Genome Editing: A New Horizon for Oral and Craniofacial Research.

Precise and efficient genetic manipulations have enabled researchers to understand gene functions in disease and development, providing a platform to search for molecular cures. Over the past decade, the unprecedented advancement of genome editing techniques has revolutionized the biological research fields. Early genome editing strategies involved many naturally occurring nucleases, including meganucleases, zinc finger nucleases, and transcription activator-like effector-based nucleases. More recently, the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nucleases (CRISPR/Cas) system has greatly enriched genetic manipulation methods in conducting research. Those nucleases generate double-strand breaks in the target gene sequences and then utilize DNA repair mechanisms to permit precise yet versatile genetic manipulations. The oral and craniofacial field harbors a plethora of diseases and developmental defects that require genetic models that can exploit these genome editing techniques. This review provides an overview of the genome editing techniques, particularly the CRISPR/Cas9 technique, for the oral and craniofacial research community. We also discuss the details about the emerging applications of genome editing in oral and craniofacial biology.

Loss of Function of Evc2 in Dental Mesenchyme Leads to Hypomorphic Enamel.

Ellis-van Creveld (EvC) syndrome is an autosomal-recessive skeletal dysplasia, characterized by short stature and postaxial polydactyly. A series of dental abnormalities, including hypomorphic enamel formation, has been reported in patients with EvC. Despite previous studies that attempted to uncover the mechanism leading to abnormal tooth development, little is known regarding how hypomorphic enamel is formed in patients with EvC. In the current study, using Evc2/ Limbin mutant mice we recently generated, we analyzed enamel formation in the mouse incisor. Consistent with symptoms in human patients, we observed that Evc2 mutant mice had smaller incisors with enamel hypoplasia. Histologic observations coupled with ameloblast marker analyses suggested that Evc2 mutant preameloblasts were capable of differentiating to secretory ameloblasts; this process, however, was apparently delayed, due to delayed odontoblast differentiation, mediated by a limited number of dental mesenchymal stem cells in Evc2 mutant mice. This concept was further supported by the observation that dental mesenchymal-specific deletion of Evc2 phenocopied the tooth abnormalities in Evc2 mutants. Overall, our findings suggest that mutations in Evc2 affect dental mesenchymal stem cell homeostasis, which further leads to hypomorphic enamel formation.

Developmental Regulation of the Growth Plate and Cranial Synchondrosis.

Long bones and the cranial base are both formed through endochondral ossification. Elongation of long bones is primarily through the growth plate, which is a cartilaginous structure at the end of long bones made up of chondrocytes. Growth plate chondrocytes are organized in columns along the longitudinal axis of bone growth. The cranial base is the growth center of the neurocranium. Synchondroses, consisting of mirror-image growth plates, are critical for cranial base elongation and development. Over the last decade, considerable progress has been made in determining the roles of the parathyroid hormone-related protein, Indian hedgehog, fibroblast growth factor, bone morphogenetic protein, and Wnt signaling pathways in various aspects of skeletal development. Furthermore, recent evidence indicates the important role of the primary cilia signaling pathway in bone elongation. Here, we review the development of the growth plate and cranial synchondrosis and the regulation by the above-mentioned signaling pathways, highlighting the similarities and differences between these 2 structures.

Activation of ß-catenin/TCF targets following loss of the tumor suppressor SNF5.

The SWI/SNF chromatin remodeling complex is a master regulator of developmental cell-fate decisions, although the key target pathways are poorly characterized. Here, we interrogated the contribution of the SWI/SNF subunit and tumor suppressor SNF5 to the regulation of developmental pathways using conditional mouse and cell culture models. We find that loss of SNF5 phenocopies ß-catenin hyperactivation and that SNF5 is essential for regulating Wnt/ß-catenin pathway target expression. These data provide insight into chromatin-based mechanisms that underlie developmental regulation and elucidate the emerging theme that mutation of this tumor suppressor complex can activate developmental pathways by uncoupling them from upstream control.

Cardiac responses to 24 hrs hyperoxia in Bmp2 and Bmp4 heterozygous mice.

BACKGROUND: Hyperoxia or clinical oxygen (O2) therapy is known to result in increased oxidative burden. Therefore, understanding susceptibility to hyperoxia exposure is clinically important. Bone morphogenetic proteins (BMPs) 2 and 4 are involved in cardiac development and may influence responses to hyperoxia. METHODS: Bmp2(+/)(-). Bmp4(+/)(-) and wild-type mice were exposed to hyperoxia (100% O2) for 24 hrs. Electrocardiograms (ECG) were recorded before and during exposure by radio-telemetry. RESULTS: At baseline, a significantly higher low frequency (LF) and total power (TP) heart rate variability (HRV) were found in Bmp2(+/)(-) mice only (p < 0.05). Twenty-four hours hyperoxia-induced strain-independent reductions in heart rate, QTcB and ST-interval and increases in QRS, LF HRV and standard deviation of RR-intervals were observed. In Bmp4(+/)(-) mice only, increased PR-interval (PR-I) (24 hrs), P-wave duration (P-d; 18 and 21-24 hrs), PR-I minus P-d (PR - Pd; 24 hrs) and root of the mean squared differences of successive RR-intervals (24 hrs) were found during hyperoxia (p < 0.05). DISCUSSION: Elevated baseline LF and TP HRV in Bmp2(+/)(-) mice suggests an altered autonomic nervous system regulation of cardiac function in these mice. However, this was not related to strain specific differences in responses to 24 hrs hyperoxia. During hyperoxia, Bmp4(+/-) mice were the most susceptible in terms of atrioventricular conduction changes and risk of atrial fibrillation, which may have important implications for patients treated with O2 who also harbor Bmp4 mutations. This study demonstrates significant ECG and HRV responses to 24 hrs hyperoxia in mice, which highlights the need to further work on the genetic mechanisms associated with cardiac susceptibility to hyperoxia.

Bmp2 is required for odontoblast differentiation and pulp vasculogenesis.

Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKO(od)) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKO(od) do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also describe an indirect effect of the Bmp2 gene in odontoblasts on formation of the vascular bed and associated pericytes in the pulp. This vascular niche and numbers of CD146+ pericytes are likely controlled by odontogenic and Bmp2-dependent VegfA production in odontoblasts. The complex roles of Bmp2, postulated to be both direct and indirect, lead to permanent defects in the teeth throughout life, and result in teeth with low quantities of dentin and dentin of poor quality.

Prevalence of gastroduodenal mucosal injury in asymptomatic patients taking antiplatelet agents in Japan.

PURPOSE: There is little knowledge regarding the prevalence of mucosal injury (MI) in Japanese patients receiving antiplatelet therapy. This study estimated the prevalence of gastroduodenal MI in asymptomatic Japanese patients taking antiplatelet agents and nonsteroidal anti-inflammatory drugs (NSAIDs). METHODS: Among patients who underwent an upper gastrointestinal endoscopy at Teikyo University Hospital (Tokyo, Japan), 382 asymptomatic patients taking either low-dose aspirin, ticlopidine, cilostazol, or other NSAIDs and 119 people not taking any of these agents were included. Endoscopic records were evaluated for the presence of MI. RESULTS: Aspirin and NSAIDs users showed a higher prevalence of MI than controls (Aspirin, OR = 2.6 (95% CI = 1.4 - 4.9), NSAIDs, 2.9 (1.4 - 4.4)). Concomitant use of aspirin and NSAIDs increased the prevalence of MI (11.2 (2.8 - 44.8)). Ticlopidine and cilostazol were less likely to cause injury than aspirin and other NSAIDs, the difference remained insignificant due to small sample number (ticlopidine, 0.8 (0.2 - 4.0), cilostazol, 1.3 (0.3 - 4.8)). CONCLUSIONS: In asymptomatic Japanese patients receiving low-dose aspirin, the prevalence of the gastroduodenal MI was the same as that in patients taking NSAIDs and higher than that in controls.

Downstream genes of Sox8 that would affect adult male fertility.

Sertoli cells provide nutritional and physical support to germ cells during spermatogenesis. Sox8 encodes a high mobility group transcription factor closely related to Sox9 and Sox10. Sertoli cells produceSOX8 protein, and its elimination results in an age-dependent deregulation of spermatogenesis resulting in male infertility. This suggests that Sox8 is a critical regulator of Sertoli cell function for the maintenance of male fertility beyond the first wave of spermatogenesis. To better understand the roles of Sox8 in testicular development and maintenance of male fertility, we have performed a detailed analysis to explore its downstream genes. We have used mRNA expression profiling to identify affected genes in Sertoli cells in the mutant testes of 2-month-old mice. Expression profiling of the heterozygous and homozygous Sox8 mutant testes indicates alterations in several important spermatogenesis and blood-testis barrier genes, providing insight into the molecular basis of the defects in Sox8(-/-) testes beyond the first wave of spermatogenesis.

Influences of reduced expression of maternal bone morphogenetic protein 2 on mouse embryonic development.

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. In the course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3' untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal mouse tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/-) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. The number of embryos exhibiting these abnormalities was increased when, due to different genotypes, expression levels of Bmp2 in maternal tissues were lower. These results suggest that the expression levels of Bmp2 in both embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds.

Functional redundancy of TGF-beta family type I receptors and receptor-Smads in mediating anti-Mullerian hormone-induced Mullerian duct regression in the mouse.

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery.

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

The Dentin matrix protein 1 (Dmp1) is specifically expressed in mineralized, but not soft, tissues during development.

Dentin Matrix Protein 1 (Dmp1) was originally identified from dentin. However, its expression and function in vivo are not clear. To clarify these two issues, we have generated mice carrying a truncated Dmp1 gene by using gene targeting to replace exon 6 with a lacZ gene. Northern blot analysis shows the expected 5.8-kb Dmp1-lacZ fusion transcript and loss of the wild-type 2.8-kb Dmp1 transcript, confirmed by a lack of immunostaining for the protein. Using heterozygous animals, we demonstrate that Dmp1 is specific for mineralized tissues. Not previously shown, Dmp1 is also expressed in pulp cells. Dmp1-deficient embryos and newborns display no apparent gross abnormal phenotype, although there are a modest expansion of the hypertrophic chondrocyte zone and a modest increase in the long bone diameter. This suggests that DMP1 is not essential for early mouse skeletal or dental development.

Gubernacular development in Müllerian inhibiting substance receptor-deficient mice.

OBJECTIVE: To determine, in mice with disrupted Müllerian inhibiting substance (MIS) receptor genes, whether MIS affects gubernacular development; MIS causes Müllerian duct regression and is proposed to be involved in the first stage of testicular descent, because gubernacular development is abnormal in humans with persistent Müllerian duct syndrome. MATERIALS AND METHODS: Ten wild-type, 11 heterozygotic and 12 homozygotic mice for MIS receptor mutations were killed at 17.5 or 18.5 days after conception or at birth, to provide serial sagittal sections of the pelvis. The amount of cremaster muscle, mitotic bodies in the gubernacular bulb, and gubernacular size were quantified by computer analysis (four mice/group). RESULTS: Müllerian ducts were present in the homozygous mutants, partially present in the heterozygotes and absent in the wild-type controls. All mice had descended testes. The cremaster muscle was significantly less developed in homozygous mutants than in wild-type controls (P < 0.001) and heterozygotes (P < 0.01) at birth. The mitotic index between the gubernacula of all groups was indistinguishable. There was no statistical difference in gubernacular area amongst the groups. Poor cremaster muscle development in homozygous mutants gave the muscle a loose mesenchymal appearance. CONCLUSIONS: Although there was an observable effect on cremaster muscle development in these mutant mice, gubernacular development and testicular descent were otherwise normal, and thus there must be other reasons for the observed differences in humans with persistent Müllerian duct syndrome.

BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb.

We demonstrate that signaling via the bone morphogenetic protein receptor IA (BMPR-IA) is required to establish two of the three cardinal axes of the limb: the proximal-distal axis and the dorsal-ventral axis. We generated a conditional knockout of the gene encoding BMPR-IA (Bmpr) that disrupted BMP signaling in the limb ectoderm. In the most severely affected embryos, this conditional mutation resulted in gross malformations of the limbs with complete agenesis of the hindlimbs. The proximal-distal axis is specified by the apical ectodermal ridge (AER), which forms from limb ectoderm at the distal tip of the embryonic limb bud. Analyses of the expression of molecular markers, such as Fgf8, demonstrate that formation of the AER was disrupted in the Bmpr mutants. Along the dorsal/ventral axis, loss of engrailed 1 (En1) expression in the non-ridge ectoderm of the mutants resulted in a dorsal transformation of the ventral limb structures. The expression pattern of Bmp4 and Bmp7 suggest that these growth factors play an instructive role in specifying dorsoventral pattern in the limb. This study demonstrates that BMPR-IA signaling plays a crucial role in AER formation and in the establishment of the dorsal/ventral patterning during limb development.

Cancer chemopreventive agents. New depsidones from Garcinia plants.

In a search for cancer chemopreventive agents from natural sources, chemical constituents of two kinds of Garcinia plants, Garcinia neglecta and Garcinia puat, collected in New Caledonia, were examined. Five new depsidones, garcinisidone-B (2), -C (3), -D (4), -E (5), and -F (6), were isolated, and their structures were determined by spectrometric analyses. Inhibitory effects of these depsidones on EBV-EA activation induced by TPA in Raji cells were also demonstrated.

Xanthone and dihydroisocoumarin from Montrouziera sphaeroidea.

A xanthone, montrouxanthone and a dihydroisocoumarin, montroumarin were isolated from the stem bark of Montrouziera sphaeroidea Pancher Ex Planchon et Triana [Guttiferae], along with two known compounds. Their structures were elucidated on the basis of spectroscopic analyses. This is the first report of the analysis of chemical constituents of Montrouziera species.

Multiple roles for activin-like kinase-2 signaling during mouse embryogenesis.

The members of the transforming growth factor-beta (TGF-beta) superfamily are secreted proteins that interact with cell-surface receptors to elicit signals that regulate a variety of biological processes during vertebrate embryogenesis. Alk2, also known as ActRIA, Tsk7L, and SKR1, encodes a type I TGF-beta family receptor for activins and BMP-7. Initially, Alk2 transcripts are detected in the visceral endoderm of gastrula stage mouse embryos, suggesting a signaling role in extraembryonic tissues during development. To study the role of Alk2 during mammalian development, Alk2 mutant mice were generated. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than controls. Morphological and molecular examination demonstrated that Alk2 mutant embryos formed a primitive streak, although abnormally thickened, and were arrested in their development around the late streak stage. These gastrulation defects were rescued in chimeric embryos generated by injection of Alk2 mutant embryonic stem (ES) cells into wild-type blastocysts. This rescue of gastrulation defects was also observed in chimeric embryos generated by aggregation of Alk2 homozygous mutant ES cells with tetraploid wild-type embryos. However, at E9.5, these embryos that were completely ES-derived also had defects. In contrast, chimeric embryos generated by injection of wild-type ES cells into Alk2 mutant blastocysts did not show rescue of the gastrulation defects. These results suggest that signaling through this type I receptor is essential in extraembryonic tissues at the time of gastrulation for normal mesoderm formation and also suggest that subsequent Alk2 signaling is essential for normal development after gastrulation.

High specificity of Müllerian-inhibiting substance signaling in vivo.

Female transgenic mice that ectopically express high levels of human Müllerian-inhibiting substance (hMIS) under the control of the mouse metallothionein (MT) promoter lack a uterus, oviducts, and ovaries. The loss of the uterus and oviducts is consistent with the known activities for MIS. However, it is not clear if the loss of the ovaries in these transgenic females is caused by interactions of MIS with its normal receptor signaling pathway or by abnormal interactions with other transforming growth factor-beta (TGF-beta) super family receptor signaling pathways. To address this question, female mice carrying the MT-hMIS transgene that were also homozygous for a targeted deletion of the MIS type II receptor gene were generated. Although these females had high levels of circulating hMIS, they had normal reproductive tracts and ovaries with germ cells. In addition, these females were able to become pregnant and gave birth to pups. These findings demonstrate that all of the abnormalities of the reproductive system that are found in female transgenic mice that ectopically express high levels of hMIS are caused by signaling through the MIS type II receptor. These in vivo data demonstrate a high specificity for MIS and its receptor.

Two visual pigments in a single photoreceptor cell: identification and histological localization of three mRNAs encoding visual pigment opsins in the retina of the butterfly Papilio xuthus.

This paper describes the localization of newly identified visual pigment opsins in the tiered retina of the Japanese yellow swallowtail Papilio xuthus. We first cloned three cDNAs encoding visual pigment opsins, PxRh1, PxRh2 and PxRh3, and then carried out histological in situ hybridization to localize their mRNAs in the retina. By combining the present data with our previous electrophysiological results, we concluded that both PxRh1 and PxRh2 correspond to visual pigments expressed in photoreceptor cells sensitive in the green wavelength region (green receptors), whereas PxRh3 corresponds to a pigment in red receptors. The in situ hybridization studies showed that some photoreceptor cells express two opsin mRNAs. In the ventral half of the eye, all green receptors in the distal tier were labelled by both PxRh1 and PxRh2 probes. The labelling by the PxRh2 and PxRh3 probes was detected throughout the eye in the proximal tier; in 18 % of ommatidia, the probes labelled the same photoreceptor cell. These results suggest that the possible co-localization of two different visual pigments will broaden the sensitivity spectrum of the photoreceptor cells.