Hedgehog and Wnt coordinate signaling in myogenic progenitors and regulate limb regeneration

Amphibians have a remarkable capacity for limb regeneration. Following a severe injury, there is complete regeneration with restoration of the patterning and cellular architecture of the amputated limb. While studies have focused on the structural anatomical changes during amphibian limb regeneration, the signaling mechanisms that govern cellular dedifferentiation and blastemal progenitors are unknown. Here, we demonstrate the temporal and spatial requirement for hedgehog (Hh) signaling and its hierarchical correlation with respect to Wnt signaling during newt limb regeneration. While the dedifferentiation process of mature lineages does not depend on Hh signaling, the proliferation and the migration of the dedifferentiated cells are dependent on Hh signaling. Temporally controlled chemical inactivation of the Hh pathway indicates that Hh-mediated antero-posterior (AP) specification occurs early during limb regeneration and that Hh is subsequently required for expansion of the blastemal progenitors. Inhibition of Hh signaling results in G0/G1 arrest with a concomitant reduction in S-phase and G2/M population in myogenic progenitors. Furthermore, Hh inhibition leads to reduced Pax7-positive cells and fewer regenerating fibers relative to control tissue. We demonstrate that activation of Wnt signaling rescues the inhibition of Hh pathway mainly by enhancing proliferative signals, possibly mediated through TCF4 activity. Collectively, our results demonstrate coordinated signaling of Hh and Wnt activities in regulating blastemal progenitors and their hierarchical positioning during limb regeneration.     

Alterations in the TGFbeta signaling pathway in myogenic progenitors with age

Myogenic progenitors in adult muscle are necessary for the repair, maintenance and hypertrophy of post-mitotic muscle fibers. With age, fat deposition and fibrosis contribute to the decline in the integrity and functional capacity of muscles. In a previous study we reported increased accumulation of lipid in myogenic progenitors obtained from aged mice, accompanied by an up-regulation of genes involved in adipogenic differentiation. The present study was designed to extend our understanding of how aging affects the fate and gene expression profile of myogenic progenitors. Affymetrix murine U74 Genechip analysis was performed using RNA extracted from myogenic progenitors isolated from adult (8-month-old) and aged (24-month-old) DBA/2JNIA mice. The cells from the aged animals exhibited major alterations in the expression level of many genes directly or indirectly involved with the TGFbeta signaling pathway. Our data indicate that with age, myogenic progenitors acquire the paradoxical phenotype of being both TGFbeta activated based on overexpression of TGFbeta-inducible genes, but resistant to the differentiation-inhibiting effects of exogenous TGFbeta. The overexpression of TGFbeta-regulated genes, such as connective tissue growth factor, may play a role in increasing fibrosis in aging muscle.     

Circulating myogenic progenitors and muscle repair

The capability of bone marrow derived cells to contribute to numerous peripheral tissues may hold tremendous promise for the field of regenerative medicine. In the context of skeletal muscle disease in particular, the ability of these cells to reach sites of damage through the circulation would overcome some key limitations of current cell therapy approaches. In muscle however, this non-classical repair process takes place at an exceedingly low frequency and fails to yield any measurable functional improvement. Recent advances regarding the cell types or mechanisms involved in this phenomenon may now provide direction for strategies aimed at increasing its efficiency to therapeutic levels.     

Loss of SHP-1 phosphatase alters cytokine expression in the mouse hindbrain following cochlear ablation

Inflammatory cytokines in the central nervous system are largely modulated by glial cells and influence neuronal responses to CNS injury. The protein tyrosine phosphatase SHP-1, an intracellular regulator of many cytokine signaling pathways, has been implicated in mediating the activation of glia. There is a direct correlation between abnormally activated microglia and neuron loss within the SHP-1 deficient motheaten (me/me) mouse auditory brainstem after afferent injury. In order to determine whether loss of SHP-1 creates an aberrant cytokine environment driving the abnormal activation of me/me microglia, the expression of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) was examined by enzyme-linked immunosorbent assay (ELISA). Normal uninjured me/me mice showed lower IL-10 but higher IL-1beta levels compared to wild-type. Following unilateral cochlear ablation, there is decreased expression of IL-4 and IL-10 in me/me brains compared to wild-type, but IL-1beta is significantly increased. These findings indicate that decreases in anti-inflammatory cytokines, in combination with increased expression of the pro-inflammatory cytokine IL-1beta, may initiate a robust inflammatory reaction within the me/me brain contributing to the neuronal degeneration in the deafferented me/me auditory brainstem. SHP-1 may therefore play a role in limiting CNS inflammation following injury and disease.     

Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.     

Isolation and characterization of the complete mouse emerin gene

Emery-Dreifuss muscular dystrophy (EMD) is an X-linked recessive disorder associated with muscle wasting, contractures, and cardiomyopathy. The responsible emerin gene has recently been identified and found to encode a serine-rich protein similar to lamina-associated protein 2 (LAP2), although the disease mechanism remains obscure. In order to pursue the pathophysiology of this disorder, we report here the isolation and characterization of the complete mouse emerin gene. The emerin cDNA was isolated from murine strain BALB/c, and the emerin gene was isolated from strain 129. The 2.9-kb mouse emerin gene was completely sequenced and found to be composed of 6 exons and encode a protein 73% identical to that of the human protein. Key similarities with LAP2 were found to be conserved, including critical LAP2 phosphorylation sites. Examination of the murine promoter revealed three previously unrecognized cAMP response elements (CRE) conserved between human and mouse. While Northern analysis shows emerin to be widely expressed in the mouse, as it is in humans, these promoter elements may indicate cAMP responsiveness. These data provide the necessary elements to further investigate EMD in a murine system. Received: 1 December 1996 / Accepted: 12 January 1997     

Skeletal myogenic progenitors in the endothelium of lung and yolk sac

We previously showed that clonable skeletal myogenic cells can be derived from the embryonic aorta but become very rare in the more mature and structured fetal aorta. The aim of this study was to investigate whether, during fetal and postnatal development, these myogenic progenitors progressively disappear or may rather associate with the microvascular district, being thus distributed to virtually all tissues. To test this hypothesis, we used F1 embryos (or mice) from a transgenic line expressing a striated muscle-specific reporter gene (LacZ) crossed with a transgenic line expressing a different endothelial-specific reporter genes (GFP). Endothelial cells were isolated from yolk sac (at E11) and lung (at E11, E17, P1, P10, and P60), two organs embryologically unrelated to paraxial mesoderm, rich in vessels, and devoid of skeletal muscle. Endothelial cells, purified by magnetic bead selection (CD31/PECAM-1(+)) or cell sorting (Tie2-GFP(+)) were then challenged for their skeletal myogenic potential in vitro and in vivo.The results demonstrated that both yolk sac and lung contain progenitor cells, which express endothelial markers and are endowed with a skeletal myogenic potential that they reveal when in the presence of differentiating myoblasts, in vitro, and regenerating muscle, in vivo.The number (or potency to generate skeletal muscle) of these vessels associated cells decreases rapidly with age and is very low in mature animals, possibly correlating with reduced regenerative capacity of adult mammalian tissues.     

Ca2+ signaling in mouse mesenteric small arteries: myogenic tone and adrenergic vasoconstriction

Arteries that have developed myogenic tone (MT) are in a markedly different physiological state compared with those that have not, with higher cytosolic [Ca(2+)] and altered activity of several signal transduction pathways. In this study, we sought to determine whether alpha(1)-adrenoceptor-induced Ca(2+) signaling is different in pressurized arteries that have spontaneously developed MT (the presumptive physiological state) compared with those that have not (a common experimental state). At 32 degrees C and intraluminal pressure of 70 mmHg, cytoplasmic [Ca(2+)] was steady in most smooth muscle cells (SMCs). In a minority of cells (34%), however, at least one propagating Ca(2+) wave occurred. alpha(1)-Adrenoceptor activation (phenylephrine, PE; 0.1-10.0 microM) caused strong vasoconstriction and markedly increased the frequency of Ca(2+) waves (in virtually all cells). However, when cytosolic [Ca(2+)] was elevated experimentally in these arteries ([K(+)] 20 mM), PE failed to elicit Ca(2+) waves, although it did elevate [Ca(2+)] (F/F(0)) further and caused further vasoconstriction. During development of MT, the cytosolic [Ca(2+)] (F/F(0)) in individual SMCs increased, Ca(2+) waves disappeared (from SMCs that had them), and small Ca(2+) ripples (frequency approximately 0.05 Hz) appeared in approximately 13% of cells. PE elicited only spatially uniform increases in [Ca(2+)] and a smaller change in diameter (than in the absence of MT). Nevertheless, when cytosolic [Ca(2+)] and MT were decreased by nifedipine (1 microM), PE did elicit Ca(2+) waves. Thus alpha(1)-adrenoceptor-mediated Ca(2+) signaling is markedly different in arteries with and without MT, perhaps due to the elevated [Ca(2+)], and may have a different molecular basis. alpha(1)-Adrenoceptor-induced vasoconstriction may be supported either by Ca(2+) waves or by steady elevation of cytoplasmic [Ca(2+)], depending on the amount of MT.     

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.     

Moderate maternal vitamin A deficiency alters myogenic regulatory protein expression and perinatal organ growth in the rat

Vitamin A deficiency is one of the most common dietary deficiencies in the developing world and is a major health concern where it is associated with increased risk of fetal and infant mortality and morbidity. Early studies in the rat demonstrated that, in addition to respiratory problems, neonates showed evidence of mobility problems in response to moderate vitamin A deficiency. This study investigated whether moderate deficiency of this vitamin plays a role in regulating key skeletal muscle regulatory pathways during development. Thirty female rats were fed vitamin A-moderate (VAM) or vitamin A-sufficient diets from weaning and throughout pregnancy. Fetal and neonatal hindlimb and muscle samples were collected on days 13.5, 15.5, 17.5, and 19.5 of pregnancy and 1 day following birth. Mothers fed the VAM diet had reduced retinol concentrations at all time points studied (P < 0.01), and neonates had reduced relative lung weights (P < 0.01). Fetal weight and survival did not differ between groups but neonatal survival was lower in the VAM group where neonates had increased relative heart weights (P < 0.05). Analysis of myogenic regulatory factor expression and calcineurin signaling in fetuses and neonates demonstrated decreased protein levels of myf5 [50% at 17.5 dg (P < 0.05)], myogenin [70% at birth (P < 0.001)], and myosin heavy chain fast [50% at birth (P < 0.05)] in response to moderate vitamin A deficiency. Overall, these changes suggest that vitamin A status during pregnancy may have important implications for fetal muscle development and subsequent muscle function in the offspring.     

Differential expression of two glucuronyltransferases synthesizing HNK-1 carbohydrate epitope in the sublineages of the rat myogenic progenitors

HNK-1 epitope is a cell-surface carbohydrate mediating various cell-cell or cell-substrate interactions. We found HNK-1 epitope in longitudinally arrayed fibers in the subpopulation of the epaxial myotome, and hypaxial myoblasts migrating into the limb bud in the rat embryo. We next investigated the expression patterns of genes encoding two glucuronyltransferases (GlcAT-P, GlcAT-D) and sulfotransferase (Sul-T), which are required for biosynthesis of HNK-1 epitope. GlcAT-P gene was expressed in the non-migrating longitudinal fibers, whereas GlcAT-D gene was expressed in the migrating myoblasts in the limb bud. Sul-T gene expression was ubiquitously observed in all these myogenic populations. Thus, differential expression of GlcAT genes may relate to the epaxial/hypaxial or migrating/non-migrating myoblast lineages.     

Integrated miRNA and mRNA expression profiling of mouse mammary tumor models identifies miRNA signatures associated with mammary tumor lineage

Background  

MicroRNAs (miRNAs) are small, non-coding, endogenous RNAs involved in regulating gene expression and protein translation. miRNA expression profiling of human breast cancers has identified miRNAs related to the clinical diversity of the disease and potentially provides novel diagnostic and prognostic tools for breast cancer therapy. In order to further understand the associations between oncogenic drivers and miRNA expression in sub-types of breast cancer, we performed miRNA expression profiling on mammary tumors from eight well-characterized genetically engineered mouse (GEM) models of human breast cancer, including MMTV-H-Ras, -Her2/neu, -c-Myc, -PymT, -Wnt1 and C3(1)/SV40 T/t-antigen transgenic mice, BRCA1 ^fl/fl ;p53 ^+/-;MMTV-cre knock-out mice and the p53 ^fl/fl ;MMTV-cre transplant model.