A transient population of neurons pioneers the olfactory pathway in the zebrafish

Abstract

Mechanisms guiding the first axons from the olfactory placode of the peripheral nervous system (PNS) to the olfactory bulb in the vertebrate CNS are unknown. We analyzed the initial outgrowth of axons from the olfactory placode in zebrafish and found a precocious transient class of pioneer neurons that prefigure the primary olfactory pathway before outgrowth of olfactory sensory axons or expression of olfactory receptor genes. Not only are the pioneers antigenically, morphologically, and spatially distinct from olfactory sensory neurons, they are also developmentally distinct; via fate mapping, we show that they arise from a more anterior region of the lateral neural plate than do the first sensory neurons. After the axons of the sensory neurons grow into the CNS, the pioneer neurons undergo apoptotic cell death. When we ablated the pioneers before axonogenesis, the following sensory axons showed severe misrouting. We propose that the pioneers provide the first necessary connection from the PNS to the CNS and that they establish an axonal scaffold for the later-arriving olfactory sensory neurons.

Fig. 1.

The pioneer neurons initiate the formation of the olfactory nerve and establish glomerular-like structures in the telencephalon. A, B, Frontal views with dorsal to the top. C–F, Dorsal views with anterior to the top. G, Ventral view with dorsal to the top. H, Diagrams labeled with letters corresponding topanels to show orientation. The second diagram is a side view, anterior to the left at a stage equivalent to the frontal view in H (A, B). The olfactory placodes are in red, and the eyes are ingray. I, A transverse section with anterior to the top. A, Frontal view of the head of a 24 h live zebrafish embryo. The olfactory placodes (op) are paired thickenings lying dorsoanteriorly. Thebroken line outlines the placode on theleft. B, Enlargement of a frontal view of a placode labeled with the zns-2 antibody at 24 h. Dendrites are absent on the apical surface (arrowhead), and the axons (arrow) exit basally and grow along the telencephalon. The broken line demarcates the edge of the placode.C, The pioneer axons (brown-blue,black arrow) meet the telencephalon in the region expressing the gene emx1 (purple,white arrow) in a 26 h embryo. D,E, A 30 h preparation showing two focal planes, labeled only with zns-2. The axons exit the placode (black arrows) and defasciculate in the telencephalon (white arrows). The broken line demarcates the posterior edge of the placode. F, At 38 h, the pioneer axons (white arrow) start to form condensations in the telencephalon. G, Axons labeled with zns-2 (black arrow) extend into the developing olfactory bulb in a 52 h embryo. Note the axonal condensations (white arrow) evident in the CNS. H, The developing olfactory placode has moved from a dorsal to a more anterior location in front of the developing eye (see A, B). As a result of this morphogenetic movement, the zns-2-labeled axons are more easily viewed from the ventral side (G,I) as they project anteriorly into the telencephalon. I, A 7.5 μm Epon section through the olfactory organ and bulb at the same developmental stage asG, with the axonal condensations indicated by thearrowhead. Scale bars: A, 40 μm;B, 25 μm; C–F, 35 μm;G, I, 40 μm.

Fig. 2.

The axons of the pioneer neurons and first sensory neurons colocalize in the CNS. A, A 44 h embryo labeled with zns-2. The pioneer axons converge to form the olfactory nerve (arrow) and then defasciculate to form axonal condensations in the olfactory bulb (arrowhead).Double cross indicates the region occupied by the (unlabeled) sensory neurons. B, Lateral-oblique view of the nose of a live 50 h fish labeled with DiI. Two cell bodies (arrow) with axons and several without axons are evident lying next to the eye. The axons extend into the developing olfactory bulb (arrowhead). Asterisk marks the region occupied by (unlabeled) pioneer neurons. C–E, The axons of primary olfactory sensory neurons colocalize with the pioneer axons in a single 1.5 μm optical section. Confocal micrographs of ventral view of an olfactory organ (outlined in purple) at 44 h showing sensory neurons labeled with DiI (arrows inC and D, red) and pioneer neurons labeled with zns-2 (D and E,green). The growth cones of the sensory neurons in the differentiating olfactory bulb colocalize (arrowhead) with the axons of the pioneer neurons. e, Eye. Anterior is to the top. This preparation is a ventral view as depicted in Figure 1H, (C–F). Scale bars: A, 30 μm; B, 20 μm;C–E, 40 μm.

Fig. 3.

The olfactory pioneer neurons die. Preparation labeled for zns-2 (green) and TUNEL (magenta) at 42 h shows pioneer neurons dying at this time. The TUNEL-labeled cells are primarily located in the basal-medial part (arrowhead) of the olfactory organ.o, Olfactory organ; ob, olfactory bulb;double cross indicates the location of the unlabeled sensory neurons. Ventral-oblique view. Serial optical sections from the olfactory organ into the bulb were superimposed. Scale bar, 20 μm.

Fig. 4.

The pioneer neurons and the olfactory sensory neurons arise from different regions of the neural plate.A, Dorsal view of the neural plate in a live 12 h embryo before the formation of the olfactory placode. Arrows 1and 2 indicate the regions in which single cells were labeled. The location of their progeny is shown in B andC, respectively. Top inset is a lateral view of an embryo, with arrowhead indicating the region shown in the micrograph. Bottom inset is a lateral view of a live embryo with a single cell labeled in position 2 at 12 h, viewed with both bright-field and fluorescence optics.B, Ventral view of live embryo at 36 h, before the onset of olfactory pioneer cell death. Single cells, labeled at the anterior end of the neural plate (A,1), gave rise to pioneer neurons, recognized by their basal-medial location in the developing olfactory organ. C, Ventral view of a live embryo at 48 h. Single cells, labeled farther posterior in the neural plate (A, 2) adjacent to the central region of the developing eye (A, e), gave rise to olfactory sensory neurons lying apically at the posterior edge of the olfactory organ. Anterior is to the top. Scale bars:A, 40 μm; B, C, 20 μm.

Fig. 5.

Ablation of pioneer neurons results in misrouting of olfactory sensory axons. Ventral views at 50 h with anterior to thetop. A, The absence of zns-2 labeling on the ablated side (right, asterisk) indicates that all the pioneer neurons were removed. Control side (left, arrowhead) shows normal pattern of zns-2 labeling. B, Olfactory sensory neurons labeled with DiI show posterior misrouting in which axons (arrow) grow posteriorly in the absence of pioneer neurons. C, Olfactory sensory neurons labeled with DiI show split misrouting in which axons grow both posteriorly (arrow) and anteriorly (arrowhead) toward telencephalon. Scale bars: A, 80 μm;B, C, 40 μm.

Fig. 6.

Sensory neurons, but not pioneer neurons, express olfactory receptor genes. A, Ventral view of a 52 h olfactory organ showing the combined expression of four olfactory receptor gene transcripts localized in the cytoplasm; axons are unlabeled. Labeled cells (dark blue) are located near the apical surface of the olfactory organ in which the sensory neurons, but not the pioneer neurons, are located. The basal edge of the olfactory organ is demarcated by the broken line. Scale bar, 20 μm. B, Cells expressing olfactory receptors are apically located. Bars represent the average (± SE) number of cells expressing olfactory receptor genes in each olfactory organ as a function of developmental time in the apical (ap) or basal (ba) region. Both olfactory organs from 22 to 28 animals were scored for each time point.

Fig. 7.

A transient population of pioneer neurons establishes the olfactory pathway. Summary diagram of the developing olfactory system from 24 to 54 h. A, The zns-2-positive pioneer neurons (green) are distinguished by their round dendriteless cell bodies. The stippled area indicates the region of emx1 gene expression in which the pioneer neurons contact the differentiating telencephalon. The sensory neurons (red) extend their axons into the CNS, following the axons of the pioneer neurons. The pioneer neurons become positive for TUNEL (purple), indicating that they die. The cells expressing olfactory receptor mRNAs (orange) are located near the apical surface in the same region that contains the olfactory sensory neurons. B, Time line showing the temporal sequence of developmental events. Thecolors correspond to the coding used inA; the arrow indicates when the olfactory placode becomes apparent with Nomarski optics in the live embryo. Thedashed lines indicate that not all preparations show labeling at the indicated time.