Boris Kablar

Myf5 and MyoD activation define independent myogenic compartments during embryonic development.

Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, we examined the sites of epaxial, hypaxial, and cephalic myogenesis at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins revealed that myogenic precursor cells were not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5(nlacZ)-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appeared to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/-2.5lacZ-expression (-20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments.

Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle

MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD−/− myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD−/− myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD−/− myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD−/− myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD−/− myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were down-regulated. Consistent with these gene expression profiles, MyoD−/− myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD−/− myoblasts. Therefore, MyoD−/− myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.

Myf5-/- :MyoD-/- amyogenic fetuses reveal the importance of early contraction and static loading by striated muscle in mouse skeletogenesis.

The mechanical loading of striated muscle is thought to play an important role in shaping bones and joints. Here, we examine skeletogenesis in late embryogenesis (embryonic day 18.5) in Myf5−/−:MyoD−/− fetuses completely lacking striated muscle. The phenotype includes enlarged and fused cervical vertebrae and postural anomalies, some viscerocranial anomalies, long bone truncation and fusion, absent deltoid tuberosity of the humerus, scapular and clavicular hypoplasia, cleft palate, and cleft sternum. In contrast, neurocranial bone development was essentially normal. While the magnitude of individual effects varied throughout the skeletal system, the results are consistent with skeletal development depending on functional muscles. Novel abnormalities in the amyogenic fetuses relative to less severely paralyzed phenotypes extend our understanding of skeletogenic dependence on embryonic muscle contraction and static loading.

MYOD AND MYF-5 DEFINE THE SPECIFICATION OF MUSCULATURE OF DISTINCT EMBRYONIC ORIGIN

Mounting evidence supports the notion that Myf-5 and MyoD play unique roles in the development of epaxial (originating in the dorso-medial half of the somite, e.g. back muscles) and hypaxial (originating in the ventro-lateral half of the somite, e.g. limb and body wall muscles) musculature. To further understand how Myf-5 and MyoD genes cooperate during skeletal muscle specification, we examined and compared the expression pattern of MyoD-lacZ (258/2.5lacZ and MD6.0-lacZ) transgenes in wild-type, Myf-5, and MyoD mutant embryos. We found that the delayed onset of muscle differentiation in the branchial arches, tongue, limbs, and diaphragm of MyoD-/- embryos was a consequence of a reduced ability of myogenic precursor cells to progress through their normal developmental program and not because of a defect in migration of muscle progenitor cells into these regions. We also found that myogenic precursor cells for back, intercostal, and abdominal wall musculature in Myf-54-/- embryos failed to undergo normal translocation or differentiation. By contrast, the myogenic precursors of intercostal and abdominal wall musculature in MyoD-/- embryos underwent normal translocation but failed to undergo timely differentiation. In conclusion, these observations strongly support the hypothesis that Myf-5 plays a unique role in the development of muscles arising after translocation of epithelial dermamyotome cells along the medial edge of the somite to the subjacent myotome (e.g., back or epaxial muscle) and that MyoD plays a unique role in the development of muscles arising from migratory precursor cells (e.g., limb and branchial arch muscles, tongue, and diaphragm). In addition, the expression pattern of MyoD-lacZ transgenes in the intercostal and abdominal wall muscles of Myf-5-/- and MyoD-/- embryos suggests that appropriate development of these muscles is dependent on both genes and, therefore, these muscles have a dual embryonic origin (epaxial and hypaxial).

Pulmonary Hypoplasia in the Connective Tissue Growth Factor (Ctgf) Null Mouse

Connective tissue growth factor (CTGF) is a mediator of growth factor activity, and Ctgf knockouts die at birth from respiratory failure due to skeletal dysplasia. Previous microarray analysis revealed Ctgf down‐regulation in the hypoplastic lungs of amyogenic mouse embryos. This study, therefore, examined pulmonary development in Ctgf−/− mouse fetuses to investigate if respiration could also have been impaired by lung abnormalities. The Ctgf−/− lungs were hypoplastic, with reduced cell proliferation and increased apoptosis. PDGF‐B, its receptor and IGF‐I, were markedly attenuated and the TTF‐1 gradient lost. Type II pneumocyte differentiation was perturbed, the cells depicting excessive glycogen retention and diminished lamellar body and nuclear size, though able to synthesize surfactant‐associated protein. However, type I pneumocyte differentiation was not affected by Ctgf deletion. Our findings indicate that the absence of Ctgf and/or its protein product, CTGF, may induce pulmonary hypoplasia by both disrupting basic lung developmental processes and restricting thoracic expansion. Developmental Dynamics 237:485–493, 2008.

The role of neurotrophins in the maintenance of the spinal cord motor neurons and the dorsal root ganglia proprioceptive sensory neurons

The aim of this study was to approach the question of neuronal dependence on neurotrophins during embryonic development in mice in a way other than gene targeting. We employed amyogenic mouse embryos and fetuses that develop without any skeletal myoblasts or skeletal muscle and consequently lose motor and proprioceptive neurons. We hypothesized that if, in spite of the complete inability to maintain motor and proprioceptive neurons, the remaining spinal and dorsal root ganglia tissues of amyogenic fetuses still contain any of the neurotrophins, that particular neurotrophin alone is not sufficient for the maintenance of motor and proprioceptive neurons. Moreover, if the remaining spinal and dorsal root ganglia tissues still contain any of the neurotrophins, that particular neurotrophin alone may be sufficient for the maintenance of the remaining neurons (i.e., mostly non‐muscle‐ and a few muscle‐innervating neurons). To test the role of the spinal cord and dorsal root ganglia tissues in the maintenance of its neurons, we performed immunohistochemistry employing double‐mutant and control tissues and antibodies against neurotrophins and their receptors. Our data suggested that: (a) during the peak of motor neuron cell death, the spinal cord and dorsal root ganglia distribution of neurotrophins was not altered; (b) the distribution of BDNF, NT‐4/5, TrkB and TrkC, and not NT‐3, was necessary for the maintenance of the spinal cord motor neurons; (c) the distribution of BDNF, NT‐4/5 and TrkC, and not NT‐3 and Trk B, was necessary for the maintenance of the DRG proprioceptive neurons; (d) NT‐3 was responsible for the maintenance of the remaining neurons and glia in the spinal cord and dorsal root ganglia (possibly via TrkB).

A significant reduction of the diaphragm in mdx:MyoD−/−9th embryos suggests a role for MyoD in the diaphragm development

To further investigate the role of MyoD during skeletal myogenesis, we backcrossed mdx mutant mice (lacking dystrophin) with MyoD knock-out mice to obtain viable mice with MyoD allele on a pure mdx background. However, after nine generations of backcrossing, it was not possible to obtain a viable mdx:MyoD-/- phenotype (designated as: mdx:MyoD-/-(9th)). The compound-mutant embryos were examined just before birth. Essentially normal Myf5-dependent and most of the MyoD-dependent musculature was observed. By contrast, the skeletal muscle compartment of the diaphragm was significantly reduced. The mesenchymal compartment of the diaphragm was intact and no herniations were observed. Other examined organs (e.g., liver, kidney, brain, etc.) showed no histological abnormalities. Pulmonary hypoplasia was determined as the cause of neonatal death. Therefore, using a different approach, our new data supplement our previous findings and suggest an essential role for MyoD in development of skeletal muscle of the diaphragm. The failure of mdx:MyoD-/-(9th) diaphragm to develop normally is not caused by a reduced number of satellite cells, but from the inability of stem cells to progress through the myogenic program. Our data also suggest that functions of MyoD and Myf5 (and the respective muscle precursor cell sub-populations) are not entirely redundant by term, as previously suggested, since Myf5 is not capable of fully substituting for MyoD in the diaphragm development.

Abnormal development of the intercostal muscles and the rib cage in Myf5-/- embryos leads to pulmonary hypoplasia

The aim of our study was to investigate the importance of pulmonary distension and fetal breathing‐like movements executed by the contractile activity of the intercostal respiratory muscles for proper lung growth and maturation. Lung development in Myf5−/− embryos, lacking the rib cage and functional intercostal musculature, was compared with wild‐type controls at embryonic days 14.5, 16.5, and 18.5. Our data revealed that Myf5−/− embryos suffered from pulmonary hypoplasia in part due to the decreased number of proliferating lung cells and in part due to the increased number of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) ‐positive cells. In addition, the proximal‐to‐distal expression gradient of thyroid transcription factor‐1 observed in wild‐type embryos was not maintained in Myf5−/− embryos. The number of lung cells expressing platelet‐derived growth factor‐BB, its receptor and insulin growth factor‐I was significantly decreased in the hypoplastic lung. By contrast, no difference in the expression pattern of surfactant associated proteins or Clara cells marker was detected between wild‐type and Myf5−/− embryos. Collectively, our data suggest that the mechanochemical signal transduction pathway used in vitro is also effective in vivo influencing lung growth but not lung cell maturation and resulting in lung hypoplasia. These data affirm the role of fetal breathing‐like movements in lung organogenesis. Developmental Dynamics 232:43–54, 2005.

Contractile activity of skeletal musculature involved in breathing is essential for normal lung cell differentiation, as revealed in Myf5-/-:MyoD-/- embryos

In the current study, the role of contractile activity of respiratory muscles in fetal lung growth and cell differentiation was examined using Myf5−/−:MyoD−/− mouse embryos. As previously found, Myf5−/−:MyoD−/− mouse embryos had no respiratory musculature. Consequently, they suffered from pulmonary hypoplasia and died shortly after birth. The hypoplastic lung had decreased proliferation and increased apoptotic index as early as embryonic day 14.5. By contrast, only at the last gestational day, the number of lung cells expressing platelet derived growth factor B and insulin growth factor I was decreased, while the gradient of the thyroid transcription factor 1 was not maintained. Type II pneumocytes had a failure in glycogen utilization and surfactant storage and secretion but were able to synthesize the surfactant‐associated proteins. Type I pneumocytes were readily detectable using an early differentiation marker (i.e., Gp38). However, the late differentiation of type I pneumocytes never occurred, as revealed by transmission electron microscopy. Together, our findings suggest that pulmonary distension due to fetal breathing‐like movements plays an important role not only in lung growth but also in lung cell differentiation. Developmental Dynamics 233:772–782, 2005.

Presence of neurotrophic factors in skeletal muscle correlates with survival of spinal cord motor neurons

To determine which combination of skeletal muscle‐derived neurotrophic factors may be important for the survival of specific subpopulations of developing spinal cord motor neurons, we used Myf5 and MyoD (myogenic regulatory factors) knockouts, containing differentially committed myogenic precursor cells (MPCc) and immunohistochemistry against several muscle‐secreted neurotrophic factors. At the peak of motor neuron cell death, skeletal muscle development is delayed in the back and body wall muscles of Myf5−/− embryos and in the limb muscles of MyoD−/− embryos. We hypothesized that, if the skeletal muscle was indeed an important source of survival factors for motor neurons, the back, the abdominal wall, and the forelimb MPCs of Myf5−/− or MyoD−/− embryos should produce at least some neurotrophic factors necessary for the survival of motor neurons. In this report, we demonstrate that (1) different MPCs lacking Myf5, MyoD, or Myf5/MyoD have different capabilities in providing factors potentially required for the survival of motor neurons and intramuscular nerve branching, (2) MPCs in double‐mutant embryos do not contain neurotrophic factors in the absence of myogenic specification, and (3) different subpopulations of MPCs contain different combinations of neurotrophic factors potentially required for the survival of the specific subpopulations of innervating motor neurons. Developmental Dynamics 234:659–669, 2005.