Secondary Palate Fusion Research Articles

Cellular mechanisms and developmental compensation during tissue fusion in the secondary palate

Cleft lip with or without cleft palate (CL/P) is the most common congenital craniofacial anomaly with considerable morbidity in affected children. Though extensive studies have established the basic histological and genetic mechanisms of palatal development, the cellular mechanisms that regulate secondary palate development are still not completely understood. Palate fusion is the crucial final step of palate development, in which the medial edge epithelial cells (MEE) make contact to form a midline epithelial seam (MES), which is subsequently removed to allow mesenchymal continuity. Though much progress has been made on understanding the cellular mechanisms of secondary palate fusion, major questions remain in part due to limitations in imaging technologies. Here we utilized new processing and imaging methods to visualize dynamic cell behaviors underlying secondary palate fusion temporally and spatially. We apply these methods to the study of mouse mutants in which apoptosis, or actomyosin contractility have been conditionally disrupted in the secondary palate epithelium. Our results indicate that apoptosis and cell extrusion contribute to MES removal, but that cellular compensation can overcome their loss. Our studies also reveal a unique form of collective epithelial cell migration that is critical for proper completion of secondary palate fusion and suggesting that secondary palate fusion may be a valuable model for understanding how collective epithelial migration occurs in other developmental and disease contexts. Finally, our observations show that genetically ablating actomyosin contractility disrupts MES removal by disrupting collective epithelial cell migration. Overall, our studies suggest a model of compensatory cellular mechanisms in epithelium removal during palate fusion.

LncRNA Meg3-mediated regulation of the Smad pathway in atRA-induced cleft palate

Palatal mesenchymal cell proliferation is essential to the process of palatogenesis, and the proliferation of mouse embryonic palate mesenchymal (MEPM) cells is impacted by both all-trans retinoic acid (atRA) and the TGF-β/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been shown to activate TGF-β/Smad signaling and to thereby regulate cell proliferation, differentiation, and related processes. Herein, we found that atRA treatment (100 mg/kg) promoted Meg3 upregulation in MEPM cells, and that such upregulation was linked to the suppression of MEPM cell proliferation in the context of secondary palate fusion on gestational day (GD) 13 and 14. Moreover, the demethylation of specific CpG sites within the lncRNA Meg3 promoter was detected in atRA-treated MEPM cells, likely explaining the observed upregulation of this lncRNA. Smad signaling was also suppressed by atRA treatment in these cells, and RNA immunoprecipitation analyses revealed that Smad2 can directly interact with Meg3 in MEPM cells following atRA treatment. Therefore, we propose a model wherein Meg3 is involved in the suppression of MEPM cell proliferation, functioning at least in part via interacting with the Smad2 protein and thereby suppressing Smad signaling in the context of atRA-induced cleft palate.

Collective epithelial cell migration in secondary palate fusion

Cleft lip with or without cleft palate (CL/P) is the most common congenital craniofacial anomaly with considerable morbidity in affected children. Though extensive studies have established the basic histological and genetic mechanisms of palatal development, the cellular mechanisms that regulate secondary palate development are still not completely understood. Palate fusion is the crucial final step of palate development, in which the medial edge epithelial cells (MEE) make contact to form a midline epithelial seam (MES), which is subsequently removed to allow mesenchymal continuity. Though much progress has been made on understanding the cellular mechanisms of secondary palate fusion, major questions remain in part due to limitations in imaging technologies. Here we utilized new processing and imaging methods to visualize dynamic cell behaviors underlying secondary palate fusion temporally and spatially. We apply these methods to the study of mouse mutants in which apoptosis, or actomyosin contractility have been conditionally disrupted in the secondary palate epithelium. Our results indicate that apoptosis and cell extrusion contribute to MES removal, but that cellular compensation can overcome their loss. Our studies also reveal a unique form of collective epithelial cell migration that is critical for proper completion of secondary palate fusion and suggesting that secondary palate fusion may be a valuable model for understanding how collective epithelial migration occurs in other developmental and disease contexts.

SMAD2 overexpression rescues the TGF-β3 null mutant mice cleft palate by increased apoptosis

During palatal development, medial edge epithelium (MEE) disappearance is one of the crucial steps in the process of fusion. The fate of these cells is still debated, and controversies remain. During secondary palate fusion, TGF-β3 signaling mediated in the cell through the SMAD2 protein plays an important role and leads to the disappearance of the midline epithelial seam (MES) and the confluence of the palatal mesenchyme. In mice, TGF-β3 knock-out is lethal and mice are born with a cleft in the secondary palate. This phenotype has been rescued by targeted overexpression of SMAD2 in the medial edge epithelium (MEE). The goal of this research was to understand the mechanism of palatal fusion in the rescue mice. MethodsThe heads of embryos with four different genotypes (wild-type, K14-SMAD2/TGF-β3(−/−), K14-SMAD2/TGF-β3(±), and TGF-β3 null) were collected at embryonic day E14.5, genotyped, fixed and embedded in paraffin. Serial sections were studied for detection of apoptosis and epithelial mesenchymal transition using immunofluorescence. ResultsTGF-β3 null mice developed a cleft in the secondary palate while both mice with K14-SMAD2 overexpression had fusion of the secondary palate. The MEE of both the rescue mice and K14-SMAD2 overexpression had a much higher ratio of apoptotic cells than wild-type mice. The increase in apoptosis was correlated with increased phospho-SMAD2 in the MEE. ConclusionSMAD2 overexpression rescued the cleft in the secondary palate by increasing apoptosis in the medial edge epithelium.

Observation of Dynamic Cellular Migration of the Medial Edge Epithelium of the Palatal Shelf in vitro.

Palatal fusion is a critical step during palatogenesis. In this fusing interface, the epithelial sheets need to be removed in order to achieve mesenchymal continuity. Epithelial cellular migration is one of the possible mechanisms, and live imaging of the labeled epithelium could provide direct evidence for it. However, the removal of medial edge epithelium (MEE) between the bilateral processes takes place in the middle of the dorso-ventral axis of the palatal shelf, and thus it is challenging to capture the cellular behavior directly. Here, we evaluate cellular behavior of MEE cells using a live imaging technique with a mouse model which expresses GFP under the promoter of Keratin14 (K14-GFP) and unpaired palatal shelf culture. Using this approach, we successfully obtained live images of epithelial behavior and detected epithelial cell migration on the surface of the secondary palatal shelf without touching of the opposing shelf. Additionally, the pattern of epithelial elimination resulted in oval-shaped exposed mesenchyme, which recapitulated the situation during secondary palate fusion in vivo. Detailed image processing revealed that most of the MEE migrated in an outward direction at the boundary regions as the oval shape of the exposed mesenchyme expanded. The migration was preceded by the bulging of MEE, and disappearance of GFP signals was not evident in bulging or migrating MEE at the boundary regions. Furthermore, the MEE migration and the subsequent mesenchymal exposure were disturbed by application of ROCK inhibitor. Together, these findings indicated that epithelial cell migration contributed importantly to the MEE removal and the subsequent exposure of the underlying mesenchyme. Furthermore, they indicated that the migration of epithelial cells was regulated in a time- and space-specific manner, since unpaired palatal shelf culture exhibited these cellular behaviors even in the absence of the opposing shelf. Altogether, present data indicated that this new experimental system combining live imaging with GFP-labeled epithelium mice and unpaired palatal shelf culture enabled direct visualization of cellular migration of MEE in vitro and could be a powerful tool to investigate its cellular and molecular mechanisms.

Live Imaging of Mouse Secondary Palate Fusion.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Computational Model of Secondary Palate Fusion and Disruption.

Morphogenetic events are driven by cell-generated physical forces and complex cellular dynamics. To improve our capacity to predict developmental effects from chemical-induced cellular alterations, we built a multicellular agent-based model in CompuCell3D that recapitulates the cellular networks and collective cell behavior underlying growth and fusion of the mammalian secondary palate. The model incorporated multiple signaling pathways (TGFβ, BMP, FGF, EGF, and SHH) in a biological framework to recapitulate morphogenetic events from palatal outgrowth through midline fusion. It effectively simulated higher-level phenotypes (e.g., midline contact, medial edge seam (MES) breakdown, mesenchymal confluence, and fusion defects) in response to genetic or environmental perturbations. Perturbation analysis of various control features revealed model functionality with respect to cell signaling systems and feedback loops for growth and fusion, diverse individual cell behaviors and collective cellular behavior leading to physical contact and midline fusion, and quantitative analysis of the TGF/EGF switch that controls MES breakdown-a key event in morphogenetic fusion. The virtual palate model was then executed with theoretical chemical perturbation scenarios to simulate switch behavior leading to a disruption of fusion following chronic (e.g., dioxin) and acute (e.g., retinoic acid) chemical exposures. This computer model adds to similar systems models toward an integrative "virtual embryo" for simulation and quantitative prediction of adverse developmental outcomes following genetic perturbation and/or environmental disruption.

Deciphering TGF-β3 function in medial edge epithelium specification and fusion during mouse secondary palate development.

Transforming growth factor-β3 (TGF-β3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF-β3 functions during secondary palate fusion are still poorly understood. We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf-β3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down-regulated in the medial edge epithelium during palatal fusion. Jagged2 down-regulation was regulated by TGF-β3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf-β3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf-β3 null mutant palatal shelves. Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF-β3 mediates palate fusion in part by down-regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF-β3 downstream target genes in palate medial edge epithelium differentiation.

Cell intercalation and migration mediated by actin contractility are key cellular behaviors in fusion of the mammalian secondary palate (344.2)

Fusion of two distinct embryonic prominences is a process common to numerous developmental contexts and often requires the removal of an intervening epithelium. Upon fusion of the mammalian secondary palatal shelves, the resulting medial epithelial seam (MES) must be removed to allow formation of a continuous secondary palate. This problem has been studied at great length, leading to three main cellular explanations. Whereas considerable investigation has been made into epithelial to mesenchymal transition (EMT) and programmed cell death of the intervening epithelium as the definitive cellular mechanisms, the contribution of cell migration of the intervening epithelium in this process is not clear. By combining confocal live imaging in explant culture with functional molecular genetics in mice, we now provide direct evidence that cell migration is a key component of removal of the MES. Further, we find that integration of a disorganized multi‐layered epithelium into a shared single layer epithelium is an essential early step in secondary palate fusion. Both integration and migration of the MES requires an actin contractility pathway involving Rho Kinase (ROCK), Myosin light chain kinase (MLCK) and culminating with activation of Non‐muscle myosin IIA (NMIIA). Together, these data provide direct support for cell migration in removal of the MES, and allow us to begin to assemble a molecular pathway controlling this action.Grant Funding Source: Supported by R03 DE022818 from NIH/NIDCR

Cell proliferation and apoptosis in the fusion of human primary and secondary palates

The markers of cell proliferation (Ki-67) and apoptosis [caspase-3, TdT-mediated biotin-dUTP nick-end labelling (TUNEL)] and the expression of syndecan-1 and heat shock protein 70 (Hsp70) were analyzed immunohistochemically in 11 developing human palates, from developmental weeks 6 to 10. During fusion of the primary palate, the proportion of proliferating cells decreased from 42 to 32% and the proportion of apoptotic cells decreased from 11 to 7% in the medial-edge epithelium. At later stages, the proportions of both types of cells decreased in the ectomesenchyme, except for proliferating cells in its non-condensing part. At developmental weeks 9-10, the epithelial seam in the secondary palate comprised 28% proliferative cells and 5% apoptotic cells. While condensing ectomesenchyme contained more apoptotic cells than proliferating cells, the opposite was observed for the non-condensing ectomesenchyme. Co-expression of syndecan-1 and Hsp70 was detected in cells budding from the epithelial seam. Our study indicates similar principles for human primary palate and secondary palate fusion, and parallel persistence of proliferation and apoptotic activity. While proliferation enables growth and fusion of different palatal primordia, apoptosis results in the removal of of large numbers epithelial cells at the fusion point. The disintegration of seam remnants seems to be executed through the processes of change in protein content and cell migration, probably leading to cell death as their final outcome.

Ephrin reverse signaling controls palate fusion via a PI3 kinase‐dependent mechanism

Secondary palate fusion requires adhesion and epithelial-to-mesenchymal transition (EMT) of the epithelial layers on opposing palatal shelves. This EMT requires transforming growth factor β3 (TGFβ3), and its failure results in cleft palate. Ephrins, and their receptors, the Ephs, are responsible for migration, adhesion, and midline closure events throughout development. Ephrins can also act as signal-transducing receptors in these processes, with the Ephs serving as ligands (termed "reverse" signaling). We found that activation of ephrin reverse signaling in chicken palates induced fusion in the absence of TGFβ3, and that PI3K inhibition abrogated this effect. Further, blockage of reverse signaling inhibited TGFβ3-induced fusion in the chicken and natural fusion in the mouse. Thus, ephrin reverse signaling is necessary and sufficient to induce palate fusion independent of TGFβ3. These data describe both a novel role for ephrins in palate morphogenesis, and a previously unknown mechanism of ephrin signaling.

A TGF‐β‐induced gene, βig‐h3, is crucial for the apoptotic disappearance of the medial edge epithelium in palate fusion

TGF-beta3, TbetaR-I, and TGF-beta-activated Smad2 has been suggested to be a series of signaling molecules for secondary palate fusion. In this article, we show that a gene induced by TGF-beta, betaig-h3, is coincidentally expressed with TGF-beta3 in medial edge epithelial (MEE) cells undergoing apoptosis during normal palatal fusion. betaig-h3 was also highly expressed in the areas of post-weaning mammary gland cells and developing phalangeal joints in which TGF-beta3 or BMP-4-induced apoptosis occurs, respectively. Blocking of betaig-h3 expression in E12.5 embryos with antisense oligodeoxynucleotides (ODN) resulted in cleft of the secondary palate in 84% of the treated mice that were born. Moreover, the antisense ODN treatment resulted in a failure of apoptosis in the MEE between palatal shelves in physical contact in organ culture. We conclude that betaig-h3 expression in the MEE is stimulated by TGF-beta3, causes cell death, and consequently results in complete fusion of the apposed palatal shelves.

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.

Gene Expression during Palate Fusion in vivo and in vitro

Failure of secondary palate fusion during embryogenesis is a cause of cleft palate. Disappearance of the medial epithelial seam (MES) is required to allow merging of the mesenchyme from both palatal shelves. This involves complex changes of the medial edge epithelial (MEE) cells and surrounding structures that are controlled by several genes whose spatio-temporal expression is tightly regulated. We have carried out morphological analyses and used a semi-quantitative RT-PCR technique to evaluate whether morphological changes and modulation in the expression of putative key genes, such as twist, snail, and E-cadherin, during the fusion process in palate organ culture parallel those observed in vivo, and show that this is indeed the case. We also show, using the organotypic model of palate fusion, that the down-regulation of the transcription factor snail that occurs with the progression of palate development is not dependent on fusion of the palatal shelves. dsg1, desmoglein1; EMT, epithelial-mesenchymal transition; MEE, medial edge epithelium; MES, medial epithelial seam; RT-PCR, reverse-transcriptase polymerase chain-reaction.

Transforming growth factor-beta 3 is required for secondary palate fusion.

Mice lacking TGF-beta 3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-beta 3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.