Synthesis of cartilage by chondrocytes is an obligatory step for endochondral ossification. Global deletion of the Runx2 gene results in complete failure of the ossification process, but the underlying cellular and molecular mechanisms are not fully known. Here, we elucidated Runx2 regulatory control distinctive to chondrocyte and cartilage tissue by generating Runx2 exon 8 floxed mice. Deletion of Runx2 in chondrocytes caused failure of endochondral ossification and lethality at birth. The limbs of Runx2ΔE8/ΔE8 mice were devoid of mature chondrocytes, vasculature, and marrow. We demonstrate that the C-terminus of Runx2 drives its biological activity. Importantly, nuclear import and DNA binding functions of Runx2 are insufficient for chondrogenesis. Molecular studies revealed that despite normal level of Sox9 and PTHrP, chondrocyte differentiation and cartilage growth is disrupted in Runx2ΔE8/ΔE8 mice. Loss of Runx2 in chondrocytes also impaired OPG-RANKL signaling and chondroclast development. Dwarfism observed in Runx2 mutants was associated with the near absence of proliferative zone in the growth plates. Finally, we show Runx2 directly regulates a unique set of cell cycle genes Gpr132, Sfn, c-Myb, and Cyclin A1 to control proliferative capacity of chondrocyte. Thus, Runx2 is obligatory for both proliferation and differentiation of chondrocytes.

Resistances to begomoviruses, including bipartite tomato mottle virus and monopartite tomato yellow leaf curl virus (TYLCV), have been introgressed to cultivated tomato (Solanum lycopersicum) from wild tomato accessions. A major gene, Ty-2 from S. habrochaites f. glabratum accession “B6013,” that confers resistance to TYLCV was previously mapped to a 19-cM region on the long arm of chromosome 11. In the present study, approximately 11,000 plants were screened and nearly 157 recombination events were identified between the flanking markers C2_At1g07960 (82.5 cM, physical distance 51.387 Mb) and T0302 (89 cM, 51.878 Mb). Molecular marker analysis of recombinants and TYLCV evaluation of progeny from these recombinants localized Ty-2 to an approximately 300,000-bp interval between markers UP8 (51.344 Mb) and M1 (51.645 Mb). No recombinants were identified between TG36 and C2_At3g52090, a region of at least 115 kb, indicating severe recombination suppression in this region. Due to the small interval, fluorescence in situ hybridization analysis failed to clarify whether recombination suppression is caused by chromosomal rearrangements. Candidate genes predicted based on tomato genome annotation were analyzed by RT-PCR and virus-induced gene silencing. Results indicate that the NBS gene family present in the Ty-2 region is likely not responsible for the Ty-2-conferred resistance and that two candidate genes might play a role in the Ty-2-conferred resistance. Several markers very tightly linked to the Ty-2 locus are presented and useful for marker-assisted selection in breeding programs to introgress Ty-2 for begomovirus resistance.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-014-0072-9) contains supplementary material, which is available to authorized users.

Background: Thrombus formed in blood vessels lead to atherothrombotic diseases such as myocardial or cerebral infarction. Thrombolytic agents are used to dissolve the already formed clots in the blood vessels; however, these drugs sometimes cause serious and fatal consequences. Herbal preparations have been used since ancient times for the treatment of several diseases although they show little toxicity in some cases. Aqueous extracts of herbs used in thrombolysis have been reported before with cytotoxic data, however, the organic extracts of herbs have not been documented. This study aims to investigate whether organic extracts possess thrombolytic properties with minimal or no toxicity. Methods: An in vitro thrombolytic model was used to check the clot lysis effect of six Bangladeshi herbal extracts viz., Ageratum conyzoides L., Clausena suffruticosa, Leea indica (Burm.f.) Merr., Leucas aspera Willd., Senna sophera L. Roxb., and Solanum torvum Swartz. using streptokinase as a positive control and water as a negative control. Briefly, venous blood drawn from twenty healthy volunteers was allowed to form clots which were weighed and treated with the test plant materials to disrupt the clots. Weight of clot after and before treatment provided a percentage of clot lysis. Cytotoxicity was screened by brine shrimp lethality bioassay using vincristine sulfate as positive control. Results: Using an in vitro thrombolytic model, Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed 18.12 ± 2.34%, 48.9 ± 2.44%, 39.30 ± 0.96%, 37.32 ± 2.00%, 31.61 ± 2.97% and 31.51 ± 0.57% and clot lysis respectively. Among the herbs studied Clausena suffruticosa, Leea indica and Leucas aspera showed very significant (p < 0.0001) percentage (%) of clot lysis compared to reference drug streptokinase (75.00 ± 3.04%). In brine shrimp cytotoxic assay, the extracts Ageratum conyzoides, Clausena suffruticosa, Leea indica, Leucas aspera, Senna sophera and Solanum torvum showed LC 50 values 508. 86 ± 6.62,41.16 ± 1.26, 2.65 ± 0.16, 181.67 ± 1.65, 233.37 ± 7.74 and 478.40 ± 3.23 μg/ml, respectively, with reference to vincristine sulfate (LC 50 0.76 ± 0.04). Conclusion: Through our study it was found that Clausena suffruticosa, Leea indica and Leucas aspera possessed effective thrombolytic properties whereas Senna sophera and Solanum torvum showed moderate to mild thrombolytic effects while Ageratum conyzoides showed no significant effect. No extract was found cytoxic compared to positive control. Clausena suffruticosa, Leea indica and Leucas aspera could be incorporated as a thrombolytic agent with in vivo effects to improve the atherothrombotic patients. However, Clausena suffruticosa could be the best one to use in this purpose.

The Runx2 transcription factor is critical for commitment to the osteoblast lineage. However, its role in committed osteoblasts and its functions during postnatal skeletogenesis remain unclear. We established a Runx2-floxed line with insertion of loxP sites around exon 8 of the Runx2 gene. Runx2 protein lacking the region encoded by exon 8 is imported into the nucleus and binds target DNA, but exhibits diminished transcriptional activity. We specifically deleted the Runx2 gene in committed osteoblasts using 2.3kb col1a-Cre transgenic mice. Surprisingly, the homozygous Runx2 mutant mice were born alive. The Runx2 heterozygous and homozygous null were grossly indistinguishable from wild-type littermates at birth. Runx2 deficiency did not alter proliferative capacity of osteoblasts during embryonic development (E18). Chondrocyte differentiation and cartilage growth in mutants was similar to wild-type mice from birth to 3 months of age. Analysis of the embryonic skeleton revealed poor calcification in homozygous mutants, which was more evident in bones formed by intramembranous ossification. Runx2 mutants showed progressive retardation in postnatal growth and exhibited significantly low bone mass by 1 month of age. Decreased bone formation was associated with decreased gene expression of osteoblast markers and impaired collagen assembly in the extracellular matrix. Consequently, Runx2 mutant bones exhibited decreased stiffness and structural integrity. By 3 months of age, bone acquisition in mutant mice was roughly half that of wild-type littermates. In addition to impaired osteoblast function, mutant mice showed markedly decreased osteoclast number and postnatal bone resorption. Taken together, functional deficiency of Runx2 in osteoblasts does not result in failed embryonic skeletogenesis, but disrupts postnatal bone formation.

Coordinated activities of multiple mesenchymal cell types contribute to the development of the mammalian skeleton formed through endochondral ossification. Synthesis of a cartilage template by chondrocytes is an obligatory step for the generation of skeletal elements during endochondral ossification. Gene ablation studies have established that Runx2 is an essential transcription factor for bone formation and the differentiation of skeletal cells. However, global gene deletion has failed to discern the tissue- and cell type-specific roles of Runx2. We generated floxed mice to elucidate the Runx2 regulatory control distinctive to cartilage tissue during bone development. Exon 8 of the Runx2 gene was selectively deleted in developing chondrocytes by utilizing Col2a-Cre mice. Cell- and tissue-specific gene recombination was confirmed by β-gal activity in R26R mice. The chondrocyte-specific loss of Runx2 caused failure of endochondral ossification, impaired craniofacial development, dwarfism, and perinatal lethality. Radiographic imaging and histochemical approaches were used to characterize the skeletal phenotype. We conclude that regulatory control of Runx2 in chondrocytes is essential for endochondral ossification, and it is independent of the role of Runx2 in osteoblasts.

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The Sp7/Osterix transcription factor is essential for bone development. Mutations of the Sp7 gene in humans are associated with craniofacial anomalies and osteogenesis imperfecta. However, the role of Sp7 in embryonic tooth development remains unknown. Here we identified the functional requirement of Sp7 for dentin synthesis and tooth development. Sp7-null mice exhibit craniofacial dysmorphogenesis and are completely void of alveolar bone. Surprisingly, initial tooth morphogenesis progressed normally in Sp7-null mice. Thus the formation of alveolar bone is not a prerequisite for tooth morphogenesis. Sp7 is required for mineralization of palatal tissue but is not essential for palatal fusion. The reduced proliferative capacity of Sp7-deficient ectomesenchyme results in small and misshapen teeth with randomly arranged cuboidal preodontoblasts and preameloblasts. Sp7 promotes functional maturation and polarization of odontoblasts. Markers of mature odontoblast (Col1a, Oc, Dspp, Dmp1) and ameloblast (Enam, Amelx, Mmp20, Amtn, Klk4) are barely expressed in incisors and molar tissues of Sp7-null mice. Consequently, dentin and enamel matrix are absent in the Sp7-null littermates. Interestingly, the Sp7 expression is restricted to cells of the dental mesenchyme indicating the effect on oral epithelium-derived ameloblasts is cell-nonautonomous. Abundant expression of Fgf3 and Fgf8 ligand was noted in the developing tooth of wild-type mice. Both ligands were remarkably absent in the Sp7-null incisor and molar, suggesting cross-signaling between mesenchyme and epithelium is disrupted. Finally, promoter-reporter assays revealed that Sp7 directly controls the expression of Fgf-ligands. Together, our data demonstrate that Sp7 is obligatory for the differentiation of both ameloblasts and odontoblasts but not for the initial tooth morphogenesis. © 2018 American Society for Bone and Mineral Research.

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