Distribution of glial cell line-derived neurotrophic factor receptor alpha-1 in the brain of adult zebrafish

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor for several types of neurons in the central and peripheral nervous systems. The biological activity of GDNF is mediated by a multicomponent receptor complex that includes a common transmembrane signaling component (the rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor) as well as a GDNF family receptor alpha (GFRalpha) subunit, a high-affinity glycosyl phosphatidylinositol (GPI)-linked binding element. Among the four known GFRalpha subunits, GFRalpha1 preferentially binds to GDNF. In zebrafish (Danio rerio) embryos, the expression of the GFRalpha1a and GFRalpha1b genes has been shown in primary motor neurons, the kidney, and the enteric nervous system. To examine the activity of GFRalpha in the adult brain of a lower vertebrate, we have investigated the localization of GFRalpha1a and GFRalpha1b mRNA and the GFRalpha1 protein in zebrafish. GFRalpha1a and GFRalpha1b transcripts were observed in brain extracts by reverse transcription-polymerase chain reaction. Whole-mount in-situ hybridization experiments revealed a wide distribution of GFRalpha1a and GFRalpha1b mRNAs in various regions of the adult zebrafish brain. These included the olfactory bulbs, dorsal and ventral telencephalic area (telencephalon), preoptic area, dorsal and ventral thalamus, posterior tuberculum and hypothalamus (diencephalon), optic tectum (mesencephalon), cerebellum, and medulla oblongata (rhombencephalon). Finally, expression patterns of the GFRalpha1 protein, detected immunohistochemically, correlated well with the mRNA expression and provided further insights into translational activity at the neuroanatomical level. In conclusion, the current study demonstrated that the presence of GFRalpha1 persists beyond the embryonic development of the zebrafish brain and, together with the GDNF ligand, is probably implicated in the brain physiology of an adult teleost fish.

Fig. 1

Reverse transcription-polymerase chain reaction profiles. (A) Glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)1a mRNA expression in adult zebrafish brain. S, molecular size standard (1-kb Plus DNA Ladder, Invitrogen); 1a, specific primers amplified a band of 800 bases in the lane of brain extracts; C, a β-actin 406-bp fragment was amplified as a control to exclude a genomic contamination and to validate the purity of cDNAs. (B) GFRalpha1b mRNA expression in adult zebrafish brain. S, molecular size standard (1-kb Plus DNA Ladder, Invitrogen); 1b, specific primers amplified a band of 760 bases in the lane of brain extracts; C, a β-actin 406-bp fragment.

Fig. 2

Photomicrographs of whole-mount glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)1a (A,B) and GFRalpha1b (C,D) in-situ hybridization. (A) Dorsal view. Expression in the dorsal and medial zone of the dorsal telencephalic area (Tel) and lobus vagus (LX). (B) Dorsal–lateral view. High magnification of expression in the olfactory bulbs (OB) and dorsal zone of the dorsal telencephalic area. (C) Dorsal view. Expression in the dorsal and lateral zone of the dorsal telencephalic area, cerebellar corpus (CCe), and lobus vagus (LX). (D) Dorsal–lateral view. Expression in olfactory bulbs (OB) and in the lateral zone of the dorsal telencephalic area. For other abbreviations see Table 1. Scale bar: 500 μm.

Fig. 3

Photomicrographs of whole-mount glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)1a (A) and GFRalpha1b (B) in-situ hybridization. (A) Ventral view. Expression in the olfactory bulbs (OB), anterior part of area preoptica (PPa), caudal (Hc) and ventral (Hv) zone of periventricular hypothalamus, diffuse nucleus of the inferior lobe of hypothalamus (DIL), and inferior olive (IO) of the medulla oblongata. (B) Ventral view. Expression in the ventral telencephalic area (Tel), anterior part of area preoptica (Ppa), caudal (Hc), dorsal (Hd) and ventral (Hv) zone of periventricular hypothalamus, and inferior olive (IO) of the medulla oblongata. For other abbreviations see Table 1. Scale bar: 500 μm.

Fig. 4

Double immunofluorescence stainings. Glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)1 (A) and neuron-specific enolase (B) immunoreactivity (IR) are co-localized (C) in neurons of the medial zone of the dorsal telencephalic area. GFRalpha1 (D) and S100 (E) IR are not co-localized (F) in cells of the optic tectum. GFRalpha1 IR is localized in superficial cells, whereas S100 IR is localized in other inner cells. For abbreviations see Table 1. Scale bar: 10 μm for A and B; 20 μm for C and D.

Fig. 5

Schematic drawings of transverse sections of adult zebrafish brain. The complete drawings show, on the left side, the cytoarchitecture of the zebrafish brain and, on the right side, the distribution of glial cell line-derived neurotrophic factor family receptor alpha1-positive neurons in gray areas. For abbreviations see Table 1. Scale bar: 200 μm. Modified from Wullimann et al. (1996).

Fig. 6

Photomicrographs of glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)1 immunoreactivity in the telencephalon, diencephalon, and mesencephalon (transversal sections). (A) GFRalpha1-positive neurons in the glomerular layer (GL) and external cellular layer of olfactory bulbs (ECL). (B) GFRalpha1-positive neurons and fibers in the dorsal medial zone of the dorsal telencephalic area. (C) GFRalpha1-positive fibers in the dorsal lateral zone (Dl) of the dorsal telencephalic area. (D) Positive parvocellular preoptic nucleus, posterior part (PPp), ventromedial (VM), and ventrolateral (VL) thalamic nucleus of diencephalon. (E) Higher magnification of ventromedial and ventrolateral thalamic nuclei. (F) Positive fibers running towards the optic tectum and positive neurons in the ventromedial thalamic nucleus (VM). (G) Positive fibers in the posterior commissure (Cpost) and positive neurons in the mesencephalic nucleus of the trigeminal nerve (MNV) and in the dorsal part of the periventricular pretectal nucleus (PPd). Insert: high magnification of the dorsal part of the mesencephalic nucleus of the trigeminal nerve. (H) Positive neurons in the superficial white and gray zone of the optic tectum (TeO). For other abbreviations see Table 1. Scale bar: 50 μm for D and F; 20 μm for A, G and H; 10 μm for B, C, E and inset G.

Fig. 7

Photomicrographs of glial cell line-derived neurotrophic factor family receptor alpha (GFRalpha)1 immunoreactivity in the medulla oblongata (transversal sections). (A–C) GFRalpha1-positive neurons in the facial motor nucleus (NVIIm) and intermediate reticular formation (IMRF). (D,E) GFRalpha1-positive neurons of lobus facialis (LVII). Scale bar: 50 μm for C; 10 μm for D; 5 μm for A, B and E.

Fig. 8

Glial cell line-derived neurotrophic factor family receptor alpha-1 immunoreactivity in the ganglion cell layer (GCL) and inner nuclear layer (INL) of the retina. Scale bar: 50 μm.