Author response:
The following is the authors’ response to the previous reviews.
Reviewer 1:
(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 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.
In the revised manuscript (revised Figure 4), we extended the observation time window as long as possible, from 24 hpf to 240 hpf. After 240 hpf, the transparency of zebrafish body decreased dramatically, which made optical imaging quite difficult.
We are confident that this 24-240 hpf time window covers the major time window during which motor neurons undergo programmed cell death during zebrafish early development. We chose the observation time window 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. 2) Most organs of zebrafish form before 48-72 hpf, and they complete hatching during 48-72 hpf. Food-seeking and active avoidance behaviors also start at 72 hpf, indicating that motor neurons are fully functional at 72 hpf.
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.
Although we are confident that 240 h tracking is long enough to measure the motor neuron death rate, several sentences have been added in the discussion part, “In our manuscript, we tracked the motor neuron death in live zebrafish until 240 hpf, which was the longest time window we could achieve. But there was still a possibility that zebrafish motor neurons might die after 240 hpf.”
We agreed that the “2%” description might not be very accurate. Thus, we have revised our title to “Zebrafish live imaging reveals a surprisingly small percentage of spinal cord motor neurons die during early development.”
(2) 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.".
Real-time imaging of live animals is quite challenging in the field. Currently, using confocal microscopy, we can only achieve minute-scale tracking. In the future, with more advanced imaging techniques, the sensor fish in the present study may provide us with more detailed information on motor neuron death. We have removed “real-time” from our revised manuscript. We also revised the mentioned sentence in the abstract.
(3) 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"
We have removed this sentence.
(4) 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.
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.
In fact, no matter what method is used, such as using antibodies to stain specific markers to label motor neurons, 100% specificity cannot be achieved. More importantly, although the mnx1 promoter might have labeled some interneurons, this will not affect our major finding that only a small percentage of spinal cord motor neurons die during the early development of zebrafish.
Reviewer 2:
(1) Title: The 50% figure of motor neurons dying through apoptosis during early vertebrate development is not precisely accurate. In papers referenced by the authors, there is a wide distribution of percentages of motor neurons that die depending on the species and the spinal cord region. In addition, the authors did not examine limb-innervating motor neurons, which are the ones best studied in motor neuron programmed cell death in other species. Thus, a better title that reflects what they actually show would be something like "A surprisingly small percentage of early developing zebrafish motor neurons die through apoptosis in non-limb innervating regions of the spinal cord."
In fish, there are no such structures as limbs, although fins may be evolutionarily related to limbs. In our manuscript, we studied the naturally occurring motor neuron death in the whole spinal cord during the early stage of zebrafish development. 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 occurs not only in limb-innervating motor neurons but also in other spinal cord motor neurons (doi: 10.1006/dbio.1999.9413).
We have revised our title to “Zebrafish live imaging reveals a surprisingly small percentage of spinal cord motor neurons die during early development.”
(2) lines 18-19: "embryonic stage of vertebrates" is very broad, since zebrafish are also vertebrates; it would be better to be more specific
lines 25-26: The authors should be more specific about which animals have widespread neuronal cell death.
We have revised our manuscript accordingly.
(3) lines 98-99; 110-111; 113; 122-123; 140-141: A cell can undergo apoptosis. But an axon, which is only part of a cell, cannot undergo apoptosis. Especially since the axon doesn't have a separate nucleus, and the definition of apoptosis usually includes nuclear fragmentation. A better subheading would describe the result, which is that caspase activation is seen in both the cell body and the axon.
We have revised the subheadings and related words in the manuscript accordingly. In the introduction, we also revised the expression of the third aim from “Which part of a neuron (cell body vs. axon) will die first?” to “Which part of a neuron (cell body vs. axon) will degrade first?”.
(4) lines 159-160; 178-179: This is an oversimplification of the literature. The authors should spell out which populations of motor neuron have been examined and say something about the similarities and difference in motor neuron death.
We have revised it accordingly.
(5) lines 200; 216: The authors did not observe macrophages engulfing motor neurons. But that does not mean that they cannot. Making the conclusion stated in this subheading would require some kind of experiment, not just observations.
We did observe few colocalizations of macrophages and dead motor neurons. To more accurately express these data, in the revised manuscript, we used “colocalization” to replace “engulfment.” The subheading has been revised to “Most dead motor neurons were not colocalized with macrophages.” Accordingly, panel C of Figure 5 has also been revised.
(6) lines 234-246: The authors seem to have missed the point about VaP motor neuron death, which was two-fold. First, VaP death has been previously described, thus it could serve as a control for the work in this paper, especially since the conditions underlying VaP death and survival have been experimentally tested. Second, they should acknowledge that previous work showed that at least some motor neuron death in zebrafish differs from that described in chick and rodents. This conclusion came from work showing that death of VaP is independent of limitations in muscle innervation area, suggesting it is not coupled to muscle-derived neurotrophic factors.
Figures: The authors should say which level of the spinal cord they examined in each figure.
We have compared our findings with previous findings in the revised manuscript. The death of VaP motor neurons is not related to neurotrophic factors, but the death of other motor neurons may be related to neurotrophic factors, which needs further study and evidence. Our study examined the overall motor neuron apoptosis regardless of the causes and locations. To avoid misunderstanding, in the revised manuscript, we removed the data and words related to neurotrophic factors.
We also extended the observation time window as long as possible, from 24 hpf to 240 hpf (revised Figure 4). After 240 hpf, the transparency of zebrafish body decreased dramatically, which made the optical imaging quite difficult.
