A FREEZE-FRACTURE STUDY ON THE DEVELOPING
SATELLITE CELLS OF SPINAL GANGLIA IN
THE CHICK EMBRYO
CLAUDIO A. FERRAZ DE CARVALHO * — CIRO F. DA SILVA **
S U M M A R Y — A f r e e z e - f r a c t u r e a n a l y s i s o f t h e s a t e l l i t e c e l l s o f s p i n a l g a n g l i a o f t h e c h i c k e m b r y o w a s p e r f o r m e d i n 8 s u c c e s s i v e s t a g e s o f d e v e l o p m e n t , f r o m t h e 5 t h i n c u b a t i o n d a y t o h a t c h i n g . T h e c h a r a c t e r i s t i c l a m i n a r d i s p o s i t i o n o f t h e c e l l s w e r e f i r s t o b s e r v e d o n t h e 7th d a y . T i g h t j u n c t i o n s w e r e f o u n d at t h e 20th i n c u b a t i o n d a y . S m a l l g r o u p s o r i r r e g u l a r a g g r e g a t e s o f p a r t i c l e s , b u t n o t g a p j u n c t i o n s , w e r e d e s c r i b e d o n t h e 7 t h a n d 8th d a y s . P i n o c y t o t i c v e s i c l e s w e r e p o i n t e d o u t i n t h e d i f f e r e n t s t a g e s c o n s i d e r e d .
Estudo com a técnica da crio-fratura das células satélites dos gânglios sensitivos do embrião de galinha.
R E S U M O — F o i f e i t a a n á l i s e p e l o m é t o d o d a c r i o - f r a t u r a d e c é l u l a s s a t é l i t e s e m 8 d i f e r e n t e s f a s e s d o d e s e n v o l v i m e n t o d o e m b r i ã o d e g a l i n h a , d o q u i n t o d i a à e c l o s ã o . A d i s p o s i ç ã o l a m i n a r c a r a c t e r í s t i c a d a s c é l u l a s f o i o b s e r v a d a i n i c i a l m e n t e n o s é t i m o d i a . F o r a m e n c o n t r a d a s j u n -ç õ e s d o t i p o « t i g h t » n o 20o
d i a d e i n c u b a ç ã o . P e q u e n o s g r u p o s o u a g r e g a d o s d e p a r t í c u l a s f o r a m d e s c r i t o s n o s é t i m o e o i t a v o d i a s , m a s n ã o t í p i c a s j u n ç õ e s « g a p » . V e s í c u l a s d e p i n o c i - ¬ t o s e f o r a m a p o n t a d a s n o s d i f e r e n t e s e s t á g i o s c o n s i d e r a d o s .
T h e satellite cells o f the s y m p a t h e t i c a n d s e n s o r y g a n g l i a h a v e b e e n e x t e n s i v e l y studied ( s e e P a n n e s e ? , f o r a r e v i e w ) . P a n n e s e et al.5,6 u s i n g the f r e e z e - f r a c t u r e t e c h n i q u e , a n a l y s e d the d e v e l o p i n g satellite cells in the spinal g a n g l i a o f c h i c k e m b r y o s , c o n s i d e r i n g p a r t i c u l a r l y the intercellular j u n c t i o n s and o t h e r m e m b r a n e s p e c i a l i z a t i o n s .
In the p r e s e n t p a p e r , w e s t u d y with the s a m e t e c h n i q u e the satellite cell m e m b r a n e s o f the l u m b a r s e n s o r y g a n g l i a o f the c h i c k e m b r y o in 8 s t a g e s o f d e v e -l o p m e n t , f r o m the 5th t o the 20th i n c u b a t i o n d a y s .
M A T E R I A L . A N D M E T H O D S
Fragments of lumbar sensory ganglia of chick embryos at the 5th, 7th, 8th, 10th, 14th, 16th, 18th and 20th days of incubation (three) specimens for each a g e ) were removed and fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer ( p H 7.2, 480 m O s ) at 4 ° C for 2 hr, frozen in liquid nitrogen-Freon 22, freeze-fractured (in a Balzers B A F 301 unit) and coated with carbon-platinum. The replicas were cleaned in 2.5% sodium hypochlorite and examined in a Phillips EM-201 electron microscope of the Heart Institute of the São Paulo Medical School.
T h i s w o r k w a s p e r f o r m e d i n t h e D e p a r t m e n t o f A n a t o m y , I n s t i t u t e o f B i o m e d i c a l S c i e n c e , U n i v e r s i t y o f S ã o P a u l o (* F u l l P r o f e s s o r , ** A s s i s t a n t P r o f e s s o r ) a n d s u p p o r t e d b y a g r a n t f r o m F A P E S P ( P B - 8 6 / 3 3 3 6 - 0 ) t o C . A . F . C . W e t h a n k P r o f . A n t o n i o S e s s o f o r t h e p r e p a r a t i o n o f t h e f r e e z e - f r a c t u r e r e p l i c a s .
R E S U L T S
Despite its apparent complexity the satellite cell surface was relatively smooth. A t the 5th incubation day the undifferentiated cells were intermingled with neuroblasts, showing
irregular shape (Fig. 1 A ) . A t the 7th incubation day the satellite cells acquired the characteristic flattened shape around the neuroblast (Fig. I B ) . Extensive laminar expansions
could be seen, forming a system of interdigitating processes already on the 8th day (Fig. 1C) and becoming more complex in later developmental stages. Tight-like junctions could
be observed at the 10th and 14th incubation days (Fig. 2 A ) . They were relatively isolated, describing slightly irregular trajectories and forming grooves on the E face whose botton
sometimes showed a single row of particles. Typical tight junctions were frequently seen close to hatching (Fig. 2 B ) . T h e y seemed to be present mainly between the laminar
processes of the satellite cells. Particle islets (gap-like junctions) (Fig. 2C) were observed at the, 7th incubation day, although they were more characteristic at the 8th day (E'ig. 2 D ) .
In other stages studied, gap or gap-like junctions were not apparent.
Pinocytotic-like vesicles were seen in variable amount in all embryonic stages, being more numerous and concentrated in certain areas, probably near to the neuroblasts or
nerve cells (Fig. 2 E ) .
C O M M E N T S
T y p i c a l tight j u n c t i o n s b e t w e e n satellite cells similar to t h o s e w e f o u n d o n the 2Cth i n c u b a t i o n d a y are not refered b y P a n n e s e et al.5.6. H o w e v e r , they mention the p r e s e n c e o f s h o r t s t r a n d s , w h i c h a p p e a r e d as r i d g e s a b o u t 15nm thick o n the P face and as thinner g r o o v e s on the E f a c e, i s o l a t e d o r g r o u p e d under v a r i o u s a n g l e s , w h i c h c o u l d c o r r e s p o n d to the structures w e d e s c r i b e d as tight-like j u n c t i o n s at the 14th d a y . Revel et al.9 and D e c k e r and Friend 3 interpreted t h o s e r i d g e s a s r e m n a n t s o f w e l l d e v e l o p e d tight j u n c t i o n s . T y p i c a l tight j u n c t i o n s did not exist in our material b e f o r e the 20th e m b r y o n i c d a y . M o r e than r e m n a n t s o r rudiments, these tight-like j u n c t i o n s 5 c o u l d r e p r e s e n t d e v e l o p i n g tight j u n c t i o n s , s i n c e they w e r e s e e n in o u r material o n l y at the 10th and 14th d a y s , w h i l e typical tight j u n c t i o n s w e r e f o u n d o n l y on the 20th d a y . Similar s t r a n d s ( g r o o v e s o n the E f a c e , a c c o r d i n g t o P a n n e s e 5 ) p r o b a b l y link m e c h a n i c a l l y a d j a c e n t satellite cells and p e r s i s t even in the adult f o w l .
T y p i c a l g a p j u n c t i o n s w e r e not o b s e r v e d in the p r e s e n t s t u d y . O n l y small g r o u p s o r i r r e g u l a r a g g r e g a t e s o f p a r t i c l e s w h i c h w e n a m e d g a p - l i k e j u n c t i o n s w e r e f o u n d at the 7th and 8th d a y s . P a n n e s e 5 o b s e r v e d these s t r u c t u r e s v e r y s e l d o m and interpreted them as a result of technical failure.
W h e t h e r such s t r u c t u r e s a s s u m e the functional r o l e of real g a p j u n c t i o n s it c o u l d n o t be d e c i d e d here. G a p j u n c t i o n s a l l o w i o n s and small m o l é c u l e s to p a s s from o n e cell to a n o t h e r 1,2,8 and m i g h t have a particular role d u r i n g d e v e l o p m e n t4
.
R E F E R E N C E S
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