Roles of Pitx, Dach and Meox genes in adult skeletal muscle stem cells

Final Report Summary - SATELLITE CELL (Roles of Pitx, Dach and Meox genes in adult skeletal muscle stem cells)

Tissue repair and the stem cells that engage in this process are of major biomedical interest. Skeletal muscle regeneration provides a paradigm of tissue regeneration, in which quiescent satellite cells, located under the basal lamina of muscle fibres become activated upon injury, proliferate and then undergo differentiation into new muscle fibres, as well as self-renewal to replenish the muscle stem cell pool. Examining the different states that a satellite cell can adopt under physiological conditions should lead to a better understanding of the mechanisms controlling quiescence, activation and differentiation of muscle stem cells. Transcriptome analysis was performed on purified satellite cells isolated from adult muscles where these cells are quiescent and from growing (postnatal 7, P7) or dystrophic muscles (mdx), where they are activated. This analysis has provided new insights into the genes differentially expressed in quiescent versus activated muscle satellite cells in vivo. The goal of this project was to follow up on this work, by focusing on two families of genes, Pitx and Pax, whose expression patterns suggest they are involved in controlling the behavior of adult skeletal muscle stem cells.

1. Analysis of Pitx2 and Pitx3 functions in adult satellite cells.

We have focused on two members of the Pitx gene family, Pitx2 and Pitx3 that encode homeodomain transcription factors. Both genes are expressed during myogenesis in the embryo, where they have partially redundant activities. They have been implicated in the activation of MyoD and entry into the myogenic programme, as well as having a role in survival of differentiating myoblasts in trunk and limb muscles, while in the head they are required for myogenic progenitor cell survival, proliferation and the onset of myogenesis. Transcriptome analysis, further confirmed by RT-qPCR and immunofluorescence has shown that both genes are expressed in satellite cells: Pitx2 is already present in adult quiescent satellite cells. This expression increase in activated MyoD-positive satellite cells, during growth or regeneration, but then decrease during late differentiation. Pitx3 transcripts are not present in quiescent satellite cells but their expression is induced upon activation of these cells. Pitx3, unlike Pitx2, continues to be expressed in adult muscle fibres. In this project, we have undertaken the analysis of single and double mutants of Pitx2 and Pitx3 thanks to a combination of in vivo and ex vivo approaches. For the in vivo approach, we have used the Pax7CRE-ERT2/+ line that permits the conditional and targeted inactivation of Pitx2/3 genes specifically in muscle satellite cells. Ex vivo experiments were performed by use of Pax3GFP/+ and R26RCRE-ERT2/+ lines that allows the conditional gene inactivation together with the direct isolation by flow cytometry of mutant and control satellite cells, and their subsequent analysis in culture. Those analyses have revealed unique and overlapping functions of Pitx2/3 in the control of satellite cell behaviour. While Pitx3 is essential for the amplification of activated satellite cells before differentiation, and by consequence for the maintenance of the satellite cell pool and of a conserved muscle fibre size, Pitx2 is required for the proliferation of activated satellite cells, as well as for the onset of differentiation through the direct regulation of the myogenin promoter. Inactivation of both genes leads to strong defects: Pitx2/3 double deficient satellite cells display a phenotype characterized by a marked deficit in both proliferation and differentiation, with evidence for cells undergoing senescence. Those defects lead to a dramatic phenotype after injury: control muscle are fully regenerated 3 weeks after injury whereas in double mutant mice muscle regeneration is still markedly compromised, indicating a strong defect in satellite cell functionality.

Project Objectives for the Grant Period:

1. Task1: description of the pattern of expression of Pitx2 and Pitx3, in vivo during quiescence, growth and regeneration, and ex vivo in culture.

It is first necessary to determine, with specific antibodies expression of Pitx2 and Pitx3 in the different satellite cell states. In particular, the laboratory uses freshly prepared and cultured single fibers preparations to follow satellite cell activation in a system which is as close as possible to the in vivo transition from quiescent to activated states. In parallel, we take use of the Pax3GFP/+mice, that permit by flow cytometry the direct isolation of quiescent (adult) or activated (postnatal and mdx) satellite cells.

2. Task2: Obtaining conditional single and double-mutants of Pitx2 and Pitx3 and analysing the functions of both Pitx2 and Pitx3 in vivo by regeneration experiments performed on single and double conditional mutants.

Pitx3flox/flox mice have been generated in J. Drouin’s lab during my first postdoct. In collaboration with J. Drouin, this line is used for postnatal analysis in the host laboratory, together with the Pitx2flox/flox mice (obtained from S. Camper). Pitx3-/- (Pitx3flox/flox;CMV-Cre) are viable and fertile but display a major postnatal muscle growth defect (unpublished result).This defect is even more pronounced in Pitx3-/-Pitx2+/- mice, suggesting a functional redundancy between these two genes. As double mutant mice are not viable, Pitx2flox/flox and Pitx3flox/flox mice will be crossed with a tamoxifen inducible-Cre line to obtain specific invalidation of both genes post-natally. As invalidation of both Pitx2/3 is expected to compromise satellite cell function, each of the following steps has to be examined: survival, proliferation and differentiation. Apoptosis is studied on muscle sections after immunostaining with activated-caspase 3 or by Tunel assay. Proliferation and differentiation are tested in vivo in the context of muscle regeneration after muscle injury.

3. Task3: Obtaining conditional single and double-mutants of Pitx2 and Pitx3 in a Pax3GFP/+ background and analysing the functions of both Pitx2 and Pitx3 ex vivo by culture of single and double-mutants satellite cells.

Satellite cell cultures and single fiber preparations from mutants and controls are used to examine ex vivo survival, proliferation and differentiation in the absence of Pitx2/3. Apoptosis is detected as above by Tunel assay, and by flow cytometry after Annexin-V staining of cultured cells. Costaining with antibodies to Ki67 or cyclin A, as well as EdU incorporation are carried out to determine the percentage of MyoD positive cycling cells and to detect abnormalities during the cell cycle. Differentiation properties of mutant cells is tested by immunodetection of markers of terminal differentiation in cultured cells. In parallel RNA interference will be used to knock-down Pitx2 and Pitx3 function by viral infection of cultured cells or single fibers ex vivo. Survival, proliferation as well as differentiation of infected cells will be analyzed as described above. The experiments described will give insight into the role of Pitx2/3 in activated satellite cells.

Semestrial planning of experiments:

1rst Semester:
Analysis of Pitx2, Pitx3, Dach1 and Meox2 expression by RT-qPCR, western-blot and immunofluorescence on quiescent versus activated satellite cells.
Breeding of Pitx3flox/flox mice and Pitx2flox/flox;Pitx3flox/flox mice with Cre-ERT2 mice.
Breeding of Pitx3flox/flox mice and Pitx2flox/flox;Pitx3flox/flox mice with Pax3GFP/+ mice.

2rnd Semester:
Breeding of (Pitx3flox/+; Cre-ERT2) mice with (Pitx3flox/flox) and (Pitx3flox/+;Pitx2flox/+; Cre-ERT2) mice with (Pitx3flox/flox;Pitx2flox/flox) mice.
Obtaining (Pitx3flox/flox; Cre-ERT2): primary cultures and analysis of cell survival, proliferation and differentiation by immunofluorescence, RT-qPCR and western-blot.
Breeding of (Pitx3flox/+; Pax3GFP/+) mice with (Pitx3flox/flox; Cre-ERT2) mice.

3rrd Semester:
Regeneration experiments on muscles of (Pitx3flox/flox; Cre-ERT2) mice by cardiotoxin injection. Analysis of regeneration by tissue histology, immunofluorescence and RT-qPCR.
Obtaining (Pitx2flox/flox;Pitx3flox/flox; Cre-ERT2) mice and controls. Primary cultures and analysis of survival, proliferation and differentiation.

4th Semester:
Breeding of (Pitx2flox/+;Pitx3flox/+; Pax3GFP/+) with (Pitx2flox/flox;Pitx3flox/flox; Cre-ERT2).
Regeneration experiments on muscles of (Pitx2flox/flox;Pitx3flox/flox; Cre-ERT2) mice by cardiotoxin injection. Analysis of regeneration by tissue histology, RT-qPCR and Western-blot.

Achievements during the complete grant period:

Task 1: description of the pattern of expression of Pitx2 and Pitx3 in satellite cells, in vivo during quiescence, growth and regeneration, and ex vivo in culture.

By immunofluorescence performed on adult muscle sections, we have shown that Pitx2, unlike Pitx3 is already present in quiescent satellite cells. Expression of both genes is increased by activation during growth (P7 muscle) or during regeneration (adult muscle 3 days post-cardiotoxin -CTX- injection, and 3 months-old mdx muscle). These results have been confirmed at mRNA level by RT-qPCR on Pax3GFP/+ cells freshly isolated from control adult, adult 3 days post-CTX and P7 muscles.

The same experiments have been performed ex vivo on isolated satellite cells (Pax3GFP/+) and cultured single fibers preparations after cytospin or after culture. We have confirmed expression of Pitx2 in quiescent satellite cells, as well as increased expression of both Pitx2/3 in activated satellite cells. These experiments have also shown that while Pitx2 expression decrease during differentiation (at both mRNA and protein levels), Pitx3 expression is maintained until late differentiation (Figure 1).

Task 2: Obtaining conditional single and double-mutants of Pitx2 and Pitx3 and analysing the functions of both Pitx2 and Pitx3 in vivo by regeneration experiments performed on single and double conditional mutants.

To recombine Pitx2/3 genes, we have used two different cre-lines R26RCRE-ERT2/+ and Pax7CRE-ERT2/+. Both are inducible by hydroxy-tamoxifen (4-OHT) (5 consecutive daily intra-peritoneal injections of 1mg). While the R26RCRE-ERT2/+ line leads to ubiquitous Cre expression, the use of the Pax7CRE-ERT2/+ line permits a specific gene inactivation in satellite cells. After multiple breedings, we have obtained the conditional and targeted single and double mutants for Pitx2 and Pitx3 genes (see below) and each mutant has been successively analysed by regeneration assays after cardiotoxin (CT) or freeze muscle injury.

Pitx3-/-
Pitx3flox/flox;Pax7CRE-ERT2/+
Pitx3flox/flox;R26RCRE-ERT2/+
Pitx3-/-;mdx

Pitx2flox/flox;Pax7CRE-ERT2/+
Pitx2flox/flox;R26RCRE-ERT2/+

Pitx2flox/flox;Pitx3flox/flox;Pax7CRE-ERT2/+
Pitx2flox/flox;Pitx3flox/flox;R26RCRE-ERT2/+

A. Pitx3 mutant.

Postnatal growth: We have confirmed and described the previously observed postnatal muscle growth defect (Pitx3-/-). This defect begins 3 days after birth and is still readily detectable in the adult. We have shown by EdU injection in P7 muscles, that this defect is due to a premature differentiation of mutant satellite cells leading to a two-fold reduction in the diameter of muscle fibers.
Adult regeneration: Cardiotoxin muscle injury assays performed in adult mutant mice (Pitx3-/-, and Pitx3flox/flox;R26RCRE-ERT2/+) showed faster regeneration leading to the formation of smaller fibers in Pitx3 mutant than in control mice. Edu injection 3 days post injury showed a two-fold reduction in the percentage of Pax7-positive cells having incorporated EdU together with a two-fold increase in the number of myogenin positive cells. These results argue in favour of a premature differentiation of Pitx3 deficient satellite cells. We have confirmed this hypothesis by repeated injury experiments. After 3 rounds of injuries (one injury every 3 weeks in the same muscle), Pitx3 mutant muscle displayed a drastic reduction of the fiber diameter as well as a major decrease in the number of satellite cells compared to control muscles. These defects were further correlated with a defective regeneration of Pitx3 mutant muscle, characterized by the appearance of fibrosis and spots of calcification. Finally, Pitx3 mutant mice line has been crossed with a dystrophic line (mdx) in order to examine the consequences of the absence of Pitx3 in a physiological situation of chronic injury/regeneration. Histological examination of Pitx3-/-;mdx muscle revealed an aggravation of the dystrophic phenotype (increase in fibrosis revealed by red Sirius staining and reduction of the muscle fibre diameter), thus confirming the results obtained in the cardiotoxin-injury assay.
All those results demonstrate that Pitx3 is required for the expansion of activated satellite cells. In absence of Pitx3, mutant satellite cells enter premature differentiation, leading to defective regeneration and progressive depletion of the satellite cell reservoir.

B. Pitx2 mutant.

Adult regeneration: Cardiotoxin muscle injury assays were performed on Pitx2flox/flox;Pax7CRE-ERT2/+ and Pitx2flox/flox;R26RCRE-ERT2/+ adult mice and injured muscles were then examined after increasing regeneration periods. Ten days after injury, while control muscle is already regenerated, mutant muscle regeneration seem compromised. This defect is compensated 3 weeks after injury, with mutant muscle being fully regenerated and similar to control. This result suggested a delay of regeneration (see task 3). This hypothesis has been tested in vivo and demonstrated by analyses of myogenin and embMyHC expression by immunofluorescence, two markers of differentiation, in injured muscle. Five days after injury, Pitx2 mutant muscle showed a three-fold reduction in the number of myogenin and embMyHC-positive cells. These defects of expression are compensated 5 days later. Similarly, RT-qPCR analysis performed on control and Pitx2 mutant muscles at different time points after injury have confirmed this delayed kinetic of Myogenin expression.

C. Pitx2/3 double mutant.

Adult regeneration: Cardiotoxin and freeze muscle injuries have been performed on Pitx2flox/flox;Pitx3flox/flox;R26RCRE-ERT2/+ and Pitx2flox/flox;Pitx3flox/flox;Pax7CRE-ERT2/+ adult mice and injured muscles were examined after increasing regeneration periods. 10 days after injury, while control muscle is already fully regenerated, mutant muscle regeneration seems compromised. In contrast to Pitx2 single mutant (B) in which regeneration is later compensated, Pitx2/3 double mutant muscle did not show any signs of regeneration, even 3 weeks after injury. At this time, mutant muscle is characterized by a massive infiltration of fibroblast, which is a mark of a defective regeneration. Results are similar with both ubiquitous (R26R) and satellite cell specific (Pax7) Cre line, thus demonstrating a complete impairment of satellite function in absence of both Pitx2 and Pitx3.
In order to confirm the essential roles of Pitx2 and Pitx3 in satellite cell during physiological injury, Pitx2flox/flox;Pitx3flox/flox;R26RCRE-ERT2/+ and Pitx2flox/flox;Pitx3flox/flox;Pax7CRE-ERT2/+ mice have been crossed with mdx line. For the two lines, invalidation of both genes was performed by 5 daily injections of 4-OHT in 6 weeks-old animals. Invalidation of Pitx2 and Pitx3 in mdx background induced a dramatic aggravation of the dystrophic phenotype, leading to muscle atrophy, acquisition of kyphosis and death of mutant animals 3 months after 4-OHT injections. Histological examination of mutant muscle revealed signs of inflammation and massive fibrosis together with few signs of regeneration. Results were similar with both ubiquitous (R26R) and satellite cell specific (Pax7) Cre line, thus demonstrating a complete impairment of satellite function in absence of both Pitx2 and Pitx3.

Task 3: Obtaining conditional single and double-mutants of Pitx2 and Pitx3 in a Pax3GFP/+ background and analysing the functions of both Pitx2 and Pitx3 ex vivo by culture of single and double-mutants satellite cells.

To reveal the mechanism responsible for the different phenotypes obtained during growth and regeneration with single and double Pitx2 and Pitx3 mutants, we have performed ex vivo analyses of mutant and control satellite cells. For their purification, we made use of the Pax3GFP allele, and first obtained single and double mutants in the Pax3GFP/+ background.
Pitx3-/-;Pax3GFP/+
Pitx3flox/flox;R26RCRE-ERT2/+;Pax3GFP/+
Pitx2flox/flox;R26RCRE-ERT2/+;Pax3GFP/+
Pitx2flox/flox;Pitx3flox/flox;R26RCRE-ERT2/+;Pax3GFP/+

After 5 consecutive daily intra-peritoneal injections, each mutant has then been analysed by satellite cell purification (Flow cytometry) and culture. Proliferation has been examined by clonal analysis, EdU incorporation or measure of the expression of the cdk inhibitor p21, while differentiation has been measured by expression of early (myogenin) and late (Troponin T) markers.

A. Pitx3 mutant.

Clonal analyses of Pitx3 mutant satellite cells revealed an expansion deficit after 3 days of culture. Measure of EdU incorporation, as well as p21, myogenin and troponin T expression (at mRNA and protein levels) has revealed a reduced number of mitosis at 3 days of culture, correlated with a precocious expression of cell cycle exit and differentiation markers. Similar results have been obtained by Pitx3 knock-down upon lentiviral transduction of siRNA. These data demonstrate that the expansion deficit observed with Pitx3 mutant cells result from their premature differentiation, and are fully correlated with in vivo results obtained during growth and regeneration (Task 2A).

B. Pitx2 mutant.

The phenotype displayed by Pitx2 mutant satellite cells in culture is first characterized by a deficit in proliferation. This defect is not due to premature differentiation, but has been correlated with defective cell cycle. Measure of EdU incorporation showed an increase number of EdU positive cells after 3 days of culture, together with a reduction of the expansion capacity, suggesting an abnormal length of S/G2 transition. This was confirmed by cell cycle analysis by flow cytometry and by quantification of Cyclin D1 and D2 expression (RT-qPCR), two known targets of Pitx2.
In addition to proliferation defects, Pitx2 mutant cells are characterized by a delay in differentiation, due to delayed myogenin expression. This defect is also revealed by siRNA mediated Pitx2 knock-down. We have identified two consensus Pitx Response Element (PRE) in the myogenin gene promoter that bind Pitx2 in vitro in gel-shit experiments. Pitx2 efficiently activates the myogenin promoter in transient transfection assay and mutation of both PRE completely abolishes this activation, demonstrating a direct transcriptional regulation of the myogenin gene by Pitx2. Chromatin immunoprecipitation assays performed on control cells after 3 days of culture have revealed a direct binding of Pitx2 on these two PRE. These results, together with in vivo experiments (Task 2B) correlate the delay of regeneration with a delay of differentiation, due to the direct regulation of myoogenin promoter by Pitx2. A manuscript based on these results will be submitted to “Stem Cells” journal.

C. Pitx2/3 double mutant.

Pitx2 and Pitx3 double deficient satellite cells display a phenotype characterized by a marked deficit in both proliferation and differentiation. In contrast to Pitx2 mutant cells that exhibit a delay in differentiation, double mutant satellite cells are characterized by a massive defect in differentiation. Time-lapse analysis has revealed that 3 days after culture, those cells stop proliferating, have a strong motility defect, do not differentiate and acquire a flat phenotype. Those cells are characterized by a high level of p21 expression (protein level) and are positive for HP1γ heterochromatic foci and Senescence-Associated-beta-galactosidase activity, demonstrating that they are undergoing senescence (Figure 2). These results are fully correlated with in vivo experiments demonstrating that Pitx2/3 double-mutant satellite cells are completely defective for regeneration (Task 2C). Accordingly, SA-βgal positive cells were also identified in Pitx2flox/flox;Pitx3flox/flox;Pax7CRE-ERT2/+;mdx muscles, demonstrating that the massive aggravation of the dystrophic phenotype observed in these mutant animals is a consequence of the defective function of mutant satellite cells that enter senescence instead of differentiation.

To further investigate the mechanism responsible for defective proliferation and differentiation and acquisition of the senescent phenotype, we performed a transcriptome analysis of mutant and control cells after 3 days of culture. This analysis first confirmed the senescent phenotype (decrease of transcription processes and acquisition of irreversible heterochromatin domain, secretion of inflammatory cytokines), together with the absence of differentiation. It also revealed a major de-regulation of the oxidative stress pathway (decrease/increase of a subset of antioxidant enzymes, and alteration of the metabolic activity), suggesting an increase of the intracellular redox activity. Quantification of this activity by immunofluorescence and flow cytometry showed a massive increase of ROS levels in mutant cells, both ex vivo after 4 days of culture, and in vivo in purified satellite cells 3 and 4 days after injury. As excessive levels of ROS have been shown to induce DNA damage, we investigated for such damages in mutant cells. Immunofluorescence analysis using markers of oxidized DNA revealed extensive DNA damage in double mutant cells. Treatment of mutant cells with N-acetyl-cysteine, a powerful antioxidant completely prevented DNA damage, and rescue the complete differentiation.

Similarly, analysis of Pitx2flox/flox;Pitx3flox/flox;Pax7CRE-ERT2/+;mdx muscles by immunofluorescence revealed extensive DNA oxidation. Mutant mice treatment with NAC not only prevented DNA damage and aggravation of the dystrophic phenotype, but also completely rescued life expectancy of Pitx2flox/flox;Pitx3flox/flox;Pax7CRE-ERT2/+;mdx mice.
Taken together, these results demonstrate an essential role of Pitx2 and Pitx3 in the regulation of the intracellular redox activity. In their absence, mutant cells accumulate excessive levels of Reactive Oxygen Species (ROS) leading to DNA damage and senescence. A manuscript is currently under preparation.

Final Report Summary - SATELLITE CELL (Roles of Pitx, Dach and Meox genes in adult skeletal muscle stem cells)

Diese Seite teilen

Herunterladen