# Delamination of chick cephalic neural crest cells requires an MMP14-dependent downregulation of Cadherin-6B

Cyril Andrieu, Cathy Danesin, Audrey Montigny, Marie Rey, Klara Baqué,

Anne Bibonne, Dominique Alfandari, Eric Theveneau

► To cite this version:

Cyril Andrieu, Cathy Danesin, Audrey Montigny, Marie Rey, Klara Baqué, et al.. Delamination of chick cephalic neural crest cells requires an MMP14-dependent downregulation of Cadherin-6B. Differentiation, 2025, 142, pp.100836. 10.1016/j.diff.2025.100836. hal-05534635

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# Delamination of chick cephalic neural crest cells requires an MMP14-dependent downregulation of Cadherin-6B

Cyril Andrieu<sup>a</sup>, Cathy Danesin<sup>a, b</sup>, Audrey Montigny<sup>a, b</sup>, Marie Rey<sup>a</sup>, Klara Baqué<sup>a</sup>, Anne Bibonne<sup>a, b</sup>, Dominique Alfandari<sup>b</sup>, Eric Theveneau<sup>a, * c</sup>

## ARTICLE INFO

**Keywords:**

Neural crest

Epithelial-mesenchymal transition

MMP14

Cadherin-6B

Chicken embryo

## ABSTRACT

Matrix Metalloproteinases (MMPs) are known for their role in matrix remodeling via their catalytic activities in the extracellular space. Interestingly, these enzymes can also play less expected roles in cell survival, polarity and motility via other substrates (e.g. receptors, chemokines), through an intracellular localization (e.g. the nucleus) or via non-catalytic functions. Most of these unconventional functions are yet to be functionally validated in a physiological context. Here, we used the delamination of the cephalic Neural Crest (NC) cells of the chicken embryo, a well described experimental model of epithelial-mesenchymal transition (EMT), to study the *in vivo* function of MMP14 (a.k.a MT1-MMP). MMP14 is a transmembrane MMP known for its importance in cell invasion and often associated with poor prognosis in cancer. We found that MMP14 is expressed and required for cephalic NC delamination. More specifically, MMP14 is necessary for the downregulation of Cadherin-6B and a co-inhibition of Cadherin-6B and MMP14 expressions is sufficient to restore NC delamination. Cadherin-6B is normally repressed by Snail2. Surprisingly, in MMP14 knockdown this lack of Cadherin-6B repression occurs in the context of a normal expression and nuclear import of Snail2. We further show that MMP14 is not detected in the nucleus and that Snail2 and MMP14 do not physically interact. These data reveals that a yet to be identified MMP14-dependent signaling event is required for the Snail2-dependent repression of Cadherin-6B. In conclusion, this work provides an *in vivo* example of atypical regulation of Cadherins by an MMP which emphasizes the importance and diversity of non-canonical functions of MMPs.

## 1. Introduction

Neural crest (NC) cells are a highly migratory multipotent embryonic stem cell population specific to vertebrates (Gouignard et al., 2018). NC cells are induced at the interface between the neural and non-neural ectoderm and undertake an extensive migration throughout the developing embryo. NC cells give rise to an extensive list of derivatives including pigment cells, neuron, glia and cartilage among many others (Le Douarin and Dupin, 2012).

The early NC development program relies on the combinatorial expression of transcription factors (e.g. members of the snail, twist, sox, ets and zeb families) that implement epithelial-mesenchymal transition (EMT) (Gouignard et al., 2018). While some variations have been observed across species and along the antero-posterior axis (Theveneau and Mayor, 2012), the main steps of NC EMT are relatively well defined. In particular, a key aspect is the qualitative change of cell-cell adhesions

in the NC domain that will subsequently allow NC cells to leave the dorsal neuroepithelium. In the anterior trunk of the chicken embryo this has been well studied. NC cells are induced in the neural folds which are expressing E-cadherin and N-cadherin at early stages of neurulation (Dady et al., 2012; Dady and Duband, 2017). E-cadherin is progressively replaced by N-cadherin such that pre-migratory NC cells are located in the dorsal-most region of an N+/E-neural tube. Then, NC cells transiently express Cadherin-6B which was deemed critical to locally sort out NC cells from the rest of the neuroepithelium (Park and Gumbiner, 2012). Concomitantly, N-cadherin levels are decreased by shedding of the extracellular domain by ADAM10 (Shoval et al., 2007). N-cadherin is then further processed to generate a short cytoplasmic fragment known as C-terminal fragment 2 (CTF2) which will contribute to the upregulation of CyclinD1 expression. In turn, CyclinD1 promotes progression of the cell cycle through the G1/S transition. Due to interkinetic movements of nuclei during cell cycle progression, the entry in S-phase acts as

<footnote><sup>a</sup> Molecular, Cellular and Developmental Biology Department (MCD), Centre de Biologie Integrative (CBI), University of Toulouse, CNRS, UPS, 31062, Toulouse, France</footnote>

<footnote><sup>b</sup> University of Massachusetts Amherst, Dept. of Veterinary and Animal Sciences, Amherst, MA, 01003, USA</footnote>

<footnote>* Corresponding author.
E-mail address: eric.theveneau@univ-tlse3.fr (E. Theveneau).</footnote>
a permissive step for delamination by positioning the nucleus at the basal pole of the cells (Burstyn-Cohen and Kalcheim, 2002). This basal positioning of the nucleus together with the weakening of cell-cell adhesion between NC cells (N-cadherin shedding) and between NC cells and the rest of the neuroepithelium (Cadherin-6B expression) favors delamination.

In the cephalic region, a similar process is at play but further scrutiny has been placed on Cadherin-6B, revealing some intriguing feedback loops between Cadherin-6B expression and processing, transcriptional regulation and proteases activity. Cadherin-6B is processed by ADAM10 and 19 and γ-secretase (Schiffmacher et al., 2014) to generate an N-terminus fragment (NTF) and a CTF. The CTF goes in the nucleus and regulates snail2 expression (Schiffmacher et al., 2016), a transcriptional repressor of Cadherin-6B itself (Taneyhill et al., 2007). Importantly, due to snail2 being a direct repressor of Cadherin-6B, their co-expression in the neural crest domain is only transient and maintenance of Cadherin-6B mRNA can only be achieved by snail2 knockdown. The NTF regulates MMP2 activity (Schiffmacher et al., 2018) which was previously described as important for chicken NC delamination and migration (Cantemir et al., 2004; Duong and Erickson, 2004). In addition to this proteolytic removal, Cadherin-6B is internalized by macropinocytosis (Padmanabhan and Taneyhill, 2015), an actin-dependent membrane ruffling process used for internalization of large cargoes (Mylvaganam et al., 2021).

One key difference between cephalic and trunk NC delamination is that the pace of cell departure from the dorsal neuroepithelium is strikingly different. In the trunk, it is a slow-paced process with NC cells exiting one at a time for 2 day at a given region along the anteroposterior axis. By contrast, in the mesencephalon, there is an all-at-once delamination with hundreds of cells delaminating in a 6 to 10-h time window. In the cephalic context, the synchrony with the S-phase of the cell cycle is no longer required (Theveneau et al., 2007). This suggests that basal positioning of nuclei in the cephalic NC cells may not be a pre-requisite for delamination. Thus, the roles of molecules involved in NC delamination that are expressed all along the anteroposterior axis may not necessarily be the same in the cephalic and the trunk regions.

This is in fact the case for Cadherin-6B which is transiently expressed in both situations. However, its expression seems to have different effects. In the trunk, it was deemed as a de-epithelialization factor whose appearance at the cell surface helps to weaken the interaction between NC cells and the neuroepithelium (Park and Gumbiner, 2010, 2012). By contrast, in the head, Cadherin-6B seems to hold NC cells together while they are located in the dorsal neural tube since preventing its down-regulation blocks delamination (Coles et al., 2007).

MMP14 is a well-known regulator of cell migration (Gifford and Itoh, 2019). It can do so by promoting the formation of tracks within the extracellular matrix, either by itself or via activation of other MMPs such as MMP2 (Hotary et al., 2000, 2006; Sato et al., 1994). Interestingly, MMP14 also has non-catalytic roles in cell migration via its cytoplasmic tail by modulating HIF-1 and Rac1 pathways (Gonzalo et al., 2010; Sakamoto and Seiki, 2009, 2010). We previously found MMP14 to be required for the basal positioning of nuclei during trunk NC delamination (Andrieu et al., 2020). Given that this process and the association with entry in S-phase appear to be dispensable in the head (Theveneau et al., 2007), we wondered what could be the function, if any, of MM14 during cephalic NC delamination. Here we found that MMP14 is expressed in the cephalic neural tube and premigratory NC cells and that its expression is maintained during delamination and migration. Inhibiting MMP14 expression led to an accumulation of NC cells in the dorsal neural tube. This effect can be rescued by expressing back full-length MMP14, its proteolytic dead version or a form lacking the cytoplasmic tail but not by expressing a form lacking the whole catalytic domain. Unlike the control cephalic NC cells, MMP14 knockdown NC cells retained cadherin-6B expression (mRNA) and accumulated Cadherin-6B protein. Importantly, a double knockdown of MMP14 and Cadherin-6B was sufficient to restore cephalic NC delamination. These results

indicate that the normal downregulation of Cadherin-6B in premigratory NC requires a non-canonical function of MMP14. Further, we show that this atypical role does not involve a nuclear accumulation of MMP14 and does not impact snail2 expression and nuclear import.

## 2. Materials and methods

### 2.1. Chicken eggs

Fertilized chicken eggs were purchased from S.C.A.L (Société commerciale avicole du Languedoc) and incubated at 38 °C until the desired stage (Hamburger and Hamilton, 1992).

### 2.2. Electroporation

Embryos were electroporated *in ovo* at stage HH8/9. The vectors for the various forms of MMP14 and the siRNA are described in detail in (Andrieu et al., 2020). Electroporation was done opening the shell with curved scissors. Then, an electroporation solution containing DNA plasmids (1–2 µg/µL), Fast Green (0.1%) and sucrose (12%) is injected in the lumen of the neural tube using a pulled glass needle. 7 square pulses of 28 V at the frequency of 5 Hz are applied to perform the transfection. PBS 1X is added on top before closing the eggs and resuming incubation. All forms of MMP14 are GFP-tagged and the siRNA vectors contain a GFP cassette. Post-electroporation, embryos are selected under a fluorescent dissecting microscope based on the GFP expression intensity and localization.

### 2.3. Cornish pasty culture

This protocol is adapted from (Nagai et al., 2011). Embryos at stage HH6-7 were harvested and placed in a Petri dish containing Pannett-Compton solution. The vitelline membrane is removed. The embryonic discs are folded such that the anteroposterior axis of the embryo is aligned with the fold, dorsal side out. Then, the edges of the embryonic disc are cut out using a scalpel which seals the pasty. After a few minutes, pasties are then placed in Pannett-Compton/albumin (1:2) with DMSO (Gibco, 61962-026) or NSC405020 (EMD Millipore, 444295) at 0.5 mM and incubated at 38 °C until the desired stage.

### 2.4. Histology, cryosections and immunostaining

The complete protocols are described in (Andrieu et al., 2020). Antibodies used: rabbit anti-snail2 (Cell Signaling, C19G7, 1 µg/mL), rabbit anti-sox10 (GeneTex GTX128374, 1 µg/mL), mouse anti-cadherin6B (DSHB, CCD6B-1, 1 µg/mL), rabbit anti-cleaved Caspase 3 (Cell Signaling, 9664T, 1 µg/mL), mouse anti-phospho H3 (Cell Signaling, MABI0312, 1 µg/mL). All secondary antibodies coupled with Alexa 488, 555/594 or 633/647 were purchased from Invitrogen (Life Technology). Embryos were fixed in PFA4% in PBS 1X for mouse anti-MMP14 and in Glyoxal 3.6%, 8% acetic acid for rabbit anti-MMP14 cytoplasmic tail.

### 2.5. In situ hybridization

Whole-mount *in situ* hybridization was performed as previously described (Theveneau et al., 2007). Probes used: Cadherin-6B (Coles et al., 2007), Cadherin-7 (Nakagawa and Takeichi, 1995), Snail2/Slug (Nieto et al., 1994), Integrin-β3 (Pietri et al., 2003), MMP14 (Andrieu et al., 2020), Sox10 (Cheng et al., 2000), Ets1 (Vandenbuider et al., 1989), rhoB (Del Barrio and Nieto, 2004) and FoxD3 (Kos et al., 2001).

### 2.6. Image acquisition and analysis

Images were taken on a stereomicroscope Leica MZF10F equipped with a camera Leica DFC450C and the LAS software, a Nikon Eclipse 80i equipped with a camera DXM1200C and the NIS-elements software, a
Zeiss AxioImager 2 equipped with a Hamamatsu ORCA Flash 4 camera and the Zen2 software, a Zeiss 710 confocal microscope. Images were processed using FIJI. For confocal images of Snail2 immunostaining the raw Snail2 levels of each individual electroporated cells were measured. Then, raw Snail2 levels were retrieved from direct non-electroporated neighbors and averaged to a single control raw levels value. The Snail2 level of each electroporated cell was then divided by the average Snail2 raw levels value of its direct neighbors and plotted. This allowed to correct for variations of quality and intensity of Snail2 staining from sample to sample and section to section.

## 2.7. Antibodies against chicken MMP14

Full length Chick MMP14-Flag was expressed in human Hek293T cells using Extreme gene HP (Sigma) and purified with anti-Flag-agarose beads (Sigma). Balbc mice (Jackson lab) were immunized with 100 µl of anti-Flag-agarose beads bound to cMMP14 using complete and the incomplete freund adjuvant. Tail bleeds were collected after 2 boosts and were tested against Hek293T cells transfected with either RFP-Flag (negative control) or cMMP14. We collected 200 µL of serum containing mouse polyclonal anti-chicken MMP14. In addition, with the company Proteogenix, we produced two rabbit polyclonal antibodies against the cytoplasmic domain of chicken MMP14 (RRRGAPKKLLRCQRSLLPRV).

## 2.8. Label-free proteomics

Stage HH12 chicken embryos were electroporated *in ovo* with MMP14-FL-GFP or MMP14-ΔCAT-GFP. The next day, the GFP-positive portions were dissected and processed for pull-down using the GFP-trap kit (Chromotek). The following amount of proteins were retrieved: MMP14-FL (8.56 µg), MMP14-ΔCAT (8.06 µg). Samples were then sent to BGI (Hong-Kong) for proteomics. The mass spectrometer used was Q-Exactive HF X. The MS data were analyzed using the Max-Quant's integrated Andromeda engine. For MMP14-GFP: total spectra 97966, identified spectra: 43453, identified peptides: 41933, identified proteins: 4863. For MMP14-ΔCAT: total spectra 95181, identified spectra 42989, identified peptides 41418, identified proteins: 4857. Pairwise comparison was then performed to identify proteins whose amount was up, down or not affected in MMP14-ΔCAT samples compared to MMP14-FL. Fold change >1.5 was used as the screening criteria for significant differences. All analyses were performed by BGI Technology. A summary of the data is presented in Fig. 5 and Supplementary Spreadsheets 1, 2 and 3. All raw data are available upon request.

## 2.9. Statistics

Statistical analyses were performed with Prism 6 (GraphPad). Datasets were tested for Gaussian distribution. Student t-tests or ANOVA followed by multiple comparisons were used with the appropriate parameters depending on the Gaussian vs non-Gaussian characteristics of the data distribution. Significance threshold was set at p < 0.05. Box and whiskers plots: the box extends from the 25th to the 75th percentile; the whiskers show the extent of the whole dataset. The median is plotted as a line inside the box.

## 2.10. Western blots

We used Subcellular Protein Fractionation Kit for Tissues (Thermo-Fisher Scientific # 87790). Protein extracts were loaded on 4–12% precast gel (Bio-Rad Mini-PROTEAN®). Proteins were transferred on PVDF-membrane and blocked for 1h with 5% BSA. The following antibodies were used: mouse anti-Tubulin (Sigma, T5201; 1:10000), rabbit anti-snail2 (Cell Signaling, C19G7, 1/1000), rabbit anti-Histone H3 (Abcam ab1791, 1/5000), anti-rabbit-HRP or anti-mouse-HRP 1:10000 (Millipore).

# 3. Results

## 3.1. MMP14 is expressed in cephalic neural crest cells together with furin, timp2 and timp3

We previously showed that the expression of MMP14 is found in the trunk neural tube and that its expression increases in the NC domain at the time of delamination (Andrieu et al., 2020). In the cephalic region, MMP14 is detected in the open neural plate (Fig. 1A, arrowhead) and the anterior neural folds (Fig. 1A, arrow). Later on, MMP14 is found in the whole closing neural tube (Fig. 1B), including the dorsal region which contains the pre-EMT NC cells (Fig. 1C–C') and is maintained in delaminating NC (Fig. 1D–D', arrowheads) and migrating NC streams (Fig. 1E and F, arrows). Interestingly, furin, which is known to process pro-MMP14 into active MMP14 (Remacle et al., 2006), is also broadly expressed in the cephalic neural tube and migratory NC cells (Fig. 1G–I), strongly suggesting that cephalic NC cells express functional MMP14. However, we also analyzed the expression of Tissue Inhibitors of Metalloproteinases 2 and 3 (TIMP2 and 3) and found them to be expressed in delaminating and migrating cephalic NC cells as well (Supplementary Fig. 1). These observations indicate that, even if processed by furin, MMP14 catalytic activity might be impaired by the presence of the TIMPs.

## 3.2. MMP14 is required for cephalic neural crest delamination and migration independently of its catalytic activity

To directly assess the functional relevance of MMP14 in cephalic NC cells, we performed whole chicken embryo *ex vivo* culture using the Cornish Pasty technique (Nagai et al., 2011). We treated embryos with either a chemical compound targeting MMP14 (NSC405020 (Remacle et al., 2012)) with DMSO as vehicle or DMSO itself as control (Fig. 2). Embryos treated with DMSO displayed a normal pre-otic (r4) and post-otic (r6) NC delamination and migration of snail2-positive cells (Fig. 2B, arrowheads). By contrast, in embryos treated with NSC405020, most NC cells were accumulated in the dorsal neural tube with only few migrating cells (Fig. 2C, arrowheads). The mean distance migrated by NC cells was shorter (Fig. 2E) and this was associated with an enlargement of the NC domain in the dorsal neural tube (Fig. 2D). These two observations indicate that blocking MMP14 with NSC405020 leads to an impairment of NC delamination. NSC405020 binds to the hemopexin domain of MMP14 and mimics the effect of a delta-hemopexin MMP14 (Remacle et al., 2012). It physically perturbs interaction between MMP14 and other proteins, including MMP14 homodimerization, but it does not inhibit its catalytic activity *per se*. Therefore, substrates whose access to the catalytic pocket of MMP14 does not involve the hemopexin domain or MMP14 homodimerization might still be processed by MMP14. In addition, as the whole embryo is treated with the compound no internal control is available in these drug assays. In order to have an internal control and a more specific knockdown of MMP14, we used a siRNA that we previously validated for efficiency and specificity (Andrieu et al., 2020). Embryos electroporated with a scrambled control of the siRNA had normal delamination and migration of NC cells, as shown by the complete absence of sox10+/foxd3+ cells in the dorsal neural tube (Fig. 2F, first panel and associated section). By contrast, in embryos electroporated with siMMP14, NC cells accumulated in the dorsal neural tube all along the electroporated side (Fig. 2F, second panel, arrowheads and associated section). Importantly, targeting MMP14 had no effect on cell proliferation (Supplementary Fig. 2) or cell survival (Supplementary Fig. 3). Altogether, these data indicate that MMP14 is required for cephalic NC delamination.

To assess which domains of MMP14 are required for its role in NC delamination, we attempted to rescue the effect of MMP14 siRNA by co-electroporating it with vectors encoding various forms of MMP14: full length (FL), a form lacking the entire catalytic domain (ΔCAT), an inactive point mutant (EA) and a form lacking the cytoplasmic domain
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** MMP14 is expressed in the cephalic neural tube, pre-migratory and migratory neural crest cells. A-F, *in situ* hybridization for chicken MMP14, representative images from more than 50 embryos between stage HH7 and HH14. Arrow in A points to the anterior neural folds. Arrowheads in A indicates the open neural plate. Arrowheads in D' indicate delaminating neural crest cells. Arrows in E and F point to streams of migratory neural crest cells. G-I, *in situ* hybridization for chicken Furin, representative images from 4 embryos. Arrows in G and I point to streams of migratory neural crest cells. E, eye; HH, hamburger-Hamilton stages of chicken development; ov, otic vesicle; r, rhombomere. Scale bars, A-B, 200 µm; C-D, 50 µm; C'-D', 20 µm; E-G, 100 µm; H-I, 50 µm.</figcaption></figure>

(ΔCYTO) (Fig. 2F, third to sixth panel, respectively). Interestingly the FL, EA and ΔCYTO forms of MMP14 were equally efficient at rescuing NC delamination (Fig. 2F and G). However, the form lacking the catalytic domain was not able to rescue MMP14 knockdown as seen by the accumulation of NC cells in the dorsal neural tube (Fig. 2F, fourth panel and associated section, 2G). Since the lack of enzymatic activity of the point mutant was previously validated (Andrieu et al., 2020), these results indicate that the complete extracellular domain of MMP14 is required for cephalic NC delamination but that its catalytic activity is dispensable.

### 3.3. MMP14 is required for the downregulation of Cadherin-6B

To investigate what might be failing in cephalic NC cells after knocking down MMP14, we monitored the expression of snail2, rhoB, cadherin-6B, ets1, cadherin-7 and integrin-β3. These factors were previously described as essential for normal cephalic NC delamination and migration or known to be specifically upregulated in migratory cephalic NC cells (Del Barrio and Nieto, 2004; Nakagawa and Takeichi, 1995, 1998; Nieto et al., 1994; Pietri et al., 2003; Tahtakran and Selleck, 2003; Theveneau et al., 2007). Snail2 and rhoB are expressed in pre-migratory and migratory NC cells, ets1 and cadherin-6B are only transiently expressed during EMT whereas cadherin-7 and integrin-β3 are expressed in migratory NC only.

We found that the inhibition of MMP14 expression by siRNA,

compared to the scrambled control, led to the accumulation of NC cells in the dorsal neural tube (Fig. 3A, enlargement of the snail2+ domain on the electroporated side, bracket). These cells expressed snail2 (Fig. 3A), but also cadherin-6B (Fig. 3C, arrowheads), rhoB (Fig. 3B, arrowheads) and integrin-β3 (Fig. 3F, arrowheads). We measured the areas of rhoB-positive (Fig. 3G) and integrin-β3-positive (Fig. 3H) cells in the dorsal neural tube after MMP14 knockdown and found a 4.1 and 3.2-fold increase compared to control respectively, similar to the 3-fold increase of the snail2 domain after treatment with the MMP14 inhibitor (Fig. 2D). By contrast, these NC cells were negative for ets1 (Fig. 3D). Given that ets1 is detected along the electroporated domain, MMP14 does not seem to be required for ets1 expression *per se*. This rather indicates that the normal post-EMT downregulation of ets1 occurs in MMP14 knockdown NC cells. The NC cells accumulated in the dorsal neural tube after MMP14 knockdown were also cadherin-7 negative (Fig. 3E) suggesting that they did not fully adopt a migratory phenotype despite their upregulation of integrin-β3 and rhoB expressions. These data show that in absence of MMP14, NC cells initiate the EMT program and reach a partial migratory expression profile with the expression of snail2, rhoB and integrin-β3. However, they fail to complete the cadherin switch by properly downregulating Cadherin-6B and upregulating Cadherin-7. Given the importance of the loss of Cadherin-6B for normal departure of cephalic NC from the dorsal neuroepithelium (Coles et al., 2007), we wondered whether it might contribute to the delamination defect observed after MMP14 knockdown.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** MMP14 is required for the delamination of cephalic neural crest cells, independently of its catalytic activity. A, protocol of the Cornish pasty culture assay. B-C, transversal section at rhombomere 4 (r4) and rhombomere 6 (r6) level in embryos treated with DMSO (B) or NSC405020 (C, 0.5 mM). D, normalized area of the snail2-positive domain in the dorsal neural tube at antero-posterior levels and experimental conditions depicted in B-C (DMSO, $n_{r4} = 4$; $n_{r6} = 4$; NSC405020, $n_{r4} = 7$; $n_{r6} = 7$); T-test r4 DMSO vs NSC, p = 0.0061 (**), T-test r6 DMSO vs NSC, p = 0.23 (ns). E, Medio-lateral migration of snail2-positive neural crest cells at antero-posterior levels and experimental conditions depicted in B-C (DMSO, $n_{r4} = 9$; $n_{r6} = 11$; NSC, $n_{r4} = 8$; $n_{r6} = 11$); T-test r4 DMSO vs NSC, p = 0.0058 (**), T-test r6 DMSO vs NSC, p = 0.0002 (***). F, *in situ* hybridization for Sox10 and Foxd3 (both probes were mixed) after electroporation of scrambled siRNA (n = 33; from 7 independent experiments), siRNA-MMP14 (n = 56; from 11 independent experiments) or siRNA-MMP14 in combination with either MMP14-FL (n = 18; from 6 independent experiments), ΔCAT (n = 14; from 3 independent experiments), EA (n = 14; from 2 independent experiments) or ΔCyto (n = 14; from 3 independent experiments). Each electroporated condition is associated with a corresponding transversal section at the level of rhombomere 6 (dotted line). G, Mean area occupied by Sox10-positive neural crest cells above the neural tube in the whole rhombencephalic regions (normalized to scrambled siRNA). ANOVA followed by multiple comparisons, ****, p < 0.0001, ***, p = 0.004. Asterisks indicate the electroporated side. Scale bars, B-C, 50 µm; F, whole mount 50 µm, sections 25 µm.</figcaption></figure>

### 3.4. Double knockdown of MMP14 and Cadherin-6B restores normal cephalic neural crest delamination

First, we checked whether the abnormal maintenance of Cadherin-6B mRNA in MMP14-knockdown NC cells translated into the accumulation of the protein Cadherin-6B. Indeed, in siMMP14 NC cells, there was a clear accumulation of Cadherin-6B compared to non-electroporated cells on the contralateral side and to cells electroporated with the scrambled control (Fig. 4A and B, arrows). Second, we tested whether this excess of Cadherin-6B is responsible for the NC delamination defect. For that, we confirmed the efficiency of a previously validated siRNA vector against Cadherin-6B (Park and Gumbiner, 2010). As expected, the control siRNA had no effect on Cadherin-6B (Fig. 4C) while the specific siRNA against Cadherin-6B led to a reduction of Cadherin-6B protein in electroporated cells (Fig. 4D, arrows). Next, we assessed whether performing a double knockdown of MMP14 and Cadherin-6B might rescue cephalic NC delamination (Fig. 4E and F). Electroporation of siMMP14 led to the accumulation of Sox10-positive NC cells in the dorsal neural tube with the NC domain on the electroporated side being twice as large as on the control side (Fig. 4G). It should be noted that Sox10 is a late NC markers found primarily in migratory NC cells. Thus, the accumulation of Sox10-positive NC cells in the dorsal neural tube after MMP14 knockdown further confirms results

shown on Fig. 3 indicating that NC progress towards a migratory phenotype but fail to exit the neural tube. By contrast, co-electroporating siMMP14 with siCadherin-6B prevented the accumulation of Sox10-positive NC cells (Fig. 4G). Altogether, our data indicate that the normal delamination of cephalic NC cells requires a MMP14-dependent downregulation of Cadherin-6B.

### 3.5. MMP14 does not physically interact with Snail2 or Cadherin-6B

Why is Cadherin-6B protein accumulating MMP14-KD NC cells? Cadherin-6B and snail2 are known to be part of a negative feedback loop such that cadherin-6B promotes the expression of snail2 that then acts as a transcriptional repressor of cadherin-6B (Taneyhill et al., 2007). As such, Cadherin-6B and snail2 mRNA are only transiently co-detected in premigratory NC cells. The fact that, after MMP14 knockdown, mRNA for both cadherin-6B and snail2 are maintained in NC cells accumulated in the dorsal neural tube indicates that MMP14 is dispensable for their respective expression but suggests that it is somehow required for the efficiency of the snail2-dependent repression of cadherin-6B. MMP14 has been previously detected into the nucleus of macrophages where it impacted transcription via direct binding to the chromatin (Shimizu-Hirota et al., 2012). This opens the possibility that, in cephalic NC cells, part of MMP14's roles might involve either a nuclear import
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** MMP14 loss-of-function leads to the accumulation of neural crest cells expressing snail2, cadherin-6B, rhoB, and integrin-β3 in the dorsal neural tube. A-F, *in situ* hybridization for snail2 (A, nscrambled = 7; nsiMMP14 = 4), rhoB (B, nscrambled = 6; nsiMMP14 = 6), cadherin-6B (C, nscrambled = 3; nsiMMP14 = 4), ets-1 (D, nscrambled = 3; nsiMMP14 = 4), cadherin-7 (E, nscrambled = 6; nsiMMP14 = 6), integrin-β3 (F, nscrambled = 8; nsiMMP14 = 6) at 12 h post electroporation. Scale bar in whole mount 100 µm, in sections 25 µm. Sections are at the level of rhombomere 6. Inserts show the electroporated region for each embryo. Asterisks indicate the electroporated side. Bracket in A indicate an expansion of the snail2 territory on the siRNA-MMP14 electroporated side. Arrowheads in C, D and F indicate neural crest cells in the dorsal neural tube expressing the corresponding gene of each panel. G-H, ratios of RhoB (G; nscrambled = 5 embryos, 22 sections; nsiRNA-MMP14 = 6 embryos, 38 sections, two-tailed Mann-Whitney test, p = 0.0173) and integrin-β3 (H; nscrambled = 6 embryos, 30 sections; nsiRNA-MMP14 = 7 embryos, 24 sections, two-tailed Mann-Whitney test, p = 0.0012) areas in electroporated and non-electroporated sides of the dorsal neural tube, averaged per embryo and normalized to the mean ratio in the scrambled siRNA condition.</figcaption></figure>

and/or some direct or indirect function in transcriptional regulation. A bold hypothesis would be that MMP14 would be imported in the nucleus and physically interact with snail2 and that this interaction would modulate snail2's ability to repress cadherin-6B expression. In addition to this, given that MMP14 is a transmembrane protein, MMP14 and Cadherin-6B could physically interact and MMP14 might be required for normal Cadherin-6B protein turnover.

To assess whether MMP14 interacts with either Cadherin-6B or snail2, we turned to pull-down assays and proteomics. We used the GFP-tagged form of full length MMP14 as bait. For comparison, we used MMP14-ΔCAT-GFP. Given that MMP14-ΔCAT-GFP was the only form of MMP14 that was unable to rescue MMP14 knockdown (Fig. 2F and G), we reasoned that proteins that differentially bind to MMP14-FL and ΔCAT might correspond to relevant co-factors involved in NC delamination. Thus, if interactions do take place, we would expect snail2 and/or Cadherin-6B to pull down with full length MMP14 but not with its ΔCAT version for instance. Both forms were electroporated in the trunk neural tube of stage HH12 embryos (Fig. 5A). At this stage of development, both cadherin-6B and snail2 are expressed in long portions of the trunk neural tube, thus providing a large amount of material for analysis. GFP-positive neural tubes were dissected and we performed a pull-

down from total protein extract using a GFP-trap assay, followed by mass spectrometry to identify proteins in each sample. We found over 4800 proteins binding to each form of MMP14 (Fig. 5B, see methods). Running a pairwise comparison between MMP14-FL and ΔCAT, we found that 3131 proteins were equally pulled down by both forms of MMP14 (Fig. 5C, Supplementary Spreadsheet 1), 698 proteins were lost upon deletion of the catalytic domain while 759 proteins were enriched in the ΔCAT sample (Fig. 5C). Among the proteins that differentially bind to the two forms of MMP14, we found proteins from various subcellular compartments such are matrix components (e.g. Fibronectin, collagen alpha2 chain), transmembrane proteins (e.g. Robo1), cytoskeletal components (e.g. alpha-actinin) or nuclear proteins (e.g. Pitx2, Olig2). However, neither cadherin-6B nor snail2 were detected as binding to either form of MMP14, which does not support a role of MMP14 in directly regulating transcriptional activity, trafficking or turnover of these factors. Interestingly, we found that a large amount of the differentially interacting proteins belonged to signal transduction, transport, translation and transcription in the KEGG classification categories (Fig. 5D, Supplementary Spreadsheet 2). Further, the subcellular analysis revealed that nuclear proteins were the most common population among the differentially interacting proteins (Fig. 5E,
<figure><img src="image_6.png" /><figcaption>Fig. 4. Knocking down Cadherin-6B is sufficient to rescue NC delamination in MMP14 loss-of-function. A-B, Immunostaining for Cadherin-6B (magenta) after electroporation of the scrambled siRNA (A, n = 6) or siRNA-MMP14 (B, n = 5), arrows indicate cells with accumulation of Cadherin-6B upon loss of MMP14. C-D, Immunostaining for Cadherin-6B (magenta) after electroporation of the siControl for Cadherin-6B (C, n = 4) or siCadherin-6B (D, n = 4), arrows indicate cells with loss of Cadherin-6B upon expression of siCadherin-6B. E-F, Immunostaining for Sox10 (magenta) after electroporation of siRNA-MMP14 (E, n = 14; from two independent experiments) or siRNA-MMP14 + siCadherin-6B (F, n = 8; from two independent experiments). Arrows indicate cells that have delaminated and are located between the neuroepithelium and the epidermis. Bracket in E indicates an example of an increase of the sox10+ area on the siRNA-MMP14 electroporated side. G, Area occupied by Sox10-positive cells in the dorsal neural tube, normalized to control (non-electroporated side). T-test, **p = 0.0015. Scale bars 50 µm.</figcaption></figure>

Supplementary Spreadsheet 3). Thus, we wondered whether MMP14 might be imported in the nucleus of cephalic NC cells.

### 3.6. MMP14 is not detected in the nucleus of delaminating cephalic neural crest cells

To assess whether MMP14 is imported in the nucleus of delaminating NC cells we performed a double immunostaining for MMP14 and Sox10. We used a custom-made mouse anti-chicken MMP14 (see methods). This antibody was previously validated for specificity (Andrieu et al., 2020). MMP14 protein is detected in the entire neural tube at pre-EMT (Fig. 6A) and EMT stage (Fig. 6B and C). Its localization encompasses the apical third of the cells which is reminiscent of the Golgi apparatus distribution (Herrera et al., 2021; Taverna et al., 2016). In Sox10-positive delaminating NC cells, this apical localization is lost and MMP14 is redistributed all along the apicobasal axis (Fig. 6B and C", asterisk and

bracket). Importantly, there is no overlap between the Sox10 nuclear staining and MMP14 staining (Fig. 6C").

To substantiate these observations, we generated a rabbit polyclonal anti-MMP14 targeting the cytoplasmic tail of MMP14 (see methods). This antibody is able to detect overexpressed full length MMP14-GFP but not MMP14-Δcytoplasmic tail-GFP (Supplementary Fig. 4). During delamination of cephalic NC cells, this antibody gives a similar staining with a Golgi-like distribution of MMP14 (Fig. 6D and E). Importantly, counterstaining with DAPI shows no overlap between DNA material and MMP14 (Fig. 6F). Altogether these data do not support the hypothesis of a nuclear import of MMP14, neither in neuroepithelial cells nor in delaminating NC cells.
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Pull-down and mass spectrometry analyses from MMP14 and MMP14-ΔCAT samples A, Procedure for identification of MMP14 binding partners (see methods). Stage HH12 embryos were electroporated *in ovo* with a plasmid to express either MMP14-FL-GFP (n = 7), MMP14-ΔCAT-GFP (n = 11). 24h later, the GFP + portions of each condition were microdissected and processed for quantitative proteomics. B, Total proteins identified in each pull-down. C, Equally and differentially interacting proteins from pairwise comparisons between MMP14 and MMP14-ΔCAT samples. D, Distribution of differentially interacting proteins per KEGG classification. E, Distribution of differentially interacting proteins per subcellular localization.</figcaption></figure>

### 3.7. MMP14 knockdown does not affect Snail2 protein level and nuclear import

Transcription factors are imported in the nucleus but their activity is often linked to a tight regulation of their shuttling between cytosol and the nucleus as previously shown for Sox10 or Twist for instance (Rehberg et al., 2002; Wei et al., 2015). Thus, even if not imported in the nucleus MMP14 might act by impacting Snail2 trafficking while in the cytosol. To assess that, we electroporated embryos with either the scrambled control siRNA or the siRNA-MMP14 and performed an immunostaining for Snail2 at 6 h (Fig. 7A and B) and 18 h (Fig. 7C and D) post-electroporation. We sectioned the region of NC delamination and searched for areas with mosaic electroporation in which transfected cells could be compared to their direct neighbors. We retrieved Snail2 intensity for electroporated cells and their adjacent controls and plotted ratios of Snail2 intensity for electroporated over control cells (Fig. 7E and F). In addition, we used a more global analysis of Snail2 protein nuclear localization by fractionation followed by Western blot. We generated nuclear extracts from embryos incubated with DMSO or the MMP14 inhibitor (NSC405020) and quantified the ratio of snail2 over histone H3. Total protein extracts from untreated control embryos were used as positive controls for all antibodies (Supplementary Fig. 5). Our

results show no reduction of Snail2 nuclear localization but instead a slight increase of nuclear snail2 after treatment with the MMP14 inhibitor. Overall, these analyses indicate that MMP14 knockdown does not prevent Snail2 protein from being produced or imported into the nucleus.

In conclusion, our study reveals a yet to be identified MMP14-dependent signaling event that is required for the normal Snail2-dependent repression of Cadherin-6B. This signaling event does not rely on MMP14 enzymatic activity but requires an intact extracellular domain of MMP14 to occur. It does not involve a physical interaction between MMP14 and Cadherin-6B/Snail2 nor a nuclear import of MMP14 but eventually affects the ability of Snail2 to act as an efficient transcriptional repressor of Cadherin-6B. Uncovering the partners of MMP14 in this process and how it affects Snail2 activity will require extensive work that is beyond the scope of this study. Importantly, the effect of MMP14-KD on Snail2 activity can be simply rescued by directly knocking down Cadherin-6B to restore NC delamination indicating that downregulation of Cadherin-6B downstream of MMP14 is a critical step in cephalic NC delamination.
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** MMP14 protein localization during cephalic neural crest delamination A-C, Double immunostaining with a mouse polyclonal antibody raised against full length MMP14 (green) and Sox10 (magenta) at pre-EMT and EMT stages, mesencephalon, stage HH8-9+. Dotted lines indicate the position of the zooms. D-F, Immunostaining with a rabbit polyclonal antibody raised against the cytoplasmic tail of MMP14 (green), mesencephalon, stage HH9. Counterstaining with DAPI (grey in D, magenta in F). Asterisk in B and bracket in B' indicate the apical loss of MMP14 staining in Sox10-positive cells. Scale bars 50 µm, 15 µm on zooms.</figcaption></figure>

## 4. Discussion

We previously showed that MMP14 is required for trunk NC delamination independently of its catalytic activity (Andrieu et al., 2020). Thus, the results of this study highlight an aspect of MMP14 biology that is conserved between cephalic and trunk NC cells in the chicken embryo. However, the delamination process between these two regions involves distinct cellular and molecular mechanisms. In particular, Cadherin-6B's role in both NC populations seems to be different, favoring mesenchymalization of trunk NC cells while maintaining epithelial status in cephalic NC cells (Coles et al., 2007; Park and Gumbiner, 2012). In addition, while all cephalic NC cells express snail2 during EMT, in the trunk, snail2 is only expressed during the first wave of delamination (Del Barrio and Nieto, 2004). These discrepancies

suggest that the functional relationship between MMP14, snail2 and Cadherin-6B might not be conserved between trunk and cephalic NC cells but this remains to be tested.

Given that MMP14's catalytic activity is dispensable for cephalic NC delamination, Cadherin-6B is unlikely to be a substrate of MMP14. This is further supported by the fact that Cadherin-6B was not pulled down by MMP14 in our assay. However, among the proteins that were significantly downregulated in MMP14-ΔCAT compared to MMP14-FL, we found CDC42, α-actinin1 (ACTN1) and 4 (ACTN4), WAVE2 (WASF2) and Abi1 which were all previously found to be involved in macro-pinocytosis (Mylvaganam et al., 2021), a key process for Cadherin-6B turnover during NC delamination (Padmanabhan and Taneyhill, 2015). Intriguingly, all these proteins are cytosolic, yet their binding to MMP14 is affected by the loss of the catalytic domain which is located
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Snail2 protein expression in control and MMP14 knockdown neural crest cells A-B, Immunostaining against Snail2 (magenta) in embryos electroporated with scrambled control siRNA (A, n = 12) or with siRNA-MMP14 (B, n = 29) at 6 h post-electroporation. Electroporated cells are GFP+ (green). C-D, Immunostaining against Snail2 (magenta) in embryos electroporated with scrambled control siRNA (C, n = 23) or with siRNA-MMP14 (D, n = 27) at 18 h post-electroporation. Electroporated cells are GFP+ (green). DAPI counterstaining for nuclei is in grey. E, Procedure to measure Snail2 intensity. F, Ratios of Snail2 raw levels in electroporated cells over neighboring control cells. Arrows indicate the position of the GFP+ cells. One-way ANOVA, uncorrected Fisher's LSD, scrambled vs siRNA at 6 h p = 0.7258, scrambled vs siRNA at 18 h p = 0.9053, siRNA at 6 h vs siRNA at 18 h p = 0.3814. Scale bars 20 µm.</figcaption></figure>
outside of the cell, suggesting that MMP14 might interact indirectly with these actin regulators through binding to another cell surface protein. Interestingly, a putative candidate for this role could be Robo1. Robo1 is found among the proteins that bind to MMP14-GFP and is significantly down-regulated in the MMP14-ΔCAT condition. In addition, Robo1 is expressed in the cephalic neural tube and NC cells at the time of delamination (Giovannone et al., 2012). Further, in other cell types, Robo1 has been shown to physically interact with MMPs (Seki et al., 2010), Cadherins (Gonda et al., 2020; Rhee et al., 2002) and the WAVE complex via its cytoplasmic tail (Chen et al., 2014). Finally, the Slit-Robo pathway can modulate macropinocytosis (Bhosle et al., 2020). All these observations suggest that there could be a molecular complex involving MMP14 and Robo1 interacting with actin regulators to mediate macropinocytosis of Cadherin-6B. While it would not explain how MMP14 might modulate snail2-dependent transcriptional repression of Cadherin-6B, it could contribute to the accumulation of Cadherin-6B protein after MMP14 knockdown.

Interaction between the actin cytoskeleton or its regulators and MMPs are not new. There are multiple levels of co-regulation including binding to actin filaments, complex feedback loops via matrix remodeling or indirect transcriptional regulation (for discussion see (Bildyug, 2016; Gucciardo et al., 2016; Terawaki et al., 2015)). Direct binding to cytoplasmic component involves the cytoplasmic tail of membrane-bound MMPs, for instance via Radixin (Terawaki et al., 2015). However, while mouse and human MMP14 have almost identical cytoplasmic tails, chicken MMP14 presents 6 mutations (Yang et al., 2008) including one removing a Tyrosine essential for the interaction with p130cas previously shown to drive the membrane localization of Rac1 (Gonzalo et al., 2010). Thus, it is not clear whether chicken MMP14 is capable of interacting with actin-related proteins by itself. This might further support the hypothesis of an indirect interaction via its extracellular domain through a relay protein (e.g. Robo1).

In conclusion, our data indicate that the normal downregulation of cadherin-6B, required for cephalic NC delamination in the chicken embryo, depends on the presence of a complete extracellular domain of the MMP14 protein. However, MMP14 catalytic activity is dispensable. Inhibition of MMP14 expression leads to an accumulation of Cadherin-6B/Snail2-positive NC cells in the dorsal neural tube indicating that, in absence of MMP14, Snail2 is no longer capable of repressing Cadherin-6B. This effect occurs without detectable nuclear import of MMP14, without physical interaction between MMP14 and Cadherin-6B/Snail2 and with no apparent impairment of Snail2 trafficking to the nucleus. MMP14 inhibition can be rescued by knocking down cadherin-6B directly, indicating that one of the main roles of MMP14 in cephalic NC delamination is to contribute to cadherin-6B downregulation. These in vivo results highlight the physiological relevance of a non-canonical function of MMP14 and establish an experimental model in which to study MMP14-dependent signaling events that affects transcriptional regulation.

### CRediT authorship contribution statement

**Cyril Andrieu:** Writing - review & editing, Validation, Investigation, Formal analysis. **Cathy Danesin:** Writing - review & editing, Validation, Investigation, Formal analysis. **Audrey Montigny:** Investigation. **Marie Rey:** Investigation. **Klara Baqué:** Investigation. **Anne Bibonne:** Investigation. **Dominique Alfandari:** Writing - review & editing, Resources. **Eric Theveneau:** Writing - review & editing, Writing - original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

### Declaration of Competing interests

No competing interests declared.

### Acknowledgements

Work in the Theveneau lab is supported by grants from the Fondation pour la Recherche Médicale (FRM AJE201224), the Region Midi-Pyrénées (13053025), Association pour la Recherche contre le Cancer (ARC, ARCPJA22020060002084) the CNRS and Université Paul Sabatier (UMR5077). ET and AB are CNRS staff. CD is a staff scientist from the French National Institute for Health and Medical Research (Inserm). CA was the recipient of a PhD fellowship from the French Ministry for Higher Education and Research, a PhD Fellowship extension from the Association pour la Recherche contre le Cancer (ARC). KB was a master's student supported by the Centre for Integrative Biology (UMR5077, CNRS, UPS). AM was a research engineer supported by the FRM grant (AJE201224). MR is a technician supported by an ANR grant (Single Crest).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.diff.2025.100836.

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