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B lood C ell Fo rm ation in C er ta in T eleost F ishes

B y W I. T .

C A T T O N , M .S C .

T

H E A D V A N C E S in our k no w ledg e of b loo d-ce ll fo rm a tion in the low er

ve rteb ra te s have been due large ly to D o w n ey ,6 and to Jo rdan and h is a sso -

c ia tes .’ {1 76 }’2 A co m preh ensive su rvey o f the “ lym pho id” tissu e in the k id neys o f

m an y fishes w as m ade by D rzew in a ,7 ’

8 and a m ore de ta iled s tu dy w as reported

by D ow ney6 on the h em atop o ietic rena l tis su e of the sp oonb ill s tu rgeon , Pol yodon

spa thu la . O n the basis o f h isto log ic exam in a tio n of the k idneys o f cer tain fish es ,

J

ordan and S pe ide l” p resen ted a hyp o thesis a s to the na tu re o f the stem ce lls an d

the p rocess o f b lo od-cell deve lop m en t. T h ey rega rded the free stem ce ll in fishes

a s b ein g sim ila r to , and on m o rph o log ic g ro und s a t lea st, a s ind istin gu ishab le

from a lym p hocy te. T h is v iew seem s in som e d eg ree to co n trad ic t th e p resen t

w o rk ing hyp o theses o f m am m alian hem a top o ie sis . O f these , the m ost w ide ly

accep ted a re the m on op hy le tic v iew , a s p ropo un ded b y M axim ow ’5 and o the rs ,

and the m od if ied po lyph y le tic o r d ua lis t v iew b ased on the w o rk o f D oan , G u n-

n in gham and S ab in .4 In the m on ophy le tic v iew there is a com m on s tem cell, the

“hem ocy tob last,” w hich d iffe rs in bo th s ize and struc tu re f rom the lym p hocy te .

In the schem e of D oan , C u nn ingh am and S ab in , the re a re tw o ind ep enden t lines

o f deve lo pm ent, th e paren t ce ll o f the ery th rocy tes ( te rm ed by th em the ‘m ega lo -

b las t” ) a ris ing as a d au gh te r ce ll o f the d iv is io n of an en do the lial ce ll o f the w all

o f a m arrow s inus, and the pa ren t ce ll o f the leuk ocy te se r ies (“p rim itiv e w hite

ce ll” ) a ris in g ex travascu la r ly from a reticu lu m cell. I t is ev iden tly n o t poss ib le

to corre la te e ith er o f these v iew s w ith the accoun ts o f Jo rdan and Sp e id el in

referen ce to fishes .

A lth ough there a re no a prio ri g roun ds fo r deny in g tha t the b lood stem ce lls

m ay be of a d iffe ren t n atu re in fishes an d m am m als, rep resen ting ex trem es o f the

verteb ra te scale , ye t the com m o nly accep ted v iew tha t th e verteb ra tes fo rm a

co m pact phy log en e tic g rou p appears to ren der su ch a co ncep tio n open to con tro -

v ersy . It w as th e a im of the presen t w o rk to re -exam ine th e qu estion of the na tu re

o f the stem ce lls in te leos t fishes in th e ligh t o f the ab ove co nsidera tions . S in ce

n o prev ious accou n t o f the ce lls o f the c ircu la ting b lo od in th e fishes s tu d ied

( roach* and tro u t) co u ld b e fo und in the lite ra tu re , a p re lim inary su rvey of the

b lo od of these fishes w as m ade .

M A T ER IA L S A N D M E T H O D S

T he m ate ria l con sis ted o f f resh ly c aug ht sp ec im e ns of E n glish bro w n tro u t

.S ’a lmo

t ru t ta , com m o ni roach

H uh /us ru /i /u s

itn i(l p erc h

P erc a flur iah ilis , ta k eti from stream s

an d

re servo irs. C e rta in o bse rva tio ns w ere also m ade on the m ar ine te leo sts,

Yleno labrus

rupestris an d T rig /a cu cu lus,

du rin g a v isit

to

th e P ly m o uth labo rato ry o f the M arin e B io -

log ical A sso cia tion of the U n ited K in gdo m .

F rom

th e D e par tm e nt o f

Phy sio logy , K ing’s C ollege , U n iv ersity o f D urham , N ew castle -

upo n-T yne , E ng land .

* A E ur op ea n

m e m b e r

of the carp fam ily .

39

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40 B L O O D C E L L F O R M A T I O N I N C E R T A I N T E L E O S T F I S H E S

The m ethods em ployed w ere m ainly his to log ic and w ere generally sim ilar to those de-

s c r i b e d by Duthie9 in

his w ork on m arine te leost fishes. Fresh bloo d w as obtained by stun-

n i n g

the fish by a sharp blo w o n the head and quickly am putating the tail. The blood w as

c o lle cte d in w axe d w atch-g lasse s, w ith h e p ar in o r so d iu m c it r a t e a s an t i-c lo t t in g ag en t .

For m icro scopic ex am ination of liv ing ce lls , suitable dilutions w ere m ade in an iso tonic

sa lin e d escr ib ed by Y oung.2 ’ For supravital staining , m ix ture s o f neutral red and janus

green w ere pre pared as fo l low s. The stains w ere disso lv ed in abso lute alcohol in eac h case

to g ive a saturated so lution; 20 to 30 drops o f the s toc k ne utral red so lutio n w ere added to

10 m l. o f ab so lu t e a lc oh o l; t o t h is w er e ad d ed 2 0 d r o p s o f j an u s gr een so lu t io n . T h e m ix t u r e

w as

t h e n

flo oded onto c lean slides , w hich w ere stood on end to dry . In use , a drop o f blo od-

saline w as plac ed on the slide and a c ove rslip added. O bse rvatio ns on surviv ing “kidne y

pulp” w e re m ade in the sam e w a y . For fixed stained film s , the film s w ere prepared

in

the

usual

wa y

and allo w ed to dry in air for five m inutes before fix ation. This w as

fo und to g ive

bette r results than fixatio n of “w et” films . Fix atives e mploy ed w e re m ethyl alco hol , the

vapo r of 40 per cent form alin and the vapor o f 2 per cent osm ium tetrox ide . For the preser-

vatio n of cy toplasmic inclusions, osm ium tetrox ide g av e the best re sults; staining w as not

adversely affec ted, co ntrary to the usual v iew w ith regard to this fix ativ e. M ethyl alco hol

and fo rm alin vapo r g av e better nuc lear fixation than osm ium tetrox ide, but w ere inferior

as re gards pre servation of c yto plasm ic s tructures. The f ixe d film s w ere stained in a dilutio n

of com m ercial G iem sa stain (G urr’s R 66 ) ,

and w ere w ashed

in w a t er b u f f er e d t o p H 7 .2 .

F o r t h e p e r ox id ase t e st t h e m et h od o f S a t o an d S ek iy a ’8 w as u s ed . M at e r ia l f o r se c t io n in g

w as f ix ed in Z e n k er - f o r m o l as a r o u t in e . F or so m e r oac h m at e r ia l, R ega u d ’s f or m o l-b ich r o -

m ate w as em plo yed, as it w as

found to preserve the coarse granulocytes better than the

r o u t in e f ix a t iv e . A f t e r R ega u d , sec t ion s w e r e s t a in ed w it h A lt m an n ’s ac id f u c h s in -m et h y l

gre en m ethod; afte r Ze nker-form ol the dilute d Gie msa w as used. In order to de mo nstrate

the peritubular capillaries and structure of the renal intertubular tissue , injec tions of

indian ink in saline w e re m ade into the he art of narc otized fish. S pe cim ens w ere killed at

periods o f up to tw e nty -fo ur hours af ter injec tio n and the kidneys then w ere rem ov ed for

se ctio ning . A bsolute red c ell c ounts w ere made w ith the standard he mo cvto m eter, using

Y o ung’s sal ine as diluting f luid. The pipets w e re pre v io us ly treate d w ith heparin to prev ent

c lo tting . D ifferential leuko cy te

cou n t s w e r e ca r r ied o u t in

the usual

way.

O B S E R V A T IO N S

Th e C e lls o f th e C ir cu la tin g Blo od

These co nsisted of nuc leated erythro cy tes , S m all and m edium -sized lym phocy tes,

throm bocytes and coarse and fine granulo cyte s. Im m ature and also senile ery thro-

cy te s w ere co m m o nly seen. The im m ature fo rm s w ere dis tinguished by their

c ircular outline and by the presence of a reticulum in the ir cy toplasm , w hich

s tained c learly w ith suprav ital cresy l blue , but could also be seen in the fixed

stained film s. The film s w ere characterized by the presence of m any m o re dis -

integrating ce lls and m ore ce ll debris than are usually seen in film s of m am m alian

blood. M o st o f this fragm entary m aterial appeared to be deriv ed from the break-

ing dow n of erythro cy tes and coarse granulo cy tes . Iso lated disintegrating nuc le i

o f ery throcytes, som etim es w ith part o f the cy to plasm still attached, w ere scat-

tered w ide ly ov er the preparations . The appearance of these nucle i w as quite

different from that of the nuc lei o f norm al ce lls , the chrom atin be ing spread out

in the fo rm of an open netw ork, w hereas in the norm al nuc leus the chrom atin

m aterial fo rm s a com pact m ass. It w as no t po ss ible to identify any ce lls resem bling

the m am m alian m ono cyte, and it w as decided to c lassify all m edium sized non-

g ranular leuko cy tes as m edium lym phocytes . In fry and yearling trout, o ccas ional









b 1

C 2

d 1

42

B L O O D C E L L F O R M A T I O N I N C E R T A I N T E L E O S T F I S H E S

F I G .

1 .-S ee leg en d , opp os ite pag e

o

0 2 0 3 0 4 , T

o

.1 :

0 7 )

b 2 b 3

C 3

d 2

d 3

e 2

f3

te

t ’









n

. :g

IO/L

w .

T . C A T TO N

4 3

1

L {216}

m

lii; . I .-- onl’il

a, -a

.

1n lI IO V t C S PI iPS

noac h , .

) i,

S n ial 1 lv n iph io id h ien iio l l:ts t .

I 1 ,

l tly an i l late

e ry t l iro l)las t

S

.

14 .

lie t icu loc y t e . z N on nal e rv t

h irocy t e. ar,,

S v n ,ile erv t h itocv t e .

a7, T linu nho cv te ro ach ).

b . f l.:nn gc lv nipho id h ien io h las ts

roach

b f2, line g ian iu lo cv tes ro ach

b. f ‘ )a rs e g na n i ml o cv t es r achi

b

se ries 1 -3 ), \eu t nal red -Jan us gre en s uprav i t al stain i .

c

s erie s, Pe n .o x i Iise ne u t ral red .

d series, ( ‘c i Is f ro m f i lm f ix e d n ie t h iv l alco ho l

,

st ai tied 0 i e m s a .

e s erie s, ‘e lls f no n ii ’t ion f ix e d IIe llv , s tain ed G ie m sa.

f

series. e l Is f rom s ect iou f ix ed H e gau l, s t ai

uie ( l

acid f uch si n i-m et liv l g ree n i.

g , Fi

tie

gran iu locv t

e

t

rou t )

.

Fix ed osm ic ac id V :tl)o l, s t ained ;m

h, o:i use gr anu l oc v t e tout) . Fi x e l o x n i i c acid v apor , st ai n ie I ;ie ns a

1, C o a n s e g na n ulo cv t

e

‘I e n ,o l al )n u s) . Fi

xe I

m e t hv l alco ho l , s t ai tie d iem sa.

j,

‘broniaf l i

ti e

cell

roach)

. From snie ar.

k,

M ac nop hage w it Ii i n iges t ed iii k pa n t id es ro ach )

1, Large lv iii pho id hen iiob las t

in n ii

t

osi.s sm ear)

ni A l)n ion n ial lv n ip h io cv te , w ith eo S itio l) liil g rat iu le s.

n,

ran iu lar an iu c le at

e

bo d ie s tro u t b loo d sm ear)

c y t o p l a s m f l o we d f r e e l y a s i n A m oeba, the f ine pro top lasm ic granu les show in g

B row n ian m ov em en ts . W ith neu tral red -Janus green su prav ital stain ing , th e

charac te ris tic granu les ap peared p ale bro w n , and in te rspersed be tw een th em w ere

nu m erous f iner particle s , stained p ale green ; these m ay hav e been m itochondria.

T he

nu cle us w as

un d iv ided or o n ly sligh tly co nstric ted in to tw o lobes, in the

r o a c h

cel ls; in tro u t c e lls it w as

d issec ted in to 3 or 4 lobes , con nected by f ine brid ges

as in th e m am m alian p o ly m o rp h n e u tro p h il c ell. T h e f in e c y to p lasm ic g ran u le s

w ere seen m os t c learly in sec tioned m aterial o f k idn ey , g ill, and in te s tin e, w h ere

they w ere s tain ed w ith eo sin . In f ix ed stained f ilm s the g ranu les w ere uns tained

w ith norm al s tain in g tim es, bu t took up eos in af te r pro lon ged stain ing . In roach









44

B L O O D C E L L FO R M A T I O N I N C E R T A I N T E L E O S T F I S H E S

and tro ut the granule s gav e a positive pero x idase reac tion. In the fishes exam ined,

f ine granulo cy tes w ere found only in the circulating blo od and hem atopo ie tic

centers, and not in the tissues generally . It is conc luded that they serv e the sam e

ro le, as scav eng ing phag ocyte s, as the m am m alian neutrophil ce lls , w hich they

so c lose ly resem ble in struc ture.

C oa r se g ra nu io cyts

(f ig .

i , b3 -f3 , h , i;

fig . 4 , j). These cells w ere rare ly seen in

film s of c irculating blood of the freshw ater spec ies exam ined; they o ccurred

fairly frequently in C t en 2 l ab r u s bloo d film s, but rarely in film s of Trig /a blood.

T h e scarc ity o f this ty pe of cell in the blood film s of fishes w as observed by m any

prev ious w orkers, and so m e w ere led t c as su m e that co arse g ranulocyte s w ere

entire ly absent fro m the blo od. If liv ing bloo d is ex am ined how ev er, these ce lls

can readily be seen, and w hen stained w ith neutral red suprav itally the g ranule s

take on a ye llow ish-brow n co lor. D ifferential counting o f the ce lls in fresh blood

is rendered difficult by the ac tiv e m ov em ents o f the ce lls , but by com pariso n w ith

the num bers o f ery thro cy te s in the sam e fields an averag e num ber o f 10 0 to 5 oo

pe r

c u. mm . w as es tim ate d.

I n

co ntrast w ith the scarc ity o f the ce lls in fix ed blo od

film s, they w ere found in larg e num bers in the hem atopo ie tic centers (kidney

and spleen), in the intestinal subm uco sa, (fig . 4 , , and in the g ill lam cllae,

(f ig .

2.

2 . They w ere also fo und in the m esenteries and peritoneal f luid. The

scarcity o f coarse granulocyte s in the blo od film s is probably to be ex plained by

the ir frag ility ; coarse granule s w ere to be seen scattered througho ut the film s,

as also w ere fragm ents o f the ce lls them se lves. In liv ing blood of trout the ce lls

m easured fro m 9 to ii when subspherical in shape (inactiv e) , and reached i8

in leng th w hen extended in am ebo id m o tio n. They adhered to the underside of

the covers lip in the sam e w ay as the fine g ranulocytes. The granules m easure

about

I /2

in diam eter and float free ly in the fluid “endoplasm ” of the cell. In

f ixed stained film s of trout bloo d the coarse granulo cy tes presented an appearance

diff icult to interpre t. In the cytoplasm w as a m ixture of granule s o f vary ing size ,

and also show ing different staining charac teristic s . M o st o f the granule s w ere

o f the larg er size s, and these w ere stained blue . The finer granule s, interspersed

am o ng them , appeared m auve in co lo r, and the sm alle st o f them appeared crim -

son. The apparent co lor differences m ay be open to a purely physical interpre ta-

tio n, as be ing due to the diffrac tion o f the light passing round the sm all partic le s .

The sm alles t partic les are o f the o rder of s ize of m itochrondria, w hich are not,

how ever, co m m only reported as staining w ith any com po nent of the R om anow sky

stain em ploy ed. In roach blood, the coarse granular ce lls w ere rarely seen in

fixed film s; w hen so preserved, the ir granule s w ere stained m auve in co lor. The

staining reac tion of the granule s is thus baso philic in tro ut, and m etachrom atic

in roach. The coarse granular cells o f

Trig /a

w ere found to be basophil and tho se

of C t en o / ab r u s ac ido phil, in agreem ent w ith D uthie .9 In the perch, the coarse

granulo cy tes , as observed in the spleen, inte stine and g ill w ere ac idophil.

G r a n u la r a nuc /ea te b od ies. (f ig . i, n) . S m all rounded m asses,

j

in diame ter, c on-

taining a m ixture of baso phil and ac idophil granules in a pale blue “cytoplasm ,”

w ere seen frequently in bloo d film s of roach and tro ut. They w ere at first cons idered

to be fragm ents o f broken dow n co arse granulo cytes , but they regularly show ed









.‘

,t

L i f l

* rb R C

Li ’

DC G

xi

Vu, ,

w .

T C A T T O N

45

FIG 2

1. O rganiz at io n of inte rtub ular hem ato poietic t issue.

2. Coarse gran iulocvtes i n gil l lam e lla (roac h).

3 . C o arse g ranulo cv tes

mi

inite stinial m uc osa (roach).

C G , C o arse granuloc y te .

( ‘I , . c. , Chromaf f in ie

cel l . ( ‘ P G , C oarse pro graniuloc vte . DGG,

D isc harging c oarse g raniulocy te .

E r e . ,

Ervthrocvte .

E r b . ,

Er throb las t. EG , Fin e g r an iu lo

c y te . I ” P G . , Fine rograniulocvte. LU , L arge ly m p ho id lie niio l)last. LUll’ , S am e in m itos is.

3 , M ac ro pl ia ge .

J IG ,

M uc ous go ble t. .1 uc , I nt es ti na l m uc os a. PT(’ , Pe ritubular c ap illary .

1W , Reticulocvte . I t ’E ’ , (‘elI o f ret iculo-e nd otheliai stronia. ASLH, Sm all ly m pho id henio -

blast .

such sm ooth unb ro ken o utlines and re lativ e uniform ity o f siz e as to sug gest that

they w ere m orp holog ic entitie s. In no case w as a nucleus seen in any of these

bod ies . N o prev ious report on these structures has b een found in the literature .









w . T. C A T T O N 47

m am m alian eo sinophil cell. I have been unable , ho w ev er, to f ind exam ples o f the

discharge pattern pheno m enon described above in any m am m alian tissue exam ined.

D ow ney6 ascribed secre to ry ac tiv ity to all the granular leuko cy tes in the gano id,

P olyo don spa th ula , and w as led to assum e that the granule s w ere a specif ic secre tio n

of the ce lls , w hich w as liberated in the ir passage throug h the c irculation. H e

c lassified these ce lls into three m ain

‘

‘series,’

‘

based on the m orphology as re-

vealed by staining w ith safranin and to luidin blue . It is diff icult to corre late his

c lass ificatio n w ith that o f m ore recent repo rts . H e re fers to S tephan’9 w ho in io6

described cyc lic chang es in the size of the granules in the g ranular leukocy tes o f

protopterus (D ipno i) . In hibernatio n, the granule s g radually shrank. In the activ e

season, on reco m m enc ing feeding , the granule s becam e enlarged.

The Site s o f H em a to po ie sis

The chie f site s o f hem atopo ie sis in these f ishes w ere in the kidney and spleen.

In the ro ach, as in

C t en o / ab r u s

and

Trig /a,

o nly the kidney show s activ ity . In the

perch, activ ity is lim ited to the spleen. In the trout, spleen and kidney are both

ac tive . The presence of hem ato po ie tic tissue in the kidney m ay be de tec ted m acro-

sco pically by the intense red co lo r o f the organ, o r o f such parts o f it w hich are

hem atopo ietically activ e . The dis tributio n o f activ e zones in the kidneys of som e

te leost fishes studied is sho w n in figure 3 . The dis tributio n w as found to be sim ilar

in m em bers of the sam e spec ies .

The Ren a l In te r tubu la r H em a top o ie tic Tissu e

This is the tissue re ferred to by D rzew ina7 as “lym pho id”, in a general survey

of the ‘lym phoid” tissue of fishes . It w as studied in m ore detail by D ow ney6

in the kidney of a gano id, P o/yodo n sp a thu /a . W hen exam ined m icroscopically ,

th e h em ato po ie ti c

activity

i s s e e n t o b e t a k i n g p l a c e i n t h i s t i s s u e wh i c h p r e s e n t s

a sim ilar degree of co m plex ity to that o f red bone m arrow . It is f irst c learly seen

that a capillary netw ork surrounds each kidney tubule; in noninjec ted spec im ens ,

these capillaries w ere recog nized by the epithe lial lining ce lls and the presence

of blo od ce lls w ithin them . A fter the injec tion of india ink into the c irculation,

the capillaries w ere v ery c learly dem onstrated, the ink partic les adhering to the

inner surfaces of the lining ce lls and also possibly being ingested by them

(f ig . 4 , i). A t certain po ints along the capillarie s the ink w as seen to fo llow nar-

ro w channe ls o f escape , w hich com m unicated w ith general spaces w ithin the

intertubular tissue , and m any partic les w ere taken up by m acrophages scattered

throughout this tis sue (fig . I, k). In s pe ci me ns in w hich the kidney w as rem o ved

for fixation w ithin fifteen m inutes o f injec ting of ink into the heart, the m acro-

phages had already taken up the ink partic les , indicating a free c irculation o f

blood w ithin the intertubular tissue . The nature of this circulation w as very

difficult to fo llo w , ow ing to the dense cro w ding o f the blo od ce lls and their pre -

cursor e lem ents. S trands of reticular ce lls w ere observed, and these in places

form ed close m eshes round the deve lo ping blo od ce lls, so that a general pic ture

w as deduced of a “spongew ork” of reticular ce lls ex tending betw een the pen-

tubular capillary nets , and form ing the suppo rting fram e of the hem ato po ie tic









‘ ;

1

F

FIG 4 .-S ee legend , opp osite page

48









w .

T . C A T F O N

49

tissue. In a few parts o f each sectio n, how ever, paralle l chains o f endo the lial ce lls

could be seen to enclo se channe ls w hich contained m ainly no rm al blo od cells . The

endothe lial w alls o f these spaces w ere alw ays incom ple te.

The spaces so de fined m ight be reg arded as prim itive representatives o f the

‘

‘open’

‘

sinuses in the bone m arrow , described by D o an3; it w as no t poss ible to

identify any struc tures correspo nding to the ‘ ‘clo sed’ ‘ or ‘ ‘formative’ ‘ s inuses o f

D o an, and as far as could be seen the blo od-form ing cells lie in m asses retained in

the re ticular spo ng ew ork of the intertubular tissue. There w as no ev idence of

segreg atio n of the precursor cells o f ery throcyte s and g ranulocyte s, these im m ature

cells be ing observed clo sely interm ing led w ith each other and bearing no w ell

de fined relation to the incom ple te s inuses described abo ve . The appearance o f the

intertubular tissue is represented in the m icrophoto graph (fig . 4 , 2 , and by the

sim plif ied diag ram , (f ig . 2.,

i

In fishes in w hich the kidney w as not ac tive in blo od-cell fo rm atio n (as in

perch), the reticular strom a w as fo und to be absent also , the tubule s be ing then

m ore c lo sely appro x im ated, and separated only by the capillary ne tw orks sur-

rounding each o ne of them . This sug gests that the re ticular stro m a is no t neces-

sanily present o nly as a supporting tissue, but m ay serve the additional func tion,

in the hem atopo ietically ac tive kidney , o f prov iding the blood stem cells .

Reg arding the c irculatio n of the intertubular tissue , D o w ney6 show ed by in-

jecting a ge latin m ass into the caudal ve in, that renal portal blood entered into

the channe ls and spaces in the tissue . The g elatin did no t pass any further into

the vascular system . M y india ink injectio ns into liv ing fish by an arterial route

hav e show n channe ls o f co m m unicatio n betw een the peritubular capillaries and

these spaces . It is to be assum ed, then, that the bloo d flo w s norm ally from the

renal portal ve ins , throug h the intertubular tis sue , and thence into the penitubular

capillaries , finally em erg ing in the renal v eins.

G ro ups of ce lls , hav ing the charac ter o f chrom affine ce lls , w ere observ ed to be

scattered thro ug hout the intertubular tissue. In unstained sec tions, these ce lls

w ere seen to have re tained a s trong brow n co lor, sug gesting an affinity fo r the

bichro m ate of the fixative (Zenker-fo rm o l). Each of these cells co ntained three

to six large c lum ps of a substance w hich too k a g reen co lor after staining w ith

Giem sa, (f ig .

I,

j The g reen co lor co uld be ascribed to a superpositio n o f the blue

com ponent o f the stain on the bro w n co lor due to the bichrom ate fixation. These

FIG. 4.- i . Roach kidney , sect ioned

after

injection of

india ink. Fixed H elly , s tained

Giem sa. X

345 .

Ink

p ar t icle s in p er itu b u lar c ap illa r ies .

2. Cte no labrus kidney . S ec tion 5 fixe d H elly , staine d G iem sa. Inte rtubular tis sue .

X 3 45 .

3. Ro ach kidney sm ear. Fix ed m ethyl alc ohol, staine d G ie msa. Group of chrom affine

cel ls ‘ hr . c . . X 575 .

4 . Cte no labrus, T. S . Intes tine. X 1 30 . C oarse granulocy tes in subm uco sa.

5.

Trout

kidne y sme ar. Fixed o sm ium tetrox ide, staine d G iem sa.

U ,

lymphocyte.

P.C. ,

pigm ent ce ll. (‘.G., co arse g ranulo cyte . Erb, e rythro blas t. X 63 5 .

6 . Trout kidne y sm ear. O sm ium te tro x ide, Gie msa. F.P.G., f in e p ro g ra nu lo c yte . S.L.H.,

small lym pho id hem oblas t. LUll . , large lym phoid hem oblast. E.G ., fine g ranulocvte .

X 63 5.










ce lls w ere identif ied in film s and sec tions o f kidneys o f trout, roach, Trig /a and

C t en o l ab r u s

(f ig . 4 , 3 .

The P hylogeny o f the Blood C e lls

The v iew is no w w idely accepted that the bloo d ce lls take the ir o rig in from

reticulo -endo the lial tissue as fo und in the hem ato po ietic o rgans , this tissue be ing

a rem nant o f the undifferentiated m esenchym e surv iv ing from em bry onic life .

These ce lls re tain the po tency for further differentiatio n, w hich is lo st in the

spec ialized tissues o f the body . The first step in blood-ce ll form ation is the re-

new al o f ac tiv ity in som e of these cells , w hich undergo certain m orpho log ic

changes. These ce lls m ay then either becom e detached from the parent tissue and

form

‘free’

s tem ce lls , or m ay rem ain attached, as ‘ ‘f ixed’

s tem ce lls , and g iv e

rise to

‘free’

stem ce lls by m ito tic div isio ns. This pro cess is no tably difficult to

fo llow in the study of m am m alian bo ne m arrow , and has prov ed no le ss so in

the study of hem atopo ies is in fishes. Ev idence of a transfo rm atio n of a “fixed”

re ticulum cell into a

‘free’

stem ce ll w as found o n tw o occasions o nly , in the

study o f m any sections of the kidney s o f different fishes . In these tw o cases, re ticu-

lum ce lls w ere o bserv ed, still form ing part o f the re ticular strom a, w hich had

underg one m orpho log ic changes approaching the struc ture o f a free s tem cell.

These changes w ere as fo llo w s : a co nsiderable increase in size o f bo th the nuc leus

and the ce ll as a w hole ; a m arkedly increased baso philia o f the cy toplasm ; and

the dev elopm ent of the prom inent chrom atin “kno ts” in the nuc leus, typical o f

the free stem ce ll to be described, w hen seen in sec tioned m aterial (f ig .

.

The rarity w ith w hich such transform ations w ere observed m ig ht im ply (a)

that the pheno m enon is diff icult to observe in the ce ll-crow ded intertubular tissue ,

or (b) that it is a rare o ccurrence in adult fishes; ev idence in favo r o f the latter

v iew w ill be g iven later.

The free stem ce ll so derived is a large ce ll o f lym pho id charac ter, reaching i

/2

in diam eter, subspherical in shape and possess ing a larg e nuc leus and a narro w

peripheral zone of cy to plasm , w hich is intense ly baso philic , ( fig .

I, d , e ).

In

fixed film s , m any ce lls o f the sam e g eneral appearance w ere seen, rang ing in s ize

fro m 7 to z /2. There w as a m arked difference in the appearance o f these ce lls

as be tw een tho se seen in film s and in sec tions. In the film s, the nuc leus show ed a

unifo rm netw o rk of f ine chro m atin strands , w ith a thin nuclear m em brane and

no ev ident nucleo li; fine baso phil granule s w ere seen in the cyto plasm . In sectio ns,

the nuc lear chro m atin w as seen to be condensed into o ne o r tw o “knots,” possibly

aro und nuc leo li no t o therw ise v isible , and the rest o f the nuc leus appeared to be

dev o id of so lid struc ture , (f ig . I, e) . It is reaso nable to suppose that this knotting

o f the nuclear chrom atin is a fixation artefact, and that w e see a truer pic ture of

the ce lls in film preparations. S tages o f m itosis w ere seen in ce lls o f this type ,

bo th in film s and sec tions , but far m ore clearly in the form er. M itoses w ere m ore

frequently observed in the larger ce lls than in the sm aller o nes. The m ost com m on

stages seen w ere m etaphases , (fig .

I,

i), and te lo phase s.

Regarding the part play ed by these lym phoid ce lls in the process o f hem ato-

po ies is , from a his to log ic s tudy alone

it

is only poss ible to pos tulate hypo the tical









N o a p u fy

0A (9L(

W . T . C A T T O N

51

f’ oi 4

{ 1 4 9 } {1 4 9 } - . . . - - -- - - - - -i

8*

S

I

Nor -mi .

F n G .

5 .-S ch en ie of de velopn ien it o f 1 )10 0(1 cel ls in T ele ost fish es

(d i a g ra mma t i c )

- ) B in d i-

ca tes hasop hilic

,

t l ie p lu s s ign s in d ic atin g in cre ase d h asoph ily . A itidicat e s fain t lv a cid o-

p h ilic . P + in d ic ate s p ero x id ase p osit ive . P - ind ica tes p ero x id ase n ega tiv e. P (0 ra n .) i n i -

dicat e s p ero x i(lase po sitive , g ra nu les on ly . a R e tic u lo -en dot he lia l c ell .

b

B lo od ste m

ce ll deve lo l)ed from R -E ce ll. c l)et ach ed 1)1 00(1 stem ce ll (larg e lym ph oid he m o bla st) .

( il) 1 )iv isiozi o f large lv n iph io id h em oblast . e S m a ll ly m p ho id h em oblasts . f M i t o s i s of

ea rly e rv t h rob last - (g ) E rv t li rob las t - Ii ) R et icu loc y t e . i E rv t h iro cv tc ’. (j )

C oa r se

pro-

gran iu locv te . I: F ine prog ran iu lo cv te . (/ an d in Progranu locy t e

nutose s .

n a nd a ) M at tire

co arse an d fine gra nu locv tes . (p ) L y m p ho cytes. (q ) T h rom bo cvte .

ac tiv ity , an d to fo llow the c lass ic m eth od o f a rrang ing the ce lls obse rved in to

se ries rep resen ting the m o st p ro bab le lines o f deve lopm ent. I p resen t m y

observa-

tions and dedu ctio ns as fo llow s.

I.

C ells o f a lym p ho id ch aracte r a re seen in a range o f size s from 7 /1 to

15 /2

d iameter .

i. T he re ticu la r strom a m ay g ive rise to th e la rg er ce lls ( tw o cases o bse rv ed to










show transitional form s); but the po ssibility o f ce lls o f any size be ing derived in

this w ay cannot be exc luded.

3 .

I t

is reasonable to suppo se that the smaller

ty pe o f cell m ay g ro w into a

larger one , but that the larg er type w ill no t dev elo p into a sm aller one by decrease

in size.

4 .

M itoses are m o re co m m only seen in the larg er cells, by casual o bservation;

this do es not necessarily im ply a higher rate o f m itosis .

5 . I n subsequent dev elo pm ent, as described belo w , the larg e cells g ive rise to

granulo cy te s, the sm all o nes to ery throcyte s, throm bocytes, and ly m phocyte s.

A t the extrem es of the s ize rang e then, w e hav e ce ll po pulations w hich differ not

o nly in size , but also in dev e lopm ental po tentialitie s .

6. I t is conv enient to designate ce lls o f the larg es t size as la rg e lympho id hemo-

b lasts; those o f the sm alle st size as small lymphoid hemob lasts .

7 . I t

m ay be deduced that the po pulation of each type o f hem oblast is m ain-

tam ed by continuous m ito tic div isio ns, and that derivation of free stem ce lls by

‘

‘recruitment’

‘

fro m the re ticular strom a is no t a norm al pro cess in the adult fish.

This w ould ex plain the rarity w ith w hich this process co uld be o bserv ed in the

sections.

The D eve lopment o f the Ery throcy te s (f ig . az-as

The erythrocyte arise s from a sm all lym pho id hem oblast, in w hich m arked

changes occur in bo th nuc leus and cytoplasm . The nuclear chro m atin becom es

gradually m o re co ndensed as deve lopm ent proceeds, and at one stag e there is a

“cartw hee l” appearance of the peripheral zone of the nuc leus (f ig .

I,

a3 , such as

has been described in late ery throblasts in m am m als. The ratio o f cy toplasm to

nuc leus increases steadily , w ith increase in size o f the ce ll as a w hole , w hich re -

tains a c ircular o utline up to and inc luding the re ticulocy te stage . The intense

basophilia o f the stem ce ll cy to plasm g ives w ay to po ly chro m atophilia in the

erythroblast a 3 , and this to ac ido philia in the m ature ery throcyte . The stages

o f dev elo pm ent m ay prov is ionally be designated as sm all lym pho id hem oblast;

pro erythro blast (faintly basophil); ery throblast (po lychrom ato phil) w ith cart-

w heel chrom atin strands; re ticulocy te ; ery throcyte . R elease into the circulation

o ccurs at the re ticulocy te s tag e. The deve lopm ent o f the ery throcyte w as m ost

clearly fo llo w ed in film s o f tro ut and ro ach kidney , but sim ilar stages w ere m ade

out in Cteno labrus and Trigla preparations . M itoses w ere frequently seen in pro-

ery thro blasts and erythroblasts , sugg es ting that the population of these ce lls

w as m aintained to som e extent by a process o f hom oplastic m aintenance , in

addition to he teroplastic derivation fro m the s tem cells .

The D eve lopment o f the Leukocyte s

i. Granulocy te s. Co m ple te series o f transitional stages linking fine and coarse

granulo cy tes w ith larg e lym pho id hem oblasts w ere m ade out in all the spec ie s

studied. The earlie st stages sho w ed the presence of a relative ly few granule s,

s ituated in the cyto plasm in the reg io n of a slight indentation o f the nuc leus. In

later stages the g ranules had increased in num ber, the nuc leus w as sm aller and









W T CAT T O N

53

d isp lac ed to th e p erip h ery o f th e c e ll, an d th e c y to p las m w as les s stro n g ly b aso -

p h ilic th an th at o f th e p are n t h em o b last. M atu re g ran u lo cy tes , as s ee n in p e-

rip h e ral b lo o d , sh o w ed co m p le te ab se n c e o f b aso p h ilia. T h e m atu rin g f o rm s w ere

t e r m e d

‘

‘progranulocytes’

‘ by D u th ie ,9 w ho describ ed the m aturation o f these

ce lls in so m e m arin e tele o s t f ish e s .

2 ..

Lymphocy te s.

It w as u su ally d if f icu lt to d is tin g u ish b e tw e en b lo o d ly m p h o cy te s

an d sm all ly m p h o id h e m o b lasts in f ilm s o f k id n e y an d s p le en . L y m p h o c y te s in

th e circu latin g b lo o d s h o w e d a les s b aso p h ilic an d m o re tran s lu c en t c y to p las m

than that o f sm all hem ob la sts from the k idney ; th e nuc leu s a lso show ed a denser

ap p e aran c e , w ith m o re c o m p acte d ch ro m atin . In d if f ere n tial c o u n tin g , n o atte m p t

w as m ad e to d is tin g u ish th e tw o c e lls . It se e m s re aso n ab le to as su m e th at ly m p h o -

cy te s are d e riv e d f ro m th e sm all h e m o b las t p o p u latio n , w ith b u t s lig h t c h an g e in

m orp h o lo g ic stru ctu re .

D if f ere n tial c o u n ts o f k id n e y an d sp lee n f ilm s o f tro u t w ere c arrie d o u t, an d th e

resu lts ap p e ar in tab le a. I t w ill b e se en th at in th e k id n e y s o f y e ar o ld f ish e s

T A B L E

i . -Dif ferent:a l C o unts o f K idne y and Spleen Films of Trout

Ce ll T 1 y ear T rou t 3 y ear T rou t 1 year T rou t

k idn ey kidn ey sp leen

L arge ly m p ho id h em ob last 3 3

12 5 2 0

Fin e p ro gran ulo cy te 15 o

C oars e p rog ran u lo cy te o .

I

.6

Fin e granu lo cy te io

17 5

6

C oarse g ranu lo cy te

0 4 I 0. 2 .

Eryth ro b las t io ii.

S m all ly m pho id c ells inc lud ing ly m p ho cy tes) 2 .9 .6 36 51 2

Figu res are p erc en tage s o f c e ll -po pu latio ns o f f ix e d f i lm s , e x c lud ing ery th roc y te s, re ticu lo cy tes

and th rom bo cy tes, an d are av erages o f six cou n ts o n three s lid es f ro m eac h s pec im e n .

th e re is a p ro n o u n ce d re d u c tio n in th e p ro p o rtio n o f larg e ly m p h o id h e m o b lasts

as co m p ared to th e y e ar o ld f ish es , w h ile th e ery th ro b lasts an d sm all ly m p h o id

c e lls rem ain at a f airly c o n stan t lev e l. T h is s ee m s to in d ic ate a d ec rease in h ete ro -

p lastic d e riv atio n o f m atu rin g ce lls f ro m s tem c ells , w ith a re lativ e in c re ase o f

h o m o p lastic m ain te n an c e am o n g ery th ro b lasts an d p ro g ran u lo c y te s in th e se o ld e r

f is h e s . In th e sp lee n , sm all ly m p h o id c e lls are se e n to o c c u r in g re ater p ro p o rtio n ,

an d larg e ly m p h o id c ells an d e ry th ro b lasts in less er p ro p o rtio n th an in th e k id n e y

o f th e y ear o ld f ish .

D I s c us s I oN

T h e f o llo w in g d is cu ssio n d o e s n o t attem p t to b e e x h au stiv e , b u t to p re se n t su c h

v ie w s e x trac te d f ro m th e w id e f ie ld o f h e m ato lo g ic lite ratu re w h ic h se em c lo se ly

re le v an t to th e arg u m e n t.

T h e e arlie r w o rk ers o n th e b lo o d an d b lo o d -f o rm in g p ro c es s in f ish e s in c lu d in g

R aw itz ,1 6 D rz e w in a,7 ’8 L an in e tm 4 ) lim ite d th e ir d e sc rip tio n s m ain ly to o b se rv atio n s

o n th e c e lls o f th e c irc u latin g b lo o d , an d in p artic u lar to th e co arse g ran u lo c y te s .’4










D rzew ina7 described so -called lym pho id tis sue in the kidney , spleen and other

organs o f certain teleost f ishes, to w hich she ascribed the functio ns o f lym pho cy te

and g ranulocyte form ation. A later paper by the sam e author8 described the

c irculating bloo d cells o f 6 8 spec ies o f fishes, and in particular drew attentio n to

the v ariability in staining charac ter o f the co arse g ranulocyte s.

D ow ney# { 17 6} m ade a care ful study of the intertubular hem atopo ietic tis sue in the

kidney of the spo onbill sturgeon, P olyodo n sp athu la . His paper dealt m ainly w ith

the nature of the c irculation through the tissue, the struc ture of the suppo rting

reticulum , and a detailed analys is o f the g ranulocyte po pulation. H e conc luded

that blo od fro m the po rtal v eins passed directly into

‘

‘lymphoid’ tissue w hich

surrounded the branches o f these ve ins. B lood m ig ht also pass direc tly into a

sy stem o f blood spaces ly ing in the m eshes o f the general reticular fram ew ork,

and thence into the

‘e fferent ve in

These spaces he did not o bserv e to be lined

w ith endothelium . B lood w hich had passed thro ugh the ly m phoid tissue flo w ed

into these sam e spaces o n its w ay to the e fferent ve ins. I have no t been able to

trace an arrangem ent o f this kind in the kidney s o f the te leo sts I hav e studied, but

hav e found ev idence of channe ls w hich are partly lined w ith endo the lium , and

of direc t com m unications from the peritubular capillaries to the general spaces

in the reticular fram ew ork. D ow ney adopted a m onophy le tic v iew o f the o rig in

of the bloo d ce lls , and described the o rig inal parent ce ll as a “large

basophilic

m ononuclear cell.” This ce ll appears to correspond to m y large ly m phoid hem o-

blast. Jordan and SpeideP’ described in detail the varie ties o f ce lls found in the

hem atopo ie tic centers o f the kidney and spleen in a num ber of elasm obranchs and

te leosts . They described large and sm all “lym pho id cells ,” w hich they referred

to in earlier w o rks as ‘ly m phocyte s,” in later w orks as “lym pho id hem o blasts”;

they regarded these cells as being

functionally and m orpho lo g ically identical w ith

the blo od ly m phocyte .The sm all lym pho cy tes w ere said to deve lop into thro m -

bo cyte s, the larger o nes into ery thro cy tes , spec ial leukocyte s (fine g ranulocyte s)

and eo sinophils (coarse granulocytes). The sm all lym phocytes w ere c laim ed to

be derived from reticulum cells and to gro w into large lym phocytes, w hich sub-

sequently underw ent am ito tic div ision, the daug hter ce lls then re inforc ing the

sm all lym phocyte population. The am ito tic div is io ns w ere observed in fixed

sectio ned m aterial, and the ir fig ures resem ble the appearance of sim ilar sec tio ns

prepared by m y se lf (fig . i, i . It is doubtful w hether they represent true am ito tic

divisions since in fixed smears evident true mitoses are seen to occur in these

cel ls .

In a paper dealing w ith the m orpho logy of the spleen in fishes, Y offey2 #{ 1 76}g av e

an acco unt of blood ce ll fo rm ation in this organ, w ith particular

refe rence to

the

dogfish (Scylio r hinus ca nicu la ). He referred to observations on the spleens of some

marine tele osts but did not study any freshwater fishes. In a description of the

c ells observed in spleen smears of

the do gfish he m entioned tw o types o f ce ll

w hich are o f spec ial interest. These ce lls he term ed “sm all ro und ce lls” and

“large ro und ce lls ,” and from his de tailed descriptions they appear to co rrespond

w ith the sm all and large lym phoid hem oblasts described in this paper. H e also

noted a co m ple te serie s o f transitional form s leading fro m one ty pe to the o ther.









w . T . C A T T O N 55

H is observations led him to adopt a m ono phy le tic v iew , and he put forw ard a

schem e of blo od cell deve lopm ent as occurring in both e lasm obranch and teleo st

spleens , w hich is the sam e as that o f B ryce2 fo r pos tlarval form s o f

Lep idosiren

p a r ad o x a (D ipno i) . In this schem e the com m on ancestor o f the blo od cells is the

‘

‘ small ro und cel l ’

‘

; it undergoes transform ation firs t into an erythroblas t, and

this in turn into an erythrocyte ; this sm all cell m ay also g row into a large round

ce ll, and fro m this ty pe the granulocyte s deve lo p. Y o ffey is thus in general agree -

m ent w ith Jordan and S pe ide l, except that he do es no t m ake the as sum ption that

the hem o blasts (sm all round ce lls) are necessarily identical w ith bloo d lym pho -

cy tes.

D uthie9 described blo od ce ll form ation in certain m arine te leost f ishes, m ainly

o f the fam ilie s Trig l idae and L ab r i d ae . H e fo und the sam e varie tie s o f large and

sm all ly m phoid ce lls as those described by Jo rdan and S pe ide l, but he term ed the

larg e ce lls ‘ ‘Granuloblasts’ ‘

,

since he deduced that they deve loped into granu-

locy te s; the sm all ce lls he term ed lym pho id hem oblasts , and he regarded them as

the com m on stem ce lls . He concluded that the sm all lym pho id ce lls g rew into the

larg er form s, and be lieved but w as not fully conv inced that they also deve loped

into ery throcyte s. He described karyokine tic figures in the lym phoid ce lls , m ore

particularly in the sm aller o nes, and did not co m m ent o n the claim of Jordan

and S peidel that the div isio ns o f these ce lls w ere m ainly am ito tic. D rzew ina8

had earlier c laim ed that both m ito tic and am ito tic div is io ns w ere to be found in

the lym pho id ce lls o f the renal hem atopo ie tic tis sue o f f ishes.

The v iew that the larg e lym phoid hem oblast is the orig inal s tem ce ll in the

blo od-ce ll- form ing process of these fishe s, as expressed in this acco unt, is based

upo n the fo llo w ing cons iderations. First, that its nuc lear structure bears a closer

resem blance to that o f the parent m esenchy m al ce lls ( in particular the reticulum

ce lls) than that o f any o ther possible form ative ce lls found in the bloo d-form ing

tissues . S eco nd, that s tages hav e been o bserv ed w hich show a trans ition betw een

apparent reticulum cells and this type of ce ll. Third, that these large lym phoid

cells appear to undergo frequent m ito tic div isions , by w hich the sm all lym pho id

hem o blasts m ay be derived. Fourth, that the larg e lym pho id hem oblast bears a

c lose resem blance to the “hem o cy toblast” described by M axim ow , Ferrata, and

the m ono phy le tic schoo l as the sing le stem cell in m am m als . It m ay be regarded

as signif icant also that in the fo rm atio n o f g ranulocyte s the first appearance of

granules is seen in these large ce lls and not in the sm all ones. S m all ce lls possessing

granules occur com m o nly , but they are of the sam e general appearance as the

m ature granulocytes, in that the cytoplasm is alm ost filled w ith characteristically -

staining granules ; they could best be regarded, then, as div ision products o f late

stages in granulocyte m aturation. If, according to prev ious autho rs, the sm all

lym pho id ce ll w ere to dev elo p into g ranulocyte s, it m ight do so in one of tw o

w ays. First, by an increase in size w ith sim ultaneo us form ation o f granule s, up

to the size o f the larg e hem oblast; this is denied by the absence o f interm ediate

stages. S econd, it m ight first increase in size, delaying the form atio n o f granule s

until it reached the size of a larg e hem oblast. It w ould be difficult then to explain

this de lay in granule fo rm atio n, since the sm all lym phoid ce ll is suff iciently m ature









56 B L O O D C E L L F O R M A T I O N I N C E R T A I N T E L E O S T FI S H E S

to be capable o f direc t m orpho log ic change into an ery throblast, i.e ., it is able to

organize a pro fo und chang e in cy toplasm ic ac tiv ity in this respect. A g ain, as-

sum ing the sm all ly m phoid ce ll to ac t as the orig inal stem ce ll,

it

w ould be re-

quired first to grow to the size o f a larg e ly m phoid hem o blast and subsequently

fo r a decrease in size to o ccur to that o f the m ature g ranulocyte .

A n alternative m ethod o f derivatio n o f the sm all lym phoid hem oblast m ight

be as sum ed to be as o ne product o f the div ision of an endothelial-type ce ll, in a

m anner com parable w ith the derivation of the ‘ ‘megaloblasts’ ‘ (proerythroblasts)

from endothe lial ce lls o f the m arrow sinuses in m am m als and birds (D oan, Cun-

ning ham and S abin4). O n this basis , the sm all lym pho id hem oblast w ould be

ho m o lo go us w ith the ‘ ‘megaloblast, ‘ ‘ and the large lym pho id hem oblast, derived

from a reticular ce ll, w o uld be ho m olo gous w ith the ‘ ‘prim itive w hite ce ll” o f

the sam e authors. This w o uld establish tw o independent lines o f ce lls , culm inating

in the productio n o f ery throcyte s on the one hand and of granular leukocyte s o n

the o ther in both teleos ts and higher vertebrates. There is , how ever, no ev idence

that sm all ly m phoid hem oblasts are deriv ed from endothe lial ce lls in te leost fishes.

J o rdan and S pe ide l’#{ 1 76}described the orig in of

‘ ‘

s mall ly mpho cy te s’

‘

fro m retic ulum

ce lls; in a later w ork,’3 they spec ifically s tated that they did not observe the den-

ivatio n o f ‘ ‘hem oblasts” fro m endothelial cells in the ho rned toad, phrynoso m a

a reptile ) , but only from reticular ce lls . D uthie9 does no t raise the questio n o f the

derivation of the stem ce lls and describes o nly their subsequent deve lopm ent.

The propo sed schem es of blo od ce ll form atio n discussed above are sum m arized

as in figure 6 , and the first schem e is show n diagram m atically in fig ure

A ttem pts to eluc idate the pattern of bloo d cell form ation in m am m alian and

av ian bone m arrow hav e g iven rise to a num ber o f different hypo theses, o f w hich

two o f the m os t w ide ly accepted are those propo sed by M axim o w ’5 and by D o an,

Cunningham and S abin.4 The latter w o rkers, em ploy ing technics o f artif icially

deple ting and thereby sim plify ing the appearance o f the bone m arro w o f pigeo ns

by starvation, and of suprav ital s taining o f the m arrow ce lls , e labo rated a theo ry

acco rding to w hich tw o chie f ty pes o f stem ce ll occur. One of these types (“m egalo -

blast”) is said to arise from endothe lial e lem ents , and to dev e lo p ins ide

“forma-

tive” sinuses in the m arro w , g iv ing rise to ery throcyte s; the o ther ty pe of s tem

ce ll is said to arise o utside the sinuses, is referred to as the ‘prim itiv e w hite

ce ll,” and g ives rise to the g ranular leuko cyte s. On this theory , the ery throcyte s

hav e an “intrav ascular” orig in and the leuko cy tes an “extrav ascular” o rig in,

and further they dev elo p from distinc t ty pes o f stem ce ll, w hose m orpho log ic

differences are best m ade o ut by suprav ital staining . S uch a theo ry is included in

the g eneral term “po lyphy le tic .”

-

The o lder (“m ono phy le tic”) schoo l, w ho se v iew s are sum m arized by M ax i-

m o w ,’5 m aintained that all blo od ce lls arose fro m a population o f m orpho log ically

identical stem ce lls (“hem ocytoblas ts”) , but that the subsequent lines o f de -

ve lopm ent w ere dec ided by the nature of the env iro nm ent in w hich the stem ce ll

first arose . Thus s tem cells (m edium ly m phocyte s) arising in the lym ph g lands

dev elo ped into lym pho cy tes , w hile m o rpho log ically sim ilar cells aris ing in the

bone m arrow sinuses gave rise to ery thro cy tes, and those o utside the sinuses to









granular leukocyte s. M axim o w also be liev ed that the lym pho cy te w as capable

o f deve loping into all types o f bloo d cells ; his “hem ocytoblast” appears to cor-

respond to Jordan’s large lym pho cy te and in a m ore lim ited w ay w ith the large

lym pho id hem oblas t described in this w o rk. Jordan’2 produced ev idence in sup-

po rt o f the v iew that the env ironm ent o f the deve loping stem ce ll ( ly m phocyte )

Jord an an d Spe ide l” 1924

R eticulum c ell o f reticulo-e ndo the lium

Ly m phoid hem oblast (sm all lym phocyte)

M ed ium lym p h oid

hemobiast

Erythrocytes

Ly mpho id he mo blasts

Large lym phoid

hemoblast

Granulocytes

r y t h r o bl s t s

Ret iculocytes

E r y t h r o c y t e s

Re t iculocyte s

Erythrocytes

R eticular ce lls

Large lym phoid he mo blasts

Fine C oarse

progranulocytes

Fine Co arse

granulocytes

W . T . C A T FO N

57

S mall ly mphoid

h em ob l a s t

B lood

Th r om boc y t e s

lymphocytes

u t b i e

i93

Granuloblasts

F in e C oa r se

B lo od pro granuloc ytes

ly mpho cytes Fine

Coarse

granulocytes

P re sen t A utho r

Re ticulo-endothe l ium

Endo the lial c ells

?

r

4 , .1 ’

S m all ly mpho id hem oblasts

B lo od Throm boc yte s Erythro blas ts

lymphocytes

Fmo . 6 .-Propose d sc hem es of blo od c ell form ation.

exerts a dec isive influence o n its subsequent deve lopm ent. In Diemyc t y l u s he fo und

that ce lls o f identical appearance in spleen and liver capsule gave ris e to ery thro -

cy tes and granulocyte s respec tive ly .

Those w o rkers w ho w ere not co nv inced o f the poss ibility o f the env ironm ent

hav ing such a dec isiv e e ffec t on the deve loping stem ce lls proposed a m odif icatio n

of the orig inal m o nophy le tic (unitarian) theory . The diverg ence of opinion aro se









58

B L O O D C E L L F O R M A T I O N I N C E R T A I N T E L E O S T FI S H E S

m ainly over the question o f the poss ible identity o f the m arrow lym pho id cells

m y eloblasts) w ith the larg e lym phocyte s o f the lym ph no des. Certain autho rs,

com m enc ing w ith N aege li, have m aintained that these ce lls w ere distinc t, the

m ye loblast g iv ing rise to all the blo od ce lls ex cepting ly m phocyte s, the large

ly m phoid cells o f the ly m ph nodes being respo nsible fo r lym phocyte production

only , under norm al conditions. M ax im ow repeatedly failed to find cons istent

differences betw een the ly m phoid cells o f the m arrow and lym ph no des; such

differences that he could de tect he ascribed to the effec ts o f the different env i-

ro nm ents. The neo -unitarian theory (N aege li, Pappenhe im , Ferrata, D o w ney )

seeks to m aintain the distinc tio n betw een the m arrow lym phoid cells (m ye loblasts

or lym pho idocy tes) and the lym pho id stem cells o f the lym ph no des, althoug h

under norm al conditions fe w stem ce lls are believ ed to be activ e , the lym pho cy te

po pulation be ing m aintained ho m oplastically . This func tional dualism breaks

do w n under patho lo g ic conditions, and all blood ce lls , including lym pho cy tes ,

are derived from the m ye loblas t.

The m o nophy le tic schoo l, then, assum es that there is o nly one ty pe of s tem

ce ll, w hich is inherently pluripo tential, and that the subsequent co urse o f its

deve lopm ent is dependent on the env iro nm ent in w hich it arises (m arrow or

lym ph node). The neo-unitarian scho o l distinguishes tw o ty pes o f stem ce ll, the

m y elo blast and the precursor ce ll o f the lym pho cy te, each under norm al conditions

hav ing its o w n lim ited potentiality o f deve lopm ent, the env iro nm ent hav ing no

particular signific anc e.

The w ork of D o an et al.4 presents a m orpho log ic pic ture of the m arrow in w hich

reasonably adequate segregation of distinc tion “physio lo g ic env ironm ents” m ay

be considered as a po ssibility , these being the env iro nm ents afforded by the in-

terior and exterio r o f the s inuses respec tive ly . B ut in te leo st f ishes it has been

sho w n in this investig atio n that the renal intertubular tissue offers no ev idence

of discre te sinuses or o f form ativ e pocke ts lim ited to one type o f ce ll; on the other

hand the deve loping ce lls appear to lie c lose ly interm ing led, precurso r ce lls o f

both the erythrocy tic and g ranular leukocytic serie s ly ing along side each o ther.

It is difficult then to conce ive any differential effec ts on the deve loping ce lls .

The schem e of bloo d-ce ll dev elo pm ent in fishes pro posed in this w ork does no t

fit c lo sely w ith any o f the accepted v iew s on m am m alian hem ato po ie sis , but

appro aches m ost c lose ly to the neo-unitarian hypothesis , in the light o f the fo l-

low ing considerations . First, that tw o types o f stem ce ll are reco gnized, the larg e

and sm all lym phoid hem oblas ts , w hich differ fro m each o ther, how ev er, only in

size. S eco nd, ow ing to the diff iculty o f prescribing segreg ated env ironm ents for

the tw o ty pes o f ce ll,

it

m ust be assum ed that each has an inherently lim ited

potentiality o f further deve lopm ent, in that distinc t lines o f deve lopm ent are

pursued by the ce lls ly ing w ithin the sam e env ironm ent. The large lym phoid

hem oblast resem bles the m am m alian m ye lo blast in s ize and appearance , but in

the fishes exam ined g ives ris e o nly to g ranulocyte s direc tly , all o ther ce lls ( in-

c luding ly m phocyte s) arising from the sm all ly m phoid hem o blast. N or can this

ce ll be clo se ly hom olog ized w ith the hem o cy toblast o f the m onophy le tic scho o l,

s ince o nly indirec tly is it the stem ce ll for ery throcyte s, ly m phocyte s and thro m -

o c y t e s









w . T . C A T T O N 59

if

ev idence can be found of the derivation of the sm all lym pho id hem oblasts

described, fro m endothe lial ce lls , w e sho uld have justification for a po lyphy le tic

theory , and be able to corre late the blood-ce ll form ing pro cess in teleos ts w ith

that in birds and m am m als, according to the theo ry of D oan, Cunningham and

Sabin.

S U M M A R Y

I.

The blo od ce lls o f trout and roach cons ist o f nuc leated erythrocyte s and re -

ticulocy te s, nuc leated thro m bocytes, co arse and fine granulocvte s, and ly m pho-

cy tes o f vary ing size s. It is difficult to distinguish any ce lls sho w ing charac teristic s

sim ilar to those o f the m onocyte s o f m am m als. Im m ature ce lls occur m ore fre -

quently in the blood than is the case in m am m als.

2. . The coarse g ranulocyte s very com m only escape fro m the bloo d vesse ls , and

have been observed in larg e num bers in the inte stinal m ucosa and subm ucosa, in

the g ill epithe lia, and in the perito neum . These ce lls m igrate to the epithe lial

surface, w here they undergo changes in struc ture leading to the form ation of a

charac teristic discharge pattern of the ir g ranules . It is propo sed that the

‘

‘gran-

u les’

‘

are in reality vesic le s w ith fluid contents , w hich are ultim ately discharged

at epithe lial surfaces.

3 . The hem atopo ie tic o rg ans o f these fishes are chie fly the intertubular tissues

of the kidney s; in the trout the spleen is also active ; in the roach, o nly the kidney

is ac tive ; in the perch, only the spleen is ac tive .

4 . Tw o alternative hy potheses o f blo od ce ll form ation are proposed. O n the

f irst hypo thesis, the co m m on stem ce ll is described as a “large ly m phoid hem o -

blast,” w hich g ives rise to granulocytes by direct transform ation and undergoes

m ito tic div is ion to g ive rise to “sm all lym pho id hem oblasts.” From the latter

deve lop the erythrocytes , thro m bocytes and blo od lym phocytes.

O n the seco nd hy pothes is , the larg e lym phoid hem oblas t, deriv ed by transfor-

m ation of re ticular cells is the precursor so lely o f the granulo cy tes , and the sm all

lym pho id hem o blast is to be deriv ed from endothelial ce lls and is the precursor

of ery throcyte s and thro m bo cy tes . In this case the large ce ll is to be com pared

w ith the “prim itive w hite ce ll” o f D oan, Cunningham and S abin, and the sm all

ce ll w ith the “m egaloblast” o f the sam e autho rs. N o ev idence how ever is avail-

able o f the de :iv atio n of sm all hem oblasts from endothe lial ce ll com ponents o f

t he re ti c ul o -e n do th e li um .

In the m aturation o f the ery throcyte in teleo st f ishes, there is a prog ressiv e

increase in the size o f the ce ll; in mammals and birds there is a decrease in size .

In both cases there is a decrease in s ize in granulocyte m aturation.

6 . There are no essential differences betw een the bloo d ce lls and hcm atopo ie tic

processes o f the freshw ater and m arine te leost f ishes exam ined.

A C K N O W L E D G M E N T S

It is w ith pleasure that I expres s m y gratitude to Pro fes sor L. E. S . las tham of the

Zoo log y D epartm ent, S heff ie ld U nive rsity , fo r prov iding m e w ith re search fac ilities in his

d e p r t me n t and f o r a llow in g me

the use

of the U niv ersity benc h at the Ply mo uth Labora-

tory . I am furthe r inde bte d to D r. 11 . B . M oo n of the Zoology D epartm ent, U niv ersity

C olleg e, Le ice ste r, fo r pro vis ion of fac ilities and assistance mi obtaining material. I w ould










like also to expres s my thanks to the m em be rs o f the S taffs of the laboratories of the M arine

B io lo g ical A ssoc iation, Plym o uth, and the Freshw ater B io log ical A ssoc iatio n, W ray C astle

for their re ady assis tance and interest and to Pro fes sor D . B urns o f the Phys io lo gy D epart-

me nt, King’s C ollege , N e w castle-upon-Ty ne , fo r e nabling me to com ple te the w o rk.

REFERENCES

n A L - H U S S A I N I , A . H . : A lkaline pho sphatase in fish g ut. N ature, Lo ndo n

161: 274 , 1948 .

2

B R Y C E ,

T. H.

: T he histo log y of the blo od of the larv a o f Lepido sire n paradox a. II. H ae -

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D O A N ,

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5

D O w N E Y ,

H. : Handboo k of Haem ato log y . N ew Y o rk, H oe ber, 1 938 .

6 -:

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C o ntribution a l’btude des

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C ontribution a l’#{ 23 3} tude des leuc ocy tes g ranule ux du sang des poisso ns. A rch. d’A nat.

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‘# { 1 7 6 }

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p e r im en t a l h em o r r h age an d h em oly t ic t ox in in t h e f r og . A m . J. Anat.

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5 29-54 6 , 19 24 .

-,

A ND

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L A N I N E , P.: D e s g lobules blancs e osinophiles dans le sang des po isso ns d’eau do uce . A rch.

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1951 6: 39-60

W. T. CATTON Blood Cell Formation in Certain Teleost Fishes

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