RESUMO
Colon cancers frequently harbor KRAS mutations, yet only a subset of KRAS mutant colon cancer cell lines are dependent upon KRAS signaling for survival. In a screen for kinases that promote survival of KRAS-dependent colon cancer cells, we found that the TAK1 kinase (MAP3K7) is required for tumor cell viability. The induction of apoptosis by RNAi-mediated depletion or pharmacologic inhibition of TAK1 is linked to its suppression of hyperactivated Wnt signaling, evident in both endogenous and genetically reconstituted cells. In APC mutant/KRAS-dependent cells, KRAS stimulates BMP-7 secretion and BMP signaling, leading to TAK1 activation and enhancement of Wnt-dependent transcription. An in vitro-derived "TAK1 dependency signature" is enriched in primary human colon cancers with mutations in both APC and KRAS, suggesting potential clinical utility in stratifying patient populations. Together, these findings identify TAK1 inhibition as a potential therapeutic strategy for a treatment-refractory subset of colon cancers exhibiting aberrant KRAS and Wnt pathway activation.
Assuntos
Neoplasias do Colo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Via de Sinalização Wnt , Proteínas ras/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Apoptose , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/química , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Perfilação da Expressão Gênica , Vida Livre de Germes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Transplante de Neoplasias , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Interferência de RNA , Ativação Transcricional , Transplante Heterólogo , Células Tumorais Cultivadas , beta Catenina/genética , Proteínas ras/genéticaRESUMO
Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible modular system. Here, we establish several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2 and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Finally, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker that is active before hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish and other models.
Assuntos
Técnicas de Transferência de Genes , Peixe-Zebra , Animais , Camundongos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais Geneticamente Modificados , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Elementos de DNA Transponíveis/genéticaRESUMO
Bcl9 and Pygopus (Pygo) are obligate Wnt/ß-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, ß-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the ß-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective ß-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.
Assuntos
Cardiopatias Congênitas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Coração/embriologia , Camundongos , Mutação , Miocárdio/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. RESULTS: Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of the two additional Switch reporters actb2:BFP-DsRed and actb2:Stop-DsRed. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. CONCLUSIONS: Our data documents the heterogeneity among lox-based Switch transgenes toward informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.
Assuntos
Integrases , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Cromatina/metabolismo , Genômica , Integrases/genética , Integrases/metabolismo , Tamoxifeno , Transgenes , Peixe-Zebra/metabolismoRESUMO
CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.
Assuntos
Sistemas CRISPR-Cas/genética , Complexos Multiproteicos/metabolismo , Mutagênese/genética , Ribonucleoproteínas/metabolismo , Alelos , Animais , Sequência de Bases , Sítios de Ligação , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fluorescência , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Morfolinos/farmacologia , Mutação/genética , Fenótipo , RNA Guia de Cinetoplastídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética/genética , Solubilidade , Fatores de Transcrição/metabolismo , Transgenes , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The casper strain of zebrafish is widely used in studies ranging from cancer to neuroscience. casper offers the advantage of relative transparency throughout adulthood, making it particularly useful for in vivo imaging by epifluorescence, confocal, and light sheet microscopy. casper was developed by selective breeding of two previously described recessive pigment mutants: 1) nacre, which harbors an inactivating mutation of the mitfa gene, rendering the fish devoid of pigmented melanocytes; and 2) roy orbison, a mutant with a so-far unidentified genetic cause that lacks reflective iridophores. To clarify the molecular nature of the roy orbison mutation, such that it can inform studies using casper, we undertook an effort to positionally clone the roy orbison mutation. We find that roy orbison is caused by an intronic defect in the gene mpv17, encoding an inner mitochondrial membrane protein that has been implicated in the human mitochondrial DNA depletion syndrome. The roy orbison mutation is phenotypically and molecularly remarkably similar to another zebrafish iridophore mutant called transparent. Using Cas9-induced crispants and germline mutants with a disrupted mpv17 open reading frame, we show in trans-heterozygote embryos that new frameshift alleles of mpv17, roy orbison, and transparent fail to complement each other. Our work provides genetic evidence that both roy orbison and transparent affect the mpv17 locus by a similar if not identical genetic lesion. Identification of mpv17 mutants will allow for further work probing the relationship between mitochondrial function and pigmentation, which has to date received little attention.
Assuntos
Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Mutação/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Alelos , Animais , Pareamento de Bases/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Mapeamento Cromossômico , DNA Mitocondrial/genética , Técnicas de Silenciamento de Genes , Loci Gênicos , Proteínas Mitocondriais/metabolismo , Morfolinos/farmacologia , Mutagênese/genética , Fenótipo , Pigmentação/efeitos dos fármacos , Pigmentação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Standard zebrafish transgenesis involves random transgene integration with resource-intensive screening. While phiC31 integrase-based attP/attB recombination has streamlined transgenesis in mice and Drosophila, validated attP-based landing sites for universal applications are lacking in zebrafish. Here, we developed phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET) as transgenesis approach, with two attP landing sites pIGLET14a and pIGLET24b from well-validated Tol2 transgenes. Both sites facilitate diverse transgenesis applications including reporters and Cre/loxP transgenes. The pIGLET14a and pIGLET24b landing sites consistently yield 25 to 50% germline transmission, substantially reducing the resources needed for transgenic line generation. Transgenesis into these sites enables reproducible expression patterns in F0 zebrafish embryos for enhancer discovery and testing of gene regulatory variants. Together, our new landing sites streamline targeted, reproducible zebrafish transgenesis as a robust platform for various applications while minimizing the workload for generating transgenic lines.
Assuntos
Animais Geneticamente Modificados , Técnicas de Transferência de Genes , Transgenes , Peixe-Zebra , Animais , Peixe-Zebra/genética , Integrases/genética , Integrases/metabolismo , Sítios de Ligação Microbiológicos/genéticaRESUMO
Defects in blood development frequently occur among syndromic congenital anomalies. Thrombocytopenia-Absent Radius (TAR) syndrome is a rare congenital condition with reduced platelets (hypomegakaryocytic thrombocytopenia) and forelimb anomalies, concurrent with more variable heart and kidney defects. TAR syndrome associates with hypomorphic gene function for RBM8A/Y14 that encodes a component of the exon junction complex involved in mRNA splicing, transport, and nonsense-mediated decay. How perturbing a general mRNA-processing factor causes the selective TAR Syndrome phenotypes remains unknown. Here, we connect zebrafish rbm8a perturbation to early hematopoietic defects via attenuated non-canonical Wnt/Planar Cell Polarity (PCP) signaling that controls developmental cell re-arrangements. In hypomorphic rbm8a zebrafish, we observe a significant reduction of cd41-positive thrombocytes. rbm8a-mutant zebrafish embryos accumulate mRNAs with individual retained introns, a hallmark of defective nonsense-mediated decay; affected mRNAs include transcripts for non-canonical Wnt/PCP pathway components. We establish that rbm8a-mutant embryos show convergent extension defects and that reduced rbm8a function interacts with perturbations in non-canonical Wnt/PCP pathway genes wnt5b, wnt11f2, fzd7a, and vangl2. Using live-imaging, we found reduced rbm8a function impairs the architecture of the lateral plate mesoderm (LPM) that forms hematopoietic, cardiovascular, kidney, and forelimb skeleton progenitors as affected in TAR Syndrome. Both mutants for rbm8a and for the PCP gene vangl2 feature impaired expression of early hematopoietic/endothelial genes including runx1 and the megakaryocyte regulator gfi1aa. Together, our data propose aberrant LPM patterning and hematopoietic defects as consequence of attenuated non-canonical Wnt/PCP signaling upon reduced rbm8a function. These results also link TAR Syndrome to a potential LPM origin and a developmental mechanism.
RESUMO
Syndromic birth defects are rare diseases that can present with seemingly pleiotropic comorbidities. Prime examples are rare congenital heart and cardiovascular anomalies that can be accompanied by forelimb defects, kidney disorders and more. Whether such multi-organ defects share a developmental link remains a key question with relevance to the diagnosis, therapeutic intervention and long-term care of affected patients. The heart, endothelial and blood lineages develop together from the lateral plate mesoderm (LPM), which also harbors the progenitor cells for limb connective tissue, kidneys, mesothelia and smooth muscle. This developmental plasticity of the LPM, which founds on multi-lineage progenitor cells and shared transcription factor expression across different descendant lineages, has the potential to explain the seemingly disparate syndromic defects in rare congenital diseases. Combining patient genome-sequencing data with model organism studies has already provided a wealth of insights into complex LPM-associated birth defects, such as heart-hand syndromes. Here, we summarize developmental and known disease-causing mechanisms in early LPM patterning, address how defects in these processes drive multi-organ comorbidities, and outline how several cardiovascular and hematopoietic birth defects with complex comorbidities may be LPM-associated diseases. We also discuss strategies to integrate patient sequencing, data-aggregating resources and model organism studies to mechanistically decode congenital defects, including potentially LPM-associated orphan diseases. Eventually, linking complex congenital phenotypes to a common LPM origin provides a framework to discover developmental mechanisms and to anticipate comorbidities in congenital diseases affecting the cardiovascular system and beyond.
Assuntos
Doenças Cardiovasculares , Cardiopatias Congênitas , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Padronização Corporal/genética , Coração , Cardiopatias Congênitas/genética , Fatores de Transcrição/metabolismo , Mesoderma/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Standard methods for transgenesis in zebrafish depend on random transgene integration into the genome followed by resource-intensive screening and validation. Targeted vector integration into validated genomic loci using phiC31 integrase-based attP/attB recombination has transformed mouse and Drosophila transgenesis. However, while the phiC31 system functions in zebrafish, validated loci carrying attP-based landing or safe harbor sites suitable for universal transgenesis applications in zebrafish have not been established. Here, using CRISPR-Cas9, we converted two well-validated single insertion Tol2-based zebrafish transgenes with long-standing genetic stability into two attP landing sites, called phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET). Generating fluorescent reporters, loxP-based Switch lines, CreERT2 drivers, and gene-regulatory variant reporters in the pIGLET14a and pIGLET24b landing site alleles, we document their suitability for transgenesis applications across cell types and developmental stages. For both landing sites, we routinely achieve 25-50% germline transmission of targeted transgene integrations, drastically reducing the number of required animals and necessary resources to generate individual transgenic lines. We document that phiC31 integrase-based transgenesis into pIGLET14a and pIGLET24b reproducibly results in representative reporter expression patterns in injected F0 zebrafish embryos suitable for enhancer discovery and qualitative and quantitative comparison of gene-regulatory element variants. Taken together, our new phiC31 integrase-based transgene landing sites establish reproducible, targeted zebrafish transgenesis for numerous applications while greatly reducing the workload of generating new transgenic zebrafish lines.
RESUMO
The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.
Assuntos
Notocorda , Peixe-Zebra , Animais , Humanos , Camundongos , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/genética , Notocorda/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The cell type-specific expression of key transcription factors is central to development. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three Brachyury-controlling notochord enhancers T3, C, and I in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, deletion of all three enhancers in mouse abolishes Brachyury/T expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. Sequence and functional conservation of Brachyury-driving notochord enhancers with the brachyury/tbxtb loci from diverse lineages of fishes dates their origin to the last common ancestor of jawed vertebrates. Our data define the enhancers for Brachyury/T/TBXTB notochord expression as ancient mechanism in axis development.
RESUMO
WTX encodes a tumor suppressor gene inactivated in Wilms tumor and recently implicated in WNT signaling through enhancement of cytoplasmic beta-catenin (CTNNB1) degradation. Here, we report that WTX translocates to the nucleus, a property that is modified by an endogenous splicing variant and is modulated by a nuclear export inhibitor. WTX is present in distinct subnuclear structures and co-localizes with the paraspeckle marker p54NRB/NONO, suggesting a role in transcriptional regulation. Notably, WTX binds WT1, another Wilms tumor suppressor and stem cell marker that encodes a zinc-finger transcription factor, and enhances WT1-mediated transcription of Amphiregulin, an endogenous target gene. Together, these observations suggest a role for WTX in nuclear pathways implicated in the transcriptional regulation of cellular differentiation programs.
Assuntos
Transporte Ativo do Núcleo Celular , Proteínas Supressoras de Tumor/metabolismo , Proteínas WT1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Anfirregulina , Linhagem Celular , Família de Proteínas EGF , Regulação da Expressão Gênica , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligação Proteica , Isoformas de Proteínas , Fatores de Transcrição , Transcrição Gênica , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas WT1/análiseRESUMO
Endothelial specification is a key event during embryogenesis; however, when, and how, endothelial cells separate from other lineages is poorly understood. In zebrafish, Npas4l is indispensable for endothelial specification by inducing the expression of the transcription factor genes etsrp, tal1, and lmo2. We generated a knock-in reporter in zebrafish npas4l to visualize endothelial progenitors and their derivatives in wild-type and mutant embryos. Unexpectedly, we find that in npas4l mutants, npas4l reporter-expressing cells contribute to the pronephron tubules. Single-cell transcriptomics and live imaging of the early lateral plate mesoderm in wild-type embryos indeed reveals coexpression of endothelial and pronephron markers, a finding confirmed by creERT2-based lineage tracing. Increased contribution of npas4l reporter-expressing cells to pronephron tubules is also observed in tal1 and lmo2 mutants and is reversed in npas4l mutants injected with tal1 mRNA. Together, these data reveal that Npas4l/Tal1/Lmo2 regulate the fate decision between the endothelial and pronephron lineages.
RESUMO
Crescentic glomerulonephritis is characterized by vascular necrosis and parietal epithelial cell hyperplasia in the space surrounding the glomerulus, resulting in the formation of crescents. Little is known about the molecular mechanisms driving this process. Inducing crescentic glomerulonephritis in two Pax2Cre reporter mouse models revealed that crescents derive from clonal expansion of single immature parietal epithelial cells. Preemptive and delayed histone deacetylase inhibition with panobinostat, a drug used to treat hematopoietic stem cell disorders, attenuated crescentic glomerulonephritis with recovery of kidney function in the two mouse models. Three-dimensional confocal microscopy and stimulated emission depletion superresolution imaging of mouse glomeruli showed that, in addition to exerting an anti-inflammatory and immunosuppressive effect, panobinostat induced differentiation of an immature hyperplastic parietal epithelial cell subset into podocytes, thereby restoring the glomerular filtration barrier. Single-cell RNA sequencing of human renal progenitor cells in vitro identified an immature stratifin-positive cell subset and revealed that expansion of this stratifin-expressing progenitor cell subset was associated with a poor outcome in human crescentic glomerulonephritis. Treatment of human parietal epithelial cells in vitro with panobinostat attenuated stratifin expression in renal progenitor cells, reduced their proliferation, and promoted their differentiation into podocytes. These results offer mechanistic insights into the formation of glomerular crescents and demonstrate that selective targeting of renal progenitor cells can attenuate crescent formation and the deterioration of kidney function in crescentic glomerulonephritis in mice.
Assuntos
Glomerulonefrite , Podócitos , Animais , Modelos Animais de Doenças , Glomerulonefrite/tratamento farmacológico , Humanos , Rim/metabolismo , Camundongos , Panobinostat/uso terapêutico , Podócitos/metabolismo , Células-Tronco/metabolismoRESUMO
The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies.
Assuntos
Mesotelioma , Peixe-Zebra , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Epitélio/metabolismo , Mesotelioma/genética , Camundongos , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The paired-box transcription factor Pax2 plays a major role in early development of the kidney and the central nervous system. It is expressed in the metanephric mesenchyme of the developing kidney, at the midbrain-hindbrain boundary and the anlagen of the inner ear. The early expression of Pax2, especially in the developing kidney, prompted us to use this locus as a novel genetic tool to introduce temporally-controlled expression of transgenes. We generated a transgenic Pax2-rtTA mouse strain through genetic recombineering using a large BAC clone which drives expression of TetO-controlled transgenes upon doxycycline treatment in natively Pax2-expressing tissues. We show that expression of a TetO-responsive lacZ gene is tightly regulated by addition of doxycycline and can be detected in all Pax2-expressing tissues. Our transgenic Pax2-rtTA mouse thus represents a suitable tool to study the cell fates and molecular pathways in Pax2-positive tissues during development, such as the kidney. We further propose that the Pax2-rtTA tool has great potential to induce time-controlled, tissue-specific alterations for tumorigenic transformation of Pax2-expressing cells for generating in vivo tumor models, such as Wilms tumor.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Doxiciclina/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição PAX2/genética , Animais , Cromossomos Artificiais Bacterianos , Proteínas de Ligação a DNA/metabolismo , Rim/embriologia , Rim/metabolismo , Óperon Lac , Camundongos , Camundongos Transgênicos , Fator de Transcrição PAX2/metabolismo , Regiões Promotoras Genéticas , TransgenesRESUMO
CRISPR-Cas9-based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms-often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.
Assuntos
Cromossomos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Drosophila/genética , Edição de Genes , Expressão Gênica , Marcação de Genes , Genes Reporter , Conformação de Ácido Nucleico , Fenótipo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The aberrant activation of developmental processes triggers diverse cancer types. Chordoma is a rare, aggressive tumor arising from transformed notochord remnants. Several potentially oncogenic factors have been found to be deregulated in chordoma, yet causation remains uncertain. In particular, sustained expression of TBXT - encoding the notochord regulator protein brachyury - is hypothesized as a key driver of chordoma, yet experimental evidence is absent. Here, we employ a zebrafish chordoma model to identify the notochord-transforming potential of implicated genes in vivo We find that Brachyury, including a form with augmented transcriptional activity, is insufficient to initiate notochord hyperplasia. In contrast, the chordoma-implicated receptor tyrosine kinases (RTKs) EGFR and Kdr/VEGFR2 are sufficient to transform notochord cells. Aberrant activation of RTK/Ras signaling attenuates processes required for notochord differentiation, including the unfolded protein response and endoplasmic reticulum stress pathways. Our results provide the first in vivo evidence against a tumor-initiating potential of Brachyury in the notochord, and imply activated RTK signaling as a possible initiating event in chordoma. Furthermore, our work points at modulating endoplasmic reticulum and protein stress pathways as possible therapeutic avenues against chordoma.
Assuntos
Cordoma/enzimologia , Proteínas Fetais/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Cordoma/genética , Notocorda/enzimologia , Notocorda/patologia , Oncogenes , Peixe-ZebraRESUMO
Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.