New Results

Connexin43 controls N-cadherin transcription during collective cell migration

Maria Kotini, Elias H. Barriga, Jonathan Leslie, Marc Gentzel, Alexandra Schambony, Roberto Mayor
doi: https://doi.org/10.1101/114371

Abstract

Connexins are the primary components of gap junctions, providing direct links between cells in many physiological processes, including cell migration and cancer metastasis. Exactly how cell migration is controlled by gap junctions remains a mystery. To shed light on this, we investigated the role of Connexin43 in collective cell migration during embryo development using the neural crest, an embryonic cell population whose migratory behavior has been likened to cancer invasion. We discovered that Connexin43 is required for contact inhibition of locomotion by directly regulating the transcription of N-cadherin. For this function, the Connexin43 carboxy tail interacts with Basic Transcription Factor 3, which mediates its translocation to the nucleus. Together, they bind to the n-cad promotor regulating n-cad transcription. Thus, we uncover an unexpected, gap junction-independent role for Connexin43 in collective migration that illustrates the possibility that connexins, in general, may be important for a wide variety of cellular processes that we are only beginning to understand.

Highlights

  • Cx43 regulates collective directional migration of neural crest cells

  • Cx43 carboxy tail controls cell polarity via n-cad regulation

  • Cx43 carboxy tail localises at the nucleus and that depends on BTF3

  • BTF3 and Cx43 carboxy tail directly interact to bind and regulate n-cad promoter activity

Introduction

Connexins, the protein components of gap junctions (GJs), have been established as pivotal molecules in morphogenesis (Liu et al., 2012; Mathias et al., 2010) and cell migration during embryonic development (Cina et al., 2009; Elias et al., 2007; Francis et al., 2011) and cancer metastasis (Ogawa et al., 2012; Stoletov et al., 2013). Yet the way that connexins regulate these processes is not fully understood. While the activity of connexins as channels has been shown critical for the regulation of tissue homeostasis (Alexander and Goldberg, 2003; Plum et al., 2000), it is not required for cell migration (Cina et al., 2009; Elias et al., 2007), an important aspect that has been emphasised in various cell types for Cx43, the most abundant and conserved connexin among vertebrates, (Elias et al., 2007, 2010; Homkajorn et al., 2010). Attemps to build a complete picture of how Cx43 functions in cell migration have been complicated by the fact that Cx43 possesses several functions. Indeed, Cx43 can work as a channel (Niessen et al., 2006) or a hemi-channel (Bruzzone et al., 2001; Ebihara et al., 1999), as an adhesive molecule (Elias et al., 2007, 2010; Elzarrad et al., 2008) and can also be involved in intracellular signaling (Cina et al., 2009). Additionally, a number of molecules interact with Cx43 (Giepmans et al., 2001; Saidi Brikci-Nigassa et al., 2012) and have been implicated in the regulation of the Cx43 activities (Ai et al., 2000; Kwak et al., 1995; Solan and Lampe, 2005). Furthermore, it has been shown that Cx43 generates small carboxy terminal isoforms (Salat-Canela et al., 2014; Smyth and Shaw, 2013), yet the biological function of these isoforms remains unknown.

Numerous studies support the notion that Cx43 is involved in cell migration via different activities (Liu et al., 2012; Mendoza-Naranjo et al., 2012; Waldo et al., 1999; Zhang et al., 2014). Interestingly, one aspect that is common to a number of cell types, including neurons, astrocytes, cardiac neural crest and metastatic cancer cells, is that loss of function of Cx43 leads to an effect on cell protrusions such as lamellipodia or filopodia and a concurrent loss in cell polarity (Elias et al., 2007; Homkajorn et al., 2010; Xu et al., 2006; Stoletov et al., 2013). In certain instances, where directional motion is involved, this loss of cell polarity may contribute to the defective migration (Elias et al., 2010; Francis et al., 2011; Xu et al., 2006). Despite this, exactly how Cx43 controls cell migration, polarity and protrusions remains unknown.

Collective cell migration is dependent on cell-cell cooperation (Friedl and Gilmour, 2009; Mayor and Ettienne-Manvile, 2016), but our knowledge on the molecular mechanism of this cell cooperation remains incomplete. For example, we know that adherens junctions and specifically N-cadherin (N-cad) is critical for establishing cohesiveness of the cells and cell polarity in a collective (Theveneau et al., 2010; Kuriyama et al., 2014). Interestingly, it has been shown that Cx43 can modify the levels of N-cad at the membrane (Giepmans, 2006; Xu et al., 2001), but the mechanism driving this regulation remains unknown.

To disentangle this problem we used Xenopus cranial neural crest (NC), which provides a well-established model for directional motion of cell collectives (Theveneau and Mayor, 2013). An essential phenomenon required for NC migration is contact inhibition of locomotion (CIL), the process by which cells polarise away from cell contacts within a cell collective (Carmona-Fontaine et al., 2008; Stramer et al., 2013). This contact dependent polarity is essential for collective motion and requires the formation of a transient cell-cell adhesion junction, with N-cad being a crucial element, which needs to be tightly regulated as excess or deficiency of N-cad is sufficient to impair NC migration (Scarpa et al., 2015; Theveneau et al., 2010). However, exactly how the levels of n-cad are controlled during collective behavior is poorly understood.

Here we propose a link between cell polarity and the regulation of N-cad levels by Cx43. We show that Cx43, a molecule primarily known for its membrane-linked activities, uses its tail isoform to control morphogenetic movements via transcriptional regulation of n-cad, an activity which is independent from its function as channel in the cell membrane. Moreover, we identify its mechanism of action by showing that Cx43 regulation of n-cad is due to a direct interaction with the basic transcription factor 3 (BTF3) and polymerase II. Adittionally we show that this complex directly binds to the n-cad promoter to modulate its transcription. Furthermore, we show that this novel activity of Cx43 as a regulator of n-cad is conserved between amphibian and mammalian cells.

Results

Cx43 modulates NC migration in vivo and in vitro via the regulation of cell polarity

Previous studies have shown that Cx43 is expressed in mouse cardiac NC and that aberrant Cx43 expression leads to migration deficiency (Ewart et al., 1997; Waldo et al., 1999). In order to study the cellular and molecular roles of Cx43 in migration we used the cephalic NC from Xenopus laevis embryos because it is an amenable system for in vivo and in vitro characterisation of cell migration. In Xenopus, cephalic NC cells can be identified by the expression of the transcription factor twist (Linker et al., 2000). Using antibody staining, we found that Cx43 is expressed in the cephalic NC migratory streams as compared with the NC marker twist (Figure 1A). In addition, Cx43 is also expressed at the neural tube and the presumptive eye (Figure 1A). In other systems, Cx43 is present in two forms: the full length molecule (Cx43FL), which is a component of GJs, and the smaller carboxy terminal fragments (Cx43iso) that are not part of the GJ structures (Salat-Canela et al., 2014; Smyth and Shaw, 2013). To identify whether these isoforms are present in Xenopus, we performed western blot analysis of Cx43 at different developmental stages. Our analysis revealed that Cx43FL (between 35kDa and 55 kDa) is expressed throughout all the stages analysed (Figure 1B, stages 16 to 25), while the expression of Cx43iso (between 15kDa and 25kDa) increases just before the NC start its migration (Figure 1B, stage 18 onwards). These results were confirmed by analysing Cx43 mRNA using RT-PCR, which shows that Cx43 mRNA is present in dissected NC and in whole embryos at stage 18 (Figure S1A).

Figure S1.
Figure S1. Related to Figure 1.

(A) RT-PCR for NC dissected from st18 embryos and for st18 whole embryos for: ubiquitous marker odc, neural crest marker twist, cx43 and n-cad. (B) Diagram of migration index analysis for ISH and NC transplants, 𝓵: measured length. (C) Diagram of MO targets and WB for Cx43 and α-tubulin. Cx43 levels normalised to α-tubulin, (N=3, mean ± SE, p*<0.05, p**<0.01, p***<0.001). (D) snail2ISH for stage 16 embryos, arrow indicates the injected side. Quantification for NC induction (nCTLMO =20, nCx43MO =15, N=4, mean ± SE). (E) Representative motility single cells tracks.

Figure 1.
Figure 1. Cx43 controls NC migration

(A) twist ISH and Cx43 immunostaining for st24 embryo; IgG control antibody; arrows indicate NC, asterisk marks the eye. Drawing: Cx43 and twist expression summary. (B) WB for Cx43 and Mapk. Cx43 temporal expression at neurula stages. (C) twist ISH for st24 embryos, arrows show normal and brackets impaired migration. Quantification of migration index (nCTLMO=31, nCx43MOATG=37, nCx43MOATGATG+Cx43FL=30, nCx43MOint=33, nCx43MOintint+Cx43FL=19, N = 3, median ± interquartile range, p***<0.001) and % of embryos (N=3, mean ± SE, p***<0.001). (D) Transplant assay summary and experiment. Migration index of grafted NC (n=15, N=3, median ± interquartile range, p***<0.001). (E) In vivo migration summary. (F) Cell tracks of NC explants towards SDF-1. Tracks for single explants and their chemotaxis index (median ± quartile, n=15, N=3, p***<0.001). (G) Directionality and velocity of NCs (nCTLMO=36, nCx43MO=31, N=4, mean ± SE, ns=non-significance). See also Figure S1.

To test whether Cx43 is required for NC migration, we blocked translation of Cx43 using two antisense morpholino variants, Cx43MOATGand Cx43MOint (see Star Methods, Figure S1C). Injection of any of these two MOs leads to a strong inhibition of NC migration when compared to the effect of a control MO (CTLMO) (Figure 1C; S1B). Moreover, co-injection of either of the MOs with human cx43FL mRNA, that does not bind the MOs, showed a rescue in NC migration confirming the specificity of these treatments (Figure 1C). To test if the observed effect is tissue-autonomous, we transplanted NC from CTLMO- or Cx43MO-injected embryos into uninjected host embryos (Figure 1D). Only embryos with Cx43MO transplanted NC showed an inhibition in migration, indicating that Cx43 is autonomously required in the NC. Importantly, our observation on migration is not an indirect result of affecting NC induction, since Cx43 downregulation does not affect the early expression of the NC marker snail2 (Mayor et al., 1995), as shown by in situ hybridisation (ISH) (Figure S1D).

To assess how Cx43 influences NC migration, we analysed the effect of its loss-of-function in vitro. In a first step, we analysed the response of the NC population towards an external source of the chemoattractant SDF-1 (Theveneau and Mayor, 2011). In comparison to CTLMO explants, Cx43MO explants failed to migrate towards SDF-1 (Figure 1F; Supplemental Movie S1). Importantly, analysis of speed and directionality of individually migrating cells show that cell motility is not affected in Cx43MO cells (Figure 1F; Supplemental Movie S2, Figure S1E). Together these results support the notion that Cx43 is involved in the collective response required for NC migration, but not in single cell motility.

To examine how loss of Cx43 might affect cells during collective migration, we analysed the effect of Cx43MO on cell morphology. It has been previously shown that NCs form large protrusions away from cell contacts and that this polarity is required for correct NC migration (Carmona-Fontaine et al., 2008; Scarpa et al., 2015). Visualisation of actin and α-tubulin cytoskeleton showed shorter protrusions in Cx43MO cell clusters (Figure 2A, B). Consistently, analysis of cell shape demonstrates a circular and less polarised phenotype in Cx43MO cells when compared to CTLMO cells (Figure 2C). An additional way to test cell polarity is the localisation of Lamellipodin (Lpd), a regulator of Rac1 activity at the cell protrusions (Law et al., 2013). In CTLMO cells, Lpd-GFP showed higher levels at the tip of the lamella and lower levels in the cytosol and contact points, as previously described (Law et al., 2013). In contrast, in Cx43MO cells, Lpd-GFP levels were lower at the free edge and higher at the contact point, confirming that cell polarity is disrupted (Figure 2D). These observations support the involvement of Cx43 in contact-dependent polarity, and suggest an implication for contact inhibition of locomotion (CIL), which is an essential activity for directional collective cell migration (Stramer and Mayor, 2017). We tested CIL on NC cells and on NC explants. First, NC cells undergoing CIL move away from each other upon collision after a short time (10min), generating small collision angles (0o corresponds to perfect repulsion). Analysis of cell collisions indicated an increase in collision duration and collision angles in Cx43 depleted NCs compared to CTLMO cells, showing an effect on CIL (Figure 2E; Supplemental Movie S3). Second, when two explants of cells that exhibit CIL are cultured a short distance apart they never overlap, as it was observed for CTLMO explants, whereas Cx43MO explants showed an increase in their area of overlap, proving an impaired CIL response (Figure 2F). Third, one of the consequences of CIL is that it promotes cell dispersion. Indeed, CTLMO cells dispersed after 4-6hrs of culture, while cells injected with Cx43MO displayed a delay in cell dispersion(Figure 2G; Supplemental Movie S4). Importantly, Cx43MO explants were able to regain their momentum and show similar levels of dispersion to CTLMO when cultured for longer times (>10hrs). Taken together, our analysis in vivo and in vitro showed that perturbed Cx43 expression inhibits migration by altering cell polarity during CIL.

Figure 2.
Figure 2. Cx43 modulates cell polarity

(A) NC cell morphology visualised by lifeactinGFP (arrows mark normal and arrowheads irregular protrusions) and by (B) Immunostaining for α-tubulin. (C) Circularity index and cell morphology diagram (nCTLMO=58, nCx43MO=39, N=3, mean ± SE, p***<0.001). (D) Heatmap of Lpd-GFP NCs. Lpd-GFP fluorescence normalised to background levels along cell length (n=8, N=3, mean ± SE). (E) Single cell collisions (0 marks collision). Blue arrow: velocity before collision; green arrow: velocity after collision; the angle between these vectors is shown in black. Quantification of CIL (n=23, N=5, mean ± SE, p**<0.01) and of collision angle (median ± interquartile range, p**<0.01). (F) Explant confrontation assay (bottom thresholded images of top panels) and quantification of overlap index (nCTLMO=12, nCx43MO=11, N=3 median ± interquartile range, p***<0.001). (G) Cell dispersion analysis using Delauney triangulation and its quantification (n=15, N=3, mean ± SE, p***<0.001, p**<0.01, p*<0.05).

Cx43 regulates n-cad expression

In order to understand how Cx43 affects cell polarity we decided to investigate other molecules linked to this process. N-cad, for example, is present at cell contacts during NC migration and is critical for cell polarisation during CIL and collective migration (Theveneau et al., 2010; Kuriyama et al., 2014). Interestingly, immunostaining of NC cells revealed that in comparison to control conditions, in which N-cad is expressed and localised at cell contacts, Cx43MO explants show an N-cad reduction at cell contacts (Figure 3A). This observation was confirmed by western blot analysis that shows a decrease in the overall N-cad protein levels in Cx43MO embryo lysates when compared to CTLMO lysates (Figure 3B).

Figure 3.
Figure 3. Cx43 regulates n-cad expression

(A) Immunostaining for N-cad. Summary of N-cad analysis. N-cad levels, 0 represents contact point (n=15, N=3, mean± SE, p***<0.001). (B) WB for N-cad and Mapk. N-cad levels normalised to Mapk (N=5, mean± SE, p**<0.01). (C) twist ISH of st24 embryos (arrowheads show normal migration, brackets impaired migration, asterisk the eye). Migration index and percentage of embryos with NC migration (nCTLMO=22, nCX43MO=24, nCx43MO+n-cad=21, N=3, median ± quartile, mean ± SE p***<0.001). (D) Cell dispersion analysis after 6hrs of culture using delauney triangulation and its quantification (n=10, mean ± SE, p***<0.001, p**<0.01). (E) NCs expressing nRFP+mRFP (white arrows show normal protrusions, arrowheads short protrusions, green arrows N-cadGFP at cell membrane) and quantification of cell morphology (nCTLMO=51, nCx43MO=32, nCx43MO+n-cadGFP=58, median ± interquartile range, p***<0.001). (F) qPCR of n-cad expression (N=8, mean ± SE, p**<0.01). (G) n-cad ISH, arrowheads indicate normal and bracket irregular expression, white arrow injected side and green segmented line the NC. Quantification of n-cad expressing embryos (nCTLMO=68, nCX43Mo=61, N=9, mean ± SE, p***<0.001). (H) Rate of n-cad degradation (N=4, mean ± SE). See also Figure S2

Figure S2.
Figure S2. Related to Figure 3.

(A) Summary of the actinomycin D treatment. MO injection at the 2-cell stage, followed by actinomycin D treatment at stage 20, and collection of samples at the indicated times. (B) qPCR plotted as relative n-cad expression over time with 4μg/ml actinomycinD treatment (N=4). (C) Immunostaining for N-cad using dmso or GJ blockers FFA and MFA. N-cad levels, 0 represents contact point (n=15, N=3, mean ± SE). (D) qPCR plotted as a relative n-cad expression (N=3, mean ± SE).

Since our experiments suggest that Cx43 acts as regulator of NC migration via n-cad, we tested whether we could rescue the absence of Cx43 by restoring n-cad levels. Co-injection of Cx43MO with n-cad mRNA leads to a perfect rescue of the in vivo migration defects induced by the Cx43MO alone (Figure 3C). Moreover, the dispersion of NC explants, inhibited by Cx43MO, was rescued by co-injection of n-cad mRNA (Figure 3D; Supplemental Movie S5). Finally, when exogenous n-cad-GFP was delivered together with Cx43MO, we observed N-cad localisation at cell contacts, restored cell polarity and cell morphology that was similar to that found in CTLMO cells (Figure 3E). In conclusion, these results support the notion that Cx43 controls cell migration by regulating n-cad expression.

To determine whether Cx43 controls N-cad by regulating the levels of n-cad mRNA, we performed qPCR and ISH against n-cad mRNA. Both methods showed a significant reduction on n-cad mRNA in Cx43MO embryos (Figure 3F, G), suggesting that Cx43 regulates n-cad mRNA levels by either stabilising n-cad mRNA or promoting n-cad transcription. To investigate the first hypothesis, we measured mRNA stability by performing qPCR in the presence of the transcriptional inhibitor actinomycin D (Figure S2A-B). We found a similar rate of n-cad mRNA degradation between CTLMO and Cx43MO embryos (Figure 3H), indicating that Cx43 does not regulate mRNA stability, and arguing for a regulation of n-cad transcription.

Since Cx43 is present in two forms: the full length molecule (Cx43FL), which are building component of GJs, and the smaller carboxy terminal fragments (Cx43iso) that are not part of the GJ structures, we asked which of these two forms was responsible for the control of n-cad expression. One possibility is that the Cx43FL form regulates n-cad expression through GJ activity. In order to test this hypothesis, we measured N-cad expression in cells or embryos treated with two different GJ blockers. By analysing immunostaining in NC cultures, N-cad appeared unaltered in NC explants treated with flufenamic acid (FFA) or meclofenamic acid (MFA) in comparison to controls incubated with DMSO (Figure S2C). Furthermore, qPCR analysis showed that n-cad mRNA was unaffected by the use of GJ blockers (Figure S2D). These results suggest that the GJ activity is not involved in n-cad regulation, and open the possibility for a role of the Cx43iso as transcriptional regulators of n-cad.

Cx43Tail controls n-cad transcriptional regulation and this activity is linked to its presence at cell nucleus

After discovering that GJ channels are not involved in the regulation of n-cad, we investigated other routes of Cx43 activity. To test which domain of Cx43 is involved in n-cad regulation, we co-injected Cx43MO with a full-length (Cx43FL), a truncated form missing the carboxy terminal (Cx43Trun), or a carboxy tail construct (Cx43Tail) (Figure 4A). Hence, the Cx43 form that is able to rescue the morphant phenotype contains important signaling involved in n-cad regulation. Co-injection of Cx43MO with Cx43FL or Cx43Tail mRNA rescued migration defects, showing a phenotype similar to the CTLMO group, while co-injection with the Cx43Trunc failed to rescue in vivo migration defects (Figure 4B). Moreover, similar to the effect on migration, n-cad deficiencies in Cx43MO embryos was rescued by the Cx43FL and Cx43Tail, but not by the Cx43Trunc mRNA (Figure 4C-4D; S3A). Importantly, when analysing cell polarity, we found that the inhibition on cell polarity induced by Cx43MO can be rescued by co-injection of Cx43FL and Cx43Tail, but not by Cx43Trunc (Figure 4E). Together, these results show that Cx43Tail is sufficient to promote migration by regulating n-cad levels and cell polarity.

Figure S3.
Figure S3. Related to Figure 4.

(A) Lateral view of embryos showing n-cad ISH of the same embryos shown in dorsal view in Figure 4D and (B) from Figure 4H. The injected and injected side of the same embryo are shown for comparison.

Figure 4.
Figure 4. Cx43Tail is the n-cad transcriptional regulator

(A) Diagram of Cx43 constructs used. (B) Quantification of normal NC migration (% of embryos), twist ISH of st24 embryos (arrowheads display normal, bracket impaired migration, asterisk the eye) and NC migration index (nCTLMO=22, nCx43MO=41, nCx43MO+FL=21, nCx43MO+Trunc=16, nCx43MO+Tail=30, N=4, mean ± SE, median ± quartile, p***<0.001). (C) qPCR plotted as normalized n-cad expression (N=4, mean ± SE, p**<0.01). (D) n-cad ISH of st24 embryos, arrow shows injected side and green line the NC area. Quantification of n-cad expressing embryos (nCTLMO=42, nCx43MO=42, nCx43MO+FL=13, nCx43MO+Trunc=15, nCx43MO+Tail=19, N=4, mean ± SE, p***<0.001). (E) NCs with nuclear (green) and membrane probes (magenta), arrows indicate normal and arrowheads short protrusions. Circularity index (nCTLMO=20, nCx43MO=32, nCx43MO+Cx43FL=32, nCx43MO+Cx43Trunc=23, nCx43MO+Cx43Tail=26, median ± quartile, p***<0.001). (F) qPCR for relative n-cad expression of animal caps (N=4, mean ± SE, p*<0.05).(G) n-cad ISH of animal caps and st9 embryos, squares in the embryos indicate the dissected animal cap area. Quantification of n-cad expressing embryos (animal caps: nCTLMO=14, nCx43Tail=45, st9 embryos: nCTL=22, nCx43Tail=39, N=3, mean ± SE, p***<0.001, p**<0.01). (H) n-cad ISH of st24 embryos, arrow shows injected side and green line the NC area. Quantification of n-cad expressing embryos (st24 embryos: nCTL=23, nCx43Tail=42, N=4, mean ± SE, p***<0.001). See also Figure S3.

To test whether Cx43Tail has the ability to induce n-cad expression, we used Cx43Tail in overexpression experiments in three different systems: in blastula embryo (when n-cad is not normally expressed), in blastula ectoderm (called animal caps, which do not express n-cad; Aybar et al., 2003; Chakrabarti et al., 1992), and in neurula embryos (which express n-cad). In all these systems we observed an upregulation of n-cad when overexpressing Cx43Tail (Figure 4G-4H; S3B). Moreover, qPCR analysis of Cx43Tail animal cap explants confirmed our results, also showing n-cad upregulation (Figure 4F). Taken together, these data show that the Cx43Tail is necessary and sufficient to regulate n-cad expression.

Cx43Tail could indirectly regulate n-cad expression, for example by modifying some signalling pathway, or it could work directly as a transcriptional regulator of n-cad. If Cx43 works as a transcriptional regulator, we would expect to find evidence of its localisation in the nucleus. We, therefore, investigated the subcellular localisation of Cx43 by expressing a low dose of Cx43Tail-GFP in the NC and observed a clear nuclear localisation of the tagged protein (Figure 5A). Similar nuclear localisation of Cx43Tail was obtained in mammalian Hela cells (Figure S4A) and amphibian XTC cells, using an antibody that reconginzes the carboxyl terminar (Figure S4F). To confirm these findings with endogenous Cx43 tail fragments (Cx43iso), we performed subcellular fractionation of Xenopus embryonic cells (Figure S5). Strikingly, Cx43iso was highly enriched at the nuclear fraction in comparison to all other fractions (Figure 5B).

Figure S4.
Figure S4. Related to Figure 5.

(A) Hela cells transfected with Cx43 constructs tagged with GFP (green). GFP nuclear fluorescence normalised to whole cell fluorescence (n=8, N=2, mean ± SE, p***<0.001). (B) PCR of Hela for Cx43 constructs using gapdh, cx43 and n-cad primers. PCR graph showing n-cad levels normalised to gapdh. (C) Immunostaining of Hela cells against N-cad. Quantification of N-cad levels (nEGFP=20, nCx43Tail=15, mean ± SE, p***<0.001, p**<0.01). (D) Hela-EGFP and Hela-Cx43TGR-GFP cells treated with dmso or dxm. Cx43TGR-GFP nuclear fluorescence normalised to whole cell fluorescence (nCx43TGR/dmSo=15, nCx43TGR/dmso =12, N=2, mean ± SE, p***<0.001). (E) PCR of Hela using gapdh, cx43 and n-cad primers. PCR graph showing n-cad levels normalised to gapdh. (F) Immunostaining of untrasfected XTC cells for Cx43. (G) Immunostaining of XTC cells against N-cad. Quantification of N-cad levels (nEGFP=17, nCx43Tail=17, mean ± SE, p**<0.01).

Figure S5.
Figure S5. Related to Figure 5.

(A) Diagram of subcellular fractionation (B) WB showing subcellular fractionation (see Figure 5B) of wild type X. embryos using (a) 400xg, (b) 600xg, (c) 800xg as the initial spin speed for nuclear pellets and Cx43, phopho-histone H3 (p-H3, nuclear control), E-cadherin (E-cad, membrane control) or Mapk (cytosolic control).

Figure 5.
Figure 5. Cx43Tail nuclear localisation is requisite for its function

(A) Cx43Tail-GFP injected NCs and fluorescence along cell length (n=6, N=2). (B) Subscellular fractionation WB for Cx43, p-H3 (nuclear marker), E-cad (membrane marker) and Mapk (cytosolic marker). Cx43iso enrichment in proportion to Cx43FL. (C) Diagram of Cx43TGR-GFP function. (D) NCs expressing Cx43TGR-GFP +/-dxm. Cx43TGR nuclear fluorescence normalised to whole cell fluorescence (ndmso=23, ndxm=29, N=3, mean ± SE, ***p<0.001). (E) twist ISH of st23 embryos (arrowheads display normal migration, brackets impaired migration, asterisk the eye) and quantification of NC migration (nCTLMO=18, nCx43MO=22, nCx43MO+Cx43TGR/dmso= 18, nCx43MO+Cx43TGR/dxm=19, N=4, mean ± SE, median ± interquartile range, p***<0.001). (F) n-cad ISH, white arrow shows injected side and green line the NC. Quantification of n-cad expressing embryos (nCTL=16, nCx43MO=13, nCx43MO+Cx43TGR/-dxm=9, nCx43MO+Cx43TGR/+dxm=13, N=3, mean ± SE, p**<0.01). (G) qPCR for relative n-cad expression (N=5, mean ± SE, p*<0.05). (H) Quantification of n-cad expressing embryos (-CHX: nCTL=35, nCx43TGR/-dxm=41, nCx43TGR/+dxm=28, +CHX: nCTL=27, nCx43TGR/-dxm=23, nCx43TGR/+dxm=25, N=3, mean ± SE, p***<0.001, p**<0.01). n-cad ISH for st9 embryos. See also Figures S4 and S5.

To investigate if the nuclear localisation of Cx43 is required for its transcriptional function, we used an inducible system that has been previously described (Tribulo et al., 2003). We fused the binding domain of the human glucocorticoid receptor (GR) with GFP tagged Cx43Tail (Cx43TGR-GFP, Figure 5C), allowing this construct to enter the nucleus only in the presence of dexamethasone (dxm). Indeed, dxm treated NCs displayed strong nuclear localisation of Cx43TGR (Figure 5D, +dxm), whereas its localisation was predominantly cytosolic in control NCs (Figure 5D, -dxm). These findings were mirrored in experiments performed on Hela cells (Figure S4D). To test the role of Cx43Tail nuclear localisation during migration, we used the inducible Cx43TGR system in NC migration and n-cad expression experiments. In both cases, we were able to rescue the morphant phenotype (reduced migration and decreased n-cad expression) when Cx43TGR was allowed to enter the nucleus using dxm treatment (Figure 5E-G). Similarly, in Hela or XTC cells, Cx43Tail overexpression led to elevated n-cad levels (Figure S4B, C, G), an effect similar to that found with the inducible system in Hela cells (Figure S5E). These data highlight the requirement for Cx43Tail in the nucleus to control n-cad transcription in different vertebrate systems, including Xenopus embryos and Xenopus and mammalian cell lines.

Our findings indicate that nuclear localisation of Cx43 is important for its function in promoting N-cad expression, yet it is not clear whether Cx43 regulates N-cad expression directly or whether it acts through an intermediary cascade of gene expresssion. To answer this question, we blocked all protein synthesis using the translation inhibitor cycloheximide (CHX), if Cx43 is not a direct regulator of N-cad transcription, it would require the transcription/translation of other proteins, and therefore any effect induced by Cx43 should be inhibited by CHX treatment. Cx43TGR-injected embryos treated with CHX, followed by dxm addition, showed an upregulation of n-cad expression, similar to non-CHX treated embryos (Figure 5H), therefore suggesting a direct effect of Cx43Tail on the n-cad transcriptional regulation.

Cx43 requires BTF3 to function and localise at the nucleus

In order to further investigate the mode of direct regulation of Cx43 on n-cad, we performed mass spectrometry analysis of Hela cells transfected with Cx43Tail construct.

This provided a list of Cx43Tail binding partners that could be involved in n-cad regulation. Using 1.3-fold change between IP and control samples as the minimal limit for consideration (Gentzel et al., 2015), we found 30 candidates that are nuclear proteins and interact with Cx43Tail (Figure 6A). Examination of the Xenopus database, Xenbase, revealed that 18 of these candidates are expressed ubiquotisly in Xenopus laevis and only one is specifically expressed in migrating NC (Figure 6A): the basic transcription factor 3 (BTF3) (Cavallini et al., 1988; Kusumawidjaja et al., 2007; Zheng et al., 1990), which is enriched more than 1.49-fold in the IP sample in comparison to control.

Figure 6.
Figure 6. Cx43Tail requires BTF3 to function

(A) Summary of mass spectrometry results. Left: Pie chart of Cx43Tail interactants. Rigth: Venn diagram of interactants found in nucleus. (B) btf3, twist and n-cad ISH of st24 embryos, arrowheads show NC, arrows the neural tube and asterisk the eye. Summary of btf3, twist and n-cad profile. (C) twist ISH and quantification of NC migration (nCTLMO=19, nBTF3MO=36, nBTf3MO+BTF3FL=23, N=4, mean ± SE, p***<0.001, p**<0.01). (D) n-cad ISH. Quantification of n-cad expressing embryos (nCTLMO=14, nBTF3MO=35, nBTf3MO+BTF3FL=13, N=3, mean ± SE, p***<0.001). (E) n-cad ISH for st9 embryos. Quantification of n-cad expressing embryos (nCTL=24, nCx43Tail=12, nCx43Tail+BTF3MO=25, N=3, mean ± SE, p***<0.001, p**<0.01). (F) Cx43TGR-GFP NCs during dxm treatment. n-RFP is used to vislaize the nucleous. Heatmap of Cx43TGR-GFP intensity in NC. Cx43TGR-GFP nuclear fluorescence normalised to whole cell fluorescence (n=10, N=2, mean ± SE, p***<0.001). (G) Heatmap Cx43Tail-GFP intensity in NCs. (H) Diagram of BTF3 constructs. Quantification of Cx43Tail-GFP nuclear localisation (nCTLMO=17, nBTF3MO=8, nBTF3MO+btf3FL=12, nBTF3MO+btf3dNLS=15, N=3, mean ± SE, p***<0.001). See also Figure S6.

Figure S6.
Figure S6. Related to Figure 6.

(A) Immonostaining for BTF3. Fluorescence levels of BTF3 normalised to background levels (nCTLMO=23, nBTF3MO=27, N=3, mean ± SE, p***<0.001, p**<0.01). (B) snail2 ISH for stage 16 X. laevis embryos, arrow indicates the injected side. Quantification of NC induction (nCTLMO =15, nBTF3MO=10, N=2, mean ± SE). (C) Lateral view of n-cad ISH from embryos from Figure 6E. (D) Immunostaining for BTF3. BTF3 nuclear fluorescence normalised to whole cell fluorescence (nCTLMO=20, nCx43MO=20, N=2, mean ± SE).

We validated btf-3 expression pattern by ISH and confirmed that btf3 is expressed at NC (Figure 6B), similar to twist, Cx43 and n-cad, with additional expression in eyes and neural folds matching the expression of n-cad and Cx43. This temporal and spatial co-localisation in the embryo and subcellular localisation in the nucleus suggests that Cx43Tail and BTF3 are potentially involved in the same pathway. To test whether BTF3 is involved in the same process as Cx43, we performed knock-down experiments using a BTF3 morpholino (BTF3MO), after confirming its efficiency (See star Methods, Figure S6A). Similar to Cx43MO, BTF3MO perturbed NC migration and n-cad expression (Figure 6C, D; S6C). Coinjection of BTF3MO with btf3FL that does not bind the MO, rescued impaired migration and n-cad expression (Figure 6C,D), thereby ensuring the specificity of BTF3MO. The effect of BTF3MO is specific for NC migration and does not affect NC induction, as shown by snail2 ISH (Figure S6B). The similar phenotypes observed with BTF3 and Cx43 loss-of-function suggest that these molecules work together. Analysis of n-cad expression in blastula embryos showed that while embryos injected with Cx43Tail present a clear induction of n-cad expression, co-injection of BTF3MO with Cx43Tail significantly decreased this effect (Figure 6E), indicating that BTF3 is required for Cx43Tail activity.

Since Cx43 nuclear localisation is requisite for n-cad expression, we investigated the effect of BTF3MO on this process. For this purpose, we performed a temporal analysis of Cx43TGR nuclear localisation after addition of dxm in presence or absence of BTF3. Control NC showed a clear increase in the leves of Cx43Tail nuclear localization after dxm addition, while these levels remain lower in BTF3 depleted cells (Figure 6F), indicating that the nuclear localisation of Cx43Tail is BTF3 dependent. The converse, however, is not true: inhibition of Cx43 by Cx43MO does not affect the nuclear localisation of BTF3 (Figure S6D). These results suggest that BTF3 is localised in the nucleus by default, while Cx43 requires a partner to control its subcellular localisation. These observations are consistent with the presence of a nuclear localisation signal (NLS) in BTF3 (Wang et al., 2014) and its absence in Cx43Tail, and prompted us to test the role of the NLS of BTF3 on the nuclear localisation of Cx43Tail. As described above, nuclear localisation of Cx43 is impaired by BTF3MO but can be rescued by BTF3FL (Figure 6G). In contrast, the expression of a BTF3 construct lacking its NLS (Figure 6H) is unable to rescue Cx43Tail nuclear localisation (Figure 6G). Altogether these data indicate that one role of BTF3 is to recruit Cx43Tail into the nucleus by providing it with an NLS.

Cx43tail and BTF3 directly interact and promote n-cad gene regulation

Co-localisation of BTF3 and Cx43Tail in the nucleus (Figure 7A) suggests interaction and a common role in that cell compartment, giving rise to the hypothesis that BTF3 interacts directly with Cx43. To test for physical interaction between Cx43 and BTF3, we used the bimolecular fluorescence complementation system (BiFC), in which two VENUS (variant of enhanced yellow fluorescent protein) components (VC and VN9m) form a fluorescent protein when are brought into close apposition (Saka et al., 2007). To build the BiFC system we fused the VENUS component (VC) to either Cx43Tail, Cx43Trunc, and the other VENUS component (VN9m) to BTF3, empty VC was used as a control. These constructs were co-injected into the NC along a nuclear marker, observing that Cx43TailVC co-injected with BTf3VN9m returned a positive signal, but not the other combinations (Figure 7B). Moreover, this signal was higher in the nucleus, where both molecules are typically found, therefore supporting our hypothesis of direct interaction between Cx43 and BTF3.

Figure 7.
Figure 7. Cx43Tail and BTF3 form a transcriptional complex

(A) Fluorescence along cell length (n=9, N=2). Anti-BTF3, Cx43Tail-GFP and Dapi in NCs. (B) BiFC system. Diagram of constructs and function. NCs coexpressing VENUS components. VENUS efficiency and BiFC nuclear signal normalised to whole cell signal (n=16, N=3, mean ± SE). (C) WB of IP for GFP, Pol II, HA and normalised intensity graph. (D) ChIP-Pol II PCR for n-cad promoter. ChIP signal normalised to input (N=2). (E) ChIP-GFP PCR for n-cad promoter. ChIP signal normalised to input (N=2). (F) Cx43Tail (green), BTF3 (red), Pol II (brown) n-cad (black). (G) Control (cyan) and Cx43 depleted (orange) NCs, N-cad (black). (H) Embryos with CTLMO NC (light blue) and Cx43MO NC (orange). See also Figure S7.

Figure S7.
Figure S7. Related to Figure 7.

Putative TATA boxes on partial n-cad promoter region form Xenopus laevis obtained using ElemeNT Analysis Resource (highlited in yellow) and binding sites for the primers used in the ChIP experiments.

To verify the biochemical interaction of Cx43Tail and BTF3, we performed immunoprecipitation (IP) using BTF3-GFP or GFP (as control) in conjunction with GFPTrap beads. We found that Cx43Tail-HA co-precipitates with GFP in BTF3-GFP expressing cells, but not in control GFP expressing cells (Figure 7C). Our experiments also confirmed that BFT3 interacts with Pol II (Figure 7C), as it has been previously shown (Zheng et al., 1987). These results support the notion that the three proteins, Cx43Tail, BTF3 and Pol II, interact and may function as a transcriptional complex.

To test this possibility, we performed chromatin immunoprecipitation (ChIP) using embryonic lysates and a Pol II antibody. We examined whether Pol II binds to the promoter of n-cad in presence or absence of Cx43, by performing PCR against the n-cad promoter region using CTLMO and Cx43MO ChIP samples as templates (see StarMethods). We detected that Pol ll is enriched at the n-cad promoter in CTLMO ChIP samples, whereas this enrichment was drastically reduced in ChIP samples collected from Cx43MO-injected embryos (Figure 7D). We conclude, therefore, that Cx43 is required for Pol II to bind at the n-cad promoter region so as to drive n-cad transcription.

To test whether Cx43Tail is directly recruited to the n-cad promoter we performed ChIP using Cx43TailGFP or Cyto-GFP (as a negative control) embryonic lysates and a GFP antibody. ChIP-PCR analysis showed that Cx43Tail is enriched at n-cad promoter region (Figure 7E). Therefore, we propose that Cx43Tail together with BTF3 and Pol II form a complex and control the transcriptional activity of n-cad.

Discussion

Although Cx43 is mostly known for its function in GJs, some studies have suggested that it may also be involved in gene regulation independent of its GJ activity (Giepmans, 2006; Kang et al., 2009). These studies propose an indirect role for Cx43 in which it acts on other gene regulators, such as members of the CCN family (Fu et al., 2004; Gupta et al., 2001) or Smads (Takamatsu et al., 2007). Our findings identify a different mechanism, in which the Cx43Tail is directly involved in gene regulation. We found that Cx43Tail fragments are present in the NC of the developing embryo, as it has been shown in other cell types (Salat-Canela et al., 2014; Smyth and Shaw, 2013; Ul-Hussain et al., 2014). Using a combination of in vivo studies in the Xenopus embryo and experiments in other vertebrate cell systems, we characterized the physiological role of Cx43Tail signalling and uncovered a direct role in regulating n-cad expression. We further identified the molecular mechanism responsible for Cx43-driven n-cad regulation and showed how this activity is crucial during tissue morphogenesis.

Our findings provide important insight into this process and identify a previously unknown role for a protein primarily associated with cell-cell signalling. Specifically, we propose the following mechanism: Cx43Tail binds to BTF3 and, together, these proteins translocate into the nucleus where they form a complex with Pol II; this, binds to the n-cad promoter and activates n-cad transcription (Figure 7F). On the cellular level, once N-cad is transcribed and translated, it localises at cell contacts where it is known to modulate the polarization of Rac1 activity during CIL (Figure 7G; Theveneau et al., 2010; Scarpa et al., 2015). CIL in turn is required for the directional and collective migration of the NC, a process which is essential for morphogenesis (Figure 7H).

It is interesting to note that Cx43 has previously been shown to function as an adhesive molecule (Elias et al., 2007, 2010; Elzarrad et al., 2008), suggesting a direct role in controlling cell movement. Our findings highlight an alternative mechanism, independent of a direct involvement of Cx43 in cell adhesions. However, it is tempting to speculate that these functions may work in concert during cell migration.

In previous studies, it has been proposed that the generation of Cx43Tail isoforms is controlled by the PI3K/AKT/mTOR pathway in mammalian cells (Smyth and Shaw, 2013), the Mnk1/2 pathway in various cancer cell lines (Salat-Canela et al., 2014) and hypoxic conditions (Ul-Hussain et al., 2014). It is plausible that similar mechanisms are at play in our system. Indeed, in NC, the PI3K/AKT/mTOR pathway is present (Nones et al., 2012) and could, therefore, be implicated for the generation of Cx43Tail. It is also possible that Cx43Tail is produced in response to hypoxia and the Hif-1alpha pathway (hypoxia-inducible transcription factor-1), which are known to regulate NC migration (Barriga et al., 2013). Our study shows that the appearance and function of the Cx43 carboxy tail isoforms in the migrating NC coincide in time and space with the activity of Hif-1alpha, supporting findings in mammalian cells (Ul-Hussain et al., 2014).

Our results show that Cx43 control cell polarity, a finding that is consistent with known activities of Cx43 in cardiac NC (Xu, 2001; Xu et al., 2006). Cx43 has also been implicated in cell migration and polarity in other cell types (Elias et al., 2010; Homkajorn et al., 2010; Xu et al., 2006). In mouse embryonic fibroblasts this was attributed to the interaction of full-length Cx43 with microtubules (Francis et al., 2011). Although we found the cytoskeleton to be affected in Cx4MO NCCs, we observed that Cx43Tail alone has the ability to recover normal cell polarity and morphology. While we cannot exclude a direct effect of full-length Cx43 on microtubule organisation in our system, our findings show a different mechanism, in which Cx43 controls cell polarity and the cytoskeleton via the regulation of n-cad levels. Thus, we have established a direct link between Cx43 and contact-dependent cell polarity, which is likely to be conserved in other cellular system.

Cx43 appears to be at a convergent point of cell communication, cell polarity and n-cad regulation, but its effect on N-cad varies depending on context. Xu and colleagues have shown that Cx43 depletion led to N-cad decrease in cardiac NC in mice (Xu, 2001), while this was not the case in mouse neuronal cells derived from the developing cortex (Elias et al., 2007). This differential response could be explained by the presence of different pathways linked to Cx43 - one involving Cx43 full-length and another involving Cx43Tail. In this situation, factors controlling Cx43Tail production would define the composition of adhesion molecules at cell contacts, and this could be tuned differently in a time- and context-dependent manner.

We have identified a novel pathway by which an adherens junction component is regulated by a protein normally associated with a different cellular function, opening the possibility of cross-talk between processes mediating intercellular communication and cell-cell interaction and a possible mechanism of cross-regulation between gap and adherens junctions at the transcriptional level. Importantly, the high conservation of Cx43 and the presence of Cx43Tail in various vertebrate systems may reflect an evolutionary mechanism, where cells employ either one or the two pathways to control cell polarity during cell migration. In this study, we uncovered the Cx43Tail pathway, which displays a distinct function independent of the activities produced by Cx43 full-length, and provides a link between the regulation of N-cad expression and polarity defects. In the light of this new evidence, the role of Cx43 in collective migration during development or disease could be re-approached.

Author Contributions

MK and RM conceived the study and designed experiments, with additional input from EHB. MK performed most experiments and data analysis. EHB helped with the bifc, promoter and Chip experiments. JL assisted with cloning. AS helped with cell line experiments; MG performed the MS analysis. MK and RM wrote the manuscript with input from all authors.

Supplemantal Movies

Movie S1. Cx43 is required for collective NC migration. Related to Figure1.

Example of chemotaxis assay with CTLMO NC explants (left side) displaying directional migration towards the chemotactic cue SDF-1 and Cx43MO NC explants that do not migrate towards the SDF-1 bead (right side). Green is membrane-GFP and red/orange is nuclear-mCherry. Frame delay 10 min. Magnification 10X.

Movie S2. Cx43 is not involved in NC cell motility. Related to Figure1.

Example of single cell tracks with CTLMO NC cells in cyan (left side) and Cx43MO in yellow(right side) and individual cell teacks. Cyan and yellow are membrane-GFP and magenta is nuclear-mCherry. Frame delay 5 min. Magnification 10X.

Movie S3. Cx43 depletion inhibits CIL. Related to Figure2.

Example of cell collision assay with CTLMO NC cells undergoing CIL (left side) and Cx43MO NC cells that stay longer in contact and do not exhibit CIL (right side). Cyan and yellow are membrane-GFP and magenta is nuclear-mCherry. Arrows display direction of each cell that is tracked. Frame delay 5 min. Magnification 20X.

Movie S4. Cx43 inhibition delays NC explant dispersion in vitro. Related to Figure2.

Example of cell dispersion assay with CTLMO NC explants dispersing over time(left side) and Cx43MO NC explants showing a later dispersion (right side). Red is nuclear-mCherry. Frame delay is 10 min. Magnfication 20X.

Movie S5. Perturbation of cell dispersion in Cx43MO explants is rescued by n-cad. Related to Figure3.

Example of cell dispersion assay. After 6hr of culture, CTLMO NC explants disperse (left), Cx43MO NC explants do not disperse (middle) and Cx43MO+n-cad mRNA NC explants disperse (right). Red is nuclear-mCherry. Frame delay is 1 hr. Magnfication 20X.

Acknowledgements

We thank A. Streit (King’s College London) for comments on the manuscript. J. Smith (Crick’s Institute) for providing us with the bifc vectors and D. Becker (University College London) for help in the initial experiments on connexins. This study was supported by grants from MRC (J000655 and M010465), BBSRC (M008517) and Wellcome Trust to RM. MK was supported by a Latsis Postgraduate Scholarship, a DAAD short term research grant and travel grants from Boehringer Ingelheim Funds and the Company of Biologists. EHB was supported by fellowships from EMBO LTF-971 and a Marie Skłodowska Curie IF-2014_ST 658536.

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Posted March 06, 2017.
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Connexin43 controls N-cadherin transcription during collective cell migration
Maria Kotini, Elias H. Barriga, Jonathan Leslie, Marc Gentzel, Alexandra Schambony, Roberto Mayor
bioRxiv 114371; doi: https://doi.org/10.1101/114371
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