Inflammation, fibrosis and skeletal muscle regeneration in LGMDR9 are orchestrated by macrophages.

AIMS: Variable degrees of inflammation, necrosis, regeneration and fibrofatty replacement are part of the pathological spectrum of the dystrophic process in alpha dystroglycanopathy LGMDR9 (FKRP-related, OMIM #607155), one of the most prevailing types of LGMDs worldwide. Inflammatory processes and their complex interplay with vascular, myogenic and mesenchymal cells may have a major impact on disease development. The purpose of our study is to describe the specific immune morphological features in muscle tissue of patients with LGMDR9 to enable a better understanding of the phenotype of muscle damage leading to disease progression. METHODS: We have analysed skeletal muscle biopsies of 17 patients genetically confirmed as having LGMDR9 by histopathological and molecular techniques. RESULTS: We identified CD206+ MHC class II+ and STAT6+ immune-repressed macrophages dominating the endomysial infiltrate in areas of myofibre regeneration and fibrosis. Additionally, PDGFRß+ pericytes were located around MHC class II+ activated capillaries residing in close proximity to areas of fibrosis and regenerating fibres. Expression of VEGF was found on many regenerating neonatal myosin+ fibres, myofibres and CD206+ macrophages also co-expressed VEGF. CONCLUSION: Our results show characteristic immune inflammatory features in LGMDR9 and more specifically shed light on the predominant role of macrophages and their function in vascular organisation, fibrosis and myogenesis. Understanding disease-specific immune phenomena potentially inform about possibilities for anti-fibrotic and anti-inflammatory therapeutic strategies, which may complement Ribitol replacement and gene therapies for LGMDR9 that may be available in the future.

Interplay of auxin, KANADI and Class III HD-ZIP transcription factors in vascular tissue formation.

Class III HD-ZIP and KANADI gene family members have complementary expression patterns in the vasculature and their gain-of-function and loss-of-function mutants have complementary vascular phenotypes. This suggests that members of the two gene families are involved in the establishment of the spatial arrangement of phloem, cambium and xylem. In this study, we have investigated the role of these two gene families in vascular tissue differentiation, in particular their interactions with the plant hormone auxin. We have analyzed the vasculature of plants that have altered expression levels of Class III HD-ZIP and KANADI transcription factors in provascular cells. Removal of either KANADI or Class III HD-ZIP expression in procambium cells led to a wider distribution of auxin in internal tissues, to an excess of procambium cell recruitment and to increased cambium activity. Ectopic expression of KANADI1 in provascular cells inhibited procambium cell recruitment due to negative effects of KANADI1 on expression and polar localization of the auxin efflux-associated protein PIN-FORMED1. Ectopic expression of Class III HD-ZIP genes promoted xylem differentiation. We propose that Class III HD-ZIP and KANADI transcription factors control cambium activity: KANADI proteins by acting on auxin transport, and Class III HD-ZIP proteins by promoting axial cell elongation and xylem differentiation.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis.

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

Dimerization of Toc-GTPases at the chloroplast protein import machinery.

Import of chloroplast precursor proteins is controlled by the coordinate action of two homologous GTPases, Toc159 and Toc33, located at the cytosol-outer membrane interface. Recent studies in Arabidopsis showed that the cytosolic form of the precursor binding protein Toc159 is targeted to its receptor at the import machinery, Toc33, via heterodimerization of their GTP-binding domains. Toc33 may also form GDP-bound homodimers, as suggested by the crystal structure of its pea ortholog. Moreover, the structural data suggested that arginine 130 (Arg130) of Arabidopsis Toc33 may function as a GTPase-activating "arginine-finger" at the other monomer in the Toc33 dimer. Here, we demonstrate that Arg130 of Toc33 does not function as an Arginine-finger. A mutant, Toc33-R130A, binds and hydrolyzes GTP like the wild type. However, we demonstrate that Arg130 is involved in both homodimerization of Toc33 and in heterodimerization with the GTP-binding domain of Toc159. The dependence of Toc33 homodimerization on Arg130 is mutual, requiring the presence of Arg130 at both monomers. As the GTPase is not activated by dimerization, it may be activated independently at either monomer, possibly even before dimerization. Independent regulation of GTPase activity may serve to coordinate the interactions of the GTPases during the import of proteins into the chloroplast.