RESUMEN
Melanocytes evolved to produce the melanin that gives colour to our hair, eyes and skin. The melanocyte lineage also gives rise to melanoma, the most lethal form of skin cancer. The melanocyte lineage differentiates from neural crest cells during development, and most melanocytes reside in the skin and hair, where they are replenished by melanocyte stem cells. Because the molecular mechanisms necessary for melanocyte specification, migration, proliferation and differentiation are co-opted during melanoma initiation and progression, studying melanocyte development is directly relevant to human disease. Here, through the lens of advances in cellular omic and genomic technologies, we review the latest findings in melanocyte development and differentiation, and how these developmental pathways become dysregulated in disease.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Melanocitos , Melanoma , Melanocitos/metabolismo , Melanocitos/citología , Humanos , Animales , Melanoma/patología , Melanoma/metabolismo , Melanoma/genética , Cresta Neural/metabolismo , Proliferación Celular , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genéticaRESUMEN
Melanocyte stem cells (McSCs) in zebrafish serve as an on-demand source of melanocytes during growth and regeneration, but metabolic programs associated with their activation and regenerative processes are not well known. Here, using live imaging coupled with scRNA-sequencing, we discovered that, during regeneration, quiescent McSCs activate a dormant embryonic neural crest transcriptional program followed by an aldehyde dehydrogenase (Aldh) 2 metabolic switch to generate progeny. Unexpectedly, although ALDH2 is well known for its aldehyde-clearing mechanisms, we find that, in regenerating McSCs, Aldh2 activity is required to generate formate - the one-carbon (1C) building block for nucleotide biosynthesis - through formaldehyde metabolism. Consequently, we find that disrupting the 1C cycle with low doses of methotrexate causes melanocyte regeneration defects. In the absence of Aldh2, we find that purines are the metabolic end product sufficient for activated McSCs to generate progeny. Together, our work reveals McSCs undergo a two-step cell state transition during regeneration, and that the reaction products of Aldh2 enzymes have tissue-specific stem cell functions that meet metabolic demands in regeneration.
Asunto(s)
Melanocitos , Pez Cebra , Animales , Diferenciación Celular , Cresta Neural , Células MadreRESUMEN
Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.
Asunto(s)
Diferenciación Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Mesencéfalo/embriología , Retina/embriología , Fase S/genética , Animales , Apoptosis , Larva/metabolismo , Mesencéfalo/metabolismo , Morfogénesis/genética , Neurogénesis/genética , ARN Ribosómico/metabolismo , Retina/metabolismo , Pez Cebra/embriologíaRESUMEN
Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.
Asunto(s)
Mesencéfalo/embriología , Mesencéfalo/metabolismo , Células-Madre Neurales/metabolismo , Retina/metabolismo , Pez Cebra/embriología , Animales , Ciclo Celular , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Mitosis , MorfogénesisRESUMEN
The highly conserved POU genes encode homeodomain transcription factors involved in various developmental events, with some, the Brn genes, playing key roles in neurogenesis. We investigated the evolutionary relationships between these genes, by studying the POU gene complement of a model teleost, the medaka (Oryzias latipes). We identified 17 POU genes and carried out a comprehensive in situ hybridization analysis focusing on the optic tectum, a cortical structure of the mesencephalon, in which cell positions and their differentiation states are spatially and temporally correlated. Six POU genes displayed patterned expression in the optic tectum: two genes were expressed in the center of the organ (a zone with differentiated neurons), two in an intermediate zone in which cells exit the cell cycle and two in the peripheral proliferation zone. These results suggest that POU genes may play key roles in both late neurogenesis and in multipotent neural progenitors.
Asunto(s)
Oryzias/anatomía & histología , Oryzias/genética , Oryzias/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Colículos Superiores/metabolismo , Animales , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Genoma , Estudio de Asociación del Genoma Completo , Datos de Secuencia Molecular , Factores del Dominio POU/clasificación , Filogenia , Somitos/embriología , Colículos Superiores/citología , Colículos Superiores/embriologíaRESUMEN
Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we applied tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed that MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage-tracing methodology in adult zebrafish cancer models.
Asunto(s)
Melanoma , Pez Cebra , Animales , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/genética , Tamoxifeno/farmacología , Proteínas de Pez CebraRESUMEN
Melanocytes, the pigment-producing cells, are replenished from multiple stem cell niches in adult tissue. Although pigmentation traits are known risk factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs and lack key lineage markers with which to identify McSCs and study their function. Here we find that Tfap2b and a select set of target genes specify an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and is essential for McSC-dependent melanocyte regeneration. Fate mapping data reveal that tfap2b+ McSCs have multifate potential, and are the cells of origin for large patches of adult melanocytes, two other pigment cell types (iridophores and xanthophores), and nerve-associated cells. Hence, Tfap2b confers McSC identity in early development, distinguishing McSCs from other neural crest and pigment cell lineages, and retains multifate potential in the adult zebrafish.
Asunto(s)
Melanocitos/metabolismo , Células Madre/clasificación , Factor de Transcripción AP-2/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Melanocitos/fisiología , Pigmentación/genética , Piel/metabolismo , Pigmentación de la Piel/genética , Células Madre/metabolismo , Factor de Transcripción AP-2/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Gain or loss-of-function mutations in fibroblast growth factor receptor 3 (FGFR3) result in cranial vault defects highlighting the protein's role in membranous ossification. Zebrafish express high levels of fgfr3 during skull development; in order to study FGFR3's role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof/lof ). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. Integrated analyses (in vivo imaging and single-cell RNA sequencing of the osteoblast lineage) of zebrafish fgfr3lof/lof revealed a delay in osteoblast expansion and differentiation, together with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis. We conclude that changes in the extracellular matrix within growing bone might impair cell-cell communication, mineralization, and new osteoblast recruitment. © 2020 American Society for Bone and Mineral Research.
Asunto(s)
Pez Cebra , Animales , Diferenciación Celular , Osteoblastos , Osteogénesis , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Cráneo , Proteínas de Pez Cebra/genéticaRESUMEN
Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.
Asunto(s)
Diferenciación Celular/genética , ARN Helicasas DEAD-box/metabolismo , Melanocitos/citología , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , ARN Helicasas DEAD-box/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Mutación , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatasas/genética , Células Madre/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The melanocyte-inducing transcription factor (MITF)-low melanoma transcriptional signature is predictive of poor outcomes for patients, but little is known about its biological significance, and animal models are lacking. Here, we used zebrafish genetic models with low activity of Mitfa (MITF-low) and established that the MITF-low state is causal of melanoma progression and a predictor of melanoma biological subtype. MITF-low zebrafish melanomas resembled human MITF-low melanomas and were enriched for stem and invasive (mesenchymal) gene signatures. MITF-low activity coupled with a p53 mutation was sufficient to promote superficial growth melanomas, whereas BRAFV600E accelerated MITF-low melanoma onset and further promoted the development of MITF-high nodular growth melanomas. Genetic inhibition of MITF activity led to rapid regression; recurrence occurred following reactivation of MITF. At the regression site, there was minimal residual disease that was resistant to loss of MITF activity (termed MITF-independent cells) with very low-to-no MITF activity or protein. Transcriptomic analysis of MITF-independent residual disease showed enrichment of mesenchymal and neural crest stem cell signatures similar to human therapy-resistant melanomas. Single-cell RNA sequencing revealed MITF-independent residual disease was heterogeneous depending on melanoma subtype. Further, there was a shared subpopulation of residual disease cells that was enriched for a neural crest G0-like state that preexisted in the primary tumor and remained present in recurring melanomas. These findings suggest that invasive and stem-like programs coupled with cellular heterogeneity contribute to poor outcomes for MITF-low melanoma patients and that MITF-independent subpopulations are an important therapeutic target to achieve long-term survival outcomes. SIGNIFICANCE: This study provides a useful model for MITF-low melanomas and MITF-independent cell populations that can be used to study the mechanisms that drive these tumors as well as identify potential therapeutic options.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/22/5769/F1.large.jpg.
Asunto(s)
Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Neoplasia Residual/genética , Transcripción Genética/genética , Pez Cebra/genética , Animales , Resistencia a Medicamentos/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Melanocitos/patología , Melanoma/patología , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Neoplasia Residual/patología , Cresta Neural/patología , Proteínas Proto-Oncogénicas B-raf/genética , Células Madre/patologíaRESUMEN
The incorporation of transition metal catalysts to the bioorthogonal toolbox has opened the possibility of producing supra-stoichiometric amounts of xenobiotics in living systems in a non-enzymatic fashion. For medical use, such metals could be embedded in implantable devices (i.e. heterogeneous catalyst) to "synthesize" drugs in desired locations (e.g. in a tumour) with high specificity and for extended periods of time, overcoming the useful life limitations of current local therapy modalities directed to specific organ sites (e.g. brachytherapy, controlled release systems). To translate this approach into a bona fide therapeutic option, it is essential to develop clinically-accessible implantation procedures and to understand and validate the activation process in relevant preclinical models. Herein we report the development of a novel Pd-activatable precursor of the red-fluorescent drug doxorubicin and Pd devices of optimized size and activity. Screening in state-of-the-art cancer models provided fundamental insights into the insertion protocols, safety and stability of the devices and into the prodrug distribution profile before and after activation.
RESUMEN
Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments.
Asunto(s)
Regeneración Nerviosa , Neuroglía/química , Neuroglía/fisiología , Notocorda/lesiones , Proteínas WT1/análisis , Cicatrización de Heridas , Animales , Movimiento Celular , Pez CebraRESUMEN
The visual systems of vertebrates and many other bilaterian clades consist of complex neural structures guiding a wide spectrum of behaviors. Homologies at the level of cell types and even discrete neural circuits have been proposed, but many questions of how the architecture of visual neuropils evolved among different phyla remain open. In this review we argue that the profound conservation of genetic and developmental steps generating the eye and its target neuropils in fish and fruit flies supports a homology between some core elements of bilaterian visual circuitries. Fish retina and tectum, and fly optic lobe, develop from a partitioned, unidirectionally proliferating neurectodermal domain that combines slowly dividing neuroepithelial stem cells and rapidly amplifying progenitors with shared genetic signatures to generate large numbers and different types of neurons in a temporally ordered way. This peculiar 'conveyor belt neurogenesis' could play an essential role in generating the topographically ordered circuitry of the visual system.
Asunto(s)
Evolución Biológica , Drosophila/fisiología , Peces/fisiología , Neurogénesis , Animales , Drosophila/crecimiento & desarrollo , Peces/crecimiento & desarrollo , Neurópilo/fisiología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Lóbulo Óptico de Animales no Mamíferos/fisiología , Retina/crecimiento & desarrollo , Retina/fisiología , Colículos Superiores/crecimiento & desarrollo , Colículos Superiores/fisiologíaRESUMEN
Although considered a 'house-keeping' function, ribosome biogenesis is regulated differently between cells and can be modulated in a cell-type-specific manner. These differences are required to generate specialized ribosomes that contribute to the translational control of gene expression by selecting mRNA subsets to be translated. Thus, differences in ribosome biogenesis between stem and differentiated cells indirectly contribute to determine cell identity. The concept of the existence of stem cell-specific mechanisms of ribosome biogenesis has progressed from an attractive theory to a useful working model with important implications for basic and medical research.