RESUMEN
Skeletal muscle is generated by the successive incorporation of primary (embryonic), secondary (fetal), and tertiary (adult) fibers into muscle. Conditional excision of Pitx2 function by an MCKCre driver resulted in animals with histological and ultrastructural defects in P30 muscles and fibers, respectively. Mutant muscle showed severe reduction in mitochondria and FoxO3-mediated mitophagy. Both oxidative and glycolytic energy metabolism were reduced. Conditional excision was limited to fetal muscle fibers after the G1-G0 transition and resulted in altered MHC, Rac1, MEF2a, and alpha-tubulin expression within these fibers. The onset of excision, monitored by a nuclear reporter gene, was observed as early as E16. Muscle at this stage was already severely malformed, but appeared to recover by P30 by the expansion of adjoining larger fibers. Our studies demonstrate that the homeodomain transcription factor Pitx2 has a postmitotic role in maintaining skeletal muscle integrity and energy homeostasis in fetal muscle fibers.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Femenino , Homeostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/embriología , Miosinas/fisiología , Proteína del Homeodomínio PITX2RESUMEN
BACKGROUND: Heart morphogenesis involves sequential anatomical changes from a linear tube of a single channel peristaltic pump to a four-chamber structure with two channels controlled by one-way valves. The developing heart undergoes continuous remodeling, including septation. RESULTS: Pitx2-null mice are characterized by cardiac septational defects of the atria, ventricles, and outflow tract. Pitx2-null mice also exhibited a short outflow tract, including unseptated conus and deformed endocardial cushions. Cushions were characterized with a jelly-like structure, rather than the distinct membrane-looking leaflets, indicating that endothelial mesenchymal transition was impaired in Pitx2(-/-) embryos. Mesoderm cells from the branchial arches and neural crest cells from the otic region contribute to the development of the endocardial cushions, and both were reduced in number. Members of the Fgf and Bmp families exhibited altered expression levels in the mutants. CONCLUSIONS: We suggest that Pitx2 is involved in the cardiac outflow tract septation by promoting and/or maintaining the number and the remodeling process of the mesoderm progenitor cells. Pitx2 influences the expression of transcription factors and signaling molecules involved in the differentiation of the cushion mesenchyme during heart development.
Asunto(s)
Corazón/embriología , Proteínas de Homeodominio/fisiología , Organogénesis/genética , Factores de Transcripción/fisiología , Animales , Muerte Celular/genética , Linaje de la Célula/genética , Proliferación Celular , Embrión de Mamíferos , Defectos de la Almohadilla Endocárdica/genética , Cojinetes Endocárdicos/embriología , Cojinetes Endocárdicos/metabolismo , Endocardio/citología , Endocardio/embriología , Regulación del Desarrollo de la Expresión Génica , Corazón/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Noqueados , Cresta Neural/embriología , Cresta Neural/metabolismo , Organogénesis/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also plays a key role in tooth development. Ctip2 is highly expressed in the ectodermal components of the developing tooth, including inner and outer enamel epithelia, stellate reticulum, stratum intermedium, and the ameloblast cell lineage. In Ctip2(-/-) mice, tooth morphogenesis appeared to proceed normally through the cap stage but developed multiple defects at the bell stage. Mutant incisors and molars were reduced in size and exhibited hypoplasticity of the stellate reticulum. An ameloblast-like cell population developed ectopically on the lingual aspect of mutant lower incisors, and the morphology, polarization, and adhesion properties of ameloblasts on the labial side of these teeth were severely disrupted. Perturbations of gene expression were also observed in the mandible of Ctip2(-/-) mice: expression of the ameloblast markers amelogenin, ameloblastin, and enamelin was down-regulated, as was expression of Msx2 and epiprofin, transcription factors implicated in the tooth development and ameloblast differentiation. These results suggest that Ctip2 functions as a critical regulator of epithelial cell fate and differentiation during tooth morphogenesis.
Asunto(s)
Ameloblastos/citología , Proteínas de Unión al ADN/fisiología , Odontogénesis , Proteínas Represoras/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Diferenciación Celular , Regulación hacia Abajo/genética , Desarrollo Embrionario , Células Epiteliales/citología , Mandíbula/crecimiento & desarrollo , Ratones , Ratones Noqueados , Diente/crecimiento & desarrollo , Factores de Transcripción/genéticaRESUMEN
The homeodomain transcription factor Pitx2 and the T-box transcription factors are essential for organogenesis. Pitx2 and T-box genes are induced by growth factors and function as transcriptional activators or repressors. Gene expression analyses on abdominal tissue were used to identify seven of the T-box genes of the genome as Pitx2 target genes in the abdomen at embryonic day.10.5. Pitx2 activated Tbx4, Tbx15, and Mga and repressed Tbx1, Tbx2, Tbx5, and Tbx6 expression. As expected, activated genes showed reduced expression patterns, and repressed T-box genes showed increased expression patterns in the abdomen of Pitx2 mutants. Pitx2 occupied chromatin sites near all of these T-box genes. Co-occupancy by coactivators, corepressors, and histone acetylation at these sites was frequently Pitx2-dependent. Genes repressed by Pitx2 generally showed increased histone acetylation and decreased histone deacetylase (HDAC)/corepressor occupancy in Pitx2 mutants. The lower N-CoR, HDAC1, and HDAC3 occupancy observed at multiple sites along Tbx1 chromatin in mutants is consistent with the model that increased histone acetylation and gene expression of Tbx1 may result from a loss of recruitment of corepressors by Pitx2. Genes activated by Pitx2 showed less consistent patterns in chromatin analyses. Reduced H4 acetylation and increased HDAC1/nuclear receptor corepressor (N-CoR) occupancy at some Tbx4 sites were accompanied by increased H3 acetylation and reduced HDAC3 occupancy at the same or other more distal chromatin sites in mutants. Pitx2-dependent occupancy by corepressors resulted in alteration of the acetylation levels of several T-box genes, whereas Pitx2-dependent occupancy by coactivators was more site-localized. These studies will provide the basic scientific underpinning to understand abdominal wall syndromes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasas/metabolismo , Proteínas de Homeodominio/fisiología , Proteínas de Dominio T Box/fisiología , Factores de Transcripción/fisiología , Animales , Cromatina/metabolismo , Femenino , Regulación de la Expresión Génica , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Dominio T Box/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.
Asunto(s)
Diferenciación Celular , Células Precursoras de Linfocitos T/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Diferenciación Celular/inmunología , Linaje de la Célula , Células Cultivadas , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Noqueados , Células Precursoras de Linfocitos T/citología , Unión Proteica , Elementos Reguladores de la Transcripción/inmunología , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Timo/citología , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Activación Transcripcional/inmunología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Flor strains of Saccharomyces cerevisiae form a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Etanol/metabolismo , Saccharomyces cerevisiae/fisiología , Vino/microbiología , Acetatos/metabolismo , Ácido Acético/metabolismo , Glicerol/metabolismo , Ácido Láctico/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismoRESUMEN
Association and relay neurons that are generated in the dorsal spinal cord play essential roles in transducing somatosensory information. During development, these two major neuronal classes are delineated by the expression of the homeodomain transcription factor Lbx1. Lbx1 is expressed in and required for the correct specification of three early dorsal interneuron populations and late-born neurons that form the substantia gelatinosa. In mice lacking Lbx1, cells types that arise in the ventral alar plate acquire more dorsal identities. This results in the loss of dorsal horn association interneurons, excess production of commissural neurons, and disrupted sensory afferent innervation of the dorsal horn. Lbx1, therefore, plays a critical role in the development of sensory pathways in the spinal cord that relay pain and touch.
Asunto(s)
Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Interneuronas/metabolismo , Proteínas Musculares/genética , Células del Asta Posterior/anomalías , Vías Aferentes/anomalías , Vías Aferentes/citología , Vías Aferentes/metabolismo , Animales , Apoptosis/genética , Axones/metabolismo , Axones/ultraestructura , Movimiento Celular/genética , Femenino , Interneuronas/citología , Masculino , Ratones , Ratones Transgénicos , Proteínas Musculares/metabolismo , Mutación/genética , Fibras Nerviosas/metabolismo , Fibras Nerviosas/ultraestructura , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Vías Nerviosas/anomalías , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Nociceptores/anomalías , Nociceptores/citología , Nociceptores/metabolismo , Células del Asta Posterior/citología , Células del Asta Posterior/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Sustancia Gelatinosa/anomalías , Sustancia Gelatinosa/citología , Sustancia Gelatinosa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The morphological events forming the body's musculature are sensitive to genetic and environmental perturbations with high incidence of congenital myopathies, muscular dystrophies and degenerations. Pattern formation generates branching series of states in the genetic regulatory network. Different states of the network specify pre-myogenic progenitor cells in the head and trunk. These progenitors reveal their myogenic nature by the subsequent onset of expression of the master switch gene MyoD and/or Myf5. Once initiated, the myogenic progression that ultimately forms mature muscle appears to be quite similar in head and trunk skeletal muscle. Several genes that are essential in specifying pre-myogenic progenitors in the trunk are known. Pax3, Lbx1, and a number of other homeobox transcription factors are essential in specifying pre-myogenic progenitors in the dermomyotome, from which the epaxial and hypaxial myoblasts, which express myogenic regulatory factors (MRFs), emerge. The proteins involved in specifying pre-myogenic progenitors in the head are just beginning to be discovered and appear to be distinct from those in the trunk. The homeobox gene Pitx2, the T-box gene Tbx1, and the bHLH genes Tcf21 and Msc encode transcription factors that play roles in specifying progenitor cells that will give rise to branchiomeric muscles of the head. Pitx2 is expressed well before the onset of myogenic progression in the first branchial arch (BA) mesodermal core and is essential for the formation of first BA derived muscle groups. Anterior-posterior patterning events that occur during gastrulation appear to initiate the Pitx2 expression domain in the cephalic and BA mesoderm. Pitx2 therefore contributes to the establishment of network states, or kernels, that specify pre-myogenic progenitors for extraocular and mastication muscles. A detailed understanding of the molecular mechanisms that regulate head muscle specification and formation provides the foundation for understanding congenital myopathies. Current technology and mouse model systems help to elucidate the molecular basis on etiology and repair of muscular degenerative diseases.
Asunto(s)
Cabeza/embriología , Desarrollo de Músculos , Músculo Esquelético/embriología , Animales , Proteínas de Homeodominio/metabolismo , Humanos , Músculos Masticadores/embriología , Desarrollo de Músculos/genética , Músculo Esquelético/citología , Músculos Oculomotores/embriología , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Late-stage Pitx2(+/LacZ) mouse embryos stained with x-gal appeared to have blue muscles, suggesting that Pitx2 expression specifically marks some phase of the myogenic progression or muscle anlagen formation. Detailed temporal and spatial analyses were undertaken to determine the extent and onset of Pitx2 expression in muscle. Pitx2 was specifically expressed in the vast majority of muscles of the head and trunk in late embryos and adults. Early Pitx2 expression in the cephalic mesoderm, first branchial arch and somatopleure preceded specification of head muscle. In contrast, Pitx2 expression appeared to follow muscle specification events in the trunk. However, Pitx2 expression was rapidly upregulated in these myogenic structures by E10.5. Upregulation correlated tightly with the apposition of a non-myogenic, Pitx2-expressing, cell cluster lateral to the dermomyotome. This cluster first appeared at the forelimb level at E10.25, gradually elongated in the posterior direction, appeared to aggregate from delaminated cells emanating from the ventrally located somatopleure, and was named the dorsal somatopleure. Immunohistochemistry on appendicular sections after E10.5 demonstrated that Pitx2 neatly marked the areas of muscle anlagen, that Pax3, Lbx1, and the muscle regulatory factors (MRFs) stained only subsets of Pitx2(+) cells within these areas, and that virtually all Pitx2(+) cells in these areas express at least one of these known myogenic markers. Taken together, the results demonstrate that, within muscle anlagen, Pitx2 marks the muscle lineage more completely that any of the known markers, and are consistent with a role for Pitx2 in muscle anlagen formation or maintenance.
Asunto(s)
Expresión Génica , Proteínas de Homeodominio/genética , Desarrollo de Músculos/genética , Músculos/embriología , Factores de Transcripción/genética , Animales , Extremidades/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/fisiología , Masculino , Desarrollo Maxilofacial , Ratones , Ratones Endogámicos C57BL , Músculos/metabolismo , Cráneo/embriología , Somitos/metabolismo , Factores de Transcripción/fisiología , Regulación hacia Arriba , Proteína del Homeodomínio PITX2RESUMEN
BACKGROUND: Genome-wide mapping reveals chromatin landscapes unique to cell states. Histone marks of regulatory genes involved in cell specification and organ development provide a powerful tool to map regulatory sequences. H3K4me3 marks promoter regions; H3K27me3 marks repressed regions, and Pol II presence indicates active transcription. The presence of both H3K4me3 and H3K27me3 characterize poised sequences, a common characteristic of genes involved in pattern formation during organogenesis. RESULTS: We used genome-wide profiling for H3K27me3, H3K4me3, and Pol II to map chromatin states in mouse embryonic day 12 forelimbs in wild type (control) and Pitx2-null mutant mice. We compared these data with previous gene expression studies from forelimb Lbx1+ migratory myoblasts and correlated Pitx2-dependent expression profiles and chromatin states. During forelimb development, several lineages including myoblast, osteoblast, neurons, angioblasts etc., require synchronized growth to form a functional limb. We identified 125 genes in the developing forelimb that are Pitx2-dependent. Genes involved in muscle specification and cytoskeleton architecture were positively regulated, while genes involved in axonal path finding were poised. CONCLUSION: Our results have established histone modification profiles as a useful tool for identifying gene regulatory states in muscle development, and identified the role of Pitx2 in extending the time of myoblast progression, promoting formation of sarcomeric structures, and suppressing attachment of neuronal axons.
RESUMEN
Phenotypic screening enables the discovery of new drug leads with novel targets. ES cells differentiate into different lineages by successively making use of different subsets of the genome's possible macromolecular interactions. If a compound effectively targets just one of these interactions, it derails the developmental pathway to produce a phenotypical change. The OTRADI microsource spectrum library of 2000 approved drug components, natural products, and bioactive components was screened for compounds that can induce phenotypic changes in ES cell cultures at 10 µM after 3 days. Twenty-one compounds that induced specific morphologies also induced unique changes to an expression profile of a dozen markers of early embryonic development, indicating that each compound has derailed the molecular developmental process in a characteristic way. Phenotypic screens conducted with ES cultures differentiating along different lineages can be used to efficiently prescreen compounds able to regulate cell differentiation lineage.
Asunto(s)
Factores Biológicos/farmacología , Productos Biológicos/farmacología , Diferenciación Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Células Madre Embrionarias de Ratones/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Biomarcadores/metabolismo , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , FenotipoRESUMEN
BACKGROUND: Cell types are defined at the molecular level during embryogenesis by a process called pattern formation and created by the selective utilization of combinations of sequence specific transcription factors. Developmental programs define the sets of genes that are available to each particular cell type, and real-time biochemical signaling interactions define the extent to which these sets are used at any given time and place. Gene expression is regulated through the integrated action of many cis-regulatory elements, including core promoters, enhancers, silencers, and insulators. The chromatin state in developing body parts provides a code to cellular populations that direct their cell fates. Chromatin profiling has been a method of choice for mapping regulatory sequences in cells that go through developmental transitions. RESULTS: We used antibodies against histone H3 lysine 4 trimethylations (H3K4me3) a modification associated with promoters and open/active chromatin, histone H3 lysine 27 trimethylations (H3K27me3) associated with Polycomb-repressed regions and RNA polymerase II (Pol2) associated with transcriptional initiation to identify the chromatin state signature of the mouse forelimb during mid-gestation, at embryonic day 12 (E12). The families of genes marked included those related to transcriptional regulation and embryogenesis. One third of the marked genes were transcriptionally active while only a small fraction were bivalent marked. Sequence specific transcription factors that were activated were involved in cell specification including bone and muscle formation. CONCLUSION: Our results demonstrate that embryonic limb cells do not exhibit the plasticity of the ES cells but are rather programmed for a finer tuning for cell lineage specification.
RESUMEN
The ventrolateral dermomyotome gives rise to all muscles of the limbs through the delamination and migration of cells into the limb buds. These cells proliferate and form myoblasts, withdraw from the cell cycle and become terminally differentiated. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Gene expression arrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1(EGFP), which resulted from the loss of Pitx2. Target genes of Pitx2 were clustered using the "David Bioinformatics Functional Annotation Tool" to bin genes according to enrichment of gene ontology keywords. This provided a way to both narrow the target genes and identify potential gene families regulated by Pitx2. Representative target genes in the most enriched bins were analyzed for the presence and evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the -20kb, intronic, and coding regions of the genes. Fifteen Pitx2 target genes were selected based on the above analysis and were identified as having functions involving cytoskeleton organization, tissue specification, and transcription factors. Data from these studies suggest that Pitx2 acts to regulate cell motility and expression of muscle specific genes in the muscle precursors during forelimb muscle development. This work provides a framework to develop the gene network leading to skeletal muscle development, growth and regeneration.
Asunto(s)
Redes Reguladoras de Genes , Modelos Genéticos , Desarrollo de Músculos/genética , Animales , Sitios de Unión/genética , Miembro Anterior/embriología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Cells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1(EGFP/+), which resulted from the loss of Pitx2. Very few genes showed changes in expression. Many small-fold, yet significant, changes were observed in genes encoding cytoskeletal and adhesion proteins which play a role in cell motility. Myogenic cells from genetically-tagged mice were cultured and subjected to live cell-tracking analysis using time-lapse imaging. Myogenic cells lacking Pitx2 were smaller, more symmetrical, and had more actin bundling. They also migrated about half of the total distance and velocity. Decreased motility may prevent myogenic cells from filling pre-patterned regions of the limb bud in a timely manner. Altered shape may prevent proper assembly of higher-order fibers within anlagen. Pitx2 therefore appears to regulate muscle anlagen development by appropriately balancing expression of cytoskeletal and adhesion molecules.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Esbozos de los Miembros/metabolismo , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Animales , Diferenciación Celular , Movimiento Celular , Polaridad Celular , Tamaño de la Célula , Embrión de Mamíferos , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/metabolismo , Esbozos de los Miembros/citología , Ratones , Ratones Transgénicos , Músculo Esquelético/citología , Imagen de Lapso de Tiempo , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene Lbx1 plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of Lbx1 in the embryonic mouse neural tube. Chmp2b was expressed and depended on Lbx1 in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor- and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Dendritas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos ICR , Neuronas Motoras/metabolismo , Fibras Nerviosas/metabolismo , Proteínas del Tejido Nervioso/genética , Tubo Neural/citología , Tubo Neural/embriología , Tubo Neural/metabolismoRESUMEN
Sequence specific transcription factors (SSTFs) combinatorially define cell types during development by forming recursively linked network kernels. Pitx2 expression begins during gastrulation, together with Hox genes, and becomes localized to the abdominal lateral plate mesoderm (LPM) before the onset of myogenesis in somites. The somatopleure of Pitx2 null embryos begins to grow abnormally outward before muscle regulatory factors (MRFs) or Pitx2 begin expression in the dermomyotome/myotome. Abdominal somites become deformed and stunted as they elongate into the mutant body wall, but maintain normal MRF expression domains. Subsequent loss of abdominal muscles is therefore not due to defects in specification, determination, or commitment of the myogenic lineage. Microarray analysis was used to identify SSTF families whose expression levels change in E10.5 interlimb body wall biopsies. All Hox9-11 paralogs had lower RNA levels in mutants, whereas genes expressed selectively in the hypaxial dermomyotome/myotome and sclerotome had higher RNA levels in mutants. In situ hybridization analyses indicate that Hox gene expression was reduced in parts of the LPM and intermediate mesoderm of mutants. Chromatin occupancy studies conducted on E10.5 interlimb body wall biopsies showed that Pitx2 protein occupied chromatin sites containing conserved bicoid core motifs in the vicinity of Hox 9-11 and MRF genes. Taken together, the data indicate that Pitx2 protein in LPM cells acts, presumably in combination with other SSTFs, to repress gene expression, that are normally expressed in physically adjoining cell types. Pitx2 thereby prevents cells in the interlimb LPM from adopting the stable network kernels that define sclerotomal, dermomyotomal, or myotomal mesenchymal cell types. This mechanism may be viewed either as lineage restriction or specification.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Homeodominio/genética , Mesodermo , Músculo Esquelético/patología , Mutación , Factores de Transcripción/genética , Animales , Genes Homeobox , Hibridación in Situ , Ratones , Proteína del Homeodomínio PITX2RESUMEN
Sequence specific transcription factors are essential for pattern formation and cell differentiation processes in mammals. The formation of the abdominal wall depends on a flawless merge of several developmental fields in time and space. The absence of Pitx2 leads to an open abdominal wall in mice, while mutations in humans result in umbilical defects, suggesting that a single homeobox transcription factor coordinates the formation and patterning of this anatomical structure. Gene expression analysis from abdominal tissue including the abdominal wall after removal of the major organs, of wild type, Pitx2 heterozygote and mutant mice, at embryonic day 10.5, identified 275 genes with altered expression levels. Pitx2 target genes were clustered using the "David Bioinformatics Functional Annotation Tool" web application, which bins genes according to gene ontology (GO) key word enrichment. This provided a way to both narrow the target gene list and to start identifying potential gene families regulated by Pitx2. Target genes in the most enriched bins were further analyzed for the presence and the evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the -20 kb, intronic and coding gene sequences. Twenty Pitx2 target genes that passed all the above criteria were classified as genes involved in cell transport and growth. Data from these studies suggest that Pitx2 acts as an inhibitor of protein transport and cell apoptosis contributing to the open body wall phenotype. This work provides the framework to which the developmental network leading to abdominal wall syndromes can be built.
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Pared Abdominal/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Pared Abdominal/anomalías , Animales , Sitios de Unión , Secuencia Conservada , Femenino , Ratones , Modelos Genéticos , Morfogénesis/genética , Familia de Multigenes , Fenotipo , Proteína del Homeodomínio PITX2RESUMEN
BACKGROUND: Genetic regulatory networks of sequence specific transcription factors underlie pattern formation in multicellular organisms. Deciphering and representing the mammalian networks is a central problem in development, neurobiology, and regenerative medicine. Transcriptional networks specify intermingled embryonic cell populations during pattern formation in the vertebrate neural tube. Each embryonic population gives rise to a distinct type of adult neuron. The homeodomain transcription factor Lbx1 is expressed in five such populations and loss of Lbx1 leads to distinct respecifications in each of the five populations. METHODOLOGY/PRINCIPAL FINDINGS: We have purified normal and respecified pools of these five populations from embryos bearing one or two copies of the null Lbx1(GFP) allele, respectively. Microarrays were used to show that expression levels of 8% of all transcription factor genes were altered in the respecified pool. These transcription factor genes constitute 20-30% of the active nodes of the transcriptional network that governs neural tube patterning. Half of the 141 regulated nodes were located in the top 150 clusters of ultraconserved non-coding regions. Generally, Lbx1 repressed genes that have expression patterns outside of the Lbx1-expressing domain and activated genes that have expression patterns inside the Lbx1-expressing domain. CONCLUSIONS/SIGNIFICANCE: Constraining epistasis analysis of Lbx1 to only those cells that normally express Lbx1 allowed unprecedented sensitivity in identifying Lbx1 network interactions and allowed the interactions to be assigned to a specific set of cell populations. We call this method ANCEA, or active node constrained epistasis analysis, and think that it will be generally useful in discovering and assigning network interactions to specific populations. We discuss how ANCEA, coupled with population partitioning analysis, can greatly facilitate the systematic dissection of transcriptional networks that underlie mammalian patterning.
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Tipificación del Cuerpo/genética , Redes Reguladoras de Genes , Modelos Genéticos , Animales , Genes Homeobox , Humanos , Mamíferos/genética , Factores de TranscripciónRESUMEN
Pitx2 expression is observed during all states of the myogenic progression in embryonic muscle anlagen and persists in adult muscle. Pitx2 mutant mice form all but a few muscle anlagen. Loss or degeneration in muscle anlagen could generally be attributed to the loss of a muscle attachment site induced by some other aspect of the Pitx2 phenotype. Muscles derived from the first branchial arch were absent, whereas muscles derived from the second branchial arch were merely distorted in Pitx2 mutants at midgestation. Pitx2 was expressed well before, and was required for, initiation of the myogenic progression in the first, but not second, branchial arch mesoderm. Pitx2 was also required for expression of premyoblast specification markers Tbx1, Tcf21, and Msc in the first, but not second, branchial arch. First, but not second, arch mesoderm of Pitx2 mutants failed to enlarge after embryonic day 9.5, well before the onset of the myogenic progression. Thus, Pitx2 contributes to specification of first, but not second, arch mesoderm. The jaw of Pitx2 mutants was vestigial by midgestation, but significant size reductions were observed as early as embryonic day 10.5. The diminutive first branchial arch of mutants could not be explained by loss of mesoderm alone, suggesting that Pitx2 contributes to the earliest specification of jaw itself.
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Región Branquial/embriología , Músculos Faciales/embriología , Proteínas de Homeodominio/fisiología , Desarrollo de Músculos , Músculo Esquelético/embriología , Factores de Transcripción/fisiología , Animales , Región Branquial/fisiología , Músculos Faciales/fisiología , Genes Homeobox , Proteínas de Homeodominio/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos ICR , Modelos Biológicos , Músculo Esquelético/fisiología , Mutación , Factores de Transcripción/genética , Proteína del Homeodomínio PITX2RESUMEN
A transcriptional network governs patterning in the developing spinal cord. As the developmental program runs, the levels of sequence-specific DNA-binding transcription factors (SSTFs) in each progenitor cell type change to ultimately define a set of postmitotic populations with combinatorial codes of expressed SSTFs. A network description of the neural tube (NT) transcriptional patterning process will require definition of nodes (SSTFs and target enhancers) and edges (interactions between nodes). There are 1,600 SSTF nodes in a given mammalian genome. To limit the complexity of a network description, it will be useful to discriminate between active and passive SSTF nodes. We define active SSTF nodes as those that are differentially expressed within the system. Our system, the developing NT, was partitioned into two pools of genetically defined populations by using flow sorting. Microarray comparisons across the partition led to an estimate of 500-700 active SSTF nodes in the transcriptional network of the developing NT. These included most of the 66 known SSTFs assembled from review articles and recent reports on NT patterning. Empirical cutoffs based on the performance of knowns were used to identify 188 further active SSTFs nodes that performed similarly. The general utility and limitations of the population-partitioning paradigm are discussed.