RESUMO
The vertebrate hindbrain is segmented into rhombomeres (r) initially defined by distinct domains of gene expression. Previous studies have shown that noise-induced gene regulation and cell sorting are critical for the sharpening of rhombomere boundaries, which start out rough in the forming neural plate (NP) and sharpen over time. However, the mechanisms controlling simultaneous formation of multiple rhombomeres and accuracy in their sizes are unclear. We have developed a stochastic multiscale cell-based model that explicitly incorporates dynamic morphogenetic changes (i.e. convergent-extension of the NP), multiple morphogens, and gene regulatory networks to investigate the formation of rhombomeres and their corresponding boundaries in the zebrafish hindbrain. During pattern initiation, the short-range signal, fibroblast growth factor (FGF), works together with the longer-range morphogen, retinoic acid (RA), to specify all of these boundaries and maintain accurately sized segments with sharp boundaries. At later stages of patterning, we show a nonlinear change in the shape of rhombomeres with rapid left-right narrowing of the NP followed by slower dynamics. Rapid initial convergence improves boundary sharpness and segment size by regulating cell sorting and cell fate both independently and coordinately. Overall, multiple morphogens and tissue dynamics synergize to regulate the sizes and boundaries of multiple segments during development.
Assuntos
Padronização Corporal/fisiologia , Modelos Biológicos , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Biologia Computacional , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Substâncias de Crescimento/fisiologia , Rombencéfalo/citologia , Rombencéfalo/embriologia , Transdução de Sinais , Processos Estocásticos , Tretinoína/fisiologia , Peixe-Zebra/genéticaRESUMO
Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed antidepressants. They regulate serotonergic neurotransmission, but it remains unclear how altered serotonergic neurotransmission may contribute to the SSRI resistance observed in approximately 30% of major depressive disorder (MDD) patients. Patient stratification based on pharmacological responsiveness and the use of patient-derived neurons may make possible the discovery of disease-relevant neural phenotypes. In our study from a large cohort of well-characterized MDD patients, we have generated induced pluripotent stem cells (iPSCs) from SSRI-remitters and SSRI-nonremitters. We studied serotonergic neurotransmission in patient forebrain neurons in vitro and observed that nonremitter patient-derived neurons displayed serotonin-induced hyperactivity downstream of upregulated excitatory serotonergic receptors, in contrast to what is seen in healthy and remitter patient-derived neurons. Our data suggest that postsynaptic forebrain hyperactivity downstream of SSRI treatment may play a role in SSRI resistance in MDD.
Assuntos
Transtorno Depressivo Resistente a Tratamento/tratamento farmacológico , Transtorno Depressivo Resistente a Tratamento/fisiopatologia , Serotonina/metabolismo , Adulto , Acatisia Induzida por Medicamentos/fisiopatologia , Antidepressivos/uso terapêutico , Estudos de Coortes , Transtorno Depressivo Maior/tratamento farmacológico , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Pessoa de Meia-Idade , Neurônios , Agitação Psicomotora/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Transmissão SinápticaRESUMO
The majority of the human genome is made of transposable elements, giving rise to interspaced repeats, including Long INterspersed Element-1s (LINE-1s or L1s). L1s are active human transposable elements involved in genomic diversity and evolution; however, they can also contribute to genomic instability and diseases. L1s require host factors to complete their life cycles, whereas the host has evolved numerous mechanisms to restrict L1-induced mutagenesis. Restriction mechanisms in somatic cells include methylation of the L1 promoter, anti-viral factors and RNA-mediated processes such as small RNAs. microRNAs (miRNAs or miRs) are small non-coding RNAs that post-transcriptionally repress multiple target genes often found in the same cellular pathways. We have recently established that miR-128 functions as a novel restriction factor inhibiting L1 mobilization in somatic cells. We have further demonstrated that miR-128 functions through a dual mechanism; by directly targeting L1 RNA for degradation and indirectly by inhibiting a cellular co-factor which L1 is dependent on to transpose to new genomic locations (TNPO1). Here, we add another piece to the puzzle of the enigmatic L1 lifecycle. We show that miR-128 also inhibits another key cellular factor, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), by significantly reducing mRNA and protein levels through direct interaction with the coding sequence (CDS) of hnRNPA1 mRNA. In addition, we demonstrate that repression of hnRNPA1 using hnRNPA1-shRNA significantly decreases de novo L1 retro-transposition and that induced hnRNPA1 expression enhances L1 mobilization. Furthermore, we establish that hnRNPA1 is a functional target of miR-128. Finally, we determine that induced hnRNPA1 expression in miR-128-overexpressing cells can partly rescue the miR-128-induced repression of L1's ability to transpose to different genomic locations. Thus, we have identified an additional mechanism by which miR-128 represses L1 retro-transposition and mediates genomic stability.
Assuntos
Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Antagomirs/metabolismo , Sequência de Bases , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1/antagonistas & inibidores , Ribonucleoproteína Nuclear Heterogênea A1/genética , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Fases de Leitura Aberta/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Alinhamento de SequênciaRESUMO
The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10) with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85%) located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51%) variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies <1% for family studies. The results identified LMNA c.357-2A>G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency.