RESUMO
Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p27/antagonistas & inibidores , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Ciclo Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fase G1/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Fase S/fisiologia , Transdução de Sinais , Xenopus laevisRESUMO
Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/ß-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding ß-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrolídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Vacúolos/metabolismo , Proteínas Wnt/metabolismo , Acilação , Animais , Embrião não Mamífero , Desenvolvimento Embrionário/efeitos dos fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Macrolídeos/isolamento & purificação , Ligação Proteica , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Vacúolos/química , Proteína Wnt3 , Proteína Wnt3A , Xenopus , Proteínas de XenopusRESUMO
Fermitin genes are highly conserved and encode cytocortex proteins that mediate integrin signalling. Fermitin 1 (Kindlin1) is implicated in Kindler syndrome, a human skin blistering disorder. We report the isolation of the three Fermitin orthologs from Xenopus laevis embryos and describe their developmental expression patterns. Fermitin 1 is expressed in the skin, otic and olfactory placodes, pharyngeal arches, pronephric duct, and heart. Fermitin 2 is restricted to the somites and neural crest. Fermitin 3 is expressed in the notochord, central nervous system, cement gland, ventral blood islands, vitelline veins, and myeloid cells. Our findings are consistent with the view that Fermitin 1 is generally expressed in the skin, Fermitin 2 in muscle, and Fermitin 3 in hematopoietic lineages. Moreover, we describe novel sites of Fermitin gene expression that extend our knowledge of this family. Our data provide a basis for further functional analysis of the Fermitin family in Xenopus laevis.
Assuntos
Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animais , Embrião não Mamífero/anatomia & histologia , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/genética , Distribuição Tecidual , Proteínas de Xenopus/genética , Xenopus laevis/anatomia & histologiaRESUMO
BACKGROUND: Team-based learning (TBL) currently relies on single best answer questions (SBAQs) to provide immediate feedback. Very short answer questions (VSAQs) are a reliable and discriminatory alternative that encourage learners to use more authentic clinical reasoning strategies compared to SBAQs. However, the challenge of marking VSAQs has limited their integration into TBL; we therefore explored the feasibility of VSAQs within a TBL session. METHODS: An online platform was developed to allow immediate marking of VSAQs during the TBL sessions. As part of the readiness assurance process, students completed VSAQs and SBAQs, which were marked in real time. RESULTS: Instructors were able to mark all VSAQs during the individual readiness assurance test (iRAT), which facilitated the provision of immediate feedback during the team readiness assurance test (tRAT). The mean time to mark five VSAQs was 422 seconds (SD 73 seconds). For VSAQs, the number of attempts to reach the correct answer ranged from 1 to 38, compared to 1 to 4 for SBAQs. In total, 71.6% of students agreed that using VSAQs in TBL helped to emphasise group discussions. DISCUSSION: The wide range of attempts at, and students' perspectives of VSAQs are suggestive of their positive impact on student discussion during TBL. We demonstrate how new technology allows VSAQs to be feasibly integrated into TBL with the potential to enrich group discussions.
Assuntos
Avaliação Educacional , Aprendizagem Baseada em Problemas , Estudos de Viabilidade , Retroalimentação , Processos Grupais , Humanos , EstudantesAssuntos
Proteínas de Ligação a DNA/genética , Proteínas Nucleares , Processos de Determinação Sexual , Sequência de Aminoácidos , Animais , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Masculino , Dados de Sequência Molecular , Fatores de Transcrição SOX9 , Homologia de Sequência de Aminoácidos , Diferenciação Sexual/genética , Proteína da Região Y Determinante do Sexo , Fatores de Transcrição/genética , Cromossomo YRESUMO
BACKGROUND: Neurogenic placodes are focal thickenings of the embryonic ectoderm that form in the vertebrate head. It is within these structures that the precursors of the majority of the sensory neurons of the cranial ganglia are specified. The trigeminal placodes, the ophthalmic and maxillomandibular, form close to the midbrain-hindbrain boundary and many lines of evidence have shown that signals emanating from this level of the neuraxis are important for the development of the ophthalmic placode. RESULTS: Here, we provide the first evidence that both the ophthalmic and maxillomandibular placodes form under the influence of isthmic Wnt and FGF signals. Activated Wnt signals direct development of the Pax3 expressing ophthalmic placodal field and induce premature differentiation of both the ophthalmic and the maxillomandibular placodes. Similarly, overexpression of Fgf8 directs premature differentiation of the trigeminal placodes. Wnt signals require FGF receptor activity to initiate Pax3 expression and, subsequently, the expression of neural markers, such as Brn3a, within the cranial ectoderm. Furthermore, fibroblast growth factor signaling via the mitogen activated protein kinase pathway is required to maintain early neuronal differentiation within the trigeminal placodes. CONCLUSION: We demonstrate the identity of inductive signals that are necessary for trigeminal ganglion formation. This is the first report that describes how isthmic derived Wnt signals act in concert with fibroblast growth factor signaling. Together, both are necessary and sufficient for the establishment and differentiation of the ophthalmic and maxillomandibular placodes and, consequently, the trigeminal ganglion.
Assuntos
Diferenciação Celular/fisiologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Tubo Neural/embriologia , Tubo Neural/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Gânglio Trigeminal/fisiologia , Proteínas Wnt/metabolismo , Animais , Western Blotting , Embrião de Galinha , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Tubo Neural/citologia , Neurônios/citologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Gânglio Trigeminal/embriologia , Gânglio Trigeminal/crescimento & desenvolvimento , Gânglio Trigeminal/metabolismo , Proteínas Wnt/genéticaRESUMO
Fibroblast growth factor 8 (FGF8) is expressed at the mid-hindbrain boundary and is an important signal emanating from the isthmic organizer. Wnt1 is expressed in the caudal midbrain juxtaposed to Fgf8 expression and has been implicated in its regulation. In this study, we examine the requirement for continuous Wnt signaling in the maintenance of Fgf8 expression at the isthmus. We demonstrate that prior to HH10, ongoing Wnt signaling is required to maintain the normal pattern of isthmic Fgf8 expression in ovo. Similarly, in explant assays, sustained Wnt signaling is essential to maintain Fgf8 expression in rhombomere 1. The mechanism by which Wnt signaling regulates isthmic Fgf8 expression is likely to be a maintenance response rather than an inductive effect. Finally, we show that Wnt maintenance of Fgf8 expression is dependent upon positive feedback by FGF signaling itself, and that rhombomere 1 does not receive instructive cues from the posterior hindbrain. In summary, these findings establish that a sustained reciprocal interaction between Wnt and FGF signaling is essential to maintain isthmic identity.