RESUMEN
Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.
Asunto(s)
Tipificación del Cuerpo/genética , Transición Epitelial-Mesenquimal/genética , Cara/embriología , Morfogénesis/genética , Nariz/embriología , Factor de Transcripción 1 de la Leucemia de Células Pre-B/fisiología , Factores de Transcripción de la Familia Snail/genética , Animales , Apoptosis/genética , Células Cultivadas , Labio Leporino/embriología , Labio Leporino/genética , Fisura del Paladar/embriología , Fisura del Paladar/genética , Embrión de Mamíferos , Cara/anomalías , Regulación del Desarrollo de la Expresión Génica , Labio/embriología , Ratones , Ratones Transgénicos , Hueso Paladar/embriología , Factor de Transcripción 1 de la Leucemia de Células Pre-B/genéticaRESUMEN
Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% (n=617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential cis-regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related Gdf5 that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with Lmx1b that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization.
Asunto(s)
Tipificación del Cuerpo/genética , Extremidades/embriología , Proteínas con Homeodominio LIM/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/metabolismo , Animales , Pollos , Inmunoprecipitación de Cromatina , Secuencia Conservada/genética , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Factor 5 de Diferenciación de Crecimiento/genética , Factor 5 de Diferenciación de Crecimiento/metabolismo , Proteínas con Homeodominio LIM/genética , Ratones Endogámicos C57BL , Modelos Biológicos , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Factores de Transcripción/genéticaRESUMEN
Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas Dishevelled/genética , ARN Polimerasa I/genética , Transcripción Genética , Proteína Wnt-5a/genética , Neoplasias de la Mama/patología , Cromatina/genética , ADN Ribosómico/genética , Proteínas Dishevelled/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Región Organizadora del Nucléolo/genética , Regiones Promotoras Genéticas , Unión Proteica , ARN Ribosómico/genética , Sirtuinas/genética , Vía de Señalización Wnt/genética , Proteína Wnt-5a/metabolismoRESUMEN
Lmx1b is a homeodomain transcription factor that regulates dorsal identity during limb development. Lmx1b knockout (KO) mice develop distal ventral-ventral limbs. Although induction of Lmx1b is linked to Wnt7a expression in the dorsal limb ectoderm, the downstream targets of Lmx1b that accomplish limb dorsalization are unknown. To identify genes targeted by Lmx1b, we compared gene arrays from Lmx1b KO and wild type mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes that were differentially expressed in all three stages. Several skeletal targets, including Emx2, Matrilin1 and Matrilin4, demonstrated a loss of scapular expression in the Lmx1b KO mice, supporting a role for Lmx1b in scapula development. Furthermore, the relative abundance of extracellular matrix-related soft tissue targets regulated by Lmx1b, such as collagens and proteoglycans, suggests a mechanism that includes changes in the extracellular matrix composition to accomplish limb dorsalization. Our study provides the most comprehensive characterization of genes regulated by Lmx1b during limb development to-date and provides targets for further investigation.
Asunto(s)
Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo , Proteína de la Matriz Oligomérica del Cartílago , Tejido Conectivo/embriología , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas Matrilinas , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Neuronas/citología , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Escápula/embriología , Factores de Transcripción/genéticaRESUMEN
Recent investigations into the mechanism of limb development have clarified the roles of several molecules, their pathways, and interactions. Characterization of the molecular pathways that orchestrate limb development has provided insight into the etiology of many limb malformations. In this review, we describe how the insights from developmental biology are related to clinically relevant anomalies and the current classification schemes used to define, categorize, and communicate patterns of upper limb malformations. We advocate an updated classification scheme for upper limb anomalies that incorporates our current molecular perspective of limb development and the pathogenetic basis for malformations using dysmorphology terminology. We anticipate that this scheme will improve the utility of a classification as a basis for diagnosis, treatment, and research.
Asunto(s)
Deformidades Congénitas de la Mano/clasificación , Deformidades Congénitas de las Extremidades Superiores/clasificación , Acrocefalosindactilia/genética , Animales , Deformidades Congénitas de la Mano/diagnóstico , Deformidades Congénitas de la Mano/genética , Deformidades Congénitas de la Mano/terapia , Proteínas de Homeodominio/genética , Humanos , Polidactilia/genética , Radio (Anatomía)/anomalías , Cúbito/anomalías , Deformidades Congénitas de las Extremidades Superiores/diagnóstico , Deformidades Congénitas de las Extremidades Superiores/genética , Deformidades Congénitas de las Extremidades Superiores/terapia , Proteínas Wnt/genéticaRESUMEN
Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Puntos de Control de la Fase G1 del Ciclo Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ribosomas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Diferenciación Celular/fisiología , Línea Celular Tumoral/trasplante , Movimiento Celular/fisiología , Nucléolo Celular/metabolismo , Embrión de Pollo , Proteínas Cromosómicas no Histona/metabolismo , ADN Ribosómico/metabolismo , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Ribosómico/metabolismo , Ribosomas/genéticaRESUMEN
During limb development, fibroblast growth factors (Fgfs) govern proximalâ»distal outgrowth and patterning. FGFs also synchronize developmental patterning between the proximalâ»distal and anteriorâ»posterior axes by maintaining Sonic hedgehog (Shh) expression in cells of the zone of polarizing activity (ZPA) in the distal posterior mesoderm. Shh, in turn, maintains Fgfs in the apical ectodermal ridge (AER) that caps the distal tip of the limb bud. Crosstalk between Fgf and Shh signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well-characterized. Implantation of Fgf beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3 h after application), while prolonged exposure (24 h) can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations accentuated genes involved in SHH regulation. Comparative analysis identified 25 candidates common to both treatments, with eight linked to SHH expression or function. Furthermore, we demonstrated that LHX2, a LIM Homeodomain transcription factor, is an intermediate in the FGF-mediated regulation of SHH. Our data suggest that LHX2 acts as a competency factor maintaining distal posterior SHH expression subjacent to the AER.
RESUMEN
Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741-52. ©2017 AACR.
Asunto(s)
Aminoácidos/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/fisiología , Glioma/tratamiento farmacológico , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Transporte Biológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Muerte Celular , Línea Celular Tumoral , Dihidropiridinas/farmacología , Glioma/metabolismo , Glioma/patología , Humanos , Ratones , Micotoxinas/farmacología , Células Madre Neoplásicas/patología , Proteómica , Sodio/metabolismoRESUMEN
Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here, we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis, we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25(ENU)). Unlike Vps25-null embryos we generated, Vps25(ENU/ENU) mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyperactivation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25(ENU/ENU) Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however, SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning.