Neurons that Migrate from the Olfactory Epithelium in ...

0013-7227/91/1283-1676$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 3 Printed in U.S.A.

Neurons that Migrate from the Olfactory Epithelium in the Chick Express Luteinizing Hormone-Releasing Hormone ROBERT B. NORCREN, Jr. and MICHAEL N. LEHMAN Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0521 ABSTRACT. In several mammalian species, luteinizing hormone-releasing hormone (LHRH) neurons have been shown to migrate from nasal regions to the brain during early development. Using immunocytochemistry, we have identified LHRH containing neurons in developing chick embryos. In embryonic day 4 (E4) and E5 animals, a small group of LHRH immunoreactive (IR) neurons were found just ventral to the olfactory pit. LHRH-IR neurons were also found within the nasal epithelium. In E6 and E7 animals, many more LHRH-IR neurons were observed in nasal epithelium, in close association with the olfactory nerve, and within the telencephalon. These data are consistent with the hypothesis that LHRH neurons in chicks originate within nasal structures and migrate into the brain. Introduction Neurons which synthesize luteinizing hormone releasing hormone (LHRH) play a pivotal role in the control of vertebrate reproduction. Recent findings in mammals suggest that these cells do not originate in the brain, but rather migrate from the olfactory epithelium to the forebrain before birth (1,2,3,4,5). This represents one of the few examples of neurons developing outside the central nervous system, then migrating into it. The failure of these cells to migrate into the CNS has been linked to human hypogonadism (Kallman's syndrome) (6) and has spurred interest in experimental studies to determine what factors guide the development and migration of LHRH neurons. Previously, the migration of LHRH neurons from the olfactory epithelium into the brain had been reported only in mammals, although work on fish and amphibian terminal nerve suggests that a similar pattern of migration of LHRH neurons might be observed in these classes of vertebrates as well (7,8). Neurons of uncertain identity migrating from the olfactory placode have been identified in the chick (9,10,11). Our objective was to determine whether these migrating cells in the chick expressed LHRH.

Labeled cells were also apparent in the epithelium of the ventral olfactory pit. In E6 and E7 chick embryos, many more labeled cells were observed. LHRH-IR neurons were observed in the nasal epithelium (Fig. 1), mostly in the medial portions, although a few were also found on the lateral side of the nasal epithelium. Some labeled cells had processes which extended towards the lumen of the olfactory pit. Other cells had processes directed away from the lumen. Both these cell types were oriented perpendicular to the surface of the lumen. Many cells at the base of the epithelium were oriented parallel to the lumen and had processes which extended towards the olfactory nerve. Clusters of LHRH-IR neurons appeared to form a chain within the olfactory nerve and dorsal-caudally in close proximity to the telencephalon. Most of the LHRH-IR neurons were found in the medial half of the olfactory nerve (Fig. 2) and along the medial edge of the telencephalon. However, a few cells were found in the lateral half of the olfactory nerve and lateral to the telencephalon. In dorsal sections, a few LHRH-IR neurons were found when the brain and had labeled processes that appeared to make contact with the lateral ventricle (Fig. 3).

Materials and Methods Chick embryos (N = 24) obtained from SPAFAS were immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, and horizontal cryostat sections were incubated for 24 hours at 4°C in a polyclonal antisera to LHRH (LR-1, gift of Dr. R. Benoit). LR-1 recognized amino acids 3,4,7,8,9 and 10 of the decapeptide and no other identified neuropeptide. Binding of the primary antibody was visualized with an avidin-biotin-immunofluorescent technique using avidin-Texas red (Vector Laboratories).

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Results

In E4 and E5 chick embryos, a small cluster of cells of LHRH-IR neurons were found just lateral to the rostral telencerjhalon, immediately ventral to the olfactory pit. Received in Iowa City November 21, 1990

Figure 1. LHRH neurons in olfactory epithelium (E7). Stai indicates lumen of olfactory pit. Scale bar = 20 /im.

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Figure 2. LHRH neurons in medial portion of olfactory nerve (E7). Arrows indicate lateral edge of olfactory nerve. Scale bar = 30 jum.

To our knowledge, this is the first report indicating the possible migration of LHRH-IR neurons from the olfactory epithelium to the brain in a non-mammal. These findings suggest that the migration of LHRH-IR neurons from nasal epithelium to the brain may be a primitive characteristic of vertebrates and not a mammalian invention. This hypothesis should be tested by examining LHRH neuron development in other classes of vertebrates. Although the results of the current study are suggestive of migration of LHRH neurons from nasal structures, ablation of the olfactory placode during early development and the subsequent absence of LHRH neurons in the adult brain would provide strong confirming evidence for this hypothesis. The developing chick embryo has many advantages as a model system in which to carry out such experiments. In particular, perturbation of embryos and subsequent survival are more easily accomplished in chicks than in mammals. Issues which remain to be resolved include the significance of early expression of LHRH in developing neurons, the relationship between LHRH-IR neurons and cells of the nasal epithelium and possible sex differences in the migrating patterns. In addition the chick embryo provides a model for investigating the cues by which the LHRH-IR neurons find their way into the brain.

References

1. Schwanzel-Fukuda M, Pfaff DW 1989 Origin of luteinizing hormone-releasing hormone neurons. Nature 338:161-164.

Figure 3. LHRH neurons along medial edge and within (arrow) telencephalon. (E7). Magnification same as Figure 1. Although some of the LHRH-IR neurons in these early embryos were round, most had an elongated appearance. The nucleus of these cells occupies an eccentric position. Short labeled processes can be seen in the E4 embryos. By E7, longer and sometimes multiple processes can be observed extending from labeled cells. Discussion

The appearance of migrating epitheliod cells from the olfactory placode in chicks (9,10) is very similar to that of migrating LHRH-IR cells observed in the current experiment. We believe that the migrating epithelioid cells from the olfactory placode observed in chicks and in various species of mammals (11,12) may be LHRH-IR neurons. In addition, the pattern of migration observed in the chick is very similar to that first reported in mouse (1,3).

2. Wray S, Grant P, Gainer H 1989 Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc Natl Acad Sci 86:8132-8136. 3. Wray S, Nieburga A, Elkabes S 1989 Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse; evidence for an embryonic origin in the olfactory placode. Dev Brain Res 46:309-318. 4. Ronnekleiv OK, Resko JA 1990 Ontogeny of gonadotropin-releasing hormone-containing neurons in early fetal development of Rhesus macaques. Endocrinology 126:498-511. 5. Daikoku-lshido H, Okamura Y, Yanaihara N, Daikoku S 1990 Development of the hypothalamic luteinizing hormone-releasing hormone-containing neurons system in the rat: In vivo and in transplantation studies. Dev Biol 140-374-387. 6. Schwanzel-Fukuda M, Bick D, Pfaff DW 1989 Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Mol Brain


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7. Demski LS 1984 The evolution of neuroantomical substrates of reproductive behavior: sex steriod and LHRH-specific pathways including the terminal nerve. Amer Zool 24:809-830. 8. Muske LE, Moore FL 1988 The nervus terminalis in amphibians: Anatomy, chemistry and relationship with the hypothalamic gonadotropin-releasing hormone system. Brain Behav Evol 32:141-150.

10. Van Campenhout E 1937 Le development du systeme nerveux cranien chez le poulet. Arch Biol 48:611-666. 11.Mendoza AS, Breipohl W, Miragall F 1982 Cell migration from the chick olfactory placode: a light and electron microscopic study. J Embryol Exp Morph 69:47-59. 12. Farbman Al 1986 Prenatal development of mammalian olfactory receptor cells. Chem Senses 11:3-18. Acknowledgments

9. Disse J 1897 Die erste Entwicklung des Riechnerven. Anat Hefte 9:257-300.

This work was supported by a Biomedical Research Support Grant (RBN) and NIH Grant HD 21968 (MNL). We are grateful to Julia Lippert for expert technical assistance.

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