Josiane Fontaine-Pérus

Blood borne macrophages are essential for the triggering of muscle regeneration following muscle transplant

The transplantation of satellite cells may constitute a strategy for rebuilding muscle fibres in inherited myopathies. However, its development requires a great understanding of the role of environmental signals in the regenerative process. It is therefore essential to identify the key events triggering and controlling this process in vivo. We investigated whether macrophages play a key role in the course of the regenerative process using skeletal muscle transplants from transgenic pHuDes-nls-LacZ mice. Before grafting, transplants were conditioned with macrophage inflammatory protein 1-beta (MIP 1-beta; stimulating the macrophages infiltration or vascular endothelial growth factor (VEGF) stimulating angiogenesis). Treatment of transplants with MIP 1-beta and VEGF both accelerated and augmented monocyte-macrophage infiltration and satellite cell differentiation and/or proliferation, as compared to controls. In addition, VEGF treatment enhanced the number of newly formed myotubes. When a complete depletion of host monocyte-macrophages was experimentally induced, no regeneration occurred in transplants. Our data suggest that the presence of blood borne macrophages is required for triggering the earliest events of skeletal muscle regeneration. The understanding of macrophage behaviour after muscle injury should allow us to develop future strategies of satellite cell transplantation as a treatment for muscular dystrophies.

Development of teeth in chick embryos after mouse neural crest transplantations

Teeth were lost in birds 70–80 million years ago. Current thinking holds that it is the avian cranial neural crest-derived mesenchyme that has lost odontogenic capacity, whereas the oral epithelium retains the signaling properties required to induce odontogenesis. To investigate the odontogenic capacity of ectomesenchyme, we have used neural tube transplantations from mice to chick embryos to replace the chick neural crest cell populations with mouse neural crest cells. The mouse/chick chimeras obtained show evidence of tooth formation showing that avian oral epithelium is able to induce a nonavian developmental program in mouse neural crest-derived mesenchymal cells.

Comparative analysis of satellite cell properties in heavy- and lightweight strains of turkey

The growth of muscle during postnatal development results partly from the proliferation of satellite cells and their fusion with muscle fibres. We analysed the properties of satellite cells in a heavyweight (HW) turkey strain characterized by high body weight and a fast growth rate, and in a lightweight farm strain (LW) characterized by low body weight and a slow growth rate. Satellite cell activation was then examined in stretched-overloaded anterior latissimus dorsi (ALD) muscle by weighting one wing in young turkeys from both strains. As early as day 1 of stretching for HW and day 2 for LW, small embryonic-like fibres expressing ventricular cardiac myosin heavy chain (MHC) isoform were observed. Following four days of stretching, the number of nascent fibres had increased in both strains but was significantly greater in HW than LW ALD muscle. The proliferation and differentiation capacities of satellite cells from HW and LW strains were investigated in culture. As judged by in vitro measurements of 3H-thymidine incorporation and DNA content, satellite cells of HW turkey exhibited a greater proliferative capability than those of LW turkey. No differences in the temporal appearance of muscle markers (desmin, MHC isoforms) were noted in vitro between the two strains. These data confirm our in vivo observations indicating that selection based on growth rate does not modify muscle fibre maturation. Our in vivo and in vitro observations suggest that variations in the postnatal muscle growth pattern between HW and LW strains may be related to a difference in the capacity of their satellite cells to proliferate.

Desmin-lacZ transgene expression and regeneration within skeletal muscle transplants

The purpose of this study was to investigate the initiation and time course of the regeneration process in fragments of skeletal muscle transplants as a function of muscle tissue age at implantation. The appearance of desmin occurs at the very beginning of myogenesis. The transgenic desminnls lacZ mice used in the study bear a transgene in which the 1 kb DNA 5′ regulatory sequence of the desmin gene is linked to a reporter gene coding for Escherichia coliβ-galactosidase. The desmin lacZ transgene labels muscle cells in which the desmin synthesis programme has commenced. We implanted pectoralis muscle fragments from fetal transgenic embryos and mature and old transgenic mice into mature non-transgenic mice. Early events of myogenesis occurring during regeneration started sooner in transplants from 4-month-old (day 3 post-implantation) muscle than in those from 24-month-old (day 5-6 post-implantation) muscle, and they lasted longer in those from young (day 17 post-implantation) than in those from old (day 14 post-implantation) muscle fragments. In adult muscle, transgene activation proceeded from the periphery toward the centre of the transplant. In transplants from fetal 18-day-old pectoralis, myotubes with transgene activity were observed from day 1 to day 19. Desmin immunoreactivity, which appeared about one day after transgene activation, was followed by myosin expression. In adult transplants, the continuity of laminin labelling was disrupted around degenerative fibres, illustrating alteration of the extracellular matrix. Our data suggest that satellite cells from old muscle tissue have lower proliferative capacity and/or less access to trophic substances released by the host (damaged fibres, vascularization) than those from fetal or young adult muscle

Limb bud colonization by somite-derived angioblasts is a crucial step for myoblast emigration

We have combined the use of mouse genetic strains and the mouse-into-chicken chimera system to determine precisely the sequence of forelimb colonization by presomitic mesoderm (PSM)-derived myoblasts and angioblasts, and the possible role of this latter cell type in myoblast guidance. By creating a new Flk1/Pax3 double reporter mouse line, we have established the precise timetable for angioblast and myoblast delamination/migration from the somite to the limb bud. This timetable was conserved when mouse PSM was grafted into a chicken host, which further validates the experimental model. The use of Pax3GFP/GFP knockout mice showed that establishment of vascular endothelial and smooth muscle cells (SMCs) is not compromised by the absence of Pax3. Of note, Pax3GFP/GFP knockout mouse PSM-derived cells can contribute to aortic, but not to limb, SMCs that are derived from the somatopleure. Finally, using the Flk1lacZ/lacZ knockout mouse, we show that, in the absence of angioblast and vascular network formation, myoblasts are prevented from migrating into the limb. Taken together, our study establishes for the first time the time schedule for endothelial and skeletal muscle cell colonization in the mouse limb bud and establishes the absolute requirement of endothelial cells for myoblast delamination and migration to the limb. It also reveals that cells delaminating from the somites display marked differentiation traits, suggesting that if a common progenitor exists, its lifespan is extremely short and restricted to the somite.

Desmin-lacZ transgene, a marker of regenerating skeletal muscle

Transgenic C57 mice bearing a transgene of the desmin gene linked to the lacZ reporter gene which encoded for the enzyme beta-galactosidase were used. In the muscle cell, a blue nuclear product appearing in the presence of the X-gal substrate for the enzyme provided evidence of the expression of the desmin gene. However, no transgene expression was observed 2 weeks postnatal in skeletal muscles, even though endogenous desmin was present. In order to investigate the regulatory mechanisms of the desmin gene during regeneration, adult pectoralis fragments (without expression of the desmin transgene) from transgenic mice were implanted into the tibialis anterior of 4 day or 6 week old Swiss mice. Adult pectoralis transplants reexpressed the transgene from day 4 to 10 after implantation. In addition, lesions were performed in adult transgenic pectoralis and transgenic expression in injured muscles was observed 2 days later. This new transgenic mouse is a powerful tool for the study of the various steps of skeletal muscle regeneration.

7 Mouse-Chick Chimera: An Experimental System for Study of Somite Development

As the mammalian embryo is implanted in the uterus and not readily accessible to direct observation or manipulation, much of our understanding of mammalian somite development is based on findings in lower vertebrates. One means of overcoming the difficulties raised by intrauterine development is to engraft mouse tissue in ovo. The experiments described in this chapter relate to the unilateral replacement of somites in chick embryo with those from mouse fetus. Mouse somites differentiate in ovo in dermis, cartilage, and skeletal muscle and are able to migrate into chick host limb. A LacZ transgenic mouse strain was used to ascertain the role of the implanted somites in forming epaxial and hypaxial muscle in the chick embryo. Myogenesis occurred normally in in ovo developing mouse somites, and muscle cells from mouse myotome formed neuromuscular contacts with chick motor axons. After fragments of fetal mouse neural primordium were transplanted into chick embryo, mouse neural tube contributed to the mechanism maintaining myogenesis in the somites of the host embryo. A recently developed double-grafting procedure involving neural tube and somites from knockout mouse strains should elucidate the molecular events involved in early somitogenesis.

Fetal muscle contains different CD34+ cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages

We have previously demonstrated that CD34(+) cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34(+) cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34(+) subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34(+) population comprised a CD31(+)/CD45(-) cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34(+)/CD31(-)/CD45(-)/Sca1(+) subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34(+)/CD31(-)/CD45(-)/Sca1(-) cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34(+) cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.

Seasonal variation in the phenotype of adult ferret (Mustela putorius furo) cremaster muscle

Using immunocytochemistry, electrophoresis and immunoblotting, we studied the expression of fast and slow myosin heavy chain isoforms in adult ferret muscles during quiescent and breeding periods. Adult cremaster muscle expressed slow and fast myosin heavy chain in relatively similar amounts during the quiescent period. During the breeding period, the expression of slow myosin heavy chain I, significantly decreased, and fast myosin heavy chain II, was predominant. No alteration of the MHC pattern in EDL and soleus muscles was detected between the quiescent and breeding periods. The possible involvement of androgens and mechanical factors in the regulation of myosin heavy chain expression in adult cremaster muscle is discussed.

Developmental Behavior of Embryonic Myogenic Progenitors Transplanted into Adult Muscle as Revealed by Desmin LacZ Recombinant Gene

We studied the behavior of myogenic progenitors from donor desmin+/– LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of β-galactosidase expression in donor myogenic cells. Immunostaining for myosin heavy chain and laminin expression was performed together with acetylcholine receptor histochemistry on sections of implanted muscle. Myogenic progenitors generated from DM, LB, and trunk were able to proliferate and adopt a myogenic pathway after transplantation into adult mouse muscle. Although their development appeared to be limited for DM and LB cell transplantation, the differentiation of myogenic progenitors occurred readily with trunk cell injection, suggesting that cell types associated with DM cells were involved in long-term myofiber differentiation (21 day). When neural tube/notochord (NTN) or sclerotomal (S) cells were co-transplanted with DM cells, myogenic nuclei were produced, indicating that both NTN and S are required for the differentiation of DMs grafted into adult muscle. These data are consistent with the differentiation of neural tissues and bone from NTN and S, respectively, and with the development of anatomic relations among all in vivo-differentiated tissues. These results suggest that embryonic trunk cells can be used to repair different types of injured tissues (especially skeletal muscle) under appropriate environmental conditions.