Shelf Elevation Research Articles

YAP/TAZ Regulate Elevation and Bone Formation of the Mouse Secondary Palate

Clefting of the secondary palate is one of the most common congenital anomalies, and the multiple corrective surgeries that individuals with isolated cleft palate undergo are associated with major costs and morbidities. Secondary palate development is a complex, multistep process that includes the elevation of the palatal shelves from a vertical to horizontal position, a process that is not well understood. The Hippo signaling cascade is a mechanosensory pathway that regulates morphogenesis, homeostasis, and regeneration by controlling cell proliferation, apoptosis, and differentiation, primarily via negative regulation of the downstream effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). We deleted Yap/Taz throughout the palatal shelf mesenchyme as well as specifically in the posterior palatal shelf mesenchyme, using the Osr2Cre and Col2Cre drivers, respectively, which resulted in palatal shelf elevation delay and clefting of the secondary palate. In addition, the deletion resulted in undersized bones of the secondary palate. We next determined downstream targets of YAP/TAZ in the posterior palatal shelves, which included Ibsp and Phex, genes involved in mineralization, and Loxl4, which encodes a lysyl oxidase that catalyzes collagen crosslinking. Ibsp, Phex, and Loxl4 were expressed at decreased levels in the ossification region in the posterior palatal shelf mesenchyme upon deletion of Yap/Taz. Furthermore, collagen levels were decreased specifically in the same region prior to elevation. Thus, our data suggest that YAP/TAZ may regulate collagen crosslinking in the palatal shelf mesenchyme, thus controlling palatal shelf elevation, as well as mineralization of the bones of the secondary palate.

SPECC1L regulates palate development downstream of IRF6.

SPECC1L mutations have been identified in patients with rare atypical orofacial clefts and with syndromic cleft lip and/or palate (CL/P). These mutations cluster in the second coiled-coil and calponin homology domains of SPECC1L and severely affect the ability of SPECC1L to associate with microtubules. We previously showed that gene-trap knockout of Specc1l in mouse results in early embryonic lethality. We now present a truncation mutant mouse allele, Specc1lΔC510, that results in perinatal lethality. Specc1lΔC510/ΔC510 homozygotes showed abnormal palate rugae but did not show cleft palate. However, when crossed with a gene-trap allele, Specc1lcGT/ΔC510 compound heterozygotes showed a palate elevation delay with incompletely penetrant cleft palate. Specc1lcGT/ΔC510 embryos exhibit transient oral epithelial adhesions at E13.5, which may delay shelf elevation. Consistent with oral adhesions, we show periderm layer abnormalities, including ectopic apical expression of adherens junction markers, similar to Irf6 hypomorphic mutants and Arhgap29 heterozygotes. Indeed, SPECC1L expression is drastically reduced in Irf6 mutant palatal shelves. Finally, we wanted to determine if SPECC1L deficiency also contributed to non-syndromic (ns) CL/P. We sequenced 62 Caucasian, 89 Filipino, 90 Ethiopian, 90 Nigerian and 95 Japanese patients with nsCL/P and identified three rare coding variants (p.Ala86Thr, p.Met91Iso and p.Arg546Gln) in six individuals. These variants reside outside of SPECC1L coiled-coil domains and result in milder functional defects than variants associated with syndromic clefting. Together, our data indicate that palate elevation is sensitive to deficiency of SPECC1L dosage and function and that SPECC1L cytoskeletal protein functions downstream of IRF6 in palatogenesis.

Disruption of the nectin-afadin complex recapitulates features of the human cleft lip/palate syndrome CLPED1.

Cleft palate (CP), one of the most common congenital conditions, arises from failures in secondary palatogenesis during embryonic development. Several human genetic syndromes featuring CP and ectodermal dysplasia have been linked to mutations in genes regulating cell-cell adhesion, yet mouse models have largely failed to recapitulate these findings. Here, we use in utero lentiviral-mediated genetic approaches in mice to provide the first direct evidence that the nectin-afadin axis is essential for proper palate shelf elevation and fusion. Using this technique, we demonstrate that palatal epithelial conditional loss of afadin (Afdn) - an obligate nectin- and actin-binding protein - induces a high penetrance of CP, not observed when Afdn is targeted later using Krt14-Cre We implicate Nectin1 and Nectin4 as being crucially involved, as loss of either induces a low penetrance of mild palate closure defects, while loss of both causes severe CP with a frequency similar to Afdn loss. Finally, expression of the human disease mutant NECTIN1W185X causes CP with greater penetrance than Nectin1 loss, suggesting this alteration may drive CP via a dominant interfering mechanism.

The KMT2D Kabuki syndrome histone methylase controls neural crest cell differentiation and facial morphology.

Kabuki syndrome (KS) is a congenital craniofacial disorder resulting from mutations in the KMT2D histone methylase (KS1) or the UTX histone demethylase (KS2). With small cohorts of KS2 patients, it is not clear whether differences exist in clinical manifestations relative to KS1. We mutated KMT2D in neural crest cells (NCCs) to study cellular and molecular functions in craniofacial development with respect to UTX. Similar to UTX, KMT2D NCC knockout mice demonstrate hypoplasia with reductions in frontonasal bone lengths. We have traced the onset of KMT2D and UTX mutant NCC frontal dysfunction to a stage of altered osteochondral progenitor differentiation. KMT2D NCC loss-of-function does exhibit unique phenotypes distinct from UTX mutation, including fully penetrant cleft palate, mandible hypoplasia and deficits in cranial base ossification. KMT2D mutant NCCs lead to defective secondary palatal shelf elevation with reduced expression of extracellular matrix components. KMT2D mutant chondrocytes in the cranial base fail to properly differentiate, leading to defective endochondral ossification. We conclude that KMT2D is required for appropriate cranial NCC differentiation and KMT2D-specific phenotypes may underlie differences between Kabuki syndrome subtypes.

Hyaluronic acid is required for palatal shelf movement and its interaction with the tongue during palatal shelf elevation

Palatal shelf elevation is an essential morphogenetic process that results from palatal shelf movement caused by an intrinsic elevating force. The nature of the elevating force remains unclear, but the accumulation of hyaluronic acid (HA) in the extracellular matrix (ECM) of the palatal shelves may play a pivotal role in developing the elevating force. In mammals, HA is synthesized by hyaluronic acid synthases (HAS) that are encoded by three genes (Has1-3). Here, we used the Wnt1-Cre driver to conditionally disrupt hyaluronic acid synthase 2 (Has2) in cranial neural crest cell lineages. All Has2 conditional knockout (cko) mice had cleft palate due to failed shelf elevation during palate development. The HA content was significantly reduced in the craniofacial mesenchyme of Has2 cko mutants. Reduced HA content affected the ECM space and shelf expansion to result in a reduced shelf area and an increased mesenchymal cell density in the palatal shelves of Has2 cko mutants. We examined palatal shelf movement by removal of the tongue and mandible from unfixed E13.5 and early E14.5 embryonic heads. Reduced shelf expansion in Has2 cko mutants altered palatal shelf movement in the medial direction resulting in a larger gap between the palatal shelves than that of littermate controls. We further examined palatal shelf movement in the intact oral cavity by culturing explants containing the maxilla, palate, mandible and tongue (MPMT explants). The palatal shelves elevated alongside morphological changes in the tongue after 24-h culture in MPMT explants of early E14.5 wild type embryos. On the contrary, shelf elevation failed to occur in MPMT explants of age-matched Has2 cko mutants because the tongue obstructs palatal shelf movement, suggesting that reduced shelf expansion could be essential for the palatal shelves to interact with the tongue and overcome tongue obstruction during shelf elevation. Has2 cko mutants also showed micrognathia due to reduced HA content in the mandibular mesenchyme including Meckel’s cartilage. Through 3D imaging and morphometric analysis, we demonstrate that mandibular growth results in a significant increase in the vertical dimension of the common oral-nasal cavity that facilitates palatal shelf movement and its interaction with the tongue during shelf elevation.

Requirement of Hyaluronan Synthase-2 in Craniofacial and Palate Development

Cleft palate is a common major birth defect resulting from disruption of palatal shelf growth, elevation, or fusion during fetal palatogenesis. Whereas the molecular mechanism controlling palatal shelf elevation is not well understood, a prevailing hypothesis is that region-specific accumulation of hyaluronan, a predominant extracellular glycosaminoglycan in developing palatal mesenchyme, plays a major role in palatal shelf elevation. However, direct genetic evidence for a requirement of hyaluronan in palate development is still lacking. In this study, we show that Has2, 1 of 3 hyaluronan synthases in mammals, plays a major role in hyaluronan synthesis in the neural crest–derived craniofacial mesenchyme during palatogenesis in mice. We analyzed developmental defects caused by tissue-specific inactivation of Has2 throughout the cranial neural crest lineage or specifically in developing palatal or mandibular mesenchyme, respectively, using Wnt1-Cre, Osr2-Cre, and Hand2-Cre transgenic mice. Inactivation of Has2 either throughout the neural crest lineage or specifically in the developing palatal mesenchyme caused reduced palatal shelf size and increased palatal mesenchyme cell density prior to the time of normal palatal shelf elevation. Whereas both Has2f/f;Wnt1-Cre and Has2f/f;Osr2-Cre mutant mice exhibit cleft palate at complete penetrance, the Has2f/f; Wnt1-Cre fetuses showed dramatically reduced mandible size and complete failure of palatal shelf elevation, whereas Has2f/f;Osr2-Cre fetuses had normal mandibles and delayed palatal shelf elevation. All Has2f/f;Hand2-Cre pups showed reduced mandible size and about 50% of them had cleft palate with disruption of palatal shelf elevation. Results from explant culture assays indicate that disruption of palatal shelf elevation in Has2f/f;Hand2-Cre mutant fetuses resulted from physical obstruction by the malformed mandible and tongue. Together, these data indicate that hyaluronan plays a crucial intrinsic role in palatal shelf expansion and timely reorientation to the horizontal position above the tongue as well as an important role in mandibular morphogenesis that secondarily affects palatal shelf elevation.

Expression of gamma-aminobutyric acid type A receptor beta3 subunit in murine cleft palate induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

Objective: To investigate the expression of gamma-aminobutyric acid type A receptor beta3 subunit (GABRB3) on cleft palate in C57BL/6J mice induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Methods: Sixty C57BL/6J pregnant mice on gestation day (GD) 10.5 were divided into two groups: one group was administered through gastric tubes one dose of 28 μg/kg TCDD (experimental group) and the other group was administered through gastric tubes one dose of 5.6 ml/kg corn oil (control group). Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13.5-17.5) and the palatal tissue studied in morphological and histological observation. The relative mRNA and protein expression of GABRB3 was measured by real-time quantitative PCR and Western blotting. Localization of GABRB3 protein was measured by immunohistochemistry or immunofluorescence. Results: The incidence of cleft palate at GD17.5 was 100% in experimental group and there was no cleft palate occurred in the control group (0); elevation of palatine processes in experimental group was completed on GD15.5 which was clearly delayed by a day compared with that in control group. On GD14.5-GD17.5, the mRNA expression (0.561±0.073, 0.728±0.104, 0.782±0.137, 0.686±0.145) and protein expression (0.288±0.013, 0.404±0.017, 0.399±0.012, 0.307±0.010) in the experimental group were significantly lower than the control group mRNA expression (0.818±0.088, 0.865±0.086, 1.021±0.054, 1.163±0.179) and protein expression (0.481±0.017, 0.456±0.009, 0.474±0.016, 0.529±0.015)(P<0.05). Immunohistochemistry and immunofluorescence showed that GABRB3 was mainly expressed in the mesenchymal cells and medial edge epithelium. Conclusions: TCDD delayed palatal shelf elevation and eventually led to cleft palate may be associated with a decrease in GABRB3. GABRB3 may play an important role in the elevation and fusion phases of the palate development.

SPECC1L‐Deficient Cells Show Impaired Collective Cell Migration Attributes that are Rescued by Upregulation of PI3K‐AKT Pathway

Clefts of the lip and/or palate (CL/P) are common anomalies that occur in 1/700 live births. Pathogenic SPECC1L variants identified in patients with rare atypical clefts and syndromic CL/P suggest the gene plays a primary role in face and palate development. Null mutants are lethal at embryonic day 9.5 with defective neural tube closure and cranial neural crest cell delamination. Hypomorphic Specc1lΔ200 mutants die perinatally, but do not show cleft palate. Specc1lnull/Δ200 compound mutants resulted in transient oral adhesions, subepidermal blebbing, and a delay in palate elevation. Palate development requires extensive mesenchymal remodeling especially during palatal shelf elevation. We posit that this remodeling involves collective cell migration (CCM) of neural crest derived palate mesenchymal cells. Live time‐lapse microscopy was performed to visualize in vitro wound‐repair assays with SPECC1L‐deficient U2OS osteosarcoma and primary mouse embryo palatal mesenchyme (MEPM) cells. SPECC1L‐deficient cells consistently showed delayed wound closure. Directional movement of neighboring cells is a hallmark of CCM. In wound‐repair assays, an increase in movement parallel to the direction of wound front propagation results can be measured as an attribute of CCM. Tracking of individual trajectories of SPECC1L deficient cells during wound‐repair indeed shows an increase in cell movement perpendicular to the direction of wound closure. This diminished directionality causes delayed wound closure even though cells are adequately motile. We previously demonstrated SPECC1L to be a novel regulator of PI3K‐AKT signaling, with in vitro and in vivo SPECC1L deficiency exhibiting reduced PI3K‐AKT signaling. Indeed, wound closure delay, and the underlying CCM defect, in SPECC1L‐deficient U2OS and MEPM cells can be rescued by activation of the PI3K‐AKT signaling pathway using 740 Y‐P small molecule activator. Our data are the first to show CCM attributes in MEPM cells, and propose that MEPM cells can be used to model mesenchymal remodeling during palatal shelf elevation. We also show a novel role for SPECC1L in CCM through modulation of P13K‐AKT signaling.Support or Funding InformationThis project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vitro Analysis of Palatal Shelf Elevation During Secondary Palate Formation.

Palatal shelf elevation is an essential morphogenetic process during secondary palate formation. It has been proposed that shelf elevation results from an intrinsic elevating force and is regulated by extrinsic factors that are associated with development of other orofacial structures. Although dynamic palate culture is a common in vitro approach for studying shelf elevation, it requires the tongue or the tongue and mandible to be removed before culture, which prevents any determination of the role of the extrinsic factors in regulating shelf elevation. We showed that ex vivo removal of the tongue and mandible from unfixed embryonic heads led to spontaneous shelf movements that were more pronounced at late E13.5 and early E14.5 than those of E12.5 and early E13.5, suggesting that the strength of the elevating force increases over time during palate development. We further used a suspension culture technique to analyze palatal shelf movement in an intact oral cavity by culturing the orofacial portion of embryonic heads that include the maxilla, palatal shelves, mandible, and tongue (MPMT). MPMT explants were cultured in the serum-free medium with slow rotation for 24-48 hr. The palatal shelves successfully elevated during culture and displayed intermediate morphologies that closely resemble those of in vivo shelf elevation. We demonstrate that the tongue and mandible facilitate shelf medial movement/growth during shelf elevation and further suggest that the interaction of the palatal shelves and tongue could be one of the extrinsic factors that regulate the elevation process. Anat Rec, 302:1594-1604, 2019. © 2019 American Association for Anatomy.

Conditional deletion of Bmp2 in cranial neural crest cells recapitulates Pierre Robin sequence in mice.

Bone morphogenetic protein (BMP) signaling plays a crucial role in the development of craniofacial organs. Mutations in numerous members of the BMP signaling pathway lead to several severe human syndromes, including Pierre Robin sequence (PRS) caused by heterozygous loss of BMP2. In this study, we generate mice carrying Bmp2-specific deletion in cranial neural crest cells using floxed Bmp2 and Wnt1-Cre alleles to mimic PRS in humans. Mutant mice exhibit severe PRS with a significantly reduced size of craniofacial bones, cleft palate, malformed tongue and micrognathia. Palate clefting is caused by the undescended tongue that prevents palatal shelf elevation. However, the tongue in Wnt1-Cre;Bmp2f/f mice does not exhibit altered rates of cell proliferation and apoptosis, suggesting contribution of extrinsic defects to the failure of tongue descent. Further studies revealed obvious reduction in cell proliferation and differentiation of osteogenic progenitors in the mandible of the mutants, attributing to the micrognathia phenotype. Our study illustrates the pathogenesis of PRS caused by Bmp2 mutation, highlights the crucial role of BMP2 in the development of craniofacial bones and emphasizes precise coordination in the morphogenesis of palate, tongue and mandible during embryonic development.

Development of Normal and Cleft Palate: A Central Role for Connective Tissue Growth Factor (CTGF)/CCN2

Development of the palate is the result of an organized series of events that require exquisite spatial and temporal regulation at the cellular level. There are a myriad of growth factors, receptors and signaling pathways that have been shown to play an important role in growth, elevation and/or fusion of the palatal shelves. Altered expression or activation of a number of these factors, receptors and signaling pathways have been shown to cause cleft palate in humans or mice with varying degrees of penetrance. This review will focus on connective tissue growth factor (CTGF) or CCN2, which was recently shown to play an essential role in formation of the secondary palate. Specifically, the absence of CCN2 in KO mice results in defective cellular processes that contribute to failure of palatal shelf growth, elevation and/or fusion. CCN2 is unique in that it has been shown to interact with a number of other factors important for palate development, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), epidermal growth factor (EGF), Wnt proteins and transforming growth factor-βs (TGF-βs), thereby influencing their ability to bind to their receptors and mediate intracellular signaling. The role that these factors play in palate development and their specific interactions with CCN2 will also be reviewed. Future studies to elucidate the precise mechanisms of action for CCN2 and its interactions with other regulatory proteins during palatogenesis are expected to provide novel information with the potential for development of new pharmacologic or genetic treatment strategies for clinical intervention of cleft palate during development.

Prdm16 and Mecom mutants exhibit cleft secondary palate as a result of perturbations that affect different stages of palatogenesis

The paralogous Prdm16 and Mecom transcription factor genes play widespread and important roles during mouse embryogenesis and postnatal development. Each gene is required for the maintenance of various stem cell populations (i.e. hematopoietic and neuronal stem cells). Our studies have focused on increasing understanding of Prdm16 function in developing orofacial structures, specifically mandible, tongue and palate. Prdm16 loss‐of‐function mutations result in cleft secondary palate (CP) due to failed palate shelf elevation secondary to micrognathia. This mechanism of clefting models that observed in human Pierre Robin Sequence CP.We are examining the requirement for Prdm16 during palate formation through neural crest‐, mandible‐ and palate‐specific ablation or rescue of Prdm16 using an invertible conditional gene trap null allele of Prdm16. These experiments largely support a palate‐extrinsic mechanism of clefting in Prdm16 mutants, since null and mandible‐specific Prdm16 mutant embryos exhibit micrognathia and CP and palate‐specific loss results in CP with reduced penetrance. To characterize the underlying cellular defects, we are assessing cell proliferation and apoptosis in the palate shelves and Meckel's cartilage. We also continue to screen craniofacial markers by in situ hybridization and have carried out RNA‐seq expression profiling in the developing mandible of wt and Prdm16 mutant embryos to identify and validate differential gene expression as a consequence of Prdm16 loss.We also hypothesized that Mecom is also required during orofacial development. Mecom or Mds1‐Evi1 complex locus includes the genes Mds1 and Evi1 and the product of intergenic splicing, Mds1‐Evi1. This study characterizes two targeted Mecom alleles, Evi1ko1 (ko1) and Evi1ko3 (ko3), which knockout Evi1 alone or both Evi1 and Mds1‐Evi1, respectively. To maintain a consistent strain background as our Prdm16 mutants, we initiated a series of successive outcrosses matings between Mecom heterozygotes and FVB/NJ mice. We observed incompletely penetrant CP in ko1 homozygotes examined at birth after the 4th generation, although this penetrance did not increase beyond 25% by the 10th generation (F10). In contrast, ko3 homozygous mutants exhibit CP with very high, but incomplete penetrance by F10. Histologic assessment of coronal head sections showed that mutant palate shelves had elevated but failed to fuse at the midline, in contrast to Prdm16 mutants that exhibit failed palate shelf elevation. To explore a potential genetic interaction between Prdm16 and Mecom, we examined Prdm16; Mecom (ko3) double heterozygous newborn pups and identified CP with high penetrance. Interestingly, these pups show both pre‐ and post‐elevation CP. These studies demonstrate a requirement for both Prdm16 and Mecom during normal palate formation, albeit via alternate mechanisms, and also evidence for the influence of Prdm16‐Mecom genetic interactions.Support or Funding InformationNIH/NIDCR R15 DE023982 and Intramural funding from the Midwestern University Office of Research and Sponsored Programs and College of Health Sciences Biomedical Sciences Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wnt6 influences the viability of mouse embryonic palatal mesenchymal cells via the β-catenin pathway.

The embryological stages of palatal shelf elongation and elevation, mainly induced by the proliferation and extracellular matrix secretion of embryonic palatal mesenchymal (MEPM) cells, are essential for normal palatal development. Wingless-related MMTV integration site gene family (Wnt) signaling pathways serve key roles in craniofacial development and palate formation. Recent studies have indicated that Wnt6 participates in embryonic development of the palate, though its exact role in palate development remains unclear. In the present study, to investigate the role of Wnt6 during the stages of palatal shelves elongation and elevation, mouse MEPM cells were cultured from dissected palatal shelves at embryonic day 13.5. Results of an MTT assay and flow cytometric analysis demonstrated that treatment with recombinant Wnt6 increased the viability of MEPM cells (P<0.01) and the proportion of cells in the S and G2/M phases (P<0.01). Meanwhile, Wnt6 activated the β-catenin signaling pathway as indicated by the dual luciferase assay result, and blockade of the WNT/β-catenin pathway reduced the cytoactivity of Wnt6 in MEPM cells (P<0.01). Collectively, these findings indicate that Wnt6 promotes the vitality of MEPM cells by increasing the S + G2/M-phase cell population, potentially through activation of the β-catenin pathway during palatal shelf elongation and elevation.

Modulating Wnt Signaling Rescues Palate Morphogenesis in Pax9 Mutant Mice

Cleft palate is a common birth defect caused by disruption of palatogenesis during embryonic development. Although mutations disrupting components of the Wnt signaling pathway have been associated with cleft lip and palate in humans and mice, the mechanisms involving canonical Wnt signaling and its regulation in secondary palate development are not well understood. Here, we report that canonical Wnt signaling plays an important role in Pax9-mediated regulation of secondary palate development. We found that cleft palate pathogenesis in Pax9-deficient embryos is accompanied by significantly reduced expression of Axin2, an endogenous target of canonical Wnt signaling, in the developing palatal mesenchyme, particularly in the posterior regions of the palatal shelves. We found that expression of Dkk2, encoding a secreted Wnt antagonist, is significantly increased whereas the levels of active β-catenin protein, the essential transcriptional coactivator of canonical Wnt signaling, is significantly decreased in the posterior regions of the palatal shelves in embryonic day 13.5 Pax9-deficent embryos in comparison with control littermates. We show that small molecule–mediated inhibition of Dickkopf (DKK) activity in utero during palatal shelf morphogenesis partly rescued secondary palate development in Pax9-deficient embryos. Moreover, we found that genetic inactivation of Wise, which is expressed in the developing palatal shelves and encodes another secreted antagonist of canonical Wnt signaling, also rescued palate morphogenesis in Pax9-deficient mice. Furthermore, whereas Pax9del/del embryos exhibit defects in palatal shelf elevation/reorientation and significant reduction in accumulation of hyaluronic acid—a high molecular extracellular matrix glycosaminoglycan implicated in playing an important role in palatal shelf elevation—80% of Pax9del/del;Wise-/- double-mutant mouse embryos exhibit rescued palatal shelf elevation/reorientation, accompanied by restored hyaluronic acid accumulation in the palatal mesenchyme. Together, these data identify a crucial role for canonical Wnt signaling in acting downstream of Pax9 to regulate palate morphogenesis.

Active subglacial lakes and channelized water flow beneath the Kamb Ice Stream

Abstract. We identify two previously unknown subglacial lakes beneath the stagnated trunk of the Kamb Ice Stream (KIS). Rapid fill-drain hydrologic events over several months are inferred from surface height changes measured by CryoSat-2 altimetry and indicate that the lakes are probably connected by a subglacial drainage network, whose structure is inferred from the regional hydraulic potential and probably links the lakes. The sequential fill-drain behavior of the subglacial lakes and concurrent rapid thinning in a channel-like topographic feature near the grounding line implies that the subglacial water repeatedly flows from the region above the trunk to the KIS grounding line and out beneath the Ross Ice Shelf. Ice shelf elevation near the hypothesized outlet is observed to decrease slowly during the study period. Our finding supports a previously published conceptual model of the KIS shutdown stemming from a transition from distributed flow to well-drained channelized flow of subglacial water. However, a water-piracy hypothesis in which the KIS subglacial water system is being starved by drainage in adjacent ice streams is also supported by the fact that the degree of KIS trunk subglacial lake activity is relatively weaker than those of the upstream lakes.

The pivotal role of CCN2 in mammalian palatogenesis.

Mammalian palatogenesis is a complex process involving a temporally and spatially regulated myriad of factors. Together these factors control the 3 vital processes of proliferation, elevation and fusion of the developing palate. In this study, we show for the first time the unequivocally vital role of CCN2 in development of the mammalian palate. We utilized CCN2 knockout (KO) mice and cranial neural crest derived mesenchymal cells from these CCN2 KO mice to investigate the 3 processes crucial to normal palatogenesis. Similar to previously published reports, the absence of CCN2 inhibits proliferation of cells in the palate specifically at the G1/S transition. Absence of CCN2 also inhibited palatal shelf elevation from the vertical to horizontal position. CCN2 KO mesenchymal cells demonstrated deficiencies in adhesion and spreading owing to an inability to activate Rac1 and RhoA. On the contrary, CCN2 KO mesenchymal cells exhibited increased rates of migration compared to WT cells. The addition of exogenous CCN2 to KO mesenchymal cells restored their ability to spread normally on fibronectin. Finally, utilizing an organ culture model we show that the palatal shelves of the CCN2 KO mice demonstrate an inability to fuse when apposed. Together, these data signify that CCN2 plays an indispensible role in normal development of the mammalian palate and warrants additional studies to determine the precise mechanism(s) responsible for these effects.

Mesenchymal Remodeling during Palatal Shelf Elevation Revealed by Extracellular Matrix and F-Actin Expression Patterns.

During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis.

Ablation of the Sox11 Gene Results in Clefting of the Secondary Palate Resembling the Pierre Robin Sequence

Mouse gene inactivation has shown that the transcription factor Sox11 is required for mouse palatogenesis. However, whether Sox11 is primarily involved in the regulation of palatogenesis still remains elusive. In this study, we explored the role ofSox11in palatogenesis by analyzing the developmental mechanism in cleft palate formation in mutants deficient in Sox11. Sox11 is expressed both in the developing palatal shelf and in the surrounding structures, including the mandible. We found that cleft palate occurs only in the mutant in which Sox11is directly deleted. As in the wild type, the palatal shelves in the Sox11 mutant undergo outgrowth in a downward direction and exhibit potential for fusion and elevation. However, mutant palatal shelves encounter clefting, which is associated with a malpositioned tongue that results in physical obstruction of palatal shelf elevation at embryonic day 14.5 (E14.5). We found that loss of Sox11led to reduced cell proliferation in the developing mandibular mesenchyme via Cyclin D1, leading to mandibular hypoplasia, which blocks tongue descent. Extensive analyses of gene expression inSox11 deficiency identified FGF9 as a potential candidate target of Sox11 in the modulation of cell proliferation both in the mandible and the palatal shelf between E12.5 and E13.5. Finally we show, using in vitro assays, that Sox11 directly regulates the expression of Fgf9 and that application of FGF9 protein to Sox11-deficient palatal shelves restores the rate of BrdU incorporation. Taken together, the palate defects presented in the Sox11 loss mutant mimic the clefting in the Pierre Robin sequence in humans.

Golgb1 regulates protein glycosylation and is crucial for mammalian palate development.

Cleft palate is a common major birth defect for which currently known causes account for less than 30% of pathology in humans. In this study, we carried out mutagenesis screening in mice to identify new regulators of palatogenesis. Through genetic linkage mapping and whole-exome sequencing, we identified a loss-of-function mutation in the Golgb1 gene that co-segregated with cleft palate in a new mutant mouse line. Golgb1 is a ubiquitously expressed large coiled-coil protein, also known as giantin, that is localized at the Golgi membrane. Using CRISPR/Cas9-mediated genome editing, we generated and analyzed developmental defects in mice carrying additional Golgb1 loss-of-function mutations, which supported a crucial requirement for Golgb1 in palate development. Through maxillary explant culture assays, we demonstrate that the Golgb1 mutant embryos have intrinsic defects in palatal shelf elevation. Just prior to the developmental stage of palatal shelf elevation in wild-type littermates, Golgb1 mutant embryos exhibit increased cell density, reduced hyaluronan accumulation and impaired protein glycosylation in the palatal mesenchyme. Together, these results demonstrate that, although it is a ubiquitously expressed Golgi-associated protein, Golgb1 has specific functions in protein glycosylation and tissue morphogenesis.

Dislocated Tongue Muscle Attachment and Cleft Palate Formation

In Pierre Robin sequence, a retracted tongue due to micrognathia is thought to physically obstruct palatal shelf elevation and thereby cause cleft palate. However, micrognathia is not always associated with palatal clefting. Here, by using the Bmp7-null mouse model presenting with cleft palate and severe micrognathia, we provide the first causative mechanism linking the two. In wild-type embryos, the genioglossus muscle, which mediates tongue protrusion, originates from the rostral process of Meckel’s cartilage and later from the mandibular symphysis, with 2 tendons positive for Scleraxis messenger RNA. In E13.5 Bmp7-null embryos, a rostral process failed to form, and a mandibular symphysis was absent at E17.5. Consequently, the genioglossus muscle fibers were diverted toward the lingual surface of Meckel’s cartilage and mandibles, where they attached in an aponeurosis that ectopically expressed Scleraxis. The deflection of genioglossus fibers from the anterior-posterior toward the medial-lateral axis alters their direction of contraction and necessarily compromises tongue protrusion. Since this muscle abnormality precedes palatal shelf elevation, it is likely to contribute to clefting. In contrast, embryos with a cranial mesenchyme-specific deletion of Bmp7 (Bmp7:Wnt1-Cre) exhibited some degree of micrognathia but no cleft palate. In these embryos, a rostral process was present, indicating that mesenchyme-derived Bmp7 is dispensable for its formation. Moreover, the genioglossus appeared normal in Bmp7:Wnt1-Cre embryos, further supporting a role of aberrant tongue muscle attachment in palatal clefting. We thus propose that in Pierre Robin sequence, palatal shelf elevation is not impaired simply by physical obstruction by the tongue but by a specific developmental defect that leads to functional changes in tongue movements.