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

Reviewer #1 (Public review):

Summary:

The authors aim at measuring the apoptotic fraction of motorneurons in developing zebrafish spinal cord to assess the extent of neuronal apoptosis during the development of 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 on the long-term. For example, at any year under 1% of human population die, 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 long term, or the new motorneuron birth rate must be estimated.

Similar argument can be applied to the macrophage results.

The conclusion regarding timing of axon and cell body caspase activation and apoptosis timing also has clear issues. The ~minutes measurement are 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 move 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 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 motorneuron, 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, mnx1 promoter is well known for its non-specific activation - while the images do suggest the authors' line is good, motorneuron 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. The author's response regarding mnx1 specificity does not mitigate the original concern.

Overall, this work does not substantiate its biological conclusions and therefore do not advance the field. The transgenic line has the potential for addressing 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 death of zebrafish spinal motor neurons.

Strengths:

Their analysis shows that the tool is a useful biosensor of motor neuron apoptosis in living zebrafish and can reveal which part of the neuron undergoes caspase activation first, achieving two of their aims.

Weaknesses:

The third aim, to provide novel insights into the spatiotemporal properties and occurrence rates of motor neuron death requires additional context and investigation, especially to understand the significance of the differences they report between zebrafish motor neuron programmed cell death and what has been previously described in chicks and rodents. For example, mnx1 expresses not only in motor neurons, but also in interneurons. However, the way the authors counted living and dead cells does not take this into consideration, potentially underestimating the percentage of motor neurons that died. Previous studies of chicks and rodents showed widespread differences in the timing of motor neuron programmed cell death and the number of cells that died depending on the spinal cord region examined. The authors have not described which spinal cord segments they examined or whether they examined motor neurons in limb-bearing segments which have been best studied in other species. Previous literature investigated the death of an identified zebrafish motor neuron and provided experimental evidence that it is independent of limitations in muscle innervation area, suggesting it is not coupled to muscle-derived neurotrophic factors. Thus, the authors need to acknowledge that even previous to their study, there was literature suggesting that programmed cell death of at least one motor neuron in zebrafish does not easily fit into the "neurotrophic hypothesis" as it is generally formulated. Finally, the authors need to be mindful that showing that something does not happen in an observational study cannot reveal the capabilities of the cells involved without an experimental test.

Author response:

The following is the authors’ response to the original reviews.

Reviewer 1:

We thank the reviewer for the time and effort in providing very useful comments and suggestions for our manuscript.

(1) 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.

We chose the time window of 24-120 hpf based on the following two reasons: 1) Previous studies showed that although the time windows of motor neuron death 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 of 24-120 hpf. 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 at 72 hpf. 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 have added this information to the revised manuscript.

We frequently used “early development” in this manuscript to describe our observation. However, we missed “early” in our title. We therefore have added this ket word of “early” in the title in the revised manuscript.

Previous studies in zebrafish 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 (doi: 10.1016/j.celrep.2015.09.050; 10.1016/j.devcel.2013.04.012; 10.1007/BF00304606; 10.3389/fcell.2021.640414). Our observation time window covers the major motor neuron production process. Therefore, we believe that neurogenesis will not affect our findings and conclusions.

We discussed the engulfment of dead motor neurons by other types of cells in the discussion section.

(2) 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.

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.

Reviewer 2:

We thank the reviewer for the time and effort in making very useful comments and suggestions for our manuscript.

The FRET-based programmed cell death biosensor described in this manuscript could be very useful. However, the authors have not considered what is already known about the development and programmed cell death of zebrafish spinal motor neurons, and potential differences between motor neuron populations innervating different types of muscles in different vertebrate models. Without this context, the application of their new biosensor tool does not provide new insights into zebrafish motor neuron programmed cell death. In addition, the authors have not carried out controls to show the efficacy and specificity of their morpholinos. Nor have they described how they counted dying motor neurons, or why they chose the specific developmental time points they addressed. These issues are addressed more specifically below.

(1) Lines 12-13: Previous studies in zebrafish showed death of identified spinal motor neurons.

Line 103: In Figure 2A the cell body in the middle is that of identified motor neuron VaP. VaP death has previously been described in several publications. The cell body on the right of the same panel appears to belong to an interneuron whose axon can be seen extending off to the left in one of the rostrocaudal axon bundles that traverse the spinal cord. Higher-resolution imaging would clarify this.

Lines 163-164: Is this the absolute number of motor neurons that died? How were the counts done? Were all the motor neurons in every segment counted? There are approximately 30 identifiable VaP motor neurons in each embryo and they have previously been reported to die between 24-36 hpf. So this analysis is likely capturing those cells.

Our study examined 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 (revised Figure 4A). 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 (revised Figure 2C). 3) There is only one or zero VaP motor neuron in each motor neuron cluster. Although our data showed that usually one motor neuron died in each motor neuron cluster, we did observe that sometimes more than one motor neuron died in the motor neuron cluster (revised Figure 2C). We included this information in the revised discussion.

(2) Lines 82-83: It is published that mnx1 is expressed in at least one type of spinal interneuron derived from the same embryonic domain as 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.

(3) Lines 161-162: Although this may be the major time window of neurogenesis, there are many more motor neurons in adults than in larvae. Neither of these references describes the increase in motor neuron numbers over this particular time span, so the rationale for this choice is unclear.

Lines 168-171: It is known that later developing motor neurons are still being generated in the spinal cord at this time, suggesting that if there is a period of programmed cell death similar to that described in chick and mouse, it would likely occur later. In addition, most of the chick and mouse studies were performed on limb-innervating motor neurons, rather than the body wall muscle-innervating motor neurons examined here.

Lines 237-238: Especially since new motor neurons are still being generated at this time.

Previous studies have shown that the production of spinal cord motor neurons largely ceases before 48 hpf in zebrafish, and then the motor neurons remain largely constant until the adulthood (doi: 10.1016/j.celrep.2015.09.050; 10.1016/j.devcel.2013.04.012; 10.1007/BF00304606; 10.3389/fcell.2021.640414). Our observation time window covers the major motor neuron production process. Therefore, we believe that neurogenesis will not affect our data and conclusions.

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 (doi: 10.1006/dbio.1999.9413). In our manuscript, we studied the naturally occurring motor neuron death in the whole spinal cord during the early stage of zebrafish development.

(4) Lines 184-187: Previous publications showed that death of VaP is independent of limitations in muscle innervation area, suggesting it is not coupled to muscle-derived neurotrophic factors.

Lines 328-334: There have been many publications describing appropriate morpholino controls. The authors need to describe their controls and show that they know that the genes they were targeting were downregulated.

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. We have removed the neurotrophic factors and morpholino-related data in the revised manuscript.