KLC4 shapes axon arbors during development and mediates adult behavior
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Evaluation Summary:
This study will be interesting to a broad audience of neuroscientists, as it reveals for the first time that mutations in klc4, which are known to cause a form of early onset hereditary spastic paraplegia in human, affect specific aspects of neuronal development and nervous system functions. High resolution movies of developing sensory neurons in vivo and behavioral assays support the key findings that klc4 plays an essential role in the control of neuronal morphogenesis and behavior. The data presented in the manuscript are overall of a descriptive nature but provide a foundation for future mechanistic studies aimed at addressing the specific functions of KLC4.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood, although the cargo-binding kinesin light chain (KLC) subunits contribute to specificity. Our work implicates one such subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches, proper microtubule (MT) dynamics, and endosomal transport. Furthermore, KLC4 is required for proper tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons. This result suggests that KLC4 patterns axonal compartments and helps establish molecular differences between central and peripheral axons. Finally, we find that klc4 mutant larva are hypersensitive to touch and adults show anxiety-like behavior in a novel tank test, implicating klc4 as a new gene involved in stress response circuits.
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Evaluation Summary:
This study will be interesting to a broad audience of neuroscientists, as it reveals for the first time that mutations in klc4, which are known to cause a form of early onset hereditary spastic paraplegia in human, affect specific aspects of neuronal development and nervous system functions. High resolution movies of developing sensory neurons in vivo and behavioral assays support the key findings that klc4 plays an essential role in the control of neuronal morphogenesis and behavior. The data presented in the manuscript are overall of a descriptive nature but provide a foundation for future mechanistic studies aimed at addressing the specific functions of KLC4.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
This study will be interesting to a broad audience of neuroscientists, as it reveals for the first time that mutations in klc4, which are known to cause a form of early onset hereditary spastic paraplegia in human, affect specific aspects of neuronal development and nervous system functions. High resolution movies of developing sensory neurons in vivo and behavioral assays support the key findings that klc4 plays an essential role in the control of neuronal morphogenesis and behavior. The data presented in the manuscript are overall of a descriptive nature but provide a foundation for future mechanistic studies aimed at addressing the specific functions of KLC4.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this study, the authors aim to analyze the functions of the motor subunit klc4 in nervous system development and function. This is an important question to address, as not much is known about the cellular functions of klc4 even though mutations in this gene cause early onset hereditary spastic paraplegia in human. The authors used CRISPR/Cas9 to generate a klc4 mutant in zebrafish and analyzed the development of sensory neurons in embryos as well as behavior in adults. The strengths of this study include the generation of a novel klc4 mutant in zebrafish, the use of high and super-resolution live microscopy over time coupled to a rigorous analysis to reveal unsuspected developmental defects in klc4 mutants, including the formation of aberrant projections by sensory neurons and an abnormal development of …
Reviewer #1 (Public Review):
In this study, the authors aim to analyze the functions of the motor subunit klc4 in nervous system development and function. This is an important question to address, as not much is known about the cellular functions of klc4 even though mutations in this gene cause early onset hereditary spastic paraplegia in human. The authors used CRISPR/Cas9 to generate a klc4 mutant in zebrafish and analyzed the development of sensory neurons in embryos as well as behavior in adults. The strengths of this study include the generation of a novel klc4 mutant in zebrafish, the use of high and super-resolution live microscopy over time coupled to a rigorous analysis to reveal unsuspected developmental defects in klc4 mutants, including the formation of aberrant projections by sensory neurons and an abnormal development of peripheral sensory axons that appear less branched and fail to repel each other. The behavioral assays conducted by the authors also yielded robust results supporting a role for klc4 in adult neural circuits regulating stress response. The data are very well quantified and support the key findings of the study. Although the study does not delineate the molecular mechanisms causing an abnormal development of sensory neurons, its findings have a high impact, as they suggest specific functions of Klcs in neuronal patterning and compartimentalization and identify klc4 as a novel gene associated with anxiety behavior.
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Reviewer #2 (Public Review):
Microtubules are the major structural elements of neuronal axons. Kinesins are motor proteins that transport cargoes along microtubules and can also regulate microtubules. The functions of kinesins in neuronal development are not well understood. Kinesins are composed of heavy chains and associated light chains (KLCs), the latter being considered to provide cargo binding specificity and control aspects of motor protein function. This study used knock out technology in zebrafish to address the consequences of the loss of KLC4, a specific isoform of KLCs, in the development of the axons of Rohon-Beard sensory neurons and adult behaviors. The data indicate that loss of KLC4 results a failure to generate normal sensory axon arbors in embryos through increased instances of branch retraction and behavioral defects …
Reviewer #2 (Public Review):
Microtubules are the major structural elements of neuronal axons. Kinesins are motor proteins that transport cargoes along microtubules and can also regulate microtubules. The functions of kinesins in neuronal development are not well understood. Kinesins are composed of heavy chains and associated light chains (KLCs), the latter being considered to provide cargo binding specificity and control aspects of motor protein function. This study used knock out technology in zebrafish to address the consequences of the loss of KLC4, a specific isoform of KLCs, in the development of the axons of Rohon-Beard sensory neurons and adult behaviors. The data indicate that loss of KLC4 results a failure to generate normal sensory axon arbors in embryos through increased instances of branch retraction and behavioral defects in adult fish. The strength of this report is in having generated an animal model of KLC4 knockout and providing an initial characterization of ensuing deficits. The manuscript does not address whether the observed effects are neuron intrinsic nor provide mechanistic insights into the specific roles played by KLC4 within neurons, but provides data sets that will serve as jumping points for future investigations.
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Reviewer #3 (Public Review):
This study reports on the phenotypes of a CRISPR-engineered zebrafish mutants in kinesin light chain 4 (KLC4). KLC4 is expressed prominently in spinal cord sensory neurons, and mutants have defects in peripheral axon branching/stabilization and branch repulsion, as well as make occasional ectopic axon branches. Imaging also demonstrates that axonal microtubule growth dynamics are altered. These axonal phenotypes are nicely characterized with beautiful light sheet time-lapse microscopy and clever image analyses methods. Additionally, the growth of adult KLC4 mutants is stunted, and they exhibit a variety of behavioral defects.
The strengths of this paper are the creation of a new mutant for studying axonal transport, the impressive imaging methods, and the development of image analysis methods for …
Reviewer #3 (Public Review):
This study reports on the phenotypes of a CRISPR-engineered zebrafish mutants in kinesin light chain 4 (KLC4). KLC4 is expressed prominently in spinal cord sensory neurons, and mutants have defects in peripheral axon branching/stabilization and branch repulsion, as well as make occasional ectopic axon branches. Imaging also demonstrates that axonal microtubule growth dynamics are altered. These axonal phenotypes are nicely characterized with beautiful light sheet time-lapse microscopy and clever image analyses methods. Additionally, the growth of adult KLC4 mutants is stunted, and they exhibit a variety of behavioral defects.
The strengths of this paper are the creation of a new mutant for studying axonal transport, the impressive imaging methods, and the development of image analysis methods for characterizing axonal trajectories across a population.
The main weaknesses is the lack of a specific mechanistic explanation for how kinesin dysfunction leads to axonal defects-what kinesin cargoes play a role in branch stabilization and branch repulsion? How does kinesin-mediate transport affect microtubule growth?
Another weakness is the lack of a connection between the cellular defects characterized in larval sensory neurons, and the behavioral defects in adults. Since the adult behavioral defects likely do not involve sensory neurons, these two parts of the paper don't fit together. The authors may want to consider moving the behavior to a different paper. Additionally, the cellular basis of the adult behavioral defects is unknown, and likely involves a complex combination of defects in multiple cell types.
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