Transcriptomic and genetic analyses identify the Krüppel-like factor dar1 as a master regulator of tube-shaped long tendon development

  1. Laurichesse Quentin
  2. Moucaud Blandine
  3. Jagla Krzysztof
  4. Soler Cédric

  • Curated by eLife

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    Evaluation Summary:

    The paper is of interest for those studying tendon development. Starting from a transcriptomic analysis of Drosophila leg tendon cells it identifies a transcription factor, Dar1, required for normal tendon morphogenesis.

    (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. Reviewer #1 agreed to share their name with the authors.)

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Abstract

To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Despite evident disparities, little is known about the molecular basis of tendon diversity. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. We provide for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, we identified the Krüppel-like factor dar1 as a critical actor of leg tendon development. Specifically expressed in leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. Our findings show that dar1 acts downstream of stripe as a critical regulator of cytoskeleton remodeling and mediates the recruitment of new stripe-positive tendon progenitors in a cell non-autonomous manner.

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  1. eLife

    Evaluation Summary:

    The paper is of interest for those studying tendon development. Starting from a transcriptomic analysis of Drosophila leg tendon cells it identifies a transcription factor, Dar1, required for normal tendon morphogenesis.

    (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. Reviewer #1 agreed to share their name with the authors.)

  2. eLife

    Reviewer #1 (Public Review):

    The well studied tendons in the fly embryo are formed by rather simple specialized epithelial cells that stay in the epithelium and attract the end of an attaching myotube. This study investigates the more complex 'long tendons' of the adult legs that form in the leg discs during pupal stages. The authors find that the long tendon precursors undergo an interesting collective migration process to re-shape into a higher order long tub-like structure. This elongation process had not been studied before.

    By performing a transcriptomics analysis of sorted leg disc tendon precursors (0h APF), followed by systematic RNAi knock-down studies of all transcription factors enriched in the tendon precursors, the authors identify the Kr-like transcriptional regulator dar1 as key regulator of long-tendon development. RNAi specificity was confirmed by analyzing 2 different lines both resulting in locomotion defects and by confirming the tendon defect by the analysis of dar1 null alleles that rarely survive until pupal stages.

    Dar1 protein is enriched in stripe positive tendon precursors of the leg discs (but not in the tendons of the wing disc). Dar1 knockdown animals show largely missing or too short internal tendons in the legs, hence strongly affecting the attachment of the adult leg muscles. This explains the observed locomotion defects of these flies.

    Excellent imaging during early stages of pupal development shows that dar1 is required for the extension of the long leg tendon precursors into a long tube. Mechanistically, dar1 organizes actin-rich long filopodial extensions during tube elongation and hence equips these cells specifically with their collective migration ability to form a long tube.

    Epistasis experiments show that dar1 acts downstream of the tendon master regulator stripe, making long tendons different from other tendons. Interestingly, the amount of stripe positive tendons cells is reduced upon dar1 knock-down, resulting in the hypothesis that dar1 is needed to recruit and specify the correct number of stripe positive tendons cells to form the larger long tendons in a non cell autonomous manner. Such a model makes sense, as likely the invagination and migration of tendon precursors into a long tube will recruit additional epithelial cells to turn on stripe and to follow the invagination as nicely illustrated in the model in figure 8.

    I am not sure if the term 'master regulator' for dar1 for the long tendons is justified. Do the authors have access to a UAS-dar1 line to see if 'normal' tendons in the wing disc or the embryo can be changed to 'long tendons' when expressing dar1? If not, the term tendon identity gene might be more appropriate.

    It would have been nice to include Dar1 antibody staining of dar1 know-down leg to confirm antibody specificity.

    Together, this is an excellent study that genetically and molecularly characterizes a novel gene controlling an interesting morphogenesis process that was not well understood. As these complex long tendons are somewhat similar to mammalian tendons, these findings could also be relevant for mammalian tendon development and diversification.

  3. eLife

    Reviewer #2 (Public Review):

    In their manuscript titled "Transcriptomic and genetic analyses identify the Krüppel-like factor dar1 as a master regulator of tube-shaped long tendon development," Quentin et al. identify Dar1 as a novel transcriptional regulator of tendon development. They present the first transcriptomic profiling of Stripe-expressing tendon progenitors in the leg disc, highlighting similarities to well-characterized tubulogenesis systems. After performing a candidate screen of 31 genes identified in this data, they focus on the transcription factor Dar1. Dar1 is selectively expressed in tendon cells of the leg and its knockdown results in both tendon and muscle defects. The long tendons are too short due to insufficient tube extension and decreased filipodial arborization. The number of tendon precursors is also greatly reduced upon knockdown of dar1. As dar1 expression is downstream of Stripe and there is little alteration of cell proliferation and death rates in the leg disc, the authors suggest Stripe positive cells are recruited during long tendon tubulogenesis and this recruitment is non-autonomously regulated by Dar1.

    There are several new observations, approaches and datasets presented in this work that are of value to the muscle field and the broader Drosophila community. First, the FACS-based RNA-Seq approach and associated protocol for tendon cells in leg can be adapted for other imaginal discs, dissectible tissues and cell types. Second, this RNA-seq dataset expands on earlier observations suggesting long-tendon development proceeds via tube-formation and thus is a valid model to study the important process of tubulogenesis. Third, the candidate climbing and survival screen combined with the RNA-Seq data provide multiple candidate genes, and notably transcription factors, that may play a role in tendon specification or development and will be useful for future studies. Lastly, this work identifies and nicely characterizes the tendon phenotype after knockdown of Dar1, ultimately placing Dar1 downstream of Stripe in the regulatory hierarchy. Given the apparent conservation of Krüpel-like factors in tendon development, this finding is of general interest and may point to a conserved mechanism fueling long tendon elongation.

    On a critical note, only select genes of interest and select GO terms are included from the RNA-Seq data, and a broader or more systematic analysis is not presented. While the title refers to Dar1 as a master regulator of tendon development, this is not addressed experimentally. The manuscript is largely descriptive, and while the characterization of Dar1 indeed raises interesting inferences notably about Stripe-positive cell recruitment, there are no experiments designed to test potential mechanisms of non-cell autonomous effects or even regulatory targets of Dar1 in tendon. The manuscript also does not address non-autonomous effects on muscle number, size and morphology in the ventral tibia, for example, and does not effectively place this result in the context of what is known about bi-directional muscle-tendon signaling. Lastly, the authors have previously characterized tendon-cell recruitment in the leg to be Notch dependent, and indeed they find enrichment of the Notch signaling pathway in their RNA-seq analysis, but the findings for Dar1 are not integrated in this context.

    In conclusion, this manuscript identifies a new transcriptional regulator of tendon development and offers an insightful and informative description of the phenotype. It arrives at mechanistic inferences that provide a framework for future studies to identify the non-cell autonomous mechanism of tendon-cell recruitment, to elucidate the regulatory mechanisms of long-tendon extension and tube formation and to investigate the transcriptional hierarchy that defines long-tendon cell identity in Drosophila.

  4. eLife

    Reviewer #3 (Public Review):

    In the introduction the authors frame some key background questions for the study concerning the specificity of muscle-tendon interactions, and the processes governing tendon development. To investigate they have set out to identify genes regulating the development of the long tendons in the Drosophila leg. These share similarities with autopod tendons in mouse and also their morphogenesis is similar to that of some tubule development.

    To investigate, the authors have profiled the RNAs (total RNA transcriptomic analysis) that are enriched in the tendon progenitors in comparison to the limb disc as a whole. From the large number of enriched genes, the consequences of knocking 31 transcription factors whose expression was >1.5-fold higher in the tendon precursors, using a viability assay. Several gave phenotypes and their homologues had been reported to have expression in mouse limb tendons. Ultimately 1 gene was chosen for further analysis, Dar 1, and conventional analysis shows that it is involved in normal tendon development and its expression is dependent on the key tendon regulator stripe. The tendons from Dar1 knock-down animals don't extend as well, have reduced amounts of actin protrusions and have slightly fewer cells. The data demonstrating this provide good evidence that Dar1 plays a role in the development of the tendons and, interestingly is specific for the leg tendons. It's not required in the wing tendons.

    The work is nicely done but it falls short of addressing the questions set. The initial RNA seq data are a good starting point and the analysis of dar1 expression and knock-down are sound. More could have been made of the primary data and the authors would need to include properly the "data not shown" about some of the other genes tested. The basic phenotypic analysis supports the conclusions that Dar1 as is an additional player in long tendon development, but its identification does not significantly add to the understanding of how tendons grow, navigate correctly and interact with the right muscle. What step is Dar1 regulating? There is a suggestion that it may regulate the cytoskeletal genes but there are no concrete data to support that. Could dar1 be involved in specifying leg versus wing tendons (the former being more tube like) given its expression in leg tendons only.

    The results identifying a new gene involved in tendon morphogenesis will be of interest to those directly in the field.

  5. Version published to 10.1101/2021.02.07.430104 on bioRxiv