RhoA and microtubule dynamics control cell–basement membrane interaction in EMT during gastrulation

There is a quite nice paper (full reference and abstract at the end of the post) I would like to present, a little bit more explicitly than usual. Most of the time I’m just dropping over here the references of papers I’m reading during coffee breaks, and sometimes there is a backstage discussion about them (people, comments on blogs are for discussions, please use them at least occasionally instead of e-mail ;-) ).

Nakaya_f8wol.jpg

At least two reasons to do so:

  • A great description, at the cellular and molecular levels, of the epithelial-mesenchymal transition, showcased at the vicinity of the primitive streak of avian gastrulas. For those don’t following my blogs I should note here that I became interested on avian gastrulas a little bit more than a year and a half, during a web discussion with the author of An Elasto-Plastic Model of Avian Gastrulation.
  • It is also a great illustration of how detailed data, at the finest available level, may be used to describe a biological phenomenon, and be used to build a model, thoroughly tested, step-by-step.

    Nakaya et al. investigate the order and regulation of epithelial-mesenchymal transition (EMT) cellular events during naturally occurring EMT in the primitive streak of gastrulating chick embryos, show that basal-membrane (BM) disassembly is the key element of this EMT and elucidate at the molecular level the intracellular mechanism leading to BM breakdown preceding cell ingression in the primitive streak.

    First, the authors observed that BM breakdown is the first recognizable event, before loss of adherens and tight junctions and apical polarity markers, of the EMT.

    Then, they investigate the signaling events involved in this process, by:

  • electroporating RhoA in medial epiblasts, which cause ingression defects characterized by laminin retention (a constituent of BM) and of integrin (a receptor that binds laminin),
  • observing that RhoA localization and activity is enriched in lateral epiblast cells, apically and basally, in comparison to medial cells, were basal RhoA localization and activity were lost,
  • found that Net1, a RhoA guanine nucleotide-exchange factor (GEF), is downregulated in cells fated to undergo EMT, that is in the medial region.

    How can RhoA promote BM stabilization? What controls Net1 basal localization? Which signal downregulates Net1 before EMT?

    RhoA’s influence on cytoskeleton organization are pleiotropic, so they dissected the system using cytoskeleton dynamics modifying drugs, and showed that microtubules are implicated in BM stabilization and that their intracellular organization define two populations, one at the apical region, constituted of a dense, stable meshwork (containing detyrosinated and acetylated tubulin), and a less stable, basal one, lost in medial cells.
    In lateral epiblast cell, they observed a subpopulation of microtubules revealed by an anti-ß-tubulin antibody, restricted basally, lost in medial cells undergoing EMT. This subpopulation being stabilized by ectopic expression of RhoA, suggesting that RhoA controls it’s presence.

    So: BM disassembly is the starting point of EMT.

    1. BM’s stability depends on a basal subpopulation of microtubules (6G7+)
    2. the stability of these 6G7+ microtubules is under control of basal RhoA
    3. basal RhoA’s activity is under control of Net1
    4. Net1 is downregulated in medial cells.

    What we have here is an elegant model explaining the onset of EMT in vivo. It will be interesting to see how these events are entangled/coordinated with the subsequent/concomitant regulation of adherens junctions.


    Now, I will not really try to explain why Nakaya’s et al. way of dealing with the question of EMT is great. Read the paper, follow the step-by-step dissection of the system, if you are not convinced I suppose you are lost and there is no much one could do for you :-) . I suppose the paper offers a great deal of material for a course on development, demonstrating how events at the cellular and subcellular levels come in play in one key developmental process.

    Let’s go back to the EPMAG proposed by Fleury, who proposed a mechanical crack of the BM. The model was greatly scratched by the publication of evidence of prepatterning of the primitive streak prior to gastrulation, which Fleury supposed to be the propagation of the crack of the BM, in a P->A direction, due to tearing apart of the BM by the epiblastic cells.
    Now we have also evidence that the BM disassembly is a cellular process driven by rearrangements of the cytoskeleton, due to RhoA local activity loss, directed by Net1 downregulation.

    So, the crack isn’t a mechanical one as proposed and the path of the primitive steak extension isn’t a crack’s propagation. The two initial steps of EPMAG don’t stand anymore and the model can be abandoned without any regret, as a curiosity born by poor understanding of the cellular and molecular mechanisms in play.

    What I would like to highlight, as relevant to a few discussions I had recently about the way to approach biological phenomena, and models/theories building, is the difference between the two approaches. One arrogantly lacking experimental testing, and thus evidence, the other carefully tested, to present facts, in an as much possible detailed way, rather then mere hypotheses, before being published.

    I always have be, and will be, fan of experimentally evidenced models, as I think that this is the way scientists have to work. And as it goes, of models that encompass every known aspect of the phenomenon under study. The last remark will be connected later to at least another mechanical interpretation of a developmental process.

    EPMAG has being abundantly cited by Fleury as a peer reviewed paper in support of histheory. Now, EPMAG doesn’t stand anymore. In a few day there will be the first anniversary of my essays to spot and position L2/R2, two vortices claimed by Fleury to be at the origin of the hind-limb buds, a feature that he failed ’till now to display, despite the fact that he is still working with avian embryos.
    Without the support of the eventuality that the EPMAG could be descriptive of the avian gastrulation (it isn’t) and lack of evidence for one of the main features to explain the tetrapods bodyplan, what remains of the basic on which Fleury based his theory?

    While discussing it with Tim and Tom, we remained at the « time will tell » position. Time passed, less than a year, and the new data allows us to have a much more evidence supported position. I’ll be happy to hear from Tim and Tom and eventually Fleury himself (for VF a reminder: comments are welcome as far as a copy is addressed to his labs head, a cc’d e-mail will do as evidence)


    RhoA and microtubule dynamics control cell–basement membrane interaction in EMT during gastrulation

    Yukiko Nakaya, Erike W. Sukowati, Yuping Wu and Guojun Sheng

    Molecular and cellular mechanisms of epithelial–mesenchymal transition (EMT), crucial in development and pathogenesis, are still poorly understood. Here we provide evidence that distinct cellular steps of EMT occur sequentially during gastrulation. Basement membrane (BM) breakdown is the first recognizable step and is controlled by loss of basally localized RhoA activity and its activator neuroepithelial-transforming-protein-1 (Net1). Failure of RhoA downregulation during EMT leads to BM retention and reduction of its activity in normal epithelium leads to BM breakdown. We also show that this is in part mediated by RhoA-regulated basal microtubule stability. Microtubule disruption causes BM breakdown and its stabilization results in BM retention. We propose that loss of Net1 before EMT reduces basal RhoA activity and destabilizes basal microtubules, causing disruption of epithelial cell–BM interaction and subsequently, breakdown of the BM.

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  • , , , , , , , , ,

    1. #1 par agathi le août 14, 2008 - 3:02

      :-) & :-*

    2. #2 par Oldcola le août 15, 2008 - 9:28

      :-P

    1. L2/R2, un an déjà… « Coffee and Sci(ence)

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