Shelf Elevation Research Articles

Cleft Palate Induced by Augmented Fibroblast Growth Factor-9 Signaling in Cranial Neural Crest Cells in Mice.

Although enhanced fibroblast growth factor (FGF) signaling has been demonstrated to be crucial in many cases of syndromic cleft palate caused by tongue malposition in humans, animal models that recapitulate this phenotype are limited, and the precise mechanisms remain elusive. Mutations in FGF9 with the effect of either loss- or gain-of-function effects have been identified to be associated with cleft palate in humans. Here, we generated a mouse model with a transgenic Fgf9 allele specifically activated in cranial neural crest cells, aiming to elucidate the gain-of-function effects of Fgf9 in palatogenesis. We observed cleft palate with 100% penetrance in mutant mice. Further analysis demonstrated that no inherent defects in the morphogenic competence of palatal shelves could be found, but a passively lifted tongue prevented the elevation of palatal shelves, leading to the cleft palate. This tongue malposition was induced by posterior spatial confinement that was exerted by temporomandibular joint (TMJ) dysplasia characterized by a reduction in Sox9+ progenitors within the condyle and a structural decrease in the posterior dimension of the lower jaw. Our findings highlight the critical role of excessive FGF signaling in disrupting spatial coordination during palate development and suggest a potential association between palatal shelf elevation and early TMJ development.

Research progress in molecular mechanism of palatal development

Cleft palate is one of the most common maxillofacial birth defects, which can occur alone or accompany with many known deformities. Palatal selves need to complete the process of vertical growth, elevation, adhesion and fusion in a specific time window of embryo development. Any abnormality in this process will lead to cleft palate. Although previous studies have identified many molecular networks that regulate the growth, location and fusion of palatal selves, there are still many unknown mechanisms for palatal development. The pathogenesis of cleft palate has not been clarified so far. In recent years, the molecular research on palate development has been deepened continuously. Here we summarize major recent advances and integrate the genes and molecular pathways with the cellular and morphogenetic processes of palatal shelf growth, patterning, elevation, adhesion, and fusion, in order to comprehensively understand the genotype-phenotype functional relationship and provide assistance in formulating effective prevention strategies for cleft palate disease.

Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

Retinoic acid delays murine palatal shelf elevation by inhibiting Wnt5a-mediated noncanonical Wnt signaling and downstream Cdc-42/F-actin remodeling in mesenchymal cells.

Mammalian palatal shelves erupted from maxillary prominences undergo vertical extention, transient elevation, and horizontal growth to fuse. Previous studies in mice reported that the retinoic acid (RA) contributed to cleft palate in high incidence by delaying the elevating procedure, but little was known about the underlying biological mechanisms. In this study, hematoxylin-eosin and immunofluorescence staining were employed to evaluate the phenotypes and the expression of related markers in the RA-treated mice model. In situ hybridization and RT-qPCR were used to detect the expression of genes involved in Wnt signaling pathway. The palatal mesenchymal cells were cultured in vitro, and stimulated with RA or CASIN, and co-treated with Foxy5. Wnt5a and Ccd42 expression were evaluated by immunofluorescence staining. Phalloidin was used to label the microfilament cytoskeleton (F-actin) in cultured cells. We revealed that RA resulted in 100% incidence of cleft palate in mouse embryos, and the expression of genes responsible for Wnt5a-mediated noncanonical Wnt signal transduction were specifically downregulated in mesenchymal palatal shelves. The in vitro study of palatal mesenchymal cells indicated that RA treatment disrupted the organized remodeling of cytoskeleton, an indicative structure of cell migration regulated by the small Rho GTPase Cdc42. Moreover, we showed that the suppression of cytoskeleton and cell migration induced by RA was partially restored using the small molecule Foxy-5-mediated activation of Wnt5A, and this restoration was attenuated by CASIN (a selective GTPase Cdc42 inhibitor) again. These data identified a crucial mechanism for Wnt5a-mediated noncanonical Wnt signaling in acting downstream of Rho GTPase Cdc42 to regulate cytoskeletal remodeling and cell migration during the process of palate elevation. Our study provided a new explanation for the cause of cleft palate induced by RA.

Gene Regulatory Networks and Signaling Pathways in Palatogenesis and Cleft Palate: A Comprehensive Review.

Palatogenesis is a complex and intricate process involving the formation of the palate through various morphogenetic events highly dependent on the surrounding context. These events comprise outgrowth of palatal shelves from embryonic maxillary prominences, their elevation from a vertical to a horizontal position above the tongue, and their subsequent adhesion and fusion at the midline to separate oral and nasal cavities. Disruptions in any of these processes can result in cleft palate, a common congenital abnormality that significantly affects patient's quality of life, despite surgical intervention. Although many genes involved in palatogenesis have been identified through studies on genetically modified mice and human genetics, the precise roles of these genes and their products in signaling networks that regulate palatogenesis remain elusive. Recent investigations have revealed that palatal shelf growth, patterning, adhesion, and fusion are intricately regulated by numerous transcription factors and signaling pathways, including Sonic hedgehog (Shh), bone morphogenetic protein (Bmp), fibroblast growth factor (Fgf), transforming growth factor beta (Tgf-β), Wnt signaling, and others. These studies have also identified a significant number of genes that are essential for palate development. Integrated information from these studies offers novel insights into gene regulatory networks and dynamic cellular processes underlying palatal shelf elevation, contact, and fusion, deepening our understanding of palatogenesis, and facilitating the development of more efficacious treatments for cleft palate.

Live imaging observation of elevation of the anterior palatal shelf in mouse embryos.

The mammalian secondary palate develops through complex processes including palatal shelf growth, elevation, and fusion. Palatal shelf elevation is a process accompanied by large-scale morphological changes over a short period. The elevation pattern changes along the anterior-posterior axis; the anterior region elevates by the "flip-up" model, and the middle and posterior regions reorient through the "flow" model; however, the mechanisms of both models are unclear because of the rapid progression of the elevation in utero. To observe palatal elevation in real-time in detail, we aimed to establish a live imaging method using explants of the anterior region of the palatal shelf in mouse embryos before the beginning of elevation. Changes in the degree of shelf orientation were measured, which showed that the palatal shelf continuously changed shape toward the lingual side. The changes in the angle between the lingual and buccal bases of the palatal shelf were different; the morphological change in the lingual side was more acute, and the buccal side was more obtuse. The timing of morphological changes of the lingual and buccal sides was nearly simultaneous, suggesting that the anterior region of the palatal shelf in vitro elevated according to the "flip-up" model. This live imaging method enables the continuous observation of palatal shelf elevation and provides new insights into palatogenesis. This article is protected by copyright. All rights reserved.

Mesenchymal β-catenin signaling affects palatogenesis by regulating α-actinin-4 and F-actin.

Our previous research have found that mesenchymal β-catenin may be involved in palatal shelf (PS) elevation by regulating F-actin. Here, we further investigated the exact mechanism of β-catenin/F-actin in the PS mesenchyme to regulate palatal reorientation. (1) Firstly, Ctnnb1ex3f (β-catenin) mice were conditionally overexpressed in the palatal mesenchyme by crossing with the Sox9-creERT2 mice (induced by Tamoxifen injections); (2) Subsequently, histology and immunohistochemistry were used to characterize the variations of PS morphology and expression of key molecules associated with developmental process; (3) Finally, experiments in vivo and ex vivo were employed to identify the critical mechanisms in β-catenin silenced and overexpressed models. We found that the Sox9CreER; Ctnnb1ex3f mice exhibited failed palatal elevation and visible cleft palate, and overexpression of β-catenin disturbed the F-actin responsible for cytoskeletal remodeling in palatal mesenchymal cells. qRT-PCR results showed mRNA levels of α-actinin4, a gene involved in F-actin cross-linking, were associated with knockdown or overexpression of β-catenin in ex vivo, respectively. Experiments in vivo revealed that mesenchymal specific inactivation or overexpression of β-catenin exhibited decreased or increased α-actinin-4 expression. Mesenchymal β-catenin/F-actin plays an essential role in PS reorientation, which mediate α-actinin-4 to regulate F-actin cytoskeleton reorganization.

Morphometric analysis of secondary palate development in human embryos.

Rapid shelf elevation and contact of the secondary palate and fusion reportedly occur due to a growth-related equilibrium change in the structures within the oro-nasal cavity. This study aimed to quantitatively evaluate complex three-dimensional morphological changes and their effects on rapid movements, such as shelf elevation and contact, and fusion. Morphological changes during secondary palate formation were analyzed using high-resolution digitalized imaging data (phase-contrast X-ray computed tomography and magnetic resonance images) obtained from 22 human embryonic and fetal samples. The three-dimensional images of the oro-nasal structures, including the maxilla, palate, pterygoid hamulus, tongue, Meckel's cartilage, nasal cavity, pharyngeal cavity, and nasal septum, were reconstructed manually. The palatal shelves were not elevated in all the samples at Carnegie stage (CS)21 and CS22 and in three samples at CS23. In contrast, the palatal shelves were elevated but not in contact in one sample at CS23. Further, the palatal shelves were elevated and fused in the remaining four samples at CS23 and all three samples from the early fetal period. For each sample, 70 landmarks were subjected to Procrustes and principal component (PC) analysis. PC-1 accounted for 67.4% of the extracted gross changes before and after shelf elevations. Notably, the PC-1 values of the negative and positive value groups differed significantly. The PC-2 value changed during the phases in which the change in the PC-1 value was unnaturally slow and stopped at CS22 and the first half of CS23. This period, defined as the "approach period", corresponds to the time before dynamic changes occur as the palatal shelves elevate, the tongue and mandibular tip change their position and shape, and secondary palatal shelves contact and fuse. During the "approach period", measurements of PC-2 changes showed that structures on the mandible (Meckel's cartilage and tongue) and maxilla (palate and nasal cavity) did not change positions, albeit both groups of structures appeared to be compressed anterior-posteriorly. However, during and after shelf elevation, measurements of PC-1 changes showed significant changes between maxillary and mandibular structures, particularly positioning of the shelves above the tongue and protrusion of the tongue and mandible. These results suggest an active role for Meckel's cartilage growth in repositioning the tongue to facilitate shelf elevation. The present data representing three distinct phases of secondary palate closure in humans can advance the understanding of morphological growth changes occurring before and after the horizontal positioning of palatal shelves and their fusion to close the secondary palate in humans successfully.

An improved algorithm for extracting crossovers of satellite ground tracks

A crossover refers to the intersection of two satellite ground tracks. Crossovers are required for performing crossover differences adjustment to remove the orbit error and for establishing the time series of elevation in surfaces, which is why crossovers are important for satellite altimetry measurements of ice sheet or ice shelf elevation changes. How to extract more crossovers precisely is the objective of this paper. On the basis of the traditional method of solving crossovers and computer graphics, this paper proposes an improved algorithm called improved rapid rejection and straddle test (IRST) for computing the position of the crossover point. This algorithm efficiently and accurately searches for two points in ascending pass and two points in descending pass that can form a crossover, and then computes crossovers’ geolocation. By using CryoSat-2 satellite altimeter data, we conducted our study on the Antarctic Ross ice shelf and Filchner–Ronne ice shelf using the fixed iteration (FI) algorithm, rapid rejection and straddle test (RST) algorithm, and the IRST algorithm. Results show that IRST is superior to the two other algorithms in terms of the number of crossovers, geolocation accuracy and computational efficiency. These advantages are more noticeable in the border areas between the ice shelf and the mountain with large terrain slope, which solves the problem that crossovers are less in these areas with poor-quality data coverage.

Spatiotemporal Gene Expression Regions along the Anterior-Posterior Axis in Mouse Embryos before and after Palatal Elevation.

The mammalian secondary palate is formed through complex developmental processes: growth, elevation, and fusion. Although it is known that the palatal elevation pattern changes along the anterior–posterior axis, it is unclear what molecules are expressed and whether their locations change before and after elevation. We examined the expression regions of molecules associated with palatal shelf elevation (Pax9, Osr2, and Tgfβ3) and tissue deformation (F-actin, E-cadherin, and Ki67) using immunohistochemistry and RT–PCR in mouse embryos at E13.5 (before elevation) and E14.5 (after elevation). Pax9 was expressed at significantly higher levels in the lingual/nasal region in the anterior and middle parts, as well as in the buccal/oral region in the posterior part at E13.5. At E14.5, Pax9 was expressed at significantly higher levels in both the lingual/nasal and buccal/oral regions in the anterior and middle parts and the buccal/oral regions in the posterior part. Osr2 was expressed at significantly higher levels in the buccal/oral region in all parts at E13.5 and was more strongly expressed at E13.5 than at E14.5 in all regions. No spatiotemporal changes were found in the other molecules. These results suggested that Pax9 and Osr2 are critical molecules leading to differences in the elevation pattern in palatogenesis.

Arhgap29 is Required for Proper Palatogenesis and its Loss in Ectodermal‐Derived Cells Results in a Kinked Tail Phenotype

Craniofacial deformities, specifically non‐syndromic cleft lip with or without palate (NSCL/P), are among the most common class of birth defects. Complex interactions between genetic and environmental factors contribute to the development of NSCL/P. Many genes have been associated with this disease, including ARHGAP29. ARHGAP29, Rho GTPase Activating Protein (GAP) 29, functions in the cyclical regulation of the small GTPase RhoA. Previous studies have identified genetic variants of ARHGAP29 in NSCL/P and have found that ARHGAP29 levels were reduced in mice deficient for Irf6. Data from these studies suggest a role for ARHGAP29 in craniofacial morphogenesis but it remains unknown if ARHGAP29 is required for, or regulates craniofacial development. ARHGAP29 is detected in many tissues, including the oral epithelium. Using the Keratin14‐driven Cre‐recombinase allele, Krt14Cre;Arhgap29fl/fl mice displayed a delay in palatal shelf elevation and did not exhibit a cleft palate. We hypothesized that the absence of a cleft palate was due to the late activation of the Krt14‐Cre driver. EctCre drives Cre recombinase specifically in the ectoderm a couple of days before the Krt14‐Cre. Our results show that EctCre;Arhgap29fl/fl mice survive to birth and display a non‐mendelian pattern of inheritance. Interestingly, EctCre;Arhgap29fl/fl mice display a fully penetrant kinked tail phenotype, which has been described in other murine models of NSCL/P, and has been attributed to neural tube and skeletal defects. Additionally, at embryonic (E) day 14.5, while all the wild‐type embryos showed adherent palatal shelves, 75% of EctCre;Arhgap29fl/fl mice displayed palatal shelves not elevated or adherent. Our preliminary data also identified one EctCre;Arhgap29fl/fl embryo out of 3 total analyzed with a cleft palate at E18.5. These data show that ARHGAP29 is required for on‐time palatal shelves movement, supporting a role for ARHGAP29 in craniofacial development.

SPECC1L deficiency causes shortened primary cilia and genetically interacts with IFT‐A deficiency to affect palatogenesis

Orofacial clefts are among the most common congenital malformations, affecting approximately 1 in 700 births. SPECC1L mutations in patients with syndromic cleft lip and/or palate cluster in the second coiled coil domain (CCD2), which facilitates interaction of SPECC1L with microtubules. We have shown that loss of SPECC1L in a null allele (Specc1l∆Ex4) resulted in perinatal lethality and delayed palatal shelf elevation, but no cleft palate (CP) or exencephaly at birth. However, mice with an in‐frame deletion of SPECC1L‐CCD2 exhibited CP and exencephaly, which are common manifestations of ciliopathies. Given that SPECC1L cytoplasmic expression includes localization around the cilia base and that SPECC1L regulates actin cytoskeleton, we investigated an association between SPECC1L and primary cilia. We observed that primary cilia were shortened relative to wild‐type in E13.5 Specc1l∆Ex4/∆Ex4 as well as Specc1l∆CCD2/∆CCD2 mutant palatal mesenchyme and epithelium. The cytoplasmic expression of SPECC1L around the cilia base was diminished in Specc1l∆CCD2/∆CCD2 cells. Thus, loss of SPECC1L association with microtubules appears sufficient to prevent its pericentriolar localization and to cause cilia shortening. Super‐resolution imaging for acetylated‐α‐tubulin and ciliary membrane protein, ARL13B, revealed bulbous structures in Specc1l mutant cilia, suggesting a defect in ciliary protein trafficking. Due to the overlapping phenotypes of shortened cilia, exencephaly and CP between the intraflagellar transport (IFT)‐A mouse mutant, Thm1aln/aln, and Specc1l∆CCD2/∆CCD2, we crossed these mutants to look for a genetic interaction. 18% of Specc1l∆CCD2/+, 0% of Thm1aln/+, and 33% of double heterozygous Specc1l∆CCD2/+;Thm1aln/+ embryos showed CP, confirming a genetic interaction. This was observed when the mother was Specc1l∆CCD2/+. However, remarkably, if the mother was Thm1aln/+, there was no CP incidence in Specc1l∆CCD2/+ and Specc1l∆CCD2/+;Thm1aln/+ embryos even though they had shortened cilia, suggesting Thm1aln/+ mothers provided a protective environment against CP phenotype. Consistent with our previous data, Specc1l∆Ex4;Thm1aln/+ mice resulted in no CP. These results suggest that shortened cilia inSpecc1l mutants do not cause but rather modulate CP phenotype. Overall, our studies indicate a novel role for SPECC1L in ciliogenesis, a novel genetic interaction between Specc1l andThm1, and a novel protective maternal genetic effect of Thm1 in palatogenesis.

Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos

Embryonic palate development involves bilateral vertical growth of palatal shelves – extensions from the maxillary processes – next to the tongue at embryonic day (E) 13.5. This vertical growth is followed by elevation and fusion above the tongue by E14.5. Recent models indicate that this process of elevation involves a complex vertical to horizontal remodeling of the palatal shelves. While earlier studies have implied that this is a rapid process, the precise timing has not been resolved. To further understand the dynamics of palatal shelf elevation, we employed time‐restricted pregnancies with a one‐hour resolution and magnetic resonance (MR) imaging of intermediate stages. Our data showed that in C57BL/6J mice, palatal shelves are unelevated in 100% of embryos at E14.0 and completely elevated in 80% of embryos at E14.25, which is only a 6‐hour timeframe. Interestingly, all E14.25 embryos with unelevated palatal shelves (20%) were female, suggesting a shift towards delayed elevation in female embryos. Even at E14.125, 25% of embryos had completely elevated palatal shelves suggesting that once initiated, the palatal shelves elevated rapidly with elevation completing in less than 3 hours in individual embryos. In contrast to C57BL/6J, in FVB/NJ mice, the elevation window was shifted earlier and was broader to E13.875‐E14.25 without any noticeable sex differences. In a proportion of embryos in both strains, we also captured an intermediate stage with unilateral palatal shelf elevation. Both right and left shelves were identified to be unilaterally elevated indicating that it was a random process. It remains to be determined whether palatal shelves elevate in a sequential manner, i.e., one‐at‐a‐time, with an obligate unilateral elevation stage. MR imaging of various stages showed a highly dynamic antero‐posterior remodeling of the shelves during elevation, beginning with the formation of bilateral bulges in the posterior palate. These bulges tapered anteriorly, however, palatal shelf fusion still began in the middle part of the palate. The bulge regions did not show changes in cell proliferation, consistent with the notion that rapid elevation was primarily due to cytoskeletal and extracellular matrix changes. We are using finite element analysis to identify the mechanical forces underlying these morphological changes. Thus, our data reveal critical novel insights into the rapid dynamic changes as well as strain and sex differences in palatal shelf elevation that lay the foundation for future studies of normal and abnormal palatogenesis.

DEM Generation with ICESat-2 Altimetry Data for the Three Antarctic Ice Shelves: Ross, Filchner–Ronne and Amery

The ice shelf is an important component of the Antarctic system, and the interaction between the ice sheet and the ocean often proceeds through mass variations of the ice shelf. The digital elevation model (DEM) of the ice shelf is particularly important for ice shelf elevation change and mass balance estimation. With the development of satellite altimetry technology, it became an important data source for DEM research of Antarctica. The National Aeronautics and Space Administration (NASA) Ice, Cloud, and Land Elevation Satellite-2 (ICESat-2) launched in 2018 is a significant improvement in along-track sampling rate and measurement accuracy compared with previous altimetry satellites. This study uses ordinary kriging interpolation to present new DEMs (ICESat-2 DEM hereinafter) for the three ice shelves (Ross, Filchner–Ronne and Amery) in Antarctica with ICESat-2 altimetry data. Two variogram models (linear and spherical) of ordinary kriging interpolation are compared in this paper. The result shows that the spherical model generally shows better performance and lower standard deviation (STD) than the linear models. The precision of the ultimate DEM was evaluated by NASA Operation IceBridge (OIB) data and compared with five previously published Antarctic DEM products (REMA, TanDEM-X PolarDEM, Slater DEM, Helm DEM, and Bamber DEM). The comparison reveals that the mean difference between ICESat-2 DEM of the Ross ice shelf and OIB is −0.016 m with a STD of 0.918 m, and the mean difference between ICESat-2 DEM of the Filchner–Ronne ice shelf and OIB is −0.533 m with a STD of 0.718 m. The three ICESat-2 DEMs show higher spatial resolution and elevation accuracy than five previously published Antarctic DEMs.

Methods of Palate Culture in Later Palatogenesis: Elevation, Horizontal Outgrowth, and Fusion.

The ex vivo culture of the palate has provided a versatile model in which to study palatogenesis. Dysmorphias of the palate remain one of the most common birth defects globally, with great scope for future research in both normal and dysmorphic palatogenesis. This process can be studied in the mouse model using both the hyperoxic rolling culture of maxillary explants and Trowell-type static cultures, which are optimal for the study of different stages of palate development respectively. Here, we describe both methods: the former for the study of palatal shelf elevation and horizontal growth, and the latter for palatal shelf fusion . Both are applicable in murine embryos cultured at embryonic day 13.5 using nonspecialist equipment.

Three-dimensional reconstruction of systematic histological sections: application to observations on palatal shelf elevation

Normal mammalian secondary palate development undergoes a series of processes, including palatal shelf (PS) growth, elevation, adhesion and fusion, and palatal bone formation. It has been estimated that more than 90% of isolated cleft palate is caused by defects associated with the elevation process. However, because of the rapidly completed elevation process, the entire process of elevation will never be easy to clarify. In this article, we present a novel method for three-dimensional (3D) reconstruction of thick tissue blocks from two-dimensional (2D) histological sections. We established multiplanar sections of the palate and tongue in coronal and sagittal directions, and further performed 3D reconstruction to observe the morphological interaction and connection between the two components prior to and during elevation. The method completes an imaging system for simultaneous morphological analysis of thick tissue samples using both synthetic and real data. The new method will provide a comprehensive picture of reorientation morphology and gene expression pattern during the palatal elevation process.

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

SPECC1L-deficient primary mouse embryonic palatal mesenchyme cells show speed and directionality defects

Cleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.

Lithium-induced overexpression of β-catenin delays murine palatal shelf elevation by Cdc-42 mediated F-actin remodeling in mesenchymal cells.

Lithium chloride (LiCl) is widely used for the treatment of manic and other psychotic disorders, but the administration of lithium can result in several congenital defects in the fetus, including cleft palate (Meng, Wang, Torensma, Jw & Bian, 2015) (Szabo, 1970). However, the mechanism of Lithium's action as a developmental toxicant in palatogenesis is not well known. In this study, hematoxylin-eosin and immunofluorescence staining were employed to evaluate the phenotypes and the expression of related markers in the LiCl-treated mice model. The palatal mesenchymal cells were cultured in vitro, and stimulated with LiCl or SKL2000, and co-treated with CASIN. β-catenin protein and other cytoskeleton associated markers were evaluated by Western blotting. We found that Lithium disrupted palate elevation by increasing the expression of β-catenin in C57BL/6J mice with the high incidence of cleft palate (62.5%). LiCl disturbed the F-actin responsible for cytoskeletal remodeling in mesenchymal cells, which proved to be essential in generating the elevating force during palatal elevation. Additionally, our Western blotting analysis revealed that the overexpression of β-catenin resulted in up-regulation of Cdc42, which mediated the downstream F-actin synthesis. We concluded the LiCl-induced β-catenin overexpression delayed murine palatal shelf elevation by disturbing Cdc42 mediated F-actin cytoskeleton synthesis in the palatal mesenchyme.

Tissue-specific analysis of Fgf18 gene function in palate development.

Previous studies showed that mice lacking Fgf18 function had cleft palate defects and that the FGF18 locus was associated with cleft lip and palate in humans, but what specific roles Fgf18 plays during palatogenesis are unclear. We show that Fgf18 exhibits regionally restricted expression in developing palatal shelves, mandible, and tongue, during palatal outgrowth and fusion in mouse embryos. Tissue-specific inactivation of Fgf18 throughout neural crest-derived craniofacial mesenchyme caused shortened mandible and reduction in ossification of the frontal, nasal, and anterior cranial base skeletal elements in Fgf18c/c ;Wnt1-Cre mutant mice. About 64% of Fgf18c/c ;Wnt1-Cre mice exhibited cleft palate. Whereas palatal shelf elevation was impaired in many Fgf18c/c ;Wnt1-Cre embryos, no significant difference in palatal cell proliferation was detected between Fgf18c/c ;Wnt1-Cre embryos and their control littermates. Embryonic maxillary explants from Fgf18c/c ;Wnt1-Cre embryos showed successful palatal shelf elevation and fusion in organ culture similar to the maxillary explants from control embryos. Furthermore, tissue-specific inactivation of Fgf18 in the early palatal mesenchyme did not cause cleft palate. These results demonstrate a critical role for Fgf18 expression in the neural crest-derived mesenchyme for the development of the mandible and multiple craniofacial bones but Fgf18 expression in the palatal mesenchyme is dispensable for palatogenesis.