DEVELOPMENTAL BRAIN

RESEARCH

ELSEVIER Developmental Brain Research 91 (1996) 237-244

Research report

Subpopulations of migrating neurons express different levels of LHRH in quail and chick embryos

Chen Gao, Reema Abou-Nasr, Robert B. Norgren Jr. *

Department of Cell Biology and Anatomy, University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-6395, USA

Accepted 24 October 1995

Abstract

LHRH neurons of the septal-preoptic area originate in the olfactory placode and migrate in the olfactory nerve into the brain during embryonic development. In adult birds, LHRH neurons have been found in the septal-preoptic area, mesencephalon and more recently in the lateral anterior nucleus of the thalamus (LA). LHRH neurons of the LA do not originate in the olfactory placode. Using immunocytochemistry, we examined the distribution of LHRH neurons in the embryonic and adult quail nervous system. The pattern of LHRH immunostaining in quail embryos was similar to that seen in chick embryos. However, there were many fewer neurons immunostained for LHRH from the olfactory placode to the septal-preoptic area in quail than in chick embryos. In contrast, there were more labeled neurons and more intense LHRH immunostaining in the thalamus of the quail than in the thalamus of chick embryos. In agreement with other studies, our data suggest that there are species differences in LHRH expression in migrating neurons. The current results should also be considered for quail-chick chimeras involving the olfactory placode.

Keywords: Migration; GnRH; Olfactory; Thalamus; Quail-chick chimera; Development

1. Introduct ion

Neurons which synthesize luteinizing hormone-releas- ing hormone (LHRH) play an important role in the control of vertebrate reproduction. LHRH neurons have been found in the septal-preoptic area and hypothalamus of many species of animals. In a recent study [10], LHRH neurons were found widely distributed in the chicken brain. In addition to the previously reported LHRH neurons found in the septal-preoptic area and mesencephalon [9,14,31], LHRH neurons were found in the lateral anterior nucleus of thalamus [10]. In Japanese quail, LHRH neurons were found in the septal-preoptic area but no mention was made of the presence of LHRH neurons in the thalamus [4,6,14,34].

Several recent studies have demonstrated that LHRH neurons of the septal-preoptic area originate in the medial olfactory placode and migrate into the brain during embry- onic development in mice, chicks and amphibians [17,22,27,35,36]. However, LHRH neurons of the LA ap- pear to originate in the brain in the chick [21].

* Corresponding author. Fax: (1) (402) 559-7328.

0165-3806/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0165-3806(95)00189-1

Ablation of chick tissue followed by transplantation of quail tissue to the chick has been used to study neuronal differentiation and migration [12,18]. Donor quail cells can be distinguished from host chick cells by differences in the appearance of heterochromatin [11] as well as with species-specific antibodies [19,25]. As we plan to use quail-chick chimeras to examine olfactory placode devel- opment and LHRH neuron differentiation and migration, it is necessary to determine the normal time course of LHRH neuron migration in the quail.

In the current work, we examine the distribution of LHRH neurons in the quail telencephalon and thalamus both during embryonic development and in the adult brain, using antibodies to LHRH. In contrast to the chick em- bryo, we found very little LHRH immunostaining in the olfactory placode of the quail embryos and relatively little LHRH immunostaining in the olfactory nerve and telen- cephalon during development, although the pattern of LHRH expression in the quail was similar to that seen in the chick. In contrast, the neurons in the thalamus of the embryonic quail showed stronger expression of LHRH than in the thalfimus of chick embryos. In addition, we report for the first time the presence of LHRH neurons in the nucleus LA of adult quail.

238 C. Gao et aL / Developmental Brain Research 91 (1996) 237-244

2. Materials and methods

2.1. Immunocytochemistry

Fertilized quail embryos (n = 22) and chick embryos (n = 20) were obtained from commercial sources (TRUSLOW farm and SPAFAS respectively) and were placed in an incubator for varying amounts of time. Both chick and quail embryos were staged according to morpho- logical criteria [5], rather than by age as chicks and quail differ in time course of development. Embyros were then either immersion-fixed in 4% paraformaldehyde overnight or perfused. Three adult quail were also perfused. Cardio- vascular perfusion with a 0.75% saline rinse was followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. After fixation, early embryo heads and brains from older animals were cryoprotected by infiltration with 20% sucrose in phosphate buffer, and cut at 20 /xm on a cryostat. Sections were incubated in primary antibody for 24 hr at 4°C. The ABC technique (Vector) and di- aminobenzidine were used to visualize immunostained tis- sues.

2.2. Antibodies

Four primary antibodies to the decapeptide LHRH were used in adult quail brain. Three antibodies were raised against mammalian LHRH. These included two polyclonal antibodies, LR-1 and HU60, and one monoclonal antibody, HU4H. In addition, a polyclonal antibody specific for chicken I LHRH (081, 3 /9 ) was used.

Both quail and chick embryos were immunostained with LR-1. LR-1 was generously provided by Dr. R. Benoit. HU4H and HU60 were gifts of Dr. H. Urbanski. 081, 3 / 9 was kindly provided by Dr. J. Millam. Speci- ficity of LR-1 was demonstrated by incubating adjacent sections in antibody that had been preabsorbed with 0.1 /zg/ml of synthetic LHRH.

2.3. Cell counts

Immunoreactive cells in quail and chick embryos were observed with a Zeiss Axiophot microscope. Each section was scanned at 200-400 magnification, and immuno- reactive cells in the telencephalon and septal-preoptic area were counted. Only cells in which the nucleus was clearly

A B C

DI

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Fig. 1. Camera lucida drawings indicating the distribution of LHRH neurons (black dots) in quail embryos at different developmental stages. Sections were taken in the plane parallel to the horizontal axis of skull. Panels A, B and C represents sections from the same animal (stage 29). Panel D represents a section from a stage 32 animal. Panel E represents a section from a stage 37 animal. In each panel, dorsal is top and ventral is bottom. CO, chiasma opticum; DI, diencephalon; LA, nucleus lateralis anterior thalami; ME, mesencephalon; OE, olfactory epithelium; ON, olfactory nerve; TEL, telencephalon; V, ventricle. Bar = 100 ~m.

present were counted. No correction was applied to these counts because the sections were taken at 20 /~m.

3. Results

All three antibodies (HU4H, HU60 and LR-1) which were directed against mammalian LHRH immunostained LHRH neurons in the telencephalon and thalamus of adult quail brain. The antibody specific for chicken I (081, 3 /9 ) immunostained cells in the telencephalon but did not im- munostain neurons in the thalamus. Thus, the thalamic neurons may express a different form of LHRH than the septal-preoptic area neurons. We have previously reported a similar finding in chick embryos [21]. In both embryos and adult quail, immunostaining in both telencephalon and

Fig. 2. Photomicrographs of LHRH neurons in quail (A, C and E) and chick (B, D and F) embryos at equivalent stages, using the same antibody (LR-1). A, B, C and D are sections from stage 29 embryos while E and F are sections from stage 32 embryos. Boxes in Fig. 1 indicate the location of the labeled neurons within sections through the quail brain represented in Fig. 2. Box (bottom) in 1C corresponds to Fig. 2A. Box in 1B corresponds to Fig. 2C. Box in 1D corresponds to Fig. 2E. All panels are at the same scale as A (Bar = 50 /.Lm). A single LHRH neuron (arrow) was found adjacent to the olfactory epithelium (oe) of a quail embryo (A), while many more labeled LHRH neurons were seen emerging from a chick embryo at the same stage (B). Fewer LHRH neurons were also found in the olfactory nerve (arrows) in quail (C) than in the chick embryo (D). LHRH neurons migrating into the telencephalon exhibited lighter immunostaining and fewer labeled LHRH neurons were seen in a quail (E) than in a chick embryo (F). The orientation is the same as in both species. Ventral is on bottom at each panel.

C. Gao et al. / Developmental Brain Research 91 (1996) 237-244 239

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240 C. Gao et al. / Developmental Brain Research 91 (1996) 237-244

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Fig. 3. Photomicrographs of LHRH neurons in thalamus of quail (A) and chick (B) embryos at stage 29. Box (top) in Fig. 1C corresponds to Fig. 3A. There were more labeled LHRH neurons and more intense LHRH immunostaining in the thalamus of the quail embryo than in the thalamus of chick embryo. Bar = 50 /xm. The magnification is the same in B.

thalamus was completely blocked by preabsorption of LR-1 with mammalian LHRH. The results described below were obtained with LR-1.

3.1. Olfactory placode and olfactory nerve

We examined immunostaining for LHRH in the olfac- tory epithelium of quail and chick embryos at the same developmental stages (HH stages 20-37). In quail em- bryos, only a few LHRH immunoreactive neurons were found in the olfactory epithelium or olfactory nerve at any of these stages. At stage 29 in the quail, a few LHRH neurons were seen emerging from the olfactory epithelium (Fig. 1C, 2A) and in the olfactory nerve (Fig. 1B, 2C). In contrast, at stage 29 in the chick, numerous LHRH neurons

were seen emerging from the rostral-medial olfactory ep- ithelium (Fig. 2B) and in the olfactory nerve (Fig. 2D).

3.2. The migration of LHRH neurons in the telencephalon

After reaching the telencephalon from the olfactory nerve, LHRH neurons migrate in two phases in the chick telencephalon [22,32]. (1) LHRH neurons migrate caudally and dorsally along the medial edge of the telencephalon. (2) LHRH neurons migrate laterally and ventrally to the septal-preoptic area. At some stages different LHRH neu- rons may be found at both phases of migration in the same animal. Fewer LHRH immunostained neurons were found in both phases of migration in the quail (Fig. 2E; Table 1) than in the chick (Fig. 2F). However, this disparity was not as great in the second phase of migration.

Table 1

Distribution of LHRH neurons in quail and chick embryos at different stages

Stage No. of animals Quail

Mean No. of cells in the TEL. a

Mean No. of cells in the SEP.-POA b

No. of animals Chick

Mean No. of cells in the TEL.

Mean No. of cells in the SEP.-POA

27 2 6 28 2 2 29 2 7 31 2 43 32 2 62 35 2 58 37 1 36

15 96 142 59

2 95 1 195 2 535 2 1069 285 2 319 1879 2 208 388

NA 1

a Telencephalon. b Septal-preoptic area. 1 Not available.

C. Gao et al. / Developmental Brain Research 91 (1996) 237-244 241

Fig. 4. Photomicrograph of LHRH neurons in the LA and in the area between the LA and the third ventricle of quail embryos at Stage 37. Box in Fig. 1E corresponds to Fig. 4. Bar = 50 /zm.

A

Fig. 5. Camera lucida drawings indicating the distribution of LHRH neurons (black dots) in adult quail. Sections were taken in the plane parallel to the horizontal axis of skull. A is rostral to B. CO, chiasma opticum; LA, nucleus lateralis anterior thalami; POA, preoptic area; S, septal area. Bar = 500 /xm.

In the adult quail brain, large numbers of intensely immunostained LHRH neurons were observed in the sep- tal-preoptic area (Figs. 5 and 6). No obvious differences in the numbers of LHRH neurons in adult chickens and adult quail were observed.

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3.3. Thalamus

LHRH neurons in the thalamus were first detected on stage 27 in quail and stage 29 in chick. These neurons

Fig. 6. Photomicrographs of LHRH neurons in adult quail. A: LHRH neurons in the septal area. Box (top) in Fig. 5A corresponds to this photomicrograph. B: LHRH neurons in the preoptic area. Box (bottom) in Fig. 5A corresponds to this photomicrograph. C: LHRH neurons in the LA. Box in Fig. 5B corresponds to this photomicrograph. Bar = 50 /zm. The magnification is the same in B and C.

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242 C. Gao et al. / Developmental Brain Research 91 (1996) 237-244

were found in the dorsal tip of the external layer of the ventral thalamus. In stage 29 quail, there was strong LHRH immunostaining in the thalamus (Fig. 1C, Fig. 3A) while immunostaining was barely detectable in the olfac- tory nerve (Fig. 2C) and telencephalon (Fig. 2E). This differs from what we observed in chick embryos where migrating neurons in the telencephalon were more densely immunostained than the LHRH neurons in thalamus (Fig. 2F, 3B). In addition, in both embryonic (starting from stage 30) and adult quail, we found a cluster of LHRH fibers and a few LHRH neurons between the third ventri- cle and lateral thalamus (Fig. 1E, Fig. 4). The thalamic LHRH neurons in adult quail were located in the lateral anterior nucleus (Fig. 5B, 6C).

4. Discussion

Considerable species differences have been observed in the level of LHRH expression during neuronal migration from the olfactory placode to the brain. In embryonic rats, LHRH neurons were rarely seen migrating from the olfac- tory placode [8,26]. In contrast, in mice, they were readily found using the same antibody to LHRH [27,35,36]. Muske and Moore [17] noted that there was no LHRH immuno- staining in the terminal nerve/olfactory nerve at all devel- opmental stages in two species of frog. In addition, no LHRH immunoreactivity was detected in the olfactory epithelium at early stages of newt embryos [15]. In the chick, LHRH is strongly expressed in the developing olfactory system [16,20,32]. We observed much less LHRH immunostaining in the olfactory system at all stages of quail embryonic development with the same antibody (LR- 1) used to immunostain chick embryos (Fig. 2). Thus quail are similar to rats in that very little LHRH immunostaining is seen during migration from the olfactory epithelium to the brain while chicks are similar to mice as many im- munostained LHRH neurons are observed during migra- tion from the olfactory epithelium to the brain.

Several lines of evidence indicate that the species dif- ferences we and others observe in the amount of LHRH expression by migrating neurons is not due to the ability of LHRH antibodies to recognize LHRH in embryos. First, the LHRH antibodies we used worked well on quail thalamic LHRH neurons in embryos and in all LHRH neurons in adults. Second, it is unlikely that LHRH anti- bodies that work on mouse embryos would not work on rat embryos because the amino acid sequence of LHRH is the same for these two closely related species [1,13]. Third, in situ hybridization experiments indicate that LHRH mes- sage is not expressed in the developing olfactory system of amphibian embryos [7] even though ablation experiments indicate that LHRH neurons must be migrating in the olfactory system during development [15,24]. Species dif- ferences in LHRH expression in the developing olfactory system may be due to differences in the level of expression

of LHRH in migrating neurons, i.e. the total number of neurons migrating to the septal-preoptic area which will ultimately express LHRH in the adult may be similar in many species of vertebrates while the percentage of these migrating cells which express LHRH during development may vary widely even among closely related species. Alternatively, the different amount of LHRH immuno- staining in the developing olfactory system may actually represent large differences in the number of LHRH neu- rons derived from the olfactory epithelium in closely re- lated species. Two lines of evidence support the first hypothesis. First, Daikoku-Ishido et al. [2] reported im- munostained neurons in the olfactory placode of 13.5 days of gestation rats with an antibody to rat gonadotropic hormone-releasing hormone associated peptide (a precur- sor molecule to LHRH) but not with an antibody to LHRH. These observations led Schwanzel-Fukuda and Pfaff [28] to propose that LHRH neurons in guinea pigs, rats and opossums originate in the olfactory placode, but do not express mature LHRH until they have migrated out of the epithelium. Second, ablation studies in amphibians indicate that even though no LHRH is expressed in the developing olfactory system, the septal-preoptic LHRH neurons must originate from the olfactory placode and migrate into the brain along the terminal nerve and/or olfactory nerve [15]. Although we saw relatively few LHRH neurons in the developing olfactory system of the quail, the pattern of expression was similar to that observed in the chick [16,22,32].

Thalamic LHRH neurons do not originate from the olfactory placode in the chick [21]. Although we first observed LHRH neurons in stage 27 quail and at stage 29 in chicks, the pattern of immunostaining was very similar. LHRH fibers and neurons were found between the third ventricle and lateral thalamus in both embryonic and adult quail (Fig. 4). These neurons were associated with fibers and were close to the neurons and fibers in the LA. Currently, the origin of thalamic LHRH neurons is un- known. If they arise from the ventricular zone of the third ventricle, as do most thalamic neurons, then they do not express LHRH until they have migrated laterally from the third ventricle.

The role of LHRH neurons in the septal-preoptic area in regulating pituitary gonadotropin release is well estab- lished [29]. However, the extensive distribution of the LHRH neuronal cell bodies and fibers and retrograde transport studies suggest that not all LHRH neurons pro- ject to the median eminence [10,30]. The function of LHRH neurons in the lateral anterior thalamic nucleus (LA) is not clear. It has been reported that the LA is a primary retinal recipient area in chick [3,23]. In addition, Kuenzel and Bl~ihser [10] suggested that since the LA receives direct retinal input, LHRH neurons in this nucleus may be involved in photoperiodic behavior. The LHRH system of the Japanese quail is also influenced by the photoperiod [4,33].

C. Gao et al. / Developmental Brain Research 91 (1996) 237-244 243

Our results on the distribution of the LHRH neurons and fibers in the septal preoptic area and hypothalamus of the adult quail are in substantial agreement with results from previous studies in quail [4,14,34]. However, this is the first report indicating the presence of thalamic LHRH neurons in both embryonic and adult quail. The very few LHRH immunostained neurons in the olfactory placode, olfactory nerve and telencephalon during development should be taken into account for quail-chick chimeras involving the olfactory placode.

Acknowledgements

We are grateful to Yongjie Ji for excellent technical assistance. This work was funded by NIH Grants R01 NS30047 (RBN) and DE06632 (D.M. Noden).

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