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Paracrine regulation of neural crest EMT by placodal MMP28 [1]

['Nadège Gouignard', 'Molecular Cellular', 'Developmental Biology Department', 'Mcd', 'Centre De Biologie Intégrative', 'Cbi', 'Université De Toulouse', 'Cnrs', 'Ups', 'Toulouse']

Date: 2023-08

Epithelial–mesenchymal transition (EMT) is an early event in cell dissemination from epithelial tissues. EMT endows cells with migratory, and sometimes invasive, capabilities and is thus a key process in embryo morphogenesis and cancer progression. So far, matrix metalloproteinases (MMPs) have not been considered as key players in EMT but rather studied for their role in matrix remodelling in later events such as cell migration per se. Here, we used Xenopus neural crest (NC) cells to assess the role of MMP28 in EMT and migration in vivo. We show that a catalytically active MMP28, expressed by neighbouring placodal cells, is required for NC EMT and cell migration. We provide strong evidence indicating that MMP28 is imported in the nucleus of NC cells where it is required for normal Twist expression. Our data demonstrate that MMP28 can act as an upstream regulator of EMT in vivo raising the possibility that other MMPs might have similar early roles in various EMT-related contexts such as cancer, fibrosis, and wound healing.

Funding: This work was supported by the Fondation pour le Recherche Medicale (FRM AJE201224 to ET; ARF20150934153 to NG), the Midi-Pyrenees Regional Council (13053025 to ET), Toulouse Cancer Sante (DynaMeca to ET), the European Marie Curie Prestiges Program (PRESTIGES 2015–4–007 to ET and NG), the National Institutes of Health (R21 DE029333 to NG; R01DE25806 to JPSJ), a pilot grant from the NYU Center for Skeletal and Craniofacial Biology, which was established by NIH (1P30DE020754 to NG), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 950254 to EHB)", EMBO (IG Project Number 4765 to EHB) and la Caixa (Junior Leader Incoming 94978 to EHB). ET receives his salary from the French National Center for Scientific Research (CNRS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Neural crest cells are multipotent stem cells that form at the interface between the neural and nonneural ectoderm [ 20 ]. They perform EMT to initiate cell migration and go on to colonize most tissues and organs of the developing embryo [ 21 ]. Neural crest EMT relies on oncogenes such as snai2 and twist [ 22 ]. The neural crest EMT program is often hijacked by invasive cells during carcinoma progression [ 23 , 24 ], making these cells an extremely relevant in vivo model to study EMT. Our data show that, during Xenopus development, MMP28 expressed in cranial placodes [ 25 ] is required for EMT of neural crest cells. MMP28 is secreted by placode cells, imported into the nucleus of adjacent neural crest cells where its catalytic activity is required for proper implementation of EMT via the maintenance of twist expression.

Here, we used Xenopus neural crest cells to assess the putative role of MMP28 in EMT. MMP28 is the last member of the MMP family to have been identified in human. It has a typical MMP structure with a secretion signal, a pro-domain that needs to be removed for complete enzymatic activity, and a hemopexin-like domain involved in cofactors binding and substrates recognition [ 17 ]. Roles and functions of MMP28 are poorly documented as compared to other members of the family. It has been shown to be involved in wound healing and nerve repair [ 17 ]. It is expressed in pulmonary fibrosis [ 18 ] and several human cancers including gastric cancer where it correlates with poor prognosis [ 19 ].

Matrix metalloproteinases (MMPs) are secreted enzymes initially discovered for their ability to remodel the extracellular matrix [ 6 ] and early evidence showed that MMPs could influence EMT via their role on the extracellular space [ 7 – 9 ]. Somehow the link between EMT and MMPs was never fully explored. This leads to the current situation where MMPs are not considered as relevant markers or regulators of EMT [ 5 ]. However, we now know that MMPs are pleiotropic players in health and diseases that can influence growth, survival, and migration [ 10 ]. MMPs have numerous noncanonical subcellular localizations (e.g., mitochondria, nucleus, cytoplasm) and several unexpected substrates have been described (e.g., cell adhesion molecules, growth factors, guidance cues) [ 6 , 11 , 12 ]. These observations suggest numerous putative functions that are not related to the regulation of the extracellular matrix but the functional and physiological relevance of these potential noncanonical functions still awaits demonstration. In particular, it is interesting to note that most MMPs have been detected in the nucleus [ 13 ] of at least 1 cell type and that some have been shown to exhibit transcriptional roles and DNA-binding abilities [ 14 – 16 ]. Given the frequent expression of MMPs by cells undergoing EMT, this calls for a re-assessment of their involvement in EMT independently of their effects on extracellular matrix.

Epithelial–mesenchymal transition (EMT) is a complex process controlled by an array of transcription factors such as members of the snai, twist, zeb, and soxE families [ 1 , 2 ]. During EMT, cells remodel their adhesion with other cells and the surrounding matrix and display increased cytoskeleton dynamics. These changes drive a change from an apicobasal polarity associated to epithelial stability to a front-rear polarity required for cell migration. EMT is essential for morphogenetic events such as ingression of mesodermal cells during gastrulation or emigration of neural crest cells from the neural tube but is also taking place in several diseases such as fibrosis and cancer [ 1 – 4 ]. EMT is an extremely complex and reversible process that is made of a series of non-obligatory steps. Therefore, despite conservation of the core changes taking place at the single-cell level, a wealth of regulatory mechanisms has been identified at the molecular level. The way cells undertake EMT appears to be highly context-dependent and renders the task of agreeing on a common definition across fields all the more challenging but common lines are starting to emerge [ 5 ].

Results

MMP28 produced by placodal cells can be imported into neural crest cells’ nuclei in vivo We next tested whether MMP28 could travel from placodes to neural crest cells in vivo within a time window compatible with normal neural crest-placodes interactions. To do so, we expressed MMP28wt-GFP, MMP28-EA-GFP, or a secreted form of GFP containing the signal peptide of MMP28 as a control, in the ectoderm of Xenopus embryos, and grafted neural crest explants labelled with rhodamine-dextran as a tracer next to the placodal region (Fig 5A). Embryos were fixed 4 h after the graft and processed for histology and confocal imaging to monitor the raw GFP signal. MMP28-GFP was detected in the cytoplasm and the nucleus of multiple neural crest cells located underneath the placodal ectoderm expressing MMP28wt-GFP or MMP28-EA-GFP (Fig 5B–5D). By contrast, neural crest cells grafted near the placodal ectoderm expressing secreted GFP had no GFP signal in their cytoplasm or nuclei, showing that GFP alone is not spontaneously endocytosed or imported in the nucleus. These data indicate that MMP28 is specifically imported and that the catalytic activity is not required for the import. To get a broader view of the distribution of MMP28-GFP in grafted neural crest cells, we performed similar grafts with MMP28wt-GFP followed by immunodetection of GFP (S6 Fig). MMP28-GFP is not restricted to cells directly underneath the MMP28-expressing placodal ectoderm and is found up to several cell diameters away from the ectoderm. By contrast, immunodetection against GFP on uninjected embryos led to no significant signals confirming the specificity of the observed staining (S6 Fig). These data show that MMP28 can travel from the placodal ectoderm to the nuclei of neural crest cells within a few hours in vivo. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. MMP28 can travel from the ectoderm to the nucleus of neural crest cells in vivo. (a) Diagram depicting the grafting procedure and sample preparation. Neural crest from a donor embryo labelled with rhodamine-dextran (grey) were grafted into a host embryo expressing MMP28wt-GFP, MMP28-EA-GFP, or secreted-GFP (green) in the ectoderm and processed for imaging. (b) Representative 1 μm-thick optical sections through the grafted area by confocal microscopy for each condition counterstained with DAPI (magenta), scale bar, 100 μm. Dash line squares indicate zoomed areas, scale bar for zooms 10 μm. No anti-GFP immunostaining was performed on these samples. (c, d) Quantification of cell internalisation and nuclear detection of MMP28-GFP in each grafted conditions shown in panels Figs 5a and 5b and S7. NC-PL MMP28wt-GFP 4 grafts, 452 cells; NC-PL MMP28-EA-GFP 2 grafts, 398 cells; NC-PL sp-GFP 4 grafts, 419 cells; NC-caudal ectoderm 7 grafts, 419 cells; PL-PL-MMP28wt-GFP 5 grafts, 259 cells; animal caps 7 sandwiches, 283 cells. Percentages of GFP-positive cells and nuclei were calculated per embryos. Means and standard deviation are plotted per experimental condition. Statistical analyses (d) comparing the proportions of cells with internal/nuclear GFP signal between NC-PL MMP28wt-GFP condition and the other conditions using contingency tables (see Materials and methods). Numerical data from all graphs can be found in the supporting S1 Data file. MMP, matrix metalloproteinase; NC, neural crest. https://doi.org/10.1371/journal.pbio.3002261.g005 Is MMP28 import depending on the neural crest-placodes interactions? To assess this, we designed several grafting experiments. We placed neural crest explants labelled with rhodamine-dextran next to caudal ectoderm producing MMP28-GFP to test MMP28 import from a non-placodal source. We grafted placodes labelled with rhodamine-dextran next to endogenous placodes producing MMP28-GFP to test whether placodes can also import MMP28. Finally, we made sandwiches of animal cap explants that are non-specified epithelia that have not yet acquired neural, neural crest, or placodal identities to further test for the universality of MMP28 import. A small animal cap explant labelled with rhodamine-dextran is engulfed in a bigger one producing MMP28-GFP (S7 Fig). In all conditions presented in Figs 5 and S7, we quantified the percentage of cells with internalised MMP28 in the cytoplasm and the nucleus (Fig 5C). We statistically compared the proportions of cells with MMP28 taken up to the cytoplasm and the nucleus between in each grafted condition to the control situation where neural crest cells receive MMP28-GFP from placodes (Fig 5D). These data show that the most favourable conditions to observe nuclear MMP28 are when neural crest cells are exposed to placodes expressing MMP28-GFP, regardless of MMP28 activity (Fig 5C and 5D, brown and black closed circles). However, internalisation and nuclear import can also be seen in neural crest cells exposed to MMP28-GFP produced by the caudal ectoderm and in placodes exposed to producing placodes, albeit at a lower efficiency (Fig 5C and 5D, open circle and open square). In addition, these experiments show that the ability to internalise MMP28-GFP does not predict its subsequent nuclear import. Indeed, the animal cap sandwiches are the experimental condition that leads to the highest rate of internalisation (Fig 5C, black cross) while having one of the lowest rates of nuclear import. In conclusion, internalisation of MMP28 in a paracrine manner is not specific to the neural crest–placodes interaction but the rate of nuclear import is higher when MMP28 is produced by placodes and received by neural crest cells than in any of the other experimental conditions tested.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002261

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