RESUMEN
The systematic classification of the cells that compose a tissue or an organ is key to understanding how these cells cooperate and interact as a functional unit. Our capacity to detect features that define cell identity has evolved from morphological and chemical analyses, through the use of predefined genetic markers, to unbiased transcriptomic and epigenetic profiling. The innovative technology of single-cell RNA sequencing (scRNA-seq) enables transcriptional profiling of thousands of individual cells. Since its development, scRNA-seq has been extensively applied to numerous organs and tissues in a wide range of animal models and human samples, thereby providing a plethora of fundamental biological insights into their development, homeostasis, and pathology. In this review, we present the findings of 3 recent studies that employed scRNA-seq to unravel the complexity of cellular composition in mammalian teeth. These findings offer an unprecedented catalogue of cell types in the mouse incisor, which is a convenient model system for studying continuous tooth growth. These studies identified novel cell types in the tooth epithelium and mesenchyme, as well as new markers for known cell types. Computational analyses of the data also uncovered the lineage and dynamics of cell states during ameloblast and odontoblast differentiation during both normal homeostasis and injury repair. The transcriptional differences between the mouse incisor and mouse and human molars uncover species-specific as well as shared features in tooth cell composition. Here, we highlight these findings and discuss important similarities and differences between these studies. We also discuss potential future applications of scRNA-seq in dental research and dentistry. Together, these studies demonstrate how the rapidly evolving technology of scRNA-seq can advance the study of tooth development and function and provide putative targets for regenerative approaches.
Asunto(s)
Análisis de la Célula Individual , Transcriptoma , Animales , Diferenciación Celular , Perfilación de la Expresión Génica , Marcadores Genéticos , Ratones , Análisis de Secuencia de ARNRESUMEN
The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural-functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.
Asunto(s)
Embrión de Mamíferos/citología , Sustancias de Crecimiento , Neuronas Motoras/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Péptidos , Animales , Axones/metabolismo , Secuencia de Bases , Cartilla de ADN , Péptidos y Proteínas de Señalización Intercelular , RatasRESUMEN
The commissural axons project toward and across the floor plate. They then turn into the longitudinal axis, extending along the contralateral side of the floor plate. F-spondin, a protein produced and secreted by the floor plate, promotes adhesion and neurite extension of commissural neurons in vitro. Injection of purified F-spondin protein into the lumen of the spinal cord of chicken embryos in ovo resulted in longitudinal turning of commissural axons before reaching the floor plate, whereas neutralizing antibody (Ab) injections caused lateral turning at the contralateral floor plate boundary. These combined in vitro and in vivo results suggest that F-spondin is required to prevent the lateral drifting of the commissural axons after having crossed the floor plate.
Asunto(s)
Axones/fisiología , Sustancias de Crecimiento , Moléculas de Adhesión de Célula Nerviosa/fisiología , Péptidos , Médula Espinal/ultraestructura , Animales , Western Blotting , Adhesión Celular/fisiología , Embrión de Pollo , Clonación Molecular , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Moléculas de Adhesión de Célula Nerviosa/farmacología , Neuritas/fisiología , Neuritas/ultraestructura , Ratas , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismoRESUMEN
Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento , Hipocampo/embriología , Moléculas de Adhesión de Célula Nerviosa/genética , Neuronas/fisiología , Péptidos , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Clonación Molecular , Drosophila , Proteínas de Drosophila , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/fisiología , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/fisiología , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Pez CebraRESUMEN
Neural crest (NC) cells migrate exclusively into the rostral half of each sclerotome, where they avoid the dermomyotome and the paranotochordal sclerotome. F-spondin is expressed in these inhibitory regions and throughout the caudal halves. In vitro bioassays of NC spreading on substrates of rostral or caudal epithelial-half somites (RS or CS, respectively) revealed that NC cells adopt on RS a fibroblastic morphology, whereas on CS they fail to flatten. F-spondin inhibited flattening of NC cells on RS. Conversely, F-spondin antibodies prevented rounding up of NC cells on CS. Addition of F-spondin to trunk explants inhibited NC migration into the sclerotome, and treatment of embryos with anti-F-spondin antibodies yielded migration into otherwise inhibitory sites. Thus, somite-derived F-spondin is an inhibitory signal involved in patterning the segmental migration of NC cells and their topographical segregation within the RS.
Asunto(s)
Movimiento Celular/fisiología , Sustancias de Crecimiento , Moléculas de Adhesión de Célula Nerviosa/fisiología , Cresta Neural/citología , Neuronas/fisiología , Péptidos , Somitos/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Técnicas de Cocultivo , Inmunohistoquímica , Hibridación in Situ , Microinyecciones , Microscopía Confocal , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Moléculas de Adhesión de Célula Nerviosa/genética , Cresta Neural/embriología , Neuronas/ultraestructura , ARN Mensajero/biosíntesis , Transducción de Señal/fisiologíaRESUMEN
F-spondin, an extracellular matrix protein, is present in peripheral nerve during embryonic development, but its amount diminishes by birth. Axotomy of adult rat sciatic nerve, however, causes a massive upregulation of both F-spondin mRNA and protein distal to the lesion. F-spondin in the distal stump of axotomized nerve promotes neurite outgrowth of sensory neurons, as revealed by protein neutralization with F-spondin-specific antibodies. Thus, F-spondin is likely to play a role in promoting axonal regeneration after nerve injury.