Coordinated cadherin functions sculpt respiratory motor circuit connectivity

  1. Alicia N Vagnozzi
  2. Matthew T Moore
  3. Minshan Lin
  4. Elyse M Brozost
  5. Ritesh KC
  6. Aambar Agarwal
  7. Lindsay A Schwarz
  8. Xin Duan
  9. Niccolò Zampieri
  10. Lynn T Landmesser
  11. Polyxeni Philippidou

  • Curated by eLife

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    eLife assessment

    This is an extremely thorough investigation of the role of cadherins in generating a functional motor circuit. The presented data support a model whereby combinations of redundant adhesion molecules create a code to wire the breathing circuit. This study advances understanding of the molecular basis of circuit wiring in the brain.

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Abstract

Breathing, and the motor circuits that control it, is essential for life. At the core of respiratory circuits are Dbx1-derived interneurons, which generate the rhythm and pattern of breathing, and phrenic motor neurons (MNs), which provide the final motor output that drives diaphragm muscle contractions during inspiration. Despite their critical function, the principles that dictate how respiratory circuits assemble are unknown. Here, we show that coordinated activity of a type I cadherin (N-cadherin) and type II cadherins (Cadherin-6, -9, and -10) is required in both MNs and Dbx1-derived neurons to generate robust respiratory motor output. Both MN- and Dbx1-specific cadherin inactivation in mice during a critical developmental window results in perinatal lethality due to respiratory failure and a striking reduction in phrenic MN bursting activity. This combinatorial cadherin code is required to establish phrenic MN cell body and dendritic topography; surprisingly, however, cell body position appears to be dispensable for the targeting of phrenic MNs by descending respiratory inputs. Our findings demonstrate that type I and II cadherins function cooperatively throughout the respiratory circuit to generate a robust breathing output and reveal novel strategies that drive the assembly of motor circuits.

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  1. Version published to 10.7554/elife.82116 on eLife
  2. eLife

    Author Response

    Reviewer 1 (Public Review):

    Weaknesses: The main conclusion that ablation of the cadherin code decreases synaptic connectivity between the rVRG and phrenic motor neurons is never directly shown. This can only be inferred by the data.

    1. Conclusion that the connectivity between rVRG premotor and phrenic nerve motor neurons is "weaker". This conclusion is inferred from several experiments but is never directly demonstrated. Alternative interpretations of the decreased amplitude of the in vitro phrenic nerve burst is that the rootlet contains fewer axons (as predicted by the fewer motor neurons in S3 and innervation of the diaphragm S2). Additionally, the intrinsic electrophysiological properties of the motor neurons might be different. To show this decisively, the authors could use electrophysiological recordings of phrenic motor neurons to directly measure a change in synaptic input (for example, mEPSPs or EPSPs after optogenetic stimulation of rVRG axon terminals). Without a direct measurement, the synaptic connectivity can only be inferred.

    We agree with the reviewer that without anatomical evidence, we can only infer the loss of synaptic connectivity. However, we believe that this is the most likely interpretation of our data (see response to the editor summary). Unfortunately, the experiment suggested (optogenetic stimulation of rVRG terminals) is not feasible at the moment, as a) a molecular tool to specifically express channelrhodopsin in rVRG does not currently exist; even if it did, it would require crossing two more alleles in our current mouse model, which contains 5 alleles, making the genetics/breeding cumbersome and b) viral-mediated channelrhodopsin expression in the rVRG is not feasible since the mice die at birth. We will continue to explore alternative approaches to directly demonstrate the loss of rVRG-PMC connectivity in the future.

    1. Conclusion that the small phenic nerve burst size in Dbx1 deleted cadherin signaling is due to less synaptic input to the motor neurons. Dbx1 is expressed in multiple compartments of the medullary breathing control circuit, like the breathing rhythm generator (preBötC). The smaller burst size could be due to altered activity between preBötC neurons to create a full burst, the transmission of this burst from the preBötC to the rVRG, etc.

    We agree with the reviewer about the alternative interpretations of the data, which we mention in the discussion. At this point, we can only conclude that cadherin signaling is required in Dbx1derived respiratory populations for proper phrenic respiratory output. We are currently developing the tools in our lab to further dissect the exact contributions of cadherins to rVRG development, connectivity, and function. As this will require significant time and effort, we believe it is outside the scope of the current work.

    1. In vitro burst size. The authors use 4 bursts from each animal to calculate the average burst size. How were the bursts chosen? Why did the authors use so few bursts? What is the variability of burst size within each animal? What parameters are used to define a burst? This analysis and the level of detail in the figure legend/methods section is inadequate to rigorously establish the conclusion that burst size is altered in the various genotypes.

    To address the reviewer’s concern, we have updated the data by analyzing 7 bursts per animal. Some control mice have burst frequencies as low as 0.2 bursts per minute (see fig. 4b), and thus acquiring 7 bursts requires 35 minutes of recording time, a substantial amount when an entire litter is being recorded in a day. All data is from 7 bursts per animal except for 4 out of 11 NMNΔ6910-/- mice, which only had 1-3 bursts total. To analyze the data, either every single burst was analyzed, or for those traces of higher frequency, bursts were selected randomly, spaced throughout the trace. Bursts were defined as activity above baseline that persists for at least 50ms. Some bursts contain pauses in activity in the middle; activity that was spaced less than 1 second apart was defined as a single burst.

    Updating the data for more bursts slightly changed some of our findings. We now find that 6910/- mice no longer exhibit significantly increased burst duration and burst activity. This was barely significant in our previous analysis, and is now just barely non-significant (p=0.065 for burst duration, p=0.059 for burst activity).

    We have included this more detailed description in the methods section. We have also included an excel sheet as source data for fig. 4 to indicate the variability of burst size within each animal and across animals.

    1. The authors state that the in vitro frequency in figure 4 is inaccurate, but then the in vitro frequency is used to claim the preBötC is not impacted in Dbx1 mutants (conclusion section "respiratory motor circuit anatomy and assembly"). To directly assess this conclusion, the bursting frequency of the in vitro preBötC rhythm should be measured.

    We have now included the quantitation of respiratory frequency data for control and βγ-catDbx1∆ mice, showing that there are no significant changes in burst frequency in βγ-catDbx1∆ mice. However, we do agree with the reviewer that the loss of excitatory drive could be due to changes either in the rVRG or the preBötC and we have toned down our conclusions to indicate that the preBötC could be impacted in βγ-catDbx1∆ mice.

    1. The burst size in picrotoxin/strychnine is used to conclude that the motor neurons intrinsic physiology is not impacted. The bursts are described, and examples are shown, but this is never quantified across many bursts within in a single recording nor in multiple animals of each genotype.

    We have now included quantification of this data, using 6-11 bursts/mouse from 3 control and 3 NMNΔ6910-/- mice. We find that both the spinal burst total duration (shown as % of recording time) and the normalized integrated spinal activity over time are not significantly different between control and NMNΔ6910-/- mice.

    Reviewer 3 (Public Review):

    Major points

    1. Page 8: 'In addition, NMNΔ and NMNΔ6910-/- mice showed a similar decrease in phrenic MN numbers, likely from the loss of trophic support due to the decrease in diaphragm innervation (Figure S3c).' This statement should be corrected: phrenic MN number in NMNΔ mice does not differ from controls, in contrast to NMNΔ6910-/- mice (Fig. S3). Similarly, diaphragm innervation is not significantly different from controls in NMNΔ (Fig. S2). Alternatively, these observations could be strengthened by increasing the number of mice analyzed to determine whether there is a significant reduction in PMN number and diaphragm innervation in NMNΔ mice.

    Following the reviewer’s suggestion, we increased the number of control mice analyzed for diaphragm innervation (n=7) and MN numbers (n=6). We now find that there is a significant reduction in both parameters in NMNΔ mice. We have modified the results section accordingly.

    1. A similar comment relates to the interpretation of the dendritic phenotype in NMNΔ and NMNΔ6910-/- mice (Fig. 3m): the authors conclude 'When directly comparing NMNΔ and NMNΔ6910-/- mice, NMNΔ6910-/- mice had a more severe loss of dorsolateral dendrites and a more significant increase in ventral dendrites (Figure 3l-m).' (page 9). The loss of dorsolateral dendrites in NMNΔ6910-/- mice indeed differs significantly from control mice, and is more severe than in NMNΔ mice, which do not differ significantly from controls. For ventral dendrites however, the increase compared to controls is significant for both NMNΔ and NMNΔ6910-/- mice, and the two genotypes do not appear to differ from each other. This suggests cooperative action of N-cadherin and cadherin 6,9,10 for dorsolateral dendrites, but suggests that N-cad is more important for ventral dendrites. This should be phrased more clearly.

    We agree with the reviewer and apologize for the lack of clarity. We have modified our description to highlight the contribution of N-cadherin to dendritic development.

    1. Related comment, page 10: 'Furthermore, the fact that phrenic MNs maintain their normal activity pattern in NMNΔ mice suggests that neither cell body position nor phrenic MN numbers significantly contribute to phrenic MN output.' This should be rephrased, phrenic MN number does not differ from control in NMNΔ mice (Fig. S2c).

    After analyzing additional control mice, we find that phrenic MN numbers are significantly reduced in NMNΔ mice.

    1. The authors conclude that spinal network activity in control and NMNΔ6910-/- mice does not differ (page 10, Fig. 4f). It is difficult to judge this from the example trace in 4f. How is this concluded from the figure and can this be quantified?

    We have now included quantification of this data, using 6-11 bursts/mouse from 3 control and 3 NMNΔ6910-/- mice. We find that both the spinal burst total duration (shown as % of recording time) and the normalized integrated spinal activity over time are not significantly different between control and NMNΔ6910-/- mice.

    1. RphiGT mice: please explain the genetic strategy better in Results section or Methods, do these mice also express the TVA receptor in a Cre-dependent manner? Crossing with the Cdh9:iCre line will then result in expression of TVA and G protein in phrenic motor neurons and presynaptic rVRG neurons in the brainstem, as well as additional Cdh9-expressing neuronal populations. How can the authors be sure that they are looking at monosynaptically connected neurons?

    We have added additional information in the methods to describe the rabies virus genetic strategy. Although the mice do express the TVA receptor, we did not include this in the description as it is not relevant to our strategy. We are using a Rabies∆G virus that is not pseudotyped with EnvA so it does not require TVA to infect cells. The specificity of primary cell (phrenic MN) infection rather comes from diaphragm injections. We only analyze mice in which we can confirm the injection was specific to the diaphragm muscle and did not leak to body wall or hypaxial muscles (about 50% of injections). We have tested different infection times to determine when monosynaptically connected neurons are labeled. We do not see any labeling at the brainstem 5 days post injection and we start to see additional labeling (possible 2nd order neurons) 10 days post injection. Thus we are confident that our analysis at 7 days post injection captures monosynaptically-connected neurons. We have also performed rabies virus tracing in ChAT::Cre mice, where the expression of G-protein is restricted to motor neurons, and we observe a similar distribution of pre-motor neurons in the brainstem, as with Cdh9::iCre, indicating that we are reproducibly labeling 1st order neurons with both genetic strategies.

    1. The authors use a Dbx1-cre strategy to inactivate cadherin signaling in multiple brainstem neuronal populations and perform analysis of burst activity in phrenic nerves. Based on the similarity in phenotype with NMNΔ6910-/- mice it is concluded that cadherin function is required in both phrenic MNs and Dbx1-derived interneurons. However, this manipulation can affect many populations including the preBötC, and the impact of this manipulation on rVRG and phrenic motor neurons (neuron number, cell body position, dendrite orientation, diaphragm innervation etc) is not described, although a model is presented in Fig. 7. These parameters should be analyzed to interpret the functional phenotype.

    We agree with the reviewer that the Dbx1-Cre mediated manipulation can affect multiple respiratory populations (see response to reviewer 1). However, Dbx1-mediated recombination does not target phrenic MNs. We have now added a figure (Figure 6-figure supplement 1), demonstrating this. Thus, we think that it is unlikely to cause any cell-autonomous changes in MN number, diaphragm innervation etc. It is plausible that there might be secondary changes in phrenic MNs as a result of changes in rVRG properties (for example, the dendritic orientation of phrenic MNs could be altered if rVRG synapses are lost), but the primary impact of this manipulation will be on Dbx1-derived neurons.

  3. eLife

    eLife assessment

    This is an extremely thorough investigation of the role of cadherins in generating a functional motor circuit. The presented data support a model whereby combinations of redundant adhesion molecules create a code to wire the breathing circuit. This study advances understanding of the molecular basis of circuit wiring in the brain.

  4. eLife

    Reviewer #1 (Public Review):

    The overarching hypothesis is that cadherin adhesion molecules specify the code that enables the premotor brainstem breathing circuits to innervate the phrenic motor neurons that control the primary breathing muscle, the diaphragm. The authors show that multiple type 1 and 2 cadherins (N-, 6, 9, 10) are expressed by phrenic motor neurons and are necessary for motor neuron development and breathing, and complementarily, that adhesion signaling in medullary breathing circuits are required for normal breathing. The presented data support a model whereby combinations of redundant adhesion molecules create a code to wire the breathing circuit.

    Strengths:

    1. The authors first use a complex, rigorous genetic approach to eliminate N, 6, 9, 10 cadherins from motor neurons and discover using whole body plethysmography that neonates do not breath.
    2. Then, the authors provide a thorough description of the anatomy of the mutant motor neurons and discover that the number of motor neurons decreases, the soma anatomical positions and dendritic arborization shift, and there is decreased innervation of the diaphragm breathing muscle.
    3. That Cdh9 medullary expressing neurons are premotor to Cdh9 expressing phrenic motor neurons.
    4. Cadherin signaling is required for normal breathing.

    Weaknesses: The main conclusion that ablation of the cadherin code decreases synaptic connectivity between the rVRG and phrenic motor neurons is never directly shown. This can only be inferred by the data.

    1. Conclusion that the connectivity between rVRG premotor and phrenic nerve motor neurons is "weaker". This conclusion is inferred from several experiments but is never directly demonstrated. Alternative interpretations of the decreased amplitude of the in vitro phrenic nerve burst is that the rootlet contains fewer axons (as predicted by the fewer motor neurons in S3 and innervation of the diaphragm S2). Additionally, the intrinsic electrophysiological properties of the motor neurons might be different. To show this decisively, the authors could use electrophysiological recordings of phrenic motor neurons to directly measure a change in synaptic input (for example, mEPSPs or EPSPs after optogenetic stimulation of rVRG axon terminals). Without a direct measurement, the synaptic connectivity can only be inferred.
    2. Conclusion that the small phenic nerve burst size in Dbx1 deleted cadherin signaling is due to less synaptic input to the motor neurons. Dbx1 is expressed in multiple compartments of the medullary breathing control circuit, like the breathing rhythm generator (preBötC). The smaller burst size could be due to altered activity between preBötC neurons to create a full burst, the transmission of this burst from the preBötC to the rVRG, etc.
    3. In vitro burst size. The authors use 4 bursts from each animal to calculate the average burst size. How were the bursts chosen? Why did the authors use so few bursts? What is the variability of burst size within each animal? What parameters are used to define a burst? This analysis and the level of detail in the figure legend/methods section is inadequate to rigorously establish the conclusion that burst size is altered in the various genotypes.
    4. The authors state that the in vitro frequency in figure 4 is inaccurate, but then the in vitro frequency is used to claim the preBötC is not impacted in Dbx1 mutants (conclusion section "respiratory motor circuit anatomy and assembly"). To directly assess this conclusion, the bursting frequency of the in vitro preBötC rhythm should be measured.
    5. The burst size in picrotoxin/strychnine is used to conclude that the motor neurons intrinsic physiology is not impacted. The bursts are described, and examples are shown, but this is never quantified across many bursts within in a single recordings nor in multiple animals of each genotype.
  5. eLife

    Reviewer #2 (Public Review):

    This is an extremely thorough investigation of the role of cadherins in generating a functional motor circuit. The work represents a major step forward in the field as it addresses several outstanding questions and verifies anatomical data with functional outcomes. First, the data show that a combination of type I (N) and type II (6, 9, 10) cadherins is needed to generate normal connectivity and function. This is novel as prior work has suggested that the two types do not work collaboratively to generate circuits. Second, the data show that cell body position (in this case) is modulated by N-cadherin but in a manner that is independent from the impact of N-cadherin on connectivity. While position and connectivity have been shown to be separable in some cases, the data support that N-cadherin plays important but separate roles toward both actions, and type II cadherins, mainly in connectivity. These findings also underscore that cadherin roles reported for hippocampus, retina, and spinal cord motor neuron pools are not generalizable across circuits. Third, while the data show that type I and type II cadherins are required for VRG to phrenic motor neuron connectivity, they also show that there are some outcomes controlled only by N-cadherin. Finally, the data reveal much about a very poorly understood and essential circuit. The approaches are sound and range from the standard (in situ, immuno, diI, breathing measurements) to the difficult (rabies-based tracing) to the impressive (challenging ephys preps, and some painstaking mouse crosses), and they incorporated strong and creative strategies for comparison and quantification. Minor questions do not detract from a really impressive piece of work.

  6. eLife

    Reviewer #3 (Public Review):

    Vagnozzi et al. analyze the role of cadherins in respiratory circuit development. The authors previously identified a combinatorial cadherin code that defines phrenic motor neurons (Vagnozzi et al., eLife 2020). Here they find that combined loss of type I N-cadherin and type II cadherins 6, 9 and 10 results in respiratory failure and reduction in phrenic motor neuron bursting activity. Furthermore, diaphragm innervation, phrenic motor neuron (MN) number, cell body position as well as dendrite orientation are all impaired in mice lacking N-cadherin and cadherins 6, 9, 10. Analysis of different genotypes indicates that phrenic MN cell body position is regulated by N-cadherin, but that dendrite orientation is regulated by the combinatorial action of N-cadherin and cadherins 6, 9, and 10. They subsequently determine that cadherin signaling in presynaptic interneurons is required for phrenic MN bursting activity. Together, the results indicate that cadherins are essential for respiratory circuit function and suggest that a combinatorial cadherin code regulates wiring specificity in this circuit.

    The manuscript is well presented with clear figures and text. My comments below mainly revolve around the interpretation of some of the findings and the correlation between phenotypes in NMNΔ6910-/- mice and βγ-catDbx1Δ mice in light of specific cadherin expression patterns and connectivity between rVRG and prenic MNs.

    Major points
    1. Page 8: 'In addition, NMNΔ and NMNΔ6910-/- mice showed a similar decrease in phrenic MN numbers, likely from the loss of trophic support due to the decrease in diaphragm innervation (Figure S3c).' This statement should be corrected: phrenic MN number in NMNΔ mice does not differ from controls, in contrast to NMNΔ6910-/- mice (Fig. S3). Similarly, diaphragm innervation is not significantly different from controls in NMNΔ (Fig. S2). Alternatively, these observations could be strengthened by increasing the number of mice analyzed to determine whether there is a significant reduction in PMN number and diaphragm innervation in NMNΔ mice.
    2. A similar comment relates to the interpretation of the dendritic phenotype in NMNΔ and NMNΔ6910-/- mice (Fig. 3m): the authors conclude 'When directly comparing NMNΔ and NMNΔ6910-/- mice, NMNΔ6910-/- mice had a more severe loss of dorsolateral dendrites and a more significant increase in ventral dendrites (Figure 3l-m).' (page 9). The loss of dorsolateral dendrites in NMNΔ6910-/- mice indeed differs significantly from control mice, and is more severe than in NMNΔ mice, which do not differ significantly from controls. For ventral dendrites however, the increase compared to controls is significant for both NMNΔ and NMNΔ6910-/- mice, and the two genotypes do not appear to differ from each other. This suggests cooperative action of N-cadherin and cadherin 6,9,10 for dorsolateral dendrites, but suggests that N-cad is more important for ventral dendrites. This should be phrased more clearly.
    3. Related comment, page 10: 'Furthermore, the fact that phrenic MNs maintain their normal activity pattern in NMNΔ mice suggests that neither cell body position nor phrenic MN numbers significantly contribute to phrenic MN output.' This should be rephrased, phrenic MN number does not differ from control in NMNΔ mice (Fig. S2c).
    4. The authors conclude that spinal network activity in control and NMNΔ6910-/- mice does not differ (page 10, Fig. 4f). It is difficult to judge this from the example trace in 4f. How is this concluded from the figure and can this be quantified?
    5. RphiGT mice: please explain the genetic strategy better in Results section or Methods, do these mice also express the TVA receptor in a Cre-dependent manner? Crossing with the Cdh9:iCre line will then result in expression of TVA and G protein in phrenic motor neurons and presynaptic rVRG neurons in the brainstem, as well as additional Cdh9-expressing neuronal populations. How can the authors be sure that they are looking at monosynaptically connected neurons?
    6. The authors use a Dbx1-cre strategy to inactivate cadherin signaling in multiple brainstem neuronal populations and perform analysis of burst activity in phrenic nerves. Based on the similarity in phenotype with NMNΔ6910-/- mice it is concluded that cadherin function is required in both phrenic MNs and Dbx1-derived interneurons. However, this manipulation can affect many populations including the preBötC, and the impact of this manipulation on rVRG and phrenic motor neurons (neuron number, cell body position, dendrite orientation, diaphragm innervation etc) is not described, although a model is presented in Fig. 7. These parameters should be analyzed to interpret the functional phenotype.
    7. Additional evidence is needed to support the model that a selective loss of excitatory rVRG to phrenic motor neuron connectivity underlies the reduced bursting activity phenotype in NMNΔ6910-/- mice, for instance by labeling the connections from rVRG to phrenic MNs and quantifying connectivity.

  7. Version published to 10.1101/2022.08.02.502511v1 on bioRxiv
  8. Version published to 10.1101/2022.08.02.502511v2 on bioRxiv