Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4.

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4Cre and Mrf4nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.

Biophysical stimuli induced by passive movements compensate for lack of skeletal muscle during embryonic skeletogenesis.

In genetically modified mice with abnormal skeletal muscle development, bones and joints are differentially affected by the lack of skeletal muscle. We hypothesise that unequal levels of biophysical stimuli in the developing humerus and femur can explain the differential effects on these rudiments when muscle is absent. We find that the expression patterns of four mechanosensitive genes important for endochondral ossification are differentially affected in muscleless limb mutants, with more extreme changes in the expression in the humerus than in the femur. Using finite element analysis, we show that the biophysical stimuli induced by muscle forces are similar in the humerus and femur, implying that the removal of muscle contractile forces should, in theory, affect the rudiments equally. However, simulations in which a displacement was applied to the end of the limb, such as could be caused in muscleless mice by movements of the mother or normal littermates, predicted higher biophysical stimuli in the femur than in the humerus. Stimuli induced by limb movement were much higher than those induced by the direct application of muscle forces, and we propose that movements of limbs caused by muscle contractions, rather than the direct application of muscle forces, provide the main mechanical stimuli for normal skeletal development. In muscleless mice, passive movement induces unequal biophysical stimuli in the humerus and femur, providing an explanation for the differential effects seen in these mice. The significance of these results is that forces originating external to the embryo may contribute to the initiation and progression of skeletal development when muscle development is abnormal.

Developing bones are differentially affected by compromised skeletal muscle formation.

Mechanical forces are essential for normal adult bone function and repair, but the impact of prenatal muscle contractions on bone development remains to be explored in depth in mammalian model systems. In this study, we analyze skeletogenesis in two 'muscleless' mouse mutant models in which the formation of skeletal muscle development is disrupted; Myf5(nlacZ/nlacZ):MyoD(-/-) and Pax3(Sp/Sp) (Splotch). Ossification centers were found to be differentially affected in the muscleless limbs, with significant decreases in bone formation in the scapula, humerus, ulna and femur, but not in the tibia. In the scapula and humerus, the morphologies of ossification centers were abnormal in muscleless limbs. Histology of the humerus revealed a decreased extent of the hypertrophic zone in mutant limbs but no change in the shape of this region. The elbow joint was also found to be clearly affected with a dramatic reduction in the joint line, while no abnormalities were evident in the knee. The humeral deltoid tuberosity was significantly reduced in size in the Myf5(nlacZ/nlacZ):MyoD(-/-) mutants while a change in shape but not in size was found in the humeral tuberosities of the Pax3(Sp/Sp) mutants. We also examined skeletal development in a 'reduced muscle' model, the Myf5(nlacZ/+):MyoD(-/-) mutant, in which skeletal muscle forms but with reduced muscle mass. The reduced muscle phenotype appeared to have an intermediate effect on skeletal development, with reduced bone formation in the scapula and humerus compared to controls, but not in other rudiments. In summary, we have demonstrated that skeletal development is differentially affected by the lack of skeletal muscle, with certain rudiments and joints being more severely affected than others. These findings indicate that the response of skeletal progenitor cells to biophysical stimuli may depend upon their location in the embryonic limb, implying a complex interaction between mechanical forces and location-specific regulatory factors affecting bone and joint development.

Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates.

Genetic regulatory networks governing skeletal myogenesis in the body are well understood, yet their hierarchical relationships in the head remain unresolved. We show that either Myf5 or Mrf4 is necessary for initiating extraocular myogenesis. Whereas Mrf4 is dispensable for pharyngeal muscle progenitor fate, Tbx1 and Myf5 act synergistically for governing myogenesis in this location. As in the body, Myod acts epistatically to the initiating cascades in the head. Thus, complementary pathways, governed by Pax3 for body, and Tbx1 for pharyngeal muscles, but absent for extraocular muscles, activate the core myogenic network. These diverse muscle progenitors maintain their respective embryonic regulatory signatures in the adult. However, these signatures are not sufficient to ensure the specific muscle phenotypes, since the expected differentiated phenotype is not manifested when satellite cells are engrafted heterotopically. These findings identify novel genetic networks that may provide insights into myopathies which often affect only subsets of muscles.

Different isoforms of BSAP regulate expression of AID in normal and chronic lymphocytic leukemia B cells.

Activation-induced cytidine deaminase (AID) is key to initiating somatic hypermutation (SHM) and class switch recombination (CSR), but its mode of action and regulation remains unclear. Since Pax-5 and Id-2 transcription factors play an opposing role in AID regulation, we have studied the expression of Pax-5, Id-2, and prdm-1 genes in 54 chronic lymphocytic leukemia (CLL) B cells. In 21 cases, presence of AID is constantly associated with high expression of the complete form of the Pax-5 gene (Pax-5a) and lower expression of the Id-2 and prdm-1 transcripts. In 33 cases, the absence of AID expression and CSR is associated with a reduction of Pax-5a and the appearance of a spliced form with a deletion in exon 8 (Pax-5/Delta-Ex8). Stimulation with CD40L+interleukin 4 (IL-4) induces CSR, the presence of AID transcripts, up-regulation of Pax-5a and down-regulation of Pax-5/Delta-Ex8, and Id-2 and prdm-1 transcripts. Pax-5a and Pax-5/Delta-Ex8 are translated into 2 isoforms of the B-cell-specific activator protein (BSAP) and both are able to bind the AID-promoter region. Overall, these results suggest that Pax-5/Delta-Ex8 could play an important role in the control of its own transcription and indirectly in AID expression and CSR.

Lower levels of surface B-cell-receptor expression in chronic lymphocytic leukemia are associated with glycosylation and folding defects of the mu and CD79a chains.

Low levels of B-cell-receptor (BCR) expression are the hallmark of tumoral B lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL). These cells also respond inadequately to stimulation through the BCR. This receptor consists of a surface immunoglobulin associated with a CD79a/CD79b heterodimer. We previously showed that the intracellular synthesis of BCR components, from transcription onward, is normal. Here, we investigated the glycosylation status and cellular localization of mu, CD79a, and CD79b chains in 10 CLL patients differing in surface immunoglobulin M (IgM) expression. We reported a severe impairment of the glycosylation and folding of mu and CD79a. These defects were associated with the retention of both chains in the endoplasmic reticulum and lower levels of surface IgM expression. In contrast, no clear impairment of glycosylation and folding was observed for CD79b. No sequence defects were identified for BCR components and for the chaperone proteins involved in BCR folding processes. These data show, for the first time, that lower levels of BCR surface expression observed in CLL are accounted for by an impaired glycosylation and folding of the mu and CD79a chains.

The LPL/ADAM29 expression ratio is a novel prognosis indicator in chronic lymphocytic leukemia.

Although the zeta-associated protein of 70 kDa (ZAP-70) is overexpressed in patients with chronic lymphocytic leukemia (CLL) displaying unmutated IGVH genes and poor prognosis, a previous microarray study from our group identified overexpression of LPL and ADAM29 genes among unmutated and mutated CLL, respectively. To assess the prognostic value of these genes, we quantified their expression by real-time quantitative polymerase chain reaction (PCR) in a cohort of 127 patients with CLL and correlated this with clinical outcome, IGVH mutational status, and ZAP-70 protein expression. IGVH mutational status, ZAP-70, and the LPL and ADAM29 mRNA ratios (L/A ratio) were predictive of event-free survival for the whole cohort and for patients with stage A disease. In patients in stage B and C, the L/A ratio was an independent prognostic factor, whereas ZAP-70 did not predict survival. Simultaneous usage of the L/A ratio and ZAP-70 expression allowed an almost perfect (99%) assessment of the IGVH status in the 80% of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). LPL and ADAM29 gene expression could also be determined by a simple competitive multiplex reverse transcription PCR assay. Overall, quantification of LPL and ADAM29 gene expression is a strong prognostic indicator in CLL, providing better prognostic assessment than ZAP-70 in advanced stages of the disease.

Transgenic B lymphocytes expressing a human cold agglutinin escape tolerance following experimental infection of mice by Mycoplasma pulmonis.

Several microbial infections, including Mycoplasma pneumoniae respiratory infection, are capable, in man, of transiently inducing the expression of anti-red blood cell autoantibody called cold agglutinins (CA). To analyze the mechanisms by which immune tolerance is broken following a mycoplasma infection, we used transgenic mice expressing a pathogenic human CA, designated CA-GAS, specific for sialylated carbohydrates. In these mice peripheral deletion of autoreactive B lymphocytes and receptor editing, prevent the development of autoimmune hemolytic anemia. Experimental infections of transgenic mice with Mycoplasma pulmonis resulted in a high anti-mycoplasma antibody response (despite a severe B cell depletion at the onset of infection), and an important induction of serum CA concentrations, reaching in some mice pathological titers. Whereas in naïve mice, only a small percentage of CA-expressing cells could be detected, in infected mice, a majority of circulating B lymphocytes were large B220(-) cells, which expressed the transgenic immunoglobulin. Immunization of the transgenic mice with keyhole limpet hemocyanin and Freund's adjuvant, to nonspecifically stimulate the expression of the passenger transgenes, only moderately increased the CA titers. These results indicate that M. pulmonis infection is capable of breaking immune tolerance in the CA-transgenic mice, in part through specific activation of CA-expressing B lymphocytes. This experimental infection mimics the induction of CA in humans and provide an animal model for studying the genesis of the autoimmune hemolytic anemia.

Somatic mutations can lead to a loss of superantigenic and polyreactive binding.

Although antibodies have been assumed to bind a specific antigen, evidence exists showing that a single antibody can bind to multiple unrelated antigens. We previously studied a human monoclonal antibody expressing a mutated form of the V(H)3-73 gene and displaying anti-tubulin activity in a patient suffering from an immunocytic lymphoma. Despite its expression of a V(H)3 family member, this immunoglobulin failed to react with protein A (SpA), suggesting that somatic mutations could account for its change in specificity. To examine this possibility, we produced recombinant Ig expressing germ-line (IgM kappa-Germ) or the mutated form (IgM kappa-PER) of the V(H)3-73 fragment. Comparison of the respective affinities of the two Ig demonstrated that IgM kappa-Germ restores its SpA-binding capacity, and shows a moderate decrease in its affinity for tubulin. Interestingly, IgM kappa-Germ displayed polyreactive specificity for different autoantigens, which contrasted to the monospecific binding of IgM kappa-PER to tubulin. These results suggest that the monoreactive IgM kappa-PER antibody may be derived from a natural polyreactive antibody through somatic mutation. In addition, both temperature modification and mild denaturation succeeded in recovering the polyreactivity of IgM kappa-PER, which favors the view that conformational modifications of the tertiary structure of antibodies may play a key role in the genesis of polyreactivity.

Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation.

In B cells, somatic hypermutation (SHM) and class switch recombination (CSR) depend on the activation-induced cytidine deaminase (AID) gene product, although the precise mode of action of AID remains unknown. Because some chronic lymphocytic leukemia (CLL) B cells can undergo CSR without SHM, it constitutes a useful model to dissect AID function. In this work, we have studied AID expression, the presence of mutations in the preswitch mu DNA region, CSR, and the SHM in 65 CLL patients. Our results demonstrate that unmutated CLL B cells can constitutively express AID and that AID expression is associated with the presence of mutations in the preswitch region and in clonally related isotype-switched transcripts. They also demonstrate that in CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Our results show a dissociation between SHM and CSR in CLL and suggest that, in this disease, AID would require additional help for carrying out the SHM process.

Idiotype-pulsed dendritic cells are able to induce antitumoral cytotoxic CD8 cells in chronic lymphocytic leukaemia.

Idiotypic structures of immunoglobulins from malignant B cells constitute tumour-specific antigens, though the function of immunoglobulin-specific CD8+ T cells in disease control and rejection remains unclear. We have studied five cases of B chronic lymphocytic leukaemia patients affected with indolent (three patients) or aggressive (two patients) disease. We showed that CD8+ T cells with major histocompatibility complex class I-restricted cytotoxicity against autologous tumour B cells could be generated following repeated stimulations with idiotype-pulsed dendritic cells in vitro. CD8+ T-cell lines were able to upregulate CD69 expression and to release interferon (IFN)-gamma upon contact with the autologous B cells, though cytolytic activity was only substantiated for patients with indolent disease. The failure of cytolytic activity in patients with aggressive disease may be explained by a skewed maturation of memory CD8 cells.