RESUMEN
Techniques allowing the precise quantification of mRNA at the cellular level are essential for understanding biological processes. Here, we present a semi-automated smiFISH (single-molecule inexpensive FISH) pipeline enabling quantification of mRNA in a small number of cells (â¼40) in fixed whole mount tissue. We describe steps for sample preparation, hybridization, image acquisition, cell segmentation, and mRNA quantification. Although the protocol was developed in Drosophila, it can be optimized for use in other organisms. For complete details on the use and execution of this protocol, please refer to Guan et al.1.
RESUMEN
How the vast array of neuronal diversity is generated remains an unsolved problem. Here, we investigate how 29 morphologically distinct leg motoneurons are generated from a single stem cell in Drosophila. We identify 19 transcription factor (TF) codes expressed in immature motoneurons just before their morphological differentiation. Using genetic manipulations and a computational tool, we demonstrate that the TF codes are progressively established in immature motoneurons according to their birth order. Comparing RNA and protein expression patterns of multiple TFs reveals that post-transcriptional regulation plays an essential role in shaping these TF codes. Two RNA-binding proteins, Imp and Syp, expressed in opposing gradients in immature motoneurons, control the translation of multiple TFs. The varying sensitivity of TF mRNAs to the opposing gradients of Imp and Syp in immature motoneurons decrypts these gradients into distinct TF codes, establishing the connectome between motoneuron axons and their target muscles.
Asunto(s)
Proteínas de Drosophila , Células-Madre Neurales , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Neuronas Motoras/metabolismo , Células-Madre Neurales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFß3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFß signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/prevención & control , Neoplasias de la Mama/prevención & control , Receptores de Estrógenos/metabolismo , Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias Óseas/inmunología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Pronóstico , Receptores de Estrógenos/genética , Transducción de Señal , Factor de Crecimiento Transformador beta3/genética , Factor de Crecimiento Transformador beta3/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Diseases of the locomotor system are at the origin of disabilities with severe social and economic consequences. The study of the neuromuscular system development and maintenance has become a key challenge for the scientific community in order to design efficient therapies. My thesis project aims to elucidate the mechanisms at the origin of the communication between motoneuron axons and their muscle targets in order to understand how specific innervations are generated during development and maintained during adulthood. The first part of the project will address the understanding of the mechanisms controlling the specific muscle-axon recognition during development. I will perform live imaging and fixed tissues experiments to visualize and understand the development of myoblasts and motoneurons at the same time. Then, I will perform transcriptomic experiments to discover molecules playing a role in the specific axon-muscle recognition. The second part of the project is meant to elucidate the mechanism controlling the system maintenance in the adult. To answer this question I will study the function of morphological transcription factors in adulthood, which are known as transcription factors controlling the morphology of motoneurons during development. To conclude, this project will lead to novel biological concepts that will increase our fundamental knowledge on developmental biology. Understanding the mechanisms that specify the muscle innervation will allow to find efficient ways to tackle neuromuscular diseases.
Asunto(s)
Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/fisiología , Regeneración/fisiología , Adulto , Animales , Axones/fisiología , Sistemas CRISPR-Cas , Regulación del Desarrollo de la Expresión Génica , Genómica/métodos , Humanos , Neuronas Motoras/fisiología , Músculo Esquelético/inervación , RNA-Seq , Regeneración/genéticaRESUMEN
Naturalists have been fascinated for centuries by animal colors and color patterns. While widely studied at the adult stage, we know little about color patterns in the embryo. Here, we study a trait consisting of coloration that is specific to the embryo and absent from postembryonic stages in water striders (Gerromorpha). By combining developmental genetics with chemical and phylogenetic analyses across a broad sample of species, we uncovered the mechanisms underlying the emergence and diversification of embryonic colors in this group of insects. We show that the pteridine biosynthesis pathway, which ancestrally produces red pigment in the eyes, has been recruited during embryogenesis in various extraocular tissues including antennae and legs. In addition, we discovered that this cooption is common to all water striders and initially resulted in the production of yellow extraocular color. Subsequently, 6 lineages evolved bright red color and 2 lineages lost the color independently. Despite the high diversity in colors and color patterns, we show that the underlying biosynthesis pathway remained stable throughout the 200 million years of Gerromorpha evolutionary time. Finally, we identified erythropterin and xanthopterin as the pigments responsible for these colors in the embryo of various species. These findings demonstrate how traits can emerge through the activation of a biosynthesis pathway in new developmental contexts.
Asunto(s)
Color , Embrión no Mamífero/metabolismo , Heterópteros/fisiología , Pigmentación/fisiología , Pigmentos Biológicos/metabolismo , Pteridinas/metabolismo , Transducción de Señal , Animales , Evolución Biológica , Embrión no Mamífero/citología , Ojo/citología , Ojo/metabolismo , Heterópteros/clasificación , Fenotipo , FilogeniaRESUMEN
Prostate cancers have a strong propensity to metastasize to bone and promote osteoblastic lesions. TMPRSS2:ERG is the most frequent gene rearrangement identified in prostate cancer, but whether it is involved in prostate cancer bone metastases is largely unknown. We exploited an intratibial metastasis model to address this issue and we found that ectopic expression of the TMPRSS2:ERG fusion enhances the ability of prostate cancer cell lines to induce osteoblastic lesions by stimulating bone formation and inhibiting the osteolytic response. In line with these in vivo results, we demonstrate that the TMPRSS2:ERG fusion protein increases the expression of osteoblastic markers, including Collagen Type I Alpha 1 Chain and Alkaline Phosphatase, as well as Endothelin-1, a protein with a documented role in osteoblastic bone lesion formation. Moreover, we determined that the TMPRSS2:ERG fusion protein is bound to the regulatory regions of these genes in prostate cancer cell lines, and we report that the expression levels of these osteoblastic markers are correlated with the expression of the TMPRSS2:ERG fusion in patient metastasis samples. Taken together, our results reveal that the TMPRSS2:ERG gene fusion is involved in osteoblastic lesion formation induced by prostate cancer cells.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Osteoblastos/metabolismo , Neoplasias de la Próstata/genética , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Línea Celular Tumoral , Cadena alfa 1 del Colágeno Tipo I , Endotelina-1/genética , Endotelina-1/metabolismo , Humanos , Masculino , Ratones SCID , Proteínas de Fusión Oncogénica/metabolismo , Osteoblastos/patología , Células PC-3 , Fenotipo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Trasplante Heterólogo , Carga Tumoral/genéticaRESUMEN
BACKGROUND: Resistance to thyroid hormone due to THRA mutations (RTHα) is a recently discovered genetic disease, displaying important variability in its clinical presentation. The mutations alter the function of TRα1, one of the two nuclear receptors for thyroid hormone. METHODS: The aim of this study was to understand the relationship between specific THRA mutations and phenotype. CRISPR/Cas9 genome editing was used to generate five new mouse models of RTHα, with frameshift or missense mutations. RESULTS: Like human patients, mutant mice displayed a hypothyroid-like phenotype, with altered development. Phenotype severity varied between the different mouse models, mainly depending on the ability of the mutant receptor to interact with transcription corepressor in the presence of thyroid hormone. CONCLUSION: The present mutant mice represent highly relevant models for the human genetic disease which will be useful for future investigations.
Asunto(s)
Genes erbA/genética , Síndrome de Resistencia a Hormonas Tiroideas/genética , Animales , Sistemas CRISPR-Cas , Ratones , Mutación , FenotipoRESUMEN
Bone metastases are one of the main complications of prostate cancer and they are incurable. We investigated whether and how estrogen receptor-related receptor alpha (ERRα) is involved in bone tumor progression associated with advanced prostate cancer. By meta-analysis, we first found that ERRα expression is correlated with castration-resistant prostate cancer (CRPC), the hallmark of progressive disease. We then analyzed tumor cell progression and the associated signaling pathways in gain-of-function/loss-of-function CRPC models in vivo and in vitro. Increased levels of ERRα in tumor cells led to rapid tumor progression, with both bone destruction and formation, and direct impacts on osteoclasts and osteoblasts. VEGF-A, WNT5A and TGFß1 were upregulated by ERRα in tumor cells and all of these factors also significantly and positively correlated withERRα expression in CRPC patient specimens. Finally, high levels of ERRα in tumor cells stimulated the pro-metastatic factor periostin expression in the stroma, suggesting that ERRα regulates the tumor stromal cell microenvironment to enhance tumor progression. Taken together, our data demonstrate that ERRα is a regulator of CRPC cell progression in bone. Therefore, inhibiting ERRα may constitute a new therapeutic strategy for prostate cancer skeletal-related events.