The Order and Place of Neuronal Differentiation Establish the Topography of Sensory Projections and the Entry Points within the Hindbrain

Abstract

Establishing topographical maps of the external world is an important but still poorly understood feature of the vertebrate sensory system. To study the selective innervation of hindbrain regions by sensory afferents in the zebrafish embryo, we mapped the fine-grained topographical representation of sensory projections at the central level by specific photoconversion of sensory neurons. Sensory ganglia located anteriorly project more medially than do ganglia located posteriorly, and this relates to the order of sensory ganglion differentiation. By single-plane illumination microscopy (SPIM) in vivo imaging, we show that (1) the sequence of arrival of cranial ganglion inputs predicts the topography of central projections, and (2) delaminated neuroblasts differentiate in close contact with the neural tube, and they never loose contact with the neural ectoderm. Afferent entrance points are established by plasma membrane interactions between primary differentiated peripheral sensory neurons and neural tube border cells with the cooperation of neural crest cells. These first contacts remain during ensuing morphological growth to establish pioneer axons. Neural crest cells and repulsive slit1/robo2 signals then guide axons from later-differentiating neurons toward the neural tube. Thus, this study proposes a new model by which the topographical representation of cranial sensory ganglia is established by entrance order, with the entry points determined by cell contact between the sensory ganglion cell bodies and the hindbrain.

Keywords: axon navigation; inner ear; neural crest cells; neuron differentiation; sensory systems; somatotopy.

Figure 1.

PhC of specific sensory neuronal pools in Tg[hspGFF53A]xTg[UAS:KAEDE] embryos. A, Depiction of the experiment: 48 hpf embryos expressed KAEDE^Green in sensory neurons; KAEDE^Green was photoconverted, and expression of KAEDE^Red in the sensory projection was assessed a few hours later. B–C, PhC of neurons from the TGg. B′, Magnification of the boxed region in B. C, Dorsal view of B′. D–E, PhC of neurons from the ALLg. D′, Magnification of the boxed region in D. E, Dorsal view of D′. F–G, PhC of neurons from the PLLg. F′, Magnification of the boxed region in F. G, Dorsal view of F′. Anterior is to the left. Axes are indicated in the figure. The contour of the otic vesicle is indicated with white circles.

Figure 2.

PhC of specific sensory neuronal pools in Tg[hspGFF53A]xTg[UAS:KAEDE] embryos. A–D, PhC of KAEDE^Green of neurons from the A-SAg (A–B) and P-SAg (C–D). A′, Magnification of the boxed region in A. B, Dorsal view of projections in A′. Note that red projection runs very ventral and medially, although it is not the most ventrally positioned, considering the allocation of the TGp. C′, Magnification of the boxed region in C. D, Dorsal view of projections in C′. Note that KAEDE^Red P-SAg projections are more dorsal and lateral than the A-SAp. E–F′, Double PhC of neurons from the ALLg and A-SAg (E, E′) and the P-SAg and PLLg (F, F′). E′, F′, Magnification of the boxed regions in E and F, respectively. G, Scheme depicting the neurosensory network with the highly ordered connectivity map, with DL/VM organization as follows: PLLg, ALLg, P-SAg, A-SAg, TGg. The right drawing represents a transverse section at the level of g. Anterior is always to the left. Axes are indicated in the figure. The contour of the otic vesicle is indicated with white circles.

Figure 3.

Sensory neuron differentiation and establishment of hindbrain afferents entrance points. A–I, Still images of the SPIM time-lapse analysis of Tg[neuroD:GFP] (A–C) or Tg[Isl3:GFP] (D–I) embryos injected with lyn-TdTomato mRNA are shown. The first differentiated sensory neurons of the TGg (A), SAg (D) and PLLg (G) are in close contact with neural tube border cells through plasma membranes, at the level of the future nerve entry point (white arrowheads). Insets in A, D, and G are z-resliced images with dorsal to the top to show as transverse views the contact point of the respective sensory neurons with the border cells of the hindbrain. Primary sensory neurons maintain contacts with the neural tube, even when they are pushed away by morphogenetic growth (B, E, H). Note that they leave trailing axons (C, F, I, white arrows). Images are single confocal planes except for PLLg images, which are MIPs of several confocal planes. J–L, Serial coronal sections of Tg[Isl3:GFP]xMu4127 embryo from dorsal to ventral. Note that the TGg entry point is located in r2, the SAg one in r4, and the PLLg one in r6. nt, Neural tube; r, rhombomere; ov, otic vesicle. Anterior is always to the left except for insets.

Figure 4.

Ablation of the first differentiated neurons results in defects in the sensory projections at the central level. Tg[Isl3:GFP] embryos were used for the ablation of the first differentiated neurons from the ALLg/SAg using the laser of a multiphoton microscope. A, C, Lateral views of control and ablated embryos, respectively, showing no ectopic entry points after pioneer axon ablation. B, D, Dorsal views of A and C, respectively, showing the sensory projections at the central levels. Note the defects in SAg nerve bundle elongation upon ablation (compare B, D, asterisks). White arrows indicate the entry point. White asterisks indicate the location of the SAp. The contour of the otic vesicle is indicated with white circles. Anterior is always to the left.

Figure 5.

Cooperation of pioneer axonal contacts and NCCs in the establishment of the entry points. A–F, Tg[Isl3:GFP] embryos were assayed for crestin in situ hybridization (blue). A, B, TGg. C, D, ALLg/SAg. E, F, PLLg. G–I, Tg[Isl3:GP] embryos were treated with DMSO (G) or leflunomide (H, I) and hybridized with crestin probe (G, H, red) or observed in vivo (I). Note that crestin expression is abolished in leflunomide-treated embryos, whereas sensory ganglia present defects in coalescence (H, white arrowheads). No effects in the entry points are observed, even in in vivo embryos (H, I, white arrows). J–L, Inhibition of NCC migration does not affect the entry point of SAg sensory axons at the central level: sdf1a^−/−Tg[cldnb:lynGFP] (K) and cxcr4b^−/−Tg[cldnb:lynGFP] (L) embryos were analyzed for ectopic entry points and compared with control embryos sdf1a^+/−Tg[cldnb:lynGFP] (J). M–N, Tg[neuroD:GFP] embryos were treated with leflunomide and the first differentiated neurons from the ALLg/SAg were ablated using multiphoton microscopy. M, Lateral view showing ectopic entry points (white arrows). M′, Magnification of boxed region in M showing the ectopic entry points (arrows) in contact with the PLLp (red asterisk). N, Dorsal view of M′ showing the TGp (yellow asterisk), PLLp (red asterisk), and a lack of ALLp/SAp nerve bundle elongation (empty space marked with white asterisk). O–O″, Different single confocal planes from medial (O) to lateral (O″) showing that ectopic entry points now contact with the PLLp (red asterisk). Anterior is always to the left. Axes are indicated in the figure. The contour of the otic vesicle is indicated in white circles. ov, otic vesicle.

Figure 6.

robo2 and slit1 are expressed in SAg neurons and afferent target fields, respectively. A–L′, Tg[Isl3:GFP] embryos were analyzed for slit1a (A–C), slit1b (D–F), robo2 (G–I′), and robo3 (J–L′) expression at 28 hpf. Note the expression of slit1a/b along the hindbrain, with zones devoid of slit1 corresponding to the places where central projection enters (A, B, E, white arrowheads). robo2 is expressed in the differentiated Isl3 sensory neurons (G′, H′, white arrows), and robo3 in the SAg neuroblasts not yet differentiated (K′, red arrows), but never in differentiated sensory neurons (blue arrow). A–A″, D–D″, Coronal sections corresponding to half-sided embryos. B, C, E, F, Transverse sections corresponding to b, c, e, and f, respectively. G–I, J–L, Serial transverse sections along the AP axis.

Figure 7.

robo2 and robo3 label different SAg neuronal populations according to their differentiation state. A, A′, Tg[Isl3:GFP] embryos analyzed for neuroD. B-D′, Tg[neuroD:GFP] embryos hybridized with neuroD (B, B′), robo2 (C, C′), and robo3 (D, D′); note that all neuroD-expressing neuroblasts display neuroD:GFP (B′, red arrow), but neuroD is not expressed in the early differentiated neuronal population (still GFP positive due to its high stability; B′, white arrow). robo2 is expressed only in a subpopulation of neuroD:GFP cells (C′, red arrows). robo3 is expressed in a subpopulation of neuro:GFP cells (D′, red arrow), but not in the earliest differentiated ones (D′, white arrow). E, E′, Tg[Isl3:GFP] embryos hybridized with robo2/neuroD. Within the robo2-positive population, some cells express Isl3:GFP (white arrows) and some neuroD (red arrow). F, F′, robo2/robo3. Note that GFP-positive cells expressing robo2 do not express robo3 (F′, red arrow), and cells expressing robo2/robo3 do not display GFP (F, white arrow). G, G′, snail in delaminating neuroblasts (black arrowhead). H, H′, cadh10 in a subpopulation of nondifferentiated neuroblasts. All images are transverse sections of embryos at the level of the otic vesicle.

Figure 8.

Robo2/Slit1 signaling regulates axonal branching and nerve bundle fasciculation. Tg[Isl3:GFP] embryos were coinjected with MO-CTRL (MO-p53), mRNA for H2B-mCherry or lyn-TdTomato, and MO-Slit1a, MO-Slit1b, MO-Robo2, or double MO-Slit1a/b. A–F, Examples of phenotypes observed at 48 hpf. Note the variety of effects ranging from ectopic entry points (white arrows), ectopic branches (white arrow heads), defasciculation (black asterisks), and combinations of primary phenotypes (E, F). G, Statistics of MO injections. H, Analyses of the percentages of different phenotypes with different MO combinations. *p < 0.1; **p < 0.01; ***p < 0.001. I, Analysis of the percentage of morphant embryos displaying different combinations of phenotypes. Orange circles correspond to embryos displaying ectopic branches, blue circles to embryos with ectopic entry points, and green circles to embryos with defasciculation. Note that many embryos display a combination of phenotypes.