THE ANAE3{IA 0]? FLEXED-TAILED .[~{ICE (M US -M USC UL US L. )

II. SIDEI-I, OCYTES

B Y H A N S ~ '" GI[UNI[BERG, De.2)tt.rtmcnt of Biomeb'y, U'~ive'rs.t;tg Cdlgeqe, Zondon ; at Pathogogff Laborato.ry, Mo.~.nt }%.r~mn tIo~'sta~, No'<thwood, M.idd~ese.~

(With Plates S and 9 and Styen Text:figures)

CONTENTS

Introduction 94:6

Mater[a.l and methods . 247

General. re~_aarks about I.he developmen~ of the blood o±" the ~nouse 249

Siderooy~es hl posbna,li;d Iitb . 252 ((~) D~ihncy 252 (b) Adul5 life . 253 (c) The tn~nsitioa fi'om the intermediate to the £efu~itive generation 256

Siderooy~es in embryotde lffb . 259 (a) The ~ransition from t, ke primitive ~o tl~e intermediate generation 259 (b) The e~rly history of t.he intermediafe gener~ion , 262 (c) The primi~b-e generation 263 (d) ~aemoe.y%oblasts . 267

Discussion 268

Summary 269

1Zeferenees 270

~NTP~O DUCTION

In a previous communication (@.r~tueberg, 19~9a), .an ac.eount has been given of the post-embryonic development of the anaemia associated with the recessive gene for flexed tail and bsIly spot (f) in the mouse. In that investiggtion, bulk me~hods were used exchsively. Such methods describe the raea~, cliaracteristies of many millions of cells in ~ given Talced. sample (Iib value, volume of packed sells, the mean volume of the cells, and the mean }It concentration of the cells). The period covered by the first rej?ort includes ~he spontaneous disappearance of the anaemia, and the results may be summarized as follows. At birth, the sel lcount is moderately reduced; the numerical deficiency of cells is made good within a week or a little over. The mean cell size is normal, but the me~m Hb concentration is considerably below normal (normoc]~ic hypochromic anaemia). The hypochromi~ disappears more slowly; i~was not detectable any more in two-months old a~imals. Bo~h in normal and anaemic mice the cell volume decreases rapidly during the first three weeks of post-natal life. The hypochromia in anaemic animals disappears at ~he same rate at which the mean cell size approaches adub level. It was concluded ' t ha t large cell size and low mean Hb concentration are inextricably linked ttp, and tha~ the same process which leads ~o the diminution of cell size also leads to the dis~p]?earan.ce of tlge anaemia'. We shall have to define this process with somewlha~ greater precision in this paper.

By means of a d.y.tlamical a.nalysis, taking' account of the growth of the blood as a whole, it was shown that the produetim~, of large and pathological cells s[ill goes on dtu'ing

gl~e first week after bbth, and possibly to a slight e x t e ~ in. the second week, after which it ceases. Finally, it was made probable tha t the ~ransRion is a gradual process~ involving ~he formation of cells wRh all h~termed~ate mean values of ceil size and of hypoohromia.

Bulk methods, such as have been used in the £rst part of this investigat.ion, are by their very n~tu°re rather insensitive, as t~ey do no~, give direc~ b_formation about the differe~ces between cells within the sample. ~{oreover, young mouse embryos are ~oo small for their application.

After the first communication ha~[ gone to the press, a new and very striking charac- teristic of ~he flexed-tail anaemia was discovered, in stained blood films. The red cells of anaem.ics contain considerable amonnts of free or easily detachable iron (as demon- strafed by the Prussian blue.reaction), a feature not ~hen known in any kind of red cell.* Such ' i ron cells ~ or 'siderocytes' (Griineberg, I941b) are: however, not confined to anaemies. They form a normal featm'e in the embryonic life of gee mouse and of the rat (Rattus norveyic~s), and their ]?resence has also been demonstrated in human embryos and new-born babies (@riix~eberg, 1941c, d); finally, they have recently been discovered in tee blood -of adult human patients (I)oniach, Graneberg & Pearson, 1943).

In dais paper we shall give the first detailed aeeoun~ of sideroeytes, both in ~he normal and the flexed-tailed mouse, in xdew of ~he apparently widespread occurrence of sidero- cymes in other orgamsms, including man in health and disease, the data will be discussed in consicierable detail. The use of this very sensitive tool has enabled us to trace the flexed~ail anaemia to its beginning i~embryonic life. I~ has further tun.bled us to cheek up the conclusions reacEed i~x the ~'s~ report by an indepez~dent and very much more delicate method. I t may be anticipated here ~hat those conclusions have stood the t.est very well, exeep~ tha t the more sensitive method has uncovered the existence of slight ab~.ormalRies in adult life, where~the cruder bulk methods have failed to do so.

The presentation of the data is somewhat diCllcnR. In developmez~tat genetics we usually ~race hack an infierRed anomaly to a well-known process in. normal development. In this case, both normal and pathological development is mnfamitiar to the reader, and ~eith.er is fully unders~a.nda]-le without ~he other.

~{ATJ~RIAL A~I3 3LI,3[['[[{ ODS

The same ~'iexed strain was used as in the previou~ work; R was kept segregating [or the :flexed gent. ,41] J~e work on embryos was done on. baekeross msthugs, i.la ~vhich ~bout one-half of the young were flexed and. the other haJf heterozygotes. Anaemic young are easily recognizable by tl~e.ir.j?aler co]our fi'om the 1ObJx da.y of gestation onwards; tlhe same criterion is fa.bly, but noi) quite, reliable on the ]5th day. In earlier stages the classification must be based on stained blood :fihns; this criterio~ is perfectly reIiable from the ]3th day ozl.wards, b~R breaks down 9x 12-da.y embryos. The timing of pregn.anc]es" wa.s oom])arat]vely rough; a male was put into a cage with non-pr%na.t~t females a~ 5 oV[oc]c in die art,crutch and removed al, 9 o'clock next morning; pregnanl; females were

* I1 ~s po.rha@s of in te res ' t to na r r a t e briefly ho~' 'these t e e s were first discovered. Sect ioned l ivers e l ' new-born ~orma.] a n d a.naemio mice wore simfned fbr iron by men,us of INe Pruss ian blue r e a c t i o n ; iron pigmen% (]baem.o~ s[derin) was d i scovered in d~e l ivers of ann.emits, bu t n o t iff ghe norma,ls. Bul, a, t i n y blue speck was also seen in a, r ed eel] sga~ading on edge in a, vessel : on being e],.ow~ to a cogeag~e, a, Very compe~enl, pa,tb.ologisl~, R was p r o m p t l y declared an artei%~eg- s ta ined blood fil.ms ]e~t;z~o doub t t h a t th is was nee so. I: iowever, on re-oxt~mlnLng the origins. | sec t ions a "f?w days labor, I N i l ed 1,o ~ n d ~ga,in ~ h a t made me sbn,ilx blood films for i ron in blue first J?asla,~ee; wlmf is vm'.y obvious in 'bile one is sea.rnely vis ible in ~5e other,

248 The c~.nae~nia, of flexed-tailed ,mice

lager picked ou~, and the begin~J.ng of pregnaney connted from tl~e time of removal of ~he male from the cage. Wb.~n, in the following, we speak of, say, a 15-day embryo, ~his means I5 completed days plus up to 1.7 hr. As most rantings l;ake place in th.e ~rsg hag of the night, most embryos :wilt be about half a day older than ~h.eir nominal age; in extreme eases they may be older by 1.7 hr. 4ks the age of the titl~ers is only approximately known, sad as the devslopm.en~aI age is not always identical with the chronological age, a-e least two liLters were sampled for every age groap. In some cases considerable diffe:t'enees coon.fred both between and within litters. However, to judge fi'om the smoothness of the develop~tent~l curves based on averages, it seems ~hat the mage.ria[ has brougk~ o~t the main ~rends in. approximately their true relation.

The pregnant :females were killed in ether vapour; the uterus and ~he embryonic membranes were opened, so far as I~ossible avoiding haemorrh~ge from maternal vessels; embryos, from 14 &~ys oztwards, were ~aken. oat of the membrames, given a thurough bath i~ physiological saline ~o remdre any ma~ern;H blood, and. then dried with. blo~ting paper; despite these preeartgions, contamination with mal;ernal blood occurred i.tl a few instances; such films are easily recognized and were discarded, in ~he ease of ]6- and tS~d.ay eml)ryos, blodd was obtained by decapitation; in 14~ and 15~day emb~:yos it proved preferable {o eat the embryo in ewe in the.region of the hea.rg; I9- and. t3-day embryos were left attached to the placenta; the amniotic fluid was ch'ai.ned as far as possible by means of strips of blo~eing paper, an.d blood was obtained d~reetly ~rom the t~earg by means of a micro-pipette.

The blood films were rapidly air<h'ied and subsequently fixed in absotu{e raethyl alcohol. Embryonic blood is very Eable {o contact ]~emolysis, and many cells always eytolise on spreading th.e film. I~ was a{ fim~ feared that this ~ende~ey ~o lysis migh~ affect d~eren~ ceil ~ypes differentially and titus in%rocluce a bias in favorer d certain. cells; however, a scrutiny of partly haemotysed cells seems ~o show ~hat all types of ceils are about equally liable to lysis, and[ hence no serious error seems to have been introduced this way. In making ctifferential counts, partially eytolys.ed cells were disregarded.

Another possible sousce of error is introd!ueed by u~even Nstribntion of the various oeI1 types over t]~e film; ~lae large prianitive cells tend to go to the edges of the film, but other types of i~J~omogensous distribution also oeenr. Before a differential count was made, ~ke slide was studied with a dry system objective, and care was taken so far as possible to counteract inho~nogsneons clis~ribntion of. ceils by an appropriate metffod of sampli~g tl~s film; no doubt this was only pa.r~ly successful, tn ~dew of ~hese sy_stem.atie sources of error, common sense is probably a safer gNde in assessing biological signJ,~cancs in ~his mageriaI ~han the usual numerical tests.

~gta,/ni~g technique

As is generally known, the i~on contained in the t tb molecule does not give the usual histochemJcal iron reactions. ]]'or this reason iron tesSs have searcety been used in haematology. IIad i~ been otl~srMse, the siderooytes would long since have been c~scovered.

(1) *r~'on test. Dried arid fixed films were :flooded with a freshly prepared sotu~ion of 1 ~/o potassium ferrooyanido i~ 1 ~ HC1. T[he ]?russian blue reaction, is complete witlfln 1 ~in; a~ room temi?erature , but as a rule smears were treated for about 2-3 min. As already mentioned in ~he preliminary report (1941 ~), HCI con,cenbrations down to 0-05 ~/o

HANS @~[fNEBE~G 249

are sufficient; still wea]<er solutions (0.09 and 0-01 ~/o HOl) bring out only a ira.orion of. the siderocytes, and in neutral solution {he reaction does not take place at all. The iron in sideroc)~es is thus not ]present in ionized form, but has to be liberated from some com- pound by the ahid. An identical result is obtained if potassium ferrieyanide is used instead of potassium ferroeyanide. The siderofic granules in siderocyte.s revealed by the iron test gradually fade under Canada balsam and cover-slip, but are permaflent otherwise; faded preparations can easily be res~ained.

(2) 6'o~z~ers~c~i~,. The usual counterstain i~.haematologieal dyes is eosin. I t is generally known that cells whie~a are fully haemoglobinized stain intensely with eosin, while cells l~oor in I-It take only a pale pink stain. We have used a counterstain which, though sometimes applied in general histology, does not seem to have been introduced into ~aematotogy. The stain is Biebrieh scarlet, a diazo compound of ~he following con- ~titutio~:

oil.

H e , s ( ) - - ~ ~ - - ( > ~ - - - - N - - ( ) "\_ / \ _ , / \ /

S 0 aH . /

Biebrieh scarlet stains red cells much like eosin, but it has the advantage tiler it does not overstain, and that the red cells retain the stain, weI1 on washing; reproducible results are tlms easily obtMned.

We conclude these remarks on method witla an explanation.. In the previous eommnnica~ tion the following stat.enaent was made: 'There is no great difference in tile appearance of sta-ined celts of notarial and anaemic mice, nor Js there a clear-cut difference between ~ormat and pathological cells in smears of annotates. . . ' (in post embryonic life). The whole of this paper will sJJow the incorrect, hess of this statement. The reason is as foIlows. Two kinds el' si~aiued films were used in the pre~dous work, ~a.me]y, Leishman's stain Mone, and smears snp.ra.~di:ally stMued with br~llh~.nt crecy], blue and subseque~r[ly eonntersba, ined with Leishm~vL I n the firs~ case the diffuse basophJlia, in the second ease the retic'.u]um of the rcticulocyees distracted my attenti on. :fro m the observe,rich o:[' the e osiu con nte~:sl;ain. The di:f~e.renee betwee~ uom>.a,] a,~d ana, em~c cells is much more ]~tarked in the absence of basic dyes, and[ indeed extremely obvious in smears of embryonic blood, as will be described below.

GEN]~I~,,~L ~E~A.t~.T(8 .a~OUT THI~ ])~VELOP~EI~T OF THE BLOOD OF TJI.E NOUS]~

In order to ,facilitate the dcscriptiom it is advisable to recaJt certain fundamental features about the development of tt~e blood of the mouse. The :first blood ce]]s are made in tile

b lood islands and in tile vess~I,~ of the yolk din. These calls are called l;be primitive generation of red bIood cells. A more detailed description w:[ll be given below. Here it may su:ft:i.ce to state that the prh~iti.ve generation consists of very large, fu.l]y haemo- globJaized cells, most of which are nudes,ted. I t has been shown by i~faximow (I927; references th ere to earlier papers) fihafi in the rat and mouse these cells are made exc]udvely in the yolk sac; and. that the yolk .sac of rat and mouse does not produce any eells belonging ~o ~he 'de:tinltive' generation.

250 ~"he anaemia of flexed-tailed mice

As :indicated d~agr~mmatically in Text-fig. 1, ~p ~o tlj.e 12th day of gestation, there are only prJmi~ive red eelIs (,'t) in ~he eis'cula~ion. On the 1.3th day, ' definitive' ceils (B, more exaotly, intermediate tells, see below) mc~ke the.it appearan.cc; these rapidly replace the primitive cells, u:aeil a~, bi.rth only very oeeasion.M primitire cells are found ie the cireu~ la~ion. "Evea .irl normat mice, most of the primitive ed]s are dearly dlsbinguishable from {he ',:lefinJ¢ive' cells; we shall see later on ~hat in flexed embryos this distinction is very much more m~rked. There seems to be agreeme~st j.n I~he 5aema~ologieM li~erat~u'e that the l~rimi~ive sad. 'deJinitive' generations are two distinct en¢ities, and that no inter- media,re eel[ ~ypes are formed, q-~he ,iat~.~ ~,o be prese:,.ted in ~bis paper are in emphatic agreen'~en~ with ~his view.

I

t

Early 13th day of gestation Birth

Text-fig. I. Diagz'~m showing t.he repl~eemeu~ of the primitive generation of red blood cells (A) by the in~er- mediate genergtion (27"}, which in tm'n is re'placed b.y the definitive genor~tion s~nsu stricto (C).

The so-cMled de~nitive generation of cells, in a nm'mal mouse, differs from the primitive generation mainly Jn two ways. ~-early all the ~definitive' cells lack a nucleus, and they are much smaller. The mean call diameter of the 1oriml{ive cells is l'oughly 12b~ or over, fha~ of the ' definitive' cells of the embryo and newborn mo~me about 8/*. The ' definitive' ceils, in the Jrs{ ins{ante, are m~cJ.e in the liver; de Aber].e (].927) has shown that the bone marrow does not become org}nJzed, a8 a haemopoietic organ until late on ~he 16th day of ges~,agion, and apparently its output of J?ed cells up to the time of bh'th remains

insignia.cant as compared with ~]l.e ~iver. Indeed., t.he liver continues to f/inoJJon a,8 haemopoietic organ for some time a:ger bir~h; as menJJo~.ed by de Aberle ~nd conf~rmcd by the presen~ writer, the liver of' ~he newborn mouse contains nnmm'ous active fool of

t{A.ss GR~SE~m~G 251

haemopoiesis; even 7 days after birth, such ceils nests are still 2ound, though they are tess extensive and probably less active.

Now we have to give the reason why we have ]persistently put in inverted commas the term 'defiNtJve' red celts. The mean diameter of tl,ese cells during embryonic life up to ~he time of birth is quite constant in the neighbourhood of S/z. Between birth and a fortnight or 3 weeks the size of the cells drops rapidly to a mean diameter of about 6/,. We have described this process (in terms of the mean cell volume) in an ea.rlier communica- tion {1989) and again in greater detail in the first paper of this series (t942@ The red ceil of the adult organism thus differs from that of the la~e embryo and newborn mouse by its size; there is a plateau at 8/z approximately up to the time of birth, a transition period durhzg the first fortnight, and again ~ plateau at (3/z thereafter (O in Text-fig. 1). _4 similar ~ransition has been described for the rat (Smith, 1939; Wintrobe & Shumacker, 1935, 1986; Brmaer, van de Erve & Carlson, 19.38) and less ex~ensively for various other m~mmals, including man. Tl~ere seem to be differences of opinion as to the interpretation of these facts amongst haenaatologists. Smifh (1932) considers th-ag the diminution of cell size during ~he transition period is a consequence of diminishing hum.bets of reticulocytes in the blood, and the same opinion has been expressed in a personal communication to me by an eminent haematologist in ~his country; as shown by Persons (1999) and by Wintrobe (193g), reticulocy~osis may raise the mean cell volume; now ff the rapid decrease of retieulooy~es in young rats and mice were its sole reasoa~, the drop in cell volume during the fi.rst fortnigJat after bir~h would merely indicate a lesser laaemopoietic activity and Jaenee be of very slight sigmficance. Another interpretation is favoured Joy Wintrobe & Shumacker (1935, 1936) and by Brunet et d . (1938); these authors consider that the decrease in cetl size is due to ~he replacement of a generation of large embryonic cells by ~he small adult cdlls; in this ~dew the difference between, the large cells of embryoand newborn animal ~nd the small cells of the adult is an intrinsic propert~y of these cells and not simply a consequ.ence of the fact that most of the earlier cells are reticulooytes.

I l~ave on. previous occasions (1939-,~:2) accepted the latter hypothesis. Unfortun~tel F, I have followed other autl~ors Jn using a somewha~ misleadiag terminology; Brunet et, cal. (19.38), for ex.'unpl<, have called the ~vho]e process ' the tra,nsit~oil from megalob]astJc erythropo]esis of the newborn rat to normol.dastio erybhropoiesis of the aduIt rat ' . The term 'megalobIastie' should be reserved :['or the prim.idve gen.erati,m of red blood cells. ]3~t what is observed separates early and late 'deEuit;ive' red ceils. I t will. be m o r e

a.pp~'opriate to call the early type of these cells the i~zZe:rvz~:dic~e 9e~,..era.Zio.~z gnc[ to restrict tlae tern?, d~Ji~ith,e ge~~,erc~io~ to the l'utly adult cells (mean d.[ameter 6to in the mouse). t t~,,]<e this opporttmity to emphasize tl~a,~ wherever in the past I have used the eo.ucept of a transition f,'om embryonic to adult cells, this refers to ~he transition from in.tern~edia,te- to defb?.it:i.ve cells,

The present paper contains a good deal of evidence for th.e existence of an intermediate generation as a, separate entity. The flexed-tail gone of the mouse a, tCa(3]]es 8. ~abel tO t]]Js generation of cells and provides clear proof' that this generatio.n di:fl}~rs %udamenbally both f'ron), its predecessors and from its successors. We shall also give so~xe evidence to show that the reticulocytosis found in young re:ice (al~d rats) is n.ot reponsfble fo~: their Jaigher cell volume. Ant:[c:ipati.~g the substance of tl~is palper, we th6re:fore hold ~hat ~he:re exist three generations of red blood corpuscles, designated as J_, Y3 and O respectively i~ Tex~figure ]. in the mouse, tlhe primi~ive generation is exclusively produced in ~the

252 The anae,rrd(~ qf flexed-tc~iled )?~ice

yolk sac; the intermediate generation, a~ ieas~ in the earlier stages, is a product of the lice:r, and ~he definitive generation, at least in later phases, is exclusively a product of the bone ma.rrow. Whereas the transition from ~I to B is disco~timtous, without any intermediate celI types, the transition from B to C is probably a gradual process.

It ~ill facilitate the d~seription if we stars the story in the middle a~d, deal ilrst with the

behavJ.onr of side.rocytes in postonatal life. In the newborn flexed mouse the majority of the cells s,re sidsrocytes (see P1. 8, figs.

17-:]0). These cells diff'er from the erythrocytes of normal newborn mice in two.ways. They are incompletely haemog[obim.sed; in ~mstained films they show very little colour, while fully haemoglobinized cells have a distil~etl.y yellowish tinge ; in stained preparations they take a pale pink countsrsts.in, whiie normal cells s~ain intensely pink or red. }Wnea stained With Biebrieh scarlet alone, the cel]s of al~aemies show no other peculiarity; in partic,:dar, no formed be&its of any ]rind are visible in them. When such films are subjected to th.e Prussian bhe test and subseciaently connterstained, a very strildag picture is obtained. Nest of the ceils, sometimes 90% or over, show a number of blue granules. Their size and nmnbsr is very variable. In the mos~ extreme cases, several dozens are present; they are lying in the cells in small grm~ps, or scattered at random tha-o~lghout the cytoplasm. The larger elements are more or less roundish blobs, which in rare eases may reach a diameter of nearly a micron. Usually ~hey are much smaller, and their precise shape cannot be recognized with oertainSy, though they-~end to be isodiams~ricai. The smallest grannies are fins dust-Hke stipples, which can only just be discovered under the oibimmersion. Some cells contain only a few ~anules, sometimes only one or awe. t f there are two granules in a cell, these granules are often lying otose t0gether lille diplocoeei. The presence of ~his siderotie material in most of ~he cells of thenewborn flexed mouse can bs cliscovered with a cLry system of tile microscope, but for exact counts the oil- immersion is always neeesfiary; in order no~ to overlook the presence of a single small granule, i~ is essential to focus np and down separately for e~ch individual cell; this is even more necessary in normal embryos and. newborn mice, where the average ammm~ of siderotic material per sideroc~s is very muph smaller; the need of carefully scamiing the whole optical thickness of each ceil often makes siderocyte counts very tiring for the eyes; however, in the newborn flexed mouse this is not the ease; as the siderotie material in most cells is q~dSe heavy and seen at first glance. In no instance has a diffnse iron stain been observed in the cytoplasm. ,Detailed counts for normal and anaemic iz~fant mice are given in Table 1..

Between bir~h and the end of the first week, ~he siderocy~e percentage of flexed mice falls from abo~t 80 to abot~t ~I0 ~ . The amount of siderotic material per sideroeyte tends to be less, and some of the cells hays aeqmred more haemoglobin, as shown by a more intense counterstain. The acquisition of more I-It is; however, apparently a gradual process. I t is not possible to sort o{~t the cells, into two distinct types, pale and fully haemoglobinized, but eli intergrades are foundl The sideroeyts percentage is lower s~ill at the age of a fortnJgh% while the haemoglobinization of the cells becomes more and more normal. From the age of ,3 weeks onwards, ~he siderocyts percentage becomes stabilized

~ r s Oi-~x~BE~e~ 253

in ~he neig}~bourhood of abou~ 3 % and, as will be si~own below, this percentage persists {hronghout life.

As show,~ in Tables 1 and 2, siderocyLes arc no~ confined ~o flexed-tailed mice. k small percentage is found in newborn zlormal mice, and a very occasional sideroey~e is found in 7-days-old-aormals. Thereafter they disappear aleogetller. In ~he preliminary aeeoun~ on sic terocytes (1941b) I left the question open whether sideroc)~es are altogether absent~ in older normal mice, or whebher they are only very rare. With the increased material and experience now available I feel ~ustified in saying that ~l~ey do not oocm- any moz'e from She age of 3 weeks onwards. Nor.reals differ from flexed mice at bixt, h n#a only by ghe

Table 1. Sid~e~'ocyte Terce)~tage in post-,;zc~taZ lif~. The co~ts of ~wr~nal ~nice are based o~ 500 ee~ts i~z each case, those of a~zae'ffdcs on 400 ceZls, e:~:eept in Zittw E, where 800 eagle were eot~,nte~; 33,000 eeg~s i,~ aft

Age in days Li t ter Normala 0-1 A 3"6 71"75

B 3-S 4.6 88'5 C' 3"8 4"2 5-8 6.2 92'0 2) 2.6 62-25 E 73"0

7 A 0 31.0 .5' O 0 ,il.75 O 0.4 0 ~2,25

0 0 36-0 (¢ 0.4 0 0 42.5

13-14. ~ 4'5 C 4"25 g 9-0 H 5.5 I 7-0

20 F 3.0 G 3,0 I #5

27-28 F 2-75 d 1-25 J

34-35 C? Y£

Ta,bte 2. 3Sea~. sidcrocyfe yrce~_lo#c 'i~, Age in days Norms.Is

0-]. 4"3 -7 0-I

13 14 0 20 0

27-28 O 36-35 0

~lexed

83"75 89'5 93-25 91.75 71-5 63-0 704 73-6

38.5 40-5 "39.75 49-5 4%0

8.75 6-25

5.75 5-75 3-0 2.25 3.25 3'5 2.25 L5

1-5 3.75 2.25 2.0 .1.75 .4,-5 4,25 1.25

2)ost~*mtcd t~ife g a r ~ S c s

81-2 -1-0"9

6.4 2.8 2.2 2-9

73-7 73-4

7.0

1.75

very much smaller pereeata,ge of siderocytes. There is also eonsiderabl,v less siderotic ma,~eriM per .sid.eroeyte; as a, rule, one or ~wo sm~,lt granutes only are fnund, azld mos~ of the sideroeytes of 1normal i?ewborn mice are more or less fully haemoglobinized (like the ceils shownin 171. 8, figs. 13-15). They are, indeed, not ea, sy Vo see, and their presence in normal newborn m~ce was overlooked for several weeks. We shall see below thae their presence in normal embryos is mueL ntore conspicuous.

(b) _,lchd/, l@

The pe.rsistenee~ in #Jexed mice, o:[' some siderocy0es inf, o adult life was somewhat, nnexpect, ed. TJae bulk n-lethods used .in the first part of th~s investiga~iun. (1942a) had :['aifed to give any iv~dieation of bi.Jod anomalies in fully grown flexed ndee, a~;d the

25'4 The a,,n~;e,~nia of f l exed- tE led ,mic.e

conchasion was drawn tha~ the anaemia disappears :~ltogether. With the much more delicate 1oo1 n.ow available R is dear that a very slight deg~:ee of anomaly persists. Under these circumstances it seemed desirable 80 cepea~ Bhe observations on achflt ~[exed mice and 8e the same time to study the behavioar of their sideroeyges.

In Ta'ble 3 are given ~he blood counts of some ad.aR normal and flexed mice raaging h~. age from 13.5 ~o 560 days. Cell couxxts, haemoglobin estima,Sun and haema~oerit vMues

Table " -D. Adtdt mice. S'iderocyle 2erce;dages ba.sed o l;~ O0~'J~t~ 0/1 10()0 CellS, exce2Jt in",ox. 10S <c~l.d 109

Ge,m- Age lib Ery~hrooytes Zb Z.]~Ib fJ, type No. Sex d~ys % per cu.mm. % 7Y 4-/f 156 :.¢ 22.0 t08 8,780,000 530 17.0 + / "} :I52 I~ 2 3 0 10~[ 7,940,000 52-0 18.1 + / f 149 c? 232 1. ] 1 9,065,000 53.0 I6.9 q / f 130 f~, 249 113 S,t?05,000 53-0 18.I +/+ 132 o 251 112 8,625,000 58-5 11.7-9 + / f 1] 9 ~ 25{:~ 120 9,955,000 52'0 t,3.6 q'/J~ 107 i~ 3138 109 9,825,000 50-0 ]5-3 +jr 110 ? 308 l l 0 8,695.000 44.0 17-5

f / f 162 c~ 135 96 9,715.000 50.5 13.6 ,, 16:3 ~l ] .35 ~ 9,1~{01(~1) 0 ,, 155 d* b26 109 8,720.000 50.0 I5.8 ,, 151 g 2%0 95 8,70.5.000 48-5 15-1 ,, 150 9 232 96 8,375,000 48.0 15.8 ,, ].$9 O" 249 69 7,305,000 36.0 13.0 ,, 131 ~ 251 96 9,000,000 48-0 14-7 ,, 12.0 9 256 103 10,040,000 53.5 14<% ,, t08 9 308 103 8,375,000 44.5 17-0 ,, 109 ~ 308 112 8,730.000 47-5 17-7 ,, t00 d 335 90 8,580,000 47.5 14,5 ,, t 0 t o 335 85 7,610,000 40"5 15.4 ,, iO0 ,~ 500 S0 8,090,000 45.5 13.6 ,, 10t ? 560 52 5,145,000 30.0 13.9

(500 calls erich}

Z.C.V.Z.CI. l-Ib.C. #' %

(}0.4 2S.1 66-5 27-6 58'5 28"9 61.6 29.4 .65"5 27,4 52-2 81'8 50.9 30-1 50-6 34-5 52"0 :36.2

57'3 27"6 55"7 27"0 57"3 :?7.6 49,3 26-5 53,3 27-6 53.3 26.6 53.1 31-9 54-4 32.5. 55-4 26.1 53,2 29-0 56-2 24.3 58"3 23~9

8iderocytes c % Per cn.mm.

5-9 573.000 6-5 594,000 2.3 201,000 0-9 78,000 2-5 .209,000 3-8 278,000 4.5 405,000 1.3 131,000 1.4 117,000 1,0 87,000 1-5 120,000 8-0 609,000 4.8 388,000 2"7 139,000

were obtained, as described in a previous commuuieation I9424). As pointed ou~ pre- viously, 5he pathologieaI feature in yoa~g flexed mice is a lowered mean corpuscular Kb concentration; this pal-ameter is de~ermined from the haemagocrR vahte in con]unction wRh the l ib value. As thesevalues are closely correlated ia normal mice, a previous kaow- ledge of the haematoez~ value might introduce a bias When estimating the Hb. For this reason, the Kb was always estimated first from a separate sample, before the haema~ocrR value was known; the same precaution had s t e a d y been used ix~ the 2-months-old azma l s in the first paper of this series. The average values obtNned are W e n in Table 4; these

Table ~ I Aser~.qe reuse derived fron~ Table 3 (l~p~)S~' ~ f ~l taEe) c~d 2-~nonths stage (lowe~ /~Ef, from @r{tneber 9, 1942~J

Siderocy~es Hb Nry~hrocy~es H~ ~f,JcIb.' f'~. M.CPV. M.C.Hb,C. ~ - ~ ,

g./100 c.c. per eu.mm. % 77 P~ % % Per ca.ram. NorraMs 15"30 8,936,250 51-7 17.2, 58-2 29"7 0 d Flexed 13-35 8,6d6,250 47"0 15-4 54-2 28,4 3'3 284,000

NormMs 15.98 10,208,000 54-6 15.7 53-5 29-3 Flexed 1 5 ÷ 0 4 9,954,500 51-1 15,2 5l-5 29-4

include alI the normal mice, but not Bhe flexed mice ~os. 162, 163, 100 and 101, as these are of ages not represented amoag ~he normals; the siderocyte averages incls.de the latter four individuals, except the re-examinations of nos. 100 and 101 in old age. For col~> parisozt we give, in ~he lower half of Table 4-, the values for 58-60 days old animals

Ha~s G ~ t L ~ c - 255

obi,ained previously. @omparing first the 2mmnths stage with the fully adtllt an.JaMs (226 308 days), it will be seen tha t cell counts, I Ib and haematocr i t valses in i,he la%er ale definitely lower, wherea.s the cellular characteristics are no t great ty di£'ereni,. The gradual decline of cell connt and } l b values with age has been noi,ed by other observez-s for deta.il.s see Griineberg, 1942b); the two oldest, individuals in Table 3 suggest t.hat later on thm:e m a y also be a decline in the mean corpuscu]ar t t b concentration.*

Comparing normals wii,h flexed mice in boi,h groups, there do no t appear to be significan~ differences in either set of data. In pari,icular, it is obvious t h a t the mean corpuscular f ib concentra t ion of flexed mice is not lower in i,he younger, and onty insignificantly so in the older group. Jclence the re-examinat ion confirms the observations of the firs~ series tha% by means of bulk me,hods and in a series of manageable size, no difference can be observed bei,ween adult flexed a~d normal mice. And ye t the presence of a small number of siderocy~es shows tha t the blood of adult flexed mice is no~ quite normM; pari,icular]y as some, ai, least, of i,he siderocy~es are incomplete ly haemoglobinized (P1. 8, fig. 23). Supposing tha t the blood of adulO flexed mice contains as m a n y as 2 ineomplei,dy haemoglobinized siderocy~es, which is p robab ly an overestimate, and assuming tha t the t t b concentrat ion of these cells i s a s low at one-half normal, the g b

• . ,concentration of all the cells, as measm'ed by bulk reel,hods, would still be 9 9 % o f the normal valu.e. One could scarcely expect ~o demonsbrate so small a difference by means .of so rough a tool.

I t is of some interest to consider ~he tota l nmnber of siderocytes in the ch'culagion, as disti~:m~ fron~ gheiz percentage. Such da ta are given in Table 5. The total number of red

Table 5. Total ~u~)~bsr of sids.rosytss in the circulation To~al ,Siderocy~e To~a]

Age. eryi.h~ocy~es percentage sidero cyrus Bh%h 939,000,000 87.2 194,000~000 ] week 705,000,000 40.9 288,000,000 2 weeks 1,8:24,000,000 @4 ]].7,000,000 3 wcd~s a,aaa,o00,0oo ,~,s 9a,000.0o0 Acl~tt~ I2,600,000,000 3"3 416,000,000

ceils in the Oi.l'Oll]a.@on of flexed mice froJ].l bird1. ~;o 3 weeks of age has beet] taken :f~rom d-~e ]94..2a ]?a]j,r: Table 9, p. 60. That of ~]m adult animals has been arrived at on the asatt]n]DbiOll tha.I) a fJ.exed tnol:lse of 20 g., with g ceil OOtlU{~ of ]0,0c)_0,000, ha8 a blood voh.lme of 6,,.- o.o.ll000 g. body weight, or ]_-2f3 o.o. (for bl~r~ b]ood volume of adM+a mice gee Oakley & Warraol< 1940), and hence abo~rb 1.2($ × 70 TM red cells; this is, if aKT~hing. , an underesdma.te, as the weight o:[ a fully adult flexed mouse wilt exceed 20 g. by a, greater a.motmt> I,]:mn the eel} count wilt fall shori, of 10 v ceils per ou.mm.

I~ will be ,see.~.~ ill Table 5 tha t t~he tota, l number of aiderocy%es in. tlie oiroulWdon increases siglfi:O_oandy (by {1:8 ~/o) between birth arid "0he end o:{" the firsO week. Tiros> even assuming thai, all 1,he siderooyges preseni, a,t bJ:,:th had era'rived,, gl,e :flexed mouse midst have p:,'oduoed ]lui]lerotlS sideroeyDes du:i:ing Who first week a,f66r birth. I n t~]je .lirsb paper of riffs series we have estab]ished by a dynamic I,:i:ea, I;ment of bul].< d a m thai, ~he flexed mon.se prodt~oes some cells deficient; in {flb durilzg the :Srsi, week after bir~,h. The da~a,

* [[?.he dM, a, pnbtlshed by tohe present~ author ~md by of, hers show I}ha,t; ~he red blood pioiaire of ghe mouse is ~ever sta'61ongry; rapid changes in h~fo.n0y ]es.d l,o pe~k values a:{, a.boug 9. 1Jooni, hs, wl_l[ch are soon followed by a. deelhm, wldch is slow ab firsl., bwC l~ter teA,hers ntomcn~nm. Blood 7~icl,ures of mice wi¢tl.ont: s,peci(yk}g I~heir ages ~re t<h.us wIueless. This, nJfforCum~t,,Ay, a.lso applies %0 l;he ot,heru.'ise careful paper by PePsi (19a3); a 7EmrttsM ol ~ his va.hms suggest,s I.b.'_~,I~ his ma.%ria.] included many vet;era.us.

256 The ~'n~tem~ic~ of f ie:ted-tailed .mic, e

presenSed here afford a completely independent confirmation of that conclusion and at t;he same time increase the confidence in the soundness of the dynamical analysis then used.

From the age of 1 week on~v~rds, the total number of sid erocytes in ~he ch'cMation decreases and reaches a mbfimum at th~ age of 3 weeks. ThereM'~er, the total number of sidero~ytes increases again, because the siderocyte percentage has now reached adult ~evel, while both g.e number of red cells per unit v o h m e ofblood and tlJ.e total blood robtree go on increasi~fg. In fully adu!t flexed mice the total number of sideroc?~es is between four and five times as great as at the bottom of the ctLrve at the age of 3 weeks, The latter fact is of importance. The stable ].evel (about 3 %) of siderocy~es in the blood of adnlt flexed mice speaks on the whole strongly ~gain,st the ~ssumption thai) these ceils are simply survivors i?om inf~mey, i-Iowever, by endowing the siderocytes with ~im.- mortality' , suc.h an a.rgum.e.~t might be upheld; their increase in absolute numbers shown i.a Table 5 irreNtably explo~Ies such a res:soming.

To sum up. An. analysis of the total number of siderocy~es in. the cilrcula~ion sinews that during the .!~rst week afk.er birth flexed mice go on producing siderocytes on a con- siderab[e scale; this confirms the results t~f a, d.ynam~cal ap.alysis of balk data, which showed the conti~nued production of }Ib~defieient celis during Shut interval. It is farther shown that itexed mice produce a small percentage of siderooytes throughout life; the enstring anomMy of the blood-is too slight to be detectable by means of bNk methods,

We have now to consider the relation, of the siderocytes to the other changes which take place in the blood of normal and flexed hxfant mice. The most prominent of these is the diminution in cell size. We have plotted, in Text-fig. 2, the siderocyte percentage (solid line), and the mean coil vohme in b~ s of flexed mice (broken line) during the first month after birth, She latter from Table ~l, p. 55 of the I9~2a paper. The general shape of these curves is remarkably similar (the ch'op below a&/l~ level of the cell volume at 3 weeks is almos~ certainly spuriou@ It has also been shown (19~2a) thaf5 ~he decline in cell volume is inextricably linked up with the attainmen~ of a normal mean corpuscular t Ib concentration. Adding the fact ~hat siderocytes are visibly Hb-deficient, there cannot be any doubt that the anaemiaof flexed4ailed mJ.ee is basica~.y due to the preennee of these siderdcytes. The a,~ae.mia of flexed-tailed ,mice is a siderocyte anae~nia and thus distinct from all other known anaemias. The return to normai (or near-norms.t) values goes parallel with the decline of cell size to adult level and a oh'asPic reduction of the siderocytes.

(c) The transitio~n j?'o'~ the inter,mediate to the defin,itivs gens~'ation

We have now to come back to the nature of the transition process, which leads .from the large cells of the newborn mouse to the small cells of the adult gnlmal. We have already mentioned that according to one view, the large cell size of newborn r~ts and mice is attributed to ~h.e presence of nume}ous retic~Jooy~es. The work of Persons (1929) has' indeed shown that the reticulocytes produced under various pathological conditions by adult humans are larger than the fully mature cells which they accompany; but it was also shown that the reticNocytes are much larger in some conditions than in others. Jclence it seemed relevant to inquire whether the reticulocytes of.newborn mine are in fact. su~%]ently large to account for a cell volume about double that of adult mice ;" c~u'iously enough, this had r~ot been done by previous authors who have adopted the 'retieulocyte hypothesis 'i

H~aNs G~-~N~BE:t¢O 257

It would be possible to refhte the validity of the 'reticuloeyCe hypoCheis' from ~he d~ta already published. The argument would be on similar lines as ~hat used. (1942a) to refute the 'two-cetLhypothesis', with whi¢]~ we shatI have to deal again below, ttowever, ~he argumen~ would be involved and i~direc~; hence it seems preferable to use this space for direct evidence which is easily obl, ainable.

Reticulocytes and fully mature cells of ~wo normal mice each at birth and 7 and 13 days of age, fz'om the same area of the same film, have been. measured by .means of an ocular gratieule. The divisions of the gra.~ic~le, wiO, the optical eqnil)men~ used, corresponded ~o 4/,; by reading .to the nearest, ciua.rter of a division, the readings were to the nearest

100

g 4~

g 6o

4 0

! OO [ \

80 \

20

\\ \k \

\ \ \ \

% \ \

% %%

_

o 0 I

x

2 3

Age iu week~

90

% 80 .~

¢1

o

- 70 =

6O

50

Te.xt fig. 7. $id~rocybe percentage (~;o]id line) and J33ea.u cell To)nine (Sroken Hne) of f~exed mice durhng l:he fir,lt mollt.h M'6et" bh'/,h. Siderocyte pm:¢cnSage of normals (dogi,ed ]in~).

ndcrom As the dii:['ercnee between zna,tm.'e, calls a,r~4 re.%i~mloc.y~es expected on the ~reticu]oey~,e hypol}hesis' is large, [,he mea.su:rement of.KEy e, ell,s of either kind per aniInal was ample.* The data are given i±1 detaii ht Table 8. The result 5s tic, ire unambiguous. ~n no case is there a significant difl%ren.oe between fully mat;are cells (normooytes) and reticulocytes;bothdeclineinpart_paripetssu. If a.n yth~ng, the reticalocytes tend to be smaller tha,z~, the no,:moey~es, though this diffe:,:ence is not .~.igJ~.ificant on the data presented. Averagin_g g.he two animals of ca,eL age group, the mean 'cell diameters of no:rmocytes are 7-87, 7-02 and 6-:4]/.z respeet.ively, sad t~hose of reticulocytes are 7-7.5, 6.95 and 6.211z respectively. These figui'es are in exeelten~ agreement with the mean ceil volume values

* ]>r~ce-Jonos recommends tlae measuring o.C .500 cells, per J'iJm; i,t!e tedieusuess of ~he ~nefihod has prevented [t,~ more general applic~gior~. The measurement or" so many tells, for ~uosl, purposes, is ~ wa.s~e of eftbr~b 1,5e inherent inn.eouraeies of fire me~hod limiI~ iihe sharpness of definil,ion of a. lnca.rt cell diem eter, aud no accumulation o[' figures, however tree.I,, will tr~mseend bhfs limil,. I Jinx.inCa.in ~ha.g for practically all proposes the mea.surement of 6.[~y cells is ,suNeiea%, and I douL'~ whether mes,suremem:s much in excess of 100 ar~ e.ver ~us~,ified.

Jenny. of C4enetics 44: 1.7

258 T h e c~ae,mia of ,flezed-tc~iled. ~nice

published previously; the ratios of the corresponding cell volumes would be 100 : 721. : 54 in the case of the normocytes and i 0 0 7 2 " 5 2 in the case of the re~iculooytes. Th.e closeness of agreement with the pnbl.ish.ed cell volumes, considering ~he small number of diameter estimations, is no doubt due to a ran of luck.

To sum up: There i.s no evidence that the regionlocyges of infant mice are appreciably larger than fully mature cells. Hence ~he decline in me~a cell size cannot be a%ribuLed ~o ~he declining number of reticrdocy~es.

The 'regiculoeyte hypoghesis' having thus collapsed, it is difgculL ~o escape the con- clusion ~hat the diminution of cell size in yonng mice (and o~her mammMs) regects some deeper physiological chalk.be i.n haemopoiesis, regardless of the fact that it happens within ~he 'definitive generation' of red corpuscles of orthodox haematology. This conclusion .is now powerfully confirmed by gee behaviour of the siderocytes. The flexed-aM.1 gone of the mouse clearly delimits an iatermed:h;ee generation of blood cells, mos~a of which are sideroeytes, from-the true d.efinigive generalXon, i~3. which these .ceils are scarce; and ~heh: decline in ml.mbe:rs is brought about by tile identical process which reduces ceil size.

Table 6. I).l~m.ete~'s of .~orv~.ocJes (N) ~zg ,retic.ulocytea (~) A g e N C e U d i a m e t e r (~ ) M e a n ce l l

hx o r ~ d i a m e t e r d ~ y s 1% 4 ,5. 6 7 8 9 I 0 1 I /.*

0 { N - - I 2 t 0 2 7 7 3 - - 7 - 9 2 ± 0 . 1 3 7 - - - - 1 12 2 8 7 :3 - - 7 - 9 4 : ~ : 0 . / 1 2

{ ~ - - - - 1~ 31 5 - - - - 7 ' 8 2 ~ 0 ' 0 8 8 0 - - 3 1 8 2 7 :2 - - - - 7 . 5 6 " 0 - 0 9 5

) N - - - - 5 21 18 ~ - - - - 7 . 2 8 ± 0 . 1 1 0 7 t i ~ - - - - 8 2 8 1 4 - - - - - - 7 - 1 2 " 0 - 0 9 3

7 1 ~ - - 2 1 8 21 8 1 - - - - 6 . 7 6 ~ 0 1 2 0 - - i 14b 3 0 5 - - - - 8 - 7 8 - I = 0 - 0 9 2

1 3 { ~ - - 5 2.9 1 3 3 - - ~ - - 6 - 2 8 ± 0 - 1 0 3 - - 6 33 ii . . . . 6-10±0,082

{ ~ - - 1 2 6 1 8 5' - - - - - - 6 - 5 4 4 ~ 0 - 0 9 5 13 - - 2 31 16 i -- -- -- 6-32±0"08~

The mechanism of this ~ransition has ah'eady been discussed (I9~2@ Tw 9 hypotheses are possible (see Text-fig. 3). The 'two-ceIl-hypo~hesis' assumes two discontinaotis distributions of ceil size .(and in flexed mice of I-Ib concentration), whieE replace each other without the formation of intermediates; the 'mean cell voiume (and in flexed mice the mean I-Ib concentration) of the mixVure changing solely wi~h the proportion of tke two components (g in Text~fig..3). The Mbernative is a continuous process, with a gradual shift due to .the formation of true intermediates for ~n.ean cell volume (and in flexed mice of mea~ HD concentration), as symbolized in Tex~-fig. 3 B. The iz~clirect argument in fa'vour of the latter hypothesis (19~2~) is now strengthened by the observation tha t in- flexed mice, dining the ~ransition period, all .kinds of intermediates between. Hb-d;ficient and fully haemoglobhaized ceils are found.; i6 is impossible to sore out the cells into two distinct types. We shall see below th.a~ the situation is very different in embryonic life, where the anaemia comes in.to being by a sharp hiatus according to a 'two-cell hypothesis '.

It has already been pointed out that the gi'a&uat transition probably indicates a general physiologicM change i~ the o~:ganism, not a local difference between organs. I f all the sells of the intermediate generation originated in the liver and all the cells, of the definitive genera}ion in the bone marrow, there should be two d.isconCinuous distributions. As ~he transitioa seems Go be gradual, it probably takes place wherever haem0poiesis happens

H_~NB GgikNm~mga 259

++o be going on at tlae time. Nevertheless, in the mouse, at any rate, most of ~he cells of the intermediate generation come from the liver, and. a]l the later de.ff~fi~ive cells are produe~s of the bone marrow. The latter undoubtedly applies to the siderocytes of adult flexed mice. as their liver ha.emopoiesis dies out jhs~ as in a normal mouse.

A @ ® @ @ @ ® ® e

@ @ ®

.B @ @ e

@ @ @ e e @ ® ® e

Texbfig. 3. Expl~.nation in the J~eM,.

In tJae preceding sections the behavioar of the sideroey~es in infancy and adult life has been described, The ]?cried covered the tra, nsition from the intermediate to the defimtive generation .of red cells. We shall now consider the development of the blood of nennal and flexed mice in embryonic life. BM'erenee to Text-fig.l above shows that during the week preceding bird5., I~l~e primitive generation of red blood cells is repla, eed by the intern~ed~ate generation. We sha,lt first desm:il)e t, he transition as such; this is in effect the shape of the curve which divides t,he territories A and B in Text~fig. 1 ; we shall then tra.ce back the intermedia,ge generation to its beginning and finally describe %he phmit, ive ge~lera,t%n which precedes .b.

(e) T/te ~ra)7,st',gio)z from t/+~', p.r#)~iti.ve, to tT~e iv(,er?ned,~&te ge~erc4io~z

In Table 7 are given the averages of ~]ie embryomc counks; to save space the in.dividua] values will t~oe be given separaeely. Three gro~lps of cells are distinguished. Tile inter- mediate generation, in the 1S-day embryos, may possibly contain a few celts of l:he definitive generation. In the n.ormal embryos it also includes (f['om the t3th day onwards) ~he non.-nuc]eated cells of the primitive generation; ~n a£aemic eml)J:yos p~imitive and intermediate p]as~:id.s ( - r e d cells lacking a m~cleus) can always easily be distinguished; in normal mice this is not always possible; hence the small group of prind~ive plast, ids }~.a,s been included u.nder the intermediate phl,stids. The haemocytoblasts form a group by ~hemselves; t)hey a.re never numerous in the circulation.

As will be described below, sidero~ic material may be present in alI the different cell. forms of the prlmitffve generation, in the haemocytoblases, and, of course, in the ceils of

17-2

260 The ane~emic~ of .flexed-ta, ilec[ mice

the intermediate generation.. I t seems inadvisable ~o antumber the terminology with moi'e or less eacoph.onous na,.mes like %idero-haemocytoblast', primitive 'sidero-erythro- blast', 1)riniitive 'sideroblast', etc. In. refraining from doing so,' a precedent has been followed; amongst plgstids reticulocytes are dignified[ by g separate .came, bug ~his is not d9ne when a reticalum is i'.ound in normoblasgs or primieive cells. The prese~lce or a.bsen.ce of siderotic material in ~he earlier cell types [{as been indicated by a plas or minus sign in Table 7.

Table 7. AvsrWe so.~,,~ts of nor.rood (upyr l~df of table) and anaern.ic (~o'wer/~agf of tab~e) embryos

A plies sigma ~ndiegtss t he presence , a mimas sign t h e absence o f siderotZc m~t, erial ig ~he ceils. 4= (53)' sta~nds for ' t ou r e m b r y o s o f t~#o b t t e r s ' , e tc . The individua. l cous. ts c o m p r i s e 500 ceils e a c h in normg].s of 2[2 gr id 1 5 - 1 8 d~ys ~nd in a n a e m i a s o f 12 a~.d 18 d a y s ; 1000 cells p e r e m b r y o were c m m t e d in nor iu~ls o£ 14 d a y s a n d in an~emics o f l,g-l(~ &~ys; a~ ±,he ].3-d~y sta.ge, in f i r s n o r m a l ~tnd seven ann,emit e m b r y o s 500 ceils w e r e c o u n t e d , grid h~/~v'e a n d three e m b r y o s r e spee t l ve ly 1000 cells; t h e whole e f t h e t a b l e is b~qsed, on 78,500 cells.

]?r imit ive g e n e r a t i o n In t e rmecHgte F a ge r i e r a t i on

I I a e m o e y b o b l a s t s E r y ~hrobla,~gs NornlobIas~s P l a s t i d s r " a Dt;y o f ~ . . . . - , ~ - - " - - ~ , " " - - a ~- ' ~ S idero- N o r m o -

gests ' t imx '~ k - + - + - + - cy tes ey tes 1~ 4 (2) 0.30 0-10 39,3 4-75 45-7 3-1 1-25 0.50 3-45 1-65 13 10 (4) 0.27 0-03 0.89 0+37 35.5 3.8 - - - - 39.4 19-8 I * 10 (4) 1-0 0.32 - - - - 10.7 3.7 - - - - 55-7 28.6 15 8 (2) . . . . . 2.6 1,35 -- -- ~3-~ 5~.9 16 i i (3) . . . . 0-05.5 0-073 ~ - - 36.0 63-9 18 12 {3) . . . . . . . ~ 6-5 93 '5 12 -'t (2) 0-10 0 54"0 6-0' 32,8 2,-3 1.05 0 '35 2"95 0.50 13 10 (4) 0.83 0.-~8 2-8 0-37 ~8-8 2.0 2-6 0,51 39.8 1-66 14: I 0 (~) 0.24, 0-05 -- -- ~6-3 1.3 2.6 0.34. 67.7 1,52

15 8 (2) O.1O 0-05 - - - - 8-,i 1-6 2+7 0.75 81-7 4,7 16 12 (3) 0.07 0,008 0-73 0.2"* 2-7 0.83 91.3 ~t-1 18 10 (¢) . . . . . . 1-8 0.,56 91-6 6.0

As shown in Table 7, the primitive generation of red cells consists mainly of nucleated ceils; in the 12-day stage, many of these are primitive ery~hr0blasts, while thereafter primitive normoblasts dominate %he picture. In Text-fig. ¢ the percentage of all the nucleated cells of the primitive generatio~ i~x normsl and anaemde embryos is shown as a fraction of all the cells in the circulation. These cells are in excess of 90 % in the 12-day stage; they rapidly decline in numbers, and vel;y few are found in the I6-day stage; none have been found in 18-day embryos. I t will be seen. that at every stage, the anaemics have more primitive cells in their blood than their normal sibs. i~umericaI data are given in Table 8. The data, for the 12-da,y stage have beel~ put in parentheses, a,s the distinction between normals and anaemics at this time is uncerta,in. I t will be seen tha,g the rein,tire excess of nucle,~ted primitive cells in the anaemics (A/N) increases sgadily as time goes on.

The excess of primitive cells in the a,naemics ma,y have two reasons. They m a y be o~ly relatively, increased, because tb.ere are less cells of the iutermedi~te geng.,:ation; in that case thai1- number per unit volume of blood would be normal or might even be decreased. Or they are absolntely increased in-numbers, in which case there weald be more per nnit volume of blood.

To give a, compIete answer, the number of re([ cells per on.ram, in normals and ann,emits weal(t be required. No such data, are ava~table for the 12- and IS-day stage. Xa,meno-ff's {.1935} counts show that on the 14th and Ihth days She anaemics ha,ve a, little more l,han hag ~he normal count; therea,fter the cell count rises to about three-quarters of the

261

normal values. }Iense, if we ~ssume that the de.fi.deney of rscL cells is eo~.fined ~o the in~rmsdia, te. gene.rabion (~s seems likely), ~he excess of primitive cells in the and.stoics t~p ~o ~he. 14t,h day may be s.ccoun~ecl for on ~ ~elative basis alone. Thereafter i~his is no~ possible an), longer; mos~ of the excess mns~ be due ~o absolate.ly greater numbers; thus in the. ana.emics the primitive culls sat%re longer in {he drculation; as the. anaemics are, a~ ~ha~ time., desperalely shor~ of haemoglobin, ' it is un~[srstandable t,haf they should re~ain theD ihlly haemoglobinized primitive cells as long a.s possible.

I ,

12 13 14 15 16 18 Birth

Text,~fig. ~. Pere.sn~age of nuvJ.e~f, sd prhnitiv~ ceils in nm'me.l embryos (bh ck ,~oli~ line) emd in ana.emie eJnbryos (t, hin solid liuo). Tim broke~ ]ins sbov,'s i~he percentage of ~t.] primit:ive ~eils of a.n~.~m.nics, fnehding the ptas~ids.

Table 8. Percen.to:ye o.~ wu.d, ecctec[, prf~t~i/.'~be cdl,~

Da.y ~o;'ntu,ls ( I N ) Anaemic:s (A) ! \ /~ 1: (92.8) (95.1) (1-02) ii 3 40"6 54"0 ).'33 1=~ J 4-4. 27-6 1.92 18 3-95 10-0 ~-53 I6 0.128 0.97 7.58

To sum up. The replacement of t~he primff, ivc cells by cell,~ of the ln~ermediate gene.ra.tion, ~-~s seen in diffe.re~t,ial counts, i,s de.la.ycd. The excess of pi'hnith,e cells is probably paa.'t]y dne to a rek~:tive deficiency o:[ in.termediate ceils; in ta,ter stages ~ considerabh part of t,he s:ff'ccI; mus~ be ~sc:tibe.d to ~ Ionge.r st~a'vival of primitive ce]l,~ in the. c.h:oalai;ior, of the s.n ~emics.

The broken line. in Wex~ogg. 4 gives the. values for the sngi:,.e, primit~w genera,~ion of anaem].cs inek~ding primitive plastJds. These cells canno~ ahva,ys be distinguishe.d from in~,srmedia,~e pla.sticls in nee'real mice, thoug]., this is possible for mos~ or ~hcm. Whey are sttfficiently dist,inct t:o justify t,he sta.t,emel~.~ that their proportion in. aortas.Is doe.s not, differ grea.tly from that in anaemics.

262 The anaemia of .fle:~e&tcd~a~ ~ice

(b) The a~rl// his~o'ry of the inter.mediate generation As shown by the data of Table 7, the first cells of the in~ermedi~te generatio~ make

th.eir appearance in the eil-culation in I2-day embryos (early 13th day); none have been. observed in t 1-day embryos. ~fany of t.hese eacli.est cells are tiny microoytes (PL 8, fig. 16) ~dth diameters of ¢ or 5/,; against the background of the very large primitive cells these microcyCes look extremely dwarllsh. The p.rodt~etion o f il~termediate cells rapidly gets iz~to its stride, and in normM Y3-day embryos they abeady constitute more than one-half o fall the cells (though less than a (lugrter by votun~e). The microcytes, which were common amongs~ the earliest i:atermediate cells, are ra, re from now onwards, ~z~d the mean diameter for the rest of the embryo!~ic life remMns practically co~stant in the neighbgarhood of 8/,. So far as I can see, at1 ~,he-intermediate cells in the circulation are plastids. No intermediate normoblasts have been observed in the circulating blood of embryos. As pointed out :previously (1942a), normoblas~,s are sometimes found in newbomt mice,

A e r o s ~# . . . . . . . x No,'mals na ~ e

6 0 - ,,

40 : " , , p a \\x\

20 ~ \

]2 13 J4 15 16 18 Birtth I 2 3 4 5 Adult

days weeks Text-fig. 5. Percentage o£ siderooytes amongst intermediate (and later ok amongst definitive) plastids.

Thesolid hae refers to a~aemics, the broken line to theiz norms-1 sibs.

occasionally up to 1 °/o of all ceils; the release of such showers into the circulation is possibly due to the. shook of parturition and the sudden change of the respiratory conditions. However, as intermediate normoblasts are absent from the embryonic oircnlation, that generation consists of two cell types only, normocytes and Mderocytes. Their relative frequency in normal and anaemic embryos in shown in Text-fg. 5, which is baaed on 6he numerical data of Tables 2 and 7.

In the anaemics th e percentage of siderocytes, from the 13th to the 18th day of gestation, is practically constant in the neighbouxhood of 95 °/o.. In their appearance (P]. 8, ~.gs. 17oo20) they differ in no way 5_'ore the siderocytes found at birth, The few intermediate ceils without siderotic material are just as Hb-deficicnt as the sideroeytes. It is uncertain whe*,hm: they are gm_minely free of siderotic material, or whebher it is ~?reserd; in a, form which c a ~ o t be :cecogniz'ed with the optic.a1 equipmenl> available. On account of the >aleness of eounterstain, all the intermediate cells of the anaemics are dearly dis-

1 0 0 - -

KA~S G~/)~E~ER~ 263

tinguishable from the cells of the primitive generation, which are fully haemoglobinized (PI. 8, figs. 6-10). In the ea.r]iest stage examined the distinction between the intermediate cells of normals and a.naemies becomes indistinct, as the ea.rliest cells of normals are similarly ~b-defieient as those of anaemic embryos (Pl. 8, fig. 1 i). It is uncertain whether the apparent drop of siderooyte ]percentage in I2-day embryos is a genuine phenomenon. I t might conceivably be due to the erroneous inclusion of one or more,normal embryos under the anaemics. An alternative explanation fs that t}le very small siderocy~es, on account of their tiny size, might more often escape the deposition of siderotic ma~eriM.

As already mentioned, the earlies~ members of the intermediag? generation of normal embryos are difficult to distinguish from those of anaemic embryos. They are just as tCfb-deficient, and many of them are siderocy~es with a comparable amount of siderotic material (Ph 8, fig. 11). I t may be said that every normal mouse embryo (and perhaps every normal mammalian embryo) passes through a. phase of hypochromic anaemia on first producing intermediate cells. I t looks as if the liver, in its first attempts as a h.aemopoietie organ, is not yet quite up to its task. However, in normal embryos it quieldy learns how to produce normal cells. Already in l$-day embryos, many of the cells are more or less completely haemoglobinized and contain much less siderotic material t?aan the siderocytes of anaemic embryos (like the cells shown in P1. 8, figs. 12-15); the siderocyte percentage is about 66 ~/o, and from now onwards there is never the slightest doubt as to whether a given embryo is normal or anaemic. From the l¢-day stage onwards, the sklerocyte percentage decreases steeply, and nearly all the cells are fully haemo- globinized; most of the siderocytes of normal embryos contain only*small amounts of siderotic material, usually a single grannie or two; only occasional pale cells with more siderotic pigment survive in the circulation, t~y the time of birth, the siderocytes of normal embryos have becmne scarce and inconspicuous; though their number is com- parable to ~hat ~bund in adult flexed mice, they are incomparably more dii~.cult to discover, and their enumeration is a tedious task even for an experienced observer (compare PI. 8, figs. 1,3 15 with figs. 2].. ,25).

The situation may be summed up as follows. On first producing cells of the intermediaries generation, normal mouse enabryos make red cells which are as Hbodeficient, and often as full of sidero~ic material as those of flexed-tailed adiaemics. ]But, ~,hile the normal embryo rapidly proceeds to 'tihe prodncti.on of fully haemoglobinized cells with little or no siderotie material, the ana.e:mic embryo eontb.m.es to produce the deficient cell type throughout the whole of the ~ngermedi.ate geJleration. Only on passing to the defi.t~itive generation does the flexed mouse acquire the ability 1~o make approxim~tely, thougl.~ not quRe, normal red ceils.

(c) 2r/~c pris~.igive ye~eration The primitive generation of red. cells in ~he earlier stages consists mainly of nucleated

cells; as t~ime goes or, the plastids (P1. 8, figs. 5, 10) bdcome relatively more aI~d more numerous. This is shown in Table 9 for the anaenaics, where this cell type can easily be distinguished from iatermediate pls,stJds even iia the few cases where the respecNve size d~str:ibut.ions overlap. The value for th.c ~ 12.-day stage has been put in parentheses on a, ccoant of tile uncertain.l,y of classification; the corresl)ondJJ~g vMue for supposed normals is ] '85 °i~ , the difference bei.agiusignifica.nt. The relative increase of plusSide may be due to one of two causes, or a combina.tim~ thereof; She pIastid.s may be replenished by

264 The anaemia, of flexed-tail4d ,mice

nucleated cells losing Sheir nuclei; or they ma.y survive longer. The :former process is clearly important; the peroentage of primitive plastids (in terms of all red cells) changes lit~Ie from the 13- to the 16-day s~ag¢, e-ks bo~h the number of red cells per unit voh, me of blood and the total blood volume increase considerably during this period (each of them probably at least .by a factck' tlhree), ~he total number of primitive plastids in the circulation must increase at least, tenfold. Differential survival may be an additional factor.

In the earliest s~ageg examined, many of the nucleated red ceils are primitive erythro- blasts (lq. 8, figs. i. 6 and 7). Such cells have large nuclei wi~h a relatively fine chro,natin structure a.nd sometimes one or more nucleoli. In the 1t- and 12-day embryos they divide mitotically in the blood streaml Nitoses are :rarer i~ ~he 13-day stage, and have practically disappeared in the l~-day stage. Beautifol metaphase p_lates for demon- stration purposes can be obtained with the ordinary Leishman stain; still better pre- paratio:ns are obtained by means of the geulgen method; such preparations, for which 2[ am obliged to Nr L. F. La C!our, are shown in P]. 9. The metaphases are perl'eetly regular with most of the forey chromosomes hra oh-de round the spindle (P1.9, figs. i-3). Dr £!. D. Darli~.gton kindly infbrms me that 'anaphases are tikew'ise .regular, but an important abnormality oecurs in a small proportion of them after. I2, days. One or two

Table 9. Percentage of primitive 21aerials amongst prbmitive 8egfs in ar, aemic e.mbryos Day of gesba~ion ~Percen*aage of plastids

]2 C.4~) 13 5.4 14 9.4 15 - 25,7 16 78"4 18 100

of the chromosomes fm'm .bridges which arise from union of the ends of sister chromatids (Pi. 9, figs. -¢, 5). This sister union is characteristic of chromosome division under a. variety of unfavourable conditions. Such" conditions might well arise in the last mitoses of a series which these certainly, are. Binuelea~e cells are four~d at stages when no more mitoses occur, equally amongs~ the primitive erythroblasts and the primitive normobla.sts. They are probably a consequence of the bridge formation.' -~

The primitive erythroblasts a~e transformed into primitive normoblasts (P1. 8, figs. 3, ~, 8 and 9) by a gradual process, by which.the nucleus becomes much smaller and pyknotie. In Leishman prepara~ion.s th4 -normoblast nucleus is stained dark blue. and shows tittle struetuge; in preparations stained for it'on and connterstained with Biebrich scarlet, the nucleus appears often 12he a hole punche d out of an intensely red cell, without any structure wha%oever. The priimtive normoblasts do not divide any more; they are not rarely bimmleate.

As the transforma~ion from pfimiti.ve erythroblas~s into primitive normoblasts is a gradual and eontintious ]process, they eanno~ be clearly separated into two distinct groups (see P]. 8, fig. 2). The classification of intermediates is always more or less arbitrary; hence the differences in the relative number of erythroblasts and.normoblasts between normals and anaemics in Table 7 should not be taken too seriously. While the transforma- tior~is gradual, i~ is no~e the l.ess very rapid. Two litters of the 12-day stage were examined.; in one of them the large majority of the ceils were primitive ery~hroblasts, while in the o{her tit~er most of them were ah'eady pl'imitive normoblasts. These two litters cannot

l~I~s G u i t x E ~ u e 265

hays ctiffered in age by more than 17 hr. Allowing m~ top of this a difference in developmental age, it is still trne tha t most of the transformation happens within a ~i hr. l?eriod.

As already mentioned, all members of the primitive genera.lion, both in normal and Jn anaemic miss, are fully haemoglobinized; ~his is seen by their yellowish colour in unstained films and by the intensity of the connterstain in stained preparations. As a consequence t~rsparations of ~naemio blood from the 13th clay onwards presen~ a very s~riking picture; they show a mixture of very large in~ensely stained primitive cells, most of thena nucleated, and small pate plastids of the intermediate generation; the distinction beSween the two generations by solour alone is perfectly sharp, withou~ any intermediates. In normal embryos ttfis is not the ease, as most of the cells of the inter~ mediate generation are as fully haemoglobinized as those of the primitive and of the definitive generation.

All kinds of primitive celts, erythrobl~sts, normoblasts, and plastids, may contain sidsrotie ma~eriaI (P1. 8, figs. 1-10). But on the intensely red bee)ground, many of them, particularly the smaller ones, appear less obviously blue; often they look darkish, almos~ black in_ colour. In erythroMasts the.y have a tendency to lie close to the nnelear re.era- brans; as a consequence ~hey sometimes appear to lie inside the nucleus, whereas in fact they are situated on top of it, where they a.re often diNeul~ to see (P1. 8, fig. 6). Another difficulty is added by imperfections in the films, which t have been unable to avoid; as the blood of young embryos is poor in ceils, films cannot be dried as rapidly as those of older animals ; thus in some preparations many of the nucleated cells have time to shrink on drying and develop irregularities on their surface, which appear black in preparations; it is sometimes diN.cult to be sure whether a small dark spot is such an irregularity or a siderotie granule. All these facts together make the enumeration of sial.erotic cells amongst the nucleated primitive Belie considerabIy less accurate than in the plastids. I t is thus quite possible that the figm'es of siderotic blasts may require som.e modification when rei,).~'esgigated with an improved technique, tn view of these limitations, the (!a, ta on primitive bl.asts e~t.,, only be regarded as senl.i-qua.nght, a.tive, and minor differences may well t~rn out to be spurious.

Wil,h these reservations iu mind, we may now exa.mine Text fig. 6, which gives the percentage of sffclcro~ic Bells amongst the pr.i.rnJtive ])lasts o:f normal and anaemic embryos, and amongst the primitive plas~ids of anae~Mr:s. The fo[Iowing eoaehsio~Js sse~a justified:

(1) Siderotie ceils occur connnouly in a, ll primitive cell types of both norms,1 and ana.emie embryos.

(2) The di:fference between ~he ~)lasts of .norms.is and a,naemies, if not spurious, is very mush less marked than tha, g :found in the intermediate gellerat~oll; the comparati.vely hrge difference on. the 16th day is unretia,ble, as the value for the normals is based on very few ceils.

(3) The apparen.t decline of siderotie blasts in both normals and a,naemics wi~h time may be spurious; as elms ,goes on the nnmbe:r o:f cells .per unit volume of blood increases and with. it th.e quality o:[" the :films improves;" the difference between normals and abnormMs might conceivably be due to glhs fact that-the latter always ha,ve lower counts and hence may show a larger ])roportion of artefacts. This interpretation is snggeseed by tits fact tha t the siderocytes amoxtgst pla.s~ids, which sea.rcely ever show artefacts, remain fairly steady ~hroughout.

266 TAe a.,~ae~nio~ o f f l e x e & t a i [ e d ,mi~e

The attitude ~a.ken in the ease of the pr!mitive blasts may on reinves~igation turJl out to-have been owr cautious, h i sm'veying a completely new fieli~ it has seemed advisable to err on the safe side. However, whatever the siderooy6e eomIt of the primitive cells will eventually- turn out to be, it is certain that the primitive generation of the anacmics is not Hb-deficient. Therefore, unless t]iese cells are deficient in nmnbers (and there is nothing to st~ggest this), the flexgd embryos up to the 12th day are not anaemic. Noreover, the continued presence of some fully haemogIobinized cells retards the development of the anaemia. The primitive cells have a mean diameter of 12~L or over, those of the intermediate generation of about 8 b~. Allowing for the Hb-free nuclei' of the primRive blasts, the mean effective volume uf the primitive cells is about thrice t]~at o:f their

o

IO0,

8o!

60

% %

\ \

% \

% \

\ \

\

40-

20

. . . . . . I 1 I I ........ 012 13 14 15 ]6 18

Days of emb.ryonic life

Text-fig. 6. Percentage. of siderotie cells amongst th~ nucleated primitive cells of anamnics (solid line) aug of normals (b~'ok~n lh~e). The dob~ecl line shows the perceut~ge of siderotic cells amongst the primitive pl~stids of ~naemics.

s~rceessors. If we assume that. the Hb concentration of the intermediate cells is two-thirds normal (as found at bbth), and th.at of the primitive cells unity, we can calculate the • mea,n r/orpuscular Hb concentration for the various stages of embryonic life. The result is given grapkically in Text-fig. 7. I t wilt be seen that the flexed, embryos are not serigusly anaemic before the 1,ith day, at least as judged by the mean I-~ concentration of their cells; however, the picture is differentwhen account is taken of theb seduced cell co~.mt. The data at hand do not permit a detailed[ analysis, I t is degr, however, from ][[ame~loff's Counts in conjunction with Text-fig. 7 tha~..at the 14-day stage the Hb va.Iue will be about 45 ~/o of the e.ormal~ and-that a similarly low value is maintained, u.p to the time of birth. I t seems probable tha t no detectable anaemia ( = i~b deficiency per trait yell{me

267

of blood) exists at the I2 -day stage, and only ~ eomj?ara{ively mild one (about 7 0 %

~ormal) at the i3 -day sbage.

( d) I~io.e,moo~/toblasta

The ' s t em-ce l l ' of Maximow is ca.tled the ha, emocytoblast . I n Leishman preparat ions ig is a ceil wRh a large nucleus and a nar row rim of deeply blue (basophilic) cytoplasm, closely resembling a lymphocyte . I t is never haemoglobinized. In: the embryonic stages examined, haemoeyteb las t s are never numerous in.the eh'cn]ation, being virtua,lty absent

f rom the d r c u l a i o n of some embryos.

I00

o

g

8

o

9O

N\ _ k

\k

- - N''~N N.N. \ 80

70-

60--

5O 2

t L__ 1 1 _1 13 14 t5 J6 18 Birth

Days of gests,~ion Tcxbffg. 7. Calenlated mean e.orpt~si:uhr t{[b cone,~nt.r~tion of the cells of flexed m/~bryos, ~ssuming that the

mean volume or the fuUy llaemoglobinized primbtivc cells is tl,rJee that of the internmdiaLe cells (solid lJjlc). The values of t, he br~Jken Ji~:e would be 1bend if the volume of primi~iw and intermedia,te cells were the same.

There seem to be no consistent differences in the percentage of ha.eraocyl;ob]as~s betweerl normals and ans,emics; possJ.bJ.y l;lm haemoey~oblasts sin:rive' a lit:l~te longer in the

circnla,tion of aria.emits than in normals. ARhough haen~.ocy~oblas~s are z~ever hamnoglobiniz, ed, they m g y contain siderotic

materiM. IVs a,moun.$ is, however, always very small; a :few small gra,ml.ies are often .gouud; t h e y arc ns'ually close to bhe nuclear membrane and often appear to lie on top of or n:nderneatl, the nucleus. On t]_ds account the enumerat ion of haemoc}4obla,sts con~ raining or Is,citing s[derotic gra.u u.Ies ~s very dif]~.eulL Thus, while l.{tt]e weight can be given ~,o their percentage, the occurrence o:[ sidero-llaem.ocytoblasts ' is beyon.d doubt.

268 The c~nae.~nia, qf flezed-ta.iled ~ice

IL~IscussIoN

As mos~ of the discussion has ah'ead Z beeu givea i~ the preceding secdo~:s, only a I:'e~ pgh~ts remain to be mentioned here.

The granules found in. siderocytes have nothing to do with 6he brown granular pi.gmen~ known as haemosiderin. That pigment is fotmd 9~. the form of yellowish or brownish grannies which are visible as such in unstained material. The :~i'ee' iron of siderocytes is not recognizable in unstained blood films and only made v~slble by means of the ]Prussian- bhze reaction. Haemosiderin is usually a product of haemoglobin breakdown. I~ is found, for instance, in old haemorrhages and in sites of chrSriic congestion (e.g. ;heart failure cells' in the sputum); in man it occurs commonly in small ctuant.il;ies in organs in which a physiological breakdown of blood takes place, such as spleen, bone marrow, liver, lymph follicles, etc. Many cell types may eontaiu haem.osiderin pig:men% but particularly the pb.agoc~Res of the redculo-e~x].o~]lelial system; t]~ese either phagocytize red blood corpuscles and form. eke pigment inh'acellularly by breaking down the haem6- globin, or they take up the pigment or a precursor from the blood stream, noti~bly in diseases w-Rh an increased blood destruction in ~he circulation. IIowever, haemos:ideriu has never been observed, inside intact erythrocytes. I:[aemosidedn in large cluantRles is also deposited in certain condRions, such as general haemochromatosis (v. I~eekling- hausen's disease, 'bronze diabetes'), in which there is probably no increased blood destrnetion.

Whereas haemosiderin in most cases is a product of haemoglobik breakdown, this is probably not ~he case with ~he ~free' ben in sideroeytes. We have seen above that Nlly ftedged siderocytes appearwhen the tirst cells of the intermediate generation enter the cirmtlation, i f the siderotie material were the product of s hitherto nnknown type of fib breakdo£vn, which ~akes place inside intact red blood corpuscles,, one should expect the faust ceils to be free of si¢lerotie material, and that it would increase in quantity as the cells grow otder; this is certainly not the case.

We ~hus assume ~hat the siderotic material is anabolie h-on, iron which has not been used for the I-fib synthesis.. This assumption is in agreement with the loealizati0~ of the siderodc material in primitive ery~hroblasts. Here it is often found close to the nuclear membrane, t t is virtually certain ths~ the nucleus, plays an important part in the I'fib synthesis; hence it is plausible tha~ unused buitdi~g materials shoNd be left behind at the working bench, ~hat is close Co the nucleus.

The anabolic nature of the iron is also in agreement with another fact. There is a clear negative correlation between the I:[b content of a cell and the amount of :free ~ iro.~ it contains. Such a correlation would be expected whether the iron is anabolio or catabolic. But if the siderotie material were a product of I{b breakdown, %he negative correlation should be virtually complete. A cell whieli has lost much of Rs I-It by breakdowla should always contain much 'free' iron, and a cell which is still Nl ly 5aemogtobi~zed should contain litf, le or none at all, This is, however, ~ot the case. Although most of the I-It deficient intermediate cells of anaemics are rich in ~free' h'on, ~h.ere are always some which do not contai~ any recognizable quantity. On the other h~nd, though most of the %I]y haem.oglobinized primitive cells seem to contain less siderotic material pox unit volume than the l ib deficient ceils of the intermediate generation, a few are almost stnffed to capacity ~vi~h large granules. These exceptional cases are hard to reconcile

HANS GI¢~NE:B ElCG 269

with a catabolic origin of tLe siderotio material. They are, however, understandable if ~he 'free' iron is anabolic. The %lly haemoglobini~ed primitive ceils may evidently take up more iron th~.n they can deal with; and. some Hb deficient intermediste ceils evidentIy take up only the limited .quantity of iron which they ca~ atilize for the Hb synthesis, bu~ no more, and. hence fail to show sidero~ic material.

I t seems premature at this stage to speculate about the chemical nature of tl~e 'free' iron i~ sideroeytes. One conclusion, however, seems justified. I t is unlikely chat it is identical with the 'easily detachable iron' in red blood ceils stnclied by Lemberg & Legge (see the preliminary report, 19,t-t b). That compound[ is found in the blood of adult organisms, which does not contain any siderooytes; it also seems that the Jr'on is much less easily split off ohan that found in siderocytes.

This paper has adduced much evidence for the existence of an intermediate genera¢ion of red cells as a separate entity. I t should be mentioned chat ohis conclusion is in agreement ~dth the demonstration, by Gofer (1938), of antigenic differences between the red cells of newborn and adult mice.

SUMMAI~Y

1. Siderocytes are erythroeyfles which contain easily detachable iron, as demonstrated by g~.e Prussian blue reaction: in addition to haemoglobin. Both in normal and in flexed- tailed mice there exists a negative correlation between the Hb and ' free' iron content of the cells. The siderotic material in siderocytes probably represents iron no~ utilized for the Hb synthesis.

2. The primitive generation of red blood cells is fully haemoglobinized both in normal and in flexed-tailed embryos. All primitive cells may contain sideroflie material; tha~ in flexed embryos is probably somewhat more abundant than in normal embryos.

3, Flexed embryos replace the ]?rimitive red cell generation more slowly by intermediate cells than normal embryos.

(L The first inter:mediate red cells of normal embryos are Hb deficient and contain a.ppr.ecia,ble q~la.ntitJes of sidero~ic material. Following this initial phase of ineflieient 5aemopoiesis, the liver rapidly tur.n.s out fully haemoglobiaized coils with little or no siderotic a?Ja, gerial; the Is.st sJderocytes dis~ppear fl:~,m the circulation of norm~i mice aboul; one week a.fger birth.

5. Flexed-ta.iled enabQ<,s produce lib-deficient oel]s with a, bundat~t siderotJe material throughou.t the who].e of the inte:mnediate generation of red. cells.

6. During [be £rst 3 weeks after birth, the siderooyte percentage of flexed-tailed nrice declines ra(pldly to a small vakm (about 3 ~/), which is ma.itl~ained thro~ghout life.

7. The decline of siderocyte percentage happens pari passu with a drop 5~ cell size from a diamege:r of about 8/_~ to about G/z, and with the at, tainment of a normal mean corfuseu]a:r ]:{b conceo.braOion.

8. The facts men~ioned under (7), ~ogQdJ.er wilh the demonsO.ra~ion thaO the decline in celt size is not due to a decl:h:~e in reeiculocytesis, lead to the conclusion that there e=ises a sepa:ra~e Jnternaediate genera~iou of red. ceils. This generation is labelled by the flexed-.J,a.i] gone. It consJ.s~s of large, Kb-defieient cells, most of which arc siderocybcs; the de.[initive generation sensu stricto co~sisJ;s o:[ small celis, most of w.ldch m:e %fly haeuloglobinized, and few of which are siderocytes.

9. Direst observations suppo% %direet~ evidence prodace~:[ earlier tha,t ti~e ~;:cansif, iof

2 7 0 ~ h e csnaemia, o f f l exed- ta , i l ed m i c e

from the in~ermedi~te to the definitive generation of re([ cells is a grachtal and: cm~tinumts

process. 10. The total number of siderocytes in the circulation o f flexed mice hmrtases con~

stderably during the first week aRer bir th; this co~ffirms aft earlier .finding ~hat the ~[extd mouse during this interval produces Hb-dtficien:e tells. Following ehis rise the total mm~ber of sidtrocy~es decreases arid tea:thee a min imum at the age of 3 weeks; this is followed by ano~hel' rise due to growth alone; adul t (lextd-tailed mice have 1~-5 times as m a a y siderotytes in tkeir circulation as 3-weeks otd animals. I t follows ~,hat the sida:o~ cy t t s of adult flexed mice are not suryivors f rom infancy; t hey are apparen t ly 1)ro&~ced in the boa t marrow, as the livtr haemopoiesis of flexed mice dies out as in normal mice.

11. Tl~e flexed-tail gent has a mild effect on the definitive and possibly on the primitive generat ion of red cells. I t main effect is on I;he intermediate generalhion. The pathological feature is tha¢ the initial ineffieiene haemopoiesis, which forms a, shore gransRory stage in normal embryos, is mainta ined three, ghent the whole of tha t generation.

REFERENCES :DE AB;~I~LE, S. B. (1927). A study of the heredRary anaemia of mice. Arner. J. Anat. 40, 21949. B~oo~r, W. (1938). Embryogenesis of m~mma.[ian blood, Dow,~sy's Handbool~ qf Hematology, 2, 865-922.

Londe~: t{~mish K~milton. B~w~s, ~. D., w ~ D~ E~w, & & C~a~Lso~, A. J. (tgas). The blood picture of ~ats from birth to

24 d~ys of age. Amer.J. Phgsid. 12z*, 620-6. Do~>o=, I., Gzd)~.~x¢, H. & P~a~sb> ~, J. E. g. (194La). Siderooytes ha adtdt human pa[ients. J, Path.,

Bact. (in the Press). GOB]]B, P. A. (1938). The aatigenie basis of turnout [ransphntation. J. Perth. 2~ct. 47, 23I--52. C4~B~Re, 'K . (1939). Inherited mgeroey~io anaemias in the house mouse. Genetics, 24, 777-810. C4R~lv~c.,, It. (19~15). The growth of the blood of the suckhag mouse. J. Pc~t]~. ~act. 52, 3:23-9.. O~t~n~.~o, K. (194tb). Siderozytes: a new kind of er2~hroo2tes. Natz*re, Loz~d.,' iz~8, ! l& (}gi~>m~G, K. (1941 c). 'Sidez'oeytes' iu man? Lancet, 241, p. 172. Gz~g-~y~ae, El. (t~J41d). 8ideroeytes fl~ man. z¥atu.re, Zond., I48, 469. C ~ z ~ ' ~ c ~ , ]g. (t9~25). The anaemia of ttexed-tailed mice (3flus ~r~uamdus L.}. I. Static and dymamie

h~ematology. J . Gruel 4,3, :~5268. G~i)xI~]~aa, ~. (1942b). 5~he Genetics of t/~e 2~ouse. Pp. xii+412...CambrNge University Press (in ~he

Press). Is3_~cs, g.. (1938). The eryghroeyges. D0wney:s HrO~&ook of t{ematology, t , 1-t59. t ~ m ¢ o ~ , ~. J: (1935}. Eftcogs of the flexed-±.afled gone on the deyelopme~b of the house mouse.

~2 2/[orph. 58, 117-55. 5L~13mw, A. (1927). Bindegewebe m~d. blu~bfldende gewebe, v. M/5/lendorff's Ha~dbuch get mikro-

skopisdzen Anatomic des 2ffenschen, 2, pt: 1, pp~ 232~583. OaKn~Y, C. L[ & W~d.~z~ac~, G. K. (19~t01. The blood vohme of the mouse, d. Pat£. Beet. 50, 372-7. P~I~soNs, E. L. (1929). Studies on red cell diameter, t i i . The relative diameter of immatt~re. {retieMo-

eytes) and adult red blood cells .in hea~l.th and ~nemia, espeeiaIly i,~ pernicious anemia. J. Clin. invest. 7, 615-29.

PE~'~T, 8. (1933). Morphologie ~md Zahl der Blutkhrperzhen bd 7-ca. 30 g. schweren norm~len weissen Laboratoriumsmgusen. Acts Path. MicrobioL 5can3. ~0,' 159-238.

S~r~:~/, C.m (t932). The post.embrymde devdopment of the erytl~oeytes of the a.lbbao ra.L J . Path. Bart. 35, 717-26J

Wz~a'io~, 3{. '.3{. (1934). ~ela~ion of varia~i.ons in mean eorpuseula, r vohme to number of re~ieMoeytes in pernicious a~mufia. J. Clin. 2great. 13, 669-76.

}gn4~go]3~, M. M. & S~u.z~,~o~J~.a, K. ]3. jr. (1935). Comparison; of hematoi)oiesis in the fetus and during recovery from pernicious anemia, ~ogegher with a consideration of the rglationship of fegM hema~o- poiesis tO maerooygie anemia of pregnancy and anemia in infants..Jr. C~iri./nvest. t 6 , 837-52.

WINT]~o~, M. ):[. & ,~'93~AC~N2., }f. _13. jr. (1936). Nryt]lrbeybe s~udfes in the inaluma.liau ferns arid ne{vborn. Amer. J, A,nat: 58, 313-28.

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PLAT]~ 8

Sideroeytes of normal and flexed-tailed i~ice. Prussian blue reaction, counteretained with ]3iebrich scarlet. Nagnification 2000 x .

Fig. 1. Primitive erythroblast with unusually heavy sidez'otie material; NormM t3-days otd embryo. Fig. 2. A. cell intermediate between primiSve erythroblasfl and primitive narmoblast, containing three small

siderotic granules. Same embryo. Figs. 3, 4L Two primitive normoblasts of a normal I4-days old embryo. Fig. 5. Pmmitive plastid from the same normal embryo. Figs. 6, 7, Two primitive erytbrab]asts fl'om a flexed.-tafled litter mate of the normal ]3-days old embryo

above. In the cell sho~m in Fig. 6 some of the sideretic granules are ]?dng on top of the nnelens. Figs. 8, 9- Two primitive normoblasts of a ]'4-d@ old flexed-gaffed' embryo (]55ter mate of the embryo figs, 3 5

above), Fig. i0. Primitive plas~id of the same animal, congahaing exceptionally heavy siderotic material. Figs. 11-15. Intermediate plas~ids £rom the same normal embryo as in figs, 2-5 above. Pale plastids relatively

rich in sideretic material like the cell shown ih fig. 11 dcmin'~te amongst ~he intermediate plas~ids of_ normal embryos of 13 days, but are already scanty in 14-da.ys old normal embryos, i~'rost siderocytes a~ 1~ days are fulIy haeraoglobinized and contain only a few siderotic gram~les. A~ birbh the siderocytes of normal mice are usua.ll 2 of the type shown in figs. 14 and I5.

Figs. 16-20. Intermediate pla.stids of the same flexed-tailedembryo as in figs. 8- t0 above. M.ierocytes like bhe cell shown in fig. 16 are common both in normal and anaemic embryos of i3 days, but are ah'eady rare i~ 14-days old embryos. The intemledia~e plas~ids of flexed-tailed mice up %o the t ime of birth are nearly all like those shown in figs. 17-20, except for a small percentage of cells which are pale, but lack siderotie granules.

Figs. 21-25. DefinStive plastids (sideroeyges) from a flexed-gaffed female, 335 days old. The cell shown in fig. 23 is flmompleteiy haemoglobinized; ceils ~early as pale as the intermediate sideroeytes occur: bu t are rare.

PL_~kTE 9

Chromosomes of dividing primitive erythroblasts of normal mouse embryos. Dried. smears hycbolysed in N ~C1 at '60 ° C'. for '~ rain., stained in ]eueo-basic fuchsin for 1 lu'., pressed lightly under the cover-slip in 45 % acetic acid, and mounted in Eupara L Magnification 1800 x. Preparations and photographs by ~£r L, F. La Cour.

:Figs, 1-3. ~e taphase plates. Figs. 4, 5. Fig. 4, early anaphase; fig. 5, la~e anaphase, two bridges in each.