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© The Rockefeller University Press,
0021-9525/2003/12/1291 $8.00
The Journal of Cell Biology, Volume 163, Number 6, 1291-1301
Article |
TGFß3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development
Address correspondence to Dr. Elizabeth D. Hay, Dept. of Cell Biology, Harvard Medical School, 220 Longwood Ave., B-1, Room 342, Boston, MA 02115-6092. Tel.: (617) 432-1651. Fax: (617) 432-0407. email: ehay{at}hms.harvard.edu
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Abstract |
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Epithelial mesenchymal transformation (EMT) of the medial edge epithelial (MEE) seam creates palatal confluence. This work aims to elucidate the molecular mechanisms by which TGFß3 brings about palatal seam EMT. We collected mRNA for PCR analysis from individual transforming MEE cells by laser microdissection techniques and demonstrated that TGFß3 stimulates lymphoid-enhancing factor 1 (LEF1) mRNA synthesis in MEE cells. We show with antisense ß-catenin oligonucleotides that up-regulated LEF1 is not activated by ß-catenin in palate EMT. We ruled out other TGFß3 targets, such as RhoA and MEK1/2 pathways, and we present evidence using dominant-negative Smad4 and dominant-negative LEF1 showing that TGFß3 uses Smads both to up-regulate synthesis of LEF1 and to activate LEF1 transcription during induction of palatal EMT. When phospho-Smad2 and Smad4 are present in the nucleus, LEF1 is activated without ß-catenin. Our paper is the first to show that the Smad2,4/LEF1 complex replaces ß-catenin/LEF1 during activation of EMT in vivo by TGFß3.
Key Words: LEF1 transcription; Smad2; palate confluence; ß-catenin; Smad pathways
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Introduction |
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The evidence for EMT of the MEE seam consists of cytological descriptions and cell-tracing experiments showing that the adherent seam breaks up into islands that become mesenchyme (Fitchett and Hay, 1989; Griffith and Hay, 1992; Shuler et al., 1992; Sun et al., 1998, 2000). The source of the mesenchymal cells could be identified with transmission electron microscopy (TEM) by the remnants of epithelium carried on their surfaces as they detached, e.g., desmosomes (Fitchett and Hay, 1989). The transforming epithelial cells send out filopodia that disrupt the basement membrane. The cells become elongated in cell shape and migrate into preexisting connective tissue, acquiring all the cytological features of mesenchymal cells (Fitchett and Hay, 1989). Griffith and Hay (1992) used carboxyfluorescein (CCFSE) for TEM, which is taken up by epithelia and packaged into insoluble isolation bodies. The latter allow labeled cells transforming from the MEE seam to be identified as epithelial in origin by TEM (Griffith and Hay, 1992). The advantage of CCFSE is that it is never transferred from cell to cell (Sun et al., 2000). Thus, elongated cells with mesenchymal morphology derived from MEE are easily identified by TEM some distance from the midline in confluent palates (Griffith and Hay, 1992). Shuler et al. (1992) used DiI to document this transformation by light microscopy.
Thus far, the only major signaling pathway that has been consistently been shown to be directly involved in transformation of epithelium to mesenchyme is ß-catenin/lymphoid-enhancing factor 1 (LEF1; Hay, 2003; Kim et al., 2002), but whether or not ß-catenin will activate LEF1 depends on specific isoforms of LEF/TCF transcription factor that contain an alternative COOH-terminal "E" tail (Atcha et al., 2003). This restriction raises the possibility of LEF1 activation by other non-ß-catenin mechanisms. Recent papers by Labbe et al. (2000) and Nishita et al. (2000) have shown that LEF1 can also be activated by other factors, such as Smads. Another transcription factor that has been implicated is Snail by repressing E-cadherin (Cano et al., 2000). Previous works showed that activation of ß-catenin/LEF1 is associated with c-Fos–induced EMT in mammary cells (Eger et al., 2000; El-Tanani et al., 2001) and with ILK-mediated EMT (Novak et al., 1998), as well as with metastasis in carcinomas (Morin et al., 1997; El-Tanani et al., 2001). Kim et al. (2002) demonstrated that LEF1 administered in an adenovirus directly induces EMT in colon carcinomas, and we show here that LEF1 is present in the epithelium of maxillary processes preparing to undergo EMT. LEF-/- mice have severe craniofacial deformities (Duan et al., 1999), suggesting that the LEF1 gene plays an important role in cranial embryogenesis.
TGFß is a secreted cytokine that has a diversity of biological effects including pivotal roles during embryonic development (Nakajima et al., 1994; Boyer et al., 1999). Induction of EMT by TGFß has been studied in vitro in many different epithelial cells types, including mouse mammary cell lines (Miettinen et al., 1994; Piek et al., 1999) and human keratinocytes (Zavadil et al., 2001). In vivo, TGFß plays a role in cardiac valve induction and correlates with EMT (Runyan et al., 1992). The TGFß homodimer signals through the Smad pathway using transmembrane serine/threonine kinase receptors designated as TGFß type I (TßRI) and type II (TßRII) receptors (Lin et al., 1992; Wrana et al., 1992; Lutz and Knaus, 2002). Phospho-Smad3 is not present in the MEE (Cui et al., 2003), but phospho-Smad2 and 4 are well represented. Under ideal conditions, Smad2 phosphorylation and transport into the nucleus by Smad4 (Abdollah et al., 1997) is promoted by TGFß receptors (Itoh et al., 2003), early endosomes (Panopoulou et al., 2002), and Smad anchor for receptor activation (SARA) (Tsukazaki et al., 1998; Itoh et al., 2002). PI3 kinase, which can modulate SARA via FYVE, has also been shown to be involved in palatal EMT (Kang and Svoboda, 2002). In the nucleus, Smads bind to a specific DNA site (GTCTAGAC) and cooperate with various transcription factors in regulating target gene expression (Ten Dijke et al., 2002). The induced Smad2/Smad4 heteromeric complexes in polyamine-deficient cells are able to bind to this specific DNA site, suggesting that Smads mediate transcriptional activation (Liu et al., 2003).
Non-Smad pathways have also been implicated in TGFß signaling (Bhowmick et al., 2001; Roberts, 2002). There is evidence in vitro that TGFß induces mesenchyme-like cells containing actin stress fibers independently of Smads, using the Ras-Raf-MEK-ERK (Mulder, 2000) and RhoA-Rac-MAPK-JNK-Jun pathways (Bhowmick et al., 2001; Roberts, 2002). However, production of stress fibers does not define EMT (Hay, 1995). Yu et al. (2002) demonstrated that TGFß signaling through MAPK/MEK does not initiate EMT. Kaartinen et al. (2002) reported that activation of the RhoA kinase pathway is not sufficient for palatal EMT. TGFß-induced EMT was found to require TßR, and thus to use Smad (Itoh et al., 2003). By inhibiting RhoA and MEK pathways, we show that they are not involved in palatal EMT.
Our major objective is to elucidate the roles of TGFß3 and LEF1 during palatal EMT. We show that TGFß3 acts via LEF1 to induce mouse palatal EMT not only by up-regulating LEF1 mRNA, but also by transcriptional activity of LEF1. Without Smads, palatal EMT cannot be achieved by LEF1. There is little or no involvement of ß-catenin in the activation of palatal EMT by LEF1. This paper provides the first evidence of a Smad-activated LEF1 mechanism driving EMT in development. It is tempting to hypothesize that TGFß3-initiated Smad-dependent LEF1 pathways will prove to be the major regulators of embryonic EMT.
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In addition to Smad pathways, TGFß has recently been shown to induce EMT in vitro by two non-Smad pathways, RhoGTPase-Rac-MEK-JNK (Bhowmick et al., 2001) and Ras-Raf-MEK1/2-ERK (Janda et al., 2002). We used Clostridium botulinum C3 (hereafter referred to as C3) to disrupt RhoA by inducing ADP ribosylation of RhoA protein at Asn41, which inactivates downstream signaling of Rho GTPases (Zubiaur et al., 1995). We used MEK1/2 inhibitor U0126 (Favata et al., 1998) to explore a possible role of this pathway. With C3 or U0126, palatal shelves form MEE by 12 h (Fig. 1, G and J) and proceed (Fig. 1, H, I, and K) to complete the EMT by 60 h (Fig. 1 L), the same as normal palates (Fig. 1, A–D). Thus, MEK1/2 and RhoA have no detectable effects on TGFß3-mediated palatogenesis.
We evaluated the effects of these inhibitors by quantitating morphological data (Fig. 2). Fig. 2 shows the number of cells in the MEE seam at different times (12–60 h) of palate development. They were counted in cross sections of frozen palates using Leica software for laser capture microdissection (LCM; see Materials and methods). A section of untransformed seam contains 62–96 epithelial cells with an average of 76. We staged palatogenesis according to the number of epithelial cells left in sections of the seam (see legend for Fig. 2). The untreated control is shown in Fig. 2 A (a). The intact seam is stage 6 at 12 h; 60–76 cells remain in the seam at 18 h, stage 5; 40–59 cells at 24 h, stage 4; 20–39 cells at 36 h, stage 3; 1–19 cells at 48 h, stage 2; and no cells, representing complete transformation (confluence), at 60 h, stage 1.
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ß-Catenin remains in the cytoplasm during palatal EMT
Next, we tested the hypothesis that TGFß3 induces palatal EMT via the ß-catenin/LEF1 or Wnt pathway, because it has recently been shown to directly mediate a number of EMTs (Kim et al., 2002). We found that the MEE does not transform at all in presence of dominant-negative (DN) LEF1 (Fig. 2 B, b; remain at stage 6), indicating that the ß-catenin/LEF1 pathway is needed for palatal EMT.
To examine these interrelations, we analyzed the effects of TGFß3 on LEF1 mRNA synthesis and subsequent transport of ß-catenin/LEF1 to the nucleus. To our surprise, we detected no ß-catenin in the palatal nuclei before, during, or after EMT in untreated palates (Fig. 3, A and B), nor in palates treated with either LEF1 (Fig. 3, C–F) or DN Smad4 (Fig. 3 G), indicating ß-catenin paucity characterizes normal palates. Because immunofluorescence is not a quantitative method, we cannot rule out the presence of very small amounts of ß-catenin in these nuclei. However, the data are clear cut enough to support the hypothesis that palatal LEF1 transcriptional activity depends on Smads, not ß-catenin (Labbe et al., 2000).
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LCM and quantification of LEF1 mRNA during palatal EMT
To study the process further by which TGFß3 up-regulates LEF1 during palatal EMT, we used a new technique, LCM, to obtain cells from the MEE seams to measure their metabolism by highly sensitive real-time PCR. We demonstrated that LEF1 gene expression reaches a peak in MEE cells at the onset of EMT during palate development. Endogenous LEF1 mRNA is expressed at 12 h (Fig. 4 A, a) and gradually increases to a peak by 36 h of incubation, dropping at 48 h before complete confluence at 60 h, when no MEE epithelial cells remain. The high level of LEF1 mRNA expression at 36 h is at the high point of transformation by the normal epithelial seam. Even though LEF1 levels fall after 36 h, enough is present for cells to complete transformation. When normal palates were treated with exogenous LEF1 virus, the expression of LEF1 mRNA by MEE cells increased over the control to reach peak by 24 h instead of 36 h, and decreased by 48 h (Fig. 4 A, b).
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As LEF1 mRNA synthesis and seam EMT are inhibited by TGFß3-blocking antibody, DN LEF1, or DN Smad4, we expected that exogenous LEF1 would rescue inhibition of palatal EMT by TGFß3-blocking antibody (Fig. 2 B, a), but it did not. This was a surprise because the DN LEF1 results (Fig. 2 B, b; Fig. 5 E) indicate that LEF1 is required for EMT. Correlations indicate that LEF1 mRNA peaks at 36 h and remains significantly up-regulated until EMT is completed at 60 h (Fig. 4 A, a). The amount of LEF1 mRNA is substantial (Fig. 4 B, a). This result can be explained by a need for a constant source of phospho-Smad2 not only to up-regulate levels of LEF1, but also to activate LEF1 transcription (Attisano and Tuen Lee-Hoeflich, 2001) in order to bring about EMT during palatogenesis.
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LEF1 and Smads interact to mediate the effects of TGFß3 on palatal EMT
It is well within the realm of possibility that many natural EMTs, regulated by TGFß3, use Smad/LEF1 instead of ß-catenin complexes to activate LEF1. That this is so for the palate is shown by the following data. We showed that AS ß-catenin does not inhibit normal MEE transformation (Fig. 2 B, d; Fig. 5, A–D). Thus, ß-catenin is not normally used in palatal EMT. We confirmed that morphology of the AS ß-catenin–treated palates is the same as normal palates (Fig. 5, A–D). They reach confluence by EMT at exactly the same time as normal palates (Fig. 1, A–D; Fig. 2 A, c and d). Because ß-catenin has no role in palatal EMT, it is not a reasonable candidate for the necessary interactions with LEF1 during activation of LEF1 transcription. Palate EMT must be using Smad/LEF1, which already has been shown by others to activate LEF1 transcription (Labbe et al., 2000).
To restore EMT in TGFß3-blocked palates treated with LEF1 (Fig. 2 B, a), active TGFß3 and LEF1 are required. We showed here that TGFß3 only promotes palatal EMT through Smads and LEF1-dependent pathways. Thus, the addition of TGFß3 undoubtedly adds the Smads that are needed to activate LEF1. We showed that palates treated with either DN LEF1 or DN Smad4 adenovirus do not undergo EMT (Fig. 2 B, b and c), and LEF1 mRNA expression is significantly reduced (Fig. 4 A, d; Fig. 4 B, c). The palatal shelves adhere to form normal MEE, but the EMT process is completely abolished by DN Smad4, and MEE remain untransformed (Fig. 5 F). These palates recover completely when phospho-Smads are available to interact with LEF1. This interaction has been documented and analyzed (Labbe et al., 2000), and is the only known way that LEF1 transcription can be activated in the absence of ß-catenin. Moreover, we show below that phospho-Smad2 must be in the nucleus (Fig. 6) to activate LEF1 transcription, where it interacts with LEF1.
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Discussion |
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The discussion will focus on proposed mechanisms for this newly described phenomenon (Labbe et al., 2000), whereby the TGFß3 ligand turns on a series of events activating TßRs and Smads, to up-regulate LEF1 synthesis and transcription, thereby causing palatal epithelial cells to transform into mesenchymal cells. Thus, confluence by EMT of a fusing embryonic organ in situ can be achieved in a highly sophisticated manner.
LEF1 promotion of palatal EMT and LEF1 mRNA is dependent on TGFß3 signaling
This is the first paper reporting that LEF1 mRNA is up-regulated in the MEE during palatal EMT. Expression of the LEF1 transcription factor is increased as EMT proceeds and reaches a peak between 24 and 36 h. The mRNA level then gradually falls, and the transformation is complete by 60 h. We used LCM on an embryonic organ (palate) to dissect individual MEE cells for analysis of LEF1 gene expression and quantification with real-time PCR. This is an important new technology that permits single cells to be microdissected as a pure population for chemical analysis from frozen sections of embryonic anlage.
To understand the unexpected enigma mentioned above that inhibition of palatal EMT by adding TGFß3-blocking antibody is not rescued by addition of pure LEF1, it is necessary to consider the mechanisms cells use to activate LEF1 transcription that might be in use by the palate MEE. In tumors and cell lines, it is common for ß-catenin to activate LEF1/TCF transcription (Morin et al., 1997; Eger et al., 2000; Kim et al., 2002), but an astonishing variant of this mechanism has recently been reported by Labbe et al. (2000) and Nishita et al. (2000). Smads, which can only be activated by TGFß, have been shown to activate LEF1 transcription. Thus, MEE disappearance by EMT could be dependent on both TGFß3 and LEF1 expression. Once TGFß3 ligands are bound to an appropriate receptor regime, the MEE seam is the target for TGFß3 signaling involving Smads2/4 and LEF1. Here, we show that both synthesis and transcription of LEF1 are activated by Smads as predicted by Labbe et al. (2000) and Nishita et al. (2000)
Smads, not ß-catenin, activate LEF1 mRNA synthesis and transcription during palatal EMT
ß-Catenin is an important partner in LEF1 transcriptional factor activity, and when LEF1 is bound to ß-catenin, LEF1 transports it to the nucleus (Behrens et al., 1996; Kim and Hay, 2001). Interaction of LEF1 with nuclear ß-catenin is known to effect Wnt signaling by stimulating the activity of LEF1/TCF transcription factors (Eastman and Grosschedl, 1999). ß-Catenin is such an important component of LEF1 transcriptional activity in a variety of EMTs in vitro and in vivo (Novak et al., 1998; Eger et al., 2000; El-Tanani et al., 2001) that we expected to find it well represented in the nuclei of palate cells using LEF1 for EMT.
It came as surprise, then, to find no detectable ß-catenin in the nuclei of transforming MEE cells. Abundant ß-catenin is localized in the cytoplasm and cell surface during all stages of normal palate development, but treatment with exogenous LEF1 did not move it to the nucleus. Even if a minute amount of ß-catenin is present in MEE nuclei that is not detectable by immunofluorescence, it is unlikely that significant ß-catenin signaling through LEF1 could transform in the MEE cells. Next, we looked at the possibility that normal palates are not using ß-catenin–activated LEF1 to transform the MEE to mesenchyme. We treated palates with AS ß-catenin and found it did not inhibit palatal EMT. Thus, palates must be using some other activator for LEF1
Smads are the obvious candidates to interact with LEF1 to activate its transcription (Labbe et al., 2000). We used DN Smad4 to show that Smads are indeed essential for activation of LEF1 leading to palatal EMT. Moreover, we showed by immunohistochemistry that phospho-Smad2 is present in nuclei of the normal MEE cells during EMT at the time that LEF1 is up-regulated by TGFß3. DN Smad4 abolishes all nuclear Smads. We conclude that TGFß3-mediated cell signaling during palatogenesis is not ß-catenin dependent, and that LEF1 transcriptional activity is solely Smad dependent during palatal EMT.
Considering the wide-spread and successful use of ß-catenin to activate the LEF1 transcription factor, one might ask why use Smads instead? There may be disadvantages in using ß-catenin to activate LEF1 in some systems (Ishitani et al., 2003). If ß-catenin is bound with LEF1, TGFß may not be able to dissociate the complex (Sasaki et al., 2003). It may be that in vivo, it is easier to control LEF1 with Smads during TGFß3 signaling in embryos, leaving ß-catenin under separate control in Wnt pathways. In the adult, while TGFß signaling can be involved in carcinogenesis (Oft et al., 1998; Cohen, 2003), the ß-catenin pathway is plagued by malignant mutations.
TGFß3 promotes Smad pathway, not Smad-independent pathways, in the MEE seam
Smads can only be activated by members of the TGFß superfamily using specific receptors (Piek et al., 1999; Dennler et al., 2002). TGFß induces phosphorylation of Smad2, which is known to activate the LEF1 transcription factor (Labbe et al., 2000; Nishita et al., 2000). Itoh et al. (2003) have demonstrated that TGFß-induced EMT requires intact TßRI, and thus is dependent on Smad pathway. Our data indicate that only Smad-dependent pathways regulate palatal EMT. Phospho-Smad2 is strongly expressed in the nuclei of the transforming MEE. When palates are treated with DN Smad4, Smad2 remains inactive in the cytoplasm because its nuclear translocation depends on shuttle by Smad4. With TGFß3-blocking antibody or DN Smad4, there is no expression of phospho-Smad2/4 in the MEE nuclei, raising the possibility that LEF1 mRNA up-regulation depends on both phospho-Smad2 and phospho-Smad4. Nishita et al. (2000) found that both Smad3 and Smad4 activated LEF1 transcriptional activity in the systems they analyzed. In the palate, Smad2 substitutes for Smad3 (Cui et al., 2003).
Although the Smad2/4 pathway is the only one likely to be involved in palatal EMT, several investigators recently have proposed TGFß3 signals EMT by Smad-independent signaling. These pathways are RhoA-Rac-MEK-JNK (Yu et al., 2002) and/or Ras-Raf-MEK-ERK (Mulder, 2000). C3, a specific inhibitor of RhoA kinase, blocks RhoA-Rac pathway (Kaartinen et al., 2002; Smith et al., 2003). U0126 completely inhibits MEK1/2, which takes place further downstream (Kretzschmar et al., 1999), and abolishes the pathway involving Ras-Raf-MEK (Adnane et al., 2002). The RhoA pathway of TGFß has been shown to activate in palates, but not to induce significant EMT (Bhowmick et al., 2001; Kaartinen et al., 2002). Another paper concluded that TGFß1 only induces EMT through the RhoA pathway (Bhowmick et al., 2001). We found that treatment of palates with inhibitors of RhoA (C3) and MEK1/2 (U0126) does not affect LEF1 mRNA expression or the palate EMT. The fact that DN Smad4 inhibits palatal EMT also indicates that the Smad-dependent TGFß3 signaling is required for activation of palatal EMT.
In summary, this work has investigated the remarkable cooperative roles of TGFß3 and LEF1 in bringing about EMT in palatogenesis. The discovery that LEF1 is required for palatal EMT is completely new. Also, for the first time we present evidence that TGFß3 up-regulates LEF1 mRNA and that Smads, not ß-catenin, activate LEF1 in this embryonic EMT. Our analysis of the palate extends the results of Labbe et al. (2000) and Nishita et al. (2000), and makes it tempting to speculate that embryos in general might use the TGFß pathway to turn on EMT via LEF1. This finely tuned pathway may not be subject to the extensive mutations that frequently occur in ß-catenin/LEF1 pathways.
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Materials and methods |
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Before and after placing palatal shelves together in pairs, they were treated with the following molecules alone or in combination: (1) GFP full-length LEF1 adenovirus. Full-length LEF1 with GFP was subcloned into pAdEasyTM EGFP plasmid expressing full-length active LEF1 and GFP under different CMV promoters using the adenovirus construction kit supplied by Dr. B. Vogelstein (The Johns Hopkins School of Medicine, Baltimore, MD). Generation of active LEF1 virus and DN LEF1 virus was as described previously (He et al., 1998), and all virus probes were carefully tested for activity (Kim et al., 2002). Palates transfected with 200 µl of 1.0 µg/ml LEF1 adenovirus in TSO buffer express LEF1 and undergo EMT. (2) GFP DN LEF1 adenovirus. A LEF1 construct coding aa 1–373 in the EVR-FL9b plasmid was the gift of Dr. Marian Waterman (University of California, Irvine, Irvine, CA). A Kozak sequence, KPN1 restriction site, and start site were inserted upstream of the LEF1 s amino acid, and a stop codon and Xba restriction site were added downstream of aa 313 (bp 939) to delete the NLS of B box, resulting in failure in nuclear transportation. The PCR fragment was inserted into pAdEasyTM EGFP plasmid. The resulting construct was recombined with adenoviral genome using the Vogelstein kit. Palates were transfected as above. All viruses were replication defective. (3) GFP DN Smad4 adenovirus. A recombinant adenovirus expressing a DN mutant Smad4 (AdMSmad4, pCMV5/Smad4) (1–514), a gift of Dr. D.M. Simeone (University of Michigan, Ann Arbor, MI), containing a COOH-terminal truncated DN Smad4 gene, was subcloned into the adenovirus vector. The DN Smad4 construct was bluntly ligated into the EcoRV site of the shuttle vector (pAdTrack) as described by He et al. (1998). Recombination was confirmed using multiple restriction enzyme digest analyses. The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINETM PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/µl anti–mouse TGFß3-blocking antibody (R&D Systems). (5) 2 ng/µl recombinant TGFß3 (R&D Systems). (6) AS ß-catenin oligonucleotides 5'-GGAGTTTAACCACAACAGGCAGTCC-3' and control sense 5'-CCTGACGGACAACACCAATTTGAGG-3', as described by Haertel-Wiesmann et al. (2000), used by Brunet et al. (1995), were tested in our laboratory. (7) 15 µM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem). Palates were treated with 10 µg/ml LipofectAMINETM for 2 h before C3 treatment. The C3 exoenzyme (30 µm), a toxin derived from C. botulinum, is an ADP ribosyltransferase that catalyzes mono-ADP ribosylation of the small GTP-binding proteins Rho (A, B, and C) at asparagine 41 (Wilde et al., 2002). Because ADP ribosylation occurs in the putative effector region of Rho, this modification interferes with correspondent Rho GTPase-dependent signaling pathways (Wilde et al., 2002). C3 is widely used in cell biology as a convenient tool to specifically inactivate Rho proteins (Borbiev et al., 2000).
Palatal shelves were routinely incubated 1 h before placing them together, and the medium was then changed every 24 h. Adenovirus, AS ß-catenin, and C3 treatments (Lu et al., 2001) in single palates were continued for 48 h. Palates were fixed in Bouin's fixative before embedding in paraffin or cryomold tissue freezing medium, TBS (Triangle Biomedical Sciences) for sections. We only collected prime sections from the middle one third of the palate.
Evaluation of adenovirus
All our adenovirus experiments were based on earlier published experiments (Kim and Hay, 2001) in which DLD1 cells exposed to 200 µl/ml full-length LEF1 virus were shown to undergo EMT promptly and to express LEF1 (Kim and Hay, 2001). Previously, we did tests to confirm the titration and dilution, and optimized the protocol. Adenovirus treatment continued in single palatal shelves until GFP protein is visualized by microscopy confirming viral protein production, and then shelves were placed together for further incubation. Most cultured palates were collected at 12–60 h. We found that the virus does not hurt the cells, and there are no morphological changes due to viral infection
The LEF1 construct used for the DN virus probe specifically lacks its B box for nuclear export. The DN LEF1 plasmid was also administrated in 200 µl/ml virus and effectively blocked LEF1 mRNA expression (Fig. 4 A, d). The DN Smad4 virus, which lacks COOH terminus for its nuclear translocation, abolished both Smad4 and Smad2 from the nuclei of the palate MEE cells. Palatal MEE cells were stained with Smad4 antibody to evaluate effects of DN Smad4 (Zhang et al., 2001), which shows that protein expression is exclusively in the cytoplasm of the MEE cells (Fig. 6 F). Both DN LEF1 and DN Smad4 significantly reduce LEF1 expression (Fig. 4 A, d; Fig. 4 B, c).
Evaluation of chemical inhibitors
Two chemical inhibitors were used at concentrations recommended by the manufacturer that had been shown to eliminate RhoA by C3 (Lu et al., 2001) and MEK1/2 with U0126 (Chow et al., 2001). To optimize dose and confirm the effects of TGFß3-blocking antibody, recombinant TGFß3, AS ß-catenin, and U0126 and C3, we stained the palatal sections with TGFß3 antibody (Santa Cruz Biotechnology, Inc.), anti-ß-catenin antibody (Sigma-Aldrich), phospho-p44/42 MAPK (Thr202/Tyr204) E10 antibody (Cell Signaling), and anti-RhoA antibody, respectively.
LCM
To study the EMT process in palatal MEE, uncontaminated by existing mesenchyme, analysis of pure epithelial and mesenchymal cell populations is necessary. We isolated epithelial cells dissected from the MEE of 300 pairs of palates by highlighting individual cells from the intact MEE and transforming MEE using Leica software. (1) Cutting and staining. The block is attached to the chuck in the cryostat with OCTTM. 8-µm sections were cut onto special slides (Leica) coated with plastic foil. We used HistoGeneTM LCM staining (Arcturus) for frozen sections. HistoGeneTM stains nuclei bright blue without degrading nucleic acids and yields high quality RNA. Stained sections were immediately processed for LCM. (2) Dissection. Using built-in software (IM1000) of Leica LCM, we were able to highlight MEE cells and transforming epithelial cells. Highlighted cells were cut using a laser beam (7.2 mW at 30 Hz) and collected in sterile plastic tubes. After microdissection (roughly 1,000 cells from each sample), the collecting tube containing cells in lysis buffer (ß-ME and RLT; 50 µl buffer with 0.04% proteinase K, 10 mM Tris-HCL, pH 8.0, 1 mM EDTA, and 1% Tween 20) is capped and homogenized. RNeasy® mini kit (QIAGEN) was used for total RNA extraction and purification. Extracted RNA was purified and quantified by spectrophotometry. All 260/280-nm ratios were above 1.8–2.2.
Real-time PCR
Total RNA samples of epithelial cells from different stages of palatogenesis were prepared and reverse transcribed into cDNA, and real-time PCR was performed as described by Scanlan et al. (2002).
If the 
CT values are the same, no change in the amount of the LEF1 gene in the transforming palatal MEE cells relative to single untransformed palatal MEE cells has occurred. If a difference between the two CT values is observed, the amount of the LEF1 gene molecule has increased or decreased, and this value is incorporated into a Microsoft® Excel program to generate statistics and graphs representing actual value of X, which is equal to 2-CT. Graph bar represents quantitative and numerical average value of X (n = 300), where X = -2ÓÓCT [ÓÓ CT = {CT LEF1 palate - CT GAPDH palate} - {CT LEF1 single - CT GAPDH single}]. LEF1: sense, 5'-CCCACACGGACAGTGACCTA-3'; antisense, 5'–TGGGCTCCTGCTCCTTTCT-3'. TaqMan® probe: 5'-FAM-TGCACGTGAAGCCTCAACACGAACA-VIC-3'; 5'-FAM-AGCCACATCGCTCAGACACCATGG-VIC-3'. GAPDH: sense, 5'-CCTGTTCGACAGTCAGCCG-3'; antisense, 5'-CGACCAAATCCGTTGACTCC-3'. Immunofluorescence staining was used as follows: rabbit polyclonal anti-ß-catenin antibody (1:1,000; Sigma-Aldrich), goat polyclonal anti-Smad2 antibody (1:100; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-phospho-Smad2 antibody (1:100; Cell Signaling; recognizes endogenous Smad2 of Ser 465 and 467 phosphorylation); and rabbit polyclonal anti-Smad4 antibody (1:200; Santa Cruz Biotechnology, Inc.).
Sections are blocked for 3 h at RT with 2% normal goat serum or 5% normal rabbit serum, depending on secondary antibody in PBS, and then incubated with primary antibody (e.g., mouse monoclonal anti-ß-catenin antibody at 1:1,000) at 4°C overnight. The titers of the primary antibodies are optimized by trial and error. Sections are washed in PBS three times before they are incubated at RT with secondary antibody (1:200) for 2 h. Rhodamine (Pierce Chemical Co.) was conjugated for ß-catenin antibody and phospho-Smad2 antibody or fluorescence (Pierce Chemical Co.) for ß-catenin antibody and Smad2 antibody. Sections were then washed three times in PBS followed by three times in de-ionized water and were mounted in Vectashield® (Vector Laboratories). A microscope (ES1000; Nikon) was used to image rhodamine- and fluorescein-conjugated antibody by fluorescence.
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Acknowledgments |
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This research was supported by a grant from the National Institute of Dental and Craniofacial Research (R01-DE11142) from the U.S. Public Health Service to E.D. Hay.
Submitted: 4 June 2003
Accepted: 5 November 2003
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