The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy.

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.     

Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation

During fusion of the mammalian secondary palate, it has been suggested that palatal medial edge epithelial (MEE) cells disappear by means of apoptosis, epithelial-mesenchymal transformation (EMT) and epithelial cell migration. However, it is widely believed that MEE cells never differentiate unless palatal shelves make contact and the midline epithelial seam is formed. In order to clarify the potential of MEE cells to differentiate, we cultured single (unpaired) palatal shelves of ICR mouse fetuses by using suspension and static culture methods with two kinds of gas-mixtures. We thereby found that MEE cells can disappear throughout the medial edge even without contact and adhesion to the opposing MEE in suspension culture with 95% O2/5% CO2. Careful examination of MEE cell behavior in the culture revealed that apoptosis, EMT, and epithelial cell migration all occurred at various stages of MEE cell disappearance, including the transient formation and disappearance of epithelial triangles and islets. In contrast, MEE cells showed poor differentiation in static culture in a CO2 incubator. Furthermore, mouse and human amniotic fluids were found to prevent MEE cell differentiation in the cultured single palatal shelf, although paired palatal shelves fused successfully even in the presence of amniotic fluid. We therefore conclude that terminal differentiation of MEE cells is not necessarily dependent on palatal shelf contact and midline epithelial seam formation, but such MEE cell differentiation appears to be prevented in utero by amniotic fluid unless palatal shelves make close contact and the midline epithelial seam is formed.     

Molecular and morphologic changes during the epithelial-mesenchymal transformation of palatal shelf medial edge epithelium in vitro.

The fate of the medial edge epithelial (MEE) cells during palatal fusion has been proposed to be either programmed cell death or epithelial-mesenchymal transformation. Vital cell labeling techniques were used to mark the MEE and observe their fate during palatal fusion in vitro. Fetal mouse palatal shelves were labeled with Dil and allowed to proceed through fusion while maintained in an organ culture system. The tissues were examined at several stages of palatal fusion for the distribution of Dil, presence of specific antigens and ultrastructural appearance of the cells. The MEE labeled with Dil occupied a midline position at all stages of palatal fusion. Initially the cells had keratin intermediate filaments and were separated from the underlying mesenchyme by an intact basement membrane. During the process of fusion the basement membrane was degraded and the Dil-labeled MEE were in contact with the mesenchymal-derived extracellular matrix. In the late stages of fusion the Dil-labeled MEE altered their cellular morphology, had vimentin intermediate filaments, and were not associated with an identifiable basement membrane. Dil-labeled cells, without an epithelial phenotype, remained present in the midline of the completely fused palate. The data indicate that the MEE did not die but underwent a phenotypic transformation to viable mesenchymal cell types, which were retained in the palatal mesenchyme.     

Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion

Malformations in secondary palate fusion will lead to cleft palate, a common human birth defect. Palate fusion involves the formation and subsequent degeneration of the medial edge epithelial seam. The cellular mechanisms underlying seam degeneration have been a major focus in the study of palatogenesis. Three mechanisms have been proposed for seam degeneration: lateral migration of medial edge epithelial cells; epithelial-mesenchymal trans-differentiation; and apoptosis of medial edge epithelial cells. However, there is still a great deal of controversy over these proposed mechanisms. In this study, we established a [Rosa26<-->C57BL/6] chimeric culture system, in which a Rosa26-originated ;blue' palatal shelf was paired with a C57BL/6-derived ;white' palatal shelf. Using this organ culture system, we observed the migration of medial edge epithelial cells to the nasal side, but not to the oral side. We also observed an anteroposterior migration of medial edge epithelial cells, which may play an important role in posterior palate fusion. To examine epithelial-mesenchymal transdifferentiation during palate fusion, we bred a cytokeratin 14-Cre transgenic line into the R26R background. In situ hybridization showed that the Cre transgene is expressed exclusively in the epithelium. However, beta-galactosidase staining gave extensive signals in the palatal mesenchymal region during and after palate fusion, demonstrating the occurrence of an epithelial-mesenchymal transdifferentiation mechanism during palate fusion. Finally, we showed that Apaf1 mutant mouse embryos are able to complete palate fusion without DNA fragmentation-mediated programmed cell death, indicating that this is not essential for palate fusion in vivo.     

Midline fusion in the formation of the secondary palate anticipated by upregulation of keratin K5/6 and localized expression of vimentin mRNA in medial edge epithelium.

Secondary palatal fusion is dependent on targeted removal of the epithelium between the palatal shelves. Aseptically delivered rat embryos 15 through 18 days post coitum (dpc) were probed with DIG-labeled antisense and sense ssDNA probes for spliced exon sequences flanking intron E of cytokeratins K5/6 and spliced exon sequences flanking intron F of vimentin. Cytokeratin K5/6 expression was upregulated in the medial edge epithelium (MEE) prior to rotation of the palatal shelves and in the vomerine epithelium in the region of fusion with the palate. K5/6 expression continued in the medial epithelial seam (MES) and in epithelial islands during breakdown of the MES. Vimentin expression was not detected in the MEE prior to rotation but was specifically upregulated in the MEE following rotation and prior to midline contact and continued in the MES and in epithelial cells identifiable during the breakdown of the MES. Initiation of vimentin upregulation in the MEE prior to contact of the palatal shelves was tested by serum-free organ culture of palates from embryos at 15.5 dpc with the shelves separated by a biocompatible membrane. Vimentin upregulation occurred in the epithelium specifically in the region of anticipated contact. These results are interpreted as indicating that i) cytokeratin K5/6 expression may play a critical role in the integration of the epithelial layers of the MES to ensure subsequent merging of the mesenchyme and ii) epithelial cells in the MEE are specifically 'primed' to upregulate expression of mesenchymal genes prior to integration into and breakdown of the MES.     

Expression of the epidermal growth factor receptor in developing fetal mouse palates: an immunohistochemical study

Epidermal growth factor (EGF) stimulates the growth of various tissues and, therefore, EGF receptor expression in fetal tissues may play a key role in organogenesis. We have examined immunohistochemically the ontogeny and localization of the EGF receptor in the fetal mouse palate during in vivo and in vitro palatogenesis using the anti-human EGF receptor rabbit antibody. Immunoreactive products against the EGF receptor were observed in the palatal tissue examined on days 12, 13, and 14 of gestation. On days 12 and 13, the immunoreactive products were predominantly positive on the oral and medial edge epithelia but were minimal on the epithelium of the vertical shelf. The EGF receptor immunoreactivity was less intense in the posterior palate as compared with the midpalatal region. In the fusing palate of day 14 fetuses, the cells forming the midline epithelial seam were continuously positive for EGF-R immunoreactivity. The mesenchyme of palatal shelves also showed regional heterogeneity and temporal sequence in EGF receptor expression. The localization of the EGF receptor in fetal mouse palates cultured in a serumless medium generally simulated that observed in vivo.     

Fate-mapping of the epithelial seam during palatal fusion rules out epithelial-mesenchymal transformation

During palatogenesis, fusion of the palatine shelves is a crucial event, the failure of which results in the birth defect, cleft palate. The fate of the midline epithelial seam (MES), which develops transiently upon contact of the two palatine shelves, is still strongly debated. Three major mechanisms underlying the regression of the MES upon palatal fusion have been proposed: (1) apoptosis has been evidenced by morphological and molecular criteria; (2) epithelial-mesenchymal transformation has been suggested based on ultrastructural and lipophilic dye cell labeling observations; and (3) migration of MES cells toward the oral and nasal areas has been proposed following lipophilic dye cell labeling. To verify whether epithelial-mesenchymal transformation of MES cells takes place during murine palatal fusion, we used the Cre/lox system to genetically mark Sonic hedgehog- and Keratin-14-expressing palatal epithelial cells and to identify their fate in vivo. Our analyses provide conclusive evidence that rules out the occurrence of epithelial-mesenchymal transformation of MES cells.     

Medial edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo.

Vital cell labeling techniques were used to trace the fate of the medial edge epithelial (MEE) cells during palatal fusion in vivo. Mouse palatal tissues were labeled in utero with DiI. The fetuses continued to develop in utero and tissues of the secondary palate were examined at several later stages of palatal ontogeny. The presence and distribution of DiI was correlated with the presence of cell phenotype-specific markers. During the initial stages of palatal fusion the DiI-labeled MEE were present in the midline position. These cells were attached to an intact laminin-containing basement membrane and contained keratin intermediate filaments. At later stages of palatogenesis the DiI-labeled MEE were not separated from the mesenchyme by an intact basement membrane and did not contain keratin. In late fetal development, DiI-labeled cells without an epithelial morphology were present in the mesenchyme. The transition of the DiI-labeled cells from an epithelial phenotype to a mesenchymal phenotype is consistent with a fate of epithelial-mesenchymal transformation rather than programmed cell death.     

Histogenesis of Swiss white mouse secondary palate from nine and one-half days to fifteen and one-half days In utero. I. Epithelial-mesenchymal relationships—light and electron microscopy

Development of the secondary palate in Swiss white mouse embroyos was studied from age nine-and-one-half days in utero to the stage of mesenchymal coalescence in the secondary palate (approximately fifteen-and-one-half days). The greatest changes observed occur in the mesenchyme. At early stages, mesenchymal cells underlying oral ectoderm of the head are few and only occasionally contact the ectoderm. Electron micrographs show large intercellular spaces between the ectodermal cells. As embryogenesis continues, the mesenchymal cells become more numerous, closer to each other and closer to the epithelium. Just prior to horizontal transposition of shelves, the mesenchymal cells spread farther from each other and from the palatal epithelium and epithelium of the palatal tip becomes stretched. Ultrastructurally the intercellular spaces between epithelial cells of the palate tip have become much smaller. Some mitochondria in some epithelial cells are swollen and have clear matrices and distorted cristae. The shelves become horizontal and meet in the midpalate. Cells with degeneration bodies are seen in the epithelial seam. The seam undergoes autolysis and is replaced by mesenchyme. The morphological changes described, particularly in the mesenchyme, may play an important role in determining the effect of various teratogens at different stages of palatal development. The changes in both mesenchyme and epithelial cells in the later stages may constitute part of the process of preparing shelves for fusion as postulated by Pourtois ('66).     

Immunohistochemical localization of prostaglandins E and F2 alpha in the developing murine palate

Prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) have been shown to cause changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels in a wide variety of tissues. In particular, murine palatal mesenchyme responds to PGE2 stimulation with dose-dependent increases in intracellular cAMP levels. These same mesenchymal cells also synthesize PGE2 and PGF2 alpha. The purpose of this study is to localize PGE and PGF2 alpha in the developing murine palate by using immunohistochemical techniques. Fresh frozen cryostat sections of murine C57BL/6J embryo palates (days 12-14 of gestation) were incubated with anti-PGE or PGF2 alpha monoclonal antibodies. On day 12 of gestation, PGE and PGF2 alpha, identified as 3',3-diaminobenzidine (DAB) reaction products, were localized throughout palatal mesenchyme and epithelium; on day 13 of gestation, reaction product indicative of both PGE and PGF2 alpha was detectable primarily in mesenchyme subjacent to palatal epithelium. Extracellular spaces of the adjacent mesenchyme in the central region of the day 13 palate exhibited less reaction product. Palatal epithelium, particularly the medial edge epithelium, exhibited a diminished amount of reaction product for both prostaglandins on day 13 as compared to the underlying mesenchyme. After formation of a midline epithelial seam between homologous palatal processes on day 14 of gestation, medial edge, oral, and nasal epithelium exhibited light staining for PGE or PGF2 alpha. Palate mesenchymal cells subjacent to the midline seam exhibited a diminished amount of reaction product for both PGE and PGF2 alpha as compared to day 13 of gestation. Overall, the results show local and temporal changes in the distribution of prostaglandins in the developing murine palate.     

Rescue of an in vitro palate nonfusion model using interposed embryonic mesenchyme

The authors previously established an in vitro palate nonfusion model on the basis of a spatial separation between prefusion embryonic day 13.5 mouse palates (term gestation, 19.5 days). They found that an interpalatal separation distance of 0.48 mm or greater would consistently result in nonfusion after 4 days in organ culture. In the present study, they interposed embryonic palatal mesenchymal tissue between embryonic day 13.5 mouse palatal shelves with interpalatal separation distances greater than 0.48 mm in an attempt to "rescue" this in vitro palate nonfusion phenotype. Because no medial epithelial bilayer (i.e., medial epithelial seam) could potentially form, palatal fusion in vitro was defined as intershelf mesenchymal continuity with resolution of the medial edge epithelia bilaterally. Forty-two (n = 42) palatal shelf pairs from embryonic day 13.5 CD-1 mouse embryos were isolated and placed on cell culture inserts at precisely graded distances (0, 0.67, and 0.95 mm). Positive controls consisted of shelves placed in contact (n = 6). Negative controls consisted of shelves placed at interpalatal separation distances of 0.67 mm (n = 6) and 0.95 mm (n = 7) with no interposed mesenchyme. Experimental groups consisted of embryonic day 13.5 palatal shelves separated by 0.67 mm (n = 11) and 0.95 mm (n = 12) with interposed lateral palatal mesenchyme isolated at the time of palatal shelf harvest. Specimens were cultured for 4 days (n = 19) or 10 days (n = 23), harvested, and evaluated histologically. All positive controls at 4 and 10 days in culture showed complete histologic palatal fusion. All negative controls at 4 days and 10 days in culture remained unfused. Five of six palatal shelves separated at 0.67 mm interpalatal separation distance with interposed mesenchyme were fused at 4 days, and all five were fused at 10 days. At an interpalatal separation distance of 0.95 mm with interposed mesenchyme (n = 12), no palates (zero of four) were fused at 4 days, but seven of eight were fused at 10 days. These data suggest that nonfused palatal shelves can be "rescued" with an interposed graft of endogenous embryonic mesenchyme to induce fusion in vitro.     

The distribution of syndecan during murine secondary palate morphogenesis.

The distribution of syndecan, an integral membrane proteoglycan, has been immunohistochemically mapped during the course of murine secondary palate morphogenesis, gestational days 12-15. Syndecan has been shown to mediate cell adhesion and shape change and to be involved in epithelial-mesenchymal interactions during the morphogenesis of several structures. Changes in epithelial cell architecture accompany and may serve to direct the reorientation of the murine secondary palatal shelves from a vertical position on either side of the tongue to a horizontal and adhering position above it. Using a monoclonal antibody made to the core protein of the ectodomain of syndecan, staining was observed to correlate with epithelial cell shape, packing and degree of differentiation. Staining of condensing mesenchyme was also observed. Syndecan may be involved in modulating epithelial cell shape, architecture and fates during both major phases of secondary palate morphogenesis: shelf reorientation and midline epithelial seam dissolution.     

Palatogenesis in hamster. II. Ultrastructural observations on the closure of palate

Formation of secondary palate in hamster was studied with electron microscopy. Prior to assuming horizontal position, the palatal shelves were covered by a two to three cell layer thick epithelium which was separated from the underlying mesenchyme by an intact basal lamina. Epithelial cells were attached to each other by desmosomes. Early hemidesmosomes could be identified as thickenings of the cytoplasmic membrane opposing the basal lamina. Epithelial cells, like other embryonic cells, contained only few organelles but were rich in polyribosomes. As the horizontal shelves approached each other towards the midline, lysosomes and tonofilaments appeared in the superficial and basal cells of the epithelia. Superficial cells showed degeneration and eventual lysis. Fusion of the opposing epithelia occurred between the deeper cells by means of newly formed desmosomes. The epithelial seam resulting from fusion of the epithelia was limited on each side by a continuous basal lamina. Its subsequent thining and eventual fragmentation resulted from the loss of cells by autophagy. There was no evidence of mesenchymal invasion of the epithelial seam. Mesenchymal macrophages appeared in the later stage of palatogenesis and were responsible for phagocytosis of cellular debris. Formation of the soft palate was basically similar to that of the secondary hard palate and occurred by fusion of the opposing shelves. Similarly, anterior closure of the palate occurred by fusion of the lower end of the nasal septum to the primary and secondary palates. Hyperplasia of the opposing epithelia, prior to their fusion, was often seen. It is suggested that formation of the palate occurs in predictable and coordinated fashion and that timely appearance of lysosomes causing lysis of intervening epithelia is of great significance in normal palatogenesis.     

Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE.     

TGF-beta3-induced palatogenesis requires matrix metalloproteinases

Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membrane-associated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-beta3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-beta3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-beta3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion.     

Disintegration of the medial epithelial seam: Is cell death important in palatogenesis?

During palatogenesis, the palatal medial edge epithelium (MEE) forms the medial epithelial seam (MES) on adhesion of the opposing palatal shelves. The MES eventually disappears, leading to mesenchymal confluence of the palate and completion of palatogenesis. Failure of these processes results in cleft palate, one of the most common congenital anomalies in human affecting around one case in 500-2500 live births. The cell fate of MEE has been controversial for more than 20 years. Recent studies suggest that the disappearance of MES is a complex process involving cell death, epithelial-mesenchymal transition (EMT) and epithelial migration. Interestingly, transforming growth factor-β3 (Tgf β3) expression in MEE and the tip epithelium of the nasal septum begins just before palatal shelf reorientation and lasts until MES disruption, and several works including targeted disruption of the gene have indicated that the process appears to be regulated mainly by the TGFβ3-TGFβR signaling. However, how MEE cells choose their fate and how the cell fate is altered in response to cellular environment remains to be elucidated.     

Medial edge epithelial cell fate during palatal fusion

To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice. We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.     

Medial epithelial seam cell migration during palatal fusion

The mammalian secondary palate forms from two shelves of mesenchyme sheathed in a single-layered epithelium. These shelves meet during embryogenesis to form the midline epithelial seam (MES). Failure of MES degradation prevents mesenchymal confluence and results in a cleft palate. Previous studies indicated that MES cells undergo features of epithelial-to-mesenchymal transition (EMT) and may become migratory as part of the fusion mechanism. To detect MES cell movement over the course of fusion, we imaged the midline of fusing embryonic ephrin-B2/GFP mouse palates in real time using two-photon microscopy. These mice express an ephrin-B2-driven green fluorescent protein (GFP) that labels the palatal epithelium nuclei and persists in those cells through the time window necessary for fusion. We observed collective migration of MES cells toward the oral surface of the palatal shelf over 48 hr of imaging, and we confirmed histologically that the imaged palates had fused by the end of the imaged period. We previously reported that ephrin reverse signaling in the MES is required for palatal fusion. We therefore added recombinant EphA4/Fc protein to block this signaling in imaged palates. The blockage inhibited fusion, as expected, but did not change the observed migration of GFP-labeled cells. Thus, we uncoupled migration and fusion. Our data reveal that palatal MES cells undergo a collective, unidirectional movement during palatal fusion and that ephrin reverse signaling, though required for fusion, controls aspects of the fusion mechanism independent of migration.