The focus of this review is to provide an overview of the current state of knowledge of molecular mechanisms/processes that control differentiation of vascular smooth muscle cells (SMC) during normal development and maturation of the vasculature, as well as how these mechanisms/processes are altered in vascular injury or disease. A major challenge in understanding differentiation of the vascular SMC is that this cell can exhibit a wide range of different phenotypes at different stages of development, and even in adult organisms the cell is not terminally differentiated. Indeed, the SMC is capable of major changes in its phenotype in response to changes in local environmental cues including growth factors/inhibitors, mechanical influences, cell-cell and cell-matrix interactions, and various inflammatory mediators. There has been much progress in recent years to identify mechanisms that control expression of the repertoire of genes that are specific or selective for the vascular SMC and required for its differentiated function. One of the most exciting recent discoveries was the identification of the serum response factor (SRF) coactivator gene myocardin that appears to be required for expression of many SMC differentiation marker genes, and for initial differentiation of SMC during development. However, it is critical to recognize that overall control of SMC differentiation/maturation, and regulation of its responses to changing environmental cues, is extremely complex and involves the cooperative interaction of many factors and signaling pathways that are just beginning to be understood. There is also relatively recent evidence that circulating stem cell populations can give rise to smooth muscle-like cells in association with vascular injury and atherosclerotic lesion development, although the exact role and properties of these cells remain to be clearly elucidated. The goal of this review is to summarize the current state of our knowledge in this area and to attempt to identify some of the key unresolved challenges and questions that require further study.

SummaryTargeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.

Development of anthers and pollen represents an important aspect of the life cycle in flowering plants. Genes contributing to anther and pollen development have been widely studied in many plant species. Ms26/CYP704B genes play an important role in pollen development through biosynthesis of sporopollenin for pollen exine formation. To investigate the role of Ms26/CYP704B genes in anther and pollen development of bread wheat, mutations in the A-, B-, and D-homeologs of the putative Ms26/CYP704B gene were analyzed. Single and double homozygous mutants in any of the homeologs did not affect pollen development and male fertility. Triple homozygous mutants resulted in completely male sterile plants that were defective in pollen and anther development. Additionally, double homozygous-single heterozygous mutants were also male sterile although with varying levels of residual fertility. The fertility of these triple mutants was dependent upon the homeolog contributing the wild-type allele. Two heterologous Ms26/CYP704B genes, when transformed into a triple homozygous mutant background, completely restored male fertility, whereas a single gene was unable to restore fertility. Functional analysis of Ms26/CYP704B furthers the understanding of male fertility genes which can be utilized for the development of novel hybrid seed production systems in wheat.

Although a critical component of vascular disease is modulation of the differentiated state of vascular smooth muscle cells (SMC), the mechanisms governing SMC differentiation are relatively poorly understood. We have previously shown that E-boxes and the ubiquitously expressed class I basic helix-loop-helix (bHLH) proteins, including E2-2 and E12, are important in regulation of the SMC differentiation marker gene, the SM ␣-actin gene. The aim of the present study was to identify proteins that bind to class I bHLH proteins in SMC and modulate transcriptional regulation of SMC differentiation marker genes. Herein we report that members of the protein inhibitor of activated STAT (PIAS) family interact with class I bHLH factors as well as serum response factor (SRF). PIAS1 interacted with E2-2 and E12 based on yeast two-hybrid screens, mammalian two-hybrid assays, and/or coimmunoprecipitation assays. Overexpression of PIAS1 significantly activated the SM ␣-actin promoter and mRNA expression, as well as SM myosin heavy chain and SM22␣, whereas a small interfering RNA for PIAS1 decreased activity of these promoters, as well as endogenous mRNA expression, and SRF binding to SM ␣-actin promoter within intact chromatin in cultured SMC. Of significance, PIAS1 bound to SRF and activated SM ␣-actin promoter expression in wild-type but not SRF ؊/؊ embryonic stem cells. These results provide novel evidence that PIAS1 modulates transcriptional activation of SMC marker genes through cooperative interactions with both SRF and class I bHLH proteins.Proliferation and phenotypic switching of smooth muscle cells (SMC) play important roles for development of atherosclerotic and restenotic lesions as well as development of systemic and pulmonary hypertension, which in aggregate contribute to over 50% of all deaths in Western societies (44). A key to understanding phenotypic switching of SMC is to identify the mechanisms that regulate transcription of SMC-specific or -selective genes including those for SM ␣-actin, SM myosin heavy chain (SM-MHC), SM22␣, calponin, and smoothelin (44). Expression of these genes is reduced in conjunction with activation of genes associated with enhanced cell proliferation and increased synthesis of extracellular matrix proteins during SMC phenotypic switching induced by vascular injury or in association with various pathological states. In contrast, expression levels of these SMC-specific marker genes are increased during development and maturation of SMC as well as in association with hypertension that induces vascular hypertrophy. As such, there is considerable interest in identifying mechanisms that control both normal differentiation of SMC and phenotypic switching in disease states.Several key cis elements and trans-binding factors have been identified and shown to be important in the regulation of SMC-specific gene expression. Serum response factor (SRF) has been shown to be required for expression of most SMC differentiation marker genes through cooperative interactions with the SMC/cardiomyocyte-...

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Transcriptional gene silencing (TGS) can offer a straightforward tool for functional analysis of plant genes, particularly in polyploid species such as wheat, where genetic redundancy poses a challenge in applying mutagenesis approaches, including CRISPR gene editing. In this study, we demonstrate efficient TGS in wheat, mediated by constitutive RNA expression of a promoter inverted repeat (pIR). pIR-mediated TGS of two anther-specific genes, TaMs45 and TaMs1, abolished their function resulting in male sterility. Whilst TGS of TaMs45 required transcriptional silencing of all three homoeologs, a B-genome-specific pIR for TaMs1 was sufficient to confer male sterility. We further show that the pIRs effect TGS of TaMs45 gene through DNA methylation of homologous promoter sequence, successfully suppressing transcription of all three homoeologs. Applying pIR-mediated TGS in wheat, we have generated a dominant male fertility system for production of hybrid seed and demonstrated the efficacy of this system under greenhouse and field conditions. This report describes the first successful TGS in wheat, whilst providing a dominant negative approach as alternative to gene knockout strategies for hybrid wheat breeding and seed production.

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