ABSTRACT
Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.
Subject(s)
DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Myoblasts, Skeletal/metabolism , Transcription Factors/metabolism , Animals , Apoptosis , Biomarkers , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Mice, Transgenic , Muscle Development/genetics , Muscle Development/physiology , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Myoblasts, Skeletal/cytology , PAX3 Transcription Factor , PAX7 Transcription Factor , Paired Box Transcription Factors , Transcription Factors/deficiency , Transcription Factors/geneticsABSTRACT
In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells. Two determination factors of the basic helix-loop-helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts. In the absence of these factors, progenitor cells remain multipotent and can change their fate. Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes. Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the new Myf5:Myod double-null mice only when Mrf4 expression is not compromised. This finding contradicts the widely held view that myogenic identity is conferred solely by Myf5 and Myod, and identifies Mrf4 as a determination gene. We revise the epistatic relationship of the MRFs, in which both Myf5 and Mrf4 act upstream of Myod to direct embryonic multipotent cells into the myogenic lineage.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/deficiency , Gene Deletion , Muscle Proteins/deficiency , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , MyoD Protein/genetics , Myogenic Regulatory Factors/metabolism , Trans-Activators/deficiency , Alleles , Animals , Cell Lineage , DNA-Binding Proteins/genetics , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Mice , Mice, Knockout , Muscle Proteins/genetics , Muscle, Skeletal/embryology , MyoD Protein/metabolism , Myogenic Regulatory Factor 5 , Myogenin , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/geneticsABSTRACT
OBJECT: The reliable assessment of the invasiveness of gliomas in vitro has proved elusive, because most invasion assays inadequately model in vivo invasion in its complexity. Recently, organotypical brain cultures were successfully used in short-term invasion studies on glioma cell lines. In this paper the authors report that the invasiveness of human glioma biopsy specimens directly implanted into rodent brain slices by using the intraslice implantation system (ISIS) can be quantified with precision. The model was first validated by the demonstration that, in long-term studies, established glioma cells survive in the ISIS and follow pathways of invasion similar to those in vivo. METHODS: Brain slices (400 microm thick) from newborn mice were maintained on millicell membranes for 15 days. Cells from two human and one rodent glioblastoma multiforme (GBM) cell lines injected into the ISIS were detected by immunohistochemistry or after transfection with green fluorescent protein-containing vectors. Preferential migration along blood vessels was identified using confocal and fluorescent microscopy. Freshly isolated (< or = 24 hours after removal) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-prelabeled human glioma biopsy specimens were successfully implanted in 19 (83%) of 23 cases, including 12 GBMs and seven lower grade gliomas (LGGs). Morphometric quantification of distance and density of tumor cell invasion showed that the GBMs were two to four times more invasive than the LGGs. Heterogeneity of invasion was also observed among GBMs and LGGs. Directly implanted glioma fragments were more invasive than spheroids derived from the same biopsy specimen. CONCLUSIONS: The ISIS combines a high success rate, technical simplicity, and detailed quantitative measurements and may, therefore, be used to study the invasiveness of biopsy specimens of gliomas of different grades.
Subject(s)
Brain Neoplasms/pathology , Glioma/pathology , Neoplasm Invasiveness/pathology , Animals , Biopsy , Cytological Techniques , Humans , Mice , Mice, Inbred C57BL , Organ Culture Techniques , Tumor Cells, CulturedABSTRACT
The authors used different kinds of peripheral nerve grafts to reconstruct a terminal branch of the brachial plexus (the median nerve) gap of adult Sprague-Dawley rats, including fresh or frozen autografts and allografts from Norway rats. They also performed acellular allograft repopulation by autogenous Schwann cells, to improve the environment for nerve regeneration. Three, six, nine and twelve months after grafting, rats underwent histological assessment (muscle, nerve and spinal cord) and simple functional assessment by the grasping test. Initially, the functional recovery of frozen grafts was lower than fresh graft recovery, but twelve months after surgery it was similar for both types of graft.