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1.
Med Teach ; 41(11): 1245-1251, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31282246

RESUMO

Purpose: Basic research about test-enhanced learning points towards its effectiveness to improve students' learning and is still underutilized in educational practice. Therefore, we developed an evidence-based instructional design to investigate the usefulness of test-enhanced learning within a flipped/inverted classroom approach. Materials and Methods: We developed two modes of learning material for the self-study phase of a flipped classroom for 139 students: in addition to educational films, one group of students received a reader and another group received multiple-choice questions that corresponded to the reader in content and length. An assessment of the content of the learning material was conducted at the subsequent on-site phase. Also, ratings about students' perceptions of the additional learning material were gathered. Results: At the assessment, students that prepared with films and multiple-choice questions outperformed students that prepared with films and the reader. Furthermore, students perceived the multiple-choice questions as more helpful, more motivating and felt better prepared for the assessment than students that used the reader. Conclusions: This study shows that test-enhanced learning can be utilized to promote students' learning within the self-study phase of a flipped classroom. Not only assessment scores are positively affected but also the motivation to learn and preparedness towards an assessment.

3.
BMC Med Educ ; 19(1): 64, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819178

RESUMO

BACKGROUND: The inverted classroom approach is characterized by a primary self-study phase for students followed by an on-site, face-to-face teaching phase that is used to deepen the prior acquired knowledge. Obviously, this teaching approach relies on the students preparing before the on-site phase, which in turn requires optimized preparatory material as well as defined working instructions. The major aim of this study, therefore, was to investigate the effect of different preparatory materials and working instructions for the self-study phase of an e-learning-based inverted classroom on the knowledge gained by medical students in biochemistry. Furthermore, we analyzed whether collaborative dyadic learning during the self-study phase is more effective than individual learning with respect to knowledge gain. METHODS: The study was performed in a biochemistry seminar for second semester medical students at Ulm University in Germany. This seminar was held using an e-learning-based inverted classroom. A total of 196 students were divided into three homogeneous study groups that differed in terms of the working material and instructions provided for the self-study phase. Knowledge gain was measured by formative tests at the beginning of the on-site phases. Questionnaires were also handed out asking about motivation, interest and learning time in the self-study phases. RESULTS: Students who were told to prepare in collaborating dyads during the self-study phase performed better in formative tests taken at the beginning of on-site phases than learners who were told to prepare individually. The study material that was provided was of minor importance for the differences in formative testing since almost all students prepared for the on-site phases. With the dyadic learning approach, both students benefited from this collaboration, characterized by a higher motivation and interest in the topic, as well as a longer time spent on task. CONCLUSION: Our study provides strong evidence that the study material, but more importantly the instructions provided for the self-study phase, affect students` knowledge gain in an e-learning-based inverted classroom. The instructed collaboratively working group was the most successful.


Assuntos
Bioquímica/educação , Instrução por Computador/métodos , Práticas Interdisciplinares/métodos , Estudantes de Medicina , Atitude do Pessoal de Saúde , Comportamento Cooperativo , Currículo , Alemanha , Humanos , Modelos Teóricos , Inquéritos e Questionários
4.
Dev Biol ; 449(1): 1-13, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30797757

RESUMO

Wnt proteins can activate different intracellular signaling pathways. These pathways need to be tightly regulated for proper cardiogenesis. The canonical Wnt/ß-catenin inhibitor Dkk1 has been shown to be sufficient to trigger cardiogenesis in gain-of-function experiments performed in multiple model systems. Loss-of-function studies however did not reveal any fundamental function for Dkk1 during cardiogenesis. Using Xenopus laevis as a model we here show for the first time that Dkk1 is required for proper differentiation of cardiomyocytes, whereas specification of cardiomyocytes remains unaffected in absence of Dkk1. This effect is at least in part mediated through regulation of non-canonical Wnt signaling via Wnt11. In line with these observations we also found that Isl1, a critical regulator for specification of the common cardiac progenitor cell (CPC) population, acts upstream of Dkk1.

5.
Am J Hum Genet ; 104(2): 246-259, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30661772

RESUMO

SOX4, together with SOX11 and SOX12, forms group C of SRY-related (SOX) transcription factors. They play key roles, often in redundancy, in multiple developmental pathways, including neurogenesis and skeletogenesis. De novo SOX11 heterozygous mutations have been shown to cause intellectual disability, growth deficiency, and dysmorphic features compatible with mild Coffin-Siris syndrome. Using trio-based exome sequencing, we here identify de novo SOX4 heterozygous missense variants in four children who share developmental delay, intellectual disability, and mild facial and digital morphological abnormalities. SOX4 is highly expressed in areas of active neurogenesis in human fetuses, and sox4 knockdown in Xenopus embryos diminishes brain and whole-body size. The SOX4 variants cluster in the highly conserved, SOX family-specific HMG domain, but each alters a different residue. In silico tools predict that each variant affects a distinct structural feature of this DNA-binding domain, and functional assays demonstrate that these SOX4 proteins carrying these variants are unable to bind DNA in vitro and transactivate SOX reporter genes in cultured cells. These variants are not found in the gnomAD database of individuals with presumably normal development, but 12 other SOX4 HMG-domain missense variants are recorded and all demonstrate partial to full activity in the reporter assay. Taken together, these findings point to specific SOX4 HMG-domain missense variants as the cause of a characteristic human neurodevelopmental disorder associated with mild facial and digital dysmorphism.


Assuntos
Anormalidades Múltiplas/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição SOXC/genética , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Síndrome de Coffin-Lowry/genética , Estudos de Coortes , Sequência Conservada , DNA/genética , DNA/metabolismo , Feminino , Domínios HMG-Box/genética , Heterozigoto , Humanos , Masculino , Fatores de Transcrição SOXC/química , Fatores de Transcrição SOXC/metabolismo , Ativação Transcricional , Xenopus/anatomia & histologia , Xenopus/embriologia , Xenopus/genética
6.
Dev Dyn ; 247(9): 1070-1082, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30055071

RESUMO

BACKGROUND: The nitric oxide synthase interacting protein (Nosip) has been associated with diverse human diseases including psychological disorders. In line, early neurogenesis of mouse and Xenopus is impaired upon Nosip deficiency. Nosip knockout mice show craniofacial defects and the down-regulation of Nosip in the mouse and Xenopus leads to microcephaly. Until now, the exact underlying molecular mechanisms of these malformations were still unknown. RESULTS: Here, we show that nosip is expressed in the developing ocular system as well as the anterior neural crest cells of Xenopus laevis. Furthermore, Nosip inhibition causes severe defects in eye formation in the mouse and Xenopus. Retinal lamination as well as dorso-ventral patterning of the retina were affected in Nosip-depleted Xenopus embryos. Marker gene analysis using rax, pax6 and otx2 reveals an interference with the eye field induction and differentiation. A closer look on Nosip-deficient Xenopus embryos furthermore reveals disrupted cranial cartilage structures and an inhibition of anterior neural crest cell induction and migration shown by twist, snai2, and egr2. Moreover, foxc1 as downstream factor of retinoic acid signalling is affected upon Nosip deficiency. CONCLUSIONS: Nosip is a crucial factor for the development of anterior neural tissue such the eyes and neural crest cells. Developmental Dynamics 247:1070-1082, 2018. © 2018 Wiley Periodicals, Inc.

7.
Gene Expr Patterns ; 28: 54-61, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29462671

RESUMO

The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development.


Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Receptores Acoplados a Proteínas-G/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
8.
GMS J Med Educ ; 34(3): Doc31, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28890922

RESUMO

Background: Medical students often have a problem recognising the relevance of basic science subjects for their later professional work in the pre-clinical stage of their studies. This can lead to a lower motivation to learn biochemical content and dissatisfaction in the courses amongst the students. Alternative teaching methods such as the Inverted Classroom (IC) method can address this deficiency. The goal of this study was: to analyse the motivation and satisfaction of the students in a biochemistry seminar through the use of the e-learning-based IC method, to investigate the acceptance against the IC teaching method in biochemistry, and to compare the learning success achieved using the IC approach with that of a traditional course. We also investigated how a biochemistry course in the pre-clinical stage of a human medicine course of studies can be successfully organised according to the IC method. Furthermore, we examined the benefits of the IC method over conventional teaching formats. Method: The IC method was implemented in accordance with the guidelines of the GMA committee "New Media" [30] in a biochemistry seminar for two student IC intervention groups with 42 students. A part of the factual knowledge from the on-site phase in the form of teaching videos together with self-learning control tasks were provided online before the seminar for both IC intervention groups. Exporting content to the self-learning phase creates new free time in the on-site phase, during which the content can be critically considered and processed and additional competency-based learning objectives can be taught. Identical biochemistry teaching content was taught in parallel control groups (14 student groups with n=299 students), but no material was handed out beforehand for a self-learning phase. These students only received the materials after the on-site phase. Motivation and satisfaction as well as the acceptance for the teaching methods were recorded by questionnaires, the acquisition of knowledge by MC exams. Results: On a Likert scale from 1 (strongly disagree) to 6 (strongly agree), the students in the IC intervention groups could be seen to be much more motivated (5.53) than students in the control group (4.01). Students in the IC intervention groups also recognised the relevance of the learning content much more clearly (5.44) than students in the control group (4.01). Furthermore, the IC group also observed that additional competencies were trained in addition to the biochemistry content. In addition, the IC intervention group award the event a school grade of 1.53, the traditional control group a grade of 2.96. The teaching videos were rated very positively by both groups with an average school grade of 1.3 in each case. A qualitative analysis showed that the motivation and a positive attitude of the lecturers played a decisive role in the successful implementation of the IC method. Discussion and conclusion: Pre-clinical students display a high acceptance of the e-learning-based IC method. Teaching communication competencies in a biochemistry seminar was also rated very positively by the students. The quality of the teaching video and the motivation of the lecturers were shown to be a critical parameter for the successful performance of the IC method. What's more, the IC method can contribute to implementing a competence orientation in medical studies.


Assuntos
Bioquímica/educação , Educação Baseada em Competências/organização & administração , Instrução por Computador/normas , Educação Médica/organização & administração , Logro , Adulto , Atitude do Pessoal de Saúde , Estudos de Coortes , Currículo , Feminino , Alemanha , Humanos , Masculino , Motivação , Estudantes de Medicina/psicologia
9.
Dev Biol ; 429(1): 200-212, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28663132

RESUMO

BACKGROUND: Genetic deletion of Nosip in mice causes holoprosencephaly, however, the function of Nosip in neurogenesis is currently unknown. RESULTS: We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of Nosip in neurogenesis. We found, that size and cortical thickness of the developing brain of Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of Rbp1, a regulator of retinoic acid synthesis, when Nosip was absent. We identified the homologous nosip gene to be expressed in differentiated neurons in the developing brain of Xenopus embryos. Knockdown of Nosip in Xenopus resulted in a reduction of brain size that could be rescued by reintroducing human NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon Nosip depletion. In Xenopus Nosip and Rbp1 are similarly expressed and knockdown of Nosip resulted in down regulation of Rbp1. Knockdown of Rbp1 caused a similar microcephaly phenotype as the depletion of Nosip and synergy experiments indicated that both proteins act in the same signalling pathway. CONCLUSIONS: Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of Rbp1 during early neurogenesis.


Assuntos
Neurogênese , Ubiquitina-Proteína Ligases/deficiência , Proteínas de Xenopus/deficiência , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Apoptose , Proliferação de Células , Separação Celular , Sobrevivência Celular , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Regulação para Baixo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos Knockout , Microcefalia/patologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteoma/metabolismo , Proteínas Celulares de Ligação ao Retinol/metabolismo , Esferoides Celulares/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
10.
Dev Biol ; 426(1): 69-83, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28427856

RESUMO

Formation of a functional eye during vertebrate embryogenesis requires different processes such as cell differentiation, cell migration, cell-cell interactions as well as intracellular signalling processes. It was previously shown that the non-canonical Wnt receptor Frizzled 3 (Fzd3) is required for proper eye formation, however, the underlying mechanism is poorly understood. Here we demonstrate that loss of Fzd3 induces severe malformations of the developing eye and that this defect is phenocopied by loss of the activated leukocyte cell adhesion molecule (Alcam). Promoter analysis revealed the presence of a Fzd3 responsive element within the alcam promoter, which is responsible for alcam expression during anterior neural development. In-depth analysis identified the jun N-terminal protein kinase 1 (JNK1) and the transcription factor paired box 2 (Pax2) to be important for the activation of alcam expression. Altogether our study reveals that alcam is activated through non-canonical Wnt signalling during embryonic eye development in Xenopus laevis and shows that this pathway plays a similar role in different tissues.


Assuntos
Molécula de Adesão de Leucócito Ativado/genética , Olho/embriologia , Receptores Frizzled/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Molécula de Adesão de Leucócito Ativado/metabolismo , Animais , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Olho/ultraestrutura , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Microscopia Eletrônica de Transmissão , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Morfolinos/genética , Neurogênese/genética , Neurogênese/fisiologia , Fator de Transcrição PAX2/metabolismo , Regiões Promotoras Genéticas/genética , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo
11.
Dev Biol ; 424(1): 28-39, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28237811

RESUMO

Wnt proteins are critical for embryonic cardiogenesis and cardiomyogenesis by regulating different intracellular signalling pathways. Whereas canonical Wnt/ß-catenin signalling is required for mesoderm induction and proliferation of cardiac progenitor cells, ß-catenin independent, non-canonical Wnt signalling regulates cardiac specification and terminal differentiation. Although the diverse cardiac malformations associated with the loss of non-canonical Wnt11 in mice such as outflow tract (OFT) defects, reduced ventricular trabeculation, myofibrillar disorganization and reduced cardiac marker gene expression are well described, the underlying molecular mechanisms are still not completely understood. Here we aimed to further characterize Wnt11 mediated signal transduction during vertebrate cardiogenesis. Using Xenopus as a model system, we show by loss of function and corresponding rescue experiments that the non-canonical Wnt signalling mediator Rcsd1 is required downstream of Wnt11 for ventricular trabeculation, terminal differentiation of cardiomyocytes and cardiac morphogenesis. We here place Rcsd1 downstream of Wnt11 during cardiac development thereby providing a novel mechanism for how non-canonical Wnt signalling regulates vertebrate cardiogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miocárdio/metabolismo , Organogênese , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Motivos de Aminoácidos , Animais , Diferenciação Celular , Citoplasma/metabolismo , Desenvolvimento Embrionário , Deleção de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Miocárdio/patologia , Células NIH 3T3 , Fenótipo , Ligação Proteica , Transdução de Sinais , Proteínas de Xenopus/química
12.
Dev Biol ; 423(1): 66-76, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28104388

RESUMO

The Fezzin family member Nedd4-binding protein 3 (N4BP3) is known to regulate axonal and dendritic branching. Here, we show that n4bp3 is expressed in the neural tissue of the early Xenopus laevis embryo including the eye, the brain and neural crest cells. Knockdown of N4bp3 in the Xenopus anterior neural tissue results in severe developmental impairment of the eye, the brain and neural crest derived cranial cartilage structures. Moreover, we demonstrate that N4bp3 depletion leads to a significant reduction of both eye and brain specific marker genes and reduced neural crest cell migration. Finally, we demonstrate an impact of N4bp3 deficiency on cell apoptosis and proliferation. Our studies indicate that N4bp3 is required for early anterior neural development of vertebrates. This is in line with a study implicating that genetic disruption of N4BP3 in humans might be related to neurodevelopmental disease.


Assuntos
Proteínas de Transporte/metabolismo , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Apoptose , Biomarcadores/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Transporte/genética , Cartilagem/embriologia , Cartilagem/metabolismo , Movimento Celular/genética , Proliferação de Células , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Olho/embriologia , Olho/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Marcação In Situ das Extremidades Cortadas , Crista Neural/citologia , Coloração e Rotulagem , Proteínas de Xenopus/genética
13.
Development ; 144(2): 321-333, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27993984

RESUMO

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/ß-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/ß-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.


Assuntos
Olho/embriologia , Proteínas Ativadoras de GTPase/fisiologia , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Receptor EphA4/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Catarata/genética , Diferenciação Celular/genética , Embrião não Mamífero , Olho/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cristalino/metabolismo , Organogênese/genética , Ligação Proteica , Receptor EphA4/metabolismo , Xenopus/embriologia , Xenopus/genética
14.
Dev Genes Evol ; 226(5): 369-82, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27384056

RESUMO

The signal-induced proliferation-associated (SIPA) protein family belongs to the RapGAP protein superfamily. Previous studies mainly focused on the expression and function of SIPA genes in vertebrate neuronal tissue. Only limited data about the embryonic expression pattern of the genes are currently available. Our study provides the first expression analysis of sipa1, sipa1l1, sipa1l2, and sipa1l3 during early development of the vertebrate organism Xenopus laevis. In silico, analysis revealed that all genes are highly conserved across species. Semi-quantitative RT-PCR experiments demonstrated that the RNA of all genes was maternally supplied. By whole mount in situ hybridization approaches, we showed that sipa1 is mainly expressed in various sensory organs, the respiratory and blood system, heart, neural tube, and eye. In contrast, sipa1l1 showed a broad expression during development in particular within the brain, somites, eye, and heart. Sipa1l2 was detected in the branchial arches, glomerulus, and the developing eye. In contrast, sipa1l3 revealed a tissue specific expression within the olfactory and otic vesicles, the cranial placodes and ganglia, neural tube, pronephros, retina, and lens. In summary, all sipa gene family members are expressed throughout the whole developing Xenopus organism and might play an important role during vertebrate early embryogenesis.


Assuntos
Proteínas Nucleares/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/genética , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Domínios Proteicos , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo
15.
Nucleic Acids Res ; 44(10): 4703-20, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26912830

RESUMO

The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2ß. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.


Assuntos
Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Regulação da Expressão Gênica , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transcrição Genética , Animais , Caseína Quinase II/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Código das Histonas , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Proteínas Nucleares/química , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Xenopus laevis
16.
J Neurochem ; 136(1): 28-35, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26364583

RESUMO

Rap GTPase-activating proteins (RapGAPs) are essential for synaptic function as they tightly regulate synaptic Rap signaling. Among the most abundant synaptic RapGAPs in brain are the Spine-associated RapGAPs (SPARs) Sipa1l1/SPAR and Sipa1l2/SPAR2, whereas nothing has been reported on Sipa1l3/SPAR3. In this study, we show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in the developing rat brain and is localized at excitatory postsynapses. We further demonstrate that the Sipa1l3/SPAR3 C-terminus is required for postsynaptic targeting and represents an interaction module for Fezzins such as ProSAPiP1/Lzts3, a binding partner of the postsynaptic scaffold protein Shank3. Taken together, our data imply that Sipa1l3/SPAR3 is a hitherto unknown synaptic RapGAP, which is targeted to postsynaptic specializations and interacts with Fezzins. Spine-associated RapGAPs (SPARs) are essential modulators of synaptic signaling. Our study is the first to characterize the SPAR family member Sipa1l3/SPAR3 in neuronal tissue. We show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in brain and is localized to excitatory postsynapses via its C-terminus, which represents an interaction module for other postsynaptic proteins including the Fezzin ProSAPiP1/Lzts3.


Assuntos
Proteínas de Transporte/biossíntese , Proteínas Ativadoras de GTPase/biossíntese , Proteínas de Membrana/biossíntese , Sinapses/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Animais , Encéfalo/metabolismo , Células COS , Células Cultivadas , Cercopithecus aethiops , Cães , Feminino , Humanos , Masculino , Camundongos , Pan troglodytes , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
17.
J Med Genet ; 53(3): 152-62, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26543203

RESUMO

BACKGROUND: SOX11 is a transcription factor proposed to play a role in brain development. The relevance of SOX11 to human developmental disorders was suggested by a recent report of SOX11 mutations in two patients with Coffin-Siris syndrome. Here we further investigate the role of SOX11 variants in neurodevelopmental disorders. METHODS: We used array based comparative genomic hybridisation and trio exome sequencing to identify children with intellectual disability who have deletions or de novo point mutations disrupting SOX11. The pathogenicity of the SOX11 mutations was assessed using an in vitro gene expression reporter system. Loss-of-function experiments were performed in xenopus by knockdown of Sox11 expression. RESULTS: We identified seven individuals with chromosome 2p25 deletions involving SOX11. Trio exome sequencing identified three de novo SOX11 variants, two missense (p.K50N; p.P120H) and one nonsense (p.C29*). The biological consequences of the missense mutations were assessed using an in vitro gene expression system. These individuals had microcephaly, developmental delay and shared dysmorphic features compatible with mild Coffin-Siris syndrome. To further investigate the function of SOX11, we knocked down the orthologous gene in xenopus. Morphants had significant reduction in head size compared with controls. This suggests that SOX11 loss of function can be associated with microcephaly. CONCLUSIONS: We thus propose that SOX11 deletion or mutation can present with a Coffin-Siris phenotype.


Assuntos
Anormalidades Múltiplas/genética , Face/anormalidades , Deformidades Congênitas da Mão/genética , Deficiência Intelectual/genética , Micrognatismo/genética , Pescoço/anormalidades , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição SOXC/genética , Deleção de Sequência , Anormalidades Múltiplas/fisiopatologia , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Face/fisiopatologia , Feminino , Técnicas de Silenciamento de Genes , Deformidades Congênitas da Mão/fisiopatologia , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Microcefalia , Micrognatismo/fisiopatologia , Pescoço/fisiopatologia , Transtornos do Neurodesenvolvimento/fisiopatologia , Xenopus
18.
Stem Cells ; 33(4): 1113-29, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25524439

RESUMO

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.


Assuntos
Proteínas de Homeodomínio/biossíntese , Proteínas com Homeodomínio LIM/antagonistas & inibidores , Proteínas com Homeodomínio LIM/biossíntese , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Animais , Linhagem da Célula/fisiologia , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Proteína Homeobox Nkx-2.5 , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/fisiologia , Xenopus
19.
Development ; 141(10): 2064-74, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24764076

RESUMO

Proper development of nephrons is essential for kidney function. ß-Catenin-independent Wnt signaling through Fzd8, Inversin, Daam1, RhoA and Myosin is required for nephric tubule morphogenesis. Here, we provide a novel mechanism through which non-canonical Wnt signaling contributes to tubular development. Using Xenopus laevis as a model system, we found that the cell-adhesion molecule Alcam is required for proper nephrogenesis and functions downstream of Fzd3 during embryonic kidney development. We found alcam expression to be independent of Fzd8 or Inversin, but to be transcriptionally regulated by the ß-Catenin-independent Wnt/JNK pathway involving ATF2 and Pax2 in a direct manner. These novel findings indicate that several branches of Wnt signaling are independently required for proximal tubule development. Moreover, our data indicate that regulation of morphogenesis by non-canonical Wnt ligands also involves direct transcriptional responses in addition to the effects on a post-translational level.


Assuntos
Molécula de Adesão de Leucócito Ativado/fisiologia , Desenvolvimento Embrionário/genética , Rim/embriologia , Sistema de Sinalização das MAP Quinases/genética , Via de Sinalização Wnt/genética , Molécula de Adesão de Leucócito Ativado/genética , Animais , Embrião não Mamífero , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/metabolismo , Fator de Transcrição PAX2/fisiologia , Pronefro/embriologia , Pronefro/metabolismo , Elementos de Resposta/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética
20.
PLoS One ; 9(1): e87294, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489892

RESUMO

The family of vertebrate Mef2 transcription factors is comprised of four members named Mef2a, Mef2b, Mef2c, and Mef2d. These transcription factors are regulators of the myogenic programs with crucial roles in development of skeletal, cardiac and smooth muscle cells. Mef2a and Mef2c are essential for cardiac development in mice. In Xenopus, mef2c and mef2d but not mef2a were recently shown to be expressed during cardiogenesis. We here investigated the function of Mef2c and Mef2d during Xenopus laevis cardiogenesis. Knocking down either gene by corresponding antisense morpholino oligonucleotides led to profound heart defects including morphological abnormalities, pericardial edema, and brachycardia. Marker gene expression analyses and rescue experiments revealed that (i) both genes are required for proper cardiac gene expression, (ii) Mef2d can compensate for the loss of Mef2c but not vice versa, and (iii) the γ domain of Mef2c is required for early cardiac development. Taken together, our data provide novel insights into the function of Mef2 during cardiogenesis, highlight evolutionary differences between species and might have an impact on attempts of direct reprogramming.


Assuntos
Fatores de Transcrição MEF2/fisiologia , Organogênese/genética , Animais , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Coração/embriologia , Cardiopatias Congênitas/genética , Fatores de Transcrição MEF2/metabolismo , Estrutura Terciária de Proteína , Xenopus laevis
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