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A developmental gene regulatory network for invasive differentiation of the C. elegans anchor cell

Taylor N. Medwig-Kinney, Jayson J. Smith, Nicholas J. Palmisano, Wan Zhang, David Q. Matus

Preprint posted on July 03, 2019 https://www.biorxiv.org/content/10.1101/691337v2

Article now published in Development at https://dev.biologists.org/content/early/2019/11/29/dev.185850

Breakthrough: a gene regulatory network coordinates invasion of the basement membrane by the anchor cell in C. elegans

Selected by Sina Knapp

Background

The basement membrane plays an important role in intercellular segmentation of multicellular organisms. Consisting of a deposit extracellular matrix, the basement membrane defines the tissue boundaries by separating epithelial, endothelial and mesothelial tissue from the connective tissue.  Remarkably, there are several physiological occasions when these boundaries are crossed, as it happens during pregnancy, development or an immune response, when cells demonstrate a controlled invasive behavior. In order to invade and cross the basement membrane boundary, the cells need to adhere to the basement membrane, puncture and degrade it in a concerted manner. Interestingly, this invasive potential is known to be related to cell cycle progression, since cell-cycle arrested cells have higher invasive potential than cycling cells. Remarkably, the C. elegans anchor cell (AC) constitutes a very good model for controlled invasion, as the AC breaks through the basement membrane to connect the uterus to the vulval epithelium, promoting egg laying. In the last years, four transcription factors were identified as pro-invasive in post-mitotic ACs: fos-1, nhr-67, hlh-2 and egl-43. This study tackles the question if and how these transcription factors act together to foster invasion. With the use of multiple genetic tools, Medwig-Kinney et al. could not only reveal a detailed network of the four transcription factors but could also refine the idea of a mutual dependence of cell cycle arrest and invasion by identifying an additional sub-circuit in this network promoting cell cycle independent invasion.

 

Key findings

A coherent feed-forward loop regulates AC invasion

With the use of new RNAi targeting vectors, the authors were able to successfully target the four transcription factor fos-1, nhr-67, hlh-2 and egl-42 and identified invasion failure in all four cases ranging from single ACs to multiple clustering ACs, unable to breach the basement membrane.

The authors developed a system by which in a quantitative way, interactions between the transcription factors prior to and during invasion can be followed: CRISPR-Cas9 mediated GFP labeling of the endogenous transcription factors in combination with the use of reporters for the basement membrane (laminin) and the AC (cdh-3) allowed the tracing of expression levels of the four transcription factors, respectively. With RNAi based depletion of a single transcription factor, the expression levels of the other three GFP labeled transcription factors were followed. Depletion of either egl-43 or hlh-2 did phenocopy nhr-67 knock-down by generating multiply ACs, not crossing the basement membrane, compared to fos-1 deletion, leading to a single, non-invasive AC. Interestingly, depletion of egl-42 and hlh-2 resulted in reduction of expression level of nhr-67, implying that egl-43 and hlh-2 act upstream of nhr-67 during AC invasion. The authors could identify a hierarchy, by which the transcription factors egl-43 and hlh-2 positively regulate nhr-67 activity in a feed-forward loop (Fig. 1).

 

Cell cycle arrest is necessary for AC invasion, but it is not sufficient

To understand if the depletion of egl-43 and hlh-2 also functionally phenocopies loss of nhr-67, the authors made additionally use of a cell cycle progression reporter (CDT-1) and could confirm that egl-43 and hlh-2 loss resulted in cycling, proliferative ACs.  fos-1 depletion, however, led to cell cycle arrest with a single, non-invasive AC. These results identified that egl-43, hlh-2 and nhr-67 maintain the AC in a post-mitotic, pro-invasive state. The cell-cycle independent way, in which fos-1 can regulate AC invasion was unknown before.

Further, the authors identified the ability of egl-43 to act cell cycle dependent and independently as restoring egl-43 depleted ACs to a post-mitotic state (through upregulation of the cyclin-dependent kinase inhibitor CKI-1) did stop proliferation but did not rescue invasion, just as in fos-1 depleted ACs. If egl-43 would act just in a cell-cycle dependent way, invasion would have been rescued as in hlh-2 and nhr-67 knockdown ACs upon post-mitotic cell cycle arrest. The confirmed finding that egl-43 is autoregulatory strengthens the high position of egl-43 in the intersection of several pro-invasive regulatory circuits (Fig. 1).

 

 

Fig. 1: Model of the gene regulatory network promoting anchor cell invasion.

 

Why I like this preprint

The work of Medwig-Kinney et al. is an elegant study that contributes to the understanding of controlled invasion in vivo. By integrating cutting-edge technologies and genetic tools, the authors were able to trace and quantitate the consequences of the loss of pro-invasive transcription factors. The authors not only suggested a detailed network of the four transcription factors acting in a feed-forward circuit with positive feedback but additionally, they discovered a sub-circuit that promotes cell cycle independent invasion. Further, by clarifying the hierarchy of the transcription factors, they identified egl-43 functioning in both cell cycle -dependent and -independent sub-circuits to coordinate invasion. Globally, this study provides a novel link between regulatory circuits and cellular outputs and elicits a new concept how classical cellular machinery can be used for tuning invasiveness.

 

Questions

  • Which invasion failure phenotypes would you expect following a depletion of several transcription factors simultaneously (e.g. egl-43 and hlh-2)?
  • Recent findings connect the expression of the discussed transcription factors with e.g. the regulation of adhesion proteins used for the attachment of the anchor cell to the basement membrane; are the transcription factors also linked to the establishment of F-actin rich invadosomes for punctating the basement membrane?

 

Literature

Kelley, L. C., Lohmer, L. L., Hagedorn, E. J., & Sherwood, D. R. (2014). Traversing the basement membrane in vivo: a diversity of strategies. The Journal of Cell Biology, 204(3), 291–302.

Matus, D. Q., Chang, E., Makohon-Moore, S. C., Hagedorn, M. A., Chi, Q., & Sherwood, D. R. (2014). Cell division and targeted cell cycle arrest opens and stabilizes basement membrane gaps. Nature Communications, 5(1), 4184.

Medwig, T. N., & Matus, D. Q. (2017). Breaking down barriers: the evolution of cell invasion. Current Opinion in Genetics & Development, 47, 33–40.

Sherwood, D. R., & Plastino, J. (2018). Invading, Leading and Navigating Cells in Caenorhabditis elegans: Insights into Cell Movement in Vivo. Genetics, 208(1), 53–78.

Bodofsky, S., Liberatore, K., Pioppo, L., Lapadula, D., Thompson, L., Birnbaum, S., … Wightman, B. (2018). A tissue-specific enhancer of the C. elegans nhr-67/tailless gene drives coordinated expression in uterine stem cells and the differentiated anchor cell. Gene Expression Patterns, 30, 71–81.

 

 

 

Posted on: 5th August 2019 , updated on: 11th December 2019

doi: https://doi.org/10.1242/prelights.12884

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  • Author's response

    David Matus shared

    Thanks Sina for writing this lovely preLight! We wanted to chime in on your questions you posed at the end.

    Q1: Which invasion failure phenotypes would you expect following a depletion of several transcription factors simultaneously (e.g. egl-43 and hlh-2)?

    Great question and it’s an experiment we’d love to do! Although double RNAi experiments do work in our system, what would be better would be to combine targeted genetic depletions such as either using the GFP-nanobody/ZF-tagging and/or auxin-mediated degradation. However, our RNAi penetrance when we deplete egl-43 is nearly a 100% defect in AC invasion, so there’s not much room to make the phenotype any worse if we could co-deplete egl-43 and hlh-2. What would be interesting would be to co-deplete hlh-2 and fos-1 or nhr-67 and fos-1 as we would expect then that we would perturb both pathways – the cell cycle-dependent and independent pathways that regulate invasion, as loss of nhr-67 would result in mostly cycling ACs and fos-1 is required in cell cycle arrested ACs to activate MMPs and other pro-invasive machinery to penetrate the underlying basement membrane.

    Q2: Recent findings connect the expression of the discussed transcription factors with e.g. the regulation of adhesion proteins used for the attachment of the anchor cell to the basement membrane; are the transcription factors also linked to the establishment of F-actin rich invadosomes for punctating the basement membrane?

    So this is also a great question – and ultimately what we’re really interested in doing – connecting the GRN to the underlying cell biology of invasion! Right now we haven’t identified any direct connections between TFs and F-actin polarization/activity, though we expect they’re out there! The beauty of the system is the ability to do genetic screens, so we’re looking forward to designing the right screen to ID a regulatory relationship between a pro-invasive TF and the generation of invadopodia. We know that invadopodia are only generated when the AC is in G1/G0 and that they are still generated in fos-1-deficient ACs, but that they fail to breach the basement membrane, which suggests that fos targets are required for delivering MMPs and other BM-degrading factors to the basement membrane to generate the initial breach, though MMPs are NOT required for the AC to invade, which is really extraordinary (see recent Dev Cell paper from the Sherwood lab, https://www.ncbi.nlm.nih.gov/pubmed/30686527)

     

    Update from David Matus (28 October 2019)

    Here’s the updated summary figure explaining our new findings.

    @TMedwigKinney @JayJavSmith and the rest of us were surprised to find that the GRN that mediates C. elegans AC invasion only functions after the AC has been specified, even though 3/4 TFs are reiteratively used in both specification and invasion.

    1 comment

    4 months

    Taylor Medwig-Kinney

    Hi, Sina. Thank you again for highlighting our preprint, which has now been accepted for publication at Development. As you will notice, the scope of our manuscript has expanded quite a bit following our initial submission. This was in response to the peer review process, as all three of our reviewers were interested in the temporal aspect of the gene regulatory network we described. In order to address this new point, we first examined onset of transcription factor expression in the anchor cell ancestors using endogenous GFP reporters. We were excited to see that the order of transcription factor onset (EGL-43, HLH-2, NHR-67) was consistent with the feed-forward loop hierarchy we demonstrated as functioning at the time of anchor cell invasion. We then sought to test if these three transcription factors, which each regulate the Notch-dependent cell fate decision through which the anchor cell is specified, exhibit the same network topology prior to anchor cell specification. After performing molecular epistasis experiments, we were surprised to find that EGL-43, HLH-2, and NHR-67 do not regulate each other’s expression prior to anchor cell specification. These results suggest that the gene regulatory network is assembled post-specification, possibly due to changes in chromatin state or gene expression between the pre- and post-differentiated anchor cell. We are excited to share these new data, which we believe have improved our manuscript by highlighting how dynamic gene regulatory networks can be, and are thankful for our anonymous reviewers who inspired these experiments.

    3

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