erythropoiesis in th newte triturus, cr is tat us …erythrocytes and haemoglobin, newts do not...

J. Cell Sci. la, 463-489 (i973) 463Printed in Great Britain

ERYTHROPOIESIS IN THE NEWT, TRITURUS

CR IS TAT US LAUR.

I. IDENTIFICATION OF THE 'ERYTHROID PRECURSORCELL'

J. A. GRASSODepartment of Anatomy, Boston University School of Medicine,80 East Concord Street, Boston, Massachusetts 02118, U.S.A.

SUMMARY

Phenylhydrazine injection in salamanders (Triturus sp.) produces a complete loss of all ery-throcytes and haemoglobin but does not result in an immediate erythropoietic response.Splenectomized newts survive absence of the erythron most readily. At 11-14 days afterinjection, the peripheral blood contains numerous, primitive cells which exhibit large, lepto-chromatic nuclei, wide variation in cytoplasmic basophilia, and variation in cell size. Thesecells constitute a spectrum of cells collectively called 'erythroid precursor cells' (EPC) whichterminate in the formation of basophilic erythroblasts. Earlier cell forms (at 11 days) containfew cytoplasmic ribosomes and few organelles but the ribosome concentration graduallyincreases, as does a fibrillogranular precipitate presumed to be associated with haemoglobinsynthesis. Metabolically, these cells are engaged in proliferation, RNA, haem, and proteinsynthesis. Erythroid precursor cells, which comprise 50-60% of the total blood cell popu-lation at 11-14 days, are replaced by basophilic erythroblasts at 15-18 days. The morphologicaland metabolic properties of these cells are compatible with their role in red blood cell formation.However, their similarity to mammalian marrow 'transitional' cells and the occurrence ofsimilar cells in association with macrophages suggest possible differentiative potencies alongseveral cell lines. While little is known about the origin of erythroid precursor cells, pre-liminary experiments suggest that they may be derived from circulating lymphocytes.

INTRODUCTION

The precise identity of the haemopoietic stem cell in vertebrates remains un-resolved. In mammals, the functional approach to the stem cell problem has supportedthe monophyletic - or Unitarian - concept which stressed the existence of a commonpluripotential cell capable of giving rise to all blood cells. This cell, called the haemo-cytoblast, was considered to be identical to the lymphocyte (cf. Bloom, 1938, forreview).

Recent studies show that mammalian lymphocytes represent a widely hetero-geneous cell population in terms of their developmental commitments and poten-tialities (Yoffey, 1964, 1970; Yoffey, Hudson & Osmond, 1965). The vast majorityof lymphocytes in the lymphatic organs and blood are specialized for roles in immuno-logical reactions (Everett & Tyler, 1967) and thus have a limited haemopoieticfunction. On the other hand, mammalian marrow contains a lymphocyte populationthat may serve as a haemopoietic stem cell compartment (Cudkowicz, Bennett &Shearer, 1964; Bennett & Cudkowicz, 1967).

464 J- A. Grasso

In adult urodeles, erythrocytes and thrombocytes are formed in the spleen andperipheral blood (Jordan & Speidel, 1930; Dawson, 1932; Jordan, 1938; Grasso,1973). Granulocytes are produced in the subcapsular zone of the liver (Jordan, 1938).Thus, unlike the mammal, granulocyte and erythrocyte formation occur in differentsites. Lymphocytes of various types occur in the spleen, liver, and blood, as well asin minor lymphogranulocytopoietic foci in the intestinal submucosa. As in mammals,lymphocytes, small, medium, and large, have been postulated to serve as haemo-poietic stem cells (Jordan & Speidel, 1930; Dawson, 1933; Jordan, 1938), a functionunderlined by the nomenclature applied to the haemopoietic type of lymphocyte,the lymphoid haemoblast. Unfortunately, nothing is known about the role of urodelelymphocytes in immunological responses, nor is it clear whether lymphocyte popu-lations analogous to mammalian lymphocytes in terms of their kinetic and develop-mental behaviour exist in adult urodeles.

Newts of the Triturus genus have been found to lack the total erythron afterphenylhydrazine treatment (Grasso & Shephard, 1968). Despite the absence oferythrocytes and haemoglobin, newts do not immediately restore erythrocytes in theblood but survive readily in this erythrocyte-free state. After 2 weeks, numerousprimitive cells appear in the peripheral blood and signal the occurrence of a wave oferythropoiesis. The morphological and metabolic characteristics of these 'erythroidprecursor cells' are the subject of this report.

MATERIALS AND METHODSTriturus cristatus Laur. were collected near Rome and Naples, Italy from July 1968 to

September 1969. All animals were kept in 10—15 gall. (38—57 1.) aquaria containing 2-0—6-o 1. of spring water at ambient room temperature. In initial experiments, the spleen wasremoved to stimulate erythropoietic activity in the peripheral blood (Jordan & Speidel, 1930).However, our studies indicated that the peripheral blood is a normally active erythropoieticlocus morphologically independent of the spleen. In the experiments reported here, splenec-tomy was used to limit RBC formation exclusively to the peripheral blood.

Acetylphenylhydrazine (APH), dissolved in 4% ethanol and adjusted to pH 70 witho-i N NaOH, was injected intraperitoneally for 2-3 consecutive or alternate days. A totaldosage of 075-romg was sufficient to cause the loss of all erythrocytes within a few days.Anaemic animals were kept at ambient room temperature in various sized trays or aquaria,allowing the animals sufficient swimming space and easy access to air. Sometimes air stonesor bubblers were introduced. Under these conditions, 80—100% survival was usual, althoughduring the breeding season of May-July there was an unaccountably high mortality.

For routine examination, blood was obtained by cutting the tail and smears stained withWright's or Giemsa stain. For electron microscopy, animals were bled directly into ice-cold1 % OsO4 buffered to pH 7-2-7-5 in 0-2 M Sorenson's phosphate buffer and fixed for 1 h.Aldehyde fixation (Karnovsky, 1965) of early erythroid cells in these anaemic animals wasdifficult to perform since the result was a powdery slurry which could not be easily embedded.Fixation and dehydration were done in 12-15 ml conical centrifuge tubes, taking care not todisturb the pellet during dehydration. Dehydration was effected by ethanol, or acetone at— 20 °C, and all material was embedded in Epon 812. Sections were cut with a diamond knifeand stained in uranyl acetate alone or double-stained with uranyl acetate and lead citrate.Grids were examined in a Philips 200, an AEI-6B, or RCA-EMU3g electron microscope.

Erythroid precursor cells of newt 465

Radio autographic procedure

For the demonstration of RNA synthesis, 40-50 /iCi of PH]cytidine (Schwarz: specificactivity 6 Ci/mmol) were injected into the peritoneal cavity and blood smears prepared 30—60 min after injection. Porphyrin synthesis was demonstrated by injection of 50-60 fid ofPH]-5-aminolaevulinic acid (New England Nuclear: specific activity 5-4 or 7-0 Ci/mmol)with blood samples taken 2-5-3-0 h after injection. In all experiments utilizing this isotopicprecursor, a minimum of 2-5 h exposure or incubation was required to obtain significantincorporation as measured by radioautographic means. This minimum time was required fordefinitive erythroid cells of all stages as well as for the cell type described in this report.

Protein synthesis was examined by injection of 50 /tCi of [*H]leucine (New England Nuclear:specific activity 5 Ci/mmol) with blood smears prepared after 1 h.

All smears were fixed in absolute methanol for 10-15 Tr^n and allowed to dry in air. Smearsprepared from animals receiving ['H]cytidine were subdivided into 3 groups which weretreated, respectively, as follows: Group 1, DNase extraction (0-02 % in 3 x io"5 M magnesiumsulphate, pH 6-5) for 1 h at room temperature followed by 10 min in ice-cold 5 % trichloro-acetic acid (TCA); Group 2, RNase extraction (0-02 % in distilled water, pH 6-o) for 1 h atroom temperature followed by 10 min in TCA; Group 3, 10 min in ice-cold 5% TCA.Smears from animals injected with [3H]-£-aminolaevulinic acid ('H-ALA) or PFTjleucinewere extracted in ice-cold 5 % TCA only.

After completion of extraction treatments, all slides were washed for 15 min in cold run-ning water, rinsed in distilled water, and coated with Kodak NTB-2 liquid emulsion. Slideswere stored in light-tight boxes at 3 °C. At appropriate times, radioautographs were developedin Kodak D19 developer (full strength) for 2-5 min and stained in 0-025 % azure B (pH 4-0)or Giemsa after photographic fixation and washing. Where necessary, grain counts weremade over individual cells and expressed as the average number of grains/ztm'/cell type.

RESULTS

Light-microscopic observations

In splenectomized newts treated with acetylphenylhydrazine (APH), completedestruction and loss of erythrocytes occurred within 72 h and was followed by ai-5-2-week period in which the animals failed to regenerate red cells. Some timewithin the 11-14-day interval after APH, numerous primitive cells, mainly similarto that depicted in Fig. 1, were observed in the blood and gradually decreased withthe subsequent accumulation of basophilic erythroblasts (BE) at 14-17 days. In someanimals the initial appearance of these primitive lymphocytoid cells was delayeduntil days 15-16, in which case the occurrence of basophilic erythroblasts was pro-portionally delayed. Irrespective of their initial time of appearance, the lymphocytoidcells formed 50-60 % of the total blood cell population, the remaining cells consistingof granulocytes, thrombocytes, and lymphocytes. Subsequently, basophilic erythro-blasts (Fig. 4) diluted and replaced this cell population, becoming the main blood-celltype.

Since experimental evidence has shown their direct relationship to erythropoiesis,we have named these lymphocytoid cells 'erythroid precursor cells (EPC)'. But thisterm must be tentative since identical cells may serve as progenitors of cell lines otherthan erythropoietic.

EPCs lacked morphological features which would be diagnostic of erythroid cells,i.e. their structural appearance did not betray their erythropoietic role. Significantly,these cells did not constitute a single morphological entity but instead formed a

466 J. A. Grasso

population of cells exhibiting variation both in size and extent of cytoplasmic baso-philia (Figs. 1-3,9, IO)- ̂ n Clears, their cytoplasm was very fragile and often damagedso that the cell boundaries were indistinct (Figs. 1, 9). Most cells exhibited a weaklystained, sometimes vacuolated cytoplasm which was faintly basophilic or unstained(Figs. 1, 2). In other cells, a moderate degree of cytoplasmic basophilia was observed(Figs. 3, 9), these cells exhibiting less fragile properties than their weakly stainedcounterparts. When measurable in smears, EPCs ranged from 30 to 45 fim in dia-meter. If thick smears were made, as was possible with the accumulation of basophilicerythroblasts or with the reappearance of EPCs at 22-25 days after APH, intact cellswere more frequent.

The most prominent and consistent feature in EPCs, irrespective of size or cyto-plasmic basophilia, was the nucleus (Figs. 1, 3, 9, 10), which was spherical or ovoid,occasionally indented, and approximately 22 /tm in mean diameter (range 19—25 /im).With Romanovsky stains, the nuclei were leptochromatic, possessing a loosely wovenchromatin distributed in blocks or masses (Figs. 1, 3). Occasionally, mitotic figureswere observed (Figs. 2, 9). In Romanovsky-stained smears, nucleoli were difficultto resolve. After azure B staining at pH 4-0, however, nucleoli were usually con-spicuous as 1-4 compact metachromatic bodies (Fig. 5). In these preparations, thechromatin blocks were stained a light blue-green (orthochromatic) and were separatedby thin interchromatin regions in which a faint blue-purple (metachromatic) colourwas just discernible. These staining reactions with azure B at pH 4-0 indicate thepresence of DNA and RNA, respectively (Swift, 1955), and were confirmed byextractions with appropriate nucleases (Grasso, unpublished results).

In phase-microscopic examinations of living cells suspended in plasma or of thinsections of plastic-embedded cells, the appearance of EPCs was different from thatseen in smears. Under these conditions, the cells were spherical or oval in shape,measuring 18-30 /im in diameter (Figs. 6-8). While the nuclear features remainedessentially unchanged, the cytoplasm was distinct and intact, appearing as a clearhomogeneous mass containing many dark granules, probably mitochondria (Figs. 7,8). Variation in cell size was especially evident in cell suspensions (Figs. 7, 8).

EPCs were not seen in the blood of erythropoietically inactive newts but did occurin animals exhibiting erythropoietic activity not related to APH-anaemia, such asthe bursts of erythropoiesis which take place during the spring breeding season(Figs. 9, 10). Thus, EPCs constituted a normal erythropoietic component and werenot induced solely as a result of APH administration. In both T. cristatus and T.viridescens, these cells were of identical appearance and occurred at the same timeinterval following APH injection.

In summary, erythroid precursor cells were interpreted to represent a spectrum ofsimilar, progressive cell stages leading to the formation of basophilic erythroblasts.As such, they formed a heterogeneous population marked by variably poor to moderatecytoplasmic basophilia and cytoplasmic fragility. Regardless of cytoplasmic appearance,all EPCs contained relatively large, leptochromatic nuclei in which 1-4 nucleoli werepresent. Prior to the accumulation of basophilic erythroblasts, they comprised thepredominant cell type in the peripheral circulation.


At 2-6 days after APH, free macrophages were most abundant in the blood as aresult of extensive RBC destruction. Cells similar in appearance to EPCs were alsorelatively frequent during this interval but disappeared after 6 days, concomitantwith the sharp decrease in phagocytic activity and number of macrophages. Althoughthe evidence is circumstantial, these observations suggest a possible relationship ofthese cells to the formation of macrophages.

Electron-microscopic observations

The ultrastructural appearance of 'erythroid precursor cells' was highly variable,the variability being confined to the content of the cytoplasm. As in light-microscopicobservations, the nucleus was most prominent and exhibited a relatively consistentappearance in all EPCs. Indeed, no differences were found between nuclei of EPCsand basophilic erythroblasts (Grasso, 1973). The nuclei contained masses or blocksof chromatin which were located along the inner aspect of the nuclear envelope andwithin the deeper nuclear regions (Figs. 11-14). The chromatin masses formed aninterconnecting network enclosing conspicuous interchromatin areas of variablewidth. In contrast to the coarse, dense texture of chromatin in later erythroid stages,the chromatin clumps in EPCs had a fine delicate texture (Figs. 13-15) most oftenappearing as granules or threads measuring 10-15 n m ' n width. No unusual featureswere noted in the appearance of the nuclear envelope.

An outstanding feature of the interchromatin areas was the presence of numerousdense granules whose diameter ranged from 25 to 100 nm (Figs. 11—15). Thesegranules were most often spheroidal, but some exhibited an oblong or rod form. Inmost cells, the granules were clustered about 3 size modalities: (1) a small populationin the range 25-37-5 nm, (2) a most abundant population in the 40—60 nm range, and(3) a small population in the range 65-100 nm. The larger granules were seen in asmall proportion of EPCs where they were found both in the nucleus and as masseswithin the cytoplasm. The majority of EPCs contained only a few very large granules.After ribonuclease extraction and examination in the electron microscope, most ofthe nuclear granules were abolished or greatly decreased in density (Grasso, un-published results). A similar result was obtained at the light-microscopic level sincemetachromicity of the interchromatin areas was removed in RNase—azure B prepara-tions. The size (40-60 nm), appearance, and RNase lability suggest that most of thenuclear granules in EPCs correspond to 'perichromatin granules' (Watson, 1962;Swift, 1962; Monneron & Bernhard, 1969).

The large, compact nucleoli displayed prominent fibrillar and particulate zones(Figs. 14, 15). The granules in the particulate zone measured 25-35 nrn m width andwere easily distinguished from the majority of dense granules in the interchromatinareas (Fig. 15).

At 11-12 days after APH, many 'erythroid precursor cells' typically displayed astriking lack of cytoplasmic organelles (Figs. 11, 12, 16). In individual sections, avarying number of mitochondria was seen in addition to various-sized vesicles andvacuoles. Generally, the Golgi elements were limited to a few scattered profiles oflamellae and vesicles (Figs. 11, 12, 16), although occasionally a moderately developed

468 J. A. Grasso

Golgi complex was found (Fig. 16). Typically, few or no ribosomes were present(Figs, I I , 12, 16), a finding in accordance with the weak basophilic reaction of many'erythroid precursor cells' in light-microscopic preparations of blood 11-12 daysafter APH. Instead, the cytoplasm was occupied by flocculent material (Figs. 11, 12,16) which was absent in small lymphocytes and granulocytes. At higher magnification,this precipitate appeared as a thin, thread-like network (Fig. 16), presumably con-sisting of precipitated proteins of the cell sap.

In some cells, and most frequently in 'erythroid precursor cells' at 13-14 days,varying concentrations of ribosomes were present in the cytoplasm (Figs. 13, 14,17-20). The ribosomes were disposed as long, chain-like aggregates and occasionallyas single particles or clusters of 4-6 ribosomes (Figs. 17, 18, 20). In all cells, irrespec-tive of the number of ribosomes contained, the endoplasmic reticulum was limited toa few scattered granular profiles.

With the appearance of ribosomes, many EPCs, especially at 13-14 days afterAPH, had a cytoplasm containing variable amounts of a low to moderately denseamorphous material (Figs. 17-20) which occupied the entire cytoplasm. In its leastabundant state, this amorphous precipitate appeared as a thread-like, low-densitymaterial (Fig. 17). Whether this material was similar to that seen in ribosome-poorcells could not be ascertained by the methods used in these studies. In most cells at13-14 days, the amorphous precipitate was more abundant, imparting an increaseddensity to the cells when visualized at low magnification (Figs. 18—20). At highermagnifications the appearance of this material was somewhat variable. In some cells,it consisted of a fibrillogranular network, the granular component measuring 3—20 nm in diameter. Most often, it appeared as a diffuse meshwork whose substructurewas difficult to resolve (Figs. 17, 18, 20). In this state, this amorphous substance wasvery similar to the cytoplasmic precipitate presumed to be haemoglobin in knownerythroid cells, an interpretation supported by our observations that this substancewas more evident in EPCs just prior to the accumulation of basophilic erythroblasts.In non-erythroid cells such as small lymphocytes and granulocytes, this amorphousprecipitate was not visible. The variable but ever-increasing concentration of thisamorphous substance in addition to its absence in known non-erythroid cells suggeststhat it may be specifically associated with erythroid development, perhaps the earliestsign of haemoglobin synthesis and accumulation in EPCs. Some support for thisinterpretation has been garnered from our cytochemical experiments (see below).Thus, the initial morphological signs of erythropoietic commitment in EPCs werethe appearance and increase of ribosomes accompanied by an increase in cytoplasmicdensity (Figs. 11-14, 16—20), the latter brought about by an ever-increasing concen-tration of a precipitate presumed to be haemoglobin. Despite these cytoplasmicalterations, nuclear structure remained unchanged.

While the ribosomes and the cytoplasmic amorphous precipitate were highlyvariable, the remaining cytoplasmic structures were more or less constant and couldnot be interpreted as a timed or specific sequence in erythropoietic development.Thus, the subsequent descriptions apply to all cells comprising the EPC population,regardless of ribosomal content or amount of amorphous precipitate.


Occasional aggregations of 4-5 nm filaments were observed, especially near thenucleus (Fig. 21). Less conspicuous aggregations were located in other areas of thecytoplasm. These filaments occurred only in a few cells and were not part of the cyto-plasmic precipitate described above.

Mitochondria ranged from 3-5 to 20 or more per section in different cells of similarsize and were often arranged in groups (Figs. 11—14). They were round to oblong inshape and often had a prominent matrix (Fig. 17). Frequently, the cristae wereswollen and dilated (Figs. 13, 14, 16, 18).

Variable amounts of Golgi elements were scattered in the cytoplasm. In most cellsthese consisted of small profiles of lamellae in association with a few vesicles andvacuoles (Figs. 11, 12), but occasionally the Golgi elements were more abundant(Fig. 16).

The cytoplasm of all EPCs invariably contained vesicles and vacuoles (Figs. 11,12, 16). Large vacuoles (80-150 nm), some probably derived from pinocytosis, werescattered randomly in the cytoplasm (Figs. 14, 20, 21). Numerous micropinocytoticvesicles (40-60 nm) were present in all regions of the cytoplasm but especially at ornear the cell surface (Figs. 11, 12, 16), near the Golgi complex (Fig. 16), or surround-ing multivesicular bodies. The appearance of their limiting membrane indicated 2main types of vesicles: (1) smooth-surfaced vesicles limited by a thickened membraneof 13—15 nm, consisting of, from lumen outward, a wide amorphous dense layersurrounded by a thin dense line (Figs. 17, 22, 26), and (2) coated vesicles with theirtypical triple-layer structure (Figs. 22, 23), which has been described in erythroblasts(Fawcett, 1965; Jones, 1965) and other cells. Of these 2 types, the smooth-surfacedvesicles were the most numerous in EPCs. Moreover, neither type of vesicle had anyvisible content (Figs. 17, 21-23, 26).

Ferritin particles were often seen in EPCs but were confined mainly to membrane-limited vacuoles (Fig. 24) or to dense aggregates with no apparent limiting membranes(Fig. 25). Ferritin was also enclosed within heterogeneous bodies (Figs. 22, 23), i.e.various sized bodies with a highly variable content including vesicles, membranederivatives, etc. which were embedded in a moderately dense, amorphous matrix.These bodies, relatively frequent in EPCs, were identical to heterogeneous bodiesdescribed in normal mammalian proerythroblasts (Bessis & Breton-Gorius, 1961;Grasso, Swift & Ackerman, 1962) and in abnormal human erythroblasts (Grasso &Hines, 1969). Free ferritin dispersed throughout the cytoplasm could not be identifiedwith certainty. Some of the particles associated with the amorphous precipitatedescribed earlier were of the appropriate size (5-7 nm) but difficult to identify con-clusively. If dispersed ferritin was present, it did not occur in conspicuous quantity.Moreover, there was no evidence for ferritin uptake by micropinocytosis since allpinocytotic vesicles lacked particulate content.

Cytochemical results

Although mitoses in EPCs were found occasionally, most animals exhibited variableand generally low mitotic indices (Walker, 1971). But within 1 h after injection ofPH]thymidine, 73-80 % of the EPCs were labelled (Table 1). Simultaneous micro-

47° J- A. Grasso

Table i. Cytochemical data

[•H]thymidine labelling indices, I h

Experiment 1 2 3 4Labelling index, % 737 77-1 74-5 80-5

[*H]cytidine labelling, average no. of grains///™1 of nucleus, 1 h

Experiment i2

PHJleucine, % labeUed

Experiment i2

pHj-^-aminolaevulinicExperiment i

2

3

EPC, i

acid, %

oo

•25 h

EPC•i94±o-•341 ±0-

8888

labelled EPC

40

63 475'9

153239

, 3 h

BE0-260 ±o -

0-305 ±o-1872 1 1

photometric quantitation of Feulgen-DNA amounts in individual nuclei revealed abimodal distribution of values corresponding to diploid (Gx) and tetraploid (G2)nuclei but mainly intermediate values representing cells engaged in DNA synthesis(Walker, 1971). The occurrence of predominantly intermediate values relative todiploid and tetraploid levels corresponded to the relatively high pHJthymidinelabelling indices consistently obtained in these cells. Significantly, no evidence ofpolyploidy was obtained by microphotometric measurements, indicating that EPCsare a typical dividing cell population (Walker, 1971).

With [3H]cytidine, 60—100% of the EPCs were labelled at a level roughly com-parable to that seen in basophilic erythroblasts (Table 1), the latter being the erythro-poietic cell stage most intensely engaged in RNA synthesis (Grasso, 1973). However,microphotometric estimation of azure B binding to cytoplasmic RNA in cells similarto that depicted in Fig. 1 gave levels of absorbances at or slightly higher than back-ground levels, indicating that the cytoplasm contained little RNA (Grasso, 1973).

When pHJleucine was injected into 2 animals at 13 days after APH, i.e. when mostEPCs possessed slight to moderate levels of cytoplasmic basophilia and a few baso-philic erythroblasts were present, 88 % of the EPCs were labelled (Table 1). Similarly,when 3H-ALA was used as a haem precursor, 40-76 % of the EPCs were labelled(Table 1). While it may be argued that leucine and ALA labelling represented syn-thesis of substances other than haemoglobin, several preliminary experiments lessenthis possibility. First, in animals injected with 3H-ALA at 19-22 days after APH,that is, when the blood contained mainly polychromatophilic erythroblasts, 80-90 %of the erythroid cells were labelled while non-erythroid elements in the same prepara-tions remained unlabelled. Moreover, in haemolysates prepared from erythroblastslabelled in vivo with 14C-ALA or pHJleucine for 72 h, at least 75 % of the countsextracted from acrylamide gel slices after electrophoresis were associated with thecarrier newt haemoglobin region (Grasso & Troxler, unpublished results). Secondly,

Erythroid precursor cells of netot 471

when animals were given 14C-ALA or pHJleucine at 12-13 days after APH andhaemolysates from the cells electrophoresed with carrier newt haemoglobin, 94 % ofthe counts co-electrophoresed with the haemoglobin bands (Chromey, Grasso &Troxler, unpublished results). These results, although preliminary, strongly suggestedthat the erythroid precursor cell population was engaged in haemoglobin synthesis.

In one animal, 3 (of 14 measured) EPCs exhibited measurable haem absorbancesclearly above background. In most animals, haem absorbance was generally equal to,or slightly higher than, background levels.

DISCUSSION

Erythroid precursor cells, a morphologically-primitive cell type seen in erythro-poietically active newts, appear to represent several different morphological phaseswhich, in our opinion, reflect the commitment of these cells to erythroid differentiationand their incipient differentiative expression along erythroid lines. That these cellsdevelop into erythroid cells is supported by the following observations:

(1) Erythroid precursor cells always precede the appearance of basophilic erythro-blasts in splenectomized newts and decrease as the erythroblasts increase. Erythroidprecursor cells are absent or greatly reduced in number during the 16-22-day periodafter acetylphenylhydrazine (a period characterized by the presence initially ofbasophilic and later, polychromatophilic erythroblasts) but reappear in abundance at22-25 days, initiating a second erythropoietic wave in which they again give way tobasophilic erythroblasts (Grasso, 1973). Thus erythropoiesis in the totally anaemic,splenectomized animal consists of several waves, each of which is preceded, or rather,initiated by erythroid precursor cells.

(2) Erythroid precursor cells are not found in normal, erythropoietically inactiveanimals but do occur in the blood of animals undergoing normal erythropoieticactivity typical during the spring breeding season (Figs. 9, 10). Because of theirfragility and inconspicuous appearance in smears, a result of both their weak stainingreactions and the overwhelming presence of erythroid cells, including erythrocytes,these cells were entirely overlooked or considered to be damaged, unidentifiable cellsin previous experiments (Grasso & Woodard, 1966, 1967). After their identificationin APH-treated newts, precursor cells have been seen in every animal exhibitingerythropoietic activity regardless of the stimulus causing such activity.

(3) Erythroid precursor cells contain a variable amount of a cytoplasmic precipitatewhich is absent in cells clearly not erythroid and not involved in erythropoieticdevelopment. Admittedly, at the present time, no direct evidence exists for theassociation of this precipitate with haemoglobin synthesis. Its gradual increase inEPCs and its resemblance to the cytoplasmic haemoglobin component in erythroblastsas well as to haemosiderin in mammalian reticulocytes (Matioli & Baker, 1963)suggests that it may represent the morphological expression of haemoglobin synthesisor a component involved in the haemoglobin-synthesizing process, such as an iron-containing protein. Conversely, this precipitate could be a non-specific proteincomponent of the cell sap and completely unrelated to haemoglobin production.

472 J. A. Grasso

Unfortunately, our conclusions linking this precipitate to haemoglobin synthesis mustbe tentative until we have further data of its chemical identity.

(4) Erythroid precursor cells obviously are actively engaged in metabolic processeswhich one would expect in differentiating cells. Given the anaemic condition of theseanimals, these metabolic activities are consonant with erythrocytic differentiation.Clearly, erythropoiesis in the totally anaemic newt devoid of all erythroid cells is nota steady-state system, a feature further emphasized by the successive wave propertiesof RBC development in these animals (Grasso, 1973). Under such conditions, aprecursor cell population might be expected to demonstrate intensive proliferativeactivity in order to increase the number of units differentiating along erythroid lines.At 11-14 days, EPCs and the few basophilic erythroblasts that begin to appear,are the only cells in the blood exhibiting such activity. In addition, the intense label-ling of EPCs by labelled nucleosides indicates the involvement of these cells ina differentiative event. Given the low RNA and ribosomal content of early EPCs,the expansion of these compartments within cells differentiating into erythroblastswould be necessary, a process represented by the correlative increase in cytoplasmicbasophilia, ribosomal content, and intensive labelling of RNA so characteristic ofthese cells. Finally, the majority of EPCs, especially at 13-14 days after APH, incor-porate both labelled leucine and £-aminolaevulinic acid. While the radioautographictechniques employed in this study do not demonstrate specific incorporation of theseprecursors into haem or haemoglobin, other data have strongly suggested that suchincorporation does represent haemoglobin synthesis (see Cytochemical results). Thus,the morphological and metabolic properties of the erythroid precursor cell populationare consistent with our conclusions concerning the direct relationship of these cellsto the erythropoietic process.

An important question is the analogy of newt EPCs to mammalian erythropoiesis.In foetal and neonatal rodents, a cell type closely similar to the newt EPC has beendescribed in the liver and bone marrow (Lucarelli et al. 1966; Stohlman, 1970). Itsdescription and illustration as a large cell with a faintly basophilic, finely vacuolatedcytoplasm and a large leptochromatic nucleus essentially resemble the newt EPC.In rats and mice, the frequency of these cells parallels the extent of erythropoiesis.Other authors have described populations of haematopoietic precursor cells believedresponsible for spleen 'colonization' in irradiated mice (Murphy, Bertles & Gordon,1971; De Gowin, Hoak & Miller, 1972). Their precursor cells have ultrastructuralappearances which resemble the majority of newt EPCs at 13-14 days after APH,that is, cells containing moderate, although variable, numbers of ribosomes. Incontrast, most newt EPCs at 11-12 days contain few or no ribosomes. It is possiblethat species differences account for the absence of a ribosome-poor precursor in thesemammalian systems. Alternatively, these mammalian precursor cells described inthese reports may represent stages somewhat further along in erythroid development,i.e. cells similar to those seen at 13-14 days in APH-treated newts and considered byus as intermediate stages, albeit very early, in the differentiation of EPCs into baso-philic erythroblasts.

Implicit in the identification of any primitive precursor cell is its relationship to


the haemopoietic stem cell. Various studies have shown the presence, within mam-malian bone marrow, of a cell type (or types) capable of colonizing the spleen orrestoring (by replacement) haemopoietic function in lethally irradiated animals(McCulloch, 1970). No corresponding activity exists in lymphatic cell suspensionsderived from lymph nodes or thymus, signifying that only the bone marrow containsan uncommitted, pluripotential cell that can give rise to both myeloid and lymphoidcells. While the morphological identity of this cell is uncertain, considerable opinionis in favour of the marrow lymphocyte (Cudkowicz et al. 1964; Bennett & Cudkowicz,1967; Harris & Kugler, 1967). Yoffey and his associates have described a 'transi-tional cell' believed to be derived from, or giving rise to, marrow small lymphocytes(Yoffey et al. 1965; Rosse & Yoffey, 1967). This mononuclear, lymphoidal cell differsfrom the small lymphocyte in that it is markedly proliferative, exhibits variablecytoplasmic basophilia, and varies greatly in size. Instead of a single, finite cell type,it can be envisaged as a graded series of modulations of a given population culminatingin the formation of a blast cell committed to a specific blood-cell line.

The newt erythroid precursor cell population described in this report closelycorresponds, in its morphological and metabolic properties, to the mammalian marrowtransitional cell. A high level of DNA synthesis and mitosis characterize the erythroidprecursor cell population (Walker, 1971). In this regard, it should be mentioned thatmicrophotometric measurements of Feulgen-DNA amounts in nuclei of these cellsnot only confirm the radioautographic results obtained with pHJthymidine despitelow mitotic frequencies in many animals but also show no evidence of polyploidy(Walker, 1971). Erythroid precursor cells exhibit variable cytoplasmic basophilia,varying ribosomal concentrations, and variation in cell size. These variations can beinterpreted as a sequence of changes terminating in the intensely basophilic blastform, the basophilic erythroblast - an interpretation suggested by our results thathaemoglobin and RNA accumulation mark the period preceding the appearance ofbasophilic erythroblasts (Grasso, 1973). One dissimilarity is the presence of pre-dominant, often multiple, nucleoli in newt precursor cells, structures described asabsent in mammalian transitional cells (Rosse & Yoffey, 1967). Despite this exception,the behavioural and morphological correspondence of both cell types in widelydivergent vertebrate classes is impressive.

In smears, some forms of newt erythroid precursor cells are similar to cells de-scribed as 'undifferentiated reticular cells' and 'large lymphocytes' in mammals(Sundberg & Downey, 1942; Sundberg, 1947). These similarities to cells generallyconsidered as primitive haemopoietic elements suggest that the newt precursor cellmay represent a haemopoietic stem cell. In support of this hypothesis is the observa-tion that similar cells were found in the blood of APH-treated newts during the periodof intensive macrophage increase. These cells not only paralleled the increase inmacrophage number but also declined and disappeared as the phagocytic elementsdecreased, an observation suggesting the possible transformation of these cells intoblood macrophages. In view of this possibility, the term 'erythroid precursor cell' istentative.

It is not clear if the erythroid precursor cells described herein correspond to the31 C E L 12

474 J- A. Grasso

'lymphoid haemoblast' described in lead-treated or starved newts (Jordan & Speidel,1930). Although the nuclear pattern is somewhat similar, the weak cytoplasmicbasophilia and the often extensive area of the cytoplasm in EPCs do not fit the de-scription of the typical lymphoid haemoblast (cf. p. 63, Jordan & Speidel, 1930).Also, the degree of similarity to haemoblasts in Necturus is unclear (Dawson, 1933).However, it is entirely possible that EPCs may be derived from a cell type corre-sponding to the lymphoid haemoblast or that some forms of the EPC may correspondto the haemoblast. That the erythroid precursor cells do not correspond to the electron-microscopic illustration and description of splenic reticular cells in T. cristatus(Tooze & Davies, 1968) is immediately obvious upon comparison.

The origin of the erythroid precursor cell is uncertain. This cell is absent fromblood of normal, erythropoietically inactive animals. Examination of the hepaticsubcapsular region, the kidneys, intestines, and heart in splenectomized, totallyanaemic newts has shown no evidence for the formation of EPCs in these organs.These results are in agreement with those of Jordan & Speidel (1930), demonstratingthat erythropoiesis is limited to the peripheral blood in splenectomized newts. Indeed,our studies suggest that the blood is a normal, erythropoietic locus morphologicallyindependent of the spleen (Grasso, 1973).

In initial studies attempting to determine the time parameters of the cell cycle innewt precursor cells, a consistent finding was that the percentage of labelled mitoticfigures decreased sharply between 48 and 60 h after a single dose of pHJthymidine(Walker, unpublished results). Since the pHJthymidine labelling indices of erythroidprecursor cells were in the 70-80 % range after 1 h, the decrease in labelled mitoticfigures indicates the entrance of non-cycling (Go) cells into the erythroid precursorcell population. Thus, the newt system appears to be similar to the stem-cell kineticmodel described by Lajtha (1970).

Two cell types within the haemopoietic circuit are non-cycling elements whichcould serve as progenitors of the erythroid precursor cells: primitive reticular cellsin the stroma of haemopoietic organs, and small lymphocytes. The primitive reticularcells, however, have been shown not to give rise to blast cells in rat lymphatic andmyeloid tissues, even under extreme duress (Rieke, Caffrey & Everett, 1963; Caffrey,Everett & Rieke, 1966). Moreover, in the newt, we have not observed any indicationthat either primitive reticular cells or reticulo-endothelial cells are transforming intoprecursor cells in the liver, kidneys, or intestines, suggesting that precursor cellsmay be derived from cells already present in the blood. The most likely source, inview of the requirement that it be non-proliferative, is the blood small lymphocyte.Both Jordan (1932) and Dawson (1933) concluded that lymphocytes gave origin toerythroid cells in various urodeles. In several APH-treated animals, we have observedthe appearance of numerous lymphocytes, small and medium, in the blood at 8 daysafter treatment (Grasso & Chromey, studies in progress). Some of these cells exhibitmitotic activity, especially the medium-sized lymphocytes. Thus, our present workinghypothesis is that erythroid precursor cells are the products of differentiation or,perhaps, modulation from the circulating small lymphocyte.

Erythroid precursor cells of netvt 475

This work was supported in part by Grant No. AM-IS4O3, National Institute of Arthritisand Metabolic Diseases, and from General Research Support funds granted to Boston Uni-versity School of Medicine (PHS-5-S01RR05380). The author is a Developmental CareerAwardee of the National Heart and Lung Institute, Grant No. 7 KO-4-HE-17572-O6. Theauthor acknowledges, with sincere gratitude, the kind help of Prof. Nicol6 Miani, Director,Dott. Corrado Olivieri-Sangiacomo, Dottoressa G. De Renzis, Sig. Vincenzo Panetta, andSig. Attilio Caniglia, Istituto di Anatomia Normale Umana, Universita Cattolica di SacroCuore, Rome, Italy. The courteous hospitality extended to the author during his stay in 1968will always be fondly remembered.

The author wishes to thank Dottoressa Chiara Campanella Trautteur of the Universita diNapoli for many of the animals used in these studies. He is also indebted to Dr Carolyn CraneWalker for the ["HJthymidine labelling data in Table 1.

REFERENCES

BENNETT, M. & CUDKOWICZ, G. (1967). Functional and morphological characterization of stemcells: the unipotential role of 'lymphocytes' of mouse marrow. In The Lymphocyte inImmunology and Haemopoiesis (ed. J. M. Yoffey), pp. 183-194. London: Edward Arnold.

BESSIS, M. & BRETON-GORIUS, J. (1961). Ultrastructure du pro-e'rythroblaste. Nouv. Revue fr.Hemat. 1, 529~533-

BLOOM, W. (1938). Lymphocytes and monocytes: theories of hematopoiesis. In Handbook ofHematology, vol. 1 (ed. H. Downey), pp. 374-435. New York: Hoeber.

CAFFREY, R. W., EVERETT, N. B. & RIEKE, W. O. (1966). Radioautographic studies of reticularand blast cells in the hemopoietic tissues of the rat. Anat. Rec. 155, 41-58.

CUDKOWICZ, G., BENNETT, M. & SHEARER, G. M. (1964). Pluripotent stem cell function of themouse marrow 'lymphocyte'. Science, N.Y. 144, 866-868.

DAWSON, A. B. (1932). Hemopoietic loci in Necturus maculosus. Anat. Rec. 52, 367-379.DAWSON, A. B. (1933). An experimental study of hemopoiesis in Necturus: effects of lead

poisoning on normal and splenectomized animals. J. Morph. 55, 349—385.DE GOWIN, R. L., HOAK, J. C. & MILLER, S. H. (1972). Relationship of early proliferating cells

to erythroblasts in hemopoietic colonies. Proc. Soc. exp. Biol. Med. 139, 631-635.EVERETT, N. B. & TYLER (CAFFREY), R. W. (1967). Lymphopoiesis in the thymus and other

tissues: functional implications. Int. Rev. Cytol. 22, 205-237.FAWCETT, D. W. (1965). Surface specializations of absorbing cells. J. Histochem. Cytochem. 13,

75-91-GRASSO, J. A. (1973). Erythropoiesis in the totally anaemic newt, Triturus cristatus Laur. II.

Characteristics of the erythropoietic process. J. Cell Sci. 12, 491-523.GRASSO, J. A. & HINES, J. D. (1969). A comparative electron microscopic study of refractory

and sideroblastic anaemia. Br. J. Haemat. 17, 34-44.GRASSO, J. A. & SHEPHARD, D. C. (1968). Experimental production of totally anaemic newts.

Nature, Lond. zi8, 1274-1276.GRASSO, J. A., SWIFT, H. & ACKERMAN, G. A. (1962). Observations on the development of

erythrocytes in mammalian fetal liver. J. Cell Biol. 14, 235-254.GRASSO, J. A. & WOODARD, J. W. (1966). The relationship between RNA synthesis and hemo-

globin synthesis in amphibian erythropoiesis. Cytochemical evidence. J. Cell Biol. 31,279-294-

GRASSO, J. A. & WOODARD, J. W. (1967). DNA synthesis and mitosis in erythropoietic cells.J. Cell Biol. 33, 645-655-

HARRIS, P. F. & KUGLER, J. H. (1967). Transfusion of regenerating bone marrow into irradiatedguinea-pigs. In The Lymphocyte in Immunology and Haemopoiesis (ed. J. M. Yoffey), pp.135-146. London: Edward Arnold.

JONES, O. P. (1965). Selective binding sites for the transfer of ferritin into erythroblasts. I.Preliminary report. J. natn. Cancer Inst. 35, 139—151.

JORDAN, H. E. (1932). The histology of the blood and blood-forming organs of the urodele,Proteus anguineus. Am. jf. Anat. 51, 215-252.

JORDAN, H. E. (1938). Comparative hematology. In Handbook of Hematology, vol. 2 (ed. H.Downey), pp. 700-862. New York: Hoeber.

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JORDAN, H. E. & SPEIDEL, C. C. (1930). The hemocytopoietic effect of splenectomy in thesalamander, Triturus viridescens. Am. J. Anat. 46, 55-90.

KARNOVSKY, M. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use inelectron microscopy. J. Cell Biol. 27, 137A-138A.

LAJTHA, L. G. (1970). Stem cell kinetics. In Regulation of Hematopoiesis, vol. 1 (ed. A. S.Gordon), pp. m-131 . New York: Appleton-Century-Crofts.

LUCARELLI, G., PORCELLINI, A., CARNEVALI, C , FERRARI, L., RIZZOLI, V., HOWARD, D.,STOHLMAN, F. & BUTTURINI, U. (1966). L'emopoiesi nel periodo fetale e neonatale del ratto.Ateo Parmerae 37, 293-339.

MATIOLI, G. T. & BAKER, R. F. (1963). Denaturation of ferritin and its relationship withhemosiderin. J. Ultrastruct. Res. 8, 477-490.

MCCULLOCH, E. A. (1970). Control of hematopoiesis at the cellular level. In Regulation ofHematopoiesis, vol. 1 (ed. A. S. Gordon), pp. 133-159. New York: Appleton-Century-Crofts.

MONNERON, A. & BERNHARD, W. (1969). Fine structural organization of the interphase nucleusin some mammalian cells. J. Ultrastruct. Res. 27, 266-288.

MURPHY, M. J., JR., BERTLES, J. F. & GORDON, A. S. (1971). Identifying characteristics of thehaematopoietic stem cell. J. Cell Sci. 9, 23-47.

RIEKE, W. O., CAFFREY, R. W. & EVERETT, N. B. (1963). Rates of proliferation and inter-relationship of cells in the mesenteric lymph node of the rat. Blood 22, 674-689.

ROSSE, C. & YOFFEY, J. M. (1967). The morphology of the transitional lymphocyte in guinea-pig bone marrow. J. Anat. 102, 113-124.

STOHLMAN, F. (1970). Fetal erythropoiesis. In Regulation of Hematopoiesis, vol. 1 (ed. A. S.Gordon), pp. 471-485. New York: Appleton-Century-Crofts.

SUNDBERG, R. D. (1947). Lymphocytogenesis in human lymph nodes. J. Lab. din. Med. 32,777-792-

SUNDBERG, R. D. & DOWNEY, H. (1942). Comparison of lymphoid cells of bone marrow andlymph nodes of rabbits and guinea pigs. Am. J. Anat. 70, 455-497.

SWIFT, H. (1955). Cytochemical techniques for nucleic acids. In The Nucleic Acids, vol. 2 (ed.E. Chargaff & J. N. Davidson), pp. 51—92. New York: Academic Press.

SWIFT, H. (1962). Nucleoprotein localization in electron micrographs: metal binding and radio-autography. Proc. int. Soc. Cell Biol. 1, 213-232.

TOOZE, J. & DAVIES, H. G. (1968). Light and electron microscopic observations on the spleenand splenic leukocytes of the newt Triturus cristatus. Am. J. Anat. 123, 521-556.

WALKER, C. C. (1971). An Analysis of Cell Cycle Kinetics in Erythropoiesis, Doctoral Disserta-tion, Case Western Reserve University, Cleveland, Ohio, pp. i - m .

WATSON, M. L. (1962). Observations on a granule associated with chromatin in the nuclei ofcells of rats and mouse. J. Cell Biol. 13, 162-167.

YOFFEY, J. M. (1964). The lymphocyte. A. Rev. Med. 15, 125-147.YOFFEY, J. M. (1970). Lymphocyte production in the lymphomyeloid complex. In Regulation

of Hematopoiesis, vol. 2 (ed. A. S. Gordon), pp. 1421-1454. New York: Appleton-Century-Crofts.

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(Received 3 July 1972)


Figs. i-8. For legend see p. 478.

478 J. A. Grasso

Fig. i. An 'erythroid precursor cell' in a smear prepared at 14 days after initialacetylphenylhydrazine injection. The cytoplasm appears as a faintly basophilic, finelyvacuolated mass whose limits are ill-defined. Wright's stain, x 1600.Fig. 2. An EPC in mitosis in the same smear as Fig. 1. An EPC in interphaseoccupies the lower right corner of the micrograph. Wright's stain, x 1600.Fig. 3. An EPC exhibiting moderate cytoplasmic basophilia from the same smearas preceding figure. The nucleus is similar to that seen in Fig. 1. This type of cellmost likely represents a 'proerythroblast'. Wright's stain, x 2000.Fig. 4. A basophilic erythroblast from a 16-day smear. This cell is characterized byintense cytoplasmic basophilia. Azure B stain, x 1600.Fig. 5. An EPC from the same preparation as Fig. 4. The nuclear chromatin is poorlystained and not visible in this photograph. Several probable nucleoli are indicatedby arrows, although they appear to have been damaged during smear preparation.Note the weakly basophilic, vacuolated cytoplasm. Azure B stain, x 1600.Fig. 6. Phase-contrast view of an EPC in an unstained Epon section of blood ob-tained 11—12 days after APH. The cytoplasm appears as a relatively homogeneous massin which some dark granules, probably mitochondria, can be seen; there are 2 nucleoli(arrows) in the nucleus. OsO4 fixation, x 1600.Fig. 7. Phase-contrast micrograph of an EPC in a drop of blood taken 11 days afterAPH; 2 nucleoli are indicated by arrows, x 2500.Fig. 8. Phase-contrast view of an EPC from the same preparation as Fig. 7. Notethe difference in cell size. In both micrographs the cytoplasm is homogeneous andcontains dark granules which are probably mitochondria, x 2500.

Fig. 9. Bright-field view of blood cells from a newt exhibiting erythropoietic activitynot resulting from APH administration. Most of the cells in the field are poly-chromatophilic erythroblasts; 3 (numbered) are members of the EPC population./ is in mitosis, probably in prophase. The faintly stained cytoplasm is just visible butits limits are ill-defined. 2 is in interphase again showing lightly stained cytoplasmand is smaller than cell /. 3 exhibits moderate cytoplasmic basophilia but the nucleusappears similar to that of cell 2. Cell 3 probably corresponds to a proerythroblast andcan be compared to Fig. 3. At the extreme right, a lymphocyte can be seen. Wright'sstain, x 2000.

Fig. 10. Three EPCs (arrows) from a preparation similar to that of Fig. 9. Wright'sstain, x 2000.


10

480 J. A. Grasso

Fig. 11. Electron micrograph of an EPC from an animal 11-12 days after APH.The centrally located nucleus contains distinct blocks of chromatin (c) betweenwhich interchromatin regions (ic) with many nuclear granules can be seen. Sectionsof 2 nucleoli (nc) are shown. The cytoplasm is occupied by flocculent material oflow to moderate density and also contains several mitochondria (m) and a small Golgielement (go). Small vesicles are scattered through the cytoplasm, some of which occurat or near the plasma membrane. Compare with Fig. 6. OsO4 fixation, x 9500.

Fig. 12. An EPC from the same preparation as Fig. 11. The irregularly shaped nucleusexhibits chromatin blocks (c), discrete interchromatin regions (ic) containing nucleargranules, and a single nucleolus (nc). The cytoplasm contains several small Golgielements, 2 of which are shown (go), a number of mitochondria (m), and flocculentmaterial of low to moderate density. Vesicles are distributed throughout the cyto-plasm. OsO4 fixation, x 7000.

Erythroid precursor cells of newt 4I1

J. A. Grasso

Fig. 13. An EPC from a newt 13-14 days after APH. Chromatin (c) and inter-chromatin regions (ic) with numerous nuclear granules are shown. The cytoplasmcontains some ribosomes, several mitochondria, and vesicles. Compare cytoplasmwith that in Figs. 11, 12. OsO4 fixation, x 14700.Fig. 14. An EPC 13-14 days after APH. The nucleus possesses well-defined chromatinblocks (c) and interchromatin regions (ic) with many nuclear granules. A segment ofa nucleolus (nc) is present in which the fibrillar and particulate components are clearlydelineated. The cytoplasm exhibits some ribosomes, mitochondria, small Golgielements, and vacuoles and vesicles. OsO4 fixation, x 9200.

484 J. A. Grasso

Fig. 15. Higher magnification of an EPC nucleus showing the usual appearance ofvarious nuclear components. The chromatin masses (c) consist of threads and granules10-15 nm thick but the masses lack the deep density associated with later erythroidstages. In the centre, a nucleolus is present with prominent fibrillar (fib) and particu-late (p) zones. The nucleolus-associated chromatin (c) is readily visible. In the inter-chromatin regions (ic), nuclear granules are abundant, many of which are similar tosocalled ' perichromatin granules' (arrows). OsO4 fixation, x 28 000.

Fig. 16. Higher magnification of a segment of an EPC 11-12 days after APH. Amoderately developed Golgi complex (go) is seen near the nuclear indentation whilea smaller element (go) occurs at the extreme left. Flocculent material is present in thecytoplasm but few ribosomes can be identified. Vesicles (arrows) are prominent nearor at the cell surface and near the Golgi elements. Some vesicles are clearly of thecoated type. OsO4 fixation, x 20500.

486 J. A. Grasso

Figs. 17-20. Electron micrographs of various EPCs to show the appearance of ribo-somes and the amorphous precipitate presumed to be haemoglobin. These micro-graphs should be compared with Fig. 16 as a reference point.

Fig. 17. An EPC at 13 days, exhibiting aggregates of ribosomes {rib) and the thin,wispy amorphous precipitate (arrows) which may represent the earliest signs ofhaemoglobin accumulation. A smooth-surfaced vesicle (sv) may also be seen. Themitochondrion (m) contains a prominent granular matrix (mat). A section of thenucleus (n) is in the upper left. OsO4 fixation, x 55000.

Fig. 18. An EPC at 14 days, showing an increased amount of the amorphous precipi-tate (surrounding arrows). Chain-like aggregations of ribosomes are visible alongwith several mitochondria (pi), in which swollen cristae can be seen. OsO4 fixation,

x 3c 000.

Fig. 19. Low-magnification view of an EPC 14 days after APH. The nucleus (n)contains distinct chromatin masses and nuclear granules. The cytoplasm showsincreased density (compare Figs. 9, io, or 13) which results from accumulation ofamorphous precipitate. OsO4 fixation, x 6400.

Fig. 20. Higher magnification of a cell similar to that in Fig. 19. The cytoplasmcontains ribosomal aggregations while the amorphous, moderately dense precipitatepresumed to be haemoglobin is easily visible (arrow). Several vesicles and a vacuolecan be seen. OsO4 fixation, x 27000.


*

w?^

J. A. Grasso

Fig. 21. An EPC 13 days after APH exhibiting filaments (arrows) near the nucleus.A portion of a vacuole (u) surrounded by several vesicles is included. OsO4 fixation,x 46 500.

Figs. 22-26. Electron micrographs of various EPCs demonstrating ferritin-containingstructures and vesicles.

Fig. 22. A heterogeneous body (hb) containing ferritin particles superimposed upona moderately dense matrix. A similar body (/) which may be connected to the hb isshown. A tangential section of a coated vesicle (cv) and a smooth-surfaced vesicle(sv) are indicated. OsO4 fixation, x 67 000.

Fig. 23. A heterogeneous body (hb) similar to that seen in preceding figure butcontaining more ferritin. A section through a coated vesicle (cv) lacking any contentis included. OsO4 fixation, x 46 500.

Fig. 24. A membrane-limited ferritin aggregate. The membrane of this aggregateis only partial (arrow). OsO4 fixation, x 82 000.

Fig. 25. A ferritin aggregate lacking a limiting membrane. OsO4 fixation, x 140000.Fig. 26. Smooth-surfaced vesicles (sv) devoid of apparent content. OsO4 fixation,

x 73 000.

erythropoiesis in th newte triturus, cr is tat us …erythrocytes and haemoglobin, newts do not...

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