Unit: Stems Cells and Development
Director: Shahragim Tajbakhsh
The principal interest of the laboratory is centered on how stem cells are maintained, and how their fates become restricted to specific lineages during development. Our model is skeletal muscle development at embryonic, post-natal and in the adult stages. We are using a genetic approach in the mouse, both by transgenesis and knock-ins, to localise, isolate, and characterise these stem cells in somites of the embryo, in the developing limbs, and in adult skeletal muscle satellite cells
The acquisition of skeletal muscle identity :
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. This finding coupled with the fact that Myf5 expression marks post-natal satellite cells which have stem cell-like properties, incited us to create 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 cells.
Using double mutant mice, we showed previously that the paired/homeodomain gene Pax3 and the transcription factor Myf5 act upstream of Myod. Other genes which are implicated in early skeletal muscle development are the receptor tyrosine kinase, Met, and the paralog of Pax3, Pax7. The role that Met plays within this genetic network is being examined in Met :Myf5 double mutant mice. Pax7 plays a key role in post-natal satellite cell specification - we are also investigating the role of this gene, in combination with Myf5, in adult satellite cells. Further, new Myf5 alleles which do not provoke distal rib pheontypes have been also generated in order to investigate the role of myogenic factors in this process. While investigating this phenomenon, we discovered that gene targetings in the Myf5 gene can perturb the related, and genetically linked Mrf4 gene. We investigated the role of Mrf4, which has been classified as a differentiation gene, and discovered, much to our surprise, that this regulatory factor can direct skeletal muscle identity. In other words, Myf5:Myod double mutants make skeletal muscle through the action of Mrf4. Although embryonic myogenesis proceeds, ftal myogenesis is compromised thereby providing us with a genetic model where these two programmes are uncoupled.^
The characterisation of skeletal muscle stem cells:
We are investigating muscle stem cell function using multiple strategies. An ex vivo approach involves the analysis of explants or isolated primary satellite cells from embryos and adults to investigate the stem cell to differentiated cell transitions, with an emphasis on their 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 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.
Where are skeletal muscle stem cells located, and how do they self-renew ?
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 the dermomyotome epithelium and, during foetal stages (B), most likely distributed among the skeletal muscle masses. 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 to one daughter cell during mitosis (D).
Keywords: Stem cell, asymmetric cell division, skeletal muscle, somite, satellite cell, Myf5, Pax7, Numb