Abstract Tissue interactions play an essential role in or-ganogenesis during embryonic development. However,virtually no attempts have been made to study the role oftissue interaction in pineal development. In the presentstudy we examined the inductive role of the epidermisand mesenchyme in the morphogenesis of quail pinealglands. The pineal rudiment is first observed at embry-onic day 2 (E2: 2 days of incubation) at the dorsal mid-line of the diencephalon as a short semi-spherical protru-sion. Electron microscopic observations revealed that nomesenchymal cells are found between the epidermis andthe distal end of the E2 pineal primordium but that a thinlayer of mesenchymal cells separate the epidermis fromthe pineal primordium at E3. Small pieces containing pi-neal rudiment were cut off from E2 or E3 embryos. Theywere treated with enzymes to eliminate the epidermisand/or mesenchyme, grafted into E5 chicken eyes, andcultured there for 1 week. When E3 pineal rudiment wastreated with Dispase to remove the epidermis, the pinealgland developed normally. When the rudiment was fur-
ther treated with collagenase to remove the surroundingmesenchymal cells, a multi-follicular structure was stillformed, but to a lesser extent than when rudiments weretreated with Dispase alone. When E2 quail pineal rudi-ment with the epidermis was grafted without any treat-ment, a multi-follicular structure developed which mor-phologically resembled embryonic pineal organs. Whenthe epidermis was removed from E2 rudiments by Disp-ase, a single large vesicular structure was formed. Theseresults suggest that the overlying epidermis and/or mes-enchymal cells play some inductive role in the initial pi-neal development, while the mesenchymal tissue playsan important role in pineal follicular formation later dur-ing development. Since only a few experimental studieshave been done to examine pineal morphogenesis, thepresent study provides fundamental insights into avianpineal development.
Classical studies in experimental embryology demon-strated that tissue interactions play a crucial role in orga-nogenesis of various organs, and the vertebrate eye hasbeen one of the most intensively studied organs. The in-ductive sequences have been described in eye develop-ment between the surface epidermis and the optic vesicle(reviewed in Lopashov 1963; Saha et al. 1992). The ex-traocular mesenchymal tissues also appear to have animportant role in the induction and maintenance of theneural retina and retinal pigmented epithelium (Johnstonet al. 1979; Fuhrmann et al. 2000). In spite of the numer-ous studies concerning eye formation, it is less under-stood whether tissue interaction has any role during mor-phogenesis of the pineal organ.
The pineal organ originates from the dorsal portion ofthe diencephalon and is formed by a medio-dorsal protru-sion of the rudiment during embryonic development. In
Edited by N. Satoh
Y. ShimauchiDepartment of Arts and Sciences, Osaka Kyoiku University,Kashihara, Osaka, Japan
T. Yahata · M. Araki (✉ )Department of Biology, Kyoto Prefectural University of Medicine,Kyoto, Japane-mail: [email protected]: +81-742-203411
S. MatsubaraDepartment of Obstetrics and Gynecology, Jichi Medical School,Tochigi, Japan
M. ArakiDevelopmental Neurobiology Laboratory, Department of Biological Sciences, Faculty of Science,Nara Women’s University, Nara 630–8506, Japan
Present address:Y. Shimauchi, Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka 560–0043, Japan
Dev Genes Evol (2002) 212:319–329DOI 10.1007/s00427-002-0236-1
the lower vertebrates the pineal organ contains neuronsand photoreceptors and is an important component of thephotoendocrine system (Oksche and Vollrath 1981; Voll-rath 1981). Several common features are found betweenthe pineal organ and the lateral eye with respect to theirdevelopmental and functional aspects (Araki 2001). Themost notable example of such similarity is the third eye(median eye) as seen in some lower vertebrates (Eakin1973), in which the surface epidermis develops as a lens-like organ and the diencephalic protrusion develops vari-ous photoreceptors and neurons, although no further mor-phogenesis resembling optic cup formation proceeds. Re-cent studies have revealed that several transcription fac-tors are expressed in the developing eyes and pineal or-gans, suggesting a common mechanism of morphogene-sis and a common origin in ancestral animals (Casarosa etal. 1997; Glasgow et al. 1997; Taira et al. 1993).
The development of the avian pineal organ has beenwell described at the light and electron microscopic levels, particularly in domestic fowls (Spiroff 1958; Renzoni 1970; Calvo and Boya 1978, 1979). The pinealrudiment of the chick embryo is recognized as a smallevagination in the diencephalic roof at 60 h of incubationand becomes a short semi-spherical protrusion at E3(Ohsima and Matsuo 1988). In later development, thisprotrusion enlarges and makes a secondary projection toform a multi-follicular structure (Calvo and Boya 1978).In brief, embryonic pineal development can be dividedinto three phases. In the first step the diencephalic roof atthe midline protrudes to make contact with the surfaceepidermis. Secondly, multiform follicular formation be-gins by secondary projections from the primary evagina-tion and the follicles are densely surrounded by the mes-enchymal cells. Finally, in later development, cell differ-entiation occurs to give rise to several different types ofphotoreceptors and endocrinal cells.
To analyze avian pineal morphogenesis, we have de-veloped a new experimental system involving intraoculargrafting of pineal rudiments into the anterior eye cham-ber of chick embryos. Chimerical transplantation of em-bryonic quail tissues to developing chick embryos hasbeen widely used for developmental studies because ofthe unique biological marker of the quail cell nucleus(Le Douarin 1974). Quail embryonic development is al-most the same as that of the chick embryo except that thehatching time is 3–4 days earlier than in the chick.Chick-quail chimerical transplantation is a very usefultechnique for the analysis of tissue interactions, particu-larly in cases where cells such as neural crest cells orcells of mesenchymal origin migrate during develop-ment. The present study represents the first attempt toexamine tissue interactions during pineal developmentby means of experimental manipulation.
Materials and methods
Fertilized quail and chicken eggs were purchased from several lo-cal hatcheries and were incubated at 37.8°C in a dark humidifiedincubator.
Treatment of pineal primordial grafts
Pineal primordia were isolated from quail embryos at stage 18 de-termined according to the development table of Hamburger andHamilton (1951). In some transplantation experiments, pineal pri-mordia were obtained from quail embryos at stage 15–16. Embry-os were washed with Hanks’ solution and the whole brain vesicleswere isolated from the embryos. Incisions were made around thepineal primordia and a small fragment containing the pineal pri-mordia at its center (about 500–1,000 µm–2) was obtained. Thefragments were further treated as follows:
1. Fragments were incubated in Dispase solution (Godo-ShuseiCo.; 750 units/ml in Hanks’ solution) at room temperature for20–25 min. With this treatment the epidermis and most mesen-chymal tissue were removed.
2. Fragments treated by the above procedure were further incubat-ed in collagenase solution (Sigma; 0.1% in Hanks’ solution) at37°C for 30 min to remove the remaining mesenchymal cells.
Before intraocular transplantation, graft tissues were labeled withcarbon powder to mark the donor tissue in the histological sec-tions.
Intraocular grafting
Untreated tissue fragments or those treated either with Dispase di-gestion or Dispase plus collagenase were inserted into the embry-onic host eyes. Chicken embryos at stage 25 were used as host an-imals. A small incision was made in the right host eye at the futureiris position using a tungsten needle, and quail tissue fragmentswere inserted into the host eyes with forceps. The host embryoswere further incubated for 1 week in an incubator and then sub-jected to fixation.
Histology
The heads of the host chicken embryos were briefly immersed inHanks’ saline and fixed with Bouin fixative. Eyes which receivedintraocular transplantation were examined carefully with a binocu-lar stereo microscope (Olympus, SZH-10). Fixed heads were thendehydrated and embedded in paraffin. Serial sections of 4–6 µmthickness were stained with hematoxylin and eosin.
Electron microscopy
Quail embryos were washed with Hanks’ saline and fixed for 1 hin an ice-chilled mixture of 2% paraformaldehyde and 1% glutar-aldehyde in 0.1 M cacodylate buffer (pH 7.2) followed by postfix-ation for 30 min in 1% osmium tetroxide. Semi-thin sections of1 µm thickness were cut serially and stained with toluidine blue.Ultra-thin sections were cut and double-stained with uranyl ace-tate and lead citrate.
Results
Morphologic features of quail pineal glands in early embryonic stages
In the normal development of quail pineal glands, the pine-al primordium can first be observed at approximately2 days of incubation (day E2; corresponding to stage 15 ofchick embryos according to Hamburger and Hamilton) as asmall semi-spherical protrusion at the diencephalic roof(Calvo and Boya 1978; Fig. 1A–C). The E2 pineal primor-dium appeared to be in direct contact with the epidermis at
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Fig. 1A–G Embryonic development of the quail pineal organ. Allsections are stained with toluidine blue. A–C Embryonic day 2(E2) pineal primordia (48 h of incubation). A tiny protrusion isseen at the dorsal wall of the diencephalon (arrowhead in A). Thepineal primordium appears to be in direct contact with the surfaceepidermis (arrow in C). Only a few mesenchymal cells are local-ized around the primordium. D, E E 3 pineals. A few mesenchy-mal cells are occasionally found between the epidermis and the
primordium (arrowhead in E). F E4.5 pineal organ. Mesenchymaltissues clearly separate the epidermis from the primordium. At thedistal end of the primordium, areas of secondary cell proliferationare seen (arrowheads), which later develop as follicles. G E8 pi-neal gland which consists of a large primary follicle (asterisk) andnumerous secondary follicles. Bar in A is 300 µm. Bar in B is50 µm and also applies to D and G. Bar in C is 10 µm and also ap-plies to E. Bar in F is 30 µm
the distal end, and only a few mesenchymal cells werefound to surround the primordium. These observationswere confirmed by electron microscopic observations(Fig. 2): the layer of epidermal cells was very thin at thedistal end of the pineal primordium and no mesenchymalcells were found between the epidermis and pineal primor-dium. The basement membrane of the presumptive pinealcells was not well developed (Fig. 2B). In areas other thanthe pineal distal end, few mesenchymal cells were found.Between days E2 and E3 (at about stage 18) the pineal pri-mordium became a fairly conspicuous structure and ap-peared to be in close contact with the epidermis (Fig. 1D,
E). More mesenchymal cells surrounded the pineal primor-dium. Electron microscopic observations clearly showedthat E3 pineal primordia were separated from the epider-mis by a thin layer of mesenchymal cells (Fig. 3A). Lightlystained materials were observed in the matrix between thepineal primordium and the epidermis (Fig. 3B). The pinealcells were arranged in a pseudo-stratified epithelial layerand showed a well-developed basement membrane(Fig. 3B). At day E4 (about stage 21, 24 h later than stage18), the pineal gland was clearly separated from the epider-mis by a mesenchymal cell layer (Fig. 1F). A few cellularmammilliform projections (Fig. 1F, arrowheads) were
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Fig. 2A, B Electron micro-graphs of an E2 quail pinealgland. A The pineal primordi-um comes very close to the epidermal epithelium (arrow-heads) at its distal end. B A similar region to thatshown in A is shown at a higher magnification. Bars in A and B are 5 µm and 2 µm, respectively
formed at the distal end of the pineal outline. They devel-oped into secondary follicles at E8 (Fig. 1G).
Dispase treatment of E2 or E3 pineal primordia re-moved the epidermis and some of the mesenchymal cellssurrounding the primordium, but a few mesenchymalcells still remained adhering to the pineal outline(Fig. 4A, D). Subsequent treatment of E3 pineal rudi-ments with collagenase removed most of these mesen-chymal cells and also appeared to partly digest somebasement membrane components, as indicated by thefact that the basal surface of pineal cells had a fuzzy ap-pearance (Fig. 4B).
Intraocular transplantation of pineal primordiumfrom stage 18 embryos
After grafting, the incision made in the host eyes for in-traocular graft insertion was sealed and the host eyesusually developed normally after this operation. Graftsin such host eye chambers were often found around thelens or iris (Fig. 5). However, in some cases the hosteyes showed abnormal developmental features after theoperation; some of them were rather small and in somecases the neural retinal layer was found to sprawl outthrough the incision. In the latter cases, the grafts were
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Fig. 3A, B Electron micro-graphs of an E3 quail pinealgland. A A thin layer of mesen-chymal cells (arrowheads) separates the surface epidermalcells (e) from the pineal primordium, which shows apseudostratified epithelial layer. B The distal end of theprimordium in A is shown at ahigher magnification. Very thincytoplasm (arrowheads) ofmesenchymal cells (m) is always found between the epidermal cells (e) and primor-dium. Bars in A and B are5 µm and 1 µm, respectively
located outside the host eyeballs. Although the nourish-ment of grafted tissues in the host eyes was not alwaysidentical to each other, it seemed that there was no rela-tion between the condition of the host chambers and theresults described below.
Untreated primordium
In 8 out of 10 specimens, the grafted primordia devel-oped as a pineal-like structure with numerous follicles,
and occasionally a pineal recess was observed, too(Fig. 5A, B). The number of follicles ranged between 5and more than 20, as roughly estimated by examinationof the serial sections. The follicles were surrounded bymany mesenchymal cells with a distinct nucleolus intheir nucleus, which indicates that these cells were de-rived from the graft quail tissue. In the other 2 cases, thegraft tissues formed an epithelial sheet without any fol-licular structure.
Dispase-treated primordium (without epidermis)
In 9 out of 13 grafts, follicular formation was observed.The number and shape of follicles differed considerablydepending on the grafts, and it appeared that such varia-tion was related to the amount of surrounding mesenchy-mal tissue. In 4 cases that had only a few follicles, a fewmesenchymal cells of the donor quail tissues were at-tached to the basal side of follicles, although they did notdevelop into an obvious mesenchymal tissue. The con-figuration of the follicles was usually irregular. In theother 5 cases, numerous follicles were formed, and therewere many mesenchymal cells surrounding the follicles(Fig. 5C). Interestingly, in the cases where the follicles
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Fig. 4A–D Pineal primordia treated by enzymatic digestion. A The surface epidermis has been removed from the E3 pineal ru-diment by Dispase treatment. Many mesenchymal cells (arrow-heads) are still found attached to the basal surface of the primordi-um. B The pineal primordium has been treated by sequential enzy-matic treatment with Dispase and collagenase. No connective cellsare found around the primordium. C Tissue fragments for intraoc-ular transplantation; the epidermis has been removed by Dispasetreatment. Arrowheads indicate pineal primordia. Fragments willbe further trimmed along the indicated lines for transplantation. D E2 pineal rudiment has been treated with Dispase, and the sur-face ectoderm has been removed. Only a few mesenchymal cellsare seen attached to the primordium (arrowheads). The arrow in-dicates the approximate area of the presumptive pineal primordi-um. Bar in A is 20 µm and also applies to B. Bar in D is 20 µm
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Fig. 5A–F Intraocular transplantation of E3 pineal primordia.Cells from graft tissue can be discriminated by the nuclear markerand are indicated by arrowheads. A, B Intact pineal primordia in-cluding the epidermis and mesenchyme were transplanted withoutany enzymatic treatment. Numerous follicles are observed in thevicinity of the iris (i) and are surrounded by connective tissues. C,D Grafts of Dispase-treated pineal primordia. In C, many folliclesare seen near the iris as observed in the case of intact primordia.At a higher magnification in D, most of the surrounding connec-
tive cells (arrows) are seen to be derived from the host chickencells. E, F Grafts of primordia after double (Dispase plus collage-nase) enzymatic treatment. Arrowheads indicate the boundary ofthe graft. In some cases (E), very few follicles were formed,whereas in other cases (F), numerous follicles were formed. Inboth cases the follicular structure was surrounded by numerousconnective cells derived from the host chicken embryos. Bar in Ais 100 µm and also applies to B and C. Bar in D is 10 µm. Bar inE) is 50 µm and also applies to F. Hematoxylin and eosin staining
were most well developed, they were surrounded bywell-developed mesenchymal tissues which were mostlyderived from the host ocular mesenchymal tissues(Fig. 5D).
Dispase plus collagenase-treated primordium (without most mesenchymal cells)
In 1 out of 8 specimens, no follicular formation was ob-served, and instead, a large luminal structure coveredwith an epithelial sheet was found. In 1 specimen, awell-developed follicular formation was observed andfollicles were embedded in the host conjunctiva stroma(Fig. 5F). In the other 6 cases, the follicular formationwas very poor and only a few follicles were formed,ranging in number from a few to ten (Fig. 5E). The num-ber of follicles was much lower than that found in Dis-pase-treated pineal primordia. In all cases, the surround-
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Fig. 6A–E Intraocular transplantation of E2 pineal primordia. A, B Intact pineal primordia with the epidermis were transplantedwithout any enzymatic treatment. Numerous follicles were formedin the host tissue between the iris (i) and cornea (c). At a highermagnification (B), two types of pineal cells are seen in the folli-cles, one is lightly and the other darkly stained (arrowheads). C–EGrafts of Dispase-treated pineal primordia. In C, the graft is sur-rounded by host tissues such as the lens (le), iris (i) and retina (n).The part marked by the asterisk in D is shown at a higher magnifi-cation in E. The graft is indicated by arrowheads. No clear folli-cles were formed and, instead, a large luminous epithelial struc-ture is seen, and only a few mesenchymal cells surround the pri-mordium. Bars in A and B are 100 µm and 10 µm, and also applyto C and E, respectively. Bar in D is 50 µm. Hematoxylin and eo-sin staining
ing mesenchymal cells lacked a distinct nucleolus, indi-cating that they were mostly derived from host chickentissues.
Intraocular transplantation of pineal primordiumfrom stage 15 embryos
Untreated primordium
Donor quail tissues could be observed among the serialsections of the host eyes in only 3 out of 7 specimens. Inthe rest of the cases, the donor tissue appeared to havebeen lost from the host eye chamber immediately afterthe implantation procedure due to the extremely smallsize of the donor tissue.
The transplanted pineal primordia attained fairly well-developed morphology, and numerous follicles wereformed and surrounded by dense mesenchymal cells,suggesting that the pineal primordium at such an earlystage of development has already attained the capacityfor full morphogenesis (Fig. 6A, B). Cells in the follicu-lar epithelium were often seen to extend apical protru-sions into the luminal cavity, a characteristic feature ofdifferentiated pineal cells (Calvo and Boya 1978).
Dispase-treated primordium (without epidermis)
Donor tissues could be observed in only 2 out of 8 speci-mens. It seemed that the grafts were more easily lost be-cause the tissues became very fragile and delicate afterthe epidermis was removed by enzymatic treatment. Inthe 2 successful cases, instead of clustered follicular for-mation, a large luminous follicle was formed which oc-casionally branched into a few follicles (Fig. 6C–E). Thefollicular epithelial cells did not show any characteristicmorphologic properties such as filiform apical projec-tions. Only a few mesenchymal cells were seen to bescattered around these follicular structures.
Discussion
Developmental studies on the avian pineal gland havemostly been based on morphological observations at thelight (Renzoni 1970; Calvo and Boya 1978) and electronmicroscopic (Omura 1977; Calvo and Boya 1979) levels,and include descriptions of the expression patterns ofgenes such as rhodopsin (Robinson et al. 1995), HIOMT(Grechez-Cassiau et al. 1995) AA-NAT (Herichova et al.2001) and the transcription factors. Only a limited num-ber of studies aiming to clarify the mechanisms involvedin pineal development have been reported (Cameron1903; Bargmann 1943; Holmgnen 1965). This is in clearcontrast with the enormous volume of work that hasdealt with the mechanisms of eye development, one ofthe classical subjects of embryonic induction. During eyedevelopment, inductive sequences between neighboring
tissues occur progressively and give rise to tissues withnew characteristics. The surface epidermis induces opticcup formation and is essential for the development of theneural retina (Webster et al. 1984), and it has beenshown that FGF signaling is involved in this process(McAvoy et al. 1991; Hyer et al. 1998; Le and Musil2001). The surrounding mesenchymal cells also appearto play an important role in the regionalization of the op-tic cup (Lopashov 1963; Fuhrmann et al. 2000).
The present electron microscopic observations revealseveral new findings about the distal interface betweenthe epidermis and pineal primordium: E2 pineal primor-dium comes very close to the epidermis, while mesenchy-mal cells thrust between them and interfere with the closecontact at E3. Our intraocular transplantation studies indi-cated that tissue interactions between the pineal primordi-um and its neighboring tissues have an important role inpineal morphogenesis, and suggested that E2 pineal pri-mordium needs inductive signal(s) provided from the epi-dermal cells to undergo appropriate morphogenesis andthat the E3 pineal rudiment needs an adequate amount ofmesenchymal cells to form the follicular structure.
Morphological features of avian pineal developmentin terms of tissue interaction
With respect to the structural relation between the pinealprimordium and neighboring tissues, E2 is a discriminat-ing stage in the pineal development of quail embryos. AtE2, the pineal primordium is observed as a small hemi-spherical protrusion, and the overlying epidermis comesvery close to the primordium, as shown by electron mi-croscopic observations. At E3, however, there is a thinlayer of mesenchymal cells apparently separating the epi-dermis from the pineal organ. In contrast, the optic vesi-cle continues to be in direct contact with the surface epi-dermis during early development and no mesenchymalcells exist between the two tissues, supporting the ideathat there is a direct inductive signaling between the pre-sumptive lens placode and the optic vesicle (Webster etal. 1983, 1984). At around E4 and E5, pineal follicularformation begins by secondary protrusion or branching atthe distal end from the primitive tubular structure, and theepidermis has been clearly separated from the pineal or-gan by growing connective tissue. These observationssuggest a possibility that the epidermis does not play arole in the formation of secondary protrusions which de-velop into follicular structures, but that mesenchymalcells are involved in the process. In accordance with thisview, during further development, numerous mesenchy-mal cells come to surround the follicles, which increasein number as development proceeds.
E2 pineal rudiments require tissue interaction
In the present study, we have shown that E2 pineal pri-mordia isolated from neighboring brain vesicles can de-
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velop multiple follicles when incubated with surroundingtissues such as the ectodermal sheet and mesenchymalcells. The results suggest that differentiation of the E2pineal primordia is not influenced by adjacent regions ofthe brain vesicle.
When E2 pineal primordia were treated with Dispaseto remove the epidermis and were transplanted intraocu-larly, the follicular structures were not well developedand, instead, a large luminous structure with occasional-ly branching small follicles was formed; cells in the fol-licular epithelium appeared to be poorly developed. Thisis in contrast to the case of E3 pineal transplantation,where well-developed follicular structures were formedwithout the epidermis. The simplest interpretation forthese observations is that the epidermis plays a substan-tial role in the early phase of pineal development but be-comes nonessential by E3.
As described above, there are some mesenchymalcells around the E2 pineal primordium, and more mesen-chymal cells are distributed around the primordium asdevelopment proceeds. Since some of these mesenchy-mal cells are also removed by Dispase treatment, it ispossible that these cells also have some inductive role inthe early pineal development. It can be speculated that,between E2 and E3, the pineal primordium receivessome signals from either the midline epidermal cells ormesenchymal cells, which induce the determination ofthe pineal primordium and its development into the pine-al organ.
E3 pineal rudiments need mesenchymal cells to produce follicular structures
In the intraocular transplantation of E 3 pineal primordia,most Dispase-treated grafts formed a well-developed fol-licular structure which was surrounded by a dense mes-enchymal tissue. In contrast, in cases in which follicleswere not well developed, only a few mesenchymal cellswere observed around the follicles. These observationsindicate that mesenchymal cells play an essential role inthe formation of pineal follicles. It is interesting to notethat the effect of mesenchymal cells on follicular forma-tion is the same whether the cells are derived from thegraft or host tissue. It may be that the ocular (mostly irisand cornea stroma) and pineal mesenchymal cells havesome properties in common.
When the primordia were transplanted after sequen-tial treatment with Dispase and collagenase, less-devel-oped follicular structures were formed than those ob-tained with transplantation of Dispase-treated primordia.With this double-enzymatic treatment, most of the mes-enchymal cells were removed from the primordium and,in addition, some factors in the extracellular matrix(ECM) were digested. It is considered that these factorsin the ECM are essential for the migration of the hostmesenchymal cells into the grafts, and that their absencefrom the grafts may result in less-developed follicularstructures.
The branching morphogenesis of mouse embryonicsalivary glands has been examined extensively in theanalysis of epithelial-mesenchymal interactions. Somecollagen subtypes have been shown to work as key sub-stances for cleft initiation of the submandibular epitheli-um, an initial step in the formation of the branching aci-nar structure. In accordance with this observation, colla-genase inhibitor has also been shown to stimulate cleftformation (Nakanishi et al. 1986). A hypothetical modelhas been presented in which a furrow is made in the lob-ule of the glandular epithelium by contracting a bundleof collagen fibrils by the traction of mesenchymal cells(Nakanishi et al. 1988). It is not known whether a similarmechanism is involved in the pineal follicular formation.
Constructing experimental strategy to analyze tissue in-teraction during pineal organogenesis
The present study has demonstrated that intraocularlytransplanted pineal primordia undergo organogenetic de-velopment. The chimerical combination also enables usto discriminate the donor pineal cells from the host cells.This is important particularly for the identification of theorigin of mesenchymal cells. In a previous study, underorgan culture conditions for E8 pineal glands, photore-ceptor differentiation (expression of several photopig-ment genes) proceeds, but in the culture of the early pi-neal rudiments from E2 or E3 embryos, normal process-es of morphogenesis such as follicular formation are not observed (Yamao et al. 1999). Aige-Gli and Murillo-Ferrol (1991) reported that an isolated diencephalonfrom early chick embryos (stage 13 according to Ham-burger and Hamilton) can form a single small protrusionbut does not undergo further development when it isgrafted onto the chorioallantoic membrane.
In contrast to these previous studies, the pineal pri-mordium in the present study continued to develop in theintraocular milieu like a normally developing pinealgland. It is possible that by intraocular transplantationmore mesenchymal cells were provided which can drivefollicular formation. Both eyes and pineal glands are de-rived from the presumptive diencephalic region of neuraltube, and there may be common properties in the mesen-chymal tissues surrounding those two organs. The natureof mesenchymal influence on pineal development mustbe studied further and it is also important to investigatewhether the mesenchymal tissue from other parts of theembryo can support pineal morphogenesis.
Pineal cytodifferentiation was observed in the grafts:apical projections extended toward the luminal space,and two different cell types containing either darklystained or lightly stained cytoplasm were observed, asreported in normally developing avian pineal glands(Calvo and Boya 1978, 1979). Previous immunohisto-chemical studies have shown that a variety of cell typesdifferentiate in embryonic quail pineal organs, includingseveral different photoreceptors and neural cells withdifferent properties (Araki et al. 1992, 1993). These
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properties are expressed only at the late embryonic stag-es (after E13–E14). Further study is necessary to screenthe conditions for longer tissue culture in order to exam-ine whether pineal cells in the grafts differentiate to ex-press some photoreceptor or neural cell properties.
Acknowledgements We are deeply grateful to Professor EmeritusDr. K. Kato for valuable discussion. We also thank Ms. S. Honmafor excellent technical assistance. This work was supported in partby a Grant-in-Aid from the Ministry of Education, Science, Cul-ture, Sports and Technology of Japan to M. Araki.
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