The migration of neuroblasts in the developing cerebral cortex

19 Pages·2005·4.07 MB·English

J. Anoat. (1965), 99, 4, pp. 691-709 691
With 12 figures
Prinited inl Great Britain
The migration of neuroblasts in the developing
cerebral cortex
BY M. BERRY AND A. W. ROGERS*
Department ofAniatomy, Birminghamt
INTRODUCTION
Tilney (1933) was the first to make a detailed histological study of Nissl-stained
preparations of the developing cortex of the rat. He concluded that, after their
formation in the ependymal and mantle layers, the neuroblasts migrated to take up
definitive positions in the developing cerebrum. He described the cellular migra-
tions as occurring in three distinct waves; the first over the period from the 16th
to the 17th day, the second from the 18th to the 19th, and the third from the 20th
to the 21st, or last day of gestation, respectively. The neuroblasts involved in the
first migration formed a lamina of cells below the pial surface called the 'primary
granular lamina' which was later seen togive rise to the two superficial layers ofthe
adult cortex, layers II and III. A 'secondary granular lamina' was described as
being formed in a similar manner, by a second migration of cells, and was said to
differentiate later into layer IV of the adult cortex. Lastly, the neuroblasts of the
third migration established the most deeply situated laminae, layers V and VI.
Inastudy employingan autoradiographic technique inwhich tritiated thymidine
was used, Angevine & Sidman (1961) concluded that the pattern ofmigration ofthe
neuroblasts of the developing cerebral cortex was the reverse of that proposed by
Tilney. Angevine & Sidman found that tritiated thymidine was incorporated into
thecellsofthe ependymal andmantlelayers andthatthe neuroblasts labelled inthe
early stages of development were later found in the infragranular laminae (layers
V and VI), while neuroblasts labelled later subsequently appeared in the granular
and supragranular laminate (layers IV, III and II).
Berry & Eayrs (1963) studied the teratogenic effects of X-irradiation on the
developing cerebral cortex of the rat. Many of the abnormalities they observed
were also inconsistent with the sequence of events described by Tilney. For in-
stance, a dose of X-ravs delivered on the 19th day of gestation destroyed the
ependymal and mantle layers but left the migrated cells in the more superficial part
of the cortex unharmed. On Tilney's hypothesis these surviving cells should later
have formed layers II, III and IV of the adult cortex. In fact, quantitative and
qualitative examination ofthe X-irradiated cortices at 30 dayspostpartum showed
that layers II, III and IV were almost completely absent, while layers V and VI
appeared relatively normal. Like Angevine & Sidman, Berry & Eayrs also con-
cluded that the first neuroblasts to be produced form the infragranular layers, and
that the neuroblasts of later divisions must migrate through these laminae to give
rise to the more superficial cellular layers of the adult cortex.
* Present address: Molecular Enzymology Unit, Department of Biochemical Pharmacology,
State University ofNew York, Buffalo, N.Y., U.S.A.
692 M. BERRY AND A. W. ROGERS
Reported here are the results ofexperiments givingmore details ofthemigratory
pattern of cortical neuroblasts and which offer an explanation of the mechanism
underlying the processes of histogenesis and differentiation in the cerebral cortex.
Preliminary reports of some of our findings have already been published (Berry,
Rogers & Eayrs, 1964a, b).
-MATERIALS -A'NI) AIETHODS
I. Animals
Albino rats from the colonv of the Anatomy Department at Birmingham were
used. Females were placed with males and the day ofmating determined by daily
examination of vaginal smears.
Table 1. The number ofanimals usedfor autoradiography (injected animals) an(l the
number ofadult breedingfemales (V)froms which the litters were obtained (see text for
further explanation)
D)ay of Time after injection at which animals were killed
injection ,-k-- A
(post-conception) 4Ih 2d 4d (id 8 d 10 d 30 d
16 1 1 1 1 1 1 1
17 1 1 1 1 1 1 1
1 1' 1T :. 1 1 10
18 1 1 1 1 1 1 1
14 1 $ 1± 1 1 ] 1
19 1 1 1 1 1 1 1
2 1 1.
21 1 1 1 1 1 1 1
11 1 1 $1
21 1 1 1 1 1 1 1
1~<' 1 V 1 .' 1 hi'
22 1 1 1 1 1 1 1
1 1 1
Total (1) Us, 34; (2) injected animals, 49
On selected days of gestation, each female rat was anaesthetized with an intra-
peritoneal injection of Avertin, and the abdomen opened with a mid-ventral
incision. The gravid uterus was delivered through the incision and each foetus given
an injection of tritiated thymidine (Radio-chemical Centre, Amersham, product
TRA 61) through the wall of the uterus into the amniotic cavity. The uterus was
returned to the peritoneal cavity, and the abdomen closed with sutures.
The female rats were killed with chloroform at a specific time after injection, the
foetuses removed and their brains fixed for histological processing (for details see
Table 1). Neonatal animals were killed by decapitation and the brains removed
immediately for fixation.
Migration of neuroblasts in developing cortex 693
II. Tritiated thymiidilne
The tritiated thymidine was diluted with sterile distilled watertogive an activity
of0.5 mc/ml, and stored at -20 0C. Recently, Evans &Stanford (1963) have shown
that these conditions ofstorage are not optimal for tritiated thymidine, which may
undergo radiation decomposition. The majority of the injections were given within
1 month ofthe receipt ofthe thymidine, and the longest time that elapsed between
receipt and injectionwas 5 months, whichwas applicable totwofemalerats only. It
is thus unlikely that radiation decomposition during storage should have affected
the experiments significantly.
The following scale of dosages was used. Foetuses aged 16 d post-conception
(hereafter p.c.) received 10Iac each; those aged 17 and 18 d p.c. 15 jtc each; those
aged 19 and 20 d p.c. 20,uc each; and those aged 21 and 22 d p.c. and neonatal
animals, 25 ,tc each. These dosages were found empirically to give adequate
labelling in the material required for autoradiography.
Animals injected on each ofthe days listed above were sacrificed at the following
times after injection: 4 h, 2, 4, 6, 8, 10 and 30 d.
III. Histological procedures
(i) Autoradiographs. The brains were fixed in formal saline, embedded in paraffin
wax, and serial sections were cut in the coronal plane at 7,u. Those sections which
included the neocortex at about the level of the interventricular foramen were
chosen for autoradiography. These sections were mounted on gelatinized slides,
dewaxed inxylene, andtakenthroughdecreasingconcentrations ofalcoholtowater.
(ii) Nissl preparations. Brains were fixed in formal saline for 12 h, embedded in
paraffin wax, sectioned at 51t, and the sections stained with cresyl violet.
(iii) Silver preparations. Brains were either fixed in a 25% solution of chloral
hydrate in 50 0 alcohol and later impregnated by the technique ofNonidez (1939),
or fixed in formal saline and subsequently treated with Romanes's stain. In each
case the brains were embedded in paraffin wax and sectioned at 51t.
(iv) Golgi-Coxpreparations. Brains were fixed in a chromate solution, embedded
in celloidin, sectioned at 1501t, and treated by the method of Golgi-Cox as modified
by Sholl (1953).
(v) Fresh preparations. The brains, of embryos freshly removed from the uterus,
were carefully dissected to separate the cortex from the corpus striatum. The tissue
was then washed in normal saline, and disintegrated by gentle pressure applied to a
cover-slip placed on the slide. The preparations were examined immediately with
a phase-contrast microscope.
IV. Autoradiographic procedures
Initially, slides were coated with emulsion and exposed, and the sections
were stained later through the processed emulsion. Occasional batches of auto-
radiographs prepared in this manner showed high and variable levels ofbackground
grains, which were interpreted as being due to chemical interaction between the
section and the emulsion. In order to obviate these chemographic artefacts, the
43 Anat. 99
694 M. BERRY AND A. W. ROGERS
majorityofthesections werestainedfor2 mininHarris'shaematoxylinimmediately
after dewaxing, washed in running water for 10 min, and placed in distilled water.
Then, using a technique described by Sawicki & Darzynkiewicz (1963), the sections
were coated with a thin layer of polyvinyl chloride; this was thin enough not to
reduce appreciably the number of beta particles reaching the emulsion, and was
impermeable enough to prevent the chemography which affected earlier auto-
radiographs. The emulsion was subsequently applied over the polyvinyl layer.
Ilford G5 emulsion in gel form was chosen for this experiment. K2 emulsion is
sensitive enough for the detection of tritium, and has a significantly lower back-
ground than G5. But in this instance itwas desired to view the autoradiographs at
low magnification, and the larger grain size of G5 made it more suitable.
Autoradiographs were prepared in the following way. Under safe-lighting, 15 ml
of G5 emulsion were placed in a measuring cylinder standing in a thermostatically
controlled water bath at 43 'C. After 10 min gentle stirring, the molten emulsion
was added to 20 ml distilled watertowhich 0 35 ml glycerol (also at 43 'C) hadbeen
added. After gentle mixing, the molten diluted emulsion was cooled to room
temperature, the slides were dipped once in the emulsion and allowed to dry
standing upright. The slides were then placed overnight in a desiccator containing
dried silica gel and filled with carbon dioxide. The following day they were trans-
ferred to slide boxes and stored at 4 'C behind lead shielding for exposure periods
of between 2 and 3 weeks.
Development took place in an Amidol developer described by Dainton, Gattiker
& Lock (1951). This was diluted with an equal volume ofdistilled waterbefore use.
The development time was 8 min, and the temperature of the solution 18-20 'C.
Fixation followed in a 30 0 (w/v) solution ofsodium thiosulphate for 8 min. After
washing, the autoradiographs were dehydrated in increasing concentrations of
alcohol, cleared in xylene, and mounted in D.P.X.
In constructing the diagrams representing the distribution of labelled cells
(Figs. 1-5), autoradiographs were examined under the microscope, and the position
of each heavily labelled cell was marked in the appropriate region ofcortex. These
diagrams therefore indicate the relative numbers of heavily labelled cells at each
level in the cortex.
RESULTS
I. Autoradiographicfindings
In all the autoradiographs from animals sacrificed 4 h after the injection of
tritiated thymidine, labelled cells in the cerebral cortex were found only in the
ependymal and mantle layers.
(i) Foetuses injected at 16 d p.c.
Figure 1 illustrates, in schematic form, the distribution of labelled cells at the
various times after injection. At 16 d p.c., the cortex consists of little more than
ependymal and mantle layers, the latter containing the higher proportion of
labelled cells. By 18 d p.c. the cortex has become considerably thicker, and there
are labelled cells throughout its entire depth (Fig. 2). By 20 d p.c., the most
heavily labelled cells areconcentrated in aband across thecortex, at thejunctionof
Migration of neuroblasts in developing cortex 695
the superficial and middle layers. These cells are presumably the product ofthe first
cell division following the introduction of tritiated thymidine into the animal. No
cells with comparably heavy labelling remain in the ependymal or subependymal
layers. Cells with noticeably fewer silver grains over their nuclei occur throughout
the cortex, and are presumably the products oflater divisions which have migrated
more superficially.
In neonatal animals, 6 d after injection, the band of most heavily labelled cells
lies still deeperinthecortex (Fig. 2) andby20dafterbirthhas cometorestabovethe
corpus callosum, in aregion occupied by layerVI in the adult cortex. By this stage
there is practically no labelling, above the background level, in other regions ofthe
neocortex.
Zonal layer
Superficial = . - = Ill
layer Day aV
~~~~Va
-.* .
*0O
Middle
layer .* ce . Vb
Deep
layer 010~~~~~~0*~~~~~~0
0 0 *91
a .to;:' Vlb
* *0 00 :~A~~o~1~~1~' *
.... 0 __
Mantl1e. D r Corp.
layer Forv call..
Ependyma be_w__e______ _ rup o the basi ofthes Epend
Cortex 4h 2 46 8 10 36 Adult
ofthe Days after injection cortex
embryo
Fig. 1. Diagrammatic representation ofthe migration oflabelled cellsaftertheinjection
oftritiatedthymidine onthe 16th dayofgestation. Thefrequency ofdotsineachsection
ofthe diagram indicates approximately the relative numbers ofheavily labelled cells in
each corticallayer. Forvarious technical reasons strictquantitativecomparisons should
not be made between different groups on the basis ofthese data.
(ii) Foetuses injected at 17 d p.c.
Figure 3 illustrates the pattern ofdistribution oflabelled cells in animals injected
on the 17th day of gestation.
Two days after injection, labelled cells are seen throughout the cortex, but, by
4 d after injection a band ofheavily labelled cells has formed in the superficial layer
of the cortex. In all subsequent specimens, this band lies in the middle cortical
layer, whereitdevelopspredominantly intolayerVoftheadultcortex. Thelabelled
cells lying superficial to this band have progressively fewer silver grains overthem:
those lying between this band and the developing corpus callosum are unlabelled.
43-2
. BERRY AND A. W. ROGERS
k, .' Wst.G._.,
Ao- -i
V..a
....... _ .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .'V-14w 46;0 av
v._ 4"k A,: ....'.
4*
A.-4 h after injection
*
B.-2 days after injection
li2 2. Ic miiiiratintioftitiit'nllaslst Iftcr iiijelititllni Il-t>iiii(lilit
nit lIe 1(it zth'iIinI slatioll (.\litorigalizol-'a s. ianell)(iixylill
sI<liticl. x220). 1E1(11 tieirngrapilihlis c('tt piliitngraiilictl ifn (untiI-
lilt(l tIrallsiiilt(i-l and ilwi(tclnl lijidtilnj: Silver g-ra.lins lltlote
lippealr is lritilil sptl(slgahitist t1e still(it S('(tIitl. The1ptositiotn of'
IoI)"t I(.IviIv I'lI (,I(l (~tsI#xlI)(I{Ii(hecomIs)a1-c(I itII ~ItIzI II iI
til 1.. 4II .li'tcriiiJecititn 1;la(Aleli (iclls a-( si-i-rl in) thci IiairitIc
lasyti'. It. 2(I ifter iiji-ctioris lit-it-hl :ll)(Allcil ((AIS ait (lisiiise(il
lirniiloiiiiit tIllc v(olex, iti(li(iitillo t(hit the> art Iniiigntilng to the
vortieb'd smz 'lzec. wht1ere :labelle l cclea(ll^<1w Sce.-mr:vil1Xif1(i thezmml.
lavtcr. C. (i 11(f1't itljetlmu t-wI letsvlx lahielled(l 1n1clei 1t1)5 Iie
depj in 1f1i ticmt x. Note Ii rniticll silihipc ofI'(tlIleulei ini t(li
hlixnl 'ix lilcfl(Alil 111(c1 tllu( tl' sititilr. sliidlic-slilieAl i1()6lI'f the
miiir timllt lIs l wciseen Isli1)ax(iilthe -itic-er11i1lt'tiIiilltc'x.
ThC|e N<M\\>loCIr ol' ltI hI IJi'sl ol Ini>ur-'t(A( ;lallco:e-l celix is wc(1l
sliitil (fl-ro(ste). Th' ctills whicllilitxitiii (el Sitlbscilunlt liot1w
intJiii i-alitlshi1tt1)h'-st veinlili gv ieraions( lvitls l-st-(tils stillo)
teIw1a.lvXilv 1.11})el(4A (-c}1 ;llalnidlm u(i shive*tl1h lmlls eB Inve-(rrejla;illss
S0111C 11'111lioael(ivitv 1f01 SC(Tid~l oviier~aticnis. thcxes iwwXer e^(Als stll
P>SSCSS S1111111 trl(.(,s 0o the hil:lel
C.-6 days after injection
Migration of neuroblasts in developing cortex 697
(iii) Foetuses injected at 18 d p.c.
Figure 4 illustrates the pattern ofdistribution oflabelled cells in animals injected
on the 18th day p.c. It is very similar in general to the pattern seen after injection
at 17 d p.c., but the band of heavily labelled cells lies near the pial surface from
6 d after injection, and gives rise predominantly to layer IV of the adult cortex,
though a few heavily labelled cells are found in layers III and V.
Zonal layer :0. 9* ' e .
Superficial
layerA. * e e X e
C. C~~~~~~~~~~~~~~~
C C~~~~~~~~~~
Middle 9*I
layer a ieo Vb
* 0 e*
0**
4h *rme2presentation4 t 06 8 35e Adult
I
Deep * 0 V0
layer
o. VMb
Mantle ..*: * Corpus
layer or mh callosumr
Germinal layer e a 19,* 2 1 Epedym
4h 2 4 6 8 35 Adult
Days after injection Cortex
Fig. 3. Diagrammatic representation ofthe migration oflabelled cells after injection of
tritiatedthyrmidine onthe 17thdayofgestation. (Seelegendto Fig. 1 andM~aterials and
Methods for method of construction.)
(iv,) Foetuses injected at 19, 20 and 21 d p.c.
The pattern of migration and distribution of labelled cells is similar in animals
injected on these three days, and is illustrated by Fig. 5. Two days after injection
heavilylabelledcellsareseenthroughoutthecortex. By4-6d afterinjectionheavily
labelled cells form a band in the most superficial layers ofthe cortex (Fig. 6). These
labelled cells later differentiate into layers II and III. There appears to be no differ-
ence inthedistribution oflabelled cells withinlayers II and IIIbetweentheanimals
injected on the 19th, 20th or 21st days ofgestation. In all these groups ofanimals
significantly labelled cells persist in the ependymal layer over the period studied.
(v-) Animnals injected at 22 d p.c.
The pattern ofdistribution oflabelled cells in this group ofanimals is illustrated
in Fig. 7. As inothergroups, labellingat 4 hislimited totheependymal andmantle
layers, and at 2 d is found throughout the deeper layers. However, the pattern at
subsequent time intervals is quite different from that seen in all other groups of
698 M. BERRY A. W. ROGERS
AND
animals. Thus, from 4dafterinjectiononwards, labelledcellsappeartoberandomly
distributed throughout the entire cortex.
The results of the autoradiographic study are summarized in Fig. 8.
Zonal layer
Superlfical :: ** SS*
layer-*0 * * ~ : 5 ~ I
~~~Va
0. OS 55 . . *
Middle 0
0V0
layer
Deep SVIa
layer SV
Mantle :: oCorp~.
layer :~~~~~~~~~~~~~~~:.:~~S~~~~~~~~call.
Ependyma ~ pend.
Cortexof 4 h 2 4 6 8 10 34 Adult
the embryo Days after injection cortex
Fig. 4. Diagrammaticrepresentationofthemigrationoflabelledcellsaftertheinjection
oftritiatedthymidine onthe 18th day ofgestation. (See legendto Fig. 1 and Materials
andMethods for method ofconstruction.)
Zonal layer , l
Superficial
layer.0.
XIV
.__ * 0* * *
~~~~~V
a
S
Middle a
layer Vb
Deep VVa
layer *VIb
Mantle : Corpus
layer 00 callosum
Germinal layer-..,e-.-.t-. I .... 5. 0 .S-0..0' - '. .1.1'Aw*@ 0: E_pend.yma
4 h 2 4 6 8 10 33 Adult
Days after injection cortex
Fig. 5. Diagrammaticrepresentation ofthemigrationoflabelledcellsaftertheinjection
oftritiatedthymidineonthe19thdayofgestation;asimilarpatternofmigrationoccurred
onthe 20thand21stdaysofgestation. (SeelegendtoFig. 1 andMaterials andMethods
formethod ofconstruction.)
Migration of neuroblasts in developing cortex 699
AMRm
w.
A.-4 h after injection
l6.f. I 11c 11iol"Iltioll oi -,lcls 1:161el(el (m1 tiw I9!th
(1l1\ of g(stationl (AuItorlaiogrIapls IltlllIottx\lill
stttit'tli. x220). l,<lltc11((l (t1s airt Stct fll tilt' r-
oi al l 1 Ii tt l 311 l - ii li
( \) aitti (tofweltlt 'ltd1l witilill tilt stij.wagraiii iiai
la.x cs l6(I laIt(t (1). It is a1rgwed( thaIt ill o tl to
..Ithaiill thciha supo.-I-fivi.11 positioll o\(.I. thle pe(l-iwl of'
( al1ter iIterli j tlt tel :1al.tlte tll it tantit
11 O11( l lilt'l sts fmitiX' su I 11,i 11, to Ill
III'iI tIc 1;1\(,r()IlwW1XIIIt (Ic,IlN. oto(sl-I i(m.
...... .i .o
B.-6 days after injection
M. BERRY A. W. ROGERS
700 AND
Zonal layer| I
Superficial | ]II
layer
I
Va
Middle *
layer I c
Gr~rel levy - ^ ^ a______Vb
Deep
layer **Vb
Mantle e * *eCorpus
layer *e ..ecallosumn
UcermInal layer* ,. . 0 Ependyma
22 24 26 28 30 41 Adult
Days after injection cortex
Fig. 7. Diagrammatic representation ofthe migration oflabelled cells afterinjection of
tritiatedthymidineonthe22nddayofgestation. (SeelegendtoFig. 1 andMaterialsand
Methods for method ofconstruction.)
co
_-
la t
;t!
.-
.0_
W
0
a..
0-
Zonal I
Mantle la
Ependymal
I I 24 I 25 26 127 28
Days afterconception
Fig. 8. Summary of the migratory pattern of neuroblasts during the period of histo-
genesisofthecerebralcortex.Thesignals V, V, 0 and 0representtherelativepositions
of cells after labelling on the 16th, 17th, 18th, and 19th-21st days of gestation
respectively.