RESUMO
Tight control over transcription factor activity is necessary for a sensible balance between cellular proliferation and differentiation in the embryo and during tissue homeostasis by adult stem cells, but mechanistic details have remained incomplete. The homeodomain transcription factor MEIS2 is an important regulator of neurogenesis in the ventricular-subventricular zone (V-SVZ) adult stem cell niche in mice. We here identify MEIS2 as direct target of the intracellular protease calpain-2 (composed of the catalytic subunit CAPN2 and the regulatory subunit CAPNS1). Phosphorylation at conserved serine and/or threonine residues, or dimerization with PBX1, reduced the sensitivity of MEIS2 towards cleavage by calpain-2. In the adult V-SVZ, calpain-2 activity is high in stem and progenitor cells, but rapidly declines during neuronal differentiation, which is accompanied by increased stability of MEIS2 full-length protein. In accordance with this, blocking calpain-2 activity in stem and progenitor cells, or overexpression of a cleavage-insensitive form of MEIS2, increased the production of neurons, whereas overexpression of a catalytically active CAPN2 reduced it. Collectively, our results support a key role for calpain-2 in controlling the output of adult V-SVZ neural stem and progenitor cells through cleavage of the neuronal fate determinant MEIS2.
Assuntos
Células-Tronco Neurais , Fatores de Transcrição , Animais , Camundongos , Calpaína/genética , Calpaína/metabolismo , Diferenciação Celular , Proliferação de Células , Endopeptidases/metabolismo , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Peptídeo Hidrolases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Genomics data is available to the scientific community after publication of research projects and can be investigated for a multitude of research questions. However, in many cases deposited data is only assessed and used for the initial publication, resulting in valuable resources not being exploited to their full depth. MAIN: A likely reason for this is that many wetlab-based researchers are not formally trained to apply bioinformatic tools and may therefore assume that they lack the necessary experience to do so themselves. In this article, we present a series of freely available, predominantly web-based platforms and bioinformatic tools that can be combined in analysis pipelines to interrogate different types of next-generation sequencing data. Additionally to the presented exemplary route, we also list a number of alternative tools that can be combined in a mix-and-match fashion. We place special emphasis on tools that can be followed and used correctly without extensive prior knowledge in programming. Such analysis pipelines can be applied to existing data downloaded from the public domain or be compared to the results of own experiments. CONCLUSION: Integrating transcription factor binding to chromatin (ChIP-seq) with transcriptional output (RNA-seq) and chromatin accessibility (ATAC-seq) can not only assist to form a deeper understanding of the molecular interactions underlying transcriptional regulation but will also help establishing new hypotheses and pre-testing them in silico.
Assuntos
Biologia Computacional , Genômica , Biologia Computacional/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Cromatina por Imunoprecipitação , RNA-SeqRESUMO
Regulation of adult neural stem cell (NSC) number is critical for lifelong neurogenesis. Here, we identified a post-transcriptional control mechanism, centered around the microRNA 204 (miR-204), to control the maintenance of quiescent (q)NSCs. miR-204 regulates a spectrum of transcripts involved in cell cycle regulation, neuronal migration, and differentiation in qNSCs. Importantly, inhibition of miR-204 function reduced the number of qNSCs in the subependymal zone (SEZ) by inducing pre-mature activation and differentiation of NSCs without changing their neurogenic potential. Strikingly, we identified the choroid plexus of the mouse lateral ventricle as the major source of miR-204 that is released into the cerebrospinal fluid to control number of NSCs within the SEZ. Taken together, our results describe a novel mechanism to maintain adult somatic stem cells by a niche-specific miRNA repressing activation and differentiation of stem cells.
Assuntos
Plexo Corióideo/química , MicroRNAs/genética , Células-Tronco Neurais/citologia , Adulto , Animais , Ciclo Celular , Diferenciação Celular , Movimento Celular , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , MicroRNAs/líquido cefalorraquidiano , Pessoa de Meia-Idade , Células-Tronco Neurais/química , Nicho de Células-TroncoRESUMO
MEIS transcription factors are key regulators of embryonic development and cancer. Research on MEIS genes in the embryo and in stem cell systems has revealed novel and surprising mechanisms by which these proteins control gene expression. This Primer summarizes recent findings about MEIS protein activity and regulation in development, and discusses new insights into the role of MEIS genes in disease, focusing on the pathogenesis of solid cancers.
Assuntos
Desenvolvimento Embrionário , Fatores de Transcrição/fisiologia , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Proteínas de Neoplasias/genética , Neoplasias/etiologia , Neoplasias/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
TALE-homeodomain proteins function as components of heteromeric complexes that contain one member each of the PBC and MEIS/PREP subclasses. We recently showed that MEIS2 cooperates with the neurogenic transcription factor PAX6 in the control of adult subventricular zone (SVZ) neurogenesis in rodents. Expression of the PBC protein PBX1 in the SVZ has been reported, but its functional role(s) has not been investigated. Using a genetic loss-of-function mouse model, we now show that Pbx1 is an early regulator of SVZ neurogenesis. Targeted deletion of Pbx1 by retroviral transduction of Cre recombinase into Pbx2-deficient SVZ stem and progenitor cells carrying floxed alleles of Pbx1 significantly reduced the production of neurons and increased the generation of oligodendrocytes. Loss of Pbx1 expression in neuronally committed neuroblasts in the rostral migratory stream in a Pbx2 null background, by contrast, severely compromised cell survival. By chromatin immunoprecipitation from endogenous tissues or isolated cells, we further detected PBX1 binding to known regulatory regions of the neuron-specific genes Dcx and Th days or even weeks before the respective genes are expressed during the normal program of SVZ neurogenesis, suggesting that PBX1 might act as a priming factor to mark these genes for subsequent activation. Collectively, our results establish that PBX1 regulates adult neural cell fate determination in a manner beyond that of its heterodimerization partner MEIS2.
Assuntos
Envelhecimento/metabolismo , Proteínas de Homeodomínio/metabolismo , Ventrículos Laterais/metabolismo , Neurogênese , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Elementos Facilitadores Genéticos/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Proteínas de Homeodomínio/genética , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Bulbo Olfatório/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Meis homeodomain transcription factors control cell proliferation, cell fate specification and differentiation in development and disease. Previous studies have largely focused on Meis contribution to the development of non-neuronal tissues. By contrast, Meis function in the brain is not well understood. Here, we provide evidence for a dual role of the Meis family protein Meis2 in adult olfactory bulb (OB) neurogenesis. Meis2 is strongly expressed in neuroblasts of the subventricular zone (SVZ) and rostral migratory stream (RMS) and in some of the OB interneurons that are continuously replaced during adult life. Targeted manipulations with retroviral vectors expressing function-blocking forms or with small interfering RNAs demonstrated that Meis activity is cell-autonomously required for the acquisition of a general neuronal fate by SVZ-derived progenitors in vivo and in vitro. Additionally, Meis2 activity in the RMS is important for the generation of dopaminergic periglomerular neurons in the OB. Chromatin immunoprecipitation identified doublecortin and tyrosine hydroxylase as direct Meis targets in newly generated neurons and the OB, respectively. Furthermore, biochemical analyses revealed a previously unrecognized complex of Meis2 with Pax6 and Dlx2, two transcription factors involved in OB neurogenesis. The full pro-neurogenic activity of Pax6 in SVZ derived neural stem and progenitor cells requires the presence of Meis. Collectively, these results show that Meis2 cooperates with Pax6 in generic neurogenesis and dopaminergic fate specification in the adult SVZ-OB system.
Assuntos
Neurônios Dopaminérgicos/citologia , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Neurogênese/fisiologia , Bulbo Olfatório/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Proliferação de Células , Neurônios Dopaminérgicos/metabolismo , Proteínas do Domínio Duplacortina , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neuropeptídeos/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/crescimento & desenvolvimento , Fator de Transcrição PAX6 , Interferência de RNA , RNA Interferente Pequeno/genética , Fatores de Transcrição/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial-mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-ß. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-ß. PREP1 modulates the cellular sensitivity to TGF-ß by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-ß pathway, EMT, and metastasis in NSCLC.
Assuntos
Adenocarcinoma/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Neoplasias Pulmonares/genética , Camundongos , Modelos Biológicos , Metástase Neoplásica , Peptídeo Hidrolases/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad3/genética , Análise de Sobrevida , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
AIMS: The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours. METHODS: We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA). RESULTS: PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down. CONCLUSION: In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.
Assuntos
Neoplasias Cerebelares/patologia , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Fatores de Transcrição Box Pareados/biossíntese , Proteínas Wnt/metabolismo , Adolescente , Animais , Western Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/mortalidade , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Lactente , Masculino , Meduloblastoma/metabolismo , Meduloblastoma/mortalidade , Camundongos , Fator de Transcrição PAX8 , Prognóstico , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , TransfecçãoRESUMO
Our brain's cognitive performance arises from the coordinated activities of billions of nerve cells. Despite a high degree of morphological and functional differences, all neurons of the vertebrate central nervous system (CNS) arise from a common field of multipotent progenitors. Cell fate specification and differentiation are directed by multistep processes that include inductive/external cues, such as the extracellular matrix or growth factors, and cell-intrinsic determinants, such as transcription factors and epigenetic modulators of proteins and DNA. Here we review recent findings implicating TALE-homeodomain proteins in these processes. Although originally identified as HOX-cofactors, TALE proteins also contribute to many physiological processes that do not require HOX-activity. Particular focus is, therefore, given to HOX-dependent and -independent functions of TALE proteins during early vertebrate brain development. Additionally, we provide an overview about known upstream and downstream factors of TALE proteins in the developing vertebrate brain and discuss general concepts of how TALE proteins function to modulate neuronal cell fate specification.
Assuntos
Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Mesencéfalo/metabolismo , Prosencéfalo/metabolismo , Retina/metabolismo , Rombencéfalo/metabolismoRESUMO
BACKGROUND: During early stages of brain development, secreted molecules, components of intracellular signaling pathways and transcriptional regulators act in positive and negative feed-back or feed-forward loops at the mid-hindbrain boundary. These genetic interactions are of central importance for the specification and subsequent development of the adjacent mid- and hindbrain. Much less, however, is known about the regulatory relationship and functional interaction of molecules that are expressed in the tectal anlage after tectal fate specification has taken place and tectal development has commenced. RESULTS: Here, we provide experimental evidence for reciprocal regulation and subsequent cooperation of the paired-type transcription factors Pax3, Pax7 and the TALE-homeodomain protein Meis2 in the tectal anlage. Using in ovo electroporation of the mesencephalic vesicle of chick embryos we show that (i) Pax3 and Pax7 mutually regulate each other's expression in the mesencephalic vesicle, (ii) Meis2 acts downstream of Pax3/7 and requires balanced expression levels of both proteins, and (iii) Meis2 physically interacts with Pax3 and Pax7. These results extend our previous observation that Meis2 cooperates with Otx2 in tectal development to include Pax3 and Pax7 as Meis2 interacting proteins in the tectal anlage. CONCLUSION: The results described here suggest a model in which interdependent regulatory loops involving Pax3 and Pax7 in the dorsal mesencephalic vesicle modulate Meis2 expression. Physical interaction with Meis2 may then confer tectal specificity to a wide range of otherwise broadly expressed transcriptional regulators, including Otx2, Pax3 and Pax7.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Mesencéfalo/embriologia , Fatores de Transcrição Box Pareados/genética , Animais , Embrião de Galinha , Efrina-B1/genética , Efrina-B1/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Mesencéfalo/metabolismo , Modelos Genéticos , Especificidade de Órgãos , Fatores de Transcrição Box Pareados/química , Fatores de Transcrição Box Pareados/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de ProteínasRESUMO
The transcription factor Otx2 is expressed throughout the anterior neuroectoderm and is required for the formation of all forebrain- and midbrain-derived structures. The molecular determinants that cooperate with Otx2 to subdivide its expression domain into distinct functional units are, however, poorly understood at present. We show here that the TALE-homeodomain protein Meis2 is expressed in the chick tectal anlage and is both necessary and sufficient for tectal development. Unlike known tectum-inducing genes, the ability of Meis2 to initiate tectal development does not involve the formation of a secondary midbrain-hindbrain boundary organizer, but instead requires direct interaction with Otx2. Using an Otx2-dependent reporter assay we demonstrate that Meis2 competes with the Groucho co-repressor Tle4 (Grg4) for binding to Otx2 and thereby restores Otx2 transcriptional activator function. Together, our data suggest a model in which the balance between a co-repressor and a co-activator, which compete for binding to Otx2 in the mesencephalic vesicle, provides spatial and temporal control over tectal development. Controlled formation of Meis2-containing higher order protein complexes might thus serve as a general mechanism to achieve subdivision of the anterior neuroectoderm into distinct functional units during embryogenesis.
Assuntos
Proteínas de Homeodomínio/metabolismo , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Fatores de Transcrição Otx/metabolismo , Proteínas Repressoras/metabolismo , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Ligação Competitiva , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , Fatores de Transcrição Otx/genética , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
Transcription factors (TFs) guide effector proteins like chromatin-modifying or -remodeling enzymes to distinct sites in the genome and thereby fulfill important early steps in translating the genome's sequence information into the production of proteins or functional RNAs. TFs of the same family are often highly conserved in evolution, raising the question of how proteins with seemingly similar structure and DNA-binding properties can exert physiologically distinct functions or respond to context-specific extracellular cues. A good example is the TALE superclass of homeodomain-containing proteins. All TALE-homeodomain proteins share a characteristic, 63-amino acid long homeodomain and bind to similar sequence motifs. Yet, they frequently fulfill non-redundant functions even in domains of co-expression and are subject to regulation by different signaling pathways. Here we provide an overview of posttranslational modifications that are associated with murine and human TALE-homeodomain proteins and discuss their possible importance for the biology of these TFs.
RESUMO
PAX6 is a highly conserved transcription factor and key regulator of several neurogenic processes, including the continuous generation of dopaminergic/GABAergic interneurons in the adult ventricular-subventricular (V-SVZ) neurogenic system in mice. Here we report that PAX6 cooperates with the TALE-homeodomain transcription factor PBX1 in this context. Chromatin-immunoprecipitation showed that PBX1 and PAX6 co-occupy shared genomic binding sites in adult V-SVZ stem- and progenitor cell cultures and mouse embryonic stem cells, while depletion of Pbx1 revealed that association of PAX6 with these sites requires the presence of PBX1. Expression profiling together with viral overexpression or knockdown of Pax6 or Pbx1 identified novel PBX1-PAX6 co-regulated genes, including several transcription factors. Computational modeling of genome wide expression identified novel cross-regulatory networks among these very transcription factors. Taken together, the results presented here highlight the intimate link that exists between PAX6 and TALE-HD family proteins and contribute novel insights into how the orchestrated activity of transcription factors shapes adult V-SVZ neurogenesis.
Assuntos
Epistasia Genética , Regulação da Expressão Gênica , Células-Tronco Neurais/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular/genética , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Células-Tronco Neurais/citologia , Neurogênese/genética , Fator de Transcrição PAX6/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Ligação ProteicaRESUMO
The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it's up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.
RESUMO
The mid-hindbrain boundary (MHB) harbors an important organizing center for the adjacent brain regions. Here, we present evidence that the receptor protein tyrosine phosphatase lambda (RPTPlambda) is part of the complex molecular network that maintains and shapes the MHB region. RPTPlambda is expressed in a tight band of cells in the caudal midbrain, anterior to the transverse ring of Wnt1 expression. Forced expression of RPTPlambda across the mid-hindbrain region repressed expression of Wnt1, whereas RNA interference-mediated knock-down of RPTPlambda resulted in expansion and distortion of the Wnt1 domain. When ectopically expressed in the mesencephalon, RPTPlambda specifically inhibited the induction of Wnt1 expression after subsequent stimulation with Fgf8. Reduced Wnt1 expression after RPTPlambda transfection correlated with a decrease in Ras- mitogen-activated protein kinase activity at the MHB. We further show that in the embryonic midbrain, RPTPlambda can bind to beta-catenin, a central component of the canonical Wnt signaling pathway. Overexpression of RPTPlambda suppressed the activity of a beta-catenin responsive promoter in the midbrain and reduced progenitor cell proliferation. Cotransfection of Wnt1 or of a stabilized form of beta-catenin together with RPTPlambda partially rescued the RPTPlambda-mediated proliferation defect. Together, these data suggest that RPTPlambda may play a dual role in the control of midbrain development: as a negative modulator of Fgf8-induced Wnt1 expression at the MHB, which may help to confine the Wnt1 domain to it characteristic tight ring at the MHB; and as an inhibitor of canonical Wnt signaling through interaction with and presumably sequestration of beta-catenin.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesencéfalo/embriologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Proliferação de Células , Embrião de Galinha , Fator 8 de Crescimento de Fibroblasto/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Modelos Biológicos , Interferência de RNA/fisiologia , Transfecção , Proteína Wnt1/metabolismo , beta Catenina/metabolismoRESUMO
Recent experiments suggest that activation of the Ras-MAP kinase pathway at the mid-hindbrain boundary (MHB) induces cerebellar development, whereas tectal development occurs in the absence of Ras-MAP kinase activity. To test this model we have stimulated or inhibited Ras-MAP kinase signaling in chick embryos through targeted misexpression of a constitutive active (Ras(V12)) or dominant negative (Ras(N17)) form of Ras. The consequence of these manipulations on the expression of several genes that are expressed in distinct patterns at or around the MHB organizer, including En1, Pax2, Pax3, Pax5, Wnt1, Meis2, and ephrin-A2, -A5, and -B1, was assessed. Extending previous findings we show that inhibition of Ras-MAP kinase signaling differently affects Pax3 expression in different regions of the mid-hindbrain territory, inhibiting its expression in the midbrain but inducing it in the MHB region. Expression of the midbrain specific marker gene Meis2 was not affected by Ras(N17) at first but later upregulated concomitantly with the morphological transformation of hindbrain to midbrain. In addition, we show that different dosages of Ras-MAP kinase activity are required for transcriptional activation of Wnt1 and En1 at the MHB. Collectively, these results validate and extend previous findings on the molecular changes associated with Fgf8 loss-of-function or gain-of-function phenotypes at the MHB, demonstrate that gene expression at the MHB is regulated by Ras-MAP kinase signaling in a spatially and temporally distinct manner and provide evidence for a dosage dependent function of Fgf8 signaling at the MHB.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Mesencéfalo/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Rombencéfalo/metabolismo , Proteínas ras/metabolismo , Animais , Embrião de Galinha , Embrião não Mamífero , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Dosagem de Genes , Sistema de Sinalização das MAP Quinases/genética , Mesencéfalo/embriologia , Organogênese/genética , Organogênese/fisiologia , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rombencéfalo/embriologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteínas ras/genéticaRESUMO
Although the neural retina appears as a relatively uniform tissue when viewed from its surface, it is in fact highly patterned along its anterior-posterior and dorso-ventral axes. The question of how and when such patterns arise has been the subject of intensive investigations over several decades. Most studies aimed at understanding retinal pattern formation have used the retinotectal map, the ordered projections of retinal ganglion cells to the brain, as a functional readout of the pattern. However, other cell types are also topographically organized in the retina. The most commonly recognized example of such a topographic cellular organization is the differential distribution of photoreceptor types across the retina. Photoreceptor patterns are highly species-specific and may represent an important adaptation to the visual niche a given species occupies. Nevertheless, few studies have addressed this functional readout of pattern to date and our understanding of its development has remained superficial. Here, we review recent advances in understanding the molecular cascades that control regionalization of the eye anlage, relate these findings to the development of photoreceptor patterns and discuss common and unique strategies involved in both aspects of retinal pattern formation.
Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Fotorreceptoras/embriologia , Células Fotorreceptoras/metabolismo , Células Ganglionares da Retina/metabolismo , Vertebrados/embriologia , Animais , Diferenciação Celular/genética , Humanos , Vias Neurais/citologia , Vias Neurais/embriologia , Vias Neurais/metabolismo , Células Fotorreceptoras/citologia , Células Ganglionares da Retina/citologia , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
In vertebrate development, polarity of the dorsal-ventral eye axis is determined by the spatially restricted expression of transcription factors cVax/Vax2 in the ventral retina and Tbx5 in the dorsal retina. In addition, an early gradient of retinoic acid (RA) has been suggested to be involved in this process. Using retroviral transfection of the early eye anlage of chick embryos we investigated whether cVax/Vax2 and Tbx5 regulate the expression of enzymes that determine the retinal distribution of RA. Transfection of cVax/Vax2 completely abolished the spatial expression pattern of the RA synthesizing enzyme RALDH-1, of the RA degrading enzyme Cyp26A1 and caused an ectopic expression of RALDH-3 in the dorsal retina. Transfection of Tbx5 did not significantly alter the distribution of RALDH-1 and RALDH-3 but caused additional patches of Cyp26 outside of its normal domain of expression. The distribution of RA was experimentally changed either by ectopic expression of Cyp26A1, or by applications of RA. These manipulations did not affect the distribution of cVax/Vax2 or interfere with development of the retinotectal projection from dorsal retinal ganglion cells. The results show that in the chick retina the compartmentalized expression of RA metabolizing enzymes requires the spatially restricted expression of the transcription factors cVax/Vax2 and Tbx5. On the other hand, RA may not be decisive for the correct retinal projection toward the optic tectum.
Assuntos
Proteínas Aviárias/fisiologia , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Homeodomínio/fisiologia , Retina/metabolismo , Retinal Desidrogenase/genética , Proteínas com Domínio T/fisiologia , Animais , Proteínas Aviárias/genética , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Hibridização In Situ , Isoenzimas/genética , Modelos Anatômicos , Retina/efeitos dos fármacos , Retina/embriologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Ácido Retinoico 4 Hidroxilase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/genética , Fatores de Transcrição/fisiologia , Transfecção , Tretinoína/farmacologiaRESUMO
Adult neurogenesis is regulated by stem cell niche-derived extrinsic factors and cell-intrinsic regulators, yet the mechanisms by which niche signals impinge on the activity of intrinsic neurogenic transcription factors remain poorly defined. Here, we report that MEIS2, an essential regulator of adult SVZ neurogenesis, is subject to posttranslational regulation in the SVZ olfactory bulb neurogenic system. Nuclear accumulation of MEIS2 in adult SVZ-derived progenitor cells follows downregulation of EGFR signaling and is modulated by methylation of MEIS2 on a conserved arginine, which lies in close proximity to nested binding sites for the nuclear export receptor CRM1 and the MEIS dimerization partner PBX1. Methylation impairs interaction with CRM1 without affecting PBX1 dimerization and thereby allows MEIS2 nuclear accumulation, a prerequisite for neuronal differentiation. Our results describe a form of posttranscriptional modulation of adult SVZ neurogenesis whereby an extrinsic signal fine-tunes neurogenesis through posttranslational modification of a transcriptional regulator of cell fate.
Assuntos
Arginina/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Receptores ErbB/metabolismo , Proteínas de Homeodomínio/química , Carioferinas/metabolismo , Metilação , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Proteína Exportina 1RESUMO
Neuroblastoma is one of only a few human cancers that can spontaneously regress even after extensive dissemination, a poorly understood phenomenon that occurs in as many as 10% of patients. In this study, we identify the TALE-homeodomain transcription factor MEIS2 as a key contributor to this phenomenon. We identified MEIS2 as a MYCN-independent factor in neuroblastoma and showed that in this setting the alternatively spliced isoforms MEIS2A and MEIS2D exert antagonistic functions. Specifically, expression of MEIS2A was low in aggressive stage 4 neuroblastoma but high in spontaneously regressing stage 4S neuroblastoma. Moderate elevation of MEIS2A expression reduced proliferation of MYCN-amplified human neuroblastoma cells, induced neuronal differentiation and impaired the ability of these cells to form tumors in mice. In contrast, MEIS2A silencing or MEIS2D upregulation enhanced the aggressiveness of the tumor phenotype. Mechanistically, MEIS2A uncoupled a negative feedback loop that restricts accumulation of cellular retinoic acid, an effective agent in neuroblastoma treatment. Overall, our results illuminate the basis for spontaneous regression in neuroblastoma and identify an MEIS2A-specific signaling network as a potential therapeutic target in this common pediatric malignancy.Significance: This study illuminates the basis for spontaneous regressions that can occur in a common pediatric tumor, with implications for the development of new treatment strategies. Cancer Res; 78(8); 1935-47. ©2018 AACR.