Table Of ContentZOOLOGICAL SCIENCE 10: 971-976 (1993) © 1993 ZoologicalSocietyofJapan
In vitro Evidence for a Neural Factor(s) Involved in the
Proliferation of Adenohypophysial Primordial
Cells in Fetal Rats
Yuichi G. Watanabe and Manabu Shirai
Department ofBiology, Faculty ofScience, Niigata University
Niigata 950-21, Japan
—
ABSTRACT Our previous in vitro data have shown that proliferative activity of adenohypophysial
primordial cells ofthe rat drops markedly after removal ofthe developing diencephalic floor. In this
study, we transplanted the diencephalicfloorto an area ofthe adenohypophysial primordium thathad
originally not been in contact with the brain. Thistransplantation experiment was carried out on day
13.5 of fetal age. when most of the proliferating adenohypophysial cells are directed toward the
developing brain. DNA-replicating cells in the culture explants were revealed one day later by
incorporation of bromodeoxyuridine (BrDU) followed by its detection by the use of a monoclonal
antibody.
The results ofthe transplantationexperimentsdiffered, dependingon which partofthe primordium
came in contact with the neural tissue. Ifthe diencephalic floorwas attached to the dorsal halfofthe
adenohypophysial primordium, a new brain-dependent pattern ofcell proliferation took place in the
explants. Transplantation of the diencephalic floor, on the other hand, to the ventral half of the
developing adenohypophysis resulted in a decrease in the proliferative rate of the adenohypophysial
cells. The cell proliferative activity ofbrain-deprived adenohypophysial explants was very low. Thus
the results ofour study show that the presumptive neural factor(s) contained in the developing brain
affects only dorsally located cells ofthe adenohypophysial primordium.
half of the adenohypophysial primordium [15].
INTRODUCTION
This may be interpreted as 1) the restricted diffu-
The epithelio-mesenchymal interaction is the sion of the presumptive neural factor or 2) the
most well known and extensively studied subject different responsiveness of primordial cells to the
that is essential for morphogenesis of most organs neural factor. To decide which of the two holds
ofepithelialorigin. Whereasdevelopmentofsome true, we investigated whether the transplantation
epithelialtissuesis underthe inductive influenceof ofthediencephalicfloorchangesthepatternofcell
the brain. Forexample, morphogenesisofthe lens proliferation in the developing adenohypophysis.
depends on the presence ofthe opticvesicle, oran
outpocketing of the diencephalic wall [1, 9, 23]. MATERIALS AND METHODS
Since the adenohypophysis arises in close associa-
tion with the diencephalicfloor [14], the possibility
Animals
of a neural influence was investigated; and the
results revealed a crucial role of the diencephalic Sexually mature rats of the Sprague-Dawley
floor in both proliferation [15] and differentiation strain were mated at night. If spermatozoa were
[20. 21] of adenohypophysial primordial cells in found in the vaginal smears the next morning,
the rat. At the early stage of development, cell noon of that day was designated as day 0.5 of
proliferation occurs predominantly in the dorsal gestation.
Organ culture
Accepted August 9. 1993
Received March 24, 1993 Pregnant rats on day 13.5 were anesthetized
BUUrifiOBB
972 Y. G. Watanabe and M. Shirai
brain tissue was left to know the original brain-
adenohypophysial contacting site. Each explant
was placed on a piece of cellulose acetate mem-
brane and cultured in a dish for organ culture
(Falcon, no. 3037). Asdescribedelsewhere [15], a
mark was made on the acetate membrane so that
the orientation ofthe culture explants in histolog-
ical sections would be known. Cultures were
stomodeum
maintained in dvlEM containing 0.1% fetal calf
mM
Fig. 1. Diagramillustratingthetopologicalrelationship serum and 30 glucose. One day after organ
between the adenohypophysial primordium and culture, bromodeoxyuridine (BrDU) was addedto
sbirtaeisn((DBL),orVMme)setnochwhyimceh(sbtriapipnleodrarmeeas),enacnhdymtahle the medium at a concentration of6//g/ml. Three
tissue wastransplanted. DM, DLand VM indicate hours later explants were fixed overnight in
the dorso-medial, dorso-lateral and ventro-medial Bouin's solution. The cellulose acetate mem-
regions ofthe primordium, respectively. branes were removed in the course ofdehydration
in ethanol.
Immunohistochemistry
with ketamine hydrochloride solution. After
laparotomy, fetuseswere removedone by one and Afterembeddingin Paraplast, sectionswere cut
the basal part of the diencephalic vesicle with the at 2fim with the use of glass knives. Depara-
adenohypophysial primordium (Fig. 1) was iso- ffinizedsectionswereincubatedwithamonoclonal
lated in Ca- and Mg-free Hanks solutions with the antibody against BrDU for 1 hr and then with
aid of a dissecting microscope. Primordial tissue peroxidase-conjugated anti-mouse IgGfor30min.
wasthen treatedwith amixture ofan equalpartof The reaction product was then visualized with
0.3% collagenase (Sigma, type V) and serum-free 3,3'-diaminobenzidine tetrahydrochloride solution
MEM (Gibco, Grand Island, New York) at room containingH2 2. The antiseraandBrDUsolution
temperature. Separation of brain and/or mesen- were purchased from Amersham(UK). Some sec-
chyme was done under a dissecting microscope tionswere simply stained in Caracci's hematoxylin
with the aid of fine watchmaker's forceps. After solution to confirm the results of the transplanta-
about 10minutes of enzyme treatment, different tion. Ineach explant, the largest profile ofsection
combinations ofculture explants were prepared as was selected and the number of BrDU-labelled
shown in Table 1. On occasion, a fragment of nuclei was counted at a magnification of 400X.
Table1. Incidence of BrDU-labelled cells in adenohypophysial explants in different culture
conditions
Tissues co-cultured Dorsal half
Ventral
n
Brain Mesenchyme Left Right half
—
intact 6 133.0±7.6 111.3+10.4 18.9+2.5
— —
4 15.5+1.3 12.0+ 2.3 5.0±2.4
—
transplanted 3 35.3+4.3 266.0+20.5* 10.3±3.4
to area DL
—
transplanVteMd 3 8.3±3.2 6.3+ 0.9 8.7+1.2*
to area
—
intact 3 12.0+4.4 7.3+ 1.5 4.3+1.9
(area DL-VM)
Values are means±S.E.
*, brain-transplanted portion.
For explanations of areas DL and VM, see Figure 1.
Brain and Pituitary Cell Proliferation 973
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Figs. 2-7. Sectionsthroughratadenohypophysialprimordiaseparatedonfetalday 13.5andculturedfor2days. All
of these sections except that in Figure5 were immunostained with anti-BrDU that had been incorporated 3h
beforefixation. Figuresareallorientedwiththedorsalsideoftheprimordiumatthetop. X100. Fig. 2. Explant
culturedwithapartofbrain(B). MostofBrDU-labelledcellsareseeninthedorsalhalfoftheprimordium. Fig.
3. Explant cultured without brain tissue. The incidence ofBrDU-labelledcellsislow. Fig. 4. Explantcultured
with mesenchyme (M). Adenohypophysis contains a small number of labelled cells. Fig. 5. Explant after
transplantation ofthe brain (TB) to thedorso-lateralregionoftheprimordium. Inthisexplantasmallfragment
of brain tissue (arrow) was intentionally left to confirm the original site where the brain was attached to
adenohypophysis. Hematoxylin stain. Fig. 6. A consecutive section of the explant shown in Figure5.
BrDU-labelled cells are observed toward the transplanted brain (TB). The boundary of the brain and
adenohypophysis is shown by arrowheads. Fig. 7. Explant after transplantation of the brain (TB) to the
ventro-medial part of the primordium. The incidence oflabelled cells is very low.
mim
974 Y. G. Watanabe and M. Shirai
The incidence of labelled cells was compared in ment ofthe adenohypophysis and lens. The rudi-
different three regions, or the ventral and dorsal ments of both of these organs thicken and invagi-
(right and left) halves ofadenohypophysial tissue. nate keeping in contact with the outgrowth of the
diencephalic vesicle. Development ofthe lens has
been studied more frequently than that of the
RESULTS
adenohypophysis. The experimental evidence in-
When the diencephalic floor was left intact, dicates that the optic vesicle of the diencephalon
many BrDU-labelled cells were observed in ade- induces formation ofthe lens in vertebrates [1, 7,
nohypophysial tissue (Table 1). Most of these 9, 10, 18]. Onthe otherhand, mesenchymaltissue
labelled cells were in the dorso-medial (DM) re- is believed to hindernormal morphogenesis ofthe
gion ofthe adenohypophysis, where the brain was lens. In fact mesenchymal cells located between
attached (Fig. 2). Inhematoxylin-stainedsections, the opticvesicle and lens placode become necrotic
mitotic figures were frequently seen. Enzymatic and are resorbed in a normal mouse fetus [17].
removal ofthe diencephalicfloor and mesenchym- Further, in anophthalmic mice mesenchymal cells
al tissue resulted in a marked decrease in the rate fail to degenerate and consequently hinder the
of cell labelling (Fig. 3 and Table 1). This pro- intimate contact of the optic vesicle and lens
found decrease in the incidence of labelled cells placode [17] which is necessary for normal de-
was also observed in explants with mesenchyme velopment of the lens.
left intact (Fig. 4 and Table 1). In these explants, It has been generally accepted that an early
mesenchymaltissuewasincontactwithmostofthe event in the differentiation ofa rudiment exposed
margin of the adenohypophysis including the dor- to aninductivestimulusisanincreaseintherateof
so-medial region where the brain was attached. mitosis of the primordial cells [6, 12, 13, 24]. In
Although labelled cells were slightly more numer- the present study, the rate of proliferation of
ous in number in the dorsal part of the adenohy- adenohypophysial primordial cells increased
pophysis than elsewhere, the overall labellinginci- markedly in the presence of the brain.
dence was far less when compared with those In a series of in vitro experiments using fetal
explants maintained with the brain. rats, we have found that the developing dienceph-
Transplantation of the diencephalic floor to the alic floor is essential for the proliferation and dif-
dorso-lateral (DL) part of the adenohypophysial ferentiation ofadenohypophysial cells [15, 16, 20,
primordium (Fig. 5) caused a remarkable change 21]. The results of the present study further
in the pattern of cell proliferation. The dorso- suggest that the developing diencephalon contains
medial part (DM) ofthe adenohypophysial tissue, a neural factor that stimulates the early prolifera-
where the brainwasoriginally attached, contained tion of the adenohypophysial primordial cells.
only a small number oflabelled cells. In contrast, Although it is not known how this neural factor
many labelled cells were seen in the new brain- reaches the adenohypophysial tissue, it seems of
contact area of adenohypophysial tissue (Fig. 6 interesttocomparethepatternofcellproliferation
and Table 1). This change in localization of pro- of our explants with that of growth of other
liferating cells was observed only when the primordia that also require an inductive influence
diencephalic floor was transplanted to the dorsal in vitro. In a transfilter culture experiment, cell
half of the adenohypophysial primordium. The proliferation of metanephrogenic mesenchyme
incidence of BrDU-labelled cells was quite low wasnotconfinedtotheareaofthemembranefilter
when the brain was removed and attached to the whose undersurface faced the inductor; thus, 3H-
ventro-medial area (VM) of the primordium (Fig. incorporating metanephrogenic cells were distrib-
7 and Table 1). uted homogeneously in these explants [13]. On
the other hand, most of 3H-thymidine labelled
cellsinthe stimulatedpancreaticprimordiumwere
DISCUSSION
localized near the peripheral region of the ex-
There is a close similarity between the develop- plants, without any topological relation to the
Brain and Pituitary Cell Proliferation 975
inductive stimulus [24]. In view ofthese observa- Apart from the exact mechanism of such cell
tions together with our finding, it may be general proliferation, itseemsofinterestthatmosttypesof
phenomenonthatthestimuluswhichaugmentsthe hormone-producingcellsfirst appearin theventral
proliferation rate of primordial cells is somehow half of the developing adenohypophysis in the rat
transmitted even to those cells that are not in [11, 22]. The nature ofthe BrDU-labelled cells is
direct contact with the inductive tissue. not known since no cells are reactive to antiserato
It is of interest to compare the results of our adenohypophysial hormones at the developmental
experiment with studies done on the human stage examined. In a prolonged culture experi-
anencephalic fetuses whose adenohypophysial pri- ment, we have observed that some ofthese label-
mordium often failsto contactwith the brain [2-4, led cells differentiated to LH, ACTH and PRL
8]. The adenohypophysisin anencephalicbabiesis cells (Shirai and Watanabe, unpublished observa-
extremelyvariable in size [2]. The bloodsinusoids tion).
in the anencephalic hypophysis are much greater In the present transplantation experiments, our
than in normal glands [8]. This means that a mere interest was focused on the diencephalic floor
comparison of the organ weights in question is which is destined to make contact with the ade-
insufficientforevaluation oftissue growth. There- nohypophysialrudiment. Itisofinteresttoinquire
fore, the volume of the anencephalic adenohy- whetheronlythisregion ofthebrain orotherparts
pophysis was estimated after correction for the of the brain also have a stimulatory influence on
amountofbloodinthe sinusoidsanddisclosedthat proliferationofadenohypophysialprimordialcells.
the adenohypophysial malgrowth in anencephaly The size ofadenohypophysial explantsco-cultured
was not observable until 7 months offetal age [4]. withpart ofthe telencephalicroofwas remarkably
In view of the data indicating that the brain- smallerthan that ofcultures keptwith the anterior
adenohypophysial relationship develops rather or posterior diencephalic floor [5]. Our most
normally atthe earlyfetal stage [19], initialgrowth recent experiments have shown that brain tissues
oftheadenohypophysismaytakeplace atanormal other than the diencephalic floor had little effect
rate in anencephalic babies. Further studies are on cell proliferation of the adenohypophysial pri-
necessary to investigate to what extent the brain is mordium [16].
involved in growth ofthe human adenohypophysis
in congenital anomalies. ACKNOWLEDGMENTS
The developing adenohypophysial primordium
offetal rats can be roughly divided into two parts We would like to thankDr. M. Fingerman forcritical
reading ofthe manuscript.
on the basis of the rate of cell proliferation [15].
The higher rate of cell proliferation in the dorsal
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