RESUMO
The structure and mechanism of the bacterial enzyme ß-lactamase have been well-studied due to its clinical role in antibiotic resistance. ß-Lactamase is known to hydrolyze the ß-lactam ring of the cephalosporin scaffold, allowing a spontaneous self-immolation to occur. Previously, cephalosporin-based sensors have been developed to evaluate ß-lactamase expression in both mammalian cells and zebrafish embryos. Here, we present a circular caged morpholino oligonucleotide (cMO) activated by ß-lactamase-mediated cleavage of a cephalosporin motif capable of silencing the expression of T-box transcription factor Ta (tbxta), also referred to as no tail a (ntla), eliciting a distinct, observable phenotype. We explore the use of ß-lactamase to elicit a biological response in aquatic embryos for the first time and expand the utility of cephalosporin as a cleavable linker beyond targeting antibiotic-resistant bacteria. The addition of ß-lactamase to the current suite of enzymatic triggers presents unique opportunities for robust, orthogonal control over endogenous gene expression in a spatially resolved manner.
Assuntos
Oligonucleotídeos Antissenso , Peixe-Zebra , Animais , Oligonucleotídeos Antissenso/farmacologia , Peixe-Zebra/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Cefalosporinas/metabolismo , beta-Lactamases/metabolismo , Bactérias/metabolismo , Resistência Microbiana a Medicamentos , Expressão Gênica , Inibidores de beta-Lactamases , Testes de Sensibilidade Microbiana , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Caged morpholino oligonucleotides (cMOs) are synthetic tools that allow light-inducible gene silencing in live organisms. Previously reported cMOs have utilized hairpin, duplex, and cyclic structures, as well as caged nucleobases. While these antisense technologies enable efficient optical control of RNA splicing and translation, they can have limited dynamic range. A new caging strategy was developed where the two MO termini are conjugated to an internal position through a self-immolative trifunctional linker, thereby generating a bicyclic cMO that is conformationally resistant to RNA binding. The efficacy of this alternative cMO design has been demonstrated in zebrafish embryos and compared to linear MOs and monocyclic constructs.
Assuntos
Inativação Gênica , Peixe-Zebra , Animais , Morfolinos/química , Peixe-Zebra/genéticaRESUMO
Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions and yield leads for developing ALDH1B1-targeting therapies.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Aldeído Desidrogenase/química , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Aldeídos , Neoplasias do Colo/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Guanidinas , Humanos , Sondas Moleculares , Proteoma/genéticaRESUMO
Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small molecule-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger reduction. This is enabled by a highly flexible linker design that offers opportunities for the installation of chemically activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small molecule-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Animais , Corantes Fluorescentes , Técnicas de Silenciamento de Genes , Genes Controladores do Desenvolvimento , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Rodaminas , Tionucleotídeos , Peixe-Zebra/embriologiaRESUMO
The presence of organic contaminants in wastewater poses considerable risks to the health of both humans and ecosystems. Although advanced oxidation processes that rely on highly reactive radicals to destroy organic contaminants are appealing treatment options, substantial energy and chemical inputs limit their practical applications. Here we demonstrate that Cu single atoms incorporated in graphitic carbon nitride can catalytically activate H2O2 to generate hydroxyl radicals at pH 7.0 without energy input, and show robust stability within a filtration device. We further design an electrolysis reactor for the on-site generation of H2O2 from air, water and renewable energy. Coupling the single-atom catalytic filter and the H2O2 electrolytic generator in tandem delivers a wastewater treatment system. These findings provide a promising path toward reducing the energy and chemical demands of advanced oxidation processes, as well as enabling their implementation in remote areas and isolated communities.
RESUMO
ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.
Assuntos
Cílios , Proteínas Ativadoras de GTPase , Transdução de Sinais , Animais , Cílios/química , Cílios/genética , Cílios/metabolismo , Proteínas Ativadoras de GTPase/biossíntese , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Domínios Proteicos , Isoformas de Proteínas , Relação Estrutura-AtividadeRESUMO
The unique photophysical properties of lanthanides, such as europium, terbium, and ytterbium, make them versatile molecular probes of biological systems. In particular, their long-lived photoluminescence, narrow bandwidth emissions, and large Stokes shifts enable experiments that are infeasible with organic fluorophores and fluorescent proteins. The ability of these metal ions to undergo luminescence resonance energy transfer, and photon upconversion further expands the capabilities of lanthanide probes. In this review, we describe recent advances in the design of lanthanide luminophores and their application in biological research. We also summarize the latest detection systems that have been developed to fully exploit the optical properties of lanthanide luminophores. We conclude with a discussion of remaining challenges and new frontiers in lanthanide technologies. The unprecedented levels of sensitivity and multiplexing afforded by rare-earth elements illustrate how chemistry can enable new approaches in biology.
Assuntos
Corantes Fluorescentes/química , Elementos da Série dos Lantanídeos/química , Animais , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência , Nanopartículas/química , Imagem Óptica , Proteínas/química , Proteínas/metabolismoRESUMO
In principle, the long emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. However, most lanthanide-imaging systems cannot achieve sensitivities that exceed those of conventional fluorescence microscopes, since they are limited by inefficient lanthanide excitation, the low photon flux of excited lanthanide luminophores, and optics-derived background photoluminescence. We recently reported a new lanthanide-imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which overcomes each of these constraints. Here we provide a detailed procedure for visualizing endogenous protein expression in zebrafish embryos, using lanthanide-labeled antibodies, Q-switched laser illumination, and trLRET microscopy. These methods allow ultrasensitive molecular imaging in cells and organisms, establishing a new paradigm for biological exploration and discovery.
Assuntos
Elementos da Série dos Lantanídeos , Animais , Quelantes , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência , Peixe-ZebraRESUMO
Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis-regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control.
Assuntos
Optogenética/métodos , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Axônios/efeitos dos fármacos , Axônios/fisiologia , Axônios/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Expressão Gênica/efeitos da radiação , Genes Reporter , Luz , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Nitrorredutases/genética , Nitrorredutases/metabolismo , Regiões Promotoras Genéticas , Rimantadina/farmacologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Gli transcription factors within the Hedgehog (Hh) signaling pathway direct key events in mammalian development and promote a number of human cancers. Current therapies for Gli-driven tumors target Smoothened (SMO), a G protein-coupled receptor-like protein that functions upstream in the Hh pathway. Although these drugs can have remarkable clinical efficacy, mutations in SMO and downstream Hh pathway components frequently lead to chemoresistance. In principle, therapies that act at the level of Gli proteins, through direct or indirect mechanisms, would be more efficacious. We therefore screened 325 120 compounds for their ability to block the constitutive Gli activity induced by loss of Suppressor of Fused (SUFU), a scaffolding protein that directly inhibits Gli function. Our studies reveal a family of bicyclic imidazolium derivatives that can inhibit Gli-dependent transcription without affecting the ciliary trafficking or proteolytic processing of these transcription factors. We anticipate that these chemical antagonists will be valuable tools for investigating the mechanisms of Gli regulation and developing new strategies for targeting Gli-driven cancers.
Assuntos
Imidazóis/farmacologia , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Animais , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imidazóis/síntese química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Estrutura Molecular , Células NIH 3T3 , Fosforilação Oxidativa/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Mammalian spermiogenesis is a remarkable cellular transformation, during which round spermatids elongate into chromatin-condensed spermatozoa. The signaling pathways that coordinate this process are not well understood, and we demonstrate here that homeodomain-interacting protein kinase 4 (HIPK4) is essential for spermiogenesis and male fertility in mice. HIPK4 is predominantly expressed in round and early elongating spermatids, and Hipk4 knockout males are sterile, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Hipk4 mutant sperm have reduced oocyte binding and are incompetent for in vitro fertilization, but they can still produce viable offspring via intracytoplasmic sperm injection. Optical and electron microscopy of HIPK4-null male germ cells reveals defects in the filamentous actin (F-actin)-scaffolded acroplaxome during spermatid elongation and abnormal head morphologies in mature spermatozoa. We further observe that HIPK4 overexpression induces branched F-actin structures in cultured fibroblasts and that HIPK4 deficiency alters the subcellular distribution of an F-actin capping protein in the testis, supporting a role for this kinase in cytoskeleton remodeling. Our findings establish HIPK4 as an essential regulator of sperm head shaping and potential target for male contraception.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Espermatogênese/genética , Acrossomo/metabolismo , Actinas/metabolismo , Animais , Fertilidade/genética , Imunofluorescência , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutação , Fenótipo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Espermátides/citologia , Espermátides/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismoRESUMO
Caged morpholino oligonucleotides (cMOs) are useful research tools in developmental biology because they allow spatiotemporal control of gene expression in whole organisms. While cMOs are usually triggered by light of a single wavelength, the introduction of spectrally distinct chromophores can enable combinatorial regulation of multiple genes. This chapter describes the general principles and methods of wavelength-selective cMO design and synthesis from commercially available reagents. Synthetic protocols for the linkers and the two-step cMO assembly are described in detail, as well as the microinjection and photoactivation techniques. Following these protocols, spectrally separated cyclic cMOs for multiple genes of interest can be prepared, enabling their inhibition in zebrafish embryos and other animal models.
Assuntos
Inativação Gênica , Morfolinos/genética , Oligonucleotídeos Antissenso/genética , Peixe-Zebra/genética , Animais , Técnicas de Química Sintética/métodos , Luz , Microinjeções , Morfolinos/administração & dosagem , Morfolinos/síntese química , Morfolinos/química , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Processos Fotoquímicos , Peixe-Zebra/embriologiaRESUMO
Basal constriction occurs at the zebrafish midbrain-hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3ß overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.
RESUMO
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.
Assuntos
Deficiência de Glucosefosfato Desidrogenase/tratamento farmacológico , Glucosefosfato Desidrogenase/metabolismo , Indóis/uso terapêutico , Animais , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/genética , Hemólise/efeitos dos fármacos , Humanos , Indóis/química , Indóis/farmacologia , Mutação de Sentido Incorreto , Estresse Oxidativo/efeitos dos fármacos , Estabilidade Proteica , Peixe-ZebraRESUMO
Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.
Assuntos
Cílios/fisiologia , Ciliopatias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Proteínas Hedgehog/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Animais , Cílios/genética , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos , Células NIH 3T3 , Transdução de Sinais/genéticaRESUMO
In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.
Assuntos
Complexos de Coordenação/química , Elementos da Série dos Lantanídeos/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Óptica/métodos , Proteínas de Peixe-Zebra/biossíntese , Animais , Complexos de Coordenação/síntese química , Elementos da Série dos Lantanídeos/síntese química , Substâncias Luminescentes/síntese química , Sensibilidade e Especificidade , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Mutations in GLI3, which encodes a transcription factor of the Hedgehog signaling pathway, cause several developmental anomalies linked to inappropriate tissue patterning. Here, we report a novel missense variant in the fifth zinc finger domain of GLI3 (c.1826G>A; p.(Cys609Tyr)) initially identified in a proband with preaxial polydactyly type IV, developmental delay, sensorineural hearing loss, skeletal, and genitourinary anomalies. Additional family members exhibited various digital anomalies such as preaxial polydactyly, syndactyly, and postaxial polydactyly either in isolation or combined. Functional studies of Cys609Tyr GLI3 in cultured cells showed abnormal GLI3 processing leading to decreased GLI3 repressor production, increased basal transcriptional activity, and submaximal GLI reporter activity with Hedgehog pathway activation, thus demonstrating an intriguing molecular mechanism for this GLI3-related phenotype. Given the complexity of GLI3 post-translational processing and opposing biological functions as a transcriptional activator and repressor, our findings highlight the importance of performing functional studies of presumed GLI3 variants. This family also demonstrates how GLI3 variants are variably expressed.
Assuntos
Acrocefalossindactilia/genética , Dedos/anormalidades , Proteínas do Tecido Nervoso/genética , Polidactilia/genética , Polegar/anormalidades , Dedos do Pé/anormalidades , Proteína Gli3 com Dedos de Zinco/genética , Acrocefalossindactilia/diagnóstico , Sequência de Aminoácidos , Animais , Pré-Escolar , Feminino , Fibroblastos , Genes Reporter , Técnicas de Genotipagem , Humanos , Camundongos , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Polidactilia/diagnóstico , Proteínas Repressoras/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Transdução de Sinais , Dedos de ZincoRESUMO
Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.
Assuntos
Dineínas/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Pirazóis/química , Quinazolinonas/químicaRESUMO
Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.
Assuntos
Biologia do Desenvolvimento/métodos , Biologia do Desenvolvimento/tendências , Fotoquímica , Biologia do Desenvolvimento/instrumentação , Genômica , Modelos Biológicos , Sondas Moleculares/metabolismo , Estrutura Molecular , Fotoquímica/tendências , Rodopsina/químicaRESUMO
We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.