Zebrafish live imaging reveals only around 2% rather than 50% of motor neurons die through apoptosis during development

Reviewer #1 (Public Review):

Summary:

The authors aim to measure the apoptotic fraction of motorneurons in developing zebrafish spinal cord to assess the extent of neuronal apoptosis during the development of a vertebrate embryo in an in vivo context.

Strengths:

The transgenic fish line tg (mnx1:sensor C3) appears to be a good reagent for motorneuron apoptosis studies, while further validation of its motorneuron specificity should be performed.

Weaknesses:

The results do not support the conclusions. The main "selling point" as summarized in the title is that the apoptotic rate of zebrafish motorneurons during development is strikingly low (~2% ) as compared to the much higher estimate (~50%) by previous studies in other systems. The results used to support the conclusion are that only a small percentage (under 2%) of apoptotic cells were found over a large population at a variety of stages 24-120hpf. This is fundamentally flawed logic, as a short-time window measure of percentage cannot represent the percentage in the long term. For example, at any year under 1% of the human population dies, but over 100 years >99% of the starting group will have died. To find the real percentage of motorneurons that died, the motorneurons born at different times must be tracked over the long term or the new motorneuron birth rate must be estimated.

A similar argument can be applied to the macrophage results. Here the authors probably want to discuss well-established mechanisms of apoptotic neuron clearance such as by glia and microglia cells.

The conclusion regarding the timing of axon and cell body caspase activation and apoptosis timing also has clear issues. The ~minutes measurement is too long as compared to the transport/diffusion timescale between the cell body and the axon, caspase activity could have been activated in the cell body, and either caspase or the cleaved sensor moves to the axon in several seconds. The authors' results are not high-frequency enough to resolve these dynamics

Many statements suggest oversight of literature, for example, in the abstract "However, there is still no real-time observation showing this dying process in live animals.".

Many statements should use more scholarly terms and descriptions from the spinal cord or motor neuron, neuromuscular development fields, such as line 87 "their axons converged into one bundle to extend into individual somite, which serves as a functional unit for the development and contraction of muscle cells"

The transgenic line is perhaps the most meaningful contribution to the field as the work stands. However, the mnx1 promoter is well known for its non-specific activation - while the images suggest the authors' line is good, motor neuron markers should be used to validate the line. This is especially important for assessing this population later as mnx1 may be turned off in mature neurons.

Overall, this work does not substantiate its biological conclusions and therefore does not advance the field. The transgenic line has the potential to address the questions raised but requires different sets of experiments. The line and the data as reported are useful on their own by providing a short-term rate of apoptosis of the motorneuron population.

Reviewer #2 (Public Review):

Summary:

Jia and colleagues developed a fluorescence resonance energy transfer (FRET)-based biosensor to study programmed cell death in the zebrafish spinal cord. They applied this tool to study the death of zebrafish spinal motor neurons.

Strengths:
Their analysis shows that the tool is a useful biosensor of motor neuron apoptosis in living zebrafish.

Weaknesses:
However, they have ignored significant literature describing the death of an identified zebrafish motor neuron, expression of the mnx gene in interneurons that are closely related to motor neurons, the increase in number of zebrafish motor neurons over developmental time, and potential differences between the limb-innervating motor neurons whose death has been characterized in chicks and rodents and the body wall-innervating motor neurons whose death they characterized using their biosensor. Thus, although their new tool is likely to be useful in the future, it does not provide new insights into zebrafish motor neuron programmed cell death.

Author response:

We are grateful to the reviewers for recognizing the importance of our work and for their helpful suggestions. We will revise our manuscript in the revised version. However, we’d like to provide provisional responses now to answer the key questions and comments from the reviewers.

(1) Both reviewers asked why we chose 24-120 hpf to measure the apoptotic rates. We chose this time window based on the following two reasons: 1) Previous studies showed that although the motor neuron death time windows vary in chick (E5-E10), mouse (E11.5-E15.5), rat (E15-E18) and human (11-25 weeks of gestation), the common feature of these time windows is that they are all the developmental periods when motor neurons contact with muscle cells. The contact between zebrafish motor neurons and muscle cells occurs before 72 hpf, which is included in our observation time window. 2) Zebrafish complete hatching during 48-72 hpf, and most organs form before 72 hpf. More importantly, zebrafish start swimming around 72 hpf, indicating that motor neurons are fully functional.

Thus, we are confident that this 24-120 hpf time window covers the time window during which motor neurons undergo programmed cell death during zebrafish early development. We frequently used “early development” in this manuscript to describe our observation. However, we missed “early” in our title. We will add “early” in the title in the revised version.

(2) Both reviewers also asked about the neurogenesis of motor neurons. Previous studies have shown that the production of spinal cord motor neurons largely ceases before 48 hpf and then the motor neurons remain largely constant until adulthood. Our observation time window covers the major motor neuron production process. Therefore, we believe that neurogenesis will not affect our data and conclusions.

(3) Both reviewers questioned the specificity of using the mnx1 promoter to label motor neurons. The mnx1 promoter has been widely used to label motor neurons in transgenic zebrafish. Previous studies have shown that most of the cells labeled in the mnx1 transgenic zebrafish are motor neurons. In this study, we observed that the neuronal cells in our sensor zebrafish formed green cell bodies inside of the spinal cord and extended to the muscle region, which is an important morphological feature of the motor neurons. Furthermore, a few of those green cell bodies turned into blue apoptotic bodies inside the spinal cord and changed to blue axons in the muscle regions at the same time, which strongly suggests that those apoptotic neurons are not interneurons. Although the mnx1 promoter might have labeled some interneurons, this will not affect our major finding that only a small portion of motor neurons died during zebrafish early development.

(4) Reviewer 2 is concerned that the estimated 50% of motor neuron death was in limb-innervating motor neurons but not in body wall-innervating motor neurons. The death of motor neurons in limb-innervating motor neurons has been extensively studied in chicks and rodents, as it is easy to undergo operations such as amputation. However, previous studies have shown this dramatic motor neuron death does not only occur in limb-innervating motor neurons but also occurs in other spinal cord motor neurons. In our manuscript, we studied the naturally occurring motor neuron death in the whole spinal cord during the early stage of zebrafish development.

(5) Reviewer 2 mentioned that we ignored the death of an identified motor neuron. Our study was to examine the overall motor neuron apoptosis rather than a specific type of motor neuron death, so we did not emphasize the death of VaP motor neurons. We agree that the dead motor neurons observed in our manuscript contain VaP motor neurons. However, there were also other types of dead motor neurons observed in our study. The reasons are as follows: 1) VaP primary motor neurons die before 36 hpf, but our study found motor neuron cells died after 36 hpf and even at 84 hpf. 2) The position of the VaP motor neuron is together with that of the CaP motor neuron, that is, at the caudal region of the motor neuron cluster. Although it’s rare, we did observe the death of motor neurons in the rostral region of the motor neuron cluster. 3) There is only one or zero VaP motor neuron in each hemisegment. Although our data showed that usually one motor neuron died in each hemisegment, we did observe that sometimes more than one motor neuron died in the motor neuron cluster. We will include this information in the revised manuscript.

(6) For the morpholinos, we did not confirm the downregulation of the target genes. These morpholino-related data are a minor part of our manuscript and shall not affect our major findings. Thus, we didn’t think we missed “important” controls. We will perform experiments to confirm the efficiency of the morpholinos or remove these morpholino-related data from the revised version.