Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover.

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as ubiquitin E3 ligases in ubiquitin-mediated muscle protein turnover. Despite their well-characterised roles in muscle atrophy, the dynamics of MURF expression in the development and early postnatal adaptation of striated muscle is largely unknown. Here, we show that MURF2 is expressed at the very onset of mouse cardiac differentiation at embryonic day 8.5, and represents a sensitive marker for differentiating myocardium. During cardiac development, expression shifts from the 50 kDa to the 60 kDa A-isoform, which dominates postnatally. In contrast, MURF1 shows strong postnatal upregulation and MURF3 is not significantly expressed before birth. MURF2 expression parallels that of the autophagy-associated proteins LC3, p62/SQSTM1 and nbr1. SiRNA knockdown of MURF2 in neonatal rat cardiomyocytes disrupts posttranslational microtubule modification and myofibril assembly, and is only partly compensated by upregulation of MURF3 but not MURF1. Knockdown of both MURF2 and MURF3 severely disrupts the formation of ordered Z- and M-bands, likely by perturbed tubulin dynamics. These results suggest that ubiquitin-mediated protein turnover and MURF2 in particular play an unrecognised role in the earliest steps of heart muscle differentiation, and that partial complementation of MURF2 deficiency is afforded by MURF3.

A hypoplastic model of skeletal muscle development displaying reduced foetal myoblast cell numbers, increased oxidative myofibres and improved specific tension capacity.

The major component of skeletal muscle is the myofibre. Genetic intervention inducing over-enlargement of myofibres beyond a certain threshold through acellular growth causes a reduction in the specific tension generating capacity of the muscle. However the physiological parameters of a genetic model that harbours reduced skeletal muscle mass have yet to be analysed. Genetic deletion of Meox2 in mice leads to reduced limb muscle size and causes some patterning defects. The loss of Meox2 is not embryonically lethal and a small percentage of animals survive to adulthood making it an excellent model with which to investigate how skeletal muscle responds to reductions in mass. In this study we have performed a detailed analysis of both late foetal and adult muscle development in the absence of Meox2. In the adult, we show that the loss of Meox2 results in smaller limb muscles that harbour reduced numbers of myofibres. However, these fibres are enlarged. These myofibres display a molecular and metabolic fibre type switch towards a more oxidative phenotype that is induced through abnormalities in foetal fibre formation. In spite of these changes, the muscle from Meox2 mutant mice is able to generate increased levels of specific tension compared to that of the wild type.

A comparative analysis of Meox1 and Meox2 in the developing somites and limbs of the chick embryo.

We have examined the expression pattern of the avian Meox1 homeobox gene during early development and up to late limb bud stages. Its expression pattern indicates that it is involved in somite specification and differentiation. The domains of expression are similar but different to those of Meox2. Meox1 is expressed from stage 6 in the pre-somitic mesoderm and as development proceeds, in the tail bud, the dermomyotome of the rostral somites and in the dermomyotome and sclerotome of the caudal somites, the lateral rectus muscle, truncus arteriosus of the heart and the limb buds. Unlike Meox1, Meox2 is not expressed in the pre-somitic mesoderm, but is expressed first in somites formed from stage 11 onwards. In the developing limb, both genes are expressed in the dorsal and ventral limb mesoderm in adjacent domains with a small region of overlap. In the limb bud, Meox1 is co-expressed with Meox2 but neither Meox gene is co-expressed with MyoD. These expression patterns suggest that these two genes have overlapping and distinct functions in development.

Meox homeodomain proteins are required for Bapx1 expression in the sclerotome and activate its transcription by direct binding to its promoter.

The axial skeleton of vertebrates derives from the sclerotomal compartment of the somites. Genetic analysis has demonstrated that the transcription factors Pax1, Pax9, Meox1, Meox2, and Bapx1 are all required for sclerotomal differentiation. Their hierarchical relationship is, however, poorly understood. Because Bapx1 expression in the somites starts slightly later than that of the Meox genes, we asked whether Bapx1 is one of their downstream targets. Our analysis of Meox1; Meox2 mutant mice supports this hypothesis, as Bapx1 expression in the sclerotome is lost in the absence of both Meox proteins. Using transient-transfection assays, we show that Meox1 activates the Bapx1 promoter in a dose-dependent manner and that this activity is enhanced in the presence of Pax1 and/or Pax9. Furthermore, by electrophoretic mobility shift and chromatin immunoprecipitation experiments, we demonstrate that Meox1 can bind the Bapx1 promoter. The palindromic sequence TAATTA, present in the Bapx1 promoter, binds the Meox1 protein in vitro and is necessary for Meox1-induced transactivation of the Bapx1 promoter. Our data demonstrate that the Meox genes are required for Bapx1 expression in the sclerotome and suggest that the mechanism by which the Meox proteins exert this function is through direct activation of the Bapx1 gene.

Fine-tuning the onset of myogenesis by homeobox proteins that interact with the Myf5 limb enhancer.

Skeletal myogenesis in vertebrates is initiated at different sites of skeletal muscle formation during development, by activation of specific control elements of the myogenic regulatory genes. In the mouse embryo, Myf5 is the first myogenic determination gene to be expressed and its spatiotemporal regulation requires multiple enhancer sequences, extending over 120 kb upstream of the Mrf4-Myf5 locus. An enhancer, located at -57/-58 kb from Myf5, is responsible for its activation in myogenic cells derived from the hypaxial domain of the somite, that will form limb muscles. Pax3 and Six1/4 transcription factors are essential activators of this enhancer, acting on a 145-bp core element. Myogenic progenitor cells that will form the future muscle masses of the limbs express the factors necessary for Myf5 activation when they delaminate from the hypaxial dermomyotome and migrate into the forelimb bud, however they do not activate Myf5 and the myogenic programme until they have populated the prospective muscle masses. We show that Msx1 and Meox2 homeodomain-containing transcription factors bind in vitro and in vivo to specific sites in the 145-bp element, and are implicated in fine-tuning activation of Myf5 in the forelimb. Msx1, when bound between Pax and Six sites, prevents the binding of these key activators, thus inhibiting transcription of Myf5 and consequent premature myogenic differentiation. Meox2 is required for Myf5 activation at the onset of myogenesis via direct binding to other homeodomain sites in this sequence. Thus, these homeodomain factors, acting in addition to Pax3 and Six1/4, fine-tune the entry of progenitor cells into myogenesis at early stages of forelimb development.

Nop-7-associated 2 (NSA2), a candidate gene for diabetic nephropathy, is involved in the TGFß1 pathway.

We recently showed that Nop-7-associated 2 (NSA2) originally described in yeast as a nuclear protein involved in ribosomal biogenesis, is a hyperglycemia induced gene involved in diabetic nephropathy [Shahni et al., Elevated levels of renal and circulating Nop-7-associated 2 (NSA2) in rat and mouse models of diabetes, in mesangial cells in vitro and in patients with diabetic nephropathy. Diabetologia 2012;55(March(3)):825-34]. However the function of NSA2 in the cell remains unknown. In the current paper we investigate the possible mechanisms for the involvement of NSA2 in diabetic nephropathy by testing the hypothesis that NSA2 expression is linked to the TGFß1 pathway. Both TGFß1 and NSA2 mRNAs were significantly up-regulated in cultured renal mesangial cells in response to high glucose, in mouse kidneys during hyperglycemia, and in developing kidneys of mouse embryos during mesenchymal to epithelial transition. Surprisingly, the previously described nuclear NSA2 protein was predominantly located in the cytosol of cultured renal cells. Exogenous TGFß1 could elevate NSA2 mRNA/protein levels in cultured mesangial cells and could also affect the cellular localization of NSA2, causing the predominantly cytosolic NSA2 protein to rapidly translocate to the nucleus. Increased NSA2 nuclear staining was seen in diabetic mouse kidneys compared to control kidneys. Knock-down of NSA2 expression using RNA interference resulted in significantly decreased TGFß1 mRNA/protein, almost abolished TGFß1 activity, and resulted in significantly reduced mRNA levels of the TGFß1 downstream gene fibronectin. Our data suggest that NSA2 is acting upstream of the TGFß1 pathway and that NSA2 is needed for TGFß1 expression and transcriptional activity. In summary, NSA2, which increases in diabetic nephropathy, may be involved in the actions of TGFß1 and contribute to the development of diabetic nephropathy.

Transient activation of meox1 is an early component of the gene regulatory network downstream of hoxa2.

Hox genes encode transcription factors that regulate morphogenesis in all animals with bilateral symmetry. Although Hox genes have been extensively studied, their molecular function is not clear in vertebrates, and only a limited number of genes regulated by Hox transcription factors have been identified. Hoxa2 is required for correct development of the second branchial arch, its major domain of expression. We now show that Meox1 is genetically downstream from Hoxa2 and is a direct target. Meox1 expression is downregulated in the second arch of Hoxa2 mouse mutant embryos. In chromatin immunoprecipitation (ChIP), Hoxa2 binds to the Meox1 proximal promoter. Two highly conserved binding sites contained in this sequence are required for Hoxa2-dependent activation of the Meox1 promoter. Remarkably, in the absence of Meox1 and its close homolog Meox2, the second branchial arch develops abnormally and two of the three skeletal elements patterned by Hoxa2 are malformed. Finally, we show that Meox1 can specifically bind the DNA sequences recognized by Hoxa2 on its functional target genes. These results provide new insight into the Hoxa2 regulatory network that controls branchial arch identity.

Retinoic acid is both necessary for and inhibits myogenic commitment and differentiation in the chick limb.

Retinoic acid (RA) plays an essential role in the development of many embryonic tissues, including the developing tetrapod limb bud. At early stages of limb development, RA levels are highest proximally and regulate the migration of myoblasts into the limb. As the premyogenic progenitor cells migrate into the limb and accumulate in premuscle masses, they express Pax3 and Meox2. Myogenic differentiation is initiated by expression of Myf5 and MyoD, and both Pax3 and Meox2 are required for normal Myf5 expression. We show by loss of function using the inhibitor citral, that RA signalling within the limb bud is required to maintain Pax3 and Meox2 in the progenitor and Myf5 and MyoD in the differentiating myoblasts. Treatment with excess RA showed a differential effect: Meox2 and Pax3 showed localised down-regulation of expression in the limb. In contrast, there was a dramatic down-regulation of expression of MyoD, Myf5 and Meox1. The down-regulation of myogenic gene expression in response to inhibition of RA signalling, and differential response to application of excess RA, in the absence of changes to cell proliferation and apoptosis, indicate that myogenic specification and differentiation in the developing limb possess a complex sensitivity to RA concentrations.

The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation, patterning and differentiation of somites.

The paraxial mesoderm of the somites of the vertebrate embryo contains the precursors of the axial skeleton, skeletal muscles and dermis. The Meox1 and Meox2 homeobox genes are expressed in the somites and their derivatives during embryogenesis. Mice homozygous for a null mutation in Meox1 display relatively mild defects in sclerotome derived vertebral and rib bones, whereas absence of Meox2 function leads to defective differentiation and morphogenesis of the limb muscles. By contrast, mice carrying null mutations for both Meox genes display a dramatic and wide-ranging synthetic phenotype associated with extremely disrupted somite morphogenesis, patterning and differentiation. Mutant animals lack an axial skeleton and skeletal muscles are severely deficient. Our results demonstrate that Meox1 and Meox2 genes function together and upstream of several genetic hierarchies that are required for the development of somites. In particular, our studies place Meox gene function upstream of Pax genes in the regulation of chondrogenic and myogenic differentiation of paraxial mesoderm.