Unit: Stems Cells and Development
Director: Tajbakhsh Shahragim
Our principal interest is centered on how stem cells are maintained, and how their fates become restricted during development. Our model is skeletal muscle development at embryonic, foetal and post-natal stages. We are using mouse knock-ins and transgenesis to identify genetic networks which govern cell fate and self-renewal and niche character. We are also investigating how stem cells in muscle give rise to distinct cell populations, and the role of symmetric vs. asymmetric cell divisions in establishing and maintaining the lineage.
Genetic and cellular regulation of skeletal muscle identity :
Skeletal muscle development serves as a excellent paradigm for the investigation of cell fate acquisition, the study of stem to differentiated cell transitions as well as self-renewal of stem cells and tissue homeostasis. Key regulatory genes of the paired-box/homeodomain family, Pax3 and Pax7, as well as the bHLH myogenic regulatory factors Myf5, Myod, Mrf4 and Myogenin have been identified. Over the last decade, the genetic regulation of this tissue has proven to be complex, correlating partially with an underlying logic which can be explained in part by embryological criteria. For example, regional differences such as head and body proper, are mirrored by distinct epistatic pathways. Using double mutant mice, we showed previously that Pax3 and Myf5 act upstream of Myod in the embryo (Tajbakhsh et al. 1997). These findings established an genetic hierarchy in somite derived muscles in the embryo. In parallel, the presence of head muscles and absence of muscles in the body proper revealed that regional embryological territories employ distinct genetic networks. Other genes which are implicated in early skeletal muscle development which we are examining are the receptor tyrosine kinase, Met and Pax7. Pax7 plays a key role in post-natal satellite cells. We are investigating the role of this gene, in combination with Myf5, in the embryo and in adult satellite cells by creating new alleles at these loci. We also examined the role of Mrf4, which had been classified as a differentiation gene, and showed that this regulatory factor can direct skeletal muscle identity in the embryo. Thus, the inactivation of Myf5, Mrf4 and Myod is required to eliminate all skeletal muscles (Kassar-Duchossoy et al. 2004). These mutants, in combination with Pax3 null mice, provide us with models where we uncouple head from body myogenesis as well as embryonic from foetal myogenesis. This complex genetic regulation can be explained, in part, by a heterogeneity in cell populations in this lineage. However the skeletal muscle lineage has been undefined and elusive in spite of the fact that genetic networks are being unveiled. A hierarchical order which defines cell populations in skeletal muscle was proposed (Tajbakhsh, 2003, 2005), and we have genetic evidence supporting this view (Kassar-Duchossoy et. al. 2005).
Previously we showed that in the absence of Myf5, muscle progenitor cells remained multipotent and were capable of changing their fate in the embryo when located ectopically. We therefore created various knock-in alleles at the Myf5 locus to investigate these events in detail. Our repertoire includes knock-ins such as nlacZ, Myod, and GFP. In addition to investigating the genetic networks regulating skeletal myogenesis, these mice are designed to allow the molecular and cellular characterisation of skeletal muscle stem/progenitor cells. For example, Myf5GFP knock-in mice have permitted us to isolate by FACS post-natal satellite cells for engraftment studies in immuno-compromised recipient mice. Highly efficient engraftments are obtained from 500-5000 satellite cells (in collaboration with A. Cumano, J. Di Santo, V. Mouly).
From a developmental and embryological perspective, we are interested also in how cell lineages segregate in the somite. In particular, we are investigating the curious distal rib perturbations which are characteristic of some Myf5 null mice. The issue of the origins of distal rib progenitors - sclerotome vs. dermomyotome is disputed. Given that Myf5 is not expressed in the sclerotome, we have been investigating the causes of distal rib perturbations in these mutants. We have examined a number of alleles at the Myf5 locus and it appears that the regulation of this locus plays an intimate part in the outcome of progenitor cell segregation in the somite.
Identification and characterisation of skeletal muscle stem cells:
We are investigating muscle stem cell function using multiple strategies. During adult muscle regeneration, satellite cells leave the quiescent state, become activated and divide to generate precursor myoblasts which can fuse with regenerating muscle fibres. Importantly, new satellite cells are generated during homeostasis. An ex vivo approach involves the analysis of explants or isolated primary satellite cells from to investigate these events, with an emphasis on the mode of division - asymmetric vs. symmetric. We are using live imaging techniques to investigate the self-renewal and differentiation events both in the organism and in culture. Our studies using the cell fate determinant Numb reveal that the asymmetry apparatus is operational in satellite cell derived myoblasts and we have direct time-lapse images of asymmetric cell divisions taking place in this lineage. We plan to extend these studies to the organism and use a transgenic approach to manipulate stem cell fate with a goal to modify cell identity decisions and identify the key regulators involved in this critical decision.
To use an unbiased approach for the identification of upstream regulators of Myf5 and those that play a role in emergence of muscle progenitors, we have used Affymetrix microarray screens. Myf5GFP knock-in heterozygous and homozygous mice were used to isolate muscle progenitors from the embryo and satellite cells from the adult. The analysis of the microarray data is currently underway.
Where are skeletal muscle stem cells located, and how do they self-renew ?
Figure 1. During early embryonic development, skeletal muscle stem cells in the body proper are located in the somites, transitory segmented units located bilaterally along the neural tube (A). They are subsequently localised in a dorsal dermomyotome epithelium as the somite dissociates and, during foetal stages (B), they are distributed among skeletal muscle masses in the trunk and limbs. At post-natal stages and in the adult, satellite cells represent a potential stem cell population. Satellite cells can be genetically identified and distinguished from myonuclei using the Myf5nlacZ/+ knock-in mouse as seen on individual fibres (C). Satellite cell derived myoblasts can undergo asymmetric cell divisions as demonstrated by the segregation of the cell fate determinant Numb (red ; late anaphase cell, DNA blue) to one daughter cell during mitosis (D).
Figure 2. Identification of a novel population of resident Pax3+/Pax7+ primitive myogenic cells. By blocking cell fate progression and the formation of foetal muscles in Myf5GFP-P/GFP-P:Myod-/- mutants, a primitive population of myogenic cells, marked by Pax7 (A), is unveiled. These cells (red) are tightly associated with Desmin-positive differentiated embryonic fibres (green). Blue marks all nuclei (Hoechst staining). These primitive cells are detected in skeletal muscles of the trunk and limbs throughout development. At certain stages, they can be distinguished from their daughter muscle progenitor cells which will give rise to precursor myoblasts. These findings lead us to propose a cell fate progression scheme for skeletal muscle (B). At late foetal stages, stem and progenitor definitions overlap since most non-differentiated cells coexpress Pax7 and Myf5 by birth.
Keywords: stem cells, asymmetric cellular division, skeletal muscle, somites, satellite cells, Myf5, Myod, Mrf4, Pax3, Pax7, Numb