive cells resemblenonmammalian definitive erythrocytes (Koury et al.1987). As we have shown here such a resemblance isonly transitory, with both the IF network and periph-eral microtubules being removed during the final stagesof differentiation. Thus, the downregulation of vimen-tin expression and the subsequent removal of IFs hasoccurred in both murine lineages irrespective ofwhether or not the nucleus is retained. However, wehave observed that the initiation of filament removal in

Vimentln expression in erythropoiesis 93

Fig. 4. RNase protection assays of vimentin and y-actinmRNAs. Steady-state levels of vimentin and y-actinmRNAs were determined as described in Materials andmethods. Panels A and B represent two separateexperiments. (A) Lane M, ^P-end-labeled pBR322 HpaU.fragments as markers; lane P, probe in the absence ofRNases; lane C, control digestion of probe in the presenceof RNases and 10 ng of yeast tRNA; lane F, probeprotection by 5xlCr cell equivalents of murine fibroblastlysate; lanes 9 to 14, probe protection by lxlO6 cellequivalents of 9 to 14 day primitive cells. The arrowheadindicates the expected 307 nucleotide fragment protected byvimentin mRNA. Autoradiographic exposure for 5 days.(B) Lane F, protection of probe with 5X104 cell equivalentsof fibroblast lysate; lane 12, protection with 2xl06cellequivalents of 12 day primitive red cells; lane 10, protectionwith 2xlO6 cell equivalents of 10 day primitive cells.Arrowhead indicates the expected 307 nucleotide fragmentfrom vimentin mRNA. Exposure time, 7 days. The diagrambeneath A and B indicates the pGEM-4 subclonecontaining the 315 base pair (bp) murine vimentin cDNAinsert, the RNA probe of 383 nucleotides generated fromthat subclone, and the 307 nucleotide fragment specificallyprotected by vimentin mRNA. (C) Enlarged region ofautoradiogram illustrating the RNase protection assay fory-actin mRNA. Lane M, 147, 122, 110, 90, 76 and 67 bpsubset of pBR322 HpaU. markers; lane P, the 145nucleotide probe generated from the y-actin subclone in theabsence of nucleases; lanes 11 to 14, probe protection bylxlO6 cell equivalents of 11 to 14 day primitive cells.Arrowhead indicates the protected fragment of 80nucleotides.

primitive erythroblasts coincides temporally with thenucleus becoming mobile within the cytoplasm. Prior toenucleation, the nucleus must become freely mobilewithin the cell to allow its polar apposition to theplasma membrane (Geidushek and Singer, 1979). Theresults presented here argue that while IF removal initself is not sufficient for enucleation to occur, it may benecessary to allow nuclear rotation and asymmetricpositioning to occur.

In both normal and transformed murine definitiveerythroblasts, the downregulation of vimentin ex-pression and the concomitant removal of cytoplasmic

Fig. 5. Cell cycle analysis of murine primitive erythrocytesat selected days of development. AO staining of primitivecell populations was carried out as described in Materialsand methods. Each panel consists of both a histogram ofDNA content (green fluorescence) vs. cell number and atwo-color cytogram of RNA content (red fluorescence) vs.DNA content (green fluorescence). Typically 2C DNAcontent gives a mean fluorescence intensity of about 198(arbitrary units) and 4C DNA content yields a meanfluorescence of about 430 in these analyses. Panel10d+RNase represents 10 day primitive red cells treatedwith 1000 U ml"1 of RNaseA prior to AO staining. Thelower left-hand box in each cytogram (minimum RNA, 2CDNA content) represents Go cells. The upper left boxlabeled '2' (increased RNA, 2C DNA content) includes G:

cells. Cells in the middle box labeled '3' (DNA contentbetween 2C and 4C) are taken to approximate the S-phasepopulation. Cells in the right-most box labelled '4' (4CDNA content) are taken to represent G2+M cells.

10 day

1 29 49

11 day

299 449 t99 1 28 49

12 day

13 day

1 29 4« 64

1 24 49

«9« 999 1999

10 day+RNAase

-•

o- •3

DNA (green fluorescence) DNA (green fluorescence)


vimentin filaments occurs around the late CFU-e stage,since it is seen within 12 h of induced differentiation inMEL cells, before the onset of globin expression (Ngaiet al. 1984), and in early erythroblasts in vitro (Dellagi etal. 1983).

Although it remains to be determined whether theprimitive lineage progresses through the same precur-sor cell stages as the definitive pathway, it is evidentthat the downregulation of vimentin expression is anearly event in the terminal stages of primitive red celldifferentiation. Hemoglobinized primitive cells can befirst distinguished at 8 days of gestation within the bloodislands. At 9 days of gestation they are released into theembryonic circulation where they differentiatesynchronously becoming fully mature around day 14 to15 (Kovach et al. 1967; Fantoni et al. 1968, 19696; de laChapelle et al. 1969). By following the expression andsteady-state level of vimentin in primitive cells between8 and 14 days of gestation, we have shown that avimentin network exists in early primitive erythroblastsfrom day 8 to 10. However, from day 10 onwards thereis a dramatic decrease in the amount of vimentinmRNA and protein being synthesized accompanied bya loss of the vimentin IF network so that by day 13vimentin expression has ceased and filaments are nolonger detectable in these cells. These changes occurindependently of continued association with the yolksac microenvironment since identical changes occurover a similar time scale when 9 day cells are cultured invitro either together with or independently of their yolksac environment arguing that they must represent anintrinsic part of the primitive erythroid differentiationpathway.

FACS analysis of primitive cells from different daysof gestation reveals that day 12 represents the stage ofdifferentiation when murine primitive cells begin to exitfrom the mitotic cycle. This observation is in agreementwith earlier conclusions based on thymidine labeling,that these cells cease DNA synthesis and hence becomepostmitotic around day 12 (de la Chapelle et al. 1969).Therefore, vimentin downregulation and filament re-moval commences just prior to the time the cells exit themitotic cycle and enter their final phase of differen-tiation. By FACS analysis, the total RNA content ofthese primitive cells declines from day 11 onward; this isin accordance with other studies that have demon-strated a marked decline in total RNA and proteinsynthesis between day 11 and 15 (Kovach et al., 1967;Fantoni et al. 1968; de la Chapelle et al. 1969). Globinsynthesis persists relatively unchanged until day 14,presumably because of the relatively stable nature ofglobin mRNAs in these cells (Fantoni et al. 1968,1969a). Since vimentin downregulation commencesaround day 10, a stage at which the cells possess theDNA and RNA characteristics of an exponentiallygrowing population, it must represent a specific re-pression event occurring before these general changesin RNA and protein synthesis. The precipitous declinein vimentin protein synthesis suggests that the vimentinmRNA is short-lived, but the mechanisms involved inthe rapid removal of the pre-existing vimentin network

remain to be determined. Dilution through cell divisioncan not be the only contributing factor since celldivision ceases by day 12 (de la Chapelle et al. 1969).Likewise, the activation of specific proteases at thisstage is an unlikely explanation since chicken vimentinfilaments can still accumulate while the endogenousmurine vimentin is being eliminated in differentiatingMEL cells harboring the chicken vimentin gene (Ngai etal. 1987). The observation that vimentin filaments aredynamic, undergoing rapid assembly and disassembly(Ngai et al. 1990) suggests that upon initiation ofvimentin downregulation filaments begin to dis-assemble.

Assuming that this downregulation of vimentin is animportant prerequisite for the process of enucleation,the question then arises why enucleation does notproceed in murine primitive erythroblasts following therelease of mobility restraints on the nucleus. Onepossibility is that cells of the primitive lineage do notsynthesize and/or assemble the appropriate receptorsfor polarized apposition of the nucleus to the plasmamembrane and subsequent extrusion from the cyto-plasm. Alternatively, the process of enucleation mayrequire the direct interaction of these cells with aspecific subset of hematopoietic stromal cells. It hasbeen shown that erythroblasts in the process of enu-cleation within the bone marrow stroma are in closeassociation with a discrete subset of macrophages whichappear to be responsible for the phagocytosis of theextruded nuclei (Crocker et al. 1988). It is possible thatthe necessary supportive cells are not present in theyolk sac. Indeed, macrophage production is thought notto occur within the early yolk sac, but rather within theembryo around 14 days of murine embryogenesis (Rus-sell, 1979). Irrespective of the reason why primitivecells do not enucleate, the results presented here andthose of previous studies suggest that evolution of thecellular processes leading to enucleation has occurred inat least two stages. One is lineage independent andentails cytoplasmic structural changes that culminate innuclear mobility. The other is the physical process ofenucleation itself which is unique to the mammaliandefinitive lineage and may also be dependent on thestromal composition of the hematopoietic site.

We thank Rochelle Diamond for her expert assistance withFACS analysis, Jessica Dausman for help with mouse embryomanipulations, Drs Thomas Coleman and Michael Rodriguezfor valuable comments on the manuscript, and Louise Clou-tier and Dr Olivier Gandrillon for assistance with the figures.This work was supported by grants from the National Insti-tutes of Health (AGO60 78A) and from the Lucille P. MarkeyCharitable Trust to the Division of Biology at the CaliforniaInstitute of Technology. F.S. was supported by fellowshipsfrom the Anna Fuller Fund and the American Heart Associ-ation (Greater Los Angeles Affiliate). C.W. was supported bya fellowship from the American Heart Association (GreaterLos Angeles Affiliate) and by a grant from the AmericanCancer Society.

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(Accepted 25 May 1990)

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Vimentin downregulation is an inherent feature of murine ...Vimentin expression in erythropoiesis 87 described by Cudennec et al. (1981). Murine fibroblasts were isolated from the