Medical Sequencing of Candidate Genes for Nonsyndromic Cleft Lip and Palate

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations

Integration of IRF6 and Jagged2 signalling is essential for controlling palatal adhesion and fusion competence

In mammals, adhesion and fusion of the palatal shelves are essential mechanisms during the development of the secondary palate; failure of these processes leads to the congenital anomaly, cleft palate. The mechanisms that prevent pathological adhesion between the oral and palatal epithelia while permitting adhesion and subsequent fusion of the palatal shelves via their medial edge epithelia remain obscure. In humans, mutations in the transcription factor interferon regulatory factor 6 (IRF6) underlie Van der Woude syndrome and popliteal pterygium syndrome. Recently, we have demonstrated that mice homozygous for a mutation in Irf6 exhibit abnormalities of epithelial differentiation that results in cleft palate as a consequence of adhesion between the palatal shelves and the tongue. In the current paper, we demonstrate that Irf6 is essential for oral epithelial differentiation and that IRF6 and the Notch ligand Jagged2 function in convergent molecular pathways during this process. We further demonstrate that IRF6 plays a key role in the formation and maintenance of the oral periderm, spatio-temporal regulation of which is essential for ensuring appropriate palatal adhesion

BAMBI Regulates Angiogenesis and Endothelial Homeostasis through Modulation of Alternative TGFβ Signaling

BACKGROUND: BAMBI is a type I TGFβ receptor antagonist, whose in vivo function remains unclear, as BAMBI(-/-) mice lack an obvious phenotype. METHODOLOGY/PRINCIPAL FINDINGS: Identifying BAMBI's functions requires identification of cell-specific expression of BAMBI. By immunohistology we found BAMBI expression restricted to endothelial cells and by electron microscopy BAMBI(-/-) mice showed prominent and swollen endothelial cells in myocardial and glomerular capillaries. In endothelial cells over-expression of BAMBI reduced, whereas knock-down enhanced capillary growth and migration in response to TGFβ. In vivo angiogenesis was enhanced in matrigel implants and in glomerular hypertrophy after unilateral nephrectomy in BAMBI(-/-) compared to BAMBI(+/+) mice consistent with an endothelial phenotype for BAMBI(-/-) mice. BAMBI's mechanism of action in endothelial cells was examined by canonical and alternative TGFβ signaling in HUVEC with over-expression or knock-down of BAMBI. BAMBI knockdown enhanced basal and TGFβ stimulated SMAD1/5 and ERK1/2 phosphorylation, while over-expression prevented both. CONCLUSIONS/SIGNIFICANCE: Thus we provide a first description of a vascular phenotype for BAMBI(-/-) mice, and provide in vitro and in vivo evidence that BAMBI contributes to endothelial and vascular homeostasis. Further, we demonstrate that in endothelial cells BAMBI interferes with alternative TGFβ signaling, most likely through the ALK 1 receptor, which may explain the phenotype observed in BAMBI(-/-) mice. This newly described role for BAMBI in regulating endothelial function has potential implications for understanding and treating vascular disease and tumor neo-angiogenesis

The Roles of the Mn1 and Mohawk Transcription Factors in Craniofacial Development in Mice

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biomedical Genetics, 2009.Craniofacial malformations are among the most common birth defects in humans, affecting 1 in 500 to 1000 live births worldwide. The pathogenic mechanisms underlying these birth defects are largely unknown. In this thesis, I report characterization the roles of two novel transcription factors in craniofacial and other developmental processes. Mice lacking the transcription factor Mn1 exhibited cleft secondary palate and severe craniofacial skeletal defects, providing an excellent mouse model for investigating the mechanisms underlying craniofacial defects. Through expression analysis, I found that Mn1 mRNA exhibited differential expression along the anteroposterior axis of the developing secondary palate, with preferential expression in the middle and posterior regions during palatal outgrowth. By thorough histological examinations and proliferation analyses, I found that Mn1–/– mutant mice had specific growth retardation and failure of elevation of the posterior parts of the palatal shelves. Through extensive analyses of palatal gene expression, I identified Tbx22, the mouse homolog of the human X-linked cleft palate gene, as a putative downstream target of Mn1. Tbx22 expression was specifically downregulated in Mn1–/– mutant palatal shelves, and Mn1 activated the promoters of both the human and mouse Tbx22 genes. These data indicate that Mn1 acts upstream of Tbx22 and preferentially regulates posterior palate growth in mice. Since Mn1–/– mutant mice also exhibited severe craniofacial skeletal defects, further investigation was carried out to reveal the roles of Mn1 in cranial osteogenesis. I found that mineralization of certain craniofacial bones was reduced in Mn1–/– mutant embryos. By analyzing calvaria cell cultures, I found that Mn1 inactivation inhibited osteoblast differentiation. Further analyses revealed that Mn1 interacted with Runx2 to synergistically regulate the promoter activity of Osteocalcin, a marker gene for osteoblast differentiation. These results identify Mn1 as an intrinsic regulator of osteoblast differentiation and bring insights into the transcriptional control of osteoblast-specific gene expression during osteoblast differentiation in the developing craniofacial complex. During genetic mapping of a spontaneous oral cleft mutation, Twirler, a new homeobox gene Mohawk was identified. Mohawk exhibited strong expression during palate development. I generated conditional Mohawk knockout mice to investigate the roles of Mohawk in craniofacial development. While Mohawk–/– mutant mice exhibited normal palate development, they displayed curly tails. Further examination revealed that Mohawk null mutant mice exhibited smaller, fainter tendons and abnormal tendon sheath, which led us to perform a thorough expression analysis of Mohawk mRNA during mice tendon development. I found that Mohawk mRNA was expressed in tendon precursor cells and differentiated tendon cells, as well as in the tendon sheath. Further investigation revealed that tendon fibril sizes were decreased in Mohawk–/– mice. In addition, the growth pattern of tendon fibrils was altered in Mohawk–/– mutants. Molecular marker analyses revealed that Tenomodulin and Collagen I were down-regulated specifically in the tendons of Mohawk–/– mutants. These data identify Mohawk as a key regulator of tendon development

Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth

The mammalian secondary palate arises by outgrowth from the oral side of the paired maxillary processes flanking the primitive oral cavity. Palatal growth depends on reciprocal interactions between the oral ectoderm and the underlying neural-crest-derived mesenchyme. Previous studies have implicated sonic hedgehog (Shh) as an important epithelial signal for regulating palatal growth. However, the cellular and molecular mechanisms through which Shh regulates palatal development in vivo have not been directly analyzed, due in part to early embryonic lethality of mice lacking Shh or other essential components of the Shh signaling pathway. Using Cre/loxP-mediated tissue-specific inactivation of the smoothened (Smo) gene in the developing palatal mesenchyme, we show that the epithelially expressed Shh signals directly to the palatal mesenchyme to regulate palatal mesenchyme cell proliferation through maintenance of cyclin D1 (Ccnd1) and Ccnd2 expression. Moreover, we show that Shh-Smo signaling specifically regulates the expression of the transcription factors Foxf1a, Foxf2 and Osr2 in the developing palatal mesenchyme. Furthermore, we show that Shh signaling regulates Bmp2, Bmp4 and Fgf10 expression in the developing palatal mesenchyme and that specific inactivation of Smo in the palatal mesenchyme indirectly affects palatal epithelial cell proliferation. Together with previous reports that the mesenchymally expressed Fgf10 signals to the palatal epithelium to regulate Shh mRNA expression and cell proliferation, these data demonstrate that Shh signaling plays a central role in coordinating the reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth

The Odd-Skipped Family Transcription Factors Osr1 and Osr2 Have Equivalent Biochemical Activity and Play Critical but Partially Redundant Roles during Organogenesis

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2010.Osr1 and Osr2 are the only mammalian homologs of the Drosophila odd-skipped family developmental regulators. The Osr1 protein contains three zinc-finger mo- tifs whereas Osr2 exists in two isoforms, containing three (Osr2B) and five (Osr2A) zinc-finger motifs respectively, due to alternative splicing of the transcripts. Dur- ing mouse embryonic development and organogenesis, Osr1 and Osr2 exhibit dis- tinct as well as partially overlapping expression patterns. Targeted null mutations in these genes in mice resulted in distinct phenotypes, with heart and urogeni- tal developmental defects in Osr1 −/− mice and with cleft palate, supernumerary teeth and open eyelids at birth in Osr2 −/− mice. Whereas the early embryonic lethality of Osr1 −/− mutant mice precluded direct analysis of the roles of Osr1 in many developmental processes in those mutants, the correlation of developmental defects in the Osr2 −/− mutants to specific tissues that normally do not express Osr1 and the lack of phenotypes in Osr2 −/− mutants in many tissues where Osr1 and Osr2 are normally co-expressed suggest that Osr1 and Osr2 function partly redundantly during mouse embryonic development. I first tested possible functional redundancy between Osr1 and Osr2 during limb development. Osr1 and Osr2 are expressed in partially overlapping patterns in the developing limb, but no limb defect was identified during initial charac- terization of mice lacking either Osr1 or Osr2. Using the Cre/loxP-mediated tissue-specific gene inactivation strategy, I showed that deletion of both Osr1 and Osr2 from the early developing limb mesenchyme caused multiple synovial joint fusions. I found that Osr1 and Osr2 expression was activated highly specifically in the presumptive joint cells prior to any morphological signs of joint forma- tion. Molecular marker studies indicate that Osr1 and Osr2 are involved in the earliest stage of joint morphogenesis, i.e, during the formation of interzones at prospective joint sites. In the absence of both Osr1 and Osr2, joint progenitor cells failed to downregulate Col2a1 and to maintain expression of Gdf5, Wnt4 and Wnt9a, which are all critical for joint formation. Furthermore, Osr1 and Osr2 are necessary for inducing cell apoptosis in the developing joint, which is a key step in joint cavitation. Lubrication is mandatory for normal joint function. Prg4 is a marker for differentiating articular chondrocytes and encodes a proteoglycan essential for joint lubrication. Prg4 expression was dramatically downregulated in the Osr1 fneo/fneoOsr2 −/−Prx1cre mutant joint cells. While the above data indicate functional redundancy between Osr1 and Osr2 during joint development, it is possible that Osr1 and Osr2 may have distinct biochemical functions since Osr1 contains three zinc finger motifs but Osr2A has five zinc finger motifs. To investigate whether the distinct mutant phenotypes in Osr1 −/− and Osr2 −/− mutants are due to differences in their protein structure or to differential expression patterns, we generated mice in which the endoge- nous Osr2 coding region was replaced with either Osr1 cDNA or Osr2A cDNA. The knockin alleles recapitulated endogenous Osr2 mRNA expression patterns in most tissues and rescued palate, tooth and cranial skeletal developmental de- fects of Osr2 −/− mice. Mice hemizygous or homozygous for either knockin allele exhibited open-eyelids at birth, which correlated with differences in expression patterns between the knockin allele and the endogenous Osr2 gene during eyelid development. Molecular marker analyses in Osr2 −/− and Osr2 Osr1ki/Osr1ki mice revealed that Osr2 controls eyelid development through regulation of the Fgf10- Fgfr2 signaling pathway and that Osr1 rescued Osr2 function in maintaining Fgf10 expression during eyelid development in Osr2 Osr1ki/Osr1ki mice. Another transcription factor that plays an essential role in palate and tooth de- velopment is Pax9. Pax9 is a member of the paired box transcription factor family that is characterized by the paired DNA-binding domain. It was reported that homozygous Pax9 -deficient mice have a cleft secondary palate with an abnormally broadened shape and lack characteristic indentations at the lateral sides of the palatal shelves at E13.5. In the absence of Pax9, tooth development was arrested at early bud stage, with down-regulated mesenchymal expression of Bmp4, Msx1, and Lef1. Since recent study from our lab indicated that Osr2 suppressed the Msx1-Bmp4 pathway during tooth development, we investigated the mechanisms underlying the cleft palate and tooth developmental defects in Pax9 null mutant mice and whether Osr2 was involved. We found that Pax9 mRNA exhibited a unique differential pattern of expression along the anterior-posterior axis of the de- veloping secondary palate, with restricted expression in the medial mesenchyme in the anterior region and in a lateral-medial gradient in the posterior region. Molecular marker analyses demonstrated that the boundary between anterior and posterior regions of the secondary palate was disrupted and the anteriorward out- growth of the secondary palate delayed in the Pax9 null mutant embryos. In addition, the levels and patterns of expression of Osr2, Fgf10, and Bmp2 in the developing palatal mesenchyme were significantly altered in the Pax9 mutant em- bryos in comparison with control littermates. These changes correlated well with significantly altered patterns of cell proliferation in the palate mesenchyme in the mutant embryos. A dramatic decrease in cell proliferation was also found in the lateral epithelium of the secondary palate. I have also found a significant decrease in cell proliferation rate in the developing tooth mesenchyme in Pax9 null mutant embryos. In collaboration with a colleague, I am investigating whether changes in the expression of these genes also occur during tooth development in Pax9 null mutant embryos. In summary, Osr1 and Osr2 act redundantly in synovial joint development, by regulating interzone formation, joint cavitation and joint cell differentiation. The distinct roles of Osr1 and Osr2 during mouse development result from evolution- ary divergence of their cis regulatory sequences rather than distinct biochemical activities of their protein products. Pax9 regulates the expression of Osr2, Fgf10, and Bmp2 in palate mesenchyme lingual to the developing tooth bud and coordi- nates cell proliferation in the epithelium and mesenchyme to ensure normal palate morphogenesis and tooth development

Investigating the roles of Wnt/β-catenin signaling in craniofacial development

Thesis (Ph. D.)--University of Rochester. Dept. of Biology, 2009.Craniofacial malformations such as oral clefts and dental abnormalities are common congenital diseases, which arise from the disruption of various steps of normal craniofacial development. Previous studies have established important roles of several core signaling pathways, including Shh, Bmp and Fgf signaling, in secondary palate and early tooth development. However, very little is known about the role of canonical Wnt signaling in palatogenesis. Moreover, whereas canonical Wnt signaling has been shown to play critical roles in the developing dental epithelium, whether it is also required in the tooth mesenchyme for activation of the odontogenic potential is not known. In this thesis, I showed that canonical Wnt signaling was not activated in most palatal mesenchymal cells by analysis of canonical Wnt signaling reporter mice. To test the requirement of inactivation/inhibition of canonical Wnt signaling in normal palatogenesis, I utilized the Cre/Lox system to conditionally stabilize β-catein in palatal mesenchymal cells. Persistent activation of canonical Wnt signaling in palatal mesenchymal cells led to cleft palate. Detailed histological analysis indicated that elevation of palatal shelves did not occur in the β-catenin gain-of-function mutants. To investigate the cellular mechanisms underlying the cleft palate phenotype, I performed BrdU labeling and TUNEL assays to detect alterations in cell proliferation and cell death, and found that cell proliferation was reduced in both palatal epithelium and mesenchyme in mutants compared to those of control littermates at E13.5, while cell death was similar in control and mutant littermates. To investigate which downstream gene(s)/pathway(s) were affected, I analyzed the expression of genes important for secondary palate development by in-situ hybridization, and revealed that the expression of Lef1, Bmp4, Bmp2, and Osr2 was increased, while the expression of Shox2 was totally abolished in palatal mesenchyme of mutant embryos. In conclusion, our data indicate canonical Wnt signaling is actively inhibited in palatal mesenchymal cells during normal palatogenesis. I also investigated the role of mesenchymal Wnt/β-catenin in early tooth development. I found that tissue-specific inactivation of β-catenin, a central component of the canonical Wnt signaling pathway, in the developing early tooth mesenchyme, caused tooth developmental arrest at the bud stage in mice. I further showed that mesenchymal β-catenin function was required for expression of Lef1 and Fgf3 in the developing tooth mesenchyme and for induction of primary enamel knot in the developing tooth epithelium. Moreover, I found that constitutive stabilization of β-catenin in the developing palatal mesenchyme induced ectopic tooth initiation from the palatal epithelium. Together, these results revealed that Wnt ligands expressed in the presumptive dental epithelium signal directly to the developing tooth mesenchyme to activate mesenchymal odontogenic program during tooth initiation