The ontogeny of luteinizing hormone-releasing hormone (LHRH) producing neurons in the chick embryo: possible evidence for migrating LHRH neurons from the olfactory epithelium expressing a highly polysialylated neural cell adhesion molecule

Neuroscience Research, 12 (1991) 421-431

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© 1991 Elsevier Scientific Publishers Ireland, Ltd. 0168-0102/91/$03.50 NEURES 00474

The ontogeny of luteinizing hormone-releasing hormone (LHRH) producing neurons in the chick embryo: possible evidence for migrating LHRH neurons from the olfactory epithelium expressing a highly polysialylated neural cell adhesion molecule Shizuko Murakami 1, Tatsunori Seki t, Katsumi Wakabayashi 2 and Yasumasa Arai 1 I Department of Anatomy, Juntendo Unicersity School of Medicine, Hongo, Tokyo and 2 Hormone Assay Center, Institute of Endocrinology, Gunma UniL,ersity, Maebashi, Gunma (Japan) (Received 2 May 1991; Accepted 12 June 1991)

Key words: LHRH-producing neurons; Olfactory placode; Olfactory nerve; Neuronal migration; NCAM; Chick embryo

SUMMARY The development of neurons expressing luteinizing hormone-releasing hormone (LHRH) has been studied immunohistochemically in the chick embryo from the 3.5 embryonic day (ED) to the day of hatching. At ED-3.5, LHRH-immunoreactive neurons were first detected in the medial epithelium of the olfactory pit, but their appearance in the brain was delayed to ED-4.5. On EDs-6-7, cords of the LHRH-immunoreactive cells extended across the nasal septum towards the ventromedial forebrain with the olfactory nerve. By double staining for LHRH and a highly polysialylated form of neural cell adhesion molecule (NCAM-H), the LHRH-positive neurons in the olfactory-forebrain system were found strongly NCAM-H-positive. At ED-8, a marked decrease in the number of LHRH-positive cells in the olfactory epithelium and a concomitant increase in the LHRH-positive cells in the forebrain area were noted. From ED-11 to the day of hatching, the majority of LHRH-positive neurons tended to move into their usual adult position, whereas the LHRH-positive cells had almost disappeared in the olfactory epithelium. No LHRH-immunoreactive neurons were found strongly positive to NCAM-H. These results suggest that LHRH neurons originate from the olfactory placode, then as they develop they migrate across the nasal septum and enter the forebrain with the olfactory nerve. The close association of NCAM-H with the developing LHRH neurons raises the possibility that NCAM-H plays some role in guiding the migrating LHRH neurons from the olfactory epithelium to the forebrain.

INTRODUCTION

In the avian brain, neurons that produce the luteinizing hormone-releasing hormone (LHRH) have been demonstrated in the septal and preoptic nuclei and hypothalamus 5,19 LHRH controls the release of gonadotropins from the anterior pituitary, thereby playing Correspondence: Yasumasa Arai, Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 113, Japan.

422 a principal role in the regulation of the avian pituitary gonadal axis ~2,3,. In various vertebrates, LHRH-producing neurons have been found present in the olfactory bulb 15,23,37. These neurons also have been detected in the terminal nerve, a small cranial nerve that projects in most classes of vertebrates directly from the olfactory epithelium to the septal and the preoptic areas 5,6,20,24,27,311. In certain mammals, L H R H immunoreactivity can be detected within the terminal nerve prior to its appearance in the brain during embryonic development 27,30-32,41. It is therefore postulated that the L H R H neurons originate in the olfactory placode of the developing nose and then migrate into the forebrain along with the terminal nerve 4,32.41,42 However, evidence thus far has indicated that the terminal nerve is absent in birds 5a6.40. It is of interest, therefore, to investigate the ontogeny of the LHRH-producing neurons in the developing bird, in particular to examine the route that these L H R H neurons take during the neuronal migration. To facilitate this neuronal migration, some chemical environment must be involved, such as the presence of growth or trophic factors, as well as some cell adhesion molecules. A highly polysialylated form of neural cell adhesion molecule (NCAM-H), which is less adhesive than the adult form of NCAM, may be important, because it is known to be present in the embryonic stage 14,29, and it has been speculated that NCAM-H may enable these neurons to avoid forming stable junctions and to complete their migration 2~. A possible association of NCAM-H expression in the developing LHRH-producing neurons was also examined in the olfactory and forebrain regions. MATERIALS AND METHODS

Fertilized White Leghorn eggs were purchased from a commercial hatchery, and incubated at 37.6 ° C. At different times during the incubation, 65 embryos were removed and fixed overnight in Bouin's solution without acetic acid at 4°C. Each of the embryos then was immersed for 24-36 h in a phosphate buffer containing 20% sucrose. Next, serial sections of 16-/~m thickness were cut on a cryostat in transverse, sagittal or horizontal planes and mounted on glass slides coated with egg white. The embryonic stage was determined for each embryo according to Hamburger and Hamilton 10 The LHRH-producing neurons were immunohistochemically stained by the avidinbiotin peroxidase complex (ABC) method using a commercial kit (Vector Laboratories, Inc., Burlingame, CA). A monoclonal anti-LHRH serum (LRH13, immunoglobulin G) was produced by one of the authors (K.W.). In formatively, LRH13 has been demonstrated to have a high L H R H specificity and a wide cross-reactivity over animal classes 26. Thus, LRH13 was used at a dilution of 1 : 2000, and the peroxidase complex was visualized by 3,3'-diaminobenzidine tetrahydrochloride and H 2 0 2. Finally, the sections were counterstained with methylgreen or cresyl fast violet, dehydrated and mounted. For control staining, normal non-immunized horse serum was used as a primary antibody. For the immunohistochemical demonstration of NCAM-H, the monoclonal antibody MAb 12E3, immunoglobulin M, raised against the embryonic rat forebrain and produced by the authors 35, specifically recognized the polysialic acid portion of the NCAM-H. Sections obtained from ED-7 and -13 embryos were stained by the ABC method, using a MAb-12E3-containing ascitic fluid (diluted 1:5000). The staining procedures for the ABC method were almost the same as for the L H R H . In order to examine the possible association of the NCAM-H immunoreactivity with the immunoreactivity of the developing L H R H neurons, other sections were subjected

423 to a double fluorescence immunohistochemical analysis. The sections were incubated in a mixture of LRH13 (diluted 1 : 2000) and MAb-12E3 (1 : 2000) for 48 h, at 4°C, after which the sections were again incubated in a secondary antibody mixture of rhodaminlabelled anti-mouse IgG (E.Y. Labs, Inc., USA; diluted 1:20) and fluorescein isothiocyanate (FITC)-labelled anti-mouse IgM (Cappel, USA, diluted 1:50) for 2 h at room temperature. Control staining was performed by ensuring that the combinations of the primary and the secondary antisera were free of cross-reactivity. The sections were inspected with a epifluoresent microscope (Olympus AH-RFL-LB) using two-filter sets. RESULTS At stage 19 (ED-3.5), the olfactory placode began to form into the olfactory pit as a small depression on each side of the presumed face. At this stage, immunoreactive L H R H cells and fibers were not detectable in any structure of the peripheral and central nervous system. At stages 20-21 (ED-3.5), however, some cells were moderately stained, indicating the presence of L H R H in the epithelium of the medial wall of the olfactory pit (Fig. 1). Also, L H R H immunoreactivity was detected in a number of cells just outside the epithelium of the olfactory pit and within the developing axons of the olfactory nerve. These immunoreactive cells were oval or fusiform, possessing no single process or processes, and some were found to have migrated out of the epithelium with axons that extended into the olfactory nerve (Fig. 1). However, no cells or fibers containing L H RH were detected in the brain area. The olfactory pits then became deeper, forming nasal grooves with a thickened epithelium at ED-4. At stage 24 (ED-4), the number of LHRH-immunoreactive cells, based on a heightened staining intensity, increased markedly in the medial parts of the olfactory epithelium and in the olfactory nerve, and phenomena showing evidence of LHRH-positive cell migration from the epithelium were frequently encountered. Many LHRH-positive cells formed a cord-like structure or an elongated cellular band in the olfactory nerve, and coursed towards the developing forebrain (Fig. 2). The processes of the L H R H neurons within these cellular bands were either unipolar or bipolar, and were oriented parallel to the long axis of the olfactory nerve. At stages 25-26 (ED-4.5), a part of LHRH-immunoreactive cells in the cord appeared to aggregate in the olfactory nerve bundle, near the junction with the anlage of the olfactory bulb. It was at this point that a few LHRH-positive cells were first detected in the rostral telencephalon (Fig. 3). At stages 28-30 (ED-6-7), the nasal cavities became further elongated and the developing olfactory axons were found to be more numerous and arranged in a thick bundle. An increasing number of LHRH-positive ceils were observed in the medial part of the olfactory epithelium. In cross-sections of the olfactory nerve, LHRH-immunoreactive cells were seen to be not evenly distributed in the bundle, but concentrated in the medial half. Cords of LHRH-producing neurons originating in the olfactory epithelium extended dorsocaudally through the nasal septum towards the ventromedial part of the forebrain (Fig. 4). In the sagittal sections, many LHRH-immunoreactive neurons were seen to penetrate into the medial part of the basal forebrain with the olfactory nerve bundle, after which they branched from the olfactory nerve and passed obliquely upwards to form an arch through the rostral to the caudal telencephalon (Fig. 5). Some L H R H cells were found near the olfactory bulb anlage with the olfactory nerve. From this stage (ED-6), a few LHRH-immunoreactive neurons were found in the nasal branch of the ophthalmic nerve of the trigeminal nerve innervating the olfactory

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Fig. 1. At day 3.5 of incubation (ED-3.5, stage 21), LHRH-immunoreactive neurons are seen in the epithelium of the medial walt of the olfactory pit (OE). Some LHRH cells are seen migrating out from the epithelium with axons of the olfactory nerve (ON). TEL = telencephalon. Scale bar = 50/xm. Fig. 2. At ED-4 (stage 24), LHRH-immunoreactive neurons reveal a marked increase in number in the medial olfactory epithelium (OE) and the olfactory nerve. Note the immunoreactive LHRH cells migrating into the olfactory nerve bundle. Scale bar = 50/zm. Fig. 3. At ED-4.5 (stage 26), clusters of immunoreactive LHRH neurons in the olfactory nerve bundle near the junction with the anlage of the olfactory bulb. A few LHRH neurons are seen in the rostral telencephalon (arrow). Scale bar = 50 ~m.

mucosa. T h e s e L H R H - i m m u n o r e a c t i v e cells also were observed in t h e o p h t h a l m i c nerve on the day of hatching. A t ED-8, a l t h o u g h c o n s i d e r a b l e n u m b e r s of L H R H - i m m u n o r e a c t i v e cells were still p r e s e n t in the olfactory nerve, a m a r k e d decrease in the n u m b e r of L H R H positive cells was observed in the olfactory e p i t h e l i u m , while a c o n c o m i t a n t i n c r e a s e in the n u m b e r of L H R H - p o s i t i v e cells was n o t e d in the f o r e b r a i n area. T h e d i s t r i b u t i o n of L H R H - p o s i tive cells e x t e n d e d m o r e dorsally. F r o m ED-11 to the day of hatching, the d i s t r i b u t i o n of L H R H - i m m u n o r e a c t i v e cells in the b r a i n b e c a m e similar to that s e e n in the a d u l t fowl, with the majority of the L H R H - p o s i t i v e cells in the septal a n d p r e o p t i c areas, the olfactory b u l b , a n d the m e d i a l p o r t i o n of the olfactory nerve. L H R H - p o s i t i v e cells were rarely f o u n d in the olfactory

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Fig. 4. At ED-6 (stage 28), a large n u m b e r of immunoreactive L H R H n e u r o n s are seen in the medial olfactory epithelium. Cords of L H R H n e u r o n s course along the nasal s e p t u m (NS) to enter the ventromedial telencephalon together with the olfactory nerve. Scale bar = 2 5 0 / x m . For further explanation, see legend to Fig. 1.

Fig. 5. (A) A sagittal section of the forebrain at ED-7 (stage 31). The immunoreactive L H R H neurons arch into the dorsal forebrain and spread towards the septal anlage. OBA = olfactory bulb anlage. Scale bar = 300 #m. (B) A high magnification of the olfactory nerve. Arrows indicate the lateral edge of the olfactory nerve. Scale bar = 100 ~m.

epithelium. At hatching, fibers containing L H R H appeared in the external layer of the median eminence. In the ED-7 and -13 embryos, the expression of NCAM-H also was investigated immunohistochemically. At ED-7, an immunoreactivity indicating NCAM-H was found in the olfactory epithelium, with a number of strongly stained cells found in the basal layer, especially in the medial part of the epithelium. Some of these cells were seen to migrate out of the basal surface of the epithelium. The axons and the presumably migrating neurons in the olfactory nerve bundle also strongly stained. In the telencephalon, the immunoreactivity was observed in the marginal and the mantle layers, but not in the ventricular layer. In particular, clusters of cells located near the basal surface of the medial forebrain showed strong immunoreactivity. Immunohistochemical testing for L H R H in the adjacent sections showed at ED-7 that cell bodies containing L H R H were located in the same area where a strong

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427 immunoreactivity indicating that NCAM-H was present. Double staining for LHRH and NCAM-H demonstrated that in the medial part of the olfactory epithelium (Fig. 6), as well as in the olfactory nerve (Fig. 7) and the ventromedial wall of the telencephalon (Fig. 8), the distribution of the LHRH-positive cells was almost identical to that of the cells strongly positive for NCAM-H. As shown in Figures 6-8, however, all the NCAM-H-positive components were not all LHRH-positive. The fibers and neurons in the medial half of the olfactory nerve bundle were strongly NCAM-H-positive and LHRH-positive, whereas those in the remaining half were strongly NCAM-H-positive but mostly negative to L H R H (Fig. 7). At ED-13, NCAM-H immunoreactivity almost disappeared in the olfactory epithelium. However, the olfactory nerve bundle was still NCAM-H-positive. NCAM-H immunoreactivity was less prominent but widespread over the entire telencephalon and diencephalon. As has been mentioned previously, the distribution pattern of L H R H neurons at this stage became similar to that of the adult bird, and no strong NCAM-Hpositive ceils were observed in areas where L H R H was present. DISCUSSION

Previous studies of the domestic mallard have shown that LHRH-producing neurons can be detected on ED-20 within the entire septal and preoptic areas, as well as in the caudal basal hypothalamus. In the domestic fowl and the Japanese quail, however, no L H R H neurons have been found on ED-14 and -15 1. Only a brief report indicating the early occurrence of LHRH-immunoreactive cells in the chick olfactory epithelium has appeared quite recently 22 In the present paper, we have carried out a more detailed analysis of the development of the L H R H neurons in the chick embryo. Our major findings have indicated that LHRH-immunoreactive neurons appear in the epithelium of the medial olfactory placode long before LHRH-positive cells become recognizable in the brain, and that the distribution of main cell populations containing L H R H shifts from the olfactory epithelium to the forebrain area along with the olfactory nerve as developmental age progresses. The spatial-temporal appearance of these neurons during embryonic development in the chick is similar to that observed in the mouse and the rat, in which LHR H neurons first appear in the medial olfactory placode, then migrate across the nasal septum and enter the forebrain with the terminal nerve 4,32,41,42

• Fig. 6. (A) N C A M - H immunoreactivity in the medial olfactory epithelium at ED-7. Note the presence of strongly-positive cells in the epithelium and adjacent region. (B) A rhodamin fluorescent visualization of the L H R H neurons in the s a m e section. The L H R H neuronal immunoreactivity mainly corresponds to those neurons showing a strong F I T C fluorescence. Note the lack of LHRH-immunoreactivity in some of the immunoreactive N C A M - H cells (cf. Fig. 6A), Scale bar = 50 p~m (A and B). • Fig. 7. (A) The N C A M - H immunoreactivity in the olfactory nerve bundle in the same material as shown in Fig. 6. A strong FITC fluorescence is observed in the olfactory nerve fibers and the migrating cells. (B) T h e immunoreactivity of the L H R H neurons and their processes in the olfactory fiber bundle. T h e N C A M - H - i m m u n o r e a c t i v e fibers in the lateral portion of the olfactory nerve (see arrows in Fig. 7A) are mostly LHRH-negative. Scale bar = 50 p,m (A and B). • Fig. 8. (A) A cluster of N C A M - H neurons in the basal telencephalon in the same material as shown in Figs. 6 and 7. (B) A cluster of L H R H neurons. The immunoreaetivity of the L H R H neurons is identical to the strongly-immunomactive N C A M - H cells in the same section. Scale bar = 50 # m (A and B).

428 In these latter species, the terminal nerve is thought to play an important role in forming a migratory route for moving the LHRH-producing neurons towards the forebrain 32. Informatively, the Kallmann syndrome is a manifestation of an inherited hypogonadotropic hypogonadism with anosmia, and it recently has been reported that in a Kallmann fetus, LHRH-immunoreactive cells did not migrate normally into the brain because of the absence of the central projection of the terminal nerve 33. In birds, on the other hand, no report describing such terminal nerve has appeared to date 5,16.40. In the material which we inspected, however, cords of LHRH-immunoreactive neurons werc traceable along almost the entire course of the olfactory nerve bundle from stages 24 to 34. This would appear to suggest that in birds it may be the olfactory nerve, rather than the terminal nerve, that provides a favorable environment for facilitating the migration of the LHRH-producing neurons. In this regard, a report has indicated that only a few labeled fibers were detected in the basal forebrain area, caudally to the olfactory bulb, following an H R P injection into the olfactory mucosa of mallard ducklings 3s. This raises the possibility that some remnant of the terminal nerve may still be present in birds. The olfactory nerve bundle may contain some vestigeal component of the terminal nerve in birds. In the early stages of development, a strong immunoreactivity for NCAM-H was detected in most LHRH-producing neurons, whereas this strong NCAM-H immunoreactivity in the LHRH-positive cells disappeared after the L H R H neurons moved into almost the same position that they occupy in the adult brain. Similarly, in the developing mouse cerebellar cortex, NCAM-H has been reported to be expressed temporarily on all cell types, then disappears coincidentally with the cessation of granule cell migration t3 thereby suggesting that NCAM-H is involved in cell migration. NCAM-H has been found to be less adhesive than adult forms of NCAM. Probably the negatively-charged portion of polysialic acid modulates the binding property of NCAM 14,2,~.Diminished adhesiveness of NCAM-H has been speculated as enabling the neurons to avoid forming stable junctions and to remain responsive to guidance and target cues 2s. Close association between the expression of NCAM-H and the developing L H R H neurons observed in the present study may indicate the possibility that NCAM-H facilitates the migration of the L H R H neurons into the brain. With regard to this conjecture, a recent abstract has indicated that migrating LHRH-producing neurons in mice were found to be associated with the fascicles of the NCAM (not NCAM-H)-immunoreactive terminal nerve fibers in the mouse 34 The possibility may not be excluded that non-migrating cells distributed in the olfactory system or neuroblasts originated from the ventricular layer of the forebrain might express L H R H temporarily according to the time sequence of the embryonic development. From our preliminary findings, however, unilateral removal of the olfactory placode at ED-3.5 or -4 did not result in expression of L H R H on the ipsilateral side of the olfactory-forebrain axis (Akutsu et al., unpublished data). This again suggests that L H R H neurons originate in the olfactory placode and migrate into the forebrain in the chick embryo. Similar cell migration phenomena from the olfactory placode have previously been reported in the developing chick and in various mammals 2,7-9,1s.25. In essence, bundles of the olfactory axons are accompanied by these migrating cells that grow out of the olfactory epithelium, and one type of migrating cells in the olfactory nerve bundle was found to contain large cored vesicles (100-200 nm in diameter) in the cell bodies and emerging axons of the chick embryo is. It is probable that these migrating cells are capable of expressing some peptide(s), e.g. LHRH. The possible role of migrating L H R H cells in the developing olfactory system is still

429 n o t known. In t h e d e v e l o p i n g cochlea, it has b e e n s p e c u l a t e d t h a t t h e m i g r a t i n g cells f r o m t h e s e n s o r y e p i t h e l i u m act as a g u i d e in d i r e c t i n g the axons o f t h e spiral g a n g l i o n to g r o w into t h e e p i t h e l i u m , w h e r e t h e y t e r m i n a t e 3. It thus c o u l d be a s s u m e d t h a t a p o s s i b l e i n t e r a c t i o n b e t w e e n the d e v e l o p i n g o l f a c t o r y nerve axons a n d the m i g r a t i n g L H R H - a n d N C A M - H - e x p r e s s i n g n e u r o n s m a y be involved in r e g u l a t i n g b o t h t h e g r o w t h o f the o l f a c t o r y n e r v e and t h e m i g r a t i o n o f L H R H n e u r o n s into t h e f o r e b r a i n . L H R H - e x p r e s s i n g n e u r o n s were also o b s e r v e d in t h e nasal b r a n c h of t h e o p h t h a l m i c n e r v e o f t h e t r i g e m i n a l n e r v e , but n o t in t h e t r i g e m i n a l ganglion. A l t h o u g h the s e n s o r y c o m p o n e n t o f t h e t r i g e m i n a l nerve is t h o u g h t to b e d e r i v e d f r o m b o t h p l a c o d e a n d n e u r a l crest l t,21, it still r e m a i n s u n c l e a r w h e t h e r t h e fibers i n n e r v a t i n g t h e nasal cavity a r e o f p l a c o d a l o r crest origin. A c c o r d i n g to s t u d i e s investigating t h e d e v e l o p m e n t o f t h e t r i g e m i n a l n e r v e b r a n c h e s in the chick, t h e t e r m i n a l b r a n c h e s of t h e o p h t h a l m i c nerve in t h e chick e m b r y o e x t e n d e d a r o u n d t h e e x t e r n a l n a r e s at stages 2 6 - 2 7 ( E D - 4 . 5 - 5 ) 17 Since t h e o l f a c t o r y n e r v e is l o c a t e d in close p r o x i m i t y to the o p h t h a l m i c nerve at t h e c a u d a l p o l e o f t h e n a s a l b o n e 39, it is p o s s i b l e that some o f t h e m i g r a t i n g L H R H n e u r o n s from t h e o l f a c t o r y p l a c o d e m a y j o i n with the o p h t h a l m i c nerve. ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d by a G r a n t - i n - A i d for Scientific R e s e a r c h f r o m the J a p a n e s e M i n i s t r y of E d u c a t i o n , S c i e n c e a n d C u l t u r e (No. 02640585). REFERENCES 1 Bl~ihser, S. and Heinrichs, M., lmmunoreactive neuropeptide systems in avian embryos (domestic mallard, domestic fowl, japanese quail), Cell Tissue Res., 223 (1982) 287-303. 2 Bossy, Y., Development of olfactory and related structures in staged human embryos, Anat. Embryol., 161 (1980) 225-236. 3 Carney, P.R. and Silver, J,, Studies on cell migration and axon guidance in the developing distal auditory system of the mouse, J. Comp. Neurol., 215 (1983) 359-369. 4 Daikoku-lshido, H., Okamura, Y., Yanaihara, N. and Daikoku, S., Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: in vivo and in transplantation studies, Dev. Biol., 140 (1990) 374-387. 5 Demski, L.S., The evolution of neuroanatomical substrates of reproductive behavior: sex steroid and LHRH-specific pathways including the terminal nerve, Am. Zool., 24 (1984) 809-830. 6 Demski, L.S. and Schwanzel-Fukuda, M., Introduction. In L.S. Demski and M. Schwanzel-Fukuda (Eds.), The Terminal Nerce (Nervus Terminalis): Structure, Function and Evolution, Annals of the New York Academy of Sciences, 1987, pp. 1-14. 7 Disse, J., Die erste Entwicklung des Riechnerven, Anat. Hefte, 9 (1897) 257-300. 8 Farbman, A.I. and Squinto, L.M., Early development of olfactory recepter cell axons, Dev. Brain Res., 19 (1985) 205-213. 9 Filogamo, G. and Robecchi, M.G., Neuroblasts in the olfactory pits in mammals, Acta Anat. (Basel), 73 (1969) 182-187. 10 Hamburger, V. and Hamilton, H.L., A series of normal stages in the development of the chick embryo, J. Morphol., 88 (1951) 49-67. 11 Hamburger, V., Experimental analysis of the dual origin of the trigeminal ganglion in the chick embryo, J. Exp. Zool., 148 (1961) 91-123. 12 Hattori, M, and Ishii, S., Stimulation of FSH and LH releases by two chicken LHRH's and mammalian LHRH in vitro and in vivo, J. Steroid Biochem., 20 (1984) 1548. 13 Hekmat, A., Biner-Suermann, D. and Schachner, M., Immunocytological localization of the highly polysialylated form of the neural cell adhesion molecule during development of the murine cerebellar cortex, J. Comp. Neurol., 291 (1990) 457-467. 14 Hoffman, S. and Edelmann, G.M., Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule, Proc. Natl. Acad. Sci. USA, 80 (1983) 5762-5766.

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