THE ANATOMICAL RECORD 227:264-270 (1990)

Morphogenesis of the Frontal Organ in Bufo bufo During Development R.M. FARNESI, S. TEI, D. VAGNETTI, I. DI ROSA, A. FAGOTTI, AND B. SANTARELLA Istituto di Anatomia Comparata, Universita Degli Studi di Perugia, 06100 Perugia, Italia.

ABSTRACT Morphogenesis of the frontal organ in Bufo bufo was examined under transmission electron microscope. Many remarkable similarities to the frontal organ of other Amphibia Anura are observed. It originates from a diverticulum in the dorsal region of the neural tube. It is egg-shaped, has a n eccentric lumen, and is made up of three kinds of cells: 1)photoreceptors, which protrude into the lumen; 2) supportive cells; and 3) ganglion cells, which make synaptic contact with the photoreceptors. Peculiar to Bufo bufo is the melanin-like pigments around the light-sensitive part of the photoreceptors. These pigments may prevent light dispersion. The frontal organ in Bufo bufo starts degenerating during the early premetamorphic stages. Morhpogenesis of the posterior parietal region in after metamorphosis, whereas in some species it may Amphibia Anura begins during the early stages of em- degenerate (Von Haffner, 1950; Korf et al., 19811, but bryonic development, as in all vertebrates. A vesicle is this would not lead to drastic behavioural changes as formed on the dorsal wall of the neural tube in the the other component of this photoneuroendocrinic proencephalic area, and its lumen is connected to the unit-the epiphysis-remains functional. Even though studies on the morphology and ultrathird ventricle. As development progresses, the vesicle subdivides into a n anterior frontal organ, which mi- structure of the frontal organ in Amphibia Anura are grates from the meninges to just under the epidermis somewhat scarce, noteworthy are those on adult Rana and posterior epiphysis, which remains on the roof of pipiens (Kelly and Smith, 1964; Eldren and Nolte, the neural tube, These two structures are joined by the 1981) and on Hyla regilla and Xenopus laevis during pineal nerve (Eakin and Westfall, 1961a; Oksche, the larval and adult stages (Eakin and Westfall, 1961b; 1965; Oksche and Vaupel-von Harnack, 1965; Beau- Eakin et al., 1963; Korf e t al., 1981). To date there have been no studies on the developmont and Cassier, 1974; Padoa, 1988). They form the pineal complex and are believed to be light sensitive ment of the frontal organ in Bufonidae, nor on the efdue to their position and to their sensory cells, which fects metamorphosis might have on it. The present are similar to retinal photoreceptors Won Haffner, study examines the ultrastructure of this organ in Bufo 1951; Bagnara, 1963; Beaumont and Cassier, 1974). It bufo during embryonic and larval development. has been suggested that the pineal organ behaves like MATERIALS AND METHODS a gland producing melanin-like substances. A specific function of the frontal organ has not yet been discovBufo bufo eggs were collected near Perugia immediered. Tests carried out to determine the physiology of ately after fertilization and kept in aquaria. Upon the pineal complex in Xenopus laevis have shown that reaching the desired stages of development (stage 17: the neural fibers emerging from the frontal organ are muscuIar contraction; stage 18: muscular response; light sensitive only in the larval stage (Korf et al., stage 19: heart beat; stage 20: branchial circulation; 1981). stage 21: stomodeum rupture; VIII larval stage: beginSeveral authors (Bagnara, 1960, 1963; Korf et al., ning of premetamorphosis stages; Rossi, 19591, the 1981) have suggested that in Anura larvae the pineal cephalic region was removed from 200 embryos and complex is involved in pigmentation control and that it examined under light microscopy and transmission works as a single functional unit: should light be lack- electron microscopy (TEM). For examination under ing, the frontal organ would stimulate the epiphysis to light microscopy, the specimens were fixed in Bouin's release melanin or similar substances. Once these sub- fluid, embedded in paraffin, and sectioned for staining stances are in circulation, they would cause concentra- with hematoxylin and eosin. tion of the melanin pigment and thus blanching of the For observation under TEM, the specimens were tadpoles. In the presence of light, the frontal organ fixed for 24 hours in 5% glutaraldehyde in 0.1 M (pH would instead inhibit the epiphysis; therefore, the mel- 7.5) cacodylate buffer a t about 4"C, washed repeatedly anin pigment would spread, producing a dark colour in 0.1 M cacodylate buffer, and postfixed for 2 hours in typical of tadpoles. Confirming this hypothesis, Bagnara (1963) observed that the blanking reaction in tadpoles disappears when the parietal organs are destroyed by cautery. In most Amphibia Anura, the frontal organ remains Received April 18, 1989; accepted September 21, 1989 0 1990 WILEY-LISS, INC.

MORPHOGENESIS OF T H E FRONTAL ORGAN I N BUFO

1% OsO, in 0.1 M cacodylate buffer. The material then was rapidly dehydrated in graded ethanols and embedded in Epon-Araldite according to Mollenhauer's (1964) method. Ultrathin sections stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) were examined under a Philips 400 T TEM at 80 KV'. RESULTS

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The outer segment, the fourth part of the phtoreceptors, originates from this cilium. This segment is made up of series of flattened and heaped sacs that rise from the numerous introflexions of the external membrane of the cilium; the sac cavities remain open in the lumen of the frontal organ (Fig. 9). Large cells are observed a t the base of the frontal organ. They appear to have the same characteristics as ganglion cells: they contain a large nucleus, a welldeveloped Golgi apparatus, and a n abundant rough reticulum similar to Nissl bodies. These cells are grouped in neuropil areas that are rich in nerve fibers (Fig. 10). The supportive cells are characterized by long cytoplasmatic processes containing lipid droplets (Fig. 6), protein granules, and glycogen. They surround the photoreceptors and the ganglion cells. The supportive cells situated near the lumen of the organ have fingershaped cytoplasmatic extroversions a t the apex. Some appear to secrete electron-dense pigment granules into the lumen. These granules appear to gather around the outer segments of the photoreceptors (Fig. 11).The supportive cells that bound the ventral part of the organ have cytoplasmatic finger-like structures close to the wall of the underlying blood vessel (Fig. 12). At the VIII larval stage (beginning of the premetamorphic stages), the Bufo bufo frontal organ appears to degenerate. Wide vacuolized spaces are observed between the various cells (Fig. 13). The photoreceptor cells show signs of degeneration due to the presence of lysosomes, lipofuscin, and a large quantity of vesicular material in the nuclear segment and ellipsoid. The outer segment no longer has its typical structure since the heaped sacs dilate and fragment, forming scattered vesicle-like clusters in the lumen (Fig. 14). Also, the supportive cells and neuron cells appear to degenerate. They contain large lipid droplets, masses of lipofuscin, and large quantities of glycogen (Fig. 15).Macrophages are present in the blood vessel close to the frontal organ (Fig. 16).

During the initial stages of embryonic development (stages 17,181, a diverticulum originating in the ventricular chamber (Fig. 1)is observed in the dorsal region of the neural tube. At the heartbeat stage (stage 191, the diverticulum assumes the shape of a disk. It expands and is subdivided into a superficial portion going beyond the meninges under the epidermis-the frontal organ-and into a deeper portion-the pineal organ-which remains on the roof of the neural tube in the diencephalon (Fig. 2). At embryonic stage 20 (branchial circulation), the frontal organ is egg-shaped and lies just beneath the epidermis. Two types of cells were identified during the ultrastructural examination of this organ, although they were only slightly differentiated. Morphological evidence indicates that a cell division was ongoing a t this stage when the tissue was harvested. The first type of cells is characterized by nuclei and cytoplasm containing abundant lipid droplets, protein granules, and rough endoplasmic reticulum. Hence they are to be considered trophic and supportive cells. The outer ones tend to flatten and form a thin wall around the frontal organ. The second type contains lobed nucleus with clusters of chromatin and a visible nucleolus. Its cytoplasm is accumulated mainly around the cell pole and contains numerous dense and transparent vesicles. Because of their appearance, these cells should be considered neuronal elements at the early stages of differentiation (Fig. 3). At stage 21 (stomodeum rupture), the frontal organ is fully developed and has a n unevenly shaped eccenDISCUSSION tric lumen. Three kinds of cells can be identified: phoOur findings on the embryonic and larval stages in toreceptors protruding into the lumen and ganglion neurons and supportive cells which, with their cyto- Bufo bufo have shed light in the morphology and ulplasmatic extensions, surround the photoreceptors and trastructure of the frontal organ. Its structure and development stages are very similar to those of other Amthe neurons (Fig. 4). The photoreceptors are composed of four well-defined phibia Anura (Eakin and Westfall, 1961b; Eakin e t al., segments (Fig. 5 ) . The first, the subnuclear segment, is 1963; Korf et al., 1981). Indeed, the frontal organ in made up of a protruding axon cone that makes synaptic Bufo originates from a diverticulum in the dorsal recontact with neighbouring ganglion cells (Figs. 6, 7) gion of the neural tube. Subsequently, the organ is sepand are to be the site of pineal nerve formation. The arated from the epiphysis and migrates out of the second, the nuclear segment, is composed of a large meninges to just under the epidermis. When the frontal organ is fully developed, its funcegg-shaped nucleus with visible nucleolus and clusters of chromatin and a scanty cytoplasm with a visible tion is demonstrated by the presence of photoreceptors rough reticulum, Golgi apparatus, and some protein cells, which have a n ultrastructure similar to the cones granules (Fig. 8). The third segment, the so-called el- of the lateral eye. Cells presenting the same morphollipsoid, is separated from the nuclear region by a no- ogy have been observed in the frontal organ of larvae of ticeable constriction. The ellipsoid segment is round other Amphibia (Eakin and Westfall, 1961b; Eakin et and the cytoplasm contains abundant mitochondria, a al., 1963; Korf et al., 1981). Two kinds of ganglion cells, which are the second rough reticulum, transparent vesicles, glycogen, and lysosomes (Fig. 8).Tight junctions are formed between type of cell present in the organ, were observed in X e adjacent photoreceptors a t the ellipsoid, and junctions nopus laevis under light microscopy using the acetylbetween photoreceptors and supportive cells are situ- cholinesterase reaction (Korf e t al., 1981). However, ated a t the base of the ellipsoid. The basal apparatus of Eakin et al. (1963) failed to identify them clearly in the cilium, composed of two perpendicular centrioles, Hyla regilla under electron microscopy, although they can be observed in the apex cytoplasm of the ellipsoid. do describe large cells stained with methylene blue on

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Fig. 1 . Light micrograph of Bufo bufo embryo at stage 18. Diverticulum in dorsal area of neural tube (arrow). X 90.

Fig. 3. Electron micrograph of frontal organ at differentiation stages. Nerve cells (nc) and supportive cells (sc) can be identified. At this stage, mitotic figures are also visible (arrow). x 1,700.

Fig. 2. Light micrograph of stage 19 embryo. Subepidermal frontal organ (fo) and pineal organ (PO) on the roof of neural tube. x 135.

the floor of the frontal organ. In Bufo bufo we observed large cells which, in our opinion, may be considered ganglion cells, given their ultrastructural appearance

and their arrangement within the neuropil area in the frontal organ. Between the receptor and ganglion cells are the sup-

MORPHOGENESIS O F T H E FRONTAL ORGAN I N BUFO

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5 Fig. 4. Completely differentiated frontal organ. Photoreceptors (PI protruding in the lumen (L), supportive cells (sc) adhering to the photoreceptors, and ganglion cells (gc) are all visible. A blood vessel (v) is present in the ventral part of the organ. x 1,800. Fig, 5. Diagram illustrating the three cell elements of the frontal organ: 1) the photoreceptor with an outer segment (os), ellipsoid ( e ) ,a nuclear segment (ns), and synaptic area (sa); 2) ganglion cells (gc); and 3 ) supportive cells (sc) with pigment granules (pg).

Fig. 6. Details of the synaptic area (sa) of the photoreceptor. A supportive cell (sc) and a ganglion cell (gc) are visible. x 5,700. Fig. 7. Details of synaptic contacts (arrows)between a photoreceptor

(P) and a ganglion cell (gc). x 8,700.

R.M. FARNESI ET AL.

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Fig. 8. Nuclear segment (ns) and the ellipsoid (e) of photoreceptors. Junction (arrow) between ellipsoids of two adjacent photoreceptors. x 4,900.

the ellipsoid (el of a photoreceptor. Cilium and clusters of glycogen (g) granules are visible. x 7,600. Fig. 9. Detail of outer segment

( 0 s ) and

Fig. 10. Ganglion cell situated in neuropil area (arrows).Nissl bodies (N) and nucleus (n). x 4,200. Fig. 11. Pigment granules secreted from supportive cells and spilling into lumen of frontal organ. They settle around the outer segment of the photoreceptor. x 6,000.

MORPHOGENESIS OF THE FRONTAL ORGAN I N BUFO

Fig. 12. Supportive cell in close relationship with blood vessel (v) through cytoplasmatic finger-like formation (arrows). X 5,300.

Fig. 13. View of degenerating frontal organ. Large vacuolated spaces can be seen between the cells. x 1,500. Fig. 14. Details of degenerating lumen in the frontal organ. The morphology of the outer segment of the photoreceptors is completely altered. x 4,700.

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Fig. 15. Details of degenerating frontal organ. The supportive cells contain lipofuscin granules (Ig), lipid droplets (Id), and glycogen (g). x 5,200. Fig. 16. Detail of degenerating frontal organ. Large clusters of glycogen (g) can be observed, and a macrophage (M) is visible in the underlying blood vessel. X 6,300.

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ACKNOWLEDGMENTS portive cells, characterized by cytoplasmic processes and by the presence of reserve material as lipid dropThese investigations were carried out with the techlets, protein granules, and glycogen. These character- nical assistance of the Electron Microscope Laboratory istics suggest that, besides being supportive, the cells of the University of Perugia. may also have a trophic behaviour toward the other LITERATURE CITED two types of cells. The supportive cells situated near the lumen of the Bagnara, J.T.1960 Pineal regulation of the body lightening reaction in amphibian larvae. Science, 132t1481-1483. organ establish close contact with the ellipsoid of the photoreceptors by of junctions thus forming a Bagnara, J.T. 1963 The pineal and the body lightening reaction of larval amphibians. Gen. Comp. Endocrinol., 3t86-100. the lumen. There has Beaumont, layer A., and P. Cassier 1974 Anatomia comparata dei Vertebeen a n attempt to identify the Supportive cells in the brati. Casa Editrice Ambrosiana, Milano. Eakin, R. M., and J. A. Westfall 1961a Photoreceptors in the stirnorfrontal organ of Hyla regdla (Eakin et al., 1963). Algan oftadpoles of the treefrog, Hyla regilla. Am. Zool., 1;352. though the description is rather brief, the cells do apR.M., and J.A. Westfall 1961b The development of photoreceppear to be similar to the supportive cells we observed in Eakin, tors in the stirnorgan of the treefrog, Hyla regilla. Embryologia, Bufo. 6t84-98. From our observations it appears that the supportive Eakin R.M., W.B. Quay, and J.A. Westfall 1963 Cytological and cytochemical studies on the frontal and pineal organs of the treecells situated near the lumen of the frontal organ frog, Hyla regzlla. Z. Zellforsch., 59t663-683. secrete pigment granules, which gather around the Eldren, D.W., and J . Nolte 1981 Multiple classes of photoreceptors outer segment of the photoreceptors. The pigments, and neurons in the frontal organ of Rana pipiens. J. Comp. Neuprobably of a melanin nature, are likely to be a shield rol., 203:269-296. around the light-sensitive part of the photoreceptor to Kelly, E.D., and S.W. Smith 1964 Fine structure of the pineal organs the adult frog Rana pipiens. J. Cell Biol., 22:653-674. prevent light dispersion. the pigments Korf,ofH.V., R. Liesner, H. Meissl, and A. Kirk 1981 Pineal complex of could be compared with the granules the clawed toad, Xenopus laeuzs Daud.: Structure and function. in the pigmented retinic epithelium of the lateral eye Cell Tissue Res., 216:113-130. (Patt and Patt, 1969; Beaumont and Gassier, 1974). Mollenhauer, H.H. 1964 Plastic embedding mixtures for use in electron microscopy. Stain Technol., 39t111. structures have not been Observed in Other Oksche, A. 1965 Survey of the development and comparative morstudies on Amphibia; hence they are peculiar to phology of the pineal organ. Prog. Brain Res., 10:3-29. Bufo. Oksche, A., and M. Vaupel-von Harnack 1965 Vergleichende elektronenmikroskopische Studien am Pineal Organ. In: Progress in The frontal organ in Bufo bufo begins to degenerate Brain Research. Structure and Function of the Epiphysis Cerebri. prior to metamorphosis. At the VIII larval stage, wide J. Ariens Kappers and J.P. Schade, eds. Elsevier, Amsterdam, vacuolized spaces are observed between the different vOl. pp. 237-258. cells, suggesting a process of autophagocytosis. The Cy- Padoa, E. 1988 Manuale di Anatomia Comparata dei Vertebrati. Feltrinelli ed., Milano. toplasm of all of the cells contains large lipid droplets, D.J., and G.R. Patt 1969 Comparative Vertebrate Histology. lipofuscin granules, ~ysosomes,and myelin Patt,Harper & Row, Publishers, New York, pp. 363-381. forms- The Outer segment Of the photoreceptors Reynolds, E.S. 1963 The use of lead citrate at high pH as an electron loses its integrity, becoming vesicle clusters scattered opaque stain in electron microscopy. J. Cell Biol., I 7t208-212. in the lumen. The immense glycogen accumulation ob- Rossi, A. 1959 Tavole cronologiche dello sviluppo embrionale e larvale del Bufo bufo (L.1. Monit. Zool. Ital., LXVI:1-17. served in degenerating supportive further ‘OnVon Haffner, K. 1950 h e r die progressive und regressive Entwickfirms a loss of photoreceptor activity (Eakin et al., lung der Pinealblase (Parietalorgan) des Krallenfrosches ( X e 1963). nopus laeuis Daud). Verh. Zool. Gesell., pp. 93-100. Therefore, Bufo bufo is to be considered one of the Von Haffner, K. 1951Die Pinealblase (Stirnorgan, Parietal Organ) von Xenopus laeuis Daud. und ihre Entwicklung, Verlagerung und few species of Amphibia A~~~~ whose adults do not Degeneration. Zool. Jahrb. Abt. Anat. Ontog. Tiere, 71t375-421. have the Organ, as it regresses during Watson, H.L. 1958 Staining of tissue sections for electron microsphosis. copy with heavy metals. J. Biophys. Biochem. Cytol., 4t475-478.

Morphogenesis of the frontal organ in Bufo bufo during development.

Morphogenesis of the frontal organ in Bufo bufo was examined under transmission electron microscope. Many remarkable similarities to the frontal organ...
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