Extraocular motoneuron pools develop along a dorsoventral axis in zebrafish, Danio rerio

Abstract

Both spatial and temporal cues determine the fate of immature neurons. A major challenge at the interface of developmental and systems neuroscience is to relate this spatiotemporal trajectory of maturation to circuit-level functional organization. This study examined the development of two extraocular motor nuclei (nIII and nIV), structures in which a motoneuron's identity, or choice of muscle partner, defines its behavioral role. We used retro-orbital dye fills, in combination with fluorescent markers for motoneuron location and birthdate, to probe spatial and temporal organization of the oculomotor (nIII) and trochlear (nIV) nuclei in the larval zebrafish. We describe a dorsoventral organization of the four nIII motoneuron pools, in which inferior and medial rectus motoneurons occupy dorsal nIII, while inferior oblique and superior rectus motoneurons occupy distinct divisions of ventral nIII. Dorsal nIII motoneurons are, moreover, born before motoneurons of ventral nIII and nIV. The order of neurogenesis can therefore account for the dorsoventral organization of nIII and may play a primary role in determining motoneuron identity. We propose that the temporal development of extraocular motoneurons plays a key role in assembling a functional oculomotor circuit. J. Comp. Neurol. 525:65-78, 2017. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Keywords: RRID:SCR_002285; RRID:SCR_010279; RRID:ZFIN_ZDB-GENO-030919-1; RRID:ZFIN_ZDB-GENO-060619-2; cranial nerve; cranial nucleus; development; nIII; nucleogenesis; oculomotor.

Figure 1

Labeling of extraocular motoneurons by retro‐orbital dye fill. (A) Retro‐orbital fill procedure. Larvae were anesthetized and immobilized in 2% agar, target eye near the surface (A1). Agar was cleared from eye and a tungsten needle used to make an incision of angle (Θ) in the vicinity of one/more extraocular muscle (A2). Crystallized dye was placed at incision site (A3); somata were imaged later. (B) Targeted position and angle (Θ) of incision around eye to label a given motoneuron population. (C) Schematic of extraocular muscle innervation by ipsilaterally projecting motoneurons. LR motoneurons located in nVI were not targeted for dye fills. (D) Schematic of extraocular muscle innervation by contralaterally projecting motoneurons. MR, IR, SR, LR: medial, inferior, superior, lateral rectus. SO, IO: superior, inferior oblique. nIII: oculomotor nucleus. nIV: trochlear nucleus. nVI: abducens nucleus.

Figure 2

Identification of dye‐filled motoneuron subtypes by projection pattern and Tg(isl1:GFP) expression. (A) Filled motoneurons in ventral nIII (plane z4, or 5th of 15 6‐μm‐thick planes counting dorsally from z0, dashed line). Yellow arrowheads indicate dye‐filled motoneurons that were also GFP+ (contralateral: SR motoneurons; ipsilateral: IR/MR motoneurons). (B) Filled motoneurons in ventral nIII (plane z5, dashed line). White arrowheads indicate dye‐filled motoneurons without GFP (IO motoneurons); yellow arrowhead indicates a dye‐filled motoneuron that was also GFP+ (IR/MR motoneuron). Color bars represent photons detected in photon‐counting mode (IO data only); green range: 0–198; magenta range: 0–231. Green = GFP; magenta = Alexa Fluor 647 dye. Scale bars = 10 μm.

Figure 3

Fluorescent somata in Tg(isl1:GFP) zebrafish define the spatial extent of extraocular motoneurons in nIII and nIV. (A) Positions of fluorescent motoneurons in dorsal nIII/nIV (z12) relative to anatomical landmarks. nIII: rostral to MHB; nIV: caudal to MHB. (B) Positions of fluorescent motoneurons in ventral nIII (z5) relative to anatomical landmarks. Yellow dotted line represents the distance between the center of intensity of green fluorescence right of the midline and the centroid of the right PMCtA. Mid: midline. MHB: midbrain–hindbrain boundary. MLF: medial longitudinal fasciculus. PMCtA: posterior mesencephalic central artery. CCtA: cerebellar central artery. Scale bars = 20 μm.

Figure 4

Birthdating Analysis by photoconverted fluorescent Protein Tracing In vivo, with Subpopulation Markers (BAPTISM) method (Caron et al., 2008) for identifying the time of terminal differentiation (birthdate) of motoneurons in Tg(huC:Kaede; isl1:GFP) zebrafish, which express photolabile fluorescent protein Kaede pan‐neuronally. (A) Neurons born by the time of initial photoconversion (one of five timepoints shown) contain converted Kaede in initial image, taken at 5 dpf. Second photoconversion converts remaining Kaede, leaving GFP as the only green signal in Final image. Images are compared to determine birthdate of GFP+ motoneurons. (B) Dorsal plane (z12, dashed line) from a 5 dpf Tg(huC:Kaede; isl1:GFP) larva initially photoconverted at 36 hpf. Yellow arrowheads indicate an nIII motoneuron (isl:GFP+, final image) born by 36 hpf (converted huC:Kaede+, initial image). White arrowheads indicate a neuron born by 36 hpf not belonging to nIII/nIV. Orange arrowheads indicate an nIV motoneuron not born by 36 hpf. Scale bars = 20 μm.

Figure 5

Distribution of labeled motoneuron somata in 5–7‐day‐old Tg(isl1:GFP) zebrafish. (A) GFP+ motoneuron somata are shown as circles in nIII (light green) and nIV (dark green). The dorsoventral extent of nIII/nIV is subdivided into 15 6‐μm‐thick planes, labeled z14 (most dorsal) – z0 (most ventral). (B) Mean and individual (background traces) probability distributions from labeled motoneurons across nIII/nIV. nIII: n = 10 larvae, 2080 cells. nIV: n = 10 larvae, 936 cells.

Figure 6

Superior rectus motoneurons are located exclusively in ventral nIII in both 5–7 and 14 day old zebrafish; superior oblique motoneurons are located in nIV. (A) Location of SO (magenta) and SR (blue) motoneurons at 5–7 dpf across nIII/nIV. GFP+ neurons in nIV contralateral to the filled eye were defined as SO motoneurons; GFP+ neurons in nIII contralateral to the filled eye were defined as SR motoneurons. (B) Probability distributions of dye‐filled SO and SR motoneurons across nIII/nIV at 5–7 dpf (solid lines) and 14 dpf (dashed lines). SO motoneurons: n = 42 larvae, 606 cells (5–7 dpf); n = 19 larvae, 138 cells (14 dpf). SR motoneurons: n = 22 larvae, 229 cells (5–7 dpf); n = 14 larvae, 35 cells (14 dpf).

Figure 7

Inferior oblique motoneurons are located mainly in ventral nIII in 5–7‐day‐old zebrafish, in a distinct caudal region relative to superior rectus motoneurons, while inferior/medial rectus motoneurons are located mainly in dorsal nIII. (A) Location of IO (red), SR (blue), and IR/MR (brown) motoneurons at 5–7 dpf across nIII/nIV. GFP– neurons in nIII ipsilateral to the filled eye were defined as IO motoneurons; GFP+ neurons in nIII ipsilateral to the filled eye were defined as IR/MR motoneurons. SR data is mirrored across the midline from Fig. 4. (B) Probability distributions of dye‐filled IO and IR/MR motoneurons across nIII/nIV. IO motoneurons: n = 30 larvae (5–7 dpf), 154 cells. IR/MR motoneurons: n = 17 larvae (5–7 dpf), 160 cells.

Figure 8

Motoneurons in dorsal nIII are born before those in ventral nIII and nIV. (A) Location of motoneurons born by five initial conversion timepoints, from 22 through 50 hpf, in dorsal nIII/nIV (plane z12) and ventral nIII (plane z5) at 5 dpf. (B) Average number of motoneurons born by each timepoint across nIII and nIV.

Figure 9

Spatiotemporal organization of extraocular motoneurons in nIII and nIV. (A) Aggregate figure showing relative locations of nIII/nIV motoneurons at two exemplar planes; data from Figs. 6, 7. (B) Comparison of motoneuron subpopulation birthdates (gradient bars) with development landmarks (Clark et al., 2013) from live imaging of extraocular motoneuron projections (timeline markers; events inside gray box). Solid color bars = periods between BAPTISM initial photoconversion timepoints.