Deletion of a kinesin I motor unmasks a mechanism of homeostatic branching control by neurotrophin-3
- Centre de Recherche, France
- CNRS UMR 3215, France
- INSERM U934, France
- University of Heidelberg, Germany
- University of California San Francisco, United States
- University of California, Berkeley, United States
- Muséum National d'Histoire Naturelle, France
- Sorbonne Universités, UPMC University Paris 6, France
- Uni Research AS High Technology Centre, Norway
- National Institute of Genetics, Japan
- Max Planck Institute of Neurobiology, Germany
Figures
Figure 1 with 1 supplement
Generation of loss-of-function alleles of the anterograde motor protein Kif5aa.
(A) Employing TALENs targeting exon4 of the kif5aa open reading frame, we generated two loss-of-function alleles with a 10 bp and 13 bp deletion, respectively. These result in a frameshift at amino …
Figure 1—figure supplement 1
Melanosomes transport is not abolished in kif5aa mutants but they show no optokinetic response.
(A) Phenotype of wild-type, kif5aa*162 mutant, lakritz and blumenkohl embryos at 5 dpf. All three mutants show expanded melanosomes and appear dark. Application of norepinephrine (NA) results in …
Figure 2 with 1 supplement
Outgrowth of the optic nerve and retinotopic mapping is normal in kif5aa mutants.
(A) Confocal imaging of the Tg(pou4f3:mGFP) transgene, labeling a subpopulation of Retinal Ganglion Cells (RGCs) with membrane bound GFP, at 48 hpf reveals that outgrowth of the optic nerve formed …
Figure 2—figure supplement 1
Patterning of the mutant retina and neurogenesis is not affected in mutants.
(A) The expression of the Tg(shh:eGFP) transgene marks the onset of neurogenesis (white arrow) in the developing retina (Shkumatava et al., 2004) and its expression is not altered in kif5aa mutant …
Figure 3 with 1 supplement
RGC axons in kif5aa mutants show a delayed ingrowth into the optic tectum and grow larger arbors at later stages.
(A) Single membrane-GFP expressing RGC axons from the Tg(BGUG) transgene (left panel) and DiI injections into the contralateral retina of 3 dpf old wild-type and kif5aa mutant embryos (right panel) …
Figure 3—figure supplement 1
Mapping of the vertigos1614 locus by genetic linkage analysis.
In 1800 meioses, the vertigos1614 allele was co-segregating with the two polymorphic markers fj61a10 and tsub1g3 located on Contig 963 of linkage group 9 (LG9). Four genes encoding for …
Figure 4 with 1 supplement
Kif5aa mutant larvae show no activity in RGCs and no synaptic transmission to tectal cells.
(A) 5–7 dpf larvae were visually stimulated by bars on an LED screen running in caudal-to-rostral direction across the larva's visual field. Wild-type and kif5aa mutant larvae expressing genetically …
Figure 4—figure supplement 1
Regression-based analysis of wild-type/heterozygous vs kif5aa−/− Tg(HuC:GCaMP5G) mutants to a visual stimulus.
Kif5aa sibling and mutant Tg(HuC:GCaMP5G) larvae were stimulated with a visual bar running from caudal to rostral in the visual field. Subsequently, regression analysis of the measured Calcium …
Figure 5 with 3 supplements
Kif5aa mutant RGC arbors show the same density of presynaptic sites but are depleted of mitochondria.
(A) In vivo imaging shows the distribution of presynaptic sites marked by Synaptophysin-GFP (SypGFP) in single kif5aa mutant and wild-type RGC arbors expressing membrane localized RFP (RFPCaax). …
Figure 5—figure supplement 1
Analysis of transport dynamics of synaptic vesicles in wild-type and kif5aa mutant cells during visual system development.
(A) Synaptic vesicles were visualized by Synaptophysin-GFP (SypGFP) expression and imaged in axonal segments of wild-type and kif5aa mutant cell RGC arbors. Depending on the size of vesicles we …
Figure 5—figure supplement 2
Analysis of mitochondria localization and transport dynamics in wildtype and kif5aa mutant RGC arbors.
(A) As previously shown in other experimental systems (Obashi and Okabe, 2013) mitochondria are often localized in close proximity to synapses. In the upper panel, an axonal segment of a single RGC …
Figure 5—figure supplement 3
Retinal Ganglion Cells show a normal mitochondria distribution in blumenkohl mutants.
(A) Distribution of mitochondria (labeled by mitoGFP) in single blumenkohl mutant and wild-type RGC arbors expressing membrane localized RFP (RFPCaax) in vivo. Upper two panels: blumenkohl mutant …
Figure 6 with 1 supplement
Expression of the neurotrophic factor neurotrophin 3 in visually impaired mutants.
(A) Relative expression levels of bdnf, ntf3, ntf4, ntf7, and ngf in 4 dpf old wild-type and kif5aa mutant embryos. ntf3 is upregulated to 160% of wild-type expression levels (p < 0.05) while all …
Figure 6—figure supplement 1
Silencing of all Retinal Ganglion Cells by BoTx expression leads to Ntf3 upregulation.
(A) Expression of botulinum toxin light chain B (Brunger et al., 2008; Nevin et al., 2008) in all RGCs leads to expansion of melanosomes similar to blind mutant fish as shown in Figure 1—figure …
Figure 7 with 1 supplement
Neurotrophin 3 signaling alters axonal branch size in RGCs.
(A) In situ hybridization with ntrk3a and ntrk3b specific antisense probes shows expression of both paralogues in broad parts of the nervous system in 5 dpf larvae. Both receptors are strongly …
Figure 7—figure supplement 1
Blumenkohl mutant RGC arbors and RGC arbors growing into a Ntf3 overexpressing tectum do not show increased filopodia dynamics.
(A) Upper panel: Axonal arbor of a single blumenkohl mutant RGC at 5 and 7 dpf. (indicated by an arrow). D = dorsal, V = ventral, R = rostral, C = caudal. Lower panel: Tracings of an axonal arbor at …
Figure 8 with 2 supplements
Transplantations confirm the growth promoting-effect in kif5aa mutant tecta.
(A) Representative pictures of single in vivo imaged RGC axons after blastula stage transplantions from wild-type donors into a wild-type tectum (left panel), from kif5aa mutants into a wild-type …
Figure 8—figure supplement 1
Phenotype of transplanted RGC arbors at early stages of development.
(A) Kif5aa mutant RGC arbors show a delay of ingrowth into the optic tectum compared to wild-type cells (right column). This delay is cell autonomous as mutant RGC axons also fail to invade their …
Figure 8—figure supplement 2
Transplantation of kif5aa mutant RGCs into a blumenkohl mutant acceptor leads to an increased growth compared to transplantation into a wild-type acceptor.
(A) Representative pictures of single in vivo imaged RGC axons after blastula stage transplantions from kif5aa mutant donors into a wild-type tectum (left panel) and from kif5aa mutants into a blumen…
Videos
Video 1
In vivo timelapse imaging of the optic tectum in a Tg(HuC:GCaMP5G) transgenic 5 dpf wild-type fish.
A wild-type larva was stimulated with a visual bar running from caudal to rostral through the visual field of the contralateral eye to the imaged optic tectum. Stimulus onset is indicated by a white …
Video 2
In vivo timelapse imaging of the optic tectum in Tg(HuC:GCaMP5G) transgenic 5 dpf kif5aa mutant embryos.
A kif5aa mutant larva was stimulated with a visual bar running from caudal to rostral through the visual field of the contralateral eye to the imaged optic tectum. Stimulus onset is indicated by a …
Video 3
In vivo timelapse imaging of Synaptophysin-GFP containing clusters in RGC axonal segments.
Representative RGC axonal segment in a 5 dpf old wild-type larva. SypGFP labels synaptic clusters of different sizes that were grouped in small and middle-sized plus large vesicles. Compare Figure …
Video 4
In vivo timelapse imaging of mitochondria in RGC axonal segments.
Representative RGC axonal segment in a 5 dpf old wild-type larva. Compare Figure 5—figure supplement 2B for kymogram analysis of mitochondria movements.
Additional files
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Supplementary file 1
Description of zebrafish mutant and transgenic lines used in this study.
- https://doi.org/10.7554/eLife.05061.026
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Supplementary file 2
Primers used in this study.
- https://doi.org/10.7554/eLife.05061.027
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Source code 1
Source code for moving bar stimulus.
- https://doi.org/10.7554/eLife.05061.028
