Desmosomal connectomics of all somatic muscles in an annelid larva

  1. Sanja Jasek
  2. Csaba Verasztó
  3. Emelie Brodrick
  4. Réza Shahidi
  5. Tom Kazimiers
  6. Alexandra Kerbl
  7. Gáspár Jékely

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    Evaluation Summary:

    This paper is based on digital reconstruction of a serial EM stack of a larva of the annelid Platynereis and presents a complete 3D map of all desmosomes between somatic muscle cells and their attachment partners. This resource is of interest to scientists in several fields: motor control, high-resolution anatomy, and network analyses. With the first comprehensive and complete mapping of muscle-to-body connectivity through desmosomes in an annelid larva, it has the potential to close a missing link and make progress towards understanding in a "holistic" way how a complex neural circuitry controls an equally complex pattern of movement/behavior.

    (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

Cells form networks in animal tissues through synaptic, chemical, and adhesive links. Invertebrate muscle cells often connect to other cells through desmosomes, adhesive junctions anchored by intermediate filaments. To study desmosomal networks, we skeletonised 853 muscle cells and their desmosomal partners in volume electron microscopy data covering an entire larva of the annelid Platynereis . Muscle cells adhere to each other, to epithelial, glial, ciliated, and bristle-producing cells and to the basal lamina, forming a desmosomal connectome of over 2000 cells. The aciculae – chitin rods that form an endoskeleton in the segmental appendages – are highly connected hubs in this network. This agrees with the many degrees of freedom of their movement, as revealed by video microscopy. Mapping motoneuron synapses to the desmosomal connectome allowed us to infer the extent of tissue influenced by motoneurons. Our work shows how cellular-level maps of synaptic and adherent force networks can elucidate body mechanics.

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  1. Version published to 10.7554/elife.71231 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    This paper is based on digital reconstruction of a serial EM stack of a larva of the annelid Platynereis and presents a complete 3D map of all desmosomes between somatic muscle cells and their attachment partners, including muscle cells, glia, ciliary band cells, epidermal cells and specialized epidermal cells that anchor cuticular chaetae (chaetal follicle cells) and aciculae (acicular follicle cells). The rationale is that the spatial patterning of desmosomes determines the direction of forces exerted by muscular contraction on the body wall and its appendages will determine movement of these structures, which in turn results in propulsion of the body as part of specific behaviors.

    To go a step further, if connecting this desmosome connectome with the (previously published) synaptic connectome, one may gain insight into how a specific spatio-temporal pattern of motor neuron activity will lead, via a resulting pattern of forces caused by muscles, to a specific behavior. In the authors' words: "By combining desmosomal and synaptic connectomes we can infer the impact of motoneuron activation on tissue movements".This is an interesting idea which has the potential to make progress towards understanding in a "holistic" way how a complex neural circuitry controls an equally complex behavior. The analysis of the EM data appears solid; the authors can show convincingly that desmosomes can be resolved in their EM dataset; and the technology used to plot and analyze the data is clearly up to the task. My main concern is with the way in which the desmosome pattern is entered in the analysis, which I think makes it very difficult to extract enough relevant information from the analysis that would reach the stated goal.

    1. The context of how different structures of the Platynereis larval body, by changing their position, move the body needs much more introduction than the short paragraph given at the end of the Introduction.

    -My understanding is that the larval body is segmented, and contraction of the segments can cause a certain type crawling or swimming: does it? Do the longitudinal muscles, for example, insert at segment boundaries, and alternating contraction left-right cause some sort of "wiggling" or peristalsis?

    Longitudinal muscles do not insert only at segment boundaries, but have desmosomal connections along the entire length of the cell. Individual longitudinal muscle cells can span up to 3 segments. However the cells are staggered in such a way that all longitudinal muscle cells with somas in one segment can collectively cover up to 4 segments. Longitudinal muscles are involved in turning when swimming (Randel et al., 2014). The undulatory trunk movements and parapodial walking movements are due to the contraction of oblique and parapodial muscles. The longitudinal muscles provide support during crawling (via desmosomal links) but it is unlikely that these muscles contract segmentally. Disentangling the distinct contributions of 53 types of muscles during crawling will require further studies.

    -In addition, there are segmental processes (parapodia, neuropodia), and embedded in them are long chitinous hairs (Chaetae, Acicula). Do certain types of the muscles described in the study insert at the base of the parapodia/neuropodia (coming from different angles), such that contraction would move the entire process, including the chaetae/acicula embedded in their tips?

    Yes, acicular muscles insert at the proximal base of the acicula, and by moving the acicula they move the entire noto-/neuropodia. We have presented the anatomy of all acicular and chaetal muscles types in the figures and videos.

    -Or is it that only these chaetae/acicula move, by means of muscles inserting at their base (the latter is clearly part of the story)? Or does both happen at the same time: parapodium moves relative to the trunk, and chaeta/acicula moves relative to the parapodium? How would these movements lead to different kind of behaviors?

    -Diagrams should be provided that shed light on these issues.

    We have extended Video2 to show individual muscles and their relation to the aciculae in one of the parapodia. We also clarified this in the text:

    “Several acicular muscles attach on one end to the proximal base of the aciculae and on the other end to the paratrochs and epidermal cells. Oblique muscles attach to the basal lamina, epidermal and midline cells at their proximal end, run along the anterior edge of parapodia and attach to epidermal and chaetal follicle cells at their distal tips. Both of these muscle groups are involved in moving the entire parapodium. Acicular muscles move the proximal tips of the aciculae, while oblique muscles move the parapodium by moving the tissue around the chaetae and the aciculae. All acicular movements also correspond to parapodial movements. Chaetae are embedded in the parapodium and therefore move with it, but the chaetal sac muscles can also independently retract the chaetae into the parapodium or protract them and make them fan out.”

    1. The main problem I have with the analysis is the way a muscle cell is treated, namely as a "one dimensional" node, rather than a vector.

    -In the current state of the analysis, the authors have mapped all desmosomes of a given muscle cell to its attached "target" cell. But how is that helpful? The principal way a muscle cell acts is by contracting, thereby pulling the cells it attaches to at its two end closer together. As the authors state (p.4) "...desmosomes..are enriched at the ends of muscle cells indicating that these adhesive structures transmit force upon muscle-cell contraction."

    At the level of the current analysis our data reveal which cells may be moved by the contractions of the individual muscle cells. The reviewer is right that treating a muscle as a vector (or set of vectors) would be a more accurate description, which would potentially also open up the possibility of computational modelling. We have provided such a vectorised dataset in the revised version, where each muscle-cell skeleton is subdivided into short linear segments (Figure2–source–data 2). This dataset may be useful to approach the problem with a three dimensional approach, which is beyond the scope of the current analysis. We also included an additional video (Video 7) showing examples of muscles and their partners where the cells and the desmosomes connecting them are highlighted. This reveals that the desmosomes connecting two cells are often at the very end of the muscle cell.

    -for that reason, the desmosomes at the muscle tips have to be treated as (2) special sets. Aside from these tip desmosomes there are other desmosomes (inbetween muscles, for example), but they (I would presume) have a very different function; maybe to coordinate muscle fiber contraction? Augment the force caused by contraction?

    Desmosomes between muscles only occur between muscles of different types, not for homotypic connections. There are other types of junctions (adhaerens-like junctions) that connect individual cells of a muscle bundle together (not analysed here). We clarified this in the text.

    • As far as I understand for (all of) the desmosome connectome plots, there is no differentiation made between desmosome subsets located at different positions within the muscle fiber. I therefore don't see how the plots are helpful to shed light on how the multiplicity of muscles represented in the graphs cause specific types of neurons.

    We would like to point out that the cells and structures that muscles connect to via desmosomes are very likely the parts of the body that will move during the contraction of the muscle or will provide structural support (e.g. basal lamina) for the muscle cell to contract. This is most evident in the parapodial complex. The majority of muscles in the body connect to the aciuclar folliclecells and the aciculae are the most actively moving parts in the body during crawling (see Video 4). In any case, since we provide all skeleton reconstructions and the xyz coordinates of all desmosomes, the data could be further analysed following these suggestions by the reviewer.

    • As it stands these plots "merely" help to classify muscles, based on their position and what cell type they target: but that (certainly useful) map could have probably also be achieved by light microscopic analysis.

    This has never been achieved by light microscopy analysis in the hundreds of papers on invertebrate muscle anatomy (e.g. by phalloidin staining). For an LM analysis, it would not be sufficient to label the muscle fibres, but one would also need to label the desmosomes and a multitude of non-muscle cell types including the extent of their cytoplasm. This is technically very challenging (we would nevertheless be happy to hear specific suggestions for markers etc. from the Reviewer). Currently, only EM provides the required depth of structural information and resolution. This is why we believe that our dataset and analysis is unique, despite over a century of research in invertebrate anatomy.

    1. Section "Local connectivity and modular structure of the desmosomal connectome" p.4-7" undertakes an analysis of the structure of the desmosome network, comparing it with other networks.

    -What is the rationale here? How do the conclusions help to understand how the spatial pattern of muscles and their contraction move the body?

    We hope that our analysis may also be of interest to the community of network scientists and we believe that the reconstruction of a quite large and novel type of biological network warrants a more quantitative network analysis, using the standard methods and measures of network science – as we presented e.g. in Figure 4 – even if these mathematical analyses may not directly reveal how muscles move the body. We hope that some readers with an interest in quantitative analyses will also appreciate the broader picture here.

    -Isn't, on the one hand (given that position of the desmosome was apparently not considered), the finding that desmosome networks stand out (from random networks) by their high level of connectivity ("with all cells only connecting to cells in their immediate neighbourhood forming local cliques") completely expected?

    We disagree that the result was completely expected. Even if this was the case, we think it is quite different to say that a result is expected or to thoroughly quantify certain parameters and mathematically characterise key properties of the desmosomal graph (as we have done). These network analyses help to conceptualise our findings and to think about the muscle system in more global, whole-body terms.

    -On the other hand, does this reflect the reality, given that (many?) muscle cells are quite long, connecting for example the anterior border of a segment with the posterior border.

    Indeed, a quantitative analysis helped us to identify cases where the reality deviated somewhat from what was completely expected, and we thank the reviewer for these comments. As we explain in the revised version, some longitudinal muscles show an unexpected position in the force-field layout of the graph, due to their long-range connections. We have added extra clarifications to the text: “To analyse how closely the force-field-based layout of the desmosomal connectome reflects anatomy, we coloured the nodes in the graph based on body regions (Figure 5). In the force-field layout, nodes are segregated by body side and body segment. Exceptions include the dorsolateral longitudinal muscles (MUSlongD) in segment-0. These cells connect to dorsal epidermal cells that also form desmosomes with segment-1 and segment-2 MUSlongD cells. These connections pull the MUSlongD_sg0 cells down to segment-2 in the force-field layout (Figure 5D).”

    1. In the section "Acicular movements and the unit muscle contractions that drive them" the authors record movement of the acicula and correlate it with activity (Ca imaging) of specific muscle types. This study gives insightful data, and could be extended to all movements of the larva.

    -The fact that a certain muscle is active when the acicula moves in a certain direction can be explained (in part) by the "connectivity": as shown in Fig.7L, the muscle inserts at a acicular follicle cell on the one side, and to an epithelial (epidermal?) cell and the basal lamina on the other side. But how meaningful is a description at this "cell type level" of resolution? The direction of acicula deflection depends on where (relative to the acicula base) the epithelial cell (or point in the basal lamina) is located. This information is not given in the part of the connectome network shown in Fig.7L, or any of the other graphs.

    This information is indeed not shown in the graphs, where each cell is treated as a node. However, we provide this information in the detailed anatomical figures in Figure 6 – figure supplement 1-3 and Video 7, where the individual acicular and oblique muscle types are visualised. In principle, one could subdivide aciculae into e.g. proximal and distal halves and derive a more detailed network. We have not done this but since all the EM, anatomical rendering and connectivity data are available in our public CATMAID server (https://catmaid.jekelylab.ex.ac.uk/), we hope that the interested readers will be able to further analyse the data.

    We renamed ‘epithelial’ cells to ‘epidermal’ cells.

  3. Version published to 10.1101/2021.06.09.447732v2 on bioRxiv
  4. eLife

    Evaluation Summary:

    This paper is based on digital reconstruction of a serial EM stack of a larva of the annelid Platynereis and presents a complete 3D map of all desmosomes between somatic muscle cells and their attachment partners. This resource is of interest to scientists in several fields: motor control, high-resolution anatomy, and network analyses. With the first comprehensive and complete mapping of muscle-to-body connectivity through desmosomes in an annelid larva, it has the potential to close a missing link and make progress towards understanding in a "holistic" way how a complex neural circuitry controls an equally complex pattern of movement/behavior.

    (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.)

  5. eLife

    Reviewer #1 (Public Review):

    The manuscript by Jasek et al. uses serial electron microscopy to reconstruct all 852 somatic muscle cells and their desmosomal inter-connectivity and connections to non-muscle cells and extra-cellular matrix structures in a 3-segmented larva of the nereidid Platynereis dumerilii. The study is complementary to a previous report by the same research group on the whole-body neuronal connectome using the same specimen. It is among the first studies to present a thorough and detailed analysis of the full complement of larval muscle cells and their fixations on a whole-body level and classifies them based on their morphological location and local inter-connectivity. The authors' choice to highlight the muscles around the acicula is highly justified given the pivotal role of acicula as endoskeletal anchor points for a highly diverse and complex set of muscles, thus important to understand the muscle movements controlling crawling behaviour. The authors use the morphological positions and connectivity of muscle groups to infer their role in moving acicula during crawling movements. A weakness of the manuscript is that it is quite difficult to follow how the authors inferred these acicular movements mainly due to the difficulty to represent spatial and temporal changes in a two-dimensional way. In addition, these inferred movements could only be directly tested for a small subset of muscles due to technical limitations in imaging the activity of worm muscles during locomotion. A thorough analysis of muscle functions seems however currently impossible due to technical limitations of in vivo calcium imaging of moving animals, and the complexity and speed of the muscular activities occurring during crawling movements. Altogether, the current manuscript forms a comprehensive, detailed and thorough basis for future studies aiming towards an understanding of locomotor activity from both neuronal and muscular perspectives on a whole-body level.

  6. eLife

    Reviewer #2 (Public Review):

    Attachment of muscles to exo- or endoskeleton by desmosomes and hemidesmosomes is necessary for invertebrates to transform the muscle force into body movement. Jasek et al. fully reconstructed these functional connectivity units in a 3-day annelid larva by serial electron microscopy. The final dataset includes desmosomes from 852 muscles to all non-neuromuscular cells, as well as desmosomes that connect these non-neuromuscular cells.

    The authors showed that this desmosome connectivity matrix exhibits a highly organized local community structure, and this structure matches the physical organizations of involved cells and desmosomes.

    The authors attempt to relate structures to functions. A module with the highest membership diversity that reflects an anatomical organization is the interacicular muscle complex. The connectivity map at this complexity allows the authors to infer this structure's capacity for diverse motor patterns. They verified some of these motor functions by DIC imaging and muscle calcium imaging.

    Strengths:

    1. This is an impressive trove of data, essentially an annelid anatomical atlas on how muscles connect to the body. It provides the only complete dataset for non-neuronal tissue's connection to the neuromuscular system that underline body movement.

    2. Quantitative assessment and description of the demosome network, which defines parameters to highlight its organizational difference from that of the neuromuscular system.

    3. Generation of testable hypotheses for possible motor patterns to be produced from the hub of the desmosome connectome. Some, such as the independent and coupled movement of the notopodial and neurpdodial aciculae, were corroborated with behavioral and muscle imaging data.

    Weakness:

    Behavioral and behavioral calcium imaging is at a fairly early stage, due to understandable technical challenges, such as tracking 3D movements, discerning origins of cytosolic calcium signals from a large population of muscle cells, and the imaging preparation's difficulty to perform nature movement.

  7. eLife

    Reviewer #3 (Public Review):

    This paper is based on digital reconstruction of a serial EM stack of a larva of the annelid Platynereis and presents a complete 3D map of all desmosomes between somatic muscle cells and their attachment partners, including muscle cells, glia, ciliary band cells, epidermal cells and specialized epidermal cells that anchor cuticular chaetae (circumchaetal cells) and aciculae (circumacicular cells). The rationale is that the spatial patterning of desmosomes determines the direction of forces exerted by muscular contraction on the body wall and its appendages will determine movement of these structures, which in turn results in propulsion of the body as part of specific behaviors.

    To go a step further, if connecting this desmosome connectome with the (previously published) synaptic connectome, one may gain insight into how a specific spatio-temporal pattern of motor neuron activity will lead, via a resulting pattern of forces caused by muscles, to a specific behavior. In the authors' words: "By combining desmosomal and synaptic connectomes we can infer the impact of motoneuron activation on tissue movements". This is an interesting idea which has the potential to make progress towards understanding in a "holistic" way how a complex neural circuitry controls an equally complex behavior. The analysis of the EM data appears solid; the authors can show convincingly that desmosomes can be resolved in their EM dataset; and the technology used to plot and analyze the data is clearly up to the task. My main concern is with the way in which the desmosome pattern is entered in the analysis, which I think makes it very difficult to extract enough relevant information from the analysis that would reach the stated goal.

    1.The context of how different structures of the Platynereis larval body, by changing their position, move the body needs much more introduction than the short paragraph given at the end of the Introduction.
    -My understanding is that the larval body is segmented, and contraction of the segments can cause a certain type crawling or swimming: does it? Do the longitudinal muscles, for example, insert at segment boundaries, and alternating contraction left-right cause some sort of "wiggling" or peristalsis?
    -In addition, there are segmental processes (parapodia, neuropodia), and embedded in them are long chitinous hairs (Chaetae, Acicula). Do certain types of the muscles described in the study insert at the base of the parapodia/neuropodia (coming from different angles), such that contraction would move the entire process, including the chaetae/acicula embedded in their tips?
    -Or is it that only these chaetae/acicula move, by means of muscles inserting at their base (the latter is clearly part of the story)? Or does both happen at the same time: parapodium moves relative to the trunk, and chaeta/acicula moves relative to the parapodium? How would these movements lead to different kind of behaviors?
    -Diagrams should be provided that shed light on these issues.

    2.The main problem I have with the analysis is the way a muscle cell is treated, namely as a "one dimensional" node, rather than a vector.
    -In the current state of the analysis, the authors have mapped all desmosomes of a given muscle cell to its attached "target" cell. But how is that helpful? The principal way a muscle cell acts is by contracting, thereby pulling the cells it attaches to at its two end closer together. As the authors state (p.4) "...desmosomes..are enriched at the ends of muscle cells indicating that these adhesive structures transmit force upon muscle-cell contraction."
    -for that reason, the desmosomes at the muscle tips have to be treated as (2) special sets. Aside from these tip desmosomes there are other desmosomes (inbetween muscles, for example), but they (I would presume) have a very different function; maybe to coordinate muscle fiber contraction? Augment the force caused by contraction?
    -As far as I understand for (all of) the desmosome connectome plots, there is no differentiation made between desmosome subsets located at different positions within the muscle fiber. I therefore don't see how the plots are helpful to shed light on how the multiplicity of muscles represented in the graphs cause specific types of neurons.
    -As it stands these plots "merely" help to classify muscles, based on their position and what cell type they target: but that (certainly useful) map could have probably also be achieved by light microscopic analysis.

    3.Section "Local connectivity and modular structure of the desmosomal connectome" p.4-7" undertakes an analysis of the structure of the desmosome network, comparing it with other networks.
    -What is the rationale here? How do the conclusions help to understand how the spatial pattern of muscles and their contraction move the body?
    -Isn't, on the one hand (given that position of the desmosome was apparently not considered), the finding that desmosome networks stand out (from random networks) by their high level of connectivity ("with all cells only connecting to cells in their immediate neighbourhood forming local cliques") completely expected?
    -On the other hand, does this reflect the reality, given that (many?) muscle cells are quite long, connecting for example the anterior border of a segment with the posterior border.

    4.In the section "Acicular movements and the unit muscle contractions that drive them" the authors record movement of the acicula and correlate it with activity (Ca imaging) of specific muscle types. This study gives insightful data, and could be extended to all movements of the larva.
    -The fact that a certain muscle is active when the acicula moves in a certain direction can be explained (in part) by the "connectivity": as shown in Fig.7L, the muscle inserts at a circumacicular cell on the one side, and to an epithelial (epidermal?) cell and the basal lamina on the other side. But how meaningful is a description at this "cell type level" of resolution? The direction of acicula deflection depends on where (relative to the acicula base) the epithelial cell (or point in the basal lamina) is located. This information is not given in the part of the connectome network shown in Fig.7L, or any of the other graphs.

  8. Version published to 10.1101/2021.06.09.447732v1 on bioRxiv