Two modes of cell migration in the ventral horn of the spinal cord in the chick embryo. A Golgy study
Abstract
Surnrnary. The migration process of the ventral horn in chick embryo spinal cord cells has been studied between 2.5 and 5 days of incubation (HH-17, HH-26), using the Golgi technique. Two different migratory modes are observed. Type 1.- Migration by nucleus translocation. Most of the ventral horn motor neurons migrate by nucleus translocation within the peripheral cylinder of the cytoplasm (migration by nucleus translocation). Type 11.- Free migration cells. Other cells migrate disconnected from both limiting surfaces (ventricular and pial). On the basis of shape and migratory behaviour they have been identified as smooth cells and multipodial cells.
Full text
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.
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