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[This corrects the article DOI: 10.1371/journal.pone.0185775.].
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Limb muscles derive from pax3 expressing precursor cells that migrate from the hypaxial somite into the developing limb bud. Once there they begin to differentiate and express muscle determination genes such as MyoD. This process is regulated by a combination of inductive or inhibitory signals including Fgf18, retinoic acid, HGF, Notch and IGFs. IGFs are well known to affect late stages of muscle development and to promote both proliferation and differentiation. We examined their roles in early stage limb bud myogenesis using chicken embryos as an experimental model. Grafting beads soaked in purified recombinant IGF-I, IGF-II or small molecule inhibitors of specific signaling pathways into developing chick embryo limbs showed that both IGF-I and IGF-II induce expression of the early stage myogenic markers pax3 and MyoD as well as myogenin. Their effects on pax3 and MyoD expression were blocked by inhibitors of both the IGF type I receptor (picropodophyllotoxin, PPP) and MEK (U0126). The PI3K inhibitor LY294002 blocked IGF-II, but not IGF-I, induction of pax3 mRNA as well as the IGF-I, but not IGF-II, induction of MyoD mRNA. In addition SU5402, an FGFR/ VEGFR inhibitor, blocked the induction of MyoD by both IGFs but had no effect on pax3 induction, suggesting a role for FGF or VEGF signaling in their induction of MyoD. This was confirmed by in situ hybridization showing that FGF18, a known regulator of MyoD in limb myoblasts, was induced by IGF-I. In addition to their well-known effects on later stages of myogenesis via their induction of myogenin expression, both IGF-I and IGF-II induced pax3 and MyoD expression in developing chick embryos, indicating that they also regulate early stages of myogenesis. The data suggests that the IGFs may have slightly different effects on IGF1R signal transduction via PI3K and that their stimulatory effects on MyoD expression may be indirect, possibly via induction of FGF18 expression.
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Embrión de Pollo/efectos de los fármacos , Miembro Posterior/efectos de los fármacos , Factor II del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Animales , Butadienos/farmacología , Embrión de Pollo/metabolismo , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/metabolismo , Morfolinas/farmacología , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Nitrilos/farmacología , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacología , Pirroles/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
AIMS: Maternal dietary restriction during pregnancy impairs nephron development and results in offspring with fewer nephrons. Cell turnover in the early developing kidney is altered by exposure to maternal dietary restriction and may be regulated by the LIM-kinase family of enzymes. We set out to establish whether disturbance of LIM-kinase activity might play a role in the impairment of nephron formation. MAIN METHODS: E12.5 metanephric kidneys and HK2 cells were grown in culture with the pharmacological LIM-kinase inhibitor BMS5. Organs were injected with DiI, imaged and cell numbers measured over 48h to assess growth. Cells undergoing mitosis were visualised by pH3 labelling. KEY FINDINGS: Growth of cultured kidneys reduced to 83% of controls after exposure to BMS5 and final cell number to 25% of control levels after 48h. Whilst control and BMS5 treated organs showed cells undergoing mitosis (100±11 cells/field vs 113±18 cells/field respectively) the proportion in anaphase was considerably diminished with BMS5 treatment (7.8±0.8% vs 0.8±0.6% respectively; P<0.01). This was consistent with effects on HK2 cells highlighting a severe impact of BMS5 on formation of the mitotic spindle and centriole positioning. DiI labelled cells migrated in 100% of control cultures vs 0% BMS5 treated organs. The number of nephrogenic precursor cells appeared depleted in whole organs and formation of new nephrons was blocked by exposure to BMS5. SIGNIFICANCE: Pharmacological blockade of LIM-kinase function in the early developing kidney results in failure of renal development. This is likely due to prevention of dividing cells from completion of mitosis with their resultant loss.
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Desarrollo Fetal/fisiología , Túbulos Renales Proximales , Quinasas Lim/fisiología , Mesonefro , Organogénesis/fisiología , Animales , Técnicas de Cultivo de Célula , Línea Celular , Inhibidores Enzimáticos/farmacología , Desarrollo Fetal/efectos de los fármacos , Edad Gestacional , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/enzimología , Quinasas Lim/antagonistas & inhibidores , Mesonefro/embriología , Mesonefro/enzimología , Ratones Endogámicos ICR , Mitosis/efectos de los fármacos , Organogénesis/efectos de los fármacos , Técnicas de Cultivo de TejidosRESUMEN
Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining. In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18. This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.
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Desarrollo Embrionario/genética , Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica , Animales , Embrión de Pollo , Separación Inmunomagnética , Hibridación in Situ , Fosforilación , Transducción de Señal/efectos de los fármacosRESUMEN
The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 have been analysed during the development of chicken embryo somites and limbs. In somites, Myf5 is expressed first in somites and paraxial mesoderm at HH stage 9 followed by MyoD at HH stage 12, and Mgn and MRF4 at HH stage 14. In older somites, Myf5 and MyoD are also expressed in the ventrally extending myotome prior to Mgn and MRF4 expression. In limb muscles a similar temporal sequence is observed with Myf5 expression detected first in forelimbs at HH stage 22, MyoD at HH stage 23, Mgn at HH stage 24 and MRF4 at HH stage 30. This report describes the precise time of onset of expression of each MRF in somites and limbs during chicken embryo development, and provides a detailed comparative timeline of MRF expression in different embryonic muscle groups.
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Extremidades/embriología , Músculo Esquelético/metabolismo , Factores Reguladores Miogénicos/metabolismo , Somitos/embriología , Animales , Embrión de Pollo , Extremidades/fisiología , Mesodermo/metabolismo , Organogénesis/fisiología , Somitos/metabolismoRESUMEN
Oocytes treated with the protein synthesis inhibitor cycloheximide (CHX) arrest at the germinal vesicle (GV) stage and undergo accelerated GV breakdown (GVBD) after CHX is removed. However, little is known about the underlying mechanism of accelerated meiotic maturation. Here, we investigated this mechanism and found that oocytes released from CHX arrest have higher amounts of cyclin B1 (CCNB1) and phosphorylated mitogen-activated protein kinase (pMAPK) proteins. Increased levels of these factors were not associated with mRNA polyadenylation or increased transcription rates of CCNB1 and MOS (Moloney murine sarcoma viral oncogene homolog) during CHX arrest. We found that treatment of CHX-arrested oocytes with the actin filament-stabilizing agent Jasplakinolide (Jasp) delayed GVBD following release from CHX arrest and that this was correlated with reduced maturation-promoting factor (MPF) activity. These results suggest that CCNB1 mRNAs released from actin filaments during CHX arrest increase CCNB1 transcripts available for translation after release from CHX arrest, leading to the precocious activation of MPF and accelerated meiotic progression.
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Actinas/metabolismo , Cicloheximida/farmacología , Meiosis/efectos de los fármacos , Oocitos/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Ciclina B1/metabolismo , Depsipéptidos/farmacología , Femenino , Factor Promotor de Maduración/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Virus del Sarcoma Murino de Moloney/genética , Técnicas de Transferencia Nuclear , Polimerizacion , Embarazo , OvinosRESUMEN
Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.
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Reprogramación Celular , Células Madre Embrionarias/citología , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Técnicas de Transferencia Nuclear/veterinaria , Activinas/farmacología , Animales , Blastocisto/citología , Diferenciación Celular , Células Cultivadas , Clonación de Organismos , Técnicas de Cultivo de Embriones , Expresión Génica , Proteínas de Homeodominio/metabolismo , Factores de Transcripción SOXB1/metabolismo , Oveja Doméstica , TransgenesRESUMEN
Aging is associated with a gradual loss of skeletal muscle mass and an impaired ability of this tissue to compensate for trauma. Studies in rodents and humans have also shown that resident stem cells within muscle have a reduced ability to proliferate and differentiate. In this study muscle stem cells have been isolated from two muscles, the diaphragm (DIA) and the semimembranosus (SM), from young and old pigs. The levels of three micro-RNAs (miRNAs) were measured when cells were in a proliferative phase and after 24 and 72h in differentiation medium. All three miRNAs are abundant in skeletal muscle with miR-1 and miR-206 known to regulate myogenic differentiation and miR-24 is involved in cell cycle regulation. The levels of expression of Pax7 and the myogenic regulatory factors MyoD and myogenin were also measured. There were marked differences in expression of all three miRNAs between the two age groups. Both miR-1 and miR-206 were reduced in the cells from the older animals. In contrast miR-24 expression was significantly higher in cells from older animals under differentiation conditions. There were also significant differences in the relative expression of all three miRNAs between cells from the SM and DIA in both young and old animals. The changes in miRNA expression described in this study that relate to age, may play a role in the impaired differentiation capacity of older muscle stem cells.
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Envejecimiento/metabolismo , MicroARNs/metabolismo , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Animales , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Músculo Esquelético/crecimiento & desarrollo , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/citología , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , PorcinosRESUMEN
During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD. However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development.
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Diferenciación Celular/fisiología , Extremidades/embriología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Modelos Biológicos , Mioblastos/fisiología , Tretinoina/metabolismo , Animales , Embrión de Pollo , Cartilla de ADN/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación in Situ , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/metabolismo , FosforilaciónRESUMEN
miRNAs (microRNAs) are novel post-transcriptional regulators of gene expression. Several miRNAs, expressed exclusively in muscle, play important roles during muscle development, growth and regeneration; other ubiquitously expressed miRNAs are also essential for muscle function. In the present review, we outline the miRNAs involved in embryonic muscle development and those that have been found to be dysregulated in diseases associated with skeletal muscle or are changed during muscle adaptation. miRNAs are promising biomarkers and candidates for potential therapeutic intervention. We discuss the strategies that aim to develop novel therapies through modulating miRNA activity. In time, some of these approaches may become available to treat muscle-associated diseases.
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MicroARNs/genética , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Animales , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Músculo Esquelético/citología , Músculo Esquelético/embriologíaRESUMEN
MicroRNAs are short non-coding RNAs involved in post-transcriptional regulation of multiple messenger RNA targets. The miR-1/miR-206 family is expressed during skeletal muscle differentiation and is an integral component of myogenesis. To better understand miR-1/miR-206 function during myoblast differentiation we identified novel target mRNAs by microarray and characterized their function in C2C12 myoblasts. Candidate targets from the screen were experimentally validated together with target genes that were predicted by three different algorithms. Some targets characterised have a known function in skeletal muscle development and/or differentiation and include Meox2, RARB, Fzd7, MAP4K3, CLCN3 and NFAT5, others are potentially novel regulators of myogenesis, such as the chromatin remodelling factors Smarcd2 and Smarcb1 or the anti-apoptotic protein SH3BGRL3. The expression profiles of confirmed target genes were examined during C2C12 cell myogenesis. We found that inhibition of endogenous miR-1 and miR-206 by antimiRs blocked the downregulation of most targets in differentiating cells, thus indicating that microRNA activity and target interaction is required for muscle differentiation. Finally, we show that sustained expression of validated miR-1 and/or miR-206 targets resulted in increased proliferation and inhibition of C2C12 cell myogenesis. In many cases the expression of genes related to non-muscle cell fates, such as chondrogenesis, was activated. This indicates that the concerted downregulation of multiple microRNA targets is not only crucial to the skeletal muscle differentiation program but also serves to prevent alternative cell fate choices.
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MicroARNs/genética , Mioblastos/fisiología , Animales , Diferenciación Celular/genética , Regulación de la Expresión Génica , Humanos , Ratones , MicroARNs/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Mioblastos/citología , Mioblastos/metabolismo , Células 3T3 NIH , TransfecciónRESUMEN
Commitment of progenitors in the dermomyotome to myoblast fate is the first step in establishing the body musculature. Pax3 is a crucial transcription factor, important for skeletal muscle development and expressed in myogenic progenitors in the dermomyotome of developing somites and in migratory muscle progenitors that populate the limb buds. Down-regulation of Pax3 is essential to ignite the myogenic program, including up-regulation of myogenic regulators, Myf-5 and MyoD. MicroRNAs (miRNAs) confer robustness to developmental timing by posttranscriptional repression of genetic programs that are related to previous developmental stages or to alternative cell fates. Here we demonstrate that the muscle-specific miRNAs miR-1 and miR-206 directly target Pax3. Antagomir-mediated inhibition of miR-1/miR-206 led to delayed myogenic differentiation in developing somites, as shown by transient loss of myogenin expression. This correlated with increased Pax3 and was phenocopied using Pax3-specific target protectors. Loss of myogenin after antagomir injection was rescued by Pax3 knockdown using a splice morpholino, suggesting that miR-1/miR-206 control somite myogenesis primarily through interactions with Pax3. Our studies reveal an important role for miR-1/miR-206 in providing precision to the timing of somite myogenesis. We propose that posttranscriptional control of Pax3 downstream of miR-1/miR-206 is required to stabilize myoblast commitment and subsequent differentiation. Given that mutually exclusive expression of miRNAs and their targets is a prevailing theme in development, our findings suggest that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.
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Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , MicroARNs/metabolismo , Desarrollo de Músculos/fisiología , Factores de Transcripción Paired Box/metabolismo , Animales , Western Blotting , Diferenciación Celular/genética , Células Cultivadas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Sustitución del Gen , Inmunohistoquímica , Hibridación in Situ , Ratones , Desarrollo de Músculos/genética , Miogenina/metabolismo , Oligonucleótidos/administración & dosificación , Oligonucleótidos/genética , Factor de Transcripción PAX3 , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Myogenic regulatory factors of the myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. Here we show that a mutation in the zebrafish myod gene delays and reduces early somitic and pectoral fin myogenesis, reduces miR-206 expression, and leads to a persistent reduction in somite size until at least the independent feeding stage. A mutation in myog, encoding a second MRF, has little obvious phenotype at early stages, but exacerbates the loss of somitic muscle caused by lack of Myod. Mutation of both myod and myf5 ablates all skeletal muscle. Haploinsufficiency of myod leads to reduced embryonic somite muscle bulk. Lack of Myod causes a severe reduction in cranial musculature, ablating most muscles including the protractor pectoralis, a putative cucullaris homologue. This phenotype is accompanied by a severe dysmorphology of the cartilaginous skeleton and failure of maturation of several cranial bones, including the opercle. As myod expression is restricted to myogenic cells, the data show that myogenesis is essential for proper skeletogenesis in the head.
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Huesos/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Haploinsuficiencia/genética , Desarrollo de Músculos/fisiología , Proteína MioD/genética , Cráneo/embriología , Pez Cebra/embriología , Animales , Cartílago/embriología , Haploinsuficiencia/fisiología , Inmunohistoquímica , Hibridación in Situ , Larva/fisiología , Músculo Esquelético/embriología , Mutación/genética , Proteína MioD/metabolismo , Extremidad Superior/embriología , Pez Cebra/genéticaRESUMEN
The detection of microRNAs (miRNAs) in situ presents several technical challenges. Although protocols for mRNA detection by in situ hybridization are well established, the small size of miRNAs makes their localization problematic. To overcome this, digoxygenin-labeled locked nucleic acid oligos have been used. These bind strongly and specifically to miR targets and make the identification of the precise spatiotemporal expression of these molecules possible.
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Hibridación in Situ/métodos , MicroARNs/análisis , Oligonucleótidos , Animales , Embrión de Mamíferos/química , Embrión no Mamífero/química , Músculos/química , Hibridación de Ácido NucleicoRESUMEN
The development and differentiation of vertebrate skeletal muscle provide an important paradigm to understand the inductive signals and molecular events controlling differentiation of specific cell types. Recent findings show that a core transcriptional network, initiated by the myogenic regulatory factors (MRFs; MYF5, MYOD, myogenin and MRF4), is activated by separate populations of cells in embryos in response to various signalling pathways. This review will highlight how cells from multiple distinct starting points can converge on a common set of regulators to generate skeletal muscle.
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Redes Reguladoras de Genes/fisiología , Desarrollo de Músculos/fisiología , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Transducción de Señal/fisiología , Animales , Extremidades/embriología , Cabeza/embriología , Humanos , Modelos Biológicos , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Cuello/embriología , Transducción de Señal/genética , Vertebrados/embriología , Vertebrados/metabolismoRESUMEN
Wnt signaling has been implicated in somite, limb, and branchial arch myogenesis but the mechanisms and roles are not clear. We now show that Wnt signaling via Lef1 acts to regulate the number of premyogenic cells in somites but does not regulate myogenic initiation in the limb bud or maintenance in the first or second branchial arch. We have also analysed the function and regulation of a putative downstream transcriptional target of canonical Wnt signaling, Pitx2. We show that loss-of-function of Pitx2 decreases the number of myogenic cells in the somite, whereas overexpression increases myocyte number particularly in the epaxial region of the myotome. Increased numbers of mitotic cells were observed following overexpression of Pitx2 or an activated form of Lef1, suggesting an effect on cell proliferation. In addition, we show that Pitx2 expression is regulated by canonical Wnt signaling in the epaxial somite and second branchial arch, but not in the limb or the first branchial arch. These results suggest that Wnt/Lef1 signaling regulates epaxial myogenesis via Pitx2 but that this link is uncoupled in other regions of the body, emphasizing the unique molecular networks that control the development of various muscles in vertebrates.
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Proteínas de Homeodominio/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Desarrollo de Músculos/fisiología , Transducción de Señal , Somitos/metabolismo , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , Animales , Región Branquial/citología , Región Branquial/embriología , Región Branquial/metabolismo , Recuento de Células , Diferenciación Celular , Proliferación Celular , Pollos , Extremidades/embriología , Marcación de Gen , Ratones , Unión Proteica , Somitos/citología , Somitos/embriología , Proteína del Homeodomínio PITX2RESUMEN
High throughput Solexa sequencing technology was applied to identify microRNAs in somites of developing chicken embryos. We obtained 651,273 reads, from which 340,415 were mapped to the chicken genome representing 1701 distinct sequences. Eighty-five of these were known microRNAs and 42 novel miRNA candidates were identified. Accumulation of 18 of 42 sequences was confirmed by Northern blot analysis. Ten of the 18 sequences are new variants of known miRNAs and eight short RNAs are novel miRNAs. Six of these eight have not been reported by other deep sequencing projects. One of the six new miRNAs is highly enriched in somite tissue suggesting that deep sequencing of other specific tissues has the potential to identify novel tissue specific miRNAs.
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MicroARNs/genética , Somitos/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Embrión de Pollo , Clonación MolecularRESUMEN
The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.
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MicroARNs/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Factores Reguladores Miogénicos/metabolismo , Tubo Neural/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Diferenciación Celular/fisiología , Línea Celular , Embrión de Pollo , Factores de Crecimiento de Fibroblastos/metabolismo , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
BACKGROUND: Co-ordinated cell movement is a fundamental feature of developing embryos. Massive cell movements occur during vertebrate gastrulation and during the subsequent extension of the embryonic body axis. These are controlled by cell-cell signalling and a number of pathways have been implicated. Here we use long-term video microscopy in chicken embryos to visualize the migration routes and movement behaviour of mesoderm progenitor cells as they emerge from the primitive streak (PS) between HH stages 7 and 10. RESULTS: We observed distinct cell movement behaviours along the length of the streak and determined that this is position dependent with cells responding to environmental cues. The behaviour of cells was altered by exposing embryos or primitive streak explants to cell pellets expressing Wnt3a and Wnt5a, without affecting cell fates, thus implicating these ligands in the regulation of cell movement behaviour. Interestingly younger embryos were not responsive, suggesting that Wnt3a and Wnt5a are specifically involved in the generation of posterior mesoderm, consistent with existing mouse and zebrafish mutants. To investigate which downstream components are involved mutant forms of dishevelled (dsh) and prickle1 (pk1) were electroporated into the primitive streak. These had differential effects on the behaviour of mesoderm progenitors emerging from anterior or posterior regions of the streak, suggesting that multiple Wnt pathways are involved in controlling cell migration during extension of the body axis in amniote embryos. CONCLUSION: We suggest that the distinct behaviours of paraxial and lateral mesoderm precursors are regulated by the opposing actions of Wnt5a and Wnt3a as they leave the primitive streak in neurula stage embryos. Our data suggests that Wnt5a acts via prickle to cause migration of cells from the posterior streak. In the anterior streak, this is antagonised by Wnt3a to generate non-migratory medial mesoderm.
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Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Línea Primitiva/embriología , Proteínas Wnt/genética , Animales , Movimiento Celular , Embrión de Pollo , Células Madre Embrionarias/citología , Mesodermo/citología , Morfogénesis , Línea Primitiva/citología , Transducción de Señal/genética , Factores de TiempoRESUMEN
Chicken homologues of prickle-1 (pk-1) and prickle-2 (pk-2) were isolated to gain insight into the extent of planar cell polarity signaling during avian embryogenesis. Bioinformatics analyses demonstrated homology and showed that pk-1 and pk-2 exhibited conserved synteny with ADAMTS20 and ADAMTS9, GON-related zinc metalloproteases. Expression of pk-1 and pk-2 was established during embryogenesis and early organogenesis, using in situ hybridization and sections of chicken embryos. At early stages, pk-1 was expressed in Hensen's node, primitive streak, ventral neural tube, and foregut. In older embryos, pk-1 transcripts were detected in dorsolateral epithelial somites, dorsomedial lip of dermomyotomes, and differentiating myotomes. Furthermore, pk-1 expression was seen in lateral body folds, limb buds, and ventral metencephalon. pk-2 was expressed in Hensen's node and neural ectoderm at early stages. In older embryos, pk-2 expression was restricted to ventromedial epithelial somites, except in the most recently formed somite pair, and limb bud mesenchyme.