Neuroseienee Letters, 142 (1992) 241 244 ~c' 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Origin of luteinizing hormone-releasing hormone (LHRH) neurons in the chick embryo: effect of the olfactory placode ablation S a t o r u A k u t s u ~, Michio T a k a d a ~, H i r o k o O h k i - H a m a z a k i ~, Shizuko M u r a k a m i b a n d Y a s u m a s a Arai b D~7~artments o[' "Obstetrics and Gynecology and ~'Anatomy, Juntendo University School o["Medicine. Tokvo ( Japan j and' Departnwnt q/ Basic Human Sciences, School o/' Human Sciences, Waseda University. Saitama (Japan; (Received 9 March 1992: Revised version received 12 May 1992: Accepted 12 May 1992)

Ke3 wordsv

Luteinizing hormone-releasing hormone neuron: Olfactory placodal origin; Cell migration: Placodectomy: Chick embryo

A unilateral olfactory placodectomy resulted in the absence of luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) cells in the olfactory-forebrain axis of the operated side, whereas the development of the LHRH neuronal system was not disturbed on the unoperated side. In lhe embryos in which a fragment of the medial olfactory epithelium was spared the damage, a small number of LHRH-ir cells were detected in the nasal region of the operated side, where the lack of the central projection of the olfactory nerve caused stagnation of LHRH-ir cells, no Jr-cells being found in the brain area. These results suggest that LHRH neurons originate in the olfactory placode and then migrate into the t\~rebrain along the olfactory nerve.

There is considerable evidence to suggest that luteinizing hormone-releasing hormone (LHRH) neurons do not originate in the brain, but rather migrate from the nasal region to the forebrain during embryonic development [1, 4, 8, 12]. Recently, we found that LHRH-expressing neurons were first detected in the medial part of the olfactory placode of the chick embryo prior to their appearance in the brain, and then as developmental age progressed the majority of cell populations expressing L H R H shifted from the nasal region to the forebrain along the olfactory nerve [3]. In all previous studies in avian and mammalian embryos, however, the migration of L H R H neurons is inferred from their gradual and progressive distribution changes between the olfactory and diencephalic sites. In the present study, in order to determine the origin of L H R H neurons in the chick brain and investigate the migration phenomenon of these neurons, the olfactory placode which is thought to be a possible site containing the precursor cells of L H R H neurons was surgically removed in an early stage of chick embryos. Fertilized white Leghorn eggs were incubated at 37.6°C. Embryos of 3.5-4.0 embryonic days (EDs 3.54.0) were subjected to microsurgery. The right olfactory placodal region was excised by a finely sharpened steel Correspondence." Y. Arai, Department of Anatomy, Juntendo Universit 5, School of Medicine, Hongo, Tokyo 113, Japan.

needle under a dissecting microscope. After the operation, the eggs were sealed and returned to the incubator for further development. At ED 7 or 11, operated embryos were fixed overnight in Bouin's solution without acetic acid at 4°C. Each of them was immersed for 24-36 h in a phosphate buffer containing 20% sucrose. Serial sections of 16/lm thickness were cut on a cryostat in transverse or horizontal planes and mounted on glass slides coated with egg white. L H R H expressing neurons were immunohistochemically stained by ABC methods using a commercial kit (Vector Laboratories, Inc., Burlingame, CA). A monoclonal antibody LRH13 (HAC-MM02-MSM84) was used at a dilution of 1:2,000. LRH13 has been demonstrated to have a high L H R H specificity and wide crossreactivity [5]. The peroxidase complex was visualized by 3',3-diaminobentidine tetrahydrochloride and H~O2. The sections were counterstained with methyl green or H-E. Specificity of the immunoreactions was determined by omitting the first antibody LRHI3, and by pre-incubation of LRHI 3 with synthetic L H R H ( 1/Jg/ml; Peninsula Laboratories, CA). In these cases, no immunoreactivity was found in the nasal and brain regions. Sixteen and 8 operated embryos were sacrificed at EDs 7 and 11, respectively. Unilateral olfactory placodectomy was accomplished in 4 out of these 24 embryos, and a complete ablation of the olfactory placode resulted in a complete loss of the nasal cavity, the olfactory epithe-

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lium a n d the o l f a c t o r y nerve. As shown in Fig. 1, these o l f a c t o r y c o m p o n e n t s were m o s t l y replaced by the epid e r m a l e c t o d e r m a n d m e s e n c h y m a l tissue+ A slight red u c t i o n in the size o f the o l f a c t o r y bulb was noted. H o w ever, the o t h e r m o r e p o s t e r i o r brain regions did not seem to be affected by the o p e r a t i o n , a n d the d e v e l o p m e n t o f these telencephalic a n d diencephalic structures o f the ope r a t e d side d i d n o t a p p e a r to be disturbed. In the 4 e m b r y o s with a c o m p l e t e unilateral p l a c o d e a b l a t i o n sacrificed at E D 7 o r 1l. no L H R H - i m m u n o r e a c t i v e (-ir) cells a n d fibers were detected in either the nasal region o r in the b r a i n on the o p e r a t e d side (Figs. 1-3). In the c o n t r a l a t e r a l intact side, however, L H R H - i r cells were f o u n d t h r o u g h o u t the olfactory f o r e b r a i n axis as r e p o r t e d previously [3] (Figs. 1-3). A large n u m b e r o f L H R H - i r cells were o b s e r v e d in the medial p a r t o f the o l f a c t o r y epithelium a n d in the o l f a c t o r y nerve in the two e m b r y o s sacrificed at E D 7 (Fig. 4). C o r d s o f L H R H - i r cells in the o l f a c t o r y nerve e x t e n d e d d o r s o - c a u d a l l y t h r o u g h the nasal s e p t u m t o w a r d s the v e n t r o m e d i a l p a r t o f the forebrain. M a n y L H R H - i r cells were seen to p e n e t r a t e into the medial p a r t o f the basal f o r e b r a i n with the o l f a c t o r y nerve bundle, and a n u m b e r o f L H R H - i r cells was also f o u n d to s p r e a d in the septal anlage on the u n o p e r a t e d side. In the two e m b r y o s with a c o m p l e t e p l a c o d e a b l a t i o n sacrificed at E D 1 1, the m a j o r i t y o f L H R H - i r cells were seen in the septal (Fig. 3) a n d p r e o p t i c area, the o l f a c t o r y b u l b a n d the m e d i a l p o r tion o f the o l f a c t o r y nerve o f the intact side. A t this stage, L H R H - i r cells m a r k e d l y decreased in n u m b e r in the olf a c t o r y epithelium. F o u r e m b r y o s sacrificed at E D 7 o r 1 1 were d a m a g e d in a large p a r t o f the lateral p a r t a n d the m o s t p a r t o f the m e d i a l o l f a c t o r y epithelium. In these e m b r y o s , the p o o r l y d e v e l o p e d o l f a c t o r y nerve c o u l d be traced to the r o s t r a l p a r t o f the nasal septum. O n l y a few L H R H - i r c-

Fig. 1. A transverse section of the olfactory-forebrain region in a unilaterally placodectomized ED 7 embryo. LHRH-ir neurons are seen in the medial olfactory epithelium and the olfactory nerve on the unoperated side. Note the complete loss of the nasal structure and the absence of LHRH-ir cells on the operated side. OE. olfactory epithelium: NS. nasal septum: TEL, telencephalon. Counterstained with Methyl green. Bar = 250 ¢tm. Fig. 2. A number of LHRH-ir cells are seen to enter the ventromedial telencephalon on the unoperated side, whereas no LHRH-ir cells are seen on the operated side of an ED 7 embryo. Counterstained with Methyl green. Bar = 100,am. Fig. 3. A frontal section of the septal area of a unilaterally placodectomized ED 11 embryo. Note the absence of LHRH-ir cells on the operated side (arrow). Counterstained with Methyl green. Bar = 50 ym. Fig. 4, Strongly immunoreactive cells are seen in the medial part of the olfactory nerve bundle on the intact side of an ED 7 embryo. Arrowheads indicate the lateral border of the olfactory nerve. V, blood vessel. Counterstained with H-E. Bar = 50 ¢tm.

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Fig. 5. A: a horizontal section through the septal anlage of an ED 7 embryo, in which a small fragment of the medial olfactory epithelium was spared the damage. LHRH-ir neurons are recognized in the rostral forebrain and the septal anlage on the unoperated side (arrows), whereas LHRH-ir cells are absent on the operated side. However, a few LHRH-ir cells (arrowhead) are detected in the nasal branch of the ophthalmic nerve (NB-OPN) of the operated side. No LHRH-ir cells are seen in the identical nerve of the unoperated side (arrowhead). ", right eyeball is lacking because of an artifact by sectioning. Counterstained with Methyl green. B: high magnification of the NB-OPN of the operated side from the next section to A. Nole the presence of LHRH-ir cells (arrows) in the bundle. C: high magnification of the N B-OPN of the unoperated side. Note the absence of LHRH-ir cells in the bundle. Counterstained with H-E. Bars - 25(//am (A), 25/am (B,C).

cells were detected in the o l f a c t o r y e p i t h e l i u m r e m n a n t and the thin o l f a c t o r y nerve. A few L H R H - i r cells were also f o u n d in the nasal b r a n c h o f the o p h t h a l m i c nerve o f the t r i g e m i n a l nerve i n n e r v a t i n g the o l f a c t o r y m u c o s a as s h o w n p r e v i o u s l y [3]. However, no L H R H - i r cells were seen in a n y o t h e r a r e a o f the o p e r a t e d side. In the u n o p e r a t e d side, the d i s t r i b u t i o n o f L H R H - i r cells was quite similar to that seen d u r i n g n o r m a l e m b r y o n i c d e v e l o p m e n t [3]. In two o t h e r E D 7 e m b r y o s whose lesion was rather shifted to the lateral p a r t o f the p l a c o d e , a small f r a g m e n t o f the medial o l f a c t o r y e p i t h e l i u m was f o u n d to r e m a i n intact in the o p e r a t e d side. T h e epithelial c o m p o n e n t in these e m b r y o s c o n t a i n e d a small n u m b e r o f L H R H - i r cells, However, the d e v e l o p m e n t o f the o l f a c t o r y nerve b u n d l e was d i s t u r b e d , a n d the fiber b u n d l e c o u l d be f o u n d only n e a r the epithelial structure. A small dense cluster o f L H R H - i r cells was d e t e c t e d in this o l f a c t o r y nerve r e m n a n t . However, L H R H - i r cells a n d fibers were not seen b e y o n d this a r e a a n d in the f o r e b r a i n area either

(Fig. 5A). Instead, a c o n s i d e r a b l e n u m b e r o f L H R H - i r cells were recognized in the nasal b r a n c h o f the o p h t h a l mic nerve. These L H R H - i r cells were d e t e c t a b l e a l o n g the nasal b r a n c h o f the o p h t h a l m i c nerve d o r s o c a u d a l l y to the eyeball (Fig. 5A,B). In the u n o p e r a t e d side, the d i s t r i b u t i o n o f L H R H - i r cells in the nasal region and the b r a i n was n o r m a l . In c o n t r a s t to the o p e r a t e d side, however, L H R H - i r cells in the nasal b r a n c h o f the o p h t h a l mic nerve were only rarely f o u n d in the intact side. A c t u ally no ir-cells were seen in Fig. 5A,C. In the rest o f the o p e r a t e d e m b r y o s , the lesions were m i s p l a c e d in the p e r i p l a c o d a l regions. The d e v e l o p m e n t o f L H R H - i r cells was not affected by surgery. In the present study, surgical r e m o v a l o f the o l f a c t o r y p l a c o d e resulted in the absence o f L H R H - i r cells in the o l f a c t o r y - f o r e b r a i n axis o f the o p e r a t e d side, t h o u g h the d e v e l o p m e n t o f the L H R H n e u r o n a l system was not dist u r b e d on the u n o p e r a t e d side. To o u r k n o w l e d g e , this is the first e x p e r i m e n t a l evidence suggesting that L H R H n e u r o n s in the chick e m b r y o originate in the o l f a c t o r y

244 placode, after which they migrate into the brain along the olfactory nerve. Since the development of the tetencephalic and diencephalic structures (except a slight reduction in the size of the olfactory bulb) was not affected by the operation, a lack of L H R H - i r cells in the operated side is probably not the result of some nonspecific effect(s) of the surgery, but is directly caused by the removal of the precursor cells o f the L H R H neurons in the placode and the absence of the olfactory nerve, which may provide L H R H neurons a possible migration route. This conjecture is strongly supported by the finding that the absence of the central projection of the olfactory nerve caused stagnation of L H R H - i r cells near the olfactory epithelium in the embryos with an incomplete placodectomy. For migration of L H R H neurons, some structure that forms a possible bridge between the nasal region and the forebrain seems to be prerequisite. In this regard, it is o f particular significance to note a report on the Kallmann syndrome (inherited hypogonadotropic hypogonadism with anosmia) that L H R H neurons do not migrate normally into the brain, because the olfactory nerve complex is never in contact with the brain in a Kallmann fetus [9]. In the lack o f the olfactory nerve bundle, the occurrence of a considerable number of L H R H - i r cells in the nasal branch of the ophthalmic nerve of the trigeminal nerve was noted in the embryos with an incomplete placodectomy. This again suggests the importance of availability of a route to support cell migration. These L H R H ir cells might change the regular route into the nasal branch of the ophthalmic nerve which meets the olfactory nerve near the olfactory epithelium at the caudal pole of the nasal bone [11]. We have reported that a few migrating L H R H - i r cells from the olfactory placode are also found in the nasal branch of the ophthalmic nerve during normal development [3]. To facilitate the neuronal migration, the ophthalmic nerve may also possess a chemical environment which is similar to that of the olfactory nerve. We have found that both the olfactory nerve fibers and the L H R H - i r cells are strongly immunopositive to an embryonic form of neural cell adhesion molecule ( N C A M - H ) during the time of neuronal migration [3]. From our preliminary findings, the fiber bundle of the nasal branch of the ophthalmic nerve is also N C A M - H positive (Seki et al., unpublished). Since an embryonic form of N C A M is highly polysialylated [2, 10] and less adhesive than the adult forms of N C A M [71, this diminished adhesiveness of the N C A M has been speculated to enable the neurons to avoid forming stable junctions and to remain responsive to guidance and cues [6]. It is suggested therefore

that other than the structural support for the migration of L H R H neurons, a possible interaction between the developing NCAM-H-expressing olfactory nerve axons and the migrating L H R H - and NCAM-expressing neurons may also play a critical role in regulating the migration of L H R H neurons into the forebrain. Further study is needed to elucidate these points. We would like to thank Professor K. Wakabayashi, G u n m a University, for his generous supply of L R H I 3 . This research was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture. Daikoku-Ishido,H., Okamura, Y., Yanaihara, N. and Daikoku, S., Development of the hypothalamic luteinizing hormone-releasing hormone-containingneuron system in the rat: in vivo and in transplantation studies, Dev. Biol.~ 140 (1990) 374-387. 2 Hoffman, S., Sorking, B.C., White, RC., Brackenbury, R,, Mailhammer, R., Rutishauser, U.. Cunningham, B.A. and Edelman, G.M., Chemical characterization of a neural cell adhesion molecule purified from embryonic brain membranes, J. Biol. Chem., 257 (1982) 7720--7729. 3 Murakami, S., Seki, T., Wakabayashi, K. and Arai, Y., The ontogeny of luteinizing hormone-releasinghormone(LHRH) producing neurons in the chick embryo: possible evidence tbr migrating LHRH neurons from the olfactory epithelium expressing a highly polysialylated neural cell adhesion molecule; Neurosci. Res,, 12 (1991) 421M31. 4 Norgren, R.B. and Lehman, M.N., Neurons that migrate from the olfactory epithelium in the chick express luteinizing hormone-releasing hormone, Endocrinology,128 (1991) 1676 1678. 5 Park, M,K. and Wakabayashi, K., Preparation of a monoclonal antibody to commonamino acid sequence of LHRH and its application, Endocrinol. Jpn., 33 (1986) 257 272. 6 Rutishauser, U., Polysialicacid as a regulator of cell interaction. I11 R.U. Margolis and R.K. Margolis (Eds.), Neurobiologyof Glycoconjugates, Plenum, New York. 1989,pp. 367 382. 7 Sadoul, R., Hirn, H., Deagosini-Bazin, H., Rougon, G. and Goridis, C., Adult and embryonic mouse neural celt adhesion molecules have different binding properties, Nature, 304 (1983) 347 349. 8 Schwanzel-Fukuda, M. and Pfaff, D.W., Origin of luteinizing hormone-releasing hormone neurons, Nature, 338 (1989) 161-~164. 9 Schwanzel-Fukuda, M., Bick, D, and Pfaff. D.W., Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal(Katlmann) syndrome, Mol. Brain Res., 6(1989) 311 326. 10 Seki,T. and Arai, Y., Expression of highlypolysialylatedNCAM in the neocortex and piriform cortex of the developingand the adult rat, Anat, Embryol., 184 (1991) 395 401. 11 Watanabe,T. and Yasuda, M., Comparative and topographicanatomy of the fowl. LI. Peripheral course of the olfactory nerve of fowl, Jpn. J. Vet. Sci., 30 (1968) 275 279. 12 Wray,S., Nieburgs, A. and Elkabes, S., Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode, Dev. Brain Res., 46 (1989) 309-318. 1