Cell Biology International 2001, Vol. 25, No. 12, 1229–1236doi:10.1006/cbir.2001.0800, available online at http://www.idealibrary.com on

IN VITRO MORPHOGENESIS OF AMPHIBIAN ERYTHROBLASTS

LI-FANG HUANG, LIAT LEVINHAR, MARY GINSBURG, KYENG-GEA LEE andWILLIAM D. COHEN*

Department of Biological Sciences, Hunter College, New York, U.S.A.

Received 18 April 2001; accepted 20 July 2001

Non-mammalian vertebrate erythrocytes are flattened nucleated ellipsoids containing marginalbands (MBs) of microtubules that assemble during cellular morphogenesis. Earlier worksuggested that pointed erythroid cells containing pointed MBs were intermediate stages interminal differentiation, rather than aberrant forms, but direct evidence was lacking. Here wereport on morphogenesis in individual post-cytokinetic amphibian erythroblasts in culture.Daughter cells remained adjacent in pairs, and developed pointed morphology over 1–2 h in thefollowing sequence: (a) ends opposite the cytokinetic furrow became pointed, producing aspheroidal singly-pointed stage; (b) furrow ends usually became pointed, yielding doubly-pointed cells; (c) furrow-end points disappeared, producing a second singly-pointed stage thatwas flattening. Over a longer term, the single points sometimes disappeared, yielding a flatteneddiscoid. These observations support the hypothesis that pointed cells are normal intermediatesin a biogenetic program in which post-mitotic centrosomes organize MBs while occupying thesingly-pointed ends of differentiating erythroblasts. � 2001 Academic Press

K: amphibian; culture; cytoskeleton; erythroblast; morphogenesis.A: MB, marginal band; FBS, fetal bovine serum; DAPI, 4�,6-diamidino-2-phenylindoledihydrochloride; DMEM, Dulbecco/Vogt modified Eagle’s minimal essential medium; EGTA, ethyleneglycol-bis(2-aminoethyl)-N,N,N�,N�-tetraacetic acid; PIPES, piperazine-N,N-bis(2-ethanesulfonic acid);stages: D, discoid; S, spheroid; SP, singly-pointed; DP, doubly-pointed; SP-I, first SP stage; SP-II,second SP stage.

*Author to whom correspondence should be addressed: William D.Cohen, PhD, Department of Biological Sciences, Hunter College, 695Park Avenue, New York, New York 10021, U.S.A. Email:cohen@genectr.hunter.cuny.eduThis paper is dedicated to Shai Levinhar, loving husband of Liat andfather of Sapir, whose life was cut short most tragically at the WorldTrade Centre on September 11th 2001. His memory lives on, andinspires us to strive, always, for world civility and peace.

INTRODUCTION

Nucleated erythrocytes of non-mammalian verte-brates have been utilized for many years in studiesof cytoskeletal elements, including microtubules(e.g. Fawcett and Witebsky, 1964; Barrett andScheinberg, 1972), intermediate filaments (Grangerand Lazarides, 1982), and the membrane skeleton(Lazarides and Woods, 1989; Liao et al., 2000).Their microtubules are organized into MBs in theplane of cell flattening, a feature in common withmammalian platelets, mammalian embryonicerythrocytes, adult camelid erythroblasts, and

1065–6995/01/121229+08 $35.00/0

erythrocytes and clotting cells of various inverte-brates (Cohen, 1991). In mature erythrocytes theMB underlies the membrane skeleton, acting as aflexible frame that maintains the differentiatedshape against deformation (Joseph-Silverstein andCohen, 1984). It is also a morphogenetic effector,with MB biogenesis required for conversion ofspheroidal erythroblasts to flattened ellipsoids(Barrett and Dawson, 1974). However, the mor-phological sequence and cytoskeletal mechanismsinvolved in this shape transformation are notknown.

Of considerable interest with respect to possiblemorphogenetic stages are singly- and doubly-pointed erythroid cells. These have been notedoccasionally in the circulating blood of amphib-ians, birds, embryonic mammals, and certain inver-tebrates (Lucas and Jamroz, 1961; Cohen, 1982;Cohen and Nemhauser, 1980; Cohen et al., 1990),

� 2001 Academic Press

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as well as in amphibian and avian erythroid cellcultures (Duprat and Flavin, 1976; Beug et al.,1982). Such forms, sometimes referred to as‘poikilocytes’, have often been ignored or assumedto be aberrant. However, they are consistentlypresent among splenic erythroblasts in normal de-veloping amphibian larvae, and in the new eryth-roid cell population in the circulation of adultamphibians recovering from anemia (Ginsburget al., 1989; Twersky et al., 1995). In addition, theircytoskeletal structure is suggestive of transitionalstages (Cohen, 1982; Twersky et al., 1995). In thework reported here, we sought to observe morpho-genesis in individual post-mitotic amphibian ery-throblasts in vitro, thus determining directlywhether pointed transitional stages occurred.

MATERIALS AND METHODS

Axolotl embryos in blastula and gastrula stageswere obtained from the Indiana University AxolotlColony, and were stored in spring water at �4�Cuntil use (up to 3 weeks). Embryos were transferredperiodically into Petri dishes and allowed todevelop in spring water, usually for 14–15 days at18�C. Both wild type and albinos were employed,with developing spleens easier to see in the latter.Spleens were obtained from larvae 4–9 days post-hatching, before blood vessel connection to thegeneral circulation (Bordzilovskaya and Dettlaff,1979). Briefly, larvae were anesthetized in 0.02%Tricaine methanesulfonate (=MS222) in Holt-freter’s Solution, and dissected in culture mediumcontaining anesthetic. The skin was peeled back,and the bright red spleen removed using jewelersforceps, still attached to the stomach. After wash-ing in culture medium, the spleen was torn open inthe same medium containing antibiotics, releasingerythroid cells. For other experiments, circulatinglarval blood containing erythroblasts was obtainedfrom blood vessels severed during dissection ofthe spleen. Presence of mitotic splenic cells wasassessed by DAPI staining of nuclei in spleenspre-extracted for 30 min in 100 m PIPES, 1 mMgCl2, 5 m EGTA, 0.4% Triton X-100, pH 6.8,to stabilize nuclei and remove hemoglobin.Material was stained in 1 �g/ml DAPI, squashedunder a coverslip in a fibrin clot to contain cellswithin a restricted area for counting purposes(Lee et al., 1998), and observed by fluorescencemicroscopy.

Tested variations of culture media included Lie-bovitz L-15 and DMEM/F12, with 10–20% FBSplus antibiotics. The best results were obtained

using 50% Leibovitz L-15+10% FBS+100 �g/mleach of ampicillin and streptomycin, with air as thegas phase. Cell culture temperature was 18�C, andmicroscopic observations were typically made overa course of �18 h, with temperature varying from18–25�C. Under these conditions, differentiatingerythroblasts were found in cultures maintained forup to 5 days. Individual cells were observed directlyin culture dishes or in six-well tissue culture platesby phase contrast microscopy, using a Zeiss IM35inverted microscope equipped with a DAGE-MTIvideo camera. Images were recorded perodicallyusing a Hamamatsu Argus-10 video image proces-sor with thermal printer. Cultures were allowed toremain on the microscope stage during obser-vation, so as not to disturb cells and lose theirorientation or location. Clock times were recordedon each photograph for subsequent reconstructionof sequences. To quantify flattening, changes in cellarea were determined using ‘NIH Image’ softwareby hand-tracing digital images of each cell at twospecific morphogenetic stages (‘S’ and ‘SP-II’; seebelow). To minimize the effect of cell profile tracingvariation, the % increases in area�standard devia-tions were calculated from five repeated pairs ofmeasurements per cell.

RESULTS

In addition to a preponderance of flattened ellip-soids, splenic cell populations released into culturemedia typically contained minor sub-populationsof singly- and doubly-pointed cells, flattened dis-coids, and spheroidal cells believed to be mitotic(Fig. 1a). Occurrence of mitotic stages was verifiedby fluorescence microscopy of DAPI-stained cellnuclei in larval splenic squash preparations. Forfive individual spleens at 5, 6, 7, 8, and 9 dayspost-hatching, each containing more than 2000counted cells, distinctly visible metaphase and ana-phase stages (e.g. Fig. 1b) constituted 0.14%,0.26%, 0.0%, 0.03%, and 0.04% of total cell nuclei,respectively.

The fate of amphibian larval splenic or circulat-ing erythroblasts was followed directly in culturesby observation in phase contrast, and recorded byvideography. Cells undergoing cytokinesis werelocated and examined periodically during the 2–9 hculture observation periods. Some post-cytokineticdaughter cells showed little alteration of mor-phology for considerable periods following cyto-kinesis, or underwent only brief or partial shapetransitions. However, others exhibited continuousshape alterations over a period of about 2 h and,

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somewhat unexpectedly, these daughter cellsremained adjacent in pairs during this time. Singly-and doubly-pointed morphology appeared in thesecells in a specific sequence, with both daughtererythroblasts usually behaving similarly. Pairingof cells proved to be highly advantageous forsubsequent interpretation of morphogenesis withrespect to cell polarity.

A detailed sequence for one such cell pair ispresented in Figure 2, with time (in min) shown inthe upper corners. Immediately following cytokin-esis the daughter cells were spheroidal (Fig. 2a;morphogenetic stage ‘S’). Within �20 min a slightpoint began to develop on the surface of eachdaughter cell in the region opposite the cytokineticfurrow, producing singly-pointed spheroidal cells.This stage was designated ‘single point I’ (Fig. 2d,arrowheads; stage SP-I). During the following15–20 min this point became more pronounced andthe opposite end also became pointed, yieldingdoubly-pointed cell pairs that appeared to be flat-tening (Fig. 2h, arrowheads; stage DP). The pointat the cytokinetic end then disappeared duringthe next hour, producing a second singly-pointedstage (Fig. 2m, original pointed ends at arrow-heads; stage SP-II). At final observation, theremaining points had nearly disappeared (Fig. 2o,arrowhead).

Time-course micrographs for additional cellpairs are presented in Figures 3–6. Singly- anddoubly-pointed morphology typically appearedwithin �1.5 h following cytokinesis, in the same Sto SP-I to DP to SP-II sequence. For four closelymonitored pairs of cells, the time range for theperiod from post-cytokinetic S to SP-I was �10–

20 min, S to DP�21–34 min, S to SP-II�40–120 min. During this transition the cells graduallylost their spheroidal shape and appeared to occupyincreased area associated with flattening. This wastested quantitatively for five pairs of cells bymeasurement of cell profile areas at SP-II versuspost-cytokinetic S. All of these cells exhibitedapparent area increases (Table 1), ranging from�11 to �55% and averaging �27%.

Comparison of different daughter cell pairsshowed minor variations in the sequence. In thepair shown in Figure 2, SP-I was evident immedi-ately upon completion of cytokinesis. Bothdaughters then went though the morphogeneticsequence almost synchronously, remaining at-tached throughout. For the cells in Figure 3, essen-tially the same sequence was observed, with theexception that greater daughter cell asynchronywas evident (e.g., Fig. 3b; one cell SP-I, one DP)and after �3 h the daughters separated in SP-II(Fig. 3g). A commonly observed feature at thecytokinetic end of one or both cells prior to the DPstage is illustrated in Figures 4 and 5. Whenapproaching DP, one or both cells generated a‘pronged’ appearance at the cytokinetic end (Figs4a; 5a). With daughter cell asynchrony sometimesevident (e.g., Fig. 4b, arrowhead; Fig. 5c), bothcells nevertheless ultimately progressed through DPstages to SP-II. While most cells remained at SP-IIduring typical observation periods, in a fewinstances of very long-term observation the singly-pointed ends diminished in length and disappeared,yielding flattened discoids (Fig. 6).

Occasional variant sequences are worth noting.In one instance a ‘two-pronged’ cell appeared toprogress to SP-II without a true DP stage, while thepaired daughter exhibited a typical DP stage. Inanother, both daughters in SP-I entered a typicalflattening SP-II stage after exhibiting shape distor-tions, but not a distinct point, at their cytokineticends.

Fig. 1. (a) Survey view of a field of splenic cells released intoculture medium. Most cells are flattened ellipsoids (E); otherforms include flattened discoids (D), spheroids (S), singly-pointed cells (SP), and doubly-pointed cells (DP). Phase con-trast microscopy with digital image processing; bar=20 �m. (b)a DAPI-stained splenic cell in anaphase; squash preparation,fluorescence microscopy.

DISCUSSION

Our earlier study of larval axolotl splenic erythro-blasts revealed singly- and doubly-pointed cells asnaturally-occurring members of the splenic eryth-roid population. Examination of their cytoskeletalstructure led to the hypothesis that these pointedcells might be normal intermediates in the sphere-to-flattened ellipsoid transition (Ginsburg et al.,1989). The present work attempted to test thathypothesis and establish the morphogeneticsequence. As in many other studies involving cells

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Fig. 2. Detailed time-course sequence for axolotl larval erythroblast cytokinesis and morphogenesis in vitro; culture containingboth splenic and circulating cells. Time in minutes following cytokinesis is in upper corner of each image. (a) cytokinesis nearlycomplete; cells are spheroidal (stage=S). (b–d) development of small points at ends of cells opposite plane of cytokinesis, withcells remaining attached (d, arrowheads; stage=SP-I); (e–h) genesis of a second point at cytokinetic end (h, arrowheads;stage=DP); (i–m) loss of point at cytokinetic end, with retention of original point (m, arrowheads; stage=SP-II); (n,o) gradualloss of remaining pointed end, yielding nearly discoidal cell (o, arrowhead). Note that both daughter cells go though the sequencealmost synchronously, and remain attached throughout by an as yet unidentified material. Phase contrast microscopy with digitalimage processing; bar=10 �m.

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Fig. 3. Time-course sequence for splenic erythroblast morphogenesis in vitro, with some daughter cell asynchrony. The sequencefor both cells is again (a) S, to (b) SP-I, to (c) DP, to (e) SP-II. Here, asynchrony is evident as one cell progresses to DP whilethe other is still in SP-I (b, arrowhead), but both are in DP shortly thereafter (c, arrowheads). These daughter cells remainattached for most of the sequence, but are separated in SP-II after �3 h observation (g). Phase contrast with digital imageprocessing; mag. bar as in Fig. 6.

Fig. 4. Selected stages from a splenic erythroblast morphogenetic sequence. (a) The second pointed end has a ‘pronged’appearance in one cell (arrowhead); (b) after both cells have been in DP stage, the formerly ‘pronged’ cell has regained curvatureat that end (arrowhead); (c) long-term observation; both cells remain attached and are now flattened in SP-II such that nucleibegin to be visible. Phase contrast with digital image processing; bar=10 �m.

in culture, our results are subject to the criticismthat cellular morphogenesis in vitro might differartifactually from that in vivo. While this possibilitycannot be ruled out, we believe it to be quiteunlikely with respect to our major findings. First,similar pointed cell types are present among cellsshortly after release from the experimentally-ruptured spleens (e.g., Fig. 1). Second, the appear-ance of singly- and doubly-pointed stages waspredicted based on the earlier observations. Manyother types of artifactual morphology were

potentially possible, yet these predicted pointedcells were the predominant intermediate stagesobserved, and they resembled pointed cells in initialsplenic populations. Therefore it is highly likelythat the pointed cells observed in the present workare true developmental intermediates.

We believe these to be the first direct observa-tions of terminal morphogenesis in amphibianerythroblasts, and they show that the cells gener-ate singly- and doubly-pointed stages in vitro.Essentially the same sequence, S to SP-I to DP to

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Fig. 5. Selected stages of another sequence for splenic eryth-roblast morphogenesis in which, following SP-I, both cellsexhibit ‘pronged’ morphology at the cytokinetic end (a; arrow-heads), then progress to the DP stage and beyond (b, c).Asynchrony is evident in (c), with one cell appearing to beflattened at SP-II, the other at DP. Phase contrast with digitalimage processing; bar=10 �m.

Fig. 6. An example of loss of pointed morphology, long-termobservation. (a) Points diminished in size; (b, c) points of samecells are no longer present at �5 h; cells are now separatedflattened discoids. (Note: background lines here are positionalreference scratch marks on culture dish.) Phase contrast withdigital image processing; bar=10 �m.

Table 1.Increase in apparent area of individual cells, as measuredat SP-II vs. S. Areas were determined using ‘NIH Image’

software

Cell pair, cell # % visible profile areaincrease at SP-II�SD

A 1 11�2.9A 2 12�4.1B 3 22�5.0B 4 41�6.3C 5 39�4.3C 6 55�4.6D 7 26�3.3D 8 21�1.2E 9 31�4.4E 10 15�5.1

SP-II, was observed in most cases, with continuouschanges accompanied by increased flattening(Table 1) over periods of 1.5–3 h in different cellpairs. It is important to note that the timesrecorded for appearance of specific stages areapproximate because stages were transitional, with-out distinctly defined start or end points. Stageduration times typically varied to some extent even

between paired daughter erythroblasts, evident inthe asynchrony observed at particular time points(e.g., Fig. 3b; Fig. 4b; Fig. 5c).

The transitional stages observed in vitro wereconsistent with morphology of splenic erythro-blasts obtained directly from experimentallyruptured spleens, as determined previously by scan-ning electron microscopy (Ginsburg et al., 1989).These included spheroids (S), singly-pointed sphe-roids (SP-I); doubly-pointed (DP), and flattenedsingly-pointed (SP-II) forms. Some cellsobserved by scanning electron microscopy also hadminor projections at the end opposite the majorpoint, and were thus similar to ‘pronged’ stagesseen in the cultured erythroblasts (e.g., Fig. 4a;Fig. 5a).

Daughter cells remained in pairs for some timefollowing cytokinesis, apparently tethered by acytoplasmic remnant even while changing positionslightly. DP-stage cells obtained directly fromspleens do not appear in pairs (Ginsburg et al.,1989), but this may be a matter of disruption of theconnecting material when cells are released duringmechanical fragmentation of the spleen. Althoughwe do not know whether daughter cell attachmentin culture reflects some normal feature of amphib-ian erythroblasts, it is noteworthy that cultureddaughter chicken erythroblasts have been observedto remain paired in vitro, appearing to adherelongitudinally (Barrett and Scheinberg, 1972). Inthat study, morphological differentiation beganwhile cells were attached, and, though not dis-cussed, scrutiny of the micrographs suggests thatthe chicken cells developed pointed stages. Differ-entiating HB3 chicken cell cultures also producesingly- and doubly-pointed cells in vitro, originallyinterpreted to be anomalous (Beug et al., 1982). Inaddition, mycotoxins tested on adult chickensinduced the presence of circulating immatureerythroblasts and doubly-pointed erythroid cellsthat were not preparation artifacts (Dombrink-Kurtzman et al., 1993). The original Atlas of AvianHematology reported normal presence of suchpointed cells in the adult circulation (Lucas andJamroz, 1961), and thus, based on current resultswith an amphibian, we suggest that these aresimilar stages in the terminal differentiation ofavian erythroblasts.

The results support an hypothesis that correlatessingly- and doubly-pointed terminal morpho-genetic intermediates with biogenesis of the micro-tubular cytoskeleton, as illustrated in Figure 7.Here, the observed morphogenetic sequence (dot-ted outlines) and biogenetic hypothesis (solid in-terior lines) are shown for amphibian erythroblast

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differentiation in culture. Cytokinesis (a–c) is fol-lowed by generation of singly-pointed spheroidalcells (d; SP-I), then doubly-pointed flattening cells(e; DP), then a second singly-pointed stage (f;SP-II), and continued flattening (f, g). Post-mitoticcentrosomes are predicted to generate proximalpointed ends (a–d), functioning to organize micro-tubules as indicated by the arrows (d–g). Transi-tional ‘pronged’ forms frequently observed at thecytokinetic end prior to the DP stage (e.g., Fig. 4a;Fig. 5a), or other distortions at that end, areinterpretable as being produced by growingdynamic microtubule bundles that probe that endafter initiation at the centrosomal region (Fig. 7d,e). Precedent for such surface distortion fromwithin comes from earlier work on nucleated eryth-rocytes in which the ends of experimentally linear-ized microtubule bundles produced cellular pointsor protrusions (Joseph-Silverstein and Cohen,1984; Winckler and Solomon, 1991).

The difficulty inherent in continuous individualmonitoring of these non-adherent post-cytokineticcells, without disturbing the culture medium, pre-cluded routine conduct of very long-term studies.However, in those few instances in which nearlycomplete disappearance of points was observed,the flattening cells were discoidal, i.e. circular inprofile rather than ellipsoidal (e.g., Fig. 2o; Fig. 6).Thus, despite the fact that pointed intermediatesapproach ellipsoidal shape at DP and SP-II, theobserved sequence appears to yield a discoidal cellwhich has yet to undergo final ‘maturation’ to anellipsoid. This would be in accord with studiesshowing that nucleated erythrocytes have an imma-ture flattened discoidal stage (e.g., Barrett andDawson, 1974; Dorn and Broyles, 1982), and withthe presence of some flattened discoidal cells in thelarval axolotl spleen (Fig. 1; Ginsburg et al., 1989).

The product of the proposed cytoskeletal sequence(Fig. 7, solid lines) would then be a circular MBthat establishes and maintains flattened discoidalcell shape (Fig. 7g). Mechanisms underlying disc-to-ellipse transformation remain to be demon-strated, but probably involve modification of themechanical properties of a circular MB (Cohenet al., 1998).

The morphogenetic sequence typically observedin the cultured amphibian erythroblasts (Fig. 7,dotted lines) supports an earlier model derivedfrom studies of amphibian splenic erythroblaststructure and from stages of MB reassembly inmature blood clam erythrocytes (Nemhauser et al.,1983; Ginsburg et al., 1989). It differs principally inthat the existence of two distinct SP stages is nowapparent, establishing the DP stage as an inter-mediate lasting less than 30 min. The sequence andtime-course observed in vitro predict that SP cellsshould outnumber DP cells in most larval splenicpopulations, as was observed (Ginsburg et al.,1989). In addition, since SP-II cells may lose theirpointed ends within several hours (e.g., Fig. 2o),pointed cells would be expected to constitute aminor splenic sub-population at any given time,barring a synchronous wave of differentiation. Thisis in accord with counts of pointed cells in theearlier study (Ginsburg et al., 1989). Thus, allamphibian erythrocytes, and perhaps all non-mammalian vertebrate erythrocytes, may gothrough such pointed stages, with both morphoge-netic brevity and the obscurity of hematopoieticsites contributing to their infrequent observation.

(D) SP-I(C) S(B)(A) (E) DP (F) SP-II (G) D

Fig. 7. Diagrammatic summary of the morphogenetic sequence normally observed in the cultured erythroblasts (dotted outlines),and working hypothesis for correlated causal MB biogenesis (MT bundles=solid interior lines). (a, b) cytokinesis; (c) movementof centrosomes to periphery of spheroidal (S) cells; (d) generation of singly-pointed spheroids by post-mitotic centrosomes (stageSP-I); (e) progression to doubly-pointed flattening cells (e; DP); (f) second singly-pointed stage (SP-II); (g) continued MT growtharound the periphery and flattening, yielding discoidal cells (D).

ACKNOWLEDGEMENTS

We are indebted to Sharon Bauer, Susan Carterand Yuliya Davydova for excellent technical

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assistance and helpful discussion. We also wishto thank S. Borland, S. Duhon, and Dr. G.Malacinski for the generous supply of A.mexicanum embryos from the I. U. AxolotlColony. Support for this work provided by NSF9808368, and by PSC-CUNY awards 61222 and62267, is gratefully acknowledged.

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