RESUMO
Functional micropeptides can be concealed within RNAs that appear to be noncoding. We discovered a conserved micropeptide, which we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long noncoding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca(2+) uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca(2+) handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as noncoding.
Assuntos
Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , RNA Longo não Codificante/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/química , Músculo Esquelético/citologia , Miocárdio/metabolismo , Estrutura Secundária de Proteína , Proteolipídeos/metabolismo , RNA Longo não Codificante/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Alinhamento de SequênciaRESUMO
Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.
Assuntos
Regulação da Expressão Gênica/genética , Coração/fisiologia , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Fatores Etários , Animais , Modelos Animais de Doenças , Deleção de Genes , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Fator de Resposta Sérica/genética , Transativadores/genética , Ativação TranscricionalRESUMO
Efficient repair of skeletal muscle relies upon the precise coordination of cells between the satellite cell niche and innate immune cells that are recruited to the site of injury. The expression of pro-inflammatory cytokines and chemokines such as TNFα, IFNγ, CXCL1, and CCL2, by muscle and tissue resident immune cells recruits neutrophils and M1 macrophages to the injury and activates satellite cells. These signal cascades lead to highly integrated temporal and spatial control of muscle repair. Despite the therapeutic potential of these factors for improving tissue regeneration after traumatic and chronic injuries, their transcriptional regulation is not well understood. The transcription factor Mohawk (Mkx) functions as a repressor of myogenic differentiation and regulates fiber type specification. Embryonically, Mkx is expressed in all progenitor cells of the musculoskeletal system and is expressed in human and mouse myeloid lineage cells. An analysis of mice deficient for Mkx revealed a delay in postnatal muscle repair characterized by impaired clearance of necrotic fibers and smaller newly regenerated fibers. Further, there was a delay in the expression of inflammatory signals such as Ccl2, Ifnγ, and Tgfß. This was coupled with impaired recruitment of pro-inflammatory macrophages to the site of muscle damage. These studies demonstrate that Mkx plays a critical role in adult skeletal muscle repair that is mediated through the initial activation of the inflammatory response.
Assuntos
Inflamação , Músculo Esquelético , Animais , Humanos , Camundongos , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/imunologia , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Regeneração , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Long noncoding RNAs (LncRNAs) have emerged as a diverse class of functional molecules that contribute to nearly every facet of mammalian cardiac development and disease. Recent examples show that lncRNAs can be important co-regulators of cardiac patterning and morphogenesis and modulators of the pathogenic signaling that drives heart disease. The flexibility and chemical nature of RNA allows lncRNAs to utilize diverse mechanisms, mediating their effects through their sequence, structure, and molecular interactions with DNA, protein, and other RNAs. In vivo, i.e., animal, studies of individual lncRNAs highlight their ability to balance conserved cardiac gene expression networks, serve as specific and early biomarkers, and indicate their promise as useful therapeutic targets to treat human heart disease. Here, we review recent functionally characterized lncRNAs in cardiac biology and pathology and provide a perspective on emerging approaches to decipher the role of lncRNAs in the heart.
Assuntos
Cardiopatias , RNA Longo não Codificante , Animais , Redes Reguladoras de Genes , Coração , Cardiopatias/genética , Mamíferos/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality. Hand2 expression is tightly regulated by upstream enhancers that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome, the Uph-Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromatina/genética , Coração/embriologia , Organogênese/genética , RNA Longo não Codificante/genética , Transcrição Gênica/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Perda do Embrião/genética , Elementos Facilitadores Genéticos/genética , Técnicas de Inativação de Genes , Cardiopatias Congênitas/genética , Ventrículos do Coração/anormalidades , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , RNA Longo não Codificante/biossíntese , Elongação da Transcrição GenéticaRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.
Assuntos
Envelhecimento/genética , Sistemas CRISPR-Cas/genética , Deleção de Genes , Miocárdio/metabolismo , Animais , Cardiomegalia/complicações , Cardiomegalia/patologia , Separação Celular , Dependovirus/metabolismo , Técnicas de Silenciamento de Genes , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/patologia , Camundongos Transgênicos , Modelos Animais , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Especificidade de Órgãos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Innervation of skeletal muscle by motor neurons occurs through the neuromuscular junction, a cholinergic synapse essential for normal muscle growth and function. Defects in nerve-muscle signaling cause a variety of neuromuscular disorders with features of ataxia, paralysis, skeletal muscle wasting, and degeneration. Here we show that the nuclear zinc finger protein ZFP106 is highly enriched in skeletal muscle and is required for postnatal maintenance of myofiber innervation by motor neurons. Genetic disruption of Zfp106 in mice results in progressive ataxia and hindlimb paralysis associated with motor neuron degeneration, severe muscle wasting, and premature death by 6 mo of age. We show that ZFP106 is an RNA-binding protein that associates with the core splicing factor RNA binding motif protein 39 (RBM39) and localizes to nuclear speckles adjacent to spliceosomes. Upon inhibition of pre-mRNA synthesis, ZFP106 translocates with other splicing factors to the nucleolus. Muscle and spinal cord of Zfp106 knockout mice displayed a gene expression signature of neuromuscular degeneration. Strikingly, altered splicing of the Nogo (Rtn4) gene locus in skeletal muscle of Zfp106 knockout mice resulted in ectopic expression of NOGO-A, the neurite outgrowth factor that inhibits nerve regeneration and destabilizes neuromuscular junctions. These findings reveal a central role for Zfp106 in the maintenance of nerve-muscle signaling, and highlight the involvement of aberrant RNA processing in neuromuscular disease pathogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Síndrome de Emaciação/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Células COS , Chlorocebus aethiops , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Denervação Muscular , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Síndrome de Emaciação/metabolismoRESUMO
Excitation-contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca(2+) transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (Stac3) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca(2+) release from the SR of cultured myotubes from Stac3 mutant mice could be restored by application of 4-chloro-m-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca(2+) store, and ryanodine receptors are functional in Stac3 mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.
Assuntos
Cálcio/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Potenciais de Ação/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Northern Blotting , Western Blotting , Primers do DNA/genética , Eletroporação , Genótipo , Hibridização In Situ , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , beta-GalactosidaseRESUMO
BACKGROUND: Dynamic alterations in cell shape, migration, and adhesion play a central role in tissue morphogenesis during embryonic development and congenital disease. The mesenchymal-to-epithelial transition that occurs during vertebrate somitogenesis is required for proper patterning of the axial musculoskeletal system. Somitic MET is initiated in the presomitic mesoderm by PARAXIS-dependent changes in cell adhesion, cell polarity, and the composition of the extracellular matrix. However, the target genes downstream of the transcription factor PARAXIS remain poorly described. RESULTS: A genome-wide comparison of gene expression in the anterior presomitic mesoderm and newly formed somites of Paraxis(-/-) embryos resulted in a set of deregulated genes enriched for factors associated with extracellular matrix and cytoskeletal organization and cell-cell and cell-ECM adhesion. The greatest change in expression was seen in fibroblast activation protein alpha (Fap), encoding a dipeptidyl peptidase capable of increasing fibronectin and collagen fiber organization in extracellular matrix. Further, downstream genes in the Wnt and Notch signaling pathways were downregulated, predicting that PARAXIS participates in positive feedback loops in both pathways. CONCLUSIONS: These data demonstrate that PARAXIS initiates and stabilizes somite epithelialization by integrating signals from multiple pathways to control the reorganization of the ECM, cytoskeleton, and adhesion junctions during MET.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Somitos/citologia , Somitos/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endopeptidases , Transição Epitelial-Mesenquimal/genética , Técnica Indireta de Fluorescência para Anticorpo , Gelatinases/genética , Gelatinases/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.
Assuntos
DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta/fisiologia , Motivos de Aminoácidos , Animais , Diferenciação Celular/fisiologia , DNA/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Músculo Esquelético/citologia , Células NIH 3T3 , Ligação Proteica , Proteínas Repressoras/genética , Fatores de Transcrição SOXD/biossíntese , Fatores de Transcrição SOXD/genéticaRESUMO
Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm-egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.
Assuntos
Proteínas de Transporte/metabolismo , Fatores Quimiotáticos/metabolismo , Quimiotaxia/fisiologia , Proteínas do Ovo/metabolismo , Glicoproteínas de Membrana/metabolismo , Espermatozoides/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Transporte/genética , Fatores Quimiotáticos/genética , Proteínas do Ovo/genética , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
Micropeptides function as master regulators of calcium-dependent signaling in muscle. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), the membrane pump that promotes muscle relaxation by taking up Ca2+ into the sarcoplasmic reticulum, is directly inhibited by three muscle-specific micropeptides: myoregulin (MLN), phospholamban (PLN), and sarcolipin (SLN). The widespread and essential function of SERCA across diverse cell types has raised questions as to how SERCA is regulated in cells that lack MLN, PLN, and SLN. We identified two transmembrane micropeptides, endoregulin (ELN) and another-regulin (ALN), that share key amino acids with their muscle-specific counterparts and function as direct inhibitors of SERCA pump activity. The distribution of transcripts encoding ELN and ALN mirrored that of SERCA isoform-encoding transcripts in nonmuscle cell types. Our findings identify additional members of the SERCA-inhibitory micropeptide family, revealing a conserved mechanism for the control of intracellular Ca2+ dynamics in both muscle and nonmuscle cell types.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Animais , Células COS , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/farmacologia , Chlorocebus aethiops , Masculino , Camundongos , Proteínas Musculares/química , Proteínas Musculares/farmacologia , Proteolipídeos/química , Proteolipídeos/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
Muscle contraction depends on release of Ca(2+) from the sarcoplasmic reticulum (SR) and reuptake by the Ca(2+)adenosine triphosphatase SERCA. We discovered a putative muscle-specific long noncoding RNA that encodes a peptide of 34 amino acids and that we named dwarf open reading frame (DWORF). DWORF localizes to the SR membrane, where it enhances SERCA activity by displacing the SERCA inhibitors, phospholamban, sarcolipin, and myoregulin. In mice, overexpression of DWORF in cardiomyocytes increases peak Ca(2+) transient amplitude and SR Ca(2+) load while reducing the time constant of cytosolic Ca(2+) decay during each cycle of contraction-relaxation. Conversely, slow skeletal muscle lacking DWORF exhibits delayed Ca(2+) clearance and relaxation and reduced SERCA activity. DWORF is the only endogenous peptide known to activate the SERCA pump by physical interaction and provides a means for enhancing muscle contractility.
Assuntos
Contração Muscular , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , Peptídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Musculares/metabolismo , Contração Miocárdica , Peptídeos/genética , Proteolipídeos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Retículo Sarcoplasmático/metabolismo , Transcrição GênicaRESUMO
Mohawk is an atypical homeobox gene expressed in embryonic progenitor cells of skeletal muscle, tendon, and cartilage. We demonstrate that Mohawk functions as a transcriptional repressor capable of blocking the myogenic conversion of 10T1/2 fibroblasts. The repressor activity is located in three small, evolutionarily conserved domains (MRD1-3) in the carboxy-terminal half of the protein. Point mutation analysis revealed six residues in MRD1 are sufficient for repressor function. The carboxy-terminal half of Mohawk is able to recruit components of the Sin3A/HDAC co-repressor complex (Sin3A, Hdac1, and Sap18) and a subset of Polymerase II general transcription factors (Tbp, TFIIA1 and TFIIB). Furthermore, Sap18, a protein that bridges the Sin3A/HDAC complex to DNA-bound transcription factors, is co-immunoprecipitated by MRD1. These data predict that Mohawk can repress transcription through recruitment of the Sin3A/HDAC co-repressor complex, and as a result, repress target genes required for the differentiation of cells to the myogenic lineage.
Assuntos
Regulação para Baixo , Histona Desacetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular , Linhagem Celular , Proteínas de Homeodomínio/genética , Camundongos , Modelos Moleculares , Mioblastos/citologia , Mioblastos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Complexo Correpressor Histona Desacetilase e Sin3 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 h period consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 h, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3a(tm1Amc) mutants but not in Dll3(pu) mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro.
Assuntos
Relógios Biológicos/fisiologia , Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/fisiologia , Somitos/fisiologia , Animais , Células Cultivadas , Humanos , Hibridização In Situ , Camundongos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Homeodomain-containing proteins comprise a superfamily of transcription factors that participate in the regulation of almost all aspects of embryonic development. Here, we describe the mouse embryonic expression pattern of Mohawk, a new member of the TALE superclass of atypical homeobox genes that is most-closely related to the Iroquois class. During mouse development, Mohawk was transcribed in cell lineages derived from the somites. As early as embryonic day 9.0, Mohawk was expressed in an anterior to posterior gradient in the dorsomedial and ventrolateral lips of the dermomyotome of the somites that normally give rise to skeletal muscle. Mohawk transcription in the dorsomedial region required the expression of the transcription factor paraxis. As somites matured, Mohawk transcription was observed in the tendon-specific syndetome and the sclerotome-derived condensing mesenchyme that prefigures the proximal ribs and vertebral bodies. In the limbs, Mohawk was expressed in a pattern consistent with the developing tendons that form along the dorsal and ventral aspect of the phalanges. Finally, Mohawk was detectable in the tips of the ureteric buds in the metanephric kidneys and the testis cords of the male gonad. Together, these observations suggest that Mohawk is an important regulator of vertebrate development.