Notum homolog plays a novel role in primary motor innervation

Abstract

To form complex neuronal networks, growth cones use intermediate targets as guideposts on the path to more distant targets. In the developing zebrafish (Danio rerio), the muscle pioneers (MPs) are intermediate targets for primary motor neurons (PMNs) that innervate the trunk musculature. The mechanisms regulating PMN axon guidance at the MPs are not fully understood. We have identified a new member of the Notum family in zebrafish, Notum 2, which is expressed exclusively in the MPs during primary motor innervation. While homologs of Notum, including zebrafish Notum 1a, negatively regulate the Wnt/β-catenin signaling pathway, we discovered a novel function of Notum 2 in regulating motor axon guidance. Knockdown of Notum 2 resulted in a failure of caudal primary (CaP) axons to migrate beyond the MPs, despite the proper specification of the intermediate target. In contrast, mosaic Notum 2 overexpression induced branching of PMN axons. This effect is specific to Notum 2, as overexpression of Notum 1a does not affect PMN axon trajectory. Ectopic expression of Notum 2 by cells contacting the growing CaP axon induced the highest frequency of branching, suggesting that localized Notum 2 expression affects axon behavior. We propose a model where Notum 2 expression at the MPs provides a cue to release CaP motor axons from their intermediate targets, allowing growth cones to proceed to secondary targets in the ventral muscle. This work demonstrates an unexpected role for a Notum homolog in regulating growth cone migration, separate from the well established functions of other Notum homologs in Wnt signaling.

Figure 1.

Sequence and expression of Notum 2. A, Protein sequence alignment with human NOTUM and zebrafish Notum 1a shows a predicted signal peptide sequence (purple letters) and conservation between homologs in human and zebrafish including Ser-Asp-His catalytic triad and the GXSXG active site motif (green bars). Red letters indicate identical amino acids shared by all three homologs, green letters are shared by Notum 2 and NOTUM, blue letters are shared between NOTUM and Notum 1a. B, Conserved synteny of Notum 2 with orthologs in stickleback, tetraodon, and medaka. C, Expression of notum 2 at 24 hpf is detected along the horizontal myoseptum. D, Cross section of trunk at 24 hpf, at the level indicated by dashed line in C, shows notum 2 expression in the MPs. Asterisk indicates level of HM separating dorsal myotome (DM) and ventral myotome(VM); NT is neural tube and NC is notochord. E, Expression of notum 2 at 3dpf is expanded in anterior segments. F, At 5 dpf, expression of notum 2 is reduced to the HM and surrounding slow muscle in the most anterior segments. Lateral views of zebrafish trunk stained by whole-mount in situ RNA hybridization of notum 2 at 26 hpf (G–J) are shown. G, Expression of notum 2 in wild-type embryos is restricted to the MPs (black bracket). H, Overexpression of Shha causes expansion of notum 2 expression domain (black bracket). I. Expression of notum 2 is lost in Hh-deficient disp1^tm15a mutants. J. Expression of notum 2 can be restored by treatment with Bmp inhibitor Dorsomorphin (black bracket). Scale bars (in C, E–G), 100 μm; (in D), 50 μm.

Figure 2.

Notum 2 knockdown causes CaP axon truncation. Zebrafish embryos at 5.5 hpf in bright-field (A–C) or GFP channel (D–F). A, D, Uninjected, wild-type controls do not express EGFP. B, E, Embryos injected with notum 2-EGFP mRNA show bright EGFP signal. C, F, Coinjection with AUGMO effectively blocks translation of the Notum 2-EGFP protein. G, H, Lateral views of motor axons stained with SV2 antibody at 26 hpf; arrows indicate position of the horizontal myoseptum. G, CaP motor axons in uninjected wild-type controls have crossed the HM to innervate the ventral myotome. H, Knockdown of Notum 2 using a combination of AUG and 5′UTR antisense Morpholino oligonucleotides causes axon stalling at the HM. I, Percentage of truncated axons in uninjected control or injected with 6 ng AUG-MO, 4 ng 5′UTR-MO, or 2 ng/AUG-MO+2 ng/5′UTR-MO. ***p < 0.001; + corresponds to 2 ng of MO per embryo. J, K, Lateral views of 48 hpf zebrafish trunk stained with SV2 antibody show no difference in motor nerves between wild-type (J) and AUGMO (K). Lateral views of Hb9:EGFP-positive motor neurons in 48 hpf zebrafish are shown (L–N). Dashed lines indicate the segmental borders; arrows mark the position of the horizontal myoseptum. L, In wild-type CaP, axons innervate the ventral myotome. M, Truncated CaP axons in Notum 2 morphants do not appear to recover (asterisk), while labeled RoP in the adjacent segment shows the relative position of the cell body in reference to the segmental border. N, Two clusters of Hb9:GFP-positive neurons show one innervating CaP and one truncated CaP (asterisk). Scale bars: 50 μm.

Figure 3.

Wild-type MPs rescue morpholino-induced CaP axon truncation. A, Normal innervation of ventral myotome by CaP motor axon in uninjected control embryo. B, Truncated CaP axon in 6 ng AUG-MO-injected host embryo. C, Truncated CaP axon in AUG-MO host segment with transplanted WT fast muscle cells (red). D, Rescued CaP axon in AUG-MO host with uninjected WT MPs. E, Percentage of segments with truncated axons in uninjected controls, AUG morphants with no transplanted cells, AUG morphants with transplanted non-MP muscle fibers, and AUG morphants with transplanted MPs are shown. The p value compares AUG-MO-injected segments with no transplanted cells and segments with transplanted WT MPs. The p value calculated from Fisher's exact test; *p < 0.05; + corresponds to 2 ng of MO per embryo; n = number of segments observed. Scale bar (in A), 50 μm.

Figure 4.

Notum 2 overexpression causes motor axon branching. A, Uninjected, non-heat-shocked control. B, Uninjected embryo, heat-shocked control. C, Notum 2 overexpressing embryo shows extensive axon branching. D, GFP-positive cells (green) indicate Notum 2-expressing cells. E, Hemisegments overexpressing enzyme-dead Notum 2 do not induce motor axon branching. F, GFP-positive cells (green) indicate position of mutNotum 2-expressing cells. Lateral views of primary motor axons stained with SV2 antibody. Scale bar (in E) A–F, 50 μm G, Percentage of segments with motor axon branching in heat-shocked control, Notum 2-overexpressing and mutNotum 2-overexpressing embryos. White columns indicate GFP-negative hemisegments; green columns indicate GFP-positive hemisegments. H, Schematic of data collected from segments with a single Notum 2 overexpressing cell. The numbers represent the number of observations in this relative location in the myotome. The color of the circle represents the frequency of branched axons induced by overexpressing cells in this position (bins: dashed circle, no data for this position; white, 0–24%, blue, 25–49%; green, 50–74%; red, 75–100%).

Figure 5.

Notum 2 does not affect acetylcholine receptor (AchR) clustering. A, B, Presynaptic patterning in uninjected control (A) and Notum 2 morphant (B) embryos at 18 hpf. C, D, Synaptic vesicles (C) and AchRs (D) colocalize to form NMJs in wild-type embryos at 26 hpf. E, Knockdown of Notum 2 does not affect NMJ formation (arrow). F, Segments with truncated axons do not form ectopic AchR clusters (arrow). G, H, Branched axons in Notum 2 overexpressing embryos form ectopic NMJs (arrows). Scale bar (in H), 50 μm.

Figure 6.

Knockdown of Notum 2 does not affect escape behavior. A, Zebrafish in resting position. B, C, Composite image of 30 frames over 90 ms illustrate the coiling and swimming phase of the escape response. D, Composite image of two frames at rest and peak coiling (42 ms); angled dashed lines show the amplitude of initial bend of the escape response. Dorsal view of zebrafish larvae at 48 hpf with agarose-restrained head is shown (A–D). E, F, White columns are uninjected controls; gray columns are Notum 2 morphants injected with 6 ng AUG-MO. E, Average time in each phase of the escape response. F, Amplitude of initial c-bend. N = 10 wild-type, 9 morphants, 10 trials for each embryo. Scale bar (in D): 100 μm.

Figure 7.

Notum 2 guides caudal primary axon through intermediate target. Schematic representation of CaP growth cone migrating through the MPs is shown. Red color indicates the gradient of Nrp1a/Sema3a1 repulsion. Purple marks the Notum 2 signaling center. A, CaP axon at 18 hpf uses Nrp1a/Sema3a1 repulsion to reach intermediate target. B, CaP axon at 26 hpf requires desensitization to Sema3a1 and Notum 2 expression to migrate beyond the MPs. C, At 18 hpf, CaP axon growth to the MPs is unaffected following Notum 2 knockdown. D, At 26 hpf, CaP axon growth beyond the MPs is inhibited following Notum 2 knockdown. E, At 18 hpf, ectopic Notum 2 (green and purple cell) is induced in the ventral myotome. F, At 26 hpf, contact with ectopic Notum 2 causes branching in CaP axons.