Leukocyte Receptor Tyrosine Kinase interacts with secreted midkine to promote survival of migrating neural crest cells

Neural crest cells migrate long distances throughout the embryo and rely on extracellular signals that attract, repel and/or stimulate survival to ensure proper contribution to target derivatives. Here, we show that leukocyte receptor tyrosine kinase (LTK), an ALK-type receptor tyrosine kinase, is expressed by neural crest cells during early migratory stages in chicken embryos. Loss of LTK in the cranial neural crest impairs migration and results in increased levels of apoptosis. Conversely, midkine, previously proposed as a ligand for ALK, is secreted by the non-neural ectoderm during early neural crest migratory stages and internalized by neural crest cells in vivo. Similar to loss of LTK, loss of midkine reduces survival of the migratory neural crest. Moreover, we show by proximity ligation and co-immunoprecipitation assays that midkine binds to LTK. Taken together, these results suggest that LTK in neural crest cells interacts with midkine emanating from the non-neural ectoderm to promote cell survival, revealing a new signaling pathway that is essential for neural crest development

Leukocyte Receptor Tyrosine Kinase interacts with secreted midkine to promote survival of migrating neural crest cells

Neural crest cells migrate long distances throughout the embryo and rely on extracellular signals that attract, repel and/or stimulate survival to ensure proper contribution to target derivatives. Here, we show that leukocyte receptor tyrosine kinase (LTK), an ALK-type receptor tyrosine kinase, is expressed by neural crest cells during early migratory stages in chicken embryos. Loss of LTK in the cranial neural crest impairs migration and results in increased levels of apoptosis. Conversely, midkine, previously proposed as a ligand for ALK, is secreted by the non-neural ectoderm during early neural crest migratory stages and internalized by neural crest cells in vivo. Similar to loss of LTK, loss of midkine reduces survival of the migratory neural crest. Moreover, we show by proximity ligation and co-immunoprecipitation assays that midkine binds to LTK. Taken together, these results suggest that LTK in neural crest cells interacts with midkine emanating from the non-neural ectoderm to promote cell survival, revealing a new signaling pathway that is essential for neural crest development

Transcriptome profiling of the cardiac neural crest reveals a critical role for MafB

The cardiac neural crest originates in the caudal hindbrain, migrates to the heart, and contributes to septation of the cardiac outflow tract and ventricles, an ability unique to this neural crest subpopulation. Here we have used a FoxD3 neural crest enhancer to isolate a pure population of cardiac neural crest cells for transcriptome analysis. This has led to the identification of transcription factors, signaling receptors/ligands, and cell adhesion molecules upregulated in the early migrating cardiac neural crest. We then functionally tested the role of one of the upregulated transcription factors, MafB, and found that it acts as a regulator of Sox10 expression specifically in the cardiac neural crest. Our results not only reveal the genome-wide profile of early migrating cardiac neural crest cells, but also provide molecular insight into what makes the cardiac neural crest unique

Functional analysis of Scratch2 domains: implications in the evolution of Snail transcriptional repressors

The Snail superfamily of transcription factors have a modular organization and their similarities and divergences are the basis for subdividing the superfamily into the Snail1/2 and Scratch families. As it is generally accepted that the Snail and Scratch families originated through gene duplication, understanding the functional contribution of each module could provide us with further insight about the molecular and functional evolution of the Snail superfamily. Thus, in this work, we investigated the function of the SNAG and SCRATCH domains in chicken Scratch2. Through evolutionary comparison analysis we identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. Similar to members of the Snail1/2 families, Scratch2-mediated transcriptional repression requires SNAG and nuclear localization requires the zinc-finger domain. We also identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. HINGE is highly conserved in amniotes. Single mutations of the conserved Tyrosine and Serine residues of HINGE downregulated Scratch2-mediated transcriptional repression. This effect depended on the presence of the SCRATCH domain

Functional analysis of Scratch2 domains: implications in the evolution of Snail transcriptional repressors

The Snail superfamily of transcription factors have a modular organization and their similarities and divergences are the basis for subdividing the superfamily into the Snail1/2 and Scratch families. As it is generally accepted that the Snail and Scratch families originated through gene duplication, understanding the functional contribution of each module could provide us with further insight about the molecular and functional evolution of the Snail superfamily. Thus, in this work, we investigated the function of the SNAG and SCRATCH domains in chicken Scratch2. Through evolutionary comparison analysis we identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. Similar to members of the Snail1/2 families, Scratch2-mediated transcriptional repression requires SNAG and nuclear localization requires the zinc-finger domain. We also identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. HINGE is highly conserved in amniotes. Single mutations of the conserved Tyrosine and Serine residues of HINGE downregulated Scratch2-mediated transcriptional repression. This effect depended on the presence of the SCRATCH domain

Enhanced expression of MycN/CIP2A drives neural crest toward a neural stem cell-like fate: Implications for priming of neuroblastoma

Neuroblastoma is a neural crest-derived childhood tumor of the peripheral nervous system in which MycN amplification is a hallmark of poor prognosis. Here we show that MycN is expressed together with phosphorylation-stabilizing factor CIP2A in regions of the neural plate destined to form the CNS, but MycN is excluded from the neighboring neural crest stem cell domain. Interestingly, ectopic expression of MycN or CIP2A in the neural crest domain biases cells toward CNS-like neural stem cells that express Sox2. Consistent with this, some forms of neuroblastoma have been shown to share transcriptional resemblance with CNS neural stem cells. As high MycN/CIP2A levels correlate with poor prognosis, we posit that a MycN/CIP2A-mediated cell-fate bias may reflect a possible mechanism underlying early priming of some aggressive forms of neuroblastoma. In contrast to MycN, its paralogue cMyc is normally expressed in the neural crest stem cell domain and typically is associated with better overall survival in clinical neuroblastoma, perhaps reflecting a more “normal” neural crest-like state. These data suggest that priming for some forms of aggressive neuroblastoma may occur before neural crest emigration from the CNS and well before sympathoadrenal specification

Transcription factors in the development of the early posterior neural tube.

O início da neurogênese e diferenciação neural no sistema nervoso do embrião é controlado pela expressão orquestrada de fatores de transcrição. A caracterização de novos reguladores transcricionais nestes processos é importante para o entendimento dos mecanismos responsáveis pela formação de neurônios. Neste trabalho, nós investigamos a função do fator de transcrição Scrt2 na medula espinhal do embrião de galinha. Nossos resultados indicam que Scrt2 é expresso imediatamente após a saída do ciclo celular e em conjunto com Ngn2 e NeuroM, sugerindo uma função em neurônios recém-nascidos. Para identificar potenciais alvos de Scrt2, realizamos experimentos de eletroporação in ovo no tubo neural posterior e analisamos os fenótipos transcriptômicos com RNA-Seq. Por fim, apresentamos também uma caracterização do transcriptoma do tubo neural posterior selvagem entre HH18 e HH29 (E6), provendo uma extensa base de dados de expressão gênica para futuras investigações. Com base em nossa experiência, nós discutimos o uso de RNA-Seq em diferentes abordagens experimentais.The onset of neurogenesis and neural differentiation in the embryonic nervous system is controlled by the coordinated expression of transcription factors. Identification of novel transcriptional regulators in these processes is essential for our understanding of the mechanisms underlying neuronal differentiation. Here, we used the chick embryonic spinal cord to investigate the role of the transcription factor Scrt2. Our results indicate that Scrt2 is expressed in cells that recently exited the mitotic cycle and overlaps with Ngn2 and NeuroM, suggesting a function in newborn neurons. To identify potential gene targets of Scrt2, we performed in ovo electroporation experiments in the posterior neural tube and assessed the transcriptomic phenotypes using RNA-Seq. Finally, we also present the transcriptomic profiles of the wild-type posterior neural tube from HH18 to HH29 (E6), providing an informative gene expression database for future investigations. Based on our experience, we discuss the use of RNA-Seq in distinct experimental approaches

Cloning and expression analysis of the transcription factor Scratch2 during the chicken early embryogenesis.

Em invertebrados, os genes Scratch (Scrt) codificam fatores de transcrição que promovem a neurogênese durante o desenvolvimento. A função de Scrt em vertebrados é desconhecida, mas em camundongos Scrt1 e Scrt2 são especificamente expressos em neurônios pós-mitóticos no embrião e no sistema nervoso central adulto. Neste trabalho, nós clonamos a sequência codificante de Scrt2 de galinha (cScrt2) e caracterizamos seu padrão de expressão no embrião por PCR quantitativo e hibridação in situ. A sequência codificante completa foi clonada no vetor de expressão pMES-GFP e o produto previsto da tradução é uma proteína com 276 aminoácidos. A sequência de aminoácidos compartilha identidades de 70% com Scrt2 de rato e 58% com Scrt de zebrafish. Transcritos cScrt2 são detectados primeiramente na periferia do tubo neural do rombencéfalo em HH 15 e da medula espinhal em HH 17, coincidindo com os locais onde alguns dos primeiros neurônios se diferenciam durante a embriogênese. Entre HH 19-23, a expressão no domínio motor da medula espinhal se concentra progressivamente na interface entre as zonas ventricular e do manto. Além disso, a expressão de cScrt2 também é observada nos gânglios da raíz dorsal a partir de HH 22-23, principalmente no domínio dorsomedial. O campo de expressão de cScrt2 no tubo neural é complementar aos de Notch1, que é expresso em células-tronco neurais, e SCG10, marcador de neurônios diferenciados. Nossos resultados sugerem que durante a embriogênese da medula espinhal cScrt2 é especificamente expresso em neurônios pós-mitóticos indiferenciados. A construção pMES-GFP(cScrt2) possibilita futuras análises funcionais diretas por interferência gênica no embrião de galinha, que serão de grande valor para um melhor entendimento da função dos genes Scrt em vertebrados.In invertebrates, the Scratch (Scrt) genes encode transcription factors that promote neurogenesis during development. The Scrt function in vertebrates is unknown, but in mice Scrt1 and Scrt2 are specifically expressed in post-mitotic neurons in the embryo and in the adult central nervous system. In this work, we have cloned the coding sequence of chicken Scrt2 (cScrt2) and characterized its expression pattern in the embryo with quantitative PCR and in situ hybridization. The complete coding sequence was cloned in the expression vector pMES-GFP and the predicted translation product is a 276-aminoacids protein. The aminoacid sequence shares identities of 70% with rat Scrt2 and 58% with zebrafish Scrt. cScrt2 transcripts are firstly detected in the periphery of the neural tube in the hindbrain by HH 15 and in the spinal cord by HH 17, coinciding with the places where some of the first neurons differentiate during embryogenesis. Between HH 19-23, the expression in the motor domain of the spinal cord is progressively concentrated in the interface between the ventricular and mantle zones. Furthermore, cScrt2 expression is also observed in the dorsal root ganglia after HH22-23, particularly in the dorsomedial domain. The expression pattern of cScrt2 in the neural tube is complementary to that of Notch1, which is expressed in neural stem cells, and SCG10, a marker for differentiated neurons. Our results suggest that during embryogenesis cScrt2 is specifically expressed in post-mitotic undifferentiated neurons. The construction pMES-GFP(cScrt2) makes possible future direct functional analysis by genetic interference in the chick embryo, which will be of great value for better understanding the Scrt genes function in vertebrates