Temporal requirement for SMN in motoneuron development

Abstract

Proper function of the motor unit is dependent upon the correct development of dendrites and axons. The infant/childhood onset motoneuron disease spinal muscular atrophy (SMA), caused by low levels of the survival motor neuron (SMN) protein, is characterized by muscle denervation and paralysis. Although different SMA models have shown neuromuscular junction defects and/or motor axon defects, a comprehensive analysis of motoneuron development in vivo under conditions of low SMN will give insight into why the motor unit becomes dysfunctional. We have generated genetic mutants in zebrafish expressing low levels of SMN from the earliest stages of development. Analysis of motoneurons in these mutants revealed motor axons were often shorter and had fewer branches. We also found that motoneurons had significantly fewer dendritic branches and those present were shorter. Analysis of motor axon filopodial dynamics in live embryos revealed that mutants had fewer filopodia and their average half-life was shorter. To determine when SMN was needed to rescue motoneuron development, SMN was conditionally induced in smn mutants during embryonic stages. Only when SMN was added back soon after motoneurons were born, could later motor axon development be rescued. Importantly, analysis of motor behavior revealed that animals with motor axon defects had significant deficits in motor output. We also show that SMN is required earlier for motoneuron development than for survival. These data support that SMN is needed early in development of motoneuron dendrites and axons to develop normally and that this is essential for proper connectivity and movement.

Figure 1.

Generation of Tg(hsp70:RFP-SMN) lines. (A) Western blot showing endogenous Smn and RFP-SMN protein levels from 2 dpf Tg(hsp70:RFP-SMN) os34–37 embryos after heat shocking at 1 dpf. (B) Western blot of endogenous Smn and RFP-SMN without heat shocking in line os36. (C) Survival of smn^−/− (blue line; n = 12, mean 11.1 ± 2.2 dpf) and Tg(hsp70:RFP-SMN)os36;smn^−/− (green line; n = 7, mean = 17.5 ± 2 dpf). Data are mean ± SD.

Figure 2.

RFP-SMN fully rescues smn^−/− fish. (A) Survival of Tg(hsp70:RFP-SMN);smn^−/− heat shocked twice a week (n = 21) compared with survival of smn^−/− fish (n = 40). (B) Body weight for wild-type (WT) and Tg(hsp70:RFP-SMN);smn^−/− (MT). (C) Adult WT and MT were subjected to increasing current (4.1 cm/s steps) every 5 min until fatigue as described (23). Critical swimming speed (U_crit, cm/s) was not statistically different between the groups (P > 0.5, n = 10 for each group). Data are mean ± SD.

Figure 3.

Characterization of mz-smn^−/− fish. mz-smn^−/− embryos and larvae either with the Tg(hsp70:RFP-SMN) transgene (tg) or without. (A) Lateral view of embryos/larvae at 2, 4, 7 and 10 dpf. (B) Survival of the zygotic smn^−/− fish (red line, n = 11, mean = 11 ± 2.2), mz-smn^−/− + tg fish (green line, n = 9, mean survival=9.6 ± 1.7) and mz-smn^−/− without the transgene (blue line, n = 40, mean survival = 4). (C) Levels of RFP-SMN independent of heat shock in mz-smn^−/− + tg fish and (D) mz-smn^−/− fish. Top blot in (D) is an overexposure of the RFP-SMN lane. Data are mean ± SD.

Figure 4.

Abnormal motor neurons in mz-smn mutants. Lateral views of whole-mount embryos labeled with znp1 antibody at 28 hpf. Representative images of (A) wild-type, (B) mz-smn^−/− carrying the Tg(hsp70:RFP-SMN) transgene (tg) and (C) mz-smn^−/− without the transgene. (D) Motor axon defects were analyzed and embryos characterized as severe, moderate, mild and no defects. Significance was determined by the Mann–Whitney non-parametric rank test. White arrows denote (A) a normal motor axon, (B) a branched motor axon, and (C) a truncated motor axon. Scale bar, 50 µm.

Figure 5.

Single cell analysis of motoneurons. Wild-type and mz-smn^−/− containing the Tg(hs:RFP-SMN) transgene (tg) were injected with mnx1:hsp:GFP DNA. Confocal images of 4 dpf (A–C) wild-type larvae and (D–F) mz-smn^−/− + tg larvae. (A, D) GFP-labeled CaP motoneurons. (B, E) Presynaptic anti-SV2 labeling (C, F) merge. Scale bar, 20 µm.

Figure 6.

mz-smn mutants have fewer motor axon branches. Wild-type and mz-smn^−/− containing the Tg(hs:RFP-SMN) transgene (tg) were injected with mnx1:hsp:GFP DNA. Confocal images were obtained of 2 dpf and motor axons and branches were traced using NIH image J software Fiji. (A) wild-type GFP-labeled CaP motoneuron. (B) Traced branches from (A). (C) mz-smn^−/− + tg GFP-labeled CaP motoneuron. (D) Traced branches from (C). (E) Total branch length and (F) average branch length (mean ± SD) of wild-type (WT, n = 7 neurons) and mz-smn^−/− + tg (mutant, n = 9 neurons). ***P < 0.0001, two-tailed Student’s t-test, ns = not significant. Scale bar, 25 µm.

Figure 7.

mz-smn mutants have fewer and less stable motor axon filopodia. One-cell stage wild-type and mz-smn^−/− embryos containing the Tg(hs:RFP-SMN) transgene (tg) were injected with Lifeact–GFP DNA targeted to motoneurons (see Materials and Methods). At ∼28 hpf motor axons (n = 3 motor axons from three different wild-type or mutant embryos) were imaged using 2-photon microscopy and time-lapse sequences used for the analysis of filopodial dynamics. (A) Wild-type Lifeact–GFP-labeled CaP motoneuron. (B) mz-smn^−/− + tg Lifeact–GFP-labeled CaP motoneuron. (C) Filopodia number per 10 µm of axon. (D) Filopodial lifetime. (E) Filopodial rates of extension and retraction. *P < 0.01, Wilcoxon's signed-rank test. Scale bar, 10 µm.

Figure 8.

Abnormal motoneuron and nucMLF dendrites. Confocal images of 4 dpf (A) wild-type, (B) mz-smn^−/− + tg injected with mnx1:hsp:GFP DNA showing GFP-labeled CaP motoneurons and a nucMLF midbrain neuron in Tg(mnx1:hsp:GFP) (E) wild-type and (F) mz-smn + tg larvae. Dendrites were traced using the Fiji software. (C) Total and (D) average dendritic length of motoneurons. (G) Total and (H) average dendritic length of nucMLF midbrain neurons. n = 12 neurons analyzed for each category (mean ± SD). *** P < 0.0001, **P < 0.005 two-tailed Student's t-test. Scale bar, 8 µm for (A), (B) and 15 µm for (E), (F) (main images). Arrows in inset denote axons, arrowhead denote dendrites.

Figure 9.

Motor axon defects in mz-smn mutants are rescued by early SMN induction. Representative images of znp1-labeled ∼28 hpf (A) wild-type, (B) mz-smn^−/− + tg without heat shock (hs), (C) mz-smn^−/− + tg heat shocked at 10 hpf, (D) mz-smn^−/− + tg heat shocked at 16 hpf, (E) mz-smn^−/− + tg heat shocked at 24 hpf, and (F) mz-smn^−/− + tg heat shocked at 27 hpf. (G) Motor axon defects were analyzed and embryos characterized as severe, moderate, mild and no defects. Significance was determined by a Mann–Whitney non-parametric rank test. ns, not significant. Scale bar, 25 µm.

Figure 10.

Early induction of SMN extends survival. (A) Kaplan–Meier survival plots of mz-smn^−/− + tg embryos/larvae after heat shock at different time points. No heat shock (dark purple, n = 10, mean = 9.6 ± 1.7 dpf), heat shock at 36 hpf (dark blue, n = 15, mean = 13.8 ± 1.6 dpf), heat shock at 48 hpf (green, n = 11, mean = 12.4 ± 1.7 dpf), heat shock at 60 hpf (black, n = 14, mean = 14 ± 1.3 dpf) and heat shock at 72 hpf (yellow, n = 15, mean = 10 ± 1.8 dpf). Heat shock at 10 hpf (red, n = 8, mean = 27.1 ± 1.7), heat shock at 24 hpf (light blue, n = 8, mean = 26.2 ± 1.7 dpf), heat shock at 27 hpf (pink, n = 10, mean = 24.7 ± 2.6 dpf). See Table 1 for statistics. (B) Kaplan–Meier survival plots of mz-smn^−/− + tg embryos after continuous induction starting at 24 hpf (light blue, n = 29, 69% survival > 2 years) or 36 hpf (dark blue, n = 24, mean 13.2 ± 1.4 dpf).

Figure 11.

Motor axon defects lead to motor deficits. (A) Mean gross movement of larvae over a period of 80 s. (B) Mean frequency (percentage of recorded trials) in which larvae initiated swimming and turning behaviors. (A–B) n = 32 larvae per group. Evaluation of swimming kinematic performance was determined by measuring (C) distance moved per swim, (D) number of swim half cycles, and (E) mean body curvature change per swim half cycle. For this, ∼30 fish/group were recorded and the following number of swims analyzed: WT No HS, 79; WT HS 10 hpf, 85; WT HS 24 hpf, 90; Tg (hs-RFP-SMN); mz-smn^−/− No HS, 23; Tg (hs-RFP-SMN); mz-smn^−/− HS 10 hpf, 104; Tg (hs-RFP-SMN); mz-smn^−/− HS 24 hpf, 40. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. Error bars denote SEM. HS, heat shock.