Wnt signaling plays a central regulatory role across a remarkably diverse range of functions during embryonic development, including those involved in the formation of bone and cartilage. Wnt signaling continues to play a critical role in adult osteogenic differentiation of mesenchymal stem cells. Disruptions in this highly-conserved and complex system leads to various pathological conditions, including impaired bone healing, autoimmune diseases and malignant degeneration. For reconstructive surgeons, critically sized skeletal defects represent a major challenge. These are frequently associated with significant morbidity in both the recipient and donor sites. The Wnt pathway is an attractive therapeutic target with the potential to directly modulate stem cells responsible for skeletal tissue regeneration and promote bone growth, suggesting that Wnt factors could be used to promote bone healing after trauma. This review summarizes our current understanding of the essential role of the Wnt pathway in bone regeneration and repair.

Nonsyndromic orofacial cleft (NSOFC) is a severe birth defect that occurs early in embryonic development and includes the subtypes cleft palate only (CPO), cleft lip only (CLO) and cleft lip with cleft palate (CLP). Given a lack of specific genetic factor analysis for CPO and CLO, the present study aimed to dissect the landscape of genetic factors underlying the pathogenesis of these two subtypes using 6,986 cases and 10,165 controls. By combining a genome-wide association study (GWAS) for specific subtypes of CPO and CLO, as well as functional gene network and ontology pathway analysis, we identified 18 genes/loci that surpassed genome-wide significance (P < 5 × 10−8) responsible for NSOFC, including nine for CPO, seven for CLO, two for both conditions and four that contribute to the CLP subtype. Among these 18 genes/loci, 14 are novel and identified in this study and 12 contain developmental transcription factors (TFs), suggesting that TFs are the key factors for the pathogenesis of NSOFC subtypes. Interestingly, we observed an opposite effect of the genetic variants in the IRF6 gene for CPO and CLO. Moreover, the gene expression dosage effect of IRF6 with two different alleles at the same single-nucleotide polymorphism (SNP) plays important roles in driving CPO or CLO. In addition, PAX9 is a key TF for CPO. Our findings define subtypes of NSOFC using genetic factors and their functional ontologies and provide a clue to improve their diagnosis and treatment in the future.

The sclerotium, a multicellular structure composed of the compact aggregation of vegetative hyphae, is critical for the long-term survival and sexual reproduction of the plant-pathogenic fungus Sclerotinia sclerotiorum. The development and carpogenic germination of sclerotia are regulated by integrating signals from both environmental and endogenous processes. Here, we report the regulatory functions of the S. sclerotiorum GATA-type IVb zinc-finger transcription factor SsNsd1 in these processes. SsNsd1 is orthologous to the Aspergillus nidulans NsdD (never in sexual development) and the Neurospora crassa SUB-1 (submerged protoperithecia-1) proteins. Ssnsd1 gene transcript accumulation remains relatively low, but variable, during vegetative mycelial growth and multicellular development. Ssnsd1 deletion mutants (Δnsd1-KOs) produce phialides and phialospores (spermatia) excessively in vegetative hyphae and promiscuously within the interior medulla of sclerotia. In contrast, phialospore development occurs only on the sclerotium surface in the wild-type. Loss of SsNsd1 function affects sclerotium structural integrity and disrupts ascogonia formation during conditioning for carpogenic germination. As a consequence, apothecium development is abolished. The Ssnsd1 deletion mutants are also defective in the transition from hyphae to compound appressorium formation, resulting in a loss of pathogenicity on unwounded hosts. In sum, our results demonstrate that SsNsd1 functions in a regulatory role similar to its ascomycete orthologues in regulating sexual and asexual development. Further, SsNsd1 appears to have evolved as a regulator of pre-penetration infectious development required for the successful infection of its many hosts.

The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete S. sclerotiorum genome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of S. sclerotiorum and in a second independent gene. Targeting the previously characterized Ssoah1 gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function.

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FIG. 7. The band alignment diagram of the b-Ga 2 O 3 /AlN heterojunction, along with that of GaN.

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