Analysis of Meox-2 mutant mice reveals a novel postfusion-based cleft palate.

Cleft palate represents a common human congential disease involving defects in the development of the secondary palate. Major steps in mammalian palatogenesis include vertical growth, elevation, and fusion of the palate shelves. Our current study with the homeobox gene Meox-2 during mouse secondary palate development reveals a novel postfusion-based mechanism for cleft palate. Meox-1 and Meox-2 are two functionally related homeobox genes playing important roles in somitogenesis and limb muscle differentiation. We found that the expression of Meox-2, not Meox-1, marks the specification of early mouse palatal mesenchymal cells in the maxillary processes at embryonic day 11.5 (E11.5). From E12.5 to E15.5, the expression of Meox-2 occupies only the posterior part of the palate, providing an early molecular marker for the anterior-posterior polarity in mouse secondary palate formation. A total of 35.3% of Meox-2-/- (n = 17) and 25.5% of Meox-2+/- (n = 55) mouse embryos display a cleft palate phenotype at E15.5, indicating that the reduction of Meox-2 function is associated with susceptibility to cleft palate. Unlike previously reported clefts, none of the clefts found in Meox-2 mutants contain any epithelial sheets in the medial edge areas, and detailed examination revealed that the clefts resulted from the breakdown of newly fused palates. This article is the first report of a gene required to maintain adherence of the palatal shelves after fusion.     

Regional regulation of palatal growth and patterning along the anterior-posterior axis in mice

Cleft palate is a congenital disorder arising from a failure in the multistep process of palate development. In its mildest form the cleft affects only the posterior soft palate. In more severe cases the cleft includes the soft (posterior) and hard (anterior) palate. In mice a number of genes show differential expression along the anterior-posterior axis of the palate. Mesenchymal heterogeneity is established early, as evident from Bmp4-mediated induction of Msx1 and cell proliferation exclusively in the anterior and Fgf8-specific induction of Pax9 in the posterior palate alone. In addition, the anterior palatal epithelium has the unique ability to induce Shox2 expression in the anterior mesenchyme in vivo and the posterior mesenchyme in vitro. Therefore, the induction and competence potentials of the epithelium and mesenchyme in the anterior are clearly distinct from those in the posterior. Defective growth in the anterior palate of Msx1-/- and Fgf10-/- mice leads to a complete cleft palate and supports the anterior-to-posterior direction of palatal closure. By contrast, the Shox2-/- mice exhibit incomplete clefts in the anterior presumptive hard palate with an intact posterior palate. This phenotype cannot be explained by the prevailing model of palatal closure. The ability of the posterior palate to fuse independent of the anterior palate in Shox2-/- mice underscores the intrinsic differences along the anterior-posterior axis of the palate. We must hitherto consider the heterogeneity of gene expression and function in the palate to understand better the aetiology and pathogenesis of non-syndromic cleft palate and the mechanics of normal palatogenesis.     

Extracellular Matrix in Secondary Palate Development

The secondary palate arises from outgrowths of epithelia-covered embryonic mesenchyme that grow from the maxillary prominence, remodel to meet over the tongue, and fuse at the midline. These events require the coordination of cell proliferation, migration, and gene expression, all of which take place in the context of the extracellular matrix (ECM). Palatal cells generate their ECM, and then stiffen, degrade, or otherwise modify its properties to achieve the required cell movement and organization during palatogenesis. The ECM, in turn, acts on the cells through their matrix receptors to change their gene expression and thus their phenotype. The number of ECM-related gene mutations that cause cleft palate in mice and humans is a testament to the crucial role the matrix plays in palate development and a reminder that understanding that role is vital to our progress in treating palate deformities. This article will review the known ECM constituents at each stage of palatogenesis, the mechanisms of tissue reorganization and cell migration through the palatal ECM, the reciprocal relationship between the ECM and gene expression, and human syndromes with cleft palate that arise from mutations of ECM proteins and their regulators. Anat Rec, 2019. © 2019 American Association for Anatomy     

Mapping cellular processes in the mesenchyme during palatal development in the absence of Tbx1 reveals complex proliferation changes and perturbed cell packing and polarity

The 22q11 deletion syndromes represent a spectrum of overlapping conditions including cardiac defects and craniofacial malformations. Amongst the craniofacial anomalies that are seen, cleft of the secondary palate is a common feature. Haploinsufficiency of TBX1 is believed to be a major contributor toward many of the developmental structural anomalies that occur in these syndromes, and targeted deletion of Tbx1 in the mouse reproduces many of these malformations, including cleft palate. However, the cellular basis of this defect is only poorly understood. Here, palatal development in the absence of Tbx1 has been analysed, focusing on cellular properties within the whole mesenchymal volume of the palatal shelves. Novel image analyses and data presentation tools were applied to quantify cell proliferation rates, including regions of elevated as well as reduced proliferation, and cell packing in the mesenchyme. Also, cell orientations (nucleus–Golgi axis) were mapped as a potential marker of directional cell movement. Proliferation differed only subtly between wild‐type and mutant until embryonic day (E)15.5 when proliferation in the mutant was significantly lower. Tbx1^?/? palatal shelves had slightly different cell packing than wild‐type, somewhat lower before elevation and higher at E15.5 when the wild‐type palate has elevated and fused. Cell orientation is biased towards the shelf distal edge in the mid‐palate of wild‐type embryos but is essentially random in the Tbx1^?/? mutant shelves, suggesting that polarised processes such as directed cell rearrangement might be causal for the cleft phenotype. The implications of these findings in the context of further understanding Tbx1 function during palatogenesis and of these methods for the more general analysis of genotype–phenotype functional relationships are discussed.     

Time-dependent changes of decorin in the infarct zone after experimentally induced myocardial infarction in rats: comparison with biglycan

Decorin, a small dermatan sulphate proteoglycan, has been postulated to interact with other components of the extracellular matrix. We examined time-dependent changes of decorin in the infarct zone after experimentally induced myocardial infarction in rats by Northern blotting, in situ hybridization, and immunohistochemistry. The expression of decorin mRNA was compared to that of biglycan mRNA. Northern blotting demonstrated that the decorin mRNA expression was not increased in the infarct zone on day 2, while increased biglycan mRNA was observed at that time (average 3.1-fold increase). Decorin mRNA expression was increased on day 7, and reached a peak (average 2.2-fold increase) around day 14. Biglycan mRNA expression also reached a peak level around day 14 (average 13.3-fold increase). In situ hybridization revealed that mRNA signals for decorin did not appear in the infarct zone on day 2, while biglycan mRNA signals were observed. Decorin mRNA signals were observed in spindle-shaped mesenchymal cells in the infarct peripheral zone on day 7. The decorin mRNA signals appeared later than those of biglycan. Immunopositive staining for decorin was observed in the infarct zone on day 7. The present results demonstrated a time-dependent increase in decorin mRNA expression in mesenchymal cells in the infarct zone in rats. Decorin mRNA appeared later and was increased to a lower extent in the infarct zone than biglycan mRNA.     

Remodelling of nasal mucosa in mild and severe persistent allergic rhinitis with special reference to the distribution of collagen,proteoglycans, and lymphatic vessels

Background Small leucine‐rich repeat proteoglycans (decorin, biglycan, and lumican), collagen, and lymphangiogenesis are involved in tissue remodelling of various organs with inflammatory diseases. Objective We determined the expression level and the distribution pattern of small leucine‐rich repeat proteoglycans (decorin, biglycan, and lumican), collagen and lymphatic vessels in healthy, mild, and severe persistent allergic nasal mucosa. Methods The distribution pattern of collagen, proteoglycans, and lymphatic vessels in healthy, mild, and severe persistent allergic nasal mucosa was evaluated by the van Gieson staining, immunohistochemistry, RT‐PCR, and Western blotting. Quantitative analyses of collagen deposition were calculated as the median of the total percentage area in the tissue specimen. For the evaluation of proteoglycans, the percentage area stained and median optical density were measured for each image. Lymphatic vessels were identified by D2‐40 antibody and calculated using the lymphatic vessel density and endothelial length density in tissue specimens. The expression of MMP 2 and 9, TIMP1 and 2 was evaluated with RT‐PCR and Western blotting. Results In mild and severe persistent allergic nasal mucosa, compared with healthy nasal mucosa, collagen showed more intense staining in the superficial and submucosal layer. In healthy and allergic nasal mucosa, decorin was lightly stained without significant differences in the percentage area and optical density of staining. However, lumican and biglycan showed strong immunoreactivity in mild and severe persistent allergic nasal mucosa, which was verified by Western blotting. The number and endothelial length density of lymphatic vessels were increased in mild and severe persistent allergic nasal mucosa compared with healthy nasal mucosa. The expression of MMP 9 was increased in severe persistent allergic rhinitis. Conclusion and Clinical Relevance These results suggest that the altered distribution pattern of collagen, proteoglycans, and lymphatic vessels could potentially modulate the remodelling of nasal mucosa in mild and severe persistent allergic nasal mucosa. Cite this as: T. H. Kim, J. Y. Lee, H. M. Lee, S. H. Lee, W. S. Cho, Y. H. Ju, E. H. Park, K. W. Kim and S. H. Lee, Clinical & Experimental Allergy, 2010 (40) 1742–1754.     

Kinetics of biglycan, decorin and thrombospondin-1 in mercuric chloride-induced renal tubulointerstitial fibrosis

We investigated the kinetics of decorin, biglycan and thrombospondin-1 in mercuric chloride-treated Brown Norway (BN) rats. BN rats were injected subcutaneously with 1 mg/kg b.w. of mercuric chloride one or three times. The kidney was examined histopathologically and the kinetics of decorin, biglycan and thrombospondin-1 was also examined using immunohistochemistry and real time RT-PCR. As a result, mercuric chloride induced tubular injury and subsequent tubulointerstitial fibrosis. In this lesion, the expression of thrombospondin-1 mRNA was most prominently elevated. The expression of decorin mRNA was next, but biglycan mRNA expression was not elevated. Moreover, decorin and thrombospondin-1 proteins were localized in tubular epithelial cells and peritubular interstitium. Moreover, kinetics of their mRNA expressions was relatively similar to the kinetics of TGF-beta1 mRNA expression previously reported. The present findings suggest that decorin and thrombospondin-1 may participate in the development of tubulointerstitial fibrosis and may have some relation with TGF-beta1 in mercuric chloride-treated BN rats.     

腭裂小鼠牙胚发育中β-连环蛋白的表达

背景：牙齿发育生物学中信号传导是热点问题,β-连环蛋白是Wnt信号传导通路中的关键效应因子,其在发育的牙胚中有普遍表达,并且在内釉上皮、釉结、星网状层、中间层的表达有时空变化。 目的：通过苏木精-伊红染色和免疫组化技术,观察牙胚在维甲酸诱导腭裂发生中的形态学变化及β-连环蛋白的表达变化。 方法：选取C57BL/6J近交系小鼠,按雌雄比2∶1于晚8时合笼,次日8时检查雌鼠,发现阴栓视为妊娠,定为E0 d。18只孕鼠随机分为3组：在E10 d,实验组以维甲酸100 mg/kg对孕鼠行一次性灌胃,制备腭裂动物模型;植物油对照组给予10 mL/kg橄榄油灌胃;空白对照组不做任何处理。 结果与结论：β-连环蛋白在空白对照组E13 d牙胚蕾状期、E14 d帽状期、E16 d钟状期上皮内均有表达,且空白对照组的表达随着牙胚发育的成熟而逐渐增加,实验组的变化趋势与空白对照组相同。3个时期的实验组β-连环蛋白在牙胚中的表达水平均高于空白对照组,植物油对照组和空白对照组表达无明显差异。提示维甲酸在诱导腭裂发生过程中,可能通过干扰上皮-间充质间的相互作用,上调β-连环蛋白在牙胚中的表达,使牙胚发育受阻。     

Regulation of fibrillin-1 by biglycan and decorin is important for tissue preservation in the kidney during pressure-induced injury

There is growing evidence that the two small leucine-rich proteoglycans biglycan and decorin regulate the assembly of connective tissues and alter cell behavior during development and pathological processes. In this study, we have used an experimental animal model of unilateral ureteral ligation and mice deficient in either biglycan or decorin. We discovered that pressure-induced injury to the wild-type kidneys led to overexpression of decorin, biglycan, fibrillin-1, and fibrillin-2. In contrast, in biglycan-deficient kidneys the overexpression of fibrillin-1 was markedly attenuated and this was associated with cystic dilatation of Bowman's capsule and proximal tubules. Notably, we found that in ligated kidneys from decorin-null mice, fibrillin-1 expression was initially enhanced to the same extent as in wild-type animals. However, long-term obstruction resulted in down-regulation of fibrillin-1 and concurrent cystic dilatation of Bowman's capsule in 33% of kidneys at 5 months after obstruction. In all of the genotypes, no differences in fibrillin-2 expression were observed. These in vivo data correlated with a significant induction of fibrillin-1 expression in renal fibroblasts and mesangial cells by recombinant biglycan and decorin. Our results indicate a novel role for decorin and biglycan during pressure-induced renal injury by stimulating fibrillin-1 expression.     

Expression of Wnt9b and activation of canonical Wnt signaling during midfacial morphogenesis in mice.

Cleft lip with or without cleft palate (CLP) is the most common craniofacial birth defect in humans. Recently, mutations in the WNT3 and Wnt9b genes, encoding two members of the Wnt family of signaling molecules, were found associated with CLP in human and mice, respectively. To investigate whether Wnt3 and Wnt9b directly regulate facial development, we analyzed their developmental expression patterns and found that both Wnt3 and Wnt9b are expressed in the facial ectoderm at critical stages of midfacial morphogenesis during mouse embryogenesis. Whereas Wnt3 mRNA is mainly expressed in the maxillary and medial nasal ectoderm, Wnt9b mRNA is expressed in maxillary, medial nasal, and lateral nasal ectoderm. During lip fusion, Wnt9b, but not Wnt3, is expressed in the epithelial seam between the fusing medial and lateral nasal processes. Furthermore, we found that expression of TOPGAL, a transgenic reporter of activation of canonical Wnt signaling pathway, is specifically activated in the distal regions of the medial nasal, lateral nasal, and maxillary processes prior to lip fusion. During lip fusion, the epithelial seam between the medial and lateral nasal processes as well as the facial mesenchyme directly beneath the fusing epithelia strongly expresses TOPGAL. These data, together with the CLP lip phenotype in WNT3-/- humans and Wnt9b-/- mutant mice, indicate that Wnt3 and Wnt9b signal through the canonical Wnt signaling pathway to regulate midfacial development and lip fusion.     

Corticosteroid-induced cleft lip in mice: A teratologic,topographic, and histologic investigation

Unlike cleft palate, relatively few teratogens have been found to induce cleft lip in mice. The present study was designed to assess the teratologic, topographic (SEM), and histologic effects on lip morphogenesis following the administration of triamcinolone hexacetonide on the eighth day of gestation. The frequency of cleft lip in treated A/J mice was found to be more than three times greater than the spontaneous frequency in untreated controls. Comparable studies with other murine strains suggest no association between the cleft lip response and either a maternal effect or the H-2 complex. Affected A/J embryos showed a severe reduction in the size of the lateral nasal processes; affected embryos also demonstrated localized cell type-specific alterations, particularly in the epithelia and at the interface between epithelium and mesenchyme.     

The regulation of endogenous retinoic acid level through CYP26B1 is required for elevation of palatal shelves

Background: In previous studies, we investigated the effects of excess retinoic acid (RA) during palatogenesis by RA administration to pregnant mice. In the present study, we deleted Cyp26b1, one of the RA‐degrading enzymes, to further study the effects of excess RA in the normal developing palate and to understand how endogenous levels of RA are regulated. Results: Excess RA, due to the absence of Cyp26b1, targets cells in the bend region of the palatal shelves and inhibits their horizontal elevation, leading to cleft palate. An organ culture of Cyp26b1?/? palatal shelves after tongue removal did not rescue the impaired elevation of the palatal shelves. The expression of Fgf10, Bmp2, and Tbx1, important molecules in palatal development, was down‐regulated. Cell proliferation was decreased in the bend region of palatal shelves. Tongue muscles were hypoplastic and/or missing in Cyp26b1?/? mice. Conclusions: We demonstrated that CYP26B1 is essential during palatogenesis. Excess RA due to the lack of Cyp26b1 suppresses the expression of key regulators of palate development in the bend region, resulting in a failure in the horizontal elevation of the palatal shelves. The regulation of RA signaling through CYP26B1 is also necessary for the development of tongue musculature and for tongue depression. Developmental Dynamics 241:1744–1756, 2012. © 2012 Wiley Periodicals, Inc.     

Shh和初级纤毛在腭部发育中的作用

Sonic hedgehog （ Shh）通路在脊椎动物组织和器官发育中扮演重要角色,并且有证据表明其牵涉到包括腭裂等发育缺陷。近年关于Shh及相关因子在发育中腭部的表达模式以及条件性基因敲除小鼠的相关研究着重突出了上皮和间充质细胞间的互动在腭发育中的作用。此外, Hedgehog通路还因其独特的初级纤毛依赖性备受瞩目。文章以Shh在腭部发育中的作用,以及其与初级纤毛的关系为重点来介绍近年来相关发现。     

PDGFR-alpha signaling is critical for tooth cusp and palate morphogenesis.

Platelet-derived growth factor receptor alpha (PDGFR-alpha) and PDGF ligands are key regulators for embryonic development. Although Pdgfralpha is spatially expressed in the cranial neural crest (CNC)-derived odontogenic mesenchyme, mice deficient for Pdgfralpha are embryonic lethal, making it impossible to investigate the functional significance of PDGF signaling in regulating the fate of CNC cells during tooth morphogenesis. Taking advantage of the kidney capsule assay, we investigated the biological function of PDGF signaling in regulating tooth morphogenesis. Pdgfralpha and Pdgfa are specifically and consistently expressed in the CNC-derived odontogenic mesenchyme and the dental epithelium, respectively, throughout all stages of tooth development, suggesting a paracrine function of PDGF signaling in regulating tooth morphogenesis. Highly concentrated expression patterns of Pdgfralpha and Pdgfa are associated with the developing dental cusp, suggesting possible functional importance of PDGF signaling in regulating cusp formation. Loss of the Pdgfralpha gene does not affect proper odontoblasts proliferation and differentiation in the CNC-derived odontogenic mesenchyme but perturbs the formation of extracellular matrix and the organization of odontoblast cells at the forming cusp area, resulting in dental cusp growth defect. Pdgfralpha-/- mice have complete cleft palate. We show that the cleft palate in Pdgfralpha mutant mice results from an extracellular matrix defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Collectively, our data suggests that PDGFRalpha and PDGFA are critical regulators for the continued epithelial-mesenchymal interaction during tooth and palate morphogenesis. Disruption of PDGFRalpha signaling disturbs the growth of dental cusp and interferes with the critical extension of palatal shelf during craniofacial development.     

Mesenchymal fibroblast growth factor receptor signaling regulates palatal shelf elevation during secondary palate formation

Background : Palatal shelf elevation is an essential morphogenetic process during secondary palate closure and failure or delay of palatal shelf elevation is a common cause of cleft palate, one of the most common birth defects in humans. Here, we studied the role of mesenchymal fibroblast growth factor receptor (FGFR) signaling during palate development by conditional inactivation of Fgfrs using a mesenchyme‐specific Dermo1‐Cre driver. Results : We showed that Fgfr1 is expressed throughout the palatal mesenchyme and Fgfr2 is expressed in the medial aspect of the posterior palatal mesenchyme overlapping with Fgfr1. Mesenchyme‐specific disruption of Fgfr1 and Fgfr2 affected palatal shelf elevation and resulted in cleft palate. We further showed that both Fgfr1 and Fgfr2 are expressed in mesenchymal tissues of the mandibular process but display distinct expression patterns. Loss of mesenchymal FGFR signaling reduced mandibular ossification and lower jaw growth resulting in abnormal tongue insertion in the oral‐nasal cavity. Conclusions : We propose a model to explain how redundant Fgfr1 and Fgfr2 expression in the palatal and mandibular mesenchyme regulates shelf medial wall protrusion and growth of the mandible to coordinate the craniofacial tissue movements that are required for palatal shelf elevation. Developmental Dynamics 244:1427–1438, 2015. © 2015 Wiley Periodicals, Inc.