Loss of Elp1 disrupts trigeminal ganglion neurodevelopment in a model of familial dysautonomia

  1. Carrie E Leonard
  2. Jolie Quiros
  3. Frances Lefcort
  4. Lisa A Taneyhill

  • Curated by eLife

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    Evaluation Summary:

    This study uses a combination of conditional knockout mouse embryos with targeted deletion of Elp1 in neural crest cells and neuron-specific antibodies to identify the onset of neural defects associated with the trigeminal ganglion. This manuscript is of potential interest to developmental biologists studying neurodevelopment disorders and, with additional quantification and experimentation, is likely to provide important insights into the mechanisms underlying Familial Dysautonomia in the cranial sensory ganglia.

    (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. The reviewers remained anonymous to the authors.)

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Abstract

Familial dysautonomia (FD) is a sensory and autonomic neuropathy caused by mutations in elongator complex protein 1 ( ELP1 ). FD patients have small trigeminal nerves and impaired facial pain and temperature perception. These signals are relayed by nociceptive neurons in the trigeminal ganglion, a structure that is composed of both neural crest- and placode-derived cells. Mice lacking Elp1 in neural crest derivatives (‘ Elp1 CKO’) are born with small trigeminal ganglia, suggesting Elp1 is important for trigeminal ganglion development, yet the function of Elp1 in this context is unknown. We demonstrate that Elp1, expressed in both neural crest- and placode-derived neurons, is not required for initial trigeminal ganglion formation. However, Elp1 CKO trigeminal neurons exhibit abnormal axon outgrowth and deficient target innervation. Developing nociceptors expressing the receptor TrkA undergo early apoptosis in Elp1 CKO, while TrkB- and TrkC-expressing neurons are spared, indicating Elp1 supports the target innervation and survival of trigeminal nociceptors. Furthermore, we demonstrate that specific TrkA deficits in the Elp1 CKO trigeminal ganglion reflect the neural crest lineage of most TrkA neurons versus the placodal lineage of most TrkB and TrkC neurons. Altogether, these findings explain defects in cranial gangliogenesis that may lead to loss of facial pain and temperature sensation in FD.

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  1. Version published to 10.7554/elife.71455 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    1. The Reviewer writes while the authors use an appropriate Cre line (Wnt1) to delete Elp1, they “need to consider reports that there also are Wnt1-negative neural crest cells residing in the mouse embryonic Vg, i.e., Wnt1-Cre is not expressed in every neural crest cell and therefore there will be neural crest derived cells in the Vg of their embryos in which Elp1 has not been deleted.”

    We appreciate the Reviewer pointing this out. Although Wnt1-Cre targets ~96% of Sox10-positive migratory neural crest cells in the trunk (Hari et al., 2012), we acknowledge there is a reported population of neural crest cells in the trigeminal ganglion that is not targeted by Wnt1-Cre (~30% at E9.5-E10, Karpinski et al., 2016). These analyses by Karpinski et al. were undertaken on the embryonic trigeminal ganglion before the major period of neural crest-derived neurogenesis in the trigeminal ganglion (E11-13), which is the time period we are examining. Similar analyses are not easily conducted once neural crest-derived neurogenesis begins, because Sox10 is swiftly downregulated upon neuronal differentiation. Therefore, it remained unclear what proportion of neural crest-derived trigeminal ganglion neurons are Wnt1-Cre-negative at the stages we have examined.

    These findings, along with suggestions by the other Reviewers, encouraged us to more rigorously address cellular origins of different trigeminal neuron populations from E10.5-15.5 using an additional mouse model. To this end, we crossed our Wnt1-Cre mouse with a ROSAmT/mG reporter mouse so that we could distinguish neural crest cells and their derivatives (Cre-positive, GFP-positive) from other cell types (Cre-negative, TdTomato (hereafter referred to as RFP)-positive, representing placode-derived cells and potentially other neural crest cell populations) in the trigeminal ganglion. Using this reporter, we found that Wnt1-Cre targets ~92% of Sox10-positive neural crest cells in the trigeminal ganglion at E10.5 (Figure 7). Additionally, we discovered that Six1, previously a marker attributed to the placodal lineage, labels all newly differentiating neurons in the trigeminal ganglion, irrespective of cellular origin (Figures 6 and 7). As development proceeds, it is likely that many of the RFP-positive (Cre-negative), Six1-negative neurons within the trigeminal ganglion were previously Six1-positive placodal neurons that have extinguished Six1, given what we observe with respect to Trk receptor expression. We have now carefully described our findings in the context of these previous studies and their limitations in the Results and Discussion sections.

    1. The Reviewer writes “some figures should include high magnification of the cells” to better support the claims, including Figure 1-supplemental, Figure 1P, Figure 7-supplemental, and Figure 8.”

    We thank the Reviewer for pointing this out and now provide higher magnification images in these figures as requested.

    1. The Reviewer states while “Vg appears to form normally in Elp1 CKO embryos…the language implies that the authorized analyzed neural crest migration, which they did not.” In addition, the Reviewer points out that the authors’ statement that the cells are “appropriately distributed throughout the forming ganglion” is “rather vague with no description of the data that support the conclusion.”

    We apologize for this overinterpretation of our data. We have modified the language in the manuscript to address these concerns and clarified that we did not directly analyze neural crest cell migration in this study. We have also removed language referring to cellular distribution within the trigeminal ganglion.

    To more rigorously address early trigeminal ganglion formation in the Elp1 CKO, we have now quantified the size of the trigeminal ganglion and length of the ophthalmic and mandibular nerve branches at E10.5 in control and Elp1 CKO embryos and found no statistically significant changes. Additionally, we determined the ratios of undifferentiated neural crest cells and placodal neurons present in the trigeminal ganglion at E10.5 by counting cells in sections through the forming trigeminal ganglion in control or Elp1 CKO embryos after immunohistochemistry for Sox10 (undifferentiated neural crest cells) and Six1 (placodal neurons) or Islet-1 (placodal neurons). These data are now included in the manuscript and reveal no statistically significant difference in these ratios between control or Elp1 CKO embryos (Figure 2).

    1. The Reviewer writes “it would be helpful to the reader if the composition of the “control” embryos was explained in the results/figure legends and not just in the methods” and a better description of the “number of litters examined, [whether] the images come from siblings, [and] the variance between litters.”

    We apologize for this omission and now provide this information in the Materials and Methods, Results, and Figure Legends. All analyses comparing control and Elp1 CKO embryos included sibling groups from at least two litters. Control versus Elp1 CKO images presented in the manuscript are siblings that underwent simultaneous immunohistochemistry and were imaged/processed under the exact same conditions. All graphs representing statistical comparisons of control versus Elp1 CKO embryos now include individual data points against the mean and SEM to display the variance within each genotype.

    1. The Reviewer states “there is virtually no quantitation of phenotypes” and recommends measuring, for example, the central root. Also, it is not clear if “the types and levels of abnormal axon trajectories shown in each figure found in every embryo analyzed.”

    We thank the Reviewer for bringing this to our attention. We have now quantified several aspects of trigeminal ganglion and nerve development in Elp1 CKO embryos (or littermate controls). Besides the measurements and ratios described in #3 above, we have quantified, at multiple stages, the central root diameter; size of the innervation field of the infraorbital nerve entering the whisker pad; Trk and TUNEL fluorescence; the number of Six1-positive and Trk-expressing neurons (E10.5-12.5), Sox10-positive and Cre-positive cells (E10.5), Six1-positive and Cre-positive neurons (E10.5, E12.5), and Cre-negative and Trk-expressing neurons (E15.5); and the ratio of Six1 (or Sox10)-positive to DAPI-positive cells (E11.5). The Elp1 CKO phenotype is highly penetrant and we have now clarified the presence of axon trajectory deficits in the text, indicating that all embryos exhibit these phenotypes to some degree. We hope the Reviewer finds these additional measurements to improve the manuscript.

    1. The Reviewer writes since “many of the findings for Vg are the same as what was found for trunk sensory ganglia, so impact is rather low. It could be increased significantly if other cranial ganglia were investigated for comparison…the facial nerve has one ganglion that is neural crest derived and one that is placode derived.”

    The Reviewer brings up an excellent point. We have now examined aspects of geniculate ganglion development, which is the placode-derived component of the facial nerve and, thus, not targeted for Elp1 deletion. After whole-mount immunohistochemistry for Tubb3 and TUNEL staining on tissue sections, we find no change in Elp1 CKO geniculate ganglion size or length of the chorda tympani nerve (E10.5, Figure 2) and we observe normal axon trajectories in the chorda tympani nerve compared to control embryos (E11.5, Figure 3). Moreover, we do not observe increased TUNEL staining in placode-derived geniculate neurons at E12.5 (Figure 9-figure supplement 1). Interestingly, most geniculate neurons express TrkB during this stage of development (Yamout et al., 2005; Fei and Krimm, 2013; Rios-Pilier and Krimm, 2019). Thus, the sparing of both trigeminal and geniculate placode-derived TrkB neurons in Elp1 CKO nicely aligns.

    1. The Reviewer indicates “the description in the text of whether Trk expression segregates with sensory modality and/or with neural crest versus placode origin is missing some references, for example, the apparent specific effect of the 22Q11 deletion on neural crest derived, TrkA+ Vg neurons. As stated above, it also would be useful to discern neural crest derived Vg neurons by a genetic lineage tracer such as Wnt1-GFP.”

    We thank the Reviewer for pointing us to relevant references, which we have now added to the revised manuscript, including discussion of our data as they relate to the findings from 22Q11 Deletion mice. The Reviewer raises an important question about the origin of trigeminal ganglion neurons, which we have now addressed by crossing our Wnt1-Cre line with the ROSAmT/mG reporter (see Point #1) and performing additional analyses to delineate the dynamics of normal neurogenesis and nerve growth in the trigeminal ganglion. First, we examined Trk expression from E11-E12.5 (Figure 6-supplement 1) and observed, as shown previously (Huang, Zang, et al., 1999; Huang, Wilkinson, et al., 1999), that TrkB and TrkC neurons predominate in the trigeminal ganglion early (E10.5-11), but then ultimately TrkA neurons become the majority neuronal subpopulation later (E12.5). Next, we co-labeled sections through the forming trigeminal ganglion at E10.5, E11.5, and E12.5 to identify Six1- and Trk-expressing cell populations (Figure 6). In accordance with placodal neurons differentiating first (and expressing Six1), we found that over 75% of any Trk-expressing neuron was also Six1-positive at E10.5. Surprisingly, the majority of Six1-positive cells at E11.5 and E12.5 were TrkA-positive, suggesting Six1 labels newly differentiating neurons. We subsequently confirmed this, and the cellular origin of Trk-expressing neurons, by carrying out section immunohistochemistry on the forming trigeminal ganglion in the Wnt1-Cre; ROSAmT/mG reporter mouse (Figure 7). In keeping with our initial Six1 results, only 12% of Six1-positive cells were GFP-positive at E10.5, confirming previous reports that the majority of the Six1-positive cells are placode-derived at this stage (Karpinski et al., 2016). However, 92% of Six1-expressing cells at E12.5 were GFP-positive, indicative of a neural crest origin for these neurons. Finally, we determined Trk expression at E15.5 (Figure 8) with respect to RFP-expressing (mostly placode-derived) trigeminal neurons and noted that approximately three quarters of the TrkA neurons were RFP-negative (and therefore neural crest-derived). Collectively, these data point to a neural crest origin for most of the TrkA neurons in the trigeminal ganglion, while placode cells give rise to primarily TrkB and TrkC neurons.

    Reviewer #2 (Public Review):

    1. The Reviewer states “although the authors use TrkA/B/C staining to quantify some of their data, they should have taken advantage of these specific markers in addition to the position identity of neural crest and placode-derived neurons in the ganglia to strengthen their observations of specific knockout of Elp1 in neural crest derived neurons as well as targeting defects…[These] neurons are segregated in the proximal (neural crest) and distal (placode) regions of the ganglion. The authors could also use similar location of neurons in addition to differential expression of TrkA/TrkB to confirm the absence of Elp1 in neural crest-derived neurons as opposed to placode-derived neurons of the CKO mice and to also show that neuron apoptosis occurs in only the [proximal] region of the trigeminal ganglion. Furthermore, the authors could use this differential expression of TrkA and TrkB to show the specific loss of TrkA in the target sites of Elp1 CKO mice.”

    We appreciate the point the Reviewer brings up regarding cell position within the trigeminal ganglion, particularly given our own research on chick trigeminal ganglion assembly. In the chick trigeminal ganglion, neurons are segregated by cellular origin such that placode-derived neurons reside in the distal ganglion (relative to the neural tube), while neural crest-derived neurons reside in the proximal ganglion. This pattern does not translate to the mouse trigeminal ganglion, which has been previously described as a mosaic of cellular subtypes with no preferential aggregation of any particular lineage (Karpinski et al., 2016; Motahari et al., 2021). Therefore, anatomical position is an unreliable tool for predicting placodal versus neural crest lineage in the mouse trigeminal ganglion. Our TrkA/B/C immunohistochemistry data, and the distribution of TrkA/TUNEL-double positive cells within Elp1 CKO trigeminal ganglion sections, also support this finding. Because of this, we cannot rely on position as another means to support our results. We have now addressed these differences between chick and mouse in the Discussion.

    We have conducted additional section immunohistochemistry experiments in which we have co-stained for different Trks and Elp1 in control and Elp1 CKO embryos (Figure 5, Figure 5-supplement 1 and 2, E12.5). We discovered a statistically significant decrease in TrkA fluorescence intensity in Elp1 CKO versus control trigeminal ganglia, with no change observed for TrkB or TrkC. Additionally, there were fewer TrkA-expressing nerve endings in sections through the upper lip of Elp1 CKO embryos relative to control embryos, with no change observed in TrkC-expressing nerve endings. Remarkably in the Elp1 CKO, most TrkA neurons were devoid of Elp1 protein, while the majority of Elp1-positive neurons expressed TrkB or TrkC. Altogether, these data provide further evidence to support targeting of presumptive neural crest-derived TrkA neurons in the trigeminal ganglion upon Elp1 loss in neural crest cells.

    1. The Reviewer writes the “authors state that two litters were examined per experiment, but do not provide the numbers of knockout and wildtype mice used for each experiment. In addition, quantification of the data such as the thickness of the central nerve root between control and Elp1 CKO mice would make the authors’ claim stronger.”

    We apologize for the omission of these numbers and have added them to the manuscript. In addition, we have now quantified several aspects of trigeminal ganglion and nerve development (described in detail in Reviewer 1, Point #5 above), as per the recommendations of this Reviewer and Reviewer 1. We thank the Reviewer for this comment, as the new data have strengthened the manuscript. Additionally, the numbers of animals/litters per experiment have been clarified in figure legends, and graphs representing statistical comparisons between control and Elp1 CKO embryos now include individual data points against the mean/SEM to demonstrate the number of embryos examined and the variation within genotypes.

    1. The Reviewer asks about “the expression pattern of NGF in the target tissue where the nerve bundles appear to be disformed in Elp1 CKO…[and whether] other neurotrophic factors [are present] in this region.”

    The Reviewer brings up an excellent point. To address this, we have performed immunohistochemistry to examine NGF expression and distribution in Elp1 CKO and control littermates at E12.5 (Figure 9-supplement 2). Our data reveal NGF protein in trigeminal nerve target tissues of both control and Elp1 CKO embryos. Importantly, given the role of Elp1 in translation, these findings demonstrate that Elp1 is not altering NGF protein levels. These results are consistent with previously published data showing no difference in the amount of NGF transcripts (Naftelberg et al., 2016; Morini et al., 2021) or protein (George et al., 2013) levels between control and Elp1 CKO. With regards to other neurotrophic factors, BDNF and NT-3, which can serve as ligands for TrkB and TrkC, respectively, are also expressed in the ophthalmic and maxillary regions targeted by the trigeminal nerves (Ernfors et al., 1992; Arumäe et al., 1993; Buchman et al., 1993; O’Conner and Tessier-Lavigne, 1999).

    1. Since the authors suggest that “most TrkA neurons do not express Elp1” and Elp1 is “knocked out in [neural crest]-derived neurons,” the Reviewer states “these results would be much stronger if they showed that Elp1 expression is maintained in the placode (TrkB) neurons under these conditions.”

    We thank the Reviewer for this suggestion. We now provide data from the Elp1 CKO showing that TrkA neurons are typically devoid of Elp1 protein, while TrkB and TrkC neurons still express Elp1 (Figure 5-supplement 2; see also Point #1 in this section).

    1. The Reviewer asks whether “specific targets are innervated by neural crest- or placode-derived neurons” and writes “it would be good to know what happens to placode-derived (TrkB) neurons that should be functioning normally” in Elp1 CKO.

    The Reviewer raises an excellent question regarding target tissues innervated by neural crest- vs. placode-derived neurons emanating from branches of the trigeminal ganglion. We are actively pursuing this line of research in my lab but it is currently beyond the scope of this study given the mouse work and time required to rigorously interrogate this question. We have speculated about this in the Discussion as a future direction. As a foray into this, however, we have quantified Trk fluorescence at E12.5 and find no statistically significant difference in TrkB (or TrkC) fluorescence throughout the trigeminal ganglion between control and Elp1 CKO embryos (Figure 5), while TrkA fluorescence is reduced. Additionally, we show that TrkA nerve endings are reduced in sections through the whisker pad of E12.5 Elp1 CKO embryos, but TrkC nerve endings are maintained (Figure 5).

  3. eLife

    Evaluation Summary:

    This study uses a combination of conditional knockout mouse embryos with targeted deletion of Elp1 in neural crest cells and neuron-specific antibodies to identify the onset of neural defects associated with the trigeminal ganglion. This manuscript is of potential interest to developmental biologists studying neurodevelopment disorders and, with additional quantification and experimentation, is likely to provide important insights into the mechanisms underlying Familial Dysautonomia in the cranial sensory ganglia.

    (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. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #3 (Public Review):

    Leonard et al. investigated the consequences of elp1 deletion in a mouse model of Familial Dysautonomia (FD). They used a previously published wnt-conditional knock-out mouse model that eliminates elp1 from the neural crest lineage, and they focused on the development of the trigeminal ganglia. FD patients do exhibit facial pain and temperature sensation reduction that are likely linked to trigeminal nerve defects. The authors showed elp1 expression in trigeminal ganglia and that the development of the trigeminal ganglia is affected in this FD mouse. Specifically, they showed that the TrkA-expressing nociceptors in the trigeminal ganglia is particularly affected. Additionally, they showed for the first time that TrkA neurons in the trigeminal ganglia are primarily neural crest derived, while TrkB and C neurons are placode derived.

    The conclusions of this paper are supported by the presented data.

    Strengths: The scientific question/gap in knowledge is clear and the approaches and data support the conclusions made. The data is well organized and clearly presented and lead to new insight into the two questions about the role of elp1 in trigeminal neuron development and into the composition of trigeminal ganglia and the cell’s origin. The authors present a thorough analysis of this mouse model with respect to their question.

    Weaknesses: The study is very descriptive. The vast majority of data is imaging and morphology based. Alternative, additional techniques could strengthen the results and conclusions.

  5. eLife

    Reviewer #2 (Public Review):

    In this study, Leonard et al. investigate the role of Elp1 gene deletion in the neural crest during gangliogenesis of the trigeminal ganglion and how its absence affects target innervation. They characterize the expression of Elp1 and show that it is expressed in differentiating neurons that commences with the placode-derived neurons. They show that conditional knockout of Elp1 in the neural crest component does not affect ganglion size during the onset of gangliogenesis, but defects arise during subsequent development as the nerves grow towards their targets. Defects in Elp1 CKO mice include apoptosis of TrKA-positive neurons and their loss in the target sites. Finally, they show that only TrkA-positive neurons are affected in the Elp1 CKO mice. Overall, the results from this study are informative and contribute to our understanding of Familial Dysautonomia. Although the authors use the TrKA/B/C staining to quantify some of their data, they should have taken advantage of these specific markers in addition to the position identity of neural crest and placode-derived neurons in the ganglia to strengthen their observations of specific knockdout of Elp1 in neural crest-derived neurons as well as the targeting defects. The authors would need to clarify several statements in the manuscript and quantify most of their data.

    1. In characterizing Elp1 expression during gangliogenesis, the authors use several markers to identify the placode and neural crest-derived neurons in regions where they appear to overlap. However, these neurons are segregated in the proximal (neural crest) and distal (placode) regions of the ganglion, which in addition to the markers they used would make a stronger point. The authors could also use similar location of neurons in addition to differential expression of TrKA/TrKB to confirm the absence of Elp1 in the neural crest-derived neuron as opposed to the placode-derived neurons of the CKO mice, and also to show that neuron apoptosis occurs in only the distal region of the trigeminal ganglion. Furthermore, the authors could use this differential expression of TrKA and TrKB to show the specific loss of TrKA neurons in the target sites of the Elp1 CKO mice.
    2. The authors state that two litters were examined per experiment, but do not provide the numbers of knockout and wild-type mice used for each experiment. In addition, quantification of the data such as the thickness of the central nerve root between control and Elp1 CKO mice would make the authors' claim stronger.
    3. The authors conclude that nerve defects caused by loss of Elp1 could be based on the target region. What is the expression pattern of NGF in the target tissues where the nerve bundles appear to be disformed in Elp1 CKO? Does the NGF expression overlap with other neurotrophic factors in this region?
    4. The authors suggest that in Elp1 CKO mice, most TrKA neurons do not express Elp1. Since this was knocked out in NC-derived neurons, these results would be much stronger of they showed that Elp1 expression is maintained in the placode (TrkB) neurons under these conditions.
    5. Are there specific targets innervated by neural crest- or placode-derived neurons? Given the defects observed in Elp1 CKO, it would be good to know what happens to the placode-derived neurons (TrKB neurons) that should be functioning normally.

  6. eLife

    Reviewer #1 (Public Review):

    Mutations in Elp1 is a known genetic cause of Familial Dysautonomia (FD), and many deficits in the trunk peripheral nervous system and autonomic nervous system have been described using a Wnt1-Cre driven conditional knock-out (CKO) line of Elp1. Because patients also have loss of pain and temperature perception in the craniofacial region, the authors studied the trigeminal ganglion and its nerves, which innervate the face and oropharynx, in Elp1 CKO embryos during the period of trigeminal axon outgrowth. It was of interest to compare the previous findings from the trunk sensory/autonomic system, which is derived entirely from neural crest progenitors, to their findings in the trigeminal system, which has a dual origin from neural crest and cranial placodes. The authors present data indicating that Elp1 CKO specifically affects the initial axon outgrowth and maintenance of peripheral innervation of neural crest-derived, TrkA-expressing neurons.

    Major strengths and weaknesses of methods and results:
    • The authors have used an appropriate Cre line to delete Elp1, a known causative gene of FD, in the majority of neural crest progenitors of the trigeminal ganglion (Vg). However, the authors would need to consider reports that there also are Wnt1-negative neural crest cells residing in the mouse embryonic Vg, i.e., Wnt1-Cre is not expressed in every neural crest cell and therefore there will be neural crest derived cell in the Vg of their embryos in which ELp1 has not been deleted.
    • The images are of very high quality and mostly are convincing. However, some figures should include high magnification of the cells. For example, in Figure 1-supplemental, there needs to be a high mag image of pax3+ cells to show that there is no Elp1 in the neural crest derived neurons or glia. At low mag, the data support the claim, but high mag would be much more convincing. Same comment with regards to Sox10+ cells in Figure 1P, and double-labeled cells in Figure 7-supplement and Figure 8.
    • Figure 3 shows nicely that the Vg appears to form normally in Elp1 CKO embryos, but in several places in the results and the discussion, the language implies that the authors analyzed neural crest migration, which they did not. This has been elegantly done in frog - as the authors cite for Elp3 - and in a mutant mouse using a Sox10-Venus reporter line (Karpinski et al., 2021 Dis Models Mech). Either the authors should do these types of experiments to directly address migration, or they should modify their language to state that they presume the migration pattern is normal. Similarly, the authors state in the section (p10, para2, line 7) that the cells are "appropriately distributed throughout the forming ganglion". This is a rather vague statement with no description of the data that support the conclusion other than eyeballing the picture. Others (e.g., Karpinski et al 2021) have shown that one needs to perform precise neighbor-relationships analyses to detect differences between genotypes in the diverse Vg.
    • It would be helpful to the reader if the composition of the "control" embryos were explained in the results/figure legends, and not just in the methods. Also, include the number of litters examined in each case and do the images come from siblings? What is the variance between litters?
    • The images in Figures 4, 5 and 6 are exceptional, and convincingly show that there are different axon trajectories between wild type and mutants. But disappointingly, with the exception of the Scholl analysis in Figure 5, there virtually no quantitation of the phenotypes. The descriptions (e.g., p11, para1) are descriptive and vague. Also, are the types and the levels of abnormal axon trajectories shown in each figure found in every embryo analyzed (N= only 2)? The claim that the central root in Figure 4 is smaller in Elp1 CKO, needs to be actually measured. Also, it would be useful to compare their "bulk" axon data to more recent reports using cutting edge approaches to label single Vg axons.
    • The rationale for studying a sensory ganglion that derives from two different progenitor populations is excellent because it allows the authors to compare their results to the many findings have already been described for the trunk sensory system, which is derived entirely from the neural crest. Many of the findings for Vg are the same as what was found for trunk sensory ganglia, so impact is rather low. It could be increased significantly if other cranial ganglia were investigated for comparison. As examples: VIIIg is derived exclusively from cranial placode progenitors; the facial nerve has one ganglion that is neural crest derived and one that is placode derived.
    • The authors very nicely show that the Elp1 CKO causes a decrease specifically in TrkA expression in axons and a loss of TrkA Vg neurons. This corroborates previous data for sympathetic and trunk sensory neurons. I do feel that the description in the text of whether Trk expression segregates with sensory modality and/or with neural crest versus placode origin is missing some references, for example the apparent specific effect of the 22Q11 deletion on neural crest derived, TrkA+ Vg neurons. As state above, it also would be useful to discern neural-crest derived Vg neurons by a genetic lineage tracer such as Wnt1-gfp.

  7. Version published to 10.1101/2021.06.10.447739v2 on bioRxiv
  8. Version published to 10.1101/2021.06.10.447739v1 on bioRxiv