RESUMEN

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.

Asunto(s)

Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Esbozos de los Miembros/embriología , Animales , Embrión de Pollo , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM , Esbozos de los Miembros/citología , Células Madre/fisiología , Factores de Transcripción/metabolismo

RESUMEN

In vertebrates, skeletal muscles of the body arise from cells of somitic origin. Recently, somite culture experiments have identified a set of genes, including Pax3, Six1, Eya2, and Dach2, that appear to play an important role in early myogenesis during somite development (Heanue et al. [1999] Genes Dev. 13:3231-3243). In somite culture Pax3, Six1, Eya2, and Dach2 not only function to activate myogenesis, but they form a complex network regulating each other's transcription. We sought to examine whether this putative Pax3/Six1/Eya2/Dach2 network of regulation actually functions in vivo. In particular, we tested whether Pax3 and Dach2 participate in a positive regulatory feedback loop in vivo as they do in culture. To test in vivo Pax3/Dach2 interregulation, we took advantage of the known dependence of both factors on ectodermal signals. Somites isolated from the overlying ectoderm lose expression of Pax3 and Dach2. Therefore, we attempted to rescue Pax3 or Dach2 expression in somites isolated from the ectoderm by retroviral misexpression of the complementary factor. Indeed misexpression of Pax3 or Dach2 resulted in rescue of Dach2 or Pax3, respectively. These rescue experiments demonstrate that Pax3 and Dach2 positively regulate each other's expression in vivo and support the validity of the Pax3/Six1/Eya2/Dach 2 network in regulating myogenesis.

Asunto(s)

Proteínas de Unión al ADN/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/biosíntesis , Somitos/metabolismo , Factores de Transcripción , Animales , Embrión de Pollo , Proteínas de Unión al ADN/genética , Ectodermo/metabolismo , Hibridación in Situ , Modelos Biológicos , Proteínas Nucleares/genética , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Transducción de Señal , Transcripción Genética

RESUMEN

Little is known about the genesis and patterning of tendons and other connective tissues, mostly owing to the absence of early markers. We have found that Scleraxis, a bHLH transcription factor, is a highly specific marker for all the connective tissues that mediate attachment of muscle to bone in chick and mouse, including the limb tendons, and show that early scleraxis expression marks the progenitor cell populations for these tissues. In the early limb bud, the tendon progenitor population is found in the superficial proximomedial mesenchyme. Using the scleraxis gene as a marker we show that these progenitors are induced by ectodermal signals and restricted by bone morphogenetic protein (BMP) signaling within the mesenchyme. Application of Noggin protein antagonizes this endogenous BMP activity and induces ectopic scleraxis expression. However, the presence of excess tendon progenitors does not lead to the production of additional or longer tendons, indicating that additional signals are required for the final formation of a tendon. Finally, we show that the endogenous expression of noggin within the condensing digit cartilage contributes to the induction of distal tendons.

Asunto(s)

Tendones/citología , Tendones/embriología , Factores de Transcripción/metabolismo , Animales , Proteínas Aviares , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Biomarcadores , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras , Embrión de Pollo , Tejido Conectivo/embriología , Tejido Conectivo/metabolismo , Ectodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Esbozos de los Miembros/citología , Esbozos de los Miembros/metabolismo , Proteínas/metabolismo , Transducción de Señal , Células Madre/metabolismo , Tendones/metabolismo , Factores de Transcripción/genética

Asunto(s)

Relojes Biológicos , Factores de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Factor 8 de Crecimiento de Fibroblastos , Ratones , Modelos Biológicos , Transducción de Señal

RESUMEN

A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.

Asunto(s)

Tipificación del Cuerpo , Diferenciación Celular , Inducción Embrionaria , Osteoblastos/citología , Transactivadores/farmacología , Transactivadores/fisiología , Fosfatasa Alcalina/biosíntesis , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , División Celular , Línea Celular , Embrión de Pollo , Condrocitos/citología , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Técnicas de Cultivo de Órganos , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular , Proteínas Recombinantes/farmacología , Transducción de Señal , Transactivadores/química , Alas de Animales/embriología

RESUMEN

We have isolated a new chicken gene that is a member of the cysteine-rich secreted protein family (CRISP). The CRISP family is composed of over 70 members that are found in many phyla of organisms, including: vertebrates, plants, fungi, yeast, and insects. Here we describe the cloning of a novel member of this family, SugarCrisp, and its expression pattern throughout chicken embryogenesis. We also describe its utility as a marker of thyroid and pancreatic mesoderm in the developing chicken embryo and its expression within the human and mouse in glandular tissue.

Asunto(s)

Proteínas Aviares , Cisteína/química , Mesodermo/metabolismo , Páncreas/embriología , Biosíntesis de Proteínas , Proteínas/química , Glándula Tiroides/embriología , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Clonación Molecular , ADN Complementario/metabolismo , Embrión no Mamífero/metabolismo , Humanos , Hibridación in Situ , Pulmón/embriología , Datos de Secuencia Molecular , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular

RESUMEN

The long bones of the vertebrate appendicular skeleton arise from initially continuous condensations of mesenchymal cells that subsequently segment and cavitate to form discrete elements separated by synovial joints. Little is known, however, about the molecular mechanisms of joint formation. We present evidence that Wnt-14 plays a central role in initiating synovial joint formation in the chick limb. Wnt-14 is expressed in joint-forming regions prior to the segmentation of the cartilage elements, and local misexpression of Wnt-14 induces morphological and molecular changes characteristic of the first steps of joint formation. Induction of an ectopic joint-like region by Wnt-14 suppresses the formation of the immediately adjacent endogenous joint, potentially providing insight into the spacing of joints.

Asunto(s)

Desarrollo Óseo , Cápsula Articular/embriología , Cápsula Articular/fisiología , Proteínas/fisiología , Animales , Cartílago/embriología , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Condrocitos/metabolismo , Regulación hacia Abajo , Inmunohistoquímica , Hibridación in Situ , Modelos Biológicos , Datos de Secuencia Molecular , Transducción de Señal , Factores de Tiempo

RESUMEN

Hedgehog (Hh) signaling in vertebrates controls patterning and differentiation of a broad range of tissues during development. The Hh receptor Patched (Ptc) is a critical regulator of signaling, maintaining active repression of the pathway in the absence of stimulation, limiting excess diffusion of ligand, and providing an efficient negative feedback mechanism for fine-tuning the responsiveness of receiving cells. Two distinct Ptc genes have been isolated from several vertebrates. Here, we describe the cloning of a second Ptc gene from chick (Ptc2). We show that Ptc1 and Ptc2 are both upregulated at sites of active Hh signaling but that the expression patterns of these genes only partially overlap, thus providing distinct readouts of Hh pathway stimulation. We also show that chick Ptc2 is expressed in the posterior apical ectodermal ridge (AER) of the limb bud in a pattern similar to Fgf4 and that the induction of Ptc2 within the AER, like that of Fgf4, is mediated via antagonism of BMP signaling. The differential responsiveness of cells to Hh pathway stimulation (as marked by the differential induction of Ptc genes) suggests heterogeneity in the mechanisms by which Hh signals are transduced within different populations of receiving cells.

Asunto(s)

Embrión de Pollo/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Transducción de Señal , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta , Secuencia de Aminoácidos , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Pollo/metabolismo , Clonación Molecular , Ectodermo/metabolismo , Proteínas Hedgehog , Humanos , Hibridación in Situ , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Receptores Patched , Filogenia , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores de Superficie Celular , Homología de Secuencia de Aminoácido , Transactivadores/antagonistas & inhibidores , Transcripción Genética/genética

RESUMEN

The gut organs are all derived from a simple, undifferentiated, linear gut tube. We analyzed the lineage relationships of cells derived from this gut tube in chicken embryos, determining where the progeny of a single cell are located within the gut. We find that daughter cells derived from a single progenitor can populate both the gizzard (chicken stomach) and the small intestine early in development, but that clonally related cells are restricted to a single organ by stage 12. We also find that clonally related cells can populate different mesodermal layers within the radial axis of the gut throughout all of the stages tested in these experiments. Many genes that have organ-specific expression patterns within the gut have been isolated. The onset of these restricted expression patterns correlates with the time that clonal boundaries appear to form, suggesting that these genes might be involved in the establishment of compartment boundaries, which prevent cells on one side of the boundary from intermingling with cells on the other side of the boundary.

Asunto(s)

Sistema Digestivo/embriología , Animales , Embrión de Pollo , Células Clonales , Sistema Digestivo/citología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Mesodermo/citología , Células Madre/citología , Factores de Tiempo

RESUMEN

Gli genes encode a family of zinc finger transcription factors that mediate signaling by Hedgehog proteins. We have cloned the chick Gli3 gene and studied its expression in developing chick limbs. Gli3 expression is highly similar to that of chick Gli2. Gli3 mRNA is evenly distributed in the early limb mesenchyme and subsequently downregulated in the posterior mesenchyme by the polarizing activity of Sonic hedgehog. At later stages, Gli3 is expressed in the distal limb mesenchyme.

Asunto(s)

Proteínas de Unión al ADN/genética , Extremidades/embriología , Proteínas del Tejido Nervioso , Proteínas Represoras , Transactivadores , Factores de Transcripción/genética , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Hibridación in Situ , Factores de Transcripción de Tipo Kruppel , Datos de Secuencia Molecular , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de Zinc , Dedos de Zinc/genética

RESUMEN

Patterning of the gut into morphologically distinct regions results from the appropriate factors being expressed in strict spatial and temporal patterns to assign cells their fates in development. Often, the boundaries of gene expression early in development correspond to delineations between different regions of the adult gut. For example, Bmp4 is expressed throughout the hindgut and midgut, but is not expressed in the early gizzard. Ectopic BMP4 in the gizzard caused a thinning of the muscularis. To understand this phenotype we examined the expression of the receptors transducing BMP signaling during gut development. We find that the BMP receptors are differentially expressed in distinct regions of the chicken embryonic gut. By using constitutively activated versions of the BMP type I receptors, we find that the BMP receptors act similarly to BMP4 in the gizzard when ectopically expressed. We show that the mesodermal thinning seen upon ectopic BMP signaling is due to an increase in apoptosis and a decrease in proliferation within the gizzard mesoderm. The mesodermal thinning is characterized by a disorganization and lack of differentiation of smooth muscle in the gizzard mesoderm. Further, ectopic BMP receptors cause an upregulation of Nkx2.5, the pyloric sphincter marker, similar to that seen with ectopic BMP4. This upregulation of Nkx2.5 is a cell-autonomous event within the mesoderm of the gizzard. We also find that Nkx2.5 is necessary and sufficient for establishing aspects of pyloric sphincter differentiation.

Asunto(s)

Tipificación del Cuerpo/fisiología , Proteínas Morfogenéticas Óseas/fisiología , Sistema Digestivo/embriología , Molleja de las Aves/embriología , Proteínas de Homeodominio/fisiología , Transducción de Señal , Factores de Transcripción , Proteínas de Xenopus , Animales , Apoptosis , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , División Celular , Embrión de Pollo , Perfilación de la Expresión Génica , Molleja de las Aves/metabolismo , Molleja de las Aves/fisiología , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mesodermo/fisiología , Píloro/embriología

RESUMEN

Several advances have been made in our understanding of the control of the growth and patterning of embryonic limbs. Development of the vertebrate limb is dependent on reciprocal interactions between the ectoderm and mesoderm that regulate the structure and function of the apical ectodermal ridge. One key component of this regulatory program appears to be the precise control of signaling by members of the bone morphogenetic protein family via multiple antagonistic interactions.

Asunto(s)

Extremidades/embriología , Animales , Tipificación del Cuerpo/fisiología , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Fetales/fisiología , Forminas , Proteínas de Microfilamentos , Proteínas Nucleares/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/fisiología

RESUMEN

Long bones of the appendicular skeleton are formed from a cartilage template in a process known as endochondral bone development. Chondrocytes within this template undergo a progressive program of differentiation from proliferating to postmitotic prehypertrophic to hypertrophic chondrocytes, while mesenchymal cells immediately surrounding the early cartilage template form the perichondrium. Recently, members of the Wnt family of secreted signaling molecules have been implicated in regulating chondrocyte differentiation. We find that Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb: Wnt-5a is expressed in the perichondrium, Wnt-5b is expressed in a subpopulation of prehypertrophic chondrocytes and in the outermost cell layer of the perichondrium, and Wnt-4 is expressed in cells of the joint region. Misexpression experiments demonstrate that two of these Wnt molecules, Wnt-5a and Wnt-4, have opposing effects on the differentiation of chondrocytes and that these effects are mediated through divergent signaling pathways. Specifically, Wnt-5a misexpression delays the maturation of chondrocytes and the onset of bone collar formation, while Wnt-4 misexpression accelerates these two processes. Misexpression of a stabilized form of beta-catenin also results in accelerated chondrogenesis, suggesting that a beta-catenin/TCF-LEF complex is involved in mediating the positive regulatory effect of Wnt-4. A number of the genes involved in Wnt signal tranduction, including two members of the Frizzled gene family, which are believed to encode Wnt-receptors, show very dynamic and distinct expression patterns in cartilaginous elements of developing chicken limbs. Misexpression of putative dominant-negative forms of the two Frizzled proteins results in severe shortening of the infected cartilage elements due to a delay in chondrocyte maturation, indicating that an endogenous Wnt signal does indeed function to promote chondrogenic differentiation.

Asunto(s)

Condrogénesis/fisiología , Extremidades/embriología , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G , Transducción de Señal , Transactivadores , Animales , Desarrollo Óseo/genética , Huesos/anomalías , Diferenciación Celular/genética , Embrión de Pollo , Condrocitos/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Receptores Frizzled , Regulación del Desarrollo de la Expresión Génica , Genes Dominantes , Factor de Unión 1 al Potenciador Linfoide , Proteínas Proto-Oncogénicas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Neurotransmisores/genética , Receptores de Neurotransmisores/metabolismo , Retroviridae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt , Proteína Wnt-5a , Proteína Wnt4 , beta Catenina

Asunto(s)

Tipificación del Cuerpo/fisiología , Vertebrados/embriología , Animales , Humanos , Mesodermo , Vertebrados/fisiología

RESUMEN

Although the gut is homologous among different vertebrates, morphological differences exist between different species. The most obvious variation in the guts of extant vertebrates appears in the stomach. To investigate the evolution of this structure, we compared the histology of the stomach and gastrointestinal tract in amphibian (Xenopus laevis), avian (Gallus gallus), and mammalian (Mus musculus) organisms, and defined the expression patterns of several genes within the developing guts of these lineages. In all three groups, we find that the anterior portion of the stomach has a similar glandular histology as well as a common embryonic expression of the secreted factors Wnt5a and BMP-4. Likewise, within the amniote lineages, the posterior nonglandular stomach and pyloric sphincter regions are also comparable in both histological and molecular phenotypes. The posterior stomach expresses Six2, BMPR1B, and Barx1, whereas the pyloric sphincter expresses Nkx2.5. Although the adult Xenopus stomach exhibits both glandular and aglandular regions and a distinct pyloric sphincter similar to that of the amniotic vertebrates, the histology of the Xenopus tadpole gut shows less distinct variation in differentiation in this region, which is most likely a derived condition. The molecular signature of the embryonic Xenopus gut correlates with the more derived morphology of the larval phase. We conclude that the global patterning of the gut is remarkably similar among the different vertebrate lineages. The distinct compartments of gene expression that we find in the gut be necessary for the unique morphological specializations that distinguish the stomachs from terrestrial vertebrates.

Asunto(s)

Evolución Biológica , Aves/anatomía & histología , Estómago/anatomía & histología , Xenopus laevis/anatomía & histología , Animales , Embrión de Pollo , Femenino , Hibridación in Situ , Mamíferos/anatomía & histología , Ratones , Embarazo

Asunto(s)

Inducción Embrionaria/fisiología , Gástrula/fisiología , Organizadores Embrionarios/metabolismo , Proteínas de Pez Cebra , Animales , Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/metabolismo , Inducción Embrionaria/genética , Regulación de la Expresión Génica , Mitógenos/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt , Xenopus laevis

RESUMEN

Retroviral vectors have been invaluable tools for studies of development in vertebrates. Their use has been somewhat constrained, however, by the low viral titers typically obtained with replication-incompetent vectors, particularly of the avian type. We have addressed this problem in several ways. We optimized the transient production of avian replication-incompetent viruses in a series of cell lines. One of the optimal cell lines was the mammalian line 293T, which was surprising in light of previous reports that avian viral replication was not supported by mammalian cells. We also greatly increased the efficiency of viral infection. Pseudotyping with the vesicular stomatitus virus G (VSV-G) protein led to an over 350-fold increase in the efficiency of infection in ovo relative to infection with virus particles bearing an avian retroviral envelope protein. To further increase the utility of the system, we developed new Rous sarcoma virus (RSV)-based replication-incompetent vectors, designed to express a histochemical marker gene, human placental alkaline phosphatase, as well as an additional gene. These modified retroviral vectors and the VSV-G pseudotyping technique constitute significant improvements that allow for expanded use of avian replication-incompetent viral vectors in ovo.

Asunto(s)

Virus Defectuosos/genética , Glicoproteínas de Membrana , Retroviridae/genética , Animales , Virus de la Leucosis Aviar/genética , Virus del Sarcoma Aviar/genética , Secuencia de Bases , Encéfalo/virología , Línea Celular , Embrión de Pollo , Fibroblastos/virología , Vectores Genéticos/biosíntesis , Humanos , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos , Proteínas/metabolismo , Retina/virología , Factores de Tiempo , Transfección , Transgenes , Proteínas del Envoltorio Viral/genética , Replicación Viral , Alas de Animales/virología

RESUMEN

Left-right asymmetry is initiated during chick embryogenesis in small domains near Hensen's node. Subsequently, broad asymmetric gene expression domains are established in the lateral plate mesoderm, ultimately determining the directionality of morphogenetic events. The transfer of asymmetric information from the node to the lateral plate is mediated by Caronte (Car), a novel member of the Cerberus/Dan gene family, which induces targets by antagonizing symmetrically expressed BMP signals. In addition, BMP antagonism by Car induces asymmetric expression of Lefty in the midline, preventing spread of left-sided signals to the contralateral side.

Asunto(s)

Proteínas Aviares , Regulación del Desarrollo de la Expresión Génica , Proteínas/metabolismo , Transducción de Señal , Transactivadores , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Embrión de Pollo , Proteínas Hedgehog , Humanos , Péptidos y Proteínas de Señalización Intercelular , Factores de Determinación Derecha-Izquierda , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Morfogénesis , Proteína Nodal , Proteínas/química , Homología de Secuencia de Aminoácido , Factor de Crecimiento Transformador beta/metabolismo , Xenopus/embriología , Proteínas de Xenopus

RESUMEN

The hedgehog (Hh) signaling pathway is crucial for pattern formation during metazoan development. Although originially characterized in Drosophila, vertebrate homologs have been identified for several, but not all, genes in the pathway. Analysis of mutants in Drosophila demonstrates that Suppressor of fused [Su(fu)] interacts genetically with genes encoding proteins in the Hh signal transduction pathway, and its protein product physically interacts with two of the proteins in the Hh pathway. We report here the molecular cloning and characterization of chicken and mouse homologs of Su(fu). The chick and mouse proteins are 27% identical and 53% similar at the amino acid level to the Drosophila melanogaster and Drosophila virilis proteins. Vertebrate Su(fu) is widely expressed in the developing embryo with higher levels in tissues that are known to be patterned by Hh signaling. The chick Su(fu) protein can physically interact with factors known to function in Hh signal transduction including the Drosophila serine/threonine kinase, Fused, and the vertebrate transcriptional regulators Gli1 and Gli3. This interaction may be significant for transcriptional regulation, as recombinant Su(fu) enhances the ability of Gli proteins to bind DNA in electrophoretic mobility shift assays.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Proteínas del Tejido Nervioso , Proteínas Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Pollos , Proteínas de Unión al ADN/genética , Expresión Génica , Proteínas Hedgehog , Proteínas de Insectos/metabolismo , Factores de Transcripción de Tipo Kruppel , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Oncogénicas/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Distribución Tisular , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1 , Proteína Gli3 con Dedos de Zinc

RESUMEN

To further define the role of a T-box transcription factor, Tbx5, in cardiac development, we have examined its expression in the developing mouse and chick heart and correlated this pattern with cardiac defects caused by human TBX5 mutations in Holt-Oram syndrome. Early in the developing heart, Tbx5 is uniformly expressed throughout the entire cardiac crescent. Upon formation of the linear heart tube, Tbx5 is expressed in a graded fashion, stronger near the posterior end and weaker at the anterior end. As the heart tube loops, asymmetric Tbx5 expression continues; Tbx5 is expressed in the presumptive left ventricle, but not the right ventricle or outflow tract. This pattern of expression is maintained in more mature hearts. Expression in the ventricular septum is restricted to the left side and is contiguous with left ventricular free wall expression. Trabeculae, vena cavae (inferior and superior), and the atrial aspect of the atrioventricular valves also express high levels of Tbx5. These patterns of Tbx5 expression provide an embryologic basis for the prevalence of atrial septal defects (ostium primum and secundum), ventricular muscular septal defects, and left-sided malformations (endocardial cushion defects, hypoplastic left heart, and aberrant trabeculation) observed in patients with Holt-Oram syndrome.

Asunto(s)

Cardiopatías Congénitas/embriología , Proteínas de Dominio T Box , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/genética , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Fenotipo , Alineación de Secuencia , Factores de Transcripción/química