In vitro Evidence for a Neural Factor(s) Involved in the Proliferation of Adenohypophysial Primordial Cells in Fetal Rats PDF
Preview In vitro Evidence for a Neural Factor(s) Involved in the Proliferation of Adenohypophysial Primordial Cells in Fetal Rats
ZOOLOGICAL 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 4" »* _ ^«V-" • 9 B ** ** £<3Lla s 1 •••> ;<•?*£••-_*4?f."vf> *•"*_*»,* o«^.o°";" * . . '3* * .*» * " r * "Z * <s»»p-* -d -', -• ?. . -** * <i • " . ^ • f « , '' 2 6 - - • NT- •r M w— * »aa * t>°*! "c ° t npl^& v% > - 9-? ' ' * a• * 9 4 ^ «' M - **"* - "' >l «* - -< ^ --. •«. • 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 (adneural) half of the primordium was already REFERENCES discussed as above in relation to the brain. The 1 AlexanderLE (1937) An experimentalstudyofthe ventral half of the adenohypophysial primordium, role of optic cup and overlying ectoderm in lens on the other hand, has only a small number of formation in the chick embryo. J Exp Zool 75: 41- proliferating cells. 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