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Discipline
Biological
Keywords
MND
ALS
Zebrafish
Neurodegenerative Disease
Transgenic Models
Observation Type
Standalone
Nature
Negative data
Submitted
Jan 10th, 2018
Published
Mar 19th, 2018
  • Abstract

    The E3 ubiquitin ligase protein, cyclin F (encoded by CCNF) has a role in substrate recognition for protein degradation within the ubiquitin proteasomal system. Mutations in this gene have recently been linked to the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia. To investigate the role of CCNF dysfunction in neurodegenerative disease, we aimed to develop novel transgenic zebrafish models that constitutively  overexpress CCNF fusion proteins. After several attempts at establishing these transgenic lines, with side-by-side successful controls, it became apparent that generation of constitutively  overexpressing CCNF fusion transgenic lines was not feasible. This failure appears to be a result of toxicity associated with persistent overexpression of CCNF fusion proteins in the developing embryo that precludes germ-line transmission of the transgene. The observations from our study indicate that an additional screening stage of zebrafish embryos, and/or the use of a temporal inducible system/mechanism is warranted in studies that aim to develop transgenic models expressing potentially toxic proteins, to circumvent the issues produced by expression during early developmental stages.

  • Figure
  • Introduction

    Cyclin F is a key component of the ubiquitin proteasomal system (UPS), involved in mediating the transfer of ubiquitin molecules to specific substrates, thereby tagging them for degradation by the proteasome. By regulating the degradation and consequently the expression levels of specific substrates through the UPS, cyclin F governs DNA repair and replication and controls cell cycle progression. Dysfunction of cyclin F and a subsequent dysregulation of these pathways have been linked to various forms of cancer. Additionally, mutations in CCNF have recently been reported in two clinically, pathologically and genetically linked neurodegenerative diseases- amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To date, only one in vivo model has been established as a tool to investigate CCNF dysfunction- a zebrafish model that transiently overexpressed an ALS-FTD mutation in CCNF (CCNFS621G). This model demonstrated neurological defects associated with expression of CCNFS621G, including a motor axonopathy and impaired motor function. To supplement the existing transient models, this study aimed to use the Tol2 transposase system to develop zebrafish models that constitutively expressed fluorescently labelled human CCNF under a quasi-ubiquitous (actb2) promoter.

  • Objective

    To develop novel transgenic zebrafish models that constitutively overexpress CCNF-fluorophore fusion for use in studies investigating disease-associated dysfunction of this gene.

  • Results & Discussion

    No founders were identified in the CCNF injected groups

    Offspring from 102 fish injected with a CCNF construct (32 Hsa.CCNFWT, 28 Hsa.CCNFS621G, 18 ccnfWT and 24 ccnfS6213G) were screened and no founders were identified. Offspring from 8 fish injected with the mCherry control construct were screened and 4 founders were identified (Fig. 1A). The efficiency of transgenic integration in the mCherry control line, (reported at 30–50% for Tol2 system) validated the injection and screening methods and suggested a gene-specific issue caused the failure to identify CCNF founders.

    CCNF expression is lost over the first 6 days of development

    To investigate this failure, the embryonic injections were repeated, and the screening protocol was altered. Instead of the standard screening at 2–3 days post fertilisation (dpf), screening was performed at 24 h post fertilisation (hpf) followed by subsequent analysis every 24 h up to 6 dpf. A dramatic loss of mCherry expression was observed in all CCNF-injected fish over this time period. In comparison, an increase in the number of mCherry positive cells was evident in the mCherry control fish (Fig. 1B). To quantify this, the number of mCherry positive cells in 10 fish from each injection group was estimated. This demonstrated no significant difference in the number of mCherry expressing cells between any of the injection groups at 24 hpf (p=0.08). However, significantly more mCherry positive cells were detectable in the control fish than any of the CCNF groups by 2 dpf (p<0.0001) and this difference increased over the next 5 days (Fig. 1C).

    Similar loss of expression was seen between mCherry and EGFP fused CCNF

    To eliminate an unexpected toxicity associated with mCherry fusion to CCNF, mCherry was substituted with the EGFP fluorophore in the Hsa. CCNFWT and ccnfWT constructs. Quantification of the number of fluorophore positive cells in 5 fish in each injection group demonstrated a similar loss of expression between the mCherry and the EGFP groups over the first 6 days of development (Fig. 1D).

    Higher levels of cell death were evident in CCNF injected groups compared to mCherry controls

    To investigate the mechanism responsible for the loss of cells expressing the transgene, cell death was assessed at the time of peak mCherry-CCNF expression (24 hpf) using acridine orange (AO). This analysis demonstrated significantly higher levels of cell death in all of the CCNF groups compared to sham injected controls (p<0.05). No significant difference was seen between any of the CCNF groups (n=30 per group) (Fig. 1E). Cells that both expressed mCherry fused CCNF and stained positive with AO were observed (Fig. 1F).

    This AO staining assay suggests that toxicity associated with overexpression of CCNF is an impediment to establishing transgenic lines based on this gene. It is possible that all, or the majority, of cells in which the CCNF transgene is integrated into the genome, including germline cells, undergoes apoptosis, precluding germline transmission. Given the high level of programmed cell death that occurs during early development and the regenerative capacity of the zebrafish, this CCNF-associated cell death could occur without causing detectable morphological abnormalities.

  • Conclusions

    This study demonstrates that the development of zebrafish transgenic lines that constitutively overexpress CCNF-fluorophore fusions using the described methods is not a feasible option, despite the clearly possible expression of the full-length ORF of the transgenic construct as apparent by fluorescence signal. The authors, therefore, suggest that levels of transgene expression to be re-assessed at 6–7 dpf to detect potential toxic overexpression effects at this early stage. In addition, models with temporal control of transgene expression to bypass overexpression during this period of development and cell death may be warranted.

  • Limitations

    One limitation of this study is that fluorophore fusion proteins were used instead of the native CCNF ORF. For a possible follow-up, a native CCNF ORF could be tested with a transgenesis reporter in cis (i.e. myl7:EGFP or alpha-crystallin:Venus) to uncouple transgene detection from CCNF. In addition, studies to further investigate the failure to establish CCNF transgenic lines could be performed. For example, DNA could be extracted from embryos at 24 hpf to confirm transgenic integration, then repeated at later timepoints to establish whether cells that successfully integrate CCNF survive and to confirm if the loss of expressed CCNF is at a protein or DNA level. The presence of integrated CCNF DNA may assist in the elucidation of any gene silencing effects in the model.

  • Alt. Explanations

    It is possible that some germline cells did integrate CCNF and survive, but these founders were not detected. Given the elevated level of cell death seen in embryos that mosaically express CCNF, it appears unlikely that F1 embryos with strong, ubiquitous expression would survive to time of screening at 2–3 dpf. Given the large size of zebrafish clutches and the small percentage of the F1 generation that typically carry a transgene (typically 10–15%), such an occurrence would be difficult to detect.

    An alternative explanation for the observed loss of expression in this study is a rapid silencing of the transgene. Multiple epigenetic modifications, that silence gene expression, have been identified, key amongst them being methylation. However, such rapid silencing of a transgene has not previously been reported and increased cell death would not be expected if this was the cause of the loss of expression in the CCNF-expressing embryos.

  • Conjectures

    The role of CCNF in regulating the cell cycle provides a possible explanation for the apparent toxicity of CCNF overexpression in the embryonic zebrafish. The cell cycle is highly regulated and the coordination of transcription, cell proliferation, migration, differentiation and apoptosis is essential for embryonic development. It is feasible that disruption to this highly regulated system associated with overexpression of CCNF in an organism undergoing rapid cell replication would have toxic effects.

    It is likely that the toxicity reported here extends beyond CCNF. There is the potential for toxicity to arise when overexpression is introduced into a system and this is of importance during early development when vital systems such as the nervous system are being established. In particular, the generation of models of ALS (and likely most neurodegenerative diseases) may be fraught with difficulties as many of these genes are ubiquitously expressed during development before becoming restricted to the central nervous system. This suggests that the toxicity detailed here is not specific to CCNF expression during developmental stages and may be a factor in the establishment of other models.

    To overcome this problem, we hypothesise that inducible transgenic lines, that allow for precise temporal control of gene expression will be necessary for the generation of novel models of ALS. The adoption of inducible transgenic lines such as the heat shock-inducible Cre line and Tet-on transgenic lines for doxycycline-inducible gene expression will be crucial in establishing zebrafish models of ALS. Use of such lines will allow for temporal control of CCNF (or other ALS-linked genes) expression to be delayed until the fish matures beyond the developmental stage. We believe that this delay in expression will avoid the issues of early cell toxicity and allow for stable integration of the transgene in the germline, which in turn will facilitate transmission of transgene to the next generation. This would ultimately permit the development of transgenic overexpression CCNF-based zebrafish models of ALS. These inducible models are currently being generated and it is hoped that these models will yield insights into the biological mechanisms causing ALS as well as providing a platform for testing future therapeutics.

  • Methods

    Generation of constructs

    Constructs were generated using the Tol2 Multi-site Gateway Three-Fragment Vector Construction kit (Life Technologies). The Hsa.CCNFWT (wildtype human CCNF), Hsa.CCNFS621G (mutant human CCNF), ccnfWT (wildtype zebrafish ccnf) and ccnfS623G (mutant zebrafish ccnf) att-flanked cDNA was codon optimised and synthesised by GeneArt (ThermoFisher Scientific). The CCNF cDNA sequences were cloned into a pDONR_P2R-P3 3’ entry vector (Life Technologies) using a standard BP reaction as per manufacturer’s instructions (Life Technologies). Final constructs were assembled using the LR reaction as per manufacturer’s instructions (Life Technologies) using the actb2 5’ entry vector, the mCherry and EGFP middle entry vectors, the polyA 3’ entry vector and the pDEST destination vector. All constructs were sequence validated (Macrogen).

    Zebrafish lines

    Zebrafish used in this study were AB/Tübingen wildtype (TAB_WT) and the unpigmented Casper (nacre-roy) line. Fish were bred and maintained under established conditions.

    Generation of transgenic lines

    Transposase mRNA was transcribed from a NotI-linearised pCS_zT2TP vector using the Sp6 mMESSAGE mMachine Kit (Ambion). The resulting mRNA was column purified using an RNeasy mini kit (Quiagen), eluted in RNAse-free water and stored in aliquots at -80°C until use.

    Following an initial dose-response trial, 200 pg of each Tol2 construct was co-injected with 160 pg transposase mRNA into the animal pole of zebrafish embryos at the single cell stage of development. Embryos were injected with either a mCherry control construct or 1 of 4 actb2 driven CCNF constructs with a N-terminal mCherry tag– Hsa.CCNFWT, Hsa.CCNFS621G, ccnfWT, . Injections were performed on multiple clutches on multiple days. Embryos were screened once for mCherry expression at 2–3 dpf as per standard protocol and positive embryos raised to maturity. Once mature, construct injected fish were outcrossed to wildtype fish and a minimum of 100 embryos were screened for mCherry expression across three separate lays for each mating pair at both 24 hpf and 3 dpf.

    Imaging & cell counts

    Embryos were mounted in 3% methylcellulose (Sigma Aldrich, M-0387) and imaged using a Leica M165FC stereo dissection microscope. The number of mCherry expressing cells was manually counted, however for embryos with >100 mCherry positive cells, this number was estimated.

    AO staining, imaging and quantification

    Zebrafish embryos were anesthetised in 0.2 mg/ml tricaine methanesulfonate solution (Sigma Aldrich) at 24 hpf and placed in 5 μg/ml AO solution (Sigma Aldrich) for 10 min in the dark. Embryos were briefly rinsed in E3 medium, then mounted in 3% methylcellulose and imaged on a M165FC fluorescent stereomicroscope (Leica) scope. Quantification was performed using Image J. Images were converted to 8-bit and inverted, and the number of positive cells was quantified using the Analyse Particles tool, with a size threshold of 20 mm.

  • Funding statement

    This work was supported by the Motor Neuron Disease Research Institute of Australia (MNDRIA; Mick Rodger Benalla GIA1510 and GIA1628), an MND Australia Leadership Grant, the National Health and Medical Research Council of Australia (1095215, 1107644), The Snow Foundation and European Community's Seventh Framework Programme (FP7/2007-2013) under the grant agreement number 259867.

  • Acknowledgements

    We thank D.S, R.B, T. U and J.M-P for their care of the fish in our facility. We thank the Chien and Currie Laboratories for the kind gifts of plasmids.

  • Ethics statement

    All husbandry and experimental procedures were performed in compliance with the Animal Ethics and Internal Biosafety Committees, Macquarie University (ARAs 2012/050 and 2015/034; NLRD 5201401007) (NSW, Australia).

  • References
  • 1
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    Matters11/20

    Constitutive overexpression of the ALS-linked gene CCNF fusions results in cytotoxicity to preclude generation of transgenic zebrafish models

    Affiliation listing not available.
    Abstractlink

    The E3 ubiquitin ligase protein, cyclin F (encoded by CCNF) has a role in substrate recognition for protein degradation within the ubiquitin proteasomal system. Mutations in this gene have recently been linked to the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia. To investigate the role of CCNF dysfunction in neurodegenerative disease, we aimed to develop novel transgenic zebrafish models that constitutively  overexpress CCNF fusion proteins. After several attempts at establishing these transgenic lines, with side-by-side successful controls, it became apparent that generation of constitutively  overexpressing CCNF fusion transgenic lines was not feasible. This failure appears to be a result of toxicity associated with persistent overexpression of CCNF fusion proteins in the developing embryo that precludes germ-line transmission of the transgene. The observations from our study indicate that an additional screening stage of zebrafish embryos, and/or the use of a temporal inducible system/mechanism is warranted in studies that aim to develop transgenic models expressing potentially toxic proteins, to circumvent the issues produced by expression during early developmental stages.

    Figurelink

    Figure 1. Progressive loss of transgenic CCNF expression in zebrafish embryos through early development.

    (A) F1 offspring from a actb2:mCherry founder demonstrating successful development of the transgenic line.

    (B) Representative images of a single fish from different injection groups imaged at 1, 2 and 6 dpf demonstrating a loss of expression in both CCNF-expressing groups and an increase in expression in the mCherry-expressing control.

    (C) Quantification of the average number of mCherry positive cells per injection group over the first 5 days of development (n=10 per group).

    (D) Quantification of the average number of mCherry/GFP positive cells per injection group over the first 5 days of development (n=5 per group). No difference was seen between embryos injected with GFP constructs or mCherry constructs.

    (E) Quantification of the number of acridine orange-positive cells demonstrated significantly higher levels of cell death in embryos expressing CCNF compared to controls (n=30 per group).

    (F) Example of co-expression of mCherry- tagged CCNF and an area of strong AO staining.

    (G) Representative images of acridine orange-stained fish from each of the 5 experimental groups indicating higher levels of cell death in fish expressing CCNF compared to sham injected controls.

    Introductionlink

    Cyclin F is a key component of the ubiquitin proteasomal system (UPS), involved in mediating the transfer of ubiquitin molecules to specific substrates, thereby tagging them for degradation by the proteasome. By regulating the degradation and consequently the expression levels of specific substrates through the UPS, cyclin F governs DNA repair and replication and controls cell cycle progression[1]. Dysfunction of cyclin F and a subsequent dysregulation of these pathways have been linked to various forms of cancer[2][3][4]. Additionally, mutations in CCNF have recently been reported in two clinically, pathologically and genetically linked neurodegenerative diseases- amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)[5]. To date, only one in vivo model has been established as a tool to investigate CCNF dysfunction- a zebrafish model that transiently overexpressed an ALS-FTD mutation in CCNF (CCNFS621G)[6]. This model demonstrated neurological defects associated with expression of CCNFS621G, including a motor axonopathy and impaired motor function. To supplement the existing transient models, this study aimed to use the Tol2 transposase system[7] to develop zebrafish models that constitutively expressed fluorescently labelled human CCNF under a quasi-ubiquitous (actb2) promoter.

    Objectivelink

    To develop novel transgenic zebrafish models that constitutively overexpress CCNF-fluorophore fusion for use in studies investigating disease-associated dysfunction of this gene.

    Results & Discussionlink

    No founders were identified in the CCNF injected groups

    Offspring from 102 fish injected with a CCNF construct (32 Hsa.CCNFWT, 28 Hsa.CCNFS621G, 18 ccnfWT and 24 ccnfS6213G) were screened and no founders were identified. Offspring from 8 fish injected with the mCherry control construct were screened and 4 founders were identified (Fig. 1A). The efficiency of transgenic integration in the mCherry control line, (reported at 30–50% for Tol2 system[8]) validated the injection and screening methods and suggested a gene-specific issue caused the failure to identify CCNF founders.

    CCNF expression is lost over the first 6 days of development

    To investigate this failure, the embryonic injections were repeated, and the screening protocol was altered. Instead of the standard screening at 2–3 days post fertilisation (dpf), screening was performed at 24 h post fertilisation (hpf) followed by subsequent analysis every 24 h up to 6 dpf. A dramatic loss of mCherry expression was observed in all CCNF-injected fish over this time period. In comparison, an increase in the number of mCherry positive cells was evident in the mCherry control fish (Fig. 1B). To quantify this, the number of mCherry positive cells in 10 fish from each injection group was estimated. This demonstrated no significant difference in the number of mCherry expressing cells between any of the injection groups at 24 hpf (p=0.08). However, significantly more mCherry positive cells were detectable in the control fish than any of the CCNF groups by 2 dpf (p<0.0001) and this difference increased over the next 5 days (Fig. 1C).

    Similar loss of expression was seen between mCherry and EGFP fused CCNF

    To eliminate an unexpected toxicity associated with mCherry fusion to CCNF, mCherry was substituted with the EGFP fluorophore in the Hsa. CCNFWT and ccnfWT constructs. Quantification of the number of fluorophore positive cells in 5 fish in each injection group demonstrated a similar loss of expression between the mCherry and the EGFP groups over the first 6 days of development (Fig. 1D).

    Higher levels of cell death were evident in CCNF injected groups compared to mCherry controls

    To investigate the mechanism responsible for the loss of cells expressing the transgene, cell death was assessed at the time of peak mCherry-CCNF expression (24 hpf) using acridine orange (AO). This analysis demonstrated significantly higher levels of cell death in all of the CCNF groups compared to sham injected controls (p<0.05). No significant difference was seen between any of the CCNF groups (n=30 per group) (Fig. 1E). Cells that both expressed mCherry fused CCNF and stained positive with AO were observed (Fig. 1F).

    This AO staining assay suggests that toxicity associated with overexpression of CCNF is an impediment to establishing transgenic lines based on this gene. It is possible that all, or the majority, of cells in which the CCNF transgene is integrated into the genome, including germline cells, undergoes apoptosis, precluding germline transmission. Given the high level of programmed cell death that occurs during early development[9] and the regenerative capacity of the zebrafish, this CCNF-associated cell death could occur without causing detectable morphological abnormalities.

    Conclusionslink

    This study demonstrates that the development of zebrafish transgenic lines that constitutively overexpress CCNF-fluorophore fusions using the described methods is not a feasible option, despite the clearly possible expression of the full-length ORF of the transgenic construct as apparent by fluorescence signal. The authors, therefore, suggest that levels of transgene expression to be re-assessed at 6–7 dpf to detect potential toxic overexpression effects at this early stage. In addition, models with temporal control of transgene expression to bypass overexpression during this period of development and cell death may be warranted.

    Limitationslink

    One limitation of this study is that fluorophore fusion proteins were used instead of the native CCNF ORF. For a possible follow-up, a native CCNF ORF could be tested with a transgenesis reporter in cis (i.e. myl7:EGFP or alpha-crystallin:Venus) to uncouple transgene detection from CCNF. In addition, studies to further investigate the failure to establish CCNF transgenic lines could be performed. For example, DNA could be extracted from embryos at 24 hpf to confirm transgenic integration, then repeated at later timepoints to establish whether cells that successfully integrate CCNF survive and to confirm if the loss of expressed CCNF is at a protein or DNA level. The presence of integrated CCNF DNA may assist in the elucidation of any gene silencing effects in the model.

    Alternative Explanationslink

    It is possible that some germline cells did integrate CCNF and survive, but these founders were not detected. Given the elevated level of cell death seen in embryos that mosaically express CCNF, it appears unlikely that F1 embryos with strong, ubiquitous expression would survive to time of screening at 2–3 dpf. Given the large size of zebrafish clutches and the small percentage of the F1 generation that typically carry a transgene (typically 10–15%[10]), such an occurrence would be difficult to detect.

    An alternative explanation for the observed loss of expression in this study is a rapid silencing of the transgene. Multiple epigenetic modifications, that silence gene expression, have been identified, key amongst them being methylation[11]. However, such rapid silencing of a transgene has not previously been reported and increased cell death would not be expected if this was the cause of the loss of expression in the CCNF-expressing embryos.

    Conjectureslink

    The role of CCNF in regulating the cell cycle provides a possible explanation for the apparent toxicity of CCNF overexpression in the embryonic zebrafish. The cell cycle is highly regulated and the coordination of transcription, cell proliferation, migration, differentiation and apoptosis is essential for embryonic development[9][12]. It is feasible that disruption to this highly regulated system associated with overexpression of CCNF in an organism undergoing rapid cell replication would have toxic effects.

    It is likely that the toxicity reported here extends beyond CCNF. There is the potential for toxicity to arise when overexpression is introduced into a system and this is of importance during early development when vital systems such as the nervous system are being established. In particular, the generation of models of ALS (and likely most neurodegenerative diseases) may be fraught with difficulties as many of these genes are ubiquitously expressed during development before becoming restricted to the central nervous system[6][13]. This suggests that the toxicity detailed here is not specific to CCNF expression during developmental stages and may be a factor in the establishment of other models.

    To overcome this problem, we hypothesise that inducible transgenic lines, that allow for precise temporal control of gene expression will be necessary for the generation of novel models of ALS. The adoption of inducible transgenic lines such as the heat shock-inducible Cre line[14][15] and Tet-on transgenic lines for doxycycline-inducible gene expression[16] will be crucial in establishing zebrafish models of ALS. Use of such lines will allow for temporal control of CCNF (or other ALS-linked genes) expression to be delayed until the fish matures beyond the developmental stage. We believe that this delay in expression will avoid the issues of early cell toxicity and allow for stable integration of the transgene in the germline, which in turn will facilitate transmission of transgene to the next generation. This would ultimately permit the development of transgenic overexpression CCNF-based zebrafish models of ALS. These inducible models are currently being generated and it is hoped that these models will yield insights into the biological mechanisms causing ALS as well as providing a platform for testing future therapeutics.

    Methodslink

    Generation of constructs

    Constructs were generated using the Tol2 Multi-site Gateway Three-Fragment Vector Construction kit (Life Technologies)[17]. The Hsa.CCNFWT (wildtype human CCNF), Hsa.CCNFS621G (mutant human CCNF), ccnfWT (wildtype zebrafish ccnf) and ccnfS623G (mutant zebrafish ccnf) att-flanked cDNA was codon optimised and synthesised by GeneArt (ThermoFisher Scientific). The CCNF cDNA sequences were cloned into a pDONR_P2R-P3 3’ entry vector (Life Technologies) using a standard BP reaction as per manufacturer’s instructions (Life Technologies). Final constructs were assembled using the LR reaction as per manufacturer’s instructions (Life Technologies) using the actb2 5’ entry vector, the mCherry and EGFP middle entry vectors, the polyA 3’ entry vector and the pDEST destination vector[17]. All constructs were sequence validated (Macrogen).

    Zebrafish lines

    Zebrafish used in this study were AB/Tübingen wildtype (TAB_WT) and the unpigmented Casper (nacre-roy) line[18]. Fish were bred and maintained under established conditions[19].

    Generation of transgenic lines

    Transposase mRNA was transcribed from a NotI-linearised pCS_zT2TP vector using the Sp6 mMESSAGE mMachine Kit (Ambion). The resulting mRNA was column purified using an RNeasy mini kit (Quiagen), eluted in RNAse-free water and stored in aliquots at -80°C until use.

    Following an initial dose-response trial, 200 pg of each Tol2 construct was co-injected with 160 pg transposase mRNA into the animal pole of zebrafish embryos at the single cell stage of development. Embryos were injected with either a mCherry control construct or 1 of 4 actb2 driven CCNF constructs with a N-terminal mCherry tag– Hsa.CCNFWT, Hsa.CCNFS621G, ccnfWT, . Injections were performed on multiple clutches on multiple days. Embryos were screened once for mCherry expression at 2–3 dpf as per standard protocol and positive embryos raised to maturity. Once mature, construct injected fish were outcrossed to wildtype fish and a minimum of 100 embryos were screened for mCherry expression across three separate lays for each mating pair at both 24 hpf and 3 dpf.

    Imaging & cell counts

    Embryos were mounted in 3% methylcellulose (Sigma Aldrich, M-0387) and imaged using a Leica M165FC stereo dissection microscope. The number of mCherry expressing cells was manually counted, however for embryos with >100 mCherry positive cells, this number was estimated.

    AO staining, imaging and quantification

    Zebrafish embryos were anesthetised in 0.2 mg/ml tricaine methanesulfonate solution (Sigma Aldrich) at 24 hpf and placed in 5 μg/ml AO solution (Sigma Aldrich) for 10 min in the dark. Embryos were briefly rinsed in E3 medium, then mounted in 3% methylcellulose and imaged on a M165FC fluorescent stereomicroscope (Leica) scope. Quantification was performed using Image J. Images were converted to 8-bit and inverted, and the number of positive cells was quantified using the Analyse Particles tool, with a size threshold of 20 mm.

    Funding Statementlink

    This work was supported by the Motor Neuron Disease Research Institute of Australia (MNDRIA; Mick Rodger Benalla GIA1510 and GIA1628), an MND Australia Leadership Grant, the National Health and Medical Research Council of Australia (1095215, 1107644), The Snow Foundation and European Community's Seventh Framework Programme (FP7/2007-2013) under the grant agreement number 259867.

    Acknowledgementslink

    We thank D.S, R.B, T. U and J.M-P for their care of the fish in our facility. We thank the Chien and Currie Laboratories for the kind gifts of plasmids.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    All husbandry and experimental procedures were performed in compliance with the Animal Ethics and Internal Biosafety Committees, Macquarie University (ARAs 2012/050 and 2015/034; NLRD 5201401007) (NSW, Australia).

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
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      Cyclin F-Mediated Degradation of Ribonucleotide Reductase M2 Controls Genome Integrity and DNA Repair
    2. D’angiolella Vincenzo, Donato Valerio, Vijayakumar Sangeetha,more_horiz, Pagano Michele
      SCFCyclin F controls centrosome homeostasis and mitotic fidelity through CP110 degradation
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    3. Emanuele Michael J., Elia Andrew E.H., Xu Qikai,more_horiz, Elledge Stephen J.
      Global Identification of Modular Cullin-RING Ligase Substrates
    4. Monica Kong, Elizabeth A. Barnes, Vincent Ollendorff, Daniel J. Donoghue
      Cyclin F regulates the nuclear localization of cyclin B1 through a cyclin-cyclin interaction
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    5. Kelly L. Williams, Simon Topp, Shu Yang, Bradley Smith, Jennifer A. Fifita, Sadaf T. Warraich, Katharine Y. Zhang, Natalie Farrawell, Caroline Vance, Xun Hu, Alessandra Chesi, Claire S. Leblond, Albert Lee, Stephanie L. Rayner, Vinod Sundaramoorthy, Carol Dobson-Stone, Mark P. Molloy, Marka van Blitterswijk, Dennis W. Dickson, Ronald C. Petersen, Neill R. Graff-Radford, Bradley F. Boeve, Melissa E. Murray, Cyril Pottier, Emily Don, Claire Winnick, Emily P. McCann, Alison Hogan, Hussein Daoud, Annie Levert, Patrick A. Dion, Jun Mitsui, Hiroyuki Ishiura, Yuji Takahashi, Jun Goto, Jason Kost, Cinzia Gellera, Athina Soragia Gkazi, Jack Miller, Joanne Stockton, William S. Brooks, Karyn Boundy, Meraida Polak, José Luis Muñoz-Blanco, Jesús Esteban-Pérez, Alberto Rábano, Orla Hardiman, Karen E. Morrison, Nicola Ticozzi, Vincenzo Silani, Jacqueline de Belleroche, Jonathan D. Glass, John B. J. Kwok, Gilles J. Guillemin, Roger S. Chung, Shoji Tsuji, Robert H. Brown Jr, Alberto García-Redondo, Rosa Rademakers, John E. Landers, Aaron D. Gitler, Guy A. Rouleau, Nicholas J. Cole, Justin J. Yerbury, Julie D. Atkin, Christopher E. Shaw, Garth A. Nicholson, Ian P. Blair
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      Loss of ALS-associated TDP-43 in zebrafish causes muscle degeneration, vascular dysfunction, and reduced motor neuron axon outgrowth
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