Localization of BDNF expression in the developing brain of zebrafish

Abstract

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease.

Keywords: brain-derived neurotrophic factor; central nervous system; development; real-time quantitative polymerase chain reaction; whole-mount in situ hybridization; zebrafish.

Figure 1

Relative expression of the zebrafish BDNF gene at selected developmental stages [256-cell, 24, 48, 72, 96, 120 and 192 hours post-fertilization (hpf)]. Expression levels were normalized against rpl13a and elfa1 reference genes, as described in Materials and methods. Black bars represent mean ± SD.

Figure 2

Patterns of BDNF expression at 24 hpf. Lateral (a,b,d,e), dorsal (c,f) or ventral (g) views. Anterior towards left (a,b,d–f) or up (c,g). Discrete mRNA domains across the forebrain, including (a–c) dorsal telencephalon, hypothalamus beside the preoptic/supraoptic area, pituitary gland, posterior tubercle, ventral thalamus, lateral line primordium, otic vesicle and spinal cord. Arrowheads mark telencephalic edges. Magnification of (d) lateral line primordium and (e) otic vesicle signals. BDNF expression in (c) the olfactory epithelium, (f) hindbrain and (g) heart. Abbreviations: Ce, cerebellum; Di, diencephalon; Ey, eye; HB, hindbrain; He, heart; Hy, hypothalamus; LL, lateral line primordium; MB, midbrain; OE, olfactory epithelium; OV, otic vesicle; PG, pituitary gland; Po, preoptic area; Pr, pretectum; PT, posterior tubercle; rTh, rostral thalamus; SC, spinal cord; soc, supraoptic area; Te, telencephalon; TeO, tectum; vTh, ventral thalamus. Scale bars: 50 µm.

Figure 3

Patterns of BDNF expression at 48 hpf. Lateral (a,e,e') or dorsal (b–d,f) views. Anterior towards left (a,d,e,f) or up (b,c,d'). WISH signal in (a–d) the dorsal pallium, hypothalamus, posterior tuberculum, neurosecretory preoptic area, pretectum and rhombencephalon (Rh), and olfactory bulbs. Eye signal is seen in an inner group of retinal cells (e,e'). Asterisks in (e,e') indicate non-specific precipitation of dye. BDNF expression (f) throughout Rh and in distributed groups of hair cells in the otic capsule (arrowheads). Abbreviations: ac, anterior commissure; Ce, cerebellum; cTh, caudal thalamus; Ep, epiphysis; ETh, epithalamus; Ey, eye; Hy, hypothalamus; Is, isthmus; MO, medulla oblongata; No, notochord; OB, olfactory bulbs; or, optic recess; OV, otic vesicle; P, pallium; PG, pituitary gland; Po, preoptic region; poc, preoptic area; Pr, pretectum; PT, posterior tubercle; Rh, rhombencephalon; rTh, rostral thalamus; S, subpallium; Te, telencephalon; TeO, tectum; Tg, tegmentum; tpoc, trait of the postoptic commissure. Scale bars: 50 µm.

Figure 4

Patterns of BDNF expression in post-embryonic and larval stages. Lateral (a,c,d,g,h,j–l) or dorsal (b,i) views. Anterior towards left (a,d,e,f) and up (b,c,d'). Transverse sections in (e,f). At 72 hours post-fertilization (hpf; a–f), BDNF transcript staining (a,b) in pallium, olfactory epithelium, hypothalamus and posterior tuberculum, and (c) weak labeling in the otic vesicle. (d,d') BDNF expression appears to be expanded across the retina. Black lines in (a) indicate the frontal plane of the sections in (e) and (f). At 96 hpf (g–i), mRNA labeling was prominent in a domain spanning the posterior tubercle, pretectum and optic tectum. In the eye (g'), BDNF expression was further extended towards the inner and outer cells of the retina. (j,k) Low level of expression was depicted at 120 and 196 hpf (asterisk indicates non-specific precipitation of dye). (l) Background staining of brain ventricles in 48-hpf embryo hybridized with a sense probe. Abbreviations: ac, anterior commissure; Ey, eye; Hy, hypothalamus; MO, medulla oblongata; NP, nasal pit; OB, olfactory bulbs; OV, otic vesicle; P, pallium; PE, pharyngeal epithelium; Po, preoptic region; poc, preoptic area; Pr, pretectum; PT, posterior tubercle; Rh, rhombencephalon; S, subpallium; Te, telencephalon; TeO, optic tectum; Tg, tegmentum; Th, thalamus. Scale bars: 100 µm.

Figure 5

Double WISH experiments at 24 hpf. Lateral (a,c,e,g,i) or dorsal (b,d,f,h,j) views. Anterior towards (a–i) left or (j) up. BDNF is expressed in (a,b,i,j) the mantle zone of telencephalic pallium (emx1, lhx2b, lhx9), (a,c,d,i) lateral margin of the basal plate (emx1, shh, lhx2b) and (d) lateral to the floor plate (FP; shh). Double-labeling with (e,f) pax2a and (g,h) krox20 riboprobes indicate that BDNF is not expressed in the isthmus and cerebellum (asterisk). (g,h) Note BDNF message in ventral cell clusters of all rhombomeres (krox20). Abbreviations: BP, basal plate; Ce, cerebellum; FP, floor plate; Hy, hypothalamus; Is, ishtmus; OS, optic stalks; OV, otic vesicle; r1, rhombomere 1; r3, rhombomere 3; r5, rhombomere 5; TMa, telencephalon mantle; TVz, telencephalon ventricular zone; vDi, ventral diencephalon; Zli, zona limitans intrathalamica. Scale bar: 100 µm.