RESUMO
Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2-/-;Nog+/- embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2-/- mice.
Assuntos
Fenda Labial , Fissura Palatina , Defeitos do Tubo Neural , Animais , Camundongos , Proteínas Morfogenéticas Ósseas/metabolismo , Mamíferos/metabolismo , Tubo Neural/metabolismo , Receptores Nogo/metabolismoRESUMO
Epithelia such as epidermis cover large surfaces and are crucial for survival. Maintenance of tissue homeostasis by balancing cell proliferation, cell size, and cell extrusion ensures epidermal integrity. Although the mechanisms of cell extrusion are better understood, how epithelial cells that round up under developmental or perturbed genetic conditions are reintegrated in the epithelium to maintain homeostasis remains unclear. Here, we performed live imaging in zebrafish embryos to show that epidermal cells that round up due to membrane homeostasis defects in the absence of goosepimples/myosinVb (myoVb) function, are reintegrated into the epithelium. Transcriptome analysis and genetic interaction studies suggest that the transcription factor Grainyhead-like 3 (Grhl3) induces the retention of rounded cells by regulating E-cadherin levels. Moreover, Grhl3 facilitates the survival of MyoVb deficient embryos by regulating cell adhesion, cell retention, and epidermal architecture. Our analyses have unraveled a mechanism of retention of rounded cells and its importance in epithelial homeostasis.
Assuntos
Endocitose , Células Epidérmicas/metabolismo , Estresse Fisiológico , Proteínas de Peixe-Zebra/fisiologia , Animais , Células Epidérmicas/citologia , Mutação , Transcriptoma , Regulação para Cima , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Vitamin D plays an important pleiotropic role in maintaining global homeostasis of the human body. Its functions go far beyond skeletal health, playing a crucial role in a plethora of cellular functions, as well as in extraskeletal health, ensuring the proper functioning of multiple human organs, including the skin. Genes from the Grainyhead-like (GRHL) family code for transcription factors necessary for the development and maintenance of various epithelia. Even though they are involved in many processes regulated by vitamin D, a direct link between vitamin D-mediated cellular pathways and GRHL genes has never been described. We employed various bioinformatic methods, quantitative real-time PCR, chromatin immunoprecipitation, reporter gene assays, and calcitriol treatments to investigate this issue. We report that the vitamin D receptor (VDR) binds to a regulatory region of the Grainyhead-like 1 (GRHL1) gene and regulates its expression. Ectopic expression of VDR and treatment with calcitriol alters the expression of the GRHL1 gene. The evidence presented here indicates a role of VDR in the regulation of expression of GRHL1 and correspondingly a role of GRHL1 in mediating the actions of vitamin D.
Assuntos
Regulação da Expressão Gênica , Receptores de Calcitriol , Fatores de Transcrição , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Calcitriol/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Regiões Promotoras Genéticas , Proteínas RepressorasRESUMO
This Special Issue comprises three original studies and five review articles [...].
Assuntos
Neoplasias , Fatores de Transcrição , Humanos , Neoplasias/genética , Fatores de Transcrição/genéticaRESUMO
Grainyhead-like (GRHL) factors are essential, highly conserved transcription factors (TFs) that regulate processes common to both natural cellular behaviours during embryogenesis, and de-regulation of growth and survival pathways in cancer. Serving to drive the transcription, and therefore activation of multiple co-ordinating pathways, the three GRHL family members (GRHL1-3) are a critical conduit for modulating the molecular landscape that guides cellular decision-making processes during proliferation, epithelial-mesenchymal transition (EMT) and migration. Animal models and in vitro approaches harbouring GRHL loss or gain-of-function are key research tools to understanding gene function, which gives confidence that resultant phenotypes and cellular behaviours may be translatable to humans. Critically, identifying and characterising the target genes to which these factors bind is also essential, as they allow us to discover and understand novel genetic pathways that could ultimately be used as targets for disease diagnosis, drug discovery and therapeutic strategies. GRHL1-3 and their transcriptional targets have been shown to drive comparable cellular processes in Drosophila, C. elegans, zebrafish and mice, and have recently also been implicated in the aetiology and/or progression of a number of human congenital disorders and cancers of epithelial origin. In this review, we will summarise the state of knowledge pertaining to the role of the GRHL family target genes in both development and cancer, primarily through understanding the genetic pathways transcriptionally regulated by these factors across disparate disease contexts.
Assuntos
Proteínas de Ligação a DNA , Neoplasias , Proteínas Repressoras/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Transição Epitelial-Mesenquimal/genética , Camundongos , Neoplasias/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: The highly conserved Grainyhead-like (Grhl) family of transcription factors play critical roles in the development of the neural tube and craniofacial skeleton. In particular, deletion of family member Grainyhead-like 2 (Grhl2) leads to mid-gestational embryonic lethality, maxillary clefting, abdominoschisis, and both cranial and caudal neural tube closure defects. These highly pleiotropic and systemic defects suggest that Grhl2 plays numerous critical developmental roles to ensure correct morphogenesis and patterning. RESULTS: Here, using four separate Cre-lox conditional deletion models, as well as one genetic epistasis approach (Grhl2+/- ;Edn1+/- double heterozygous mice) we have investigated tissue-specific roles of Grhl2 in embryonic development, with a particular focus on the craniofacial skeleton. We find that loss of Grhl2 in the pharyngeal epithelium (using the ShhCre driver) leads to low-penetrance micrognathia, whereas deletion of Grhl2 within the ectoderm of the pharynx (NestinCre ) leads to small, albeit significant, differences in the proximal-distal elongation of both the maxilla and mandible. Loss of Grhl2 in endoderm (Sox17-2aiCre ) resulted in noticeable lung defects and a single instance of secondary palatal clefting, although formation of other endoderm-derived organs such as the stomach, bladder and intestines was not affected. Lastly, deletion of Grhl2 in cells of the neural crest (Wnt1Cre ) did not lead to any discernible defects in craniofacial development, and similarly, our epistasis approach did not detect any phenotypic consequences of loss of a single allele of both Grhl2 and Edn1. CONCLUSION: Taken together, our study identifies a pharyngeal-epithelium intrinsic, non-cell-autonomous role for Grhl2 in the patterning and formation of the craniofacial skeleton, as well as an endoderm-specific role for Grhl2 in the formation and establishment of the mammalian lung.
Assuntos
Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Crânio/embriologia , Fatores de Transcrição/genética , Animais , Camundongos , Crista Neural/metabolismo , Tubo Neural/metabolismo , Crânio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The highly-conserved Grainyhead-like (Grhl) transcription factors are critical regulators of embryogenesis that regulate cellular survival, proliferation, migration and epithelial integrity, especially during the formation of the craniofacial skeleton. Family member Grhl2 is expressed throughout epithelial tissues during development, and loss of Grhl2 function leads to significant defects in neurulation, abdominal wall closure, formation of the face and fusion of the maxilla/palate. Whereas numerous downstream target genes of Grhl2 have been identified, very little is known about how this crucial developmental transcription factor itself is regulated. Here, using in silico and in utero expression analyses and functional deletion in mice, we have identified a novel 2.4 âkb enhancer element (mm1286) that drives reporter gene expression in a pattern that strongly recapitulates endogenous Grhl2 in the craniofacial primordia, modulates Grhl2 expression in these tissues, and augments Grhl2-mediated closure of the secondary palate. Deletion of this genomic element, in the context of inactivation of one allele of Grhl2 (through generation of double heterozygous Grhl2+/-;mm1286+/- mice), results in a significant predisposition to palatal clefting at birth. Moreover, we found that a highly conserved 325 bp region of mm1286 is both necessary and sufficient for mediating the craniofacial-specific enhancer activity of this region, and that an extremely well-conserved 12-bp sequence within this element (CTGTCAAACAGGT) substantially determines full enhancer function. Together, these data provide valuable new insights into the upstream genomic regulatory landscape responsible for transcriptional control of Grhl2 during palatal closure.
Assuntos
Elementos Facilitadores Genéticos/genética , Loci Gênicos , Neurulação/genética , Palato/embriologia , Fatores de Transcrição/genética , Alelos , Animais , Feminino , Deleção de Genes , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The transcription factor pleomorphic adenoma gene 1 (PLAG1) is required for male fertility. Mice deficient in PLAG1 exhibit decreased sperm motility and abnormal epididymal tubule elongation and coiling, indicating impaired sperm maturation during epididymal transit. However, the downstream transcriptomic profile of the Plag1 knockout (KO; Plag1-/- ) murine epididymis is currently unknown. RESULTS: In this study, the PLAG1-dependent epididymal transcriptome was characterised using RNA sequencing. Several genes important for the control of sperm maturation, motility, capacitation and the acrosome reaction were dysregulated in Plag1-/- mice. Surprisingly, several cell proliferation genes were upregulated, and Ki67 analysis indicated that cell proliferation is aberrantly upregulated in the cauda epididymis stroma of Plag1-/- mice. Gene ontology analysis showed an overall upregulation of genes encoding extracellular matrix components, and an overall downregulation of genes encoding metalloendopeptidases in the epididymides from Plag1-/- mice. CONCLUSION: Together, these results suggest a defect in the epididymal extracellular matrix in Plag1-/- mice. These results imply that in addition to maintaining epididymal integrity directly, PLAG1 may also regulate several genes involved in the regulation of sperm maturation and capacitation. Moreover, PLAG1 may also be involved in regulating tissue homeostasis and ensuring proper structure and maintenance of the extracellular matrix in the epididymis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Epididimo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Maturação do Esperma/genética , Transcriptoma , Animais , Proteínas de Ligação a DNA/genética , Proteínas da Matriz Extracelular/genética , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos KnockoutRESUMO
OBJECTIVE: Sodium valproate (VPA), the most effective antiepileptic drug for patients with genetic generalized epilepsy (GGE), is a potent human teratogen that increases the risk of a range of congenital malformations, including spina bifida. The mechanisms underlying this teratogenicity are not known, but may involve genetic risk factors. This study aimed to develop an animal model of VPA-induced birth defects. METHODS: We used three different rat strains: inbred Genetic Absence Epilepsy Rats From Strasbourg (GAERS), a model of GGE with absence seizures; inbred Non-Epileptic Controls (NEC); and outbred nonepileptic Wistars. Female rats were fed standard chow or VPA (20 g/kg food) mixed in standard chow for 2 weeks prior to conception, and then mated with same-strain males. Treatment continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 and examined for birth defects, including external assessment and spinal measurements. RESULTS: VPA-exposed pups showed significant reductions in weight, length, and whole-body development compared with controls of all three strains (P < .0001). Gestational VPA treatment altered intravertebral distances, and resulted in underdeveloped vertebral arches between thoracic region T11 and caudal region C2 in most pups (GAERS, 100%; NEC, 95%; Wistar, 80%), more frequently than in controls (9%, 13%, 19%). SIGNIFICANCE: Gestational VPA treatment results in similar developmental and morphological abnormalities in three rat strains, including one with GGE, indicating that the genetic underpinnings of epilepsy do not contribute markedly to VPA-induced birth defects. This model may be used in future studies to investigate mechanisms involved in the pathogenesis of antiepileptic drug-induced birth defects.
Assuntos
Anticonvulsivantes/toxicidade , Modelos Animais de Doenças , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Teratogênicos/toxicidade , Ácido Valproico/toxicidade , Anormalidades Induzidas por Medicamentos/patologia , Administração Oral , Animais , Feminino , Masculino , Gravidez , Ratos , Ratos Transgênicos , Ratos WistarRESUMO
Krüppel-like factor 4 (KLF4) is a transcription factor highly conserved in evolution. It is particularly well known for its role in inducing pluripotent stem cells. In addition, KLF4 plays many roles in cancer. The results of most studies suggest that KLF4 is a tumor suppressor. However, the functioning of KLF4 is regulated at many levels. These include regulation of transcription, alternative splicing, miRNA, post-translational modifications, subcellular localization, protein stability and interactions with other molecules. Simple experiments aimed at assaying transcript levels or protein levels fail to address this complexity and thus may deliver misleading results. Tumor subtypes are also important; for example, in prostate cancer KLF4 is highly expressed in indolent tumors where it impedes tumor progression, while it is absent from aggressive prostate tumors. KLF4 is important in regulating response to many known drugs, and it also plays a role in tumor microenvironment. More and more information is available about upstream regulators, downstream targets and signaling pathways associated with the involvement of KLF4 in cancer. Furthermore, KLF4 performs critical function in the overall regulation of tissue homeostasis, cellular integrity, and progression towards malignancy. Here we summarize and analyze the latest findings concerning this fascinating transcription factor.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genéticaRESUMO
The highly conserved transcription factor Grainyhead-like 2 (Grhl2) exhibits a dynamic expression pattern in lung epithelium throughout embryonic development. Using a conditional gene targeting approach to delete Grhl2 in the developing lung epithelium, our results demonstrate that Grhl2 plays multiple roles in lung morphogenesis that are essential for respiratory function. Loss of Grhl2 leads to impaired ciliated cell differentiation and perturbed formation of terminal saccules. Critically, a substantial increase in Sox9-positive distal tip progenitor cells was observed following loss of Grhl2, suggesting that Grhl2 plays an important role in branching morphogenesis. Gene transcription profiling of Grhl2-deficient lung epithelial cells revealed a significant down regulation of Elf5, a member of the Ets family of transcription factors. Furthermore, ChIP and comparative genomic analyzes confirmed that Elf5 is a direct transcriptional target of Grhl2. Taken together, these results support the hypothesis that Grhl2 controls normal lung morphogenesis by tightly regulating the activity of distal tip progenitor cells.
Assuntos
Células Epiteliais Alveolares/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Células Epiteliais Alveolares/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Epitélio/metabolismo , Perfilação da Expressão Gênica , Pulmão/embriologia , Pulmão/metabolismo , Pulmão/fisiologia , Camundongos/embriologia , Testes de Função Respiratória/métodos , Fatores de Transcrição SOX9 , Sáculo e Utrículo/metabolismoRESUMO
The two main mechanisms that expand the proteomic output of eukaryotic genes are alternative splicing and alternative translation initiation signals. Despite being essential to generate isoforms of gene products that create functional diversity during development, the impact of these mechanisms on fine-tuning regulatory gene networks is still underappreciated. In this review, we use the Grainyhead-like (Grhl) family as a case study to illustrate the importance of isoforms when investigating transcription factor family function during development and disease, and highlight the potential for differential modulation of downstream target genes. We provide insights into the importance of considering alternative gene products when designing, undertaking, and analysing primary research, and the effect that isoforms may have on development. This review also covers known mutations in Grhl family members, and postulates how genetic changes may dictate transcriptional specificity between the Grhl family members. It also contrasts and compares the available literature on the function and importance of the Grhl isoforms, and highlights current gaps in our understanding of their regulatory gene networks in development and disease.
Assuntos
Processamento Alternativo/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Animais , Humanos , Mutação/genética , Domínios Proteicos , Processamento de Proteína Pós-Traducional/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Increased apposition of the frontal and parietal bones of the skull during embryogenesis may be a risk factor for the subsequent development of premature skull fusion, or craniosynostosis. Human craniosynostosis is a prevalent, and often serious embryological and neonatal pathology. Other than known mutations in a small number of contributing genes, the aetiology of craniosynostosis is largely unknown. Therefore, the identification of novel genes which contribute to normal skull patterning, morphology and premature suture apposition is imperative, in order to fully understand the genetic regulation of cranial development. RESULTS: Using advanced imaging techniques and quantitative measurement, we show that genetic deletion of the highly-conserved transcription factor Grainyhead-like 3 (Grhl3) in mice (Grhl3 -/- ) leads to decreased skull size, aberrant skull morphology and premature apposition of the coronal sutures during embryogenesis. Furthermore, Grhl3 -/- mice also present with premature collagen deposition and osteoblast alignment at the sutures, and the physical interaction between the developing skull, and outermost covering of the brain (the dura mater), as well as the overlying dermis and subcutaneous tissue, appears compromised in embryos lacking Grhl3. Although Grhl3 -/- mice die at birth, we investigated skull morphology and size in adult animals lacking one Grhl3 allele (heterozygous; Grhl3 +/- ), which are viable and fertile. We found that these adult mice also present with a smaller cranial cavity, suggestive of post-natal haploinsufficiency in the context of cranial development. CONCLUSIONS: Our findings show that our Grhl3 mice present with increased apposition of the frontal and parietal bones, suggesting that Grhl3 may be involved in the developmental pathogenesis of craniosynostosis.
Assuntos
Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Osso Frontal/metabolismo , Osso Parietal/metabolismo , Fatores de Transcrição/genética , Animais , Suturas Cranianas/anormalidades , Suturas Cranianas/metabolismo , Craniossinostoses/embriologia , Craniossinostoses/metabolismo , Proteínas de Ligação a DNA/deficiência , Desenvolvimento Embrionário/genética , Osso Frontal/anormalidades , Osso Frontal/diagnóstico por imagem , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Camundongos Knockout , Osso Parietal/anormalidades , Osso Parietal/diagnóstico por imagem , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Risco , Crânio/anormalidades , Crânio/metabolismo , Fatores de Transcrição/deficiência , Microtomografia por Raio-XRESUMO
The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.
Assuntos
Proteínas de Transporte/metabolismo , Mesencéfalo/crescimento & desenvolvimento , Morfogênese , Rombencéfalo/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Apoptose , Proteínas de Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Mesencéfalo/metabolismo , Morfolinos/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Filogenia , Rombencéfalo/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The midbrain-hindbrain boundary (MHB) is a highly conserved vertebrate signalling centre, acting to pattern and establish neural identities within the brain. While the core signalling pathways regulating MHB formation have been well defined, novel genetic and mechanistic processes that interact with these core components are being uncovered, helping to further elucidate the complicated networks governing MHB specification, patterning and shaping. Although formation of the MHB organiser is traditionally thought of as comprising three stages, namely positioning, induction and maintenance, we propose that a fourth stage, morphogenesis, should be considered as an additional stage in MHB formation. This review will examine evidence for novel factors regulating the first three stages of MHB development and will explore the evidence for regulation of MHB morphogenesis by non-classical MHB-patterning genes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/anatomia & histologia , Rombencéfalo/anatomia & histologia , Animais , Padronização Corporal , Humanos , Mesencéfalo/metabolismo , Camundongos , Modelos Neurológicos , Morfogênese/fisiologia , Rombencéfalo/metabolismo , Transdução de Sinais , Peixe-ZebraRESUMO
This paper discusses the evidence for the role of CREB in neural stem/progenitor cell (NSPC) function and oncogenesis and how these functions may be important for the development and growth of brain tumours. The cyclic-AMP response element binding (CREB) protein has many roles in neurons, ranging from neuronal survival to higher order brain functions such as memory and drug addiction behaviours. Recent studies have revealed that CREB also has a role in NSPC survival, differentiation and proliferation. Recent work has shown that over-expression of CREB in transgenic animals can impart oncogenic properties on cells in various tissues and that aberrant CREB expression is associated with tumours in patients. It is the central position of CREB, downstream of key developmental and growth signalling pathways, which give CREB the ability to influence a spectrum of cell activities, such as cell survival, growth and differentiation in both normal and cancer cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Humanos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Neural stem-progenitor cells (NSPCs) are multipotent, self-renewing cells that generate radial glial cells (RGC). RGCs then give rise to neurons and glia during neural development. Here, we describe the process of NSPC isolation and culturing to form clonal aggregates termed neurospheres. There are multiple assays outlined in this chapter that allow us to quantify differences in proliferation, self-renewal potential, and differentiation of these cells.
Assuntos
Células-Tronco Neurais , Neurônios , Neuroglia , Diferenciação Celular/fisiologia , Células-Tronco Multipotentes , Células Cultivadas , Proliferação de CélulasRESUMO
Cryopreservation and immunohistochemistry offer a comprehensive, robust, and simple methodology to investigate neural patterning and cellular function. Rapid freezing of the whole brain allows excellent preservation of neural ultrastructure and tissue architecture without destroying sensitive protein epitopes that are often compromised following standard paraffin embedding histological techniques. Here, we present a rapid and simple protocol for employing cryosectioning and subsequent immunohistochemistry in the study of adult murine brain neural tissue.
Assuntos
Criopreservação , Crioultramicrotomia , Animais , Camundongos , Congelamento , Imuno-Histoquímica , Criopreservação/métodos , EncéfaloRESUMO
Whole-mount in situ hybridization is cable to harness the inherent advantages of zebrafish as a model organism for developmental biology, particularly when visualizing the formation of the neural tube, specifically at the level of the midbrain-hindbrain boundary. The size and transparency of developing zebrafish embryos allow for the visualization of neural markers in vivo along the length of the developing zebrafish central nervous system. In practice, this technique is useful for examining defects in neurulation and midbrain-hindbrain boundary formation that may arise following gene manipulation, for example, CRISPR mutagenesis. This method describes the process of embryo collection and preparation, RNA probe transcription, probe hybridization in vivo, as well as the process of probe detection and visualization.
Assuntos
Neurulação , Peixe-Zebra , Animais , Peixe-Zebra/genética , Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo , Rombencéfalo , Hibridização In SituRESUMO
Contaminants are increasingly accumulating in aquatic environments and biota, with potential adverse effects on individual organisms, communities and ecosystems. However, studies that explore the molecular changes in fish caused by environmentally relevant concentrations of metals, such as copper (Cu), are limited. This study uses embryos of the model organism zebrafish (Danio rerio) to investigate effect of Cu on the proteome and amino acid (AA) composition of fish. Wild-type embryos at 24 h post-fertilisation were exposed to Cu (2 µg L-1 to 120 µg L-1) for 96 h and the number of healthy larvae were determined based on larvae that had hatched and did not display loss of equilibrium (LOE). The effect concentrations where Cu caused a 10 % (EC10) or 50 % (EC50) decrease in the number of healthy larvae were calculated as 3.7 µg L-1 and 10.9 µg L-1, respectively. Proteomics analysis of embryos exposed to the EC10 and EC50 concentrations of Cu revealed the proteome to differ more strongly after 48 h than 96 h, suggesting the acclimatisation of some larvae. Exposure to excess Cu caused differentially expressed proteins (DEPs) involved in oxidative stress, mitochondrial respiration, and neural transduction as well as the modulation of the AAs (Proline, Glycine and Alanine). This is the first study to suggest that LOE displayed by Cu-stressed fish may involve the disruption to GABAergic proteins and the calcium-dependent inhibitory neurotransmitter GABA. Moreover, this study highlights that proteomics and AA analysis can be used to identify potential biomarkers for environmental monitoring.