His ol His opa h
(1990)
5:
37-42
His ology and
His opa hology
Two modes o cell mig a ion in he en al ho n o he
spinal co d in he chick emb yo.
A
Golgy s udy
M.E.
Do ado, C.E. Ch nielewski,
A.
Quesada, J.M. Genis-Gál ez and
F.A.
P ada
De elop nen al Biology Ins i u e, Facul y o Medicine, Se illa, Spain
Su n na y.
The mig a ion p ocess o he en al ho n in
chick emb yo spinal co d cells has been s udied be ween
2.5 and 5 days o incuba ion (HH-17, HH-26), using he
Golgi echnique.
Two di e en mig a o y modes a e obse ed.
Type
1.-
Mig a ion by nucleus ansloca ion. Mos o
he en al ho n mo o neu ons mig a e by nucleus
ansloca ion wi hin he pe iphe al cylinde o he
cy oplasm (mig a ion by nucleus ansloca ion).
Type
11.-
F ee mig a ion cells. O he cells mig a e
disconnec ed om bo h limi ing su aces ( en icula
and pial). On he basis o shape and mig a o y beha iou
hey ha e been iden i ied as smoo h cells and mul ipodial
cells.
Key wo ds:
Spinal co d, Mig a ion, Young mo o
neu ons
ln oduc ion
S udies on he di e en ia ion o spinal co d cells we e
ini ia ed by Ramón y Caja1 (1909, 1911) in 3-4-day-old
chick emb yos using a gen ic imp egna ion echniques.
The de elopmen o au o adiog aphic echniques
(Hambu ge , 1948; Fuji a, 1962, 1963, 1965a,b;
Fuji a and Fuji a, 1964) e ealed he ime-space sequence
o he o igin and la e localiza ion o he spinal co d cells.
In he las i e decades and due o he con ibu ion o
he educed sil e s aining me hods, new in o ma ion has
been acqui ed ega ding he di e en ia ion p ocess o he
spinal co d cells in a ious animal models. These include:
he chick (Ba on, 1946), a (Windle and Fi zge ald,
O p in eques s o:
D . M.E. Do ado Ocaña, Ins i u o de Biología del
Desa ollo, Facul ad de Medicina, A da. Sánchez Pizjuan s/n,
41009
Se illa, Spain
1936), ca (Windle e al., 1934), lamb (Ba on, 1945) and
human (Windle and Fi zge ald, 1937). The models
sugges ha he spinal co d cells di e en ia e ollowing a
en o-do sal sequen ial g adien . Mo e ecen ly, he
s udies o Wen wo h and Hinds (1978) and Wen wo h
(1980, 1984) on mice emb yo spinal co d, using Golgi's
me hod, es ablish 6 s ages in he di e en ia ion p ocess
o he spinal co d mo o cells. His ogene ic analysis in
he spinal co d has been p incipally ocused on he
di e en ia ion p ocess; while on he con a y, da a
e e ing o neu ona1 mig a ion is sca ce.
In his s udy we desc ibe he mo phological sequences
ha he en al ho n cells o he chick emb yo spinal co d
-
-
ollow du ing hei mig a ion p ocess.
Ma e ials and ne hods
Whi e Legho n chick emb yos we e incuba ed
a 37.5" C. and s aged, acco ding o Hambu ge
and Hamil on (1951), a hal -day-in e als be ween
he 2.5 h and 5 h day o incuba ion (HH-17, HH-26).
Whole emb yos we e di ec ly imme sed in S ensaas
solu ion (1967), he ixa ion ime being a ied be ween
1
and 4 days, so as o ensu e he s aining o a la ge
a ie y o cells. Fixed emb yos we e b ie ly insed
in ap wa e , hen washed in a 0.75% (wl ) sil e
ni a e solu ion, and inally imp egna ed in a la ge
olume o he same solu ion o 2 days. This was
ollowed by dehyd a ion, embedding in low iscosi y
ni ocellulose and clea ing o he blocks in ceda
wood oil be o e sec ioning. Se ial sec ions (100 pm hick)
we e collec ed in he same clea ing medium, and
moun ed wi h damma esin, ollowing he p ocedu e
published elsewhe e (P ada and López-Masca aque,
1985), o p e en he ading o p epa a ions. The
spinal co ds o 52 sucess ully s ained emb yos we e
scanned, checking by o e - and unde ocusing ha
al1 ele an cells we e wholly included wi hin he
sec ion. D awings we e made by use o a came a lucida
a achmen .
Mig a ion in he en al ho n
Resul s
Cell iden i ica ion c i e ia
Two-and-a-hal -day-old chick emb yo spinal co ds,
when s ained wi h Golgi's me hod, show ansi o y
shapes bo h o cells in he mi o ic cycle and o di e en-
ia ing neu oblas s. These wo cell ypes a e di icul o
dis inguish by ligh mic oscopy, bu he e a e some
mo phological ea u es ha allow hei dis inc ion:
a) Cells in he mi o ic cycle.
A e hose cells ha du ing he
i s de elopmen al s ages o he spinal co d a e ound,
o al. monopola andlo bipola -shaped. Cells ha a e
always a ached o he en icula su ace o o bo h
su aces ( en icula and pial), wi hou any ami ica ion
o ilopodial expansions, may be cells ha a e
in di e en
phases o he mi o ic cycle. These cha ac e is ics a e in
acco dance wi h he classical pa e ns desc ibed o he
cen al ne ous sys em (C.N.S.) by nume ous au ho s
(Fuji a, 1963: Hinds and Hinds, 1974; Jacobson, 1978)
and a e e y simila o hose es ablished by P ada e al.
(1987) o he chick e ina o 5-5.5 incuba ion days.
Following he abo e-men ioned da a, Fig. 1 and Fig. 19,
n. show cells o he spinal co d o a chick emb yo o 2.5-3
incuba ion days in he a ious s ages o he mi o ic cycle.
b)
Glial cells.
The p incipal mo phological ea u es ha
cha ac e ize he en al ho n glial cells in he spinal co d
o a chick o 2.5-3 incuba ion days a e: 1.- They a e
always a ached o he en icula and pial su aces o
he spinal co d, showing in hei ou e a achmen a
cha ac e is ic iangula shaped o la oo , (Fig. 2 and
Fig. 19, G a ow heads); 2.- The nucleus o hese cells is
loca ed in he pe i en icula laye and shows hickenings
and spines a di e en le els o i s ou e p olonga ion
om he i s di e en ia ion s ages (Fig. 2 and Fig. 19,
G
hin a ows). This p olonga ion some imes ami ies,
o ming an a ch ha also ends in he pial su ace in a
iangula oo (Fig. 19).
c) Young mo o neu ons.
Ou c i e ia o he iden i ica ion
o young mo o neu ons is mos ly based on he sp ou ing o
he axon. Following his, and independen ly o he loca ion
o he nucleus, al1 o he cells a ached o bo h limi an
su aces, o only o he pial su ace, and which p ojec a
p olonga ion ou o he incipien en al ho n o he spinal
co d, a e conside ed young mo o neu ons (Fig. 19).
Spinal co d mo o cell mig a ion (Type
1)
The mig a ion o mos o he chick ce ical spinal co d
mo o cells occu s be ween he 2.5 and 4 h di e en ia ion
days (HH-17, HH-23).
The young mo o neu ons a e disconnec ed om he
en icula su ace when hei pe ika yon is s ill loca ed
in pe i en icula zones (Fig. 9 and Fig. 19), and hey
al eady ha e a subpial a achmen . In la e phases, he
cell bodies a e adially disposed and can be ound a
di e en le els wi hin he spinal co d (Figs. 3 o
8
and
Fig. 19,
1
o 7). Du ing ansloca ion o he nucleus, mos
o he young mo o neu ons p ese e a li le cy oplasmic
s o age in he inne pole o he pe ika yon (Figs. 3,4,7,
8 and 19, 2, 3.
6
a ows), which p og essi ely e ac s.
O he s show a ounded pe ika yon (Fig. 6).
The mo phology o he young mig a o y mo o
neu on's ou e p olonga ion depends on he cell's o igin
zone a he le e1 o he en icula laye s. The e o e, hose
cells which o igina e in he mos an e io and pos e io
zones o he en al ho n show. in hei ajec o y, an
in lec ion wi h opposi e di ec ion ha allows he abo e-
men ioned p olonga ion o p ojec o he adix en al oo
(compa e Figs. 5 and 6 o 7 and 8, and Fig. 19, 3 and 7).
Those neu ons ha o igina e in a de e mined zone o he
en icula laye , which is associa ed wi h he o igin o he
adix en al oo , show a ec ilinea ou e p olonga ion
(Fig. 9 and Fig. 19,4 and 5).
Abou he 4 h day o incuba ion, mos o he cell
bodies occupy de ini i e posi ions in he p ospec i e
en al ho n o he spinal co d.
F ee mig a ion cells (Type 11)
Be ween he 3 d and he 5 h days o incuba ion he
en al ho n o he chick emb yo spinal co d has cells ha
a e disconnec ed om he en icula and pial su aces
(Figs. 10-18). These cells a e loca ed in di e en zones o
he spinal co d's hickness and, acco ding o hei shapes
and ou lines, can be classi ied in o wo di e en g oups:
smoo h cells and mul ipodial cells.
Smoo h ce1ls.-
These cells show a bipola o monopola
shape and a adial a angemen . Thei pe ika yon is o al
shaped and loca ed in he inne mos pa o he cell (Figs.
10-14 and Fig. 19, sl,
s2 and s3). A single p olonga ion
a ises om he ou e pole o he pe ika yon, ending in a
Figs. 1 o 18.
Pa ial aspec s o he ce ical spinal co d o he chick
emb yo du ing
2%
o
4%
days o incuba ion.
Fig. 1.
Cells in he mi o ic cycle a
2%
days o incuba ion.
x
400
Fig. 2.
Glial cell a
3
days o incuba ion. The a ow head shows a la
oo a ached o he pial su ace.
x
400
Figs. 3,4,6
and
7.
Young mo o neu ons loca ed in he mos an e io
zone o he en al ho n a
3-3M
days o incuba ion. The a ows show
he inne p olonga ion in e ac ion.
x
400
Figs. 5
and
8.
Young mo o neu ons loca ed in he mos pos e io
zone o he en al ho n a
3-3%
days o incuba ion (Figu e
5)
and
2%
and
3
days o incuba ion (Figu e
8).
The a ow shows he las s age o
e ac ion o he inne p olonga ion.
x
400
Fig.
9.
Pano amic iew o he en al middle o he spinal co d a
4-4%
days o incuba ion.
x
200
Figs. 10 o 12.
Bipola (Figu e
10)
and Monopola (Figu es
11
and
12)
smoo h cells a
4-4%
days o incuba ion. The a ows show he
pos e io o he inne p olonga ion, which seems damaged.
x
400
Figs. 13
and
14.
Monopola smoo h cells a
3-3%
days o incuba ion.
x
400
Figs. 15 o 18.
Di e en mul ipodial cells loca ed in he mos an e io
(Figu e
15)
and pos e io (Figu es
16
o
18)
zones o he en al ho n a
3-3%
days o incuba ion. The a ows show he po ion o he inne
p olonga ion, which seems damaged.
x
400
Mig a ion in he en al ho n
Mig a ion in he en al ho n
Mig a ion in he en al ho n
Fig.
19.
Ca ne a lucida d awings o wo ep esen a i e nodes o cell
nig a ion in he en al ho n o he ce ical spinal co d in he chick
e nb yo, du ing 2% o
4%
days o incuba ion.
Abb e a ions: n, cell in ni o ic cycle; 1 o
7,
young mo o neu ons;
G,
glial cells, sl
,
s2 and s3, s noo h cells;
nl
,
m2 and n3, nul ipodial cells.
lamellipodia (Fig. 11 and Fig. 19, s2 ames a ow) o a
ilopodial (Fig. 10 and Fig. 19, sl ames a ow) g ow h
cone.
Mul ipodial cells
.- The second g oup o ee mig a ion
cells shows a high plas ici y. These cells show g ea
a iabili y in shape and ha e mul iple cy oplasmic
p ocesses (Figs. 10-18 and 19, ml, m2 and m3), which a e
a iable in numbe and o ien a ion. Mos o hese cell's
expansions a e loca ed in he ou e pole o he pe ika yon
and hey a e gene ally ili o m; al hough some cells also
p esen lameli o m p ocesses. The inne pole o he
smoo h bipola shaped cells and o he mul ipodial cells
is con inued in a p olonga ion, which, in mos cases,
seems damaged (Figs. 10, 16, 18 and 19, sl, m3 cu ed
a ows). This could be he p olonga ion by which he
cell was a ached o he en icula su ace.
Discussion
Mos o he knowledge conce ning he spinal co d's
his ogenesis is based on he s udies o Ramón y Cajal
(1909, 1911, 1929). This au ho conside ed ha he
di e en ia ion o he spinal co d's en al ho n in he
chick emb yo occu s ollowing a mo phological sequence
which was di ided in o i e s ages.
Wen wo h (1980, 1984), basing himsel on Ramón y
Cajal's s udies, es ablished
6
s ages in he di e en ia ion
p ocess o he spinal co d's en al ho n in he mouse
emb yo.
These au ho s desc ibe, om a mo phological poin
o iew, pa ial aspec s o he neu ona1 mig a ion p ocess
in he spinal co d's en al ho n.
A p esen he e is no ag eemen on he way in
which young pos mi o ic neu ons each hei en en ual
des ina ion in he laye o he man le in he CNS. The i s
esea che s ha ook up his p oblem, His (1890) and
Ramón y Cajal (1909,1911). assumed ha he neu oblas s
mig a ed as ee amoeboid elemen s. Bu Be y and
Roge s (1965) and Mo es (1970) s a e ha he young
neu ons do no mig a e wholly, only he nucleus and i s
su ounding cy oplasm mig a es, bu wi hin he limi s o i s
cy oplasmic p olonga ion. On he o he hand, he s udies
o Rakic (1971, 1972) in he monkey's elecephalic co ex,
and he s udies o Rakic and Sidman (1973) in humans,
seem o show he exis ence o ee mig a ion cells.
La Vail and Cowan (1971), using au o adiog aphic
echniques, clea ly show he exis ence o an abundan
cell mig a ion owa ds he co ex pla e. I s pa e n is
a iable depending on he bi h-da e o he mig a o y
cells. E iden ly, he nuclea b anding o he au o adio-
g aphic echniques is no su icien o es ablish how his
mig a ion occu s. When Domesick and Mo es (1977a,b)
s udied he de elopmen o mul ipola and a ch-shaped
cells o he op ic ec um using he Golgi me hod, hey
hough ha ne e cells mig a ed exclusi ely by nucleus
ansloca ion and we e doub ul abou he ee mig a ion
model p oposed by Rakic and Sidman (1973). La e ,
Puelles and Bendala (1978) demons a ed, using Golgi's
echnic, ha in he op ic ec um o he chick emb yo,
bo h mig a o y modes exis ed.
In he chick emb yo e ina, Genis Gál ez (1977),
Genis Gál ez e al. (1978) and P ada e al. (1984) and
mo e ecen ly P ada e al. (1987) ha e demons a ed,
using he Golgi and au o adiog aphic echniques, he ee
mig a ion o young ho izon al and amac ine neu ons.
Rega ding he spinal co d, Ramón y Cajal (1909.
1911, 1929) and Wen wo h (1984) implici ly suppo ,
hough hey do no show in hei esul s, ha he young
neu ons o he spinal co d mig a e ollowing he
mig a ion by soma ansloca ion model; he young
neu on always being a ached a leas o he pial su ace.
Ou esul s suppo ha he en al ho n cells o he
chick emb yo spinal co d ollow wo di e en modes o
mig a ion:
1.- The en al ho n's mo o neu ons o he spinal
co d mos ly mig a e ansloca ing hei cell bodies
h ough he cell cy oplasm, which does no disconnec
om he pial su ace.
2.- On he o he hand, we ha e obse ed, in he chick
spinal co d, smoo h o mul ipodial shaped ee mig a ion
cells which could o igina e associa ion neu ons as occu s
in o he pa s o he C.N.S. such as in he ce ebellum
(Rakic, 1971b), co ex (Rakic, 1972), op ic ec um
(Puelles and Bendala, 1978) and in he e ina (Genis e
al., 1977; P ada e al., 1984,1987).
The mechanisms esponsible o he ee mig a ion o
he smoo h and mul ipodial cells a e also likely o be
di e en . While he mul ipodial ype o cells would

Mig a ion in he en al ho n
associa ed o amoeboid explo a o y mo emen s, he
smoo h cells would sugges he exis ence o con ac
guidance cues, such as glial guides.
The e o e, we can conclude han in he chick e nb yo
spinal co d wo neu onal mig a ion modes exis simila o
hose p oposed by se e a1 au ho s o o he pa s o he
Cen al Ne ous Sys em: soma ansloca ion and ee
nig a ion
.
Se e a1 p ocedu es a e used a p esen in ou
labo a o y o s udy he na u e o he smoo h and
mul ipodial cells.
Acknowledgemen s.
This s udy was suppo ed by g an s o n he
Comisión ln e minis e ial de Ciencia y Tecnología GG85-03 and by
Acción In eg ada Hispano-Po uguesa
no 19.37136.
Re e ences
Ba on D.H. (1945). The ole o senso y ibe s in he di e en ia ion
o he spinal co d in sheep. J. Exp. Zool. 100,431 -443.
Ba on D.H. (1946). Obse a ions o heea ly di e en ia ion o he
mo o neu oblas s in he spinal co d o he chick. J. Comp.
Neu ol. 85, 149-169.
Be y M. and Roge s A.W. (1965). The nig a ion o neu oblas s in
he de eloping ce eb al co ex. J. Ana . 99,691 -709.
Domesick V.B. and Mo es D.K. (1977a). Mig a ion and di e en ia ion
o ganglion cells in he op ic ec u n o he chick e nb yo.
Neu oscience 2,459-475.
Domesick V.B. and Mo es D.K. (1977b). Mig a ion and di e en ia ion
o shephe d's c ook cells in he op ic ec u n o he chick
e nb yo. Neu oscience 2,477-491.
Fuji a S. (1962). Kine ics o he cell p oli e a ion. Exp. Cell. Res.
28,56-60.
Fuji a S. (1 963). The na ix cell and cy ogenesis in he de eloping
cen al ne ous sys e n. J. Comp. Neu ol. 120,37-42.
Fuji a S. (1965a). The na ix cell and his ogenesis o he ne ous
sys e n. La al Med. 36,125-1 30.
Fuji a S. (1 965b). An au o adiog aphic s udy on he o igin and a e
o he subpial glioblas s in he e nb yonic chick spinal co d. J.
Comp. Neu ol. 124,51-60.
Fuji a H. and Fuji a S. (1964). Elec on nic oscopic s udies on he
di e en ia ion o he ependy nal cells and he glioblas in he
spinal co d o do nes ic owl. Z. Zell o sch. Mik osk. Ana . 64,
262-272.
Genis-Gál ez J.M., Puelles L. and P ada C. (1977). Displaced
e ina1 a nac ine cells in he chick. Exp. Neu ol. 56,151 -1 57.
Ha nbu ge V. (1 948). The ni o ic pa e ns in he spinal co d o he
chick e nb yo and hei ela ion o his ogene ic p ocesses. J.
Comp. Neu ol. 88,221 -283.
Ha nbu ge V. and Ha nil on H.L. (1 951). A se ies o no mal s ages
in he de elop nen o he chicke nb yo.
J.
Mo phol. 88,49-92.
Hinds J.W. and Hinds P.L. (1974). Ea ly ganglion cell di e en ia ion
in he nouse e ina: An elec on nic oscopic analysis u ilizing
se ial sec ions. De . Biol. 37,381 -41 6.
His W. (1890). His ogenese und Zusa n nenhang de Ne ene-
le nen e. A ch. Ana . Physiol. Leipzing. Ana . Ab . Suppl. 95,
95-1 19.
Jacobson M. (1978). De elop nen al Neu obiology. Second
Edi ion. Plenu n P ess. New Yo k, London.
La Vail J.H. and Cowan W.M. (1971). The de elop nen o he chick
op ic ec u n II. Au o adiog aphic s udies. B ain Res. 28,421 -441.
Mo es D.K. (1970). A s udy o neu ogenesis in he o eb ain o
opossu n pouch young. Z. Ana . En wicklungsgesch 130,
265-305.
P ada C. and López-Masca aque L. (1 985). Da n na esin p e en s
he ading o celloidin sec ions o Golgi i np egna ed
e nb yonic cen al ne ous sys e n. Mik oskopie 42, 146-1 47.
P ada F.A., A nengol J.A. and Genis-Gál ez J.M. (1984).
Displaced ho izon al cells in he chick e ina. J. Mo phol.
182,221 -225.
P ada C., Puelles L., Genis-Gál ez J.M. and Ra ní ez G. (1987).
Two nodes o he ee nig a ion o a nac ine cell neu oblas s
in hechick e ina. Ana . E nb yol. 175,281-287.
Puelles L. and Bendala M.C. (1 978). Di e en ia ion o neu oblas s
in he chick op ic ec u n up o eigh days incuba ion: A Golgi
s udy. Neu oscience 3,307-325.
Rakic P. (1971a). Guidance o neu ons nig a ing o he e al
nonkey neoco ex. B ain Res. 33,471 -476.
Rakic P. (1971b). Neu on-glia ela ionship du ing g anule cell
nig a ion in de eloping ce ebella co ex: A Golgi and elec on
nic oscopic s udy in Macacus hesus. J. Comp. Neu ol. 145,
283-312.
Rakic P. (1972). Mode o cell nig a ion o he supe icial laye s o
e al nonkey neoco ex. J. Comp. Neu ol. 145,61-84.
Rakic P. and Sid nan R.L. (1973). Neu onal nig a ion wi h special
e e ence o de eloping hu nan b ain: A e iew. B ain Res.
62,l-35.
Ramón y Cajal S. (1909). His ologie du Sys e ne Ne eux de
I'Ho n ne e des Ve eb es. Vol l(1952 ep in ). Ins i u o Ramón
y Cajal. Mad id.
Ramón y Cajal S. (1911). His ologie du Sys e ne Ne eux de
I'Ho n ne e des Ve eb es. Vol
11
(1955 ep in ). Ins i u o
Ramón y Cajal. Mad id.
Ramón y Cajal S. (1929). S udies in e eb a e neu ogenesis. L.
Gu h, ansl(1960), Cha les C Tho nas, Sp ing ield, III.
S ensaas L.J. (1967). The de elop nen o hippoca npal and
do sala e al pallial egions o he ce eb al he nisphe e in e al
abbi s. II. Twen y nilli ne e s age neu oblas no phology. J.
Comp. Neu ol. 129,71-84.
Wen wo h L.E. and Hinds J.W. (1 978). Ea ly no oneu on o na ion
in he ce ical spinal co d o he nouse. An elec on nic oscopic
se ial sec ion analysis. J. Comp. Neu ol. 177,611-634.
Wen wo h L.E. (1980). Golgi analysis o neu onal ela ionships in
he spinal co d o he nouse e nb yo. Neu osci: Abs . 8,287.
Wen wo h L.E. (1984). The de elop nen o he ce ical spinal
co d o he nouse e nb yo l. A Golgi analysis o en al oo
neu on di e en ia ion. J. Comp. Neu ol. 222,81-95.
Windle W.F., O D.W. and Minea W.L. (1934). The o igin and
de elop nen o e lexes in he ca du ing he hi d e al week.
Physiol. Zool. 7,600-61 7.
Windle W.F. and Bax e R.E. (1936). De elop nen o e lex
nechanis ns in he spinal co d o albino a e nb yos.
Co ela ions be ween s uc u e and unc ion and co npa ison
wi h he ca and he chick. J. Comp. Neu ol. 63, 189-209.
Windle W.F. and Fi zge ald J.E. (1 937). De elop nen o hespinal
e lex nechanis n in hu nan e nb yos. J. Comp. Neu ol. 67,
493-509.
Accep ed June 15,1989