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 Pax3(GFP/GFP) knockout mice showed that establishment of vascular endothelial and smooth muscle cells (SMCs) is not compromised by the absence of Pax3. Of note, Pax3(GFP/GFP) knockout mouse PSM-derived cells can contribute to aortic, but not to limb, SMCs that are derived from the somatopleure. Finally, using the Flk1(lacZ)(/)(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.

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.

Fetal muscle-derived cells can repair dystrophic muscles in mdx mice.

We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.

Soluble factors from neuronal cultures induce a specific proliferation and resistance to apoptosis of cognate mouse skeletal muscle precursor cells.

The mechanisms or the physiological events, which control the regeneration of skeletal muscle through muscle precursor cell multiplication and differentiation, are still largely unknown. To address the question of the involvement of neurons in this process, skeletal muscle progenitors were grown in the presence of conditioned media obtained from 3-day-old cultures of embryonic neurons (derived from either the dorsal or the ventral region of 11-day-old mouse embryos) or media conditioned with satellite cells. Strikingly, only satellite cells cultured in medium conditioned from ventral embryonic neurons exhibited increased proliferation, as well as resistance to staurosporine (STS)-induced apoptosis. Our results suggest the existence of specific anti-apoptogenic neural soluble signals, which could be involved in skeletal muscle regeneration pathways.

Mouse-chick neural chimeras.

Embryonic chimera production was used to study the developmental processes of the mouse nervous system. The difficulty of performing in situ transplantation experiments of neural primordium of mouse embryo was overcome by isotopic and isochronic grafting of mouse neural tube fragments into chick embryo. Mouse neural tube cells differentiated perfectly in ovo and neural crest cells associated with the grafted neural tube were able to migrate and reach the normal arrest sites of host neural crests. Cranial neural crest cells penetrated into chick facial areas and entered into the development of dental bud structures, participating in vibrissa formation. Depending on graft level, in ovo implanted mouse neural crest cells formed different components of the peripheral nervous system. At trunk level, they located in spinal ganglia and orthosympathetic chains and gave rise to Schwann cells lining the nerves. When implanted into the lumbosacral region, they penetrated into the enteric nervous system. At the precise 18-24 somite level, they colonized host adrenal gland. Mouse neural tube was involved in the mechanisms required to maintain myogenesis in host somites. Furthermore in ovo grafts of mouse cells from genetically modified embryos, in which many mutations induce early death, are particularly useful to investigate cellular events involved in the development of the nervous system and to identify molecular events of embryogenesis.

Endothelial cells within embryonic skeletal muscles: a potential source of myogenic progenitors.

We investigated whether the vessel-associated or endothelial cells within mouse embryo muscles can be a source of myogenic progenitors. Immunodetection of the stem cell surface markers, CD34 and Flk1, which are known to characterize the endothelial lineage, was done throughout the course of embryo muscle development. Both markers appeared to be restricted to the vessel-associated cells. On the basis of CD34 labeling, the reactive cells were purified by magnetic-bead selection from the limb muscles of 17-dpc desmin+/-LacZ mouse embryos and characterized by fluorescence-activated cell sorting. The cells in the selected CD34(+) population appeared to be approximately 95% positive for Flk1, but usually negative for CD45. We demonstrated that in vitro the CD34(+)/Flk1(+) population differentiated into endothelial cells and skeletal myofibers. When transplanted into mdx mouse muscle, this population displayed a high propensity to disperse within the recipient muscle, fuse with the host myofibers, and restore dystrophin expression. The marked ability of the embryonic muscle endothelial cells to activate myogenic program could be related to their somitic origin.

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 beta-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.

[Implications of environmental cues in cardiomyogenesis]. / Implication de l'environnement tissulaire dans la cardiomyogenèse.

The staging of murine cardiomyocyte specification and determination was investigated in cultures of tissue explants from pre- and postgastrulation embryos and after transplantation of cardiac or cardiogenic tissues from mouse embryos into chick embryos. The development of cultured and transplanted cells in cardiomyocytes was evaluated by testing the expression of several cardiac transcription factor genes (Nkx 2.5, eHAND, dHAND, GATA 4), alpha cardiac actin, and beta myosin heavy chain protein. In vitro analyses showed that cells with the potential to form cardiac muscle were present prior to gastrulation in 6.5-day postconception (dpc) epiblasts. Although, as shown by in vivo experiments, neurectodermal derived structures did not influence cardiogenesis in epiblast transplants, these transplants did not exhibit full cardiogenic cell differentiation in the chicken environment. In in vitro culture, the neurectoderm also had no effect on murine cardiomyogenesis. In contrast, the presence of endoderm in explants between mid- and late streak stages stimulated emerging mesodermal cells to adopt a myocardial pathway. Mesoderm from late streak explants (7.5 dpc) was capable of differentiating into a cardiac phenotype in the avian heterotopic environment, indicating that the specification of cardiac precursors became irreversible around the late streak stage in mouse embryo.

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.