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STAR Protoc ; 4(1): 102020, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36638016


Taming gene expression variability is critical for robust pattern formation during embryonic development. Here, we describe an optimized protocol for single-molecule fluorescence in situ hybridization and immunohistochemistry in zebrafish embryos. We detail how to count segmentation clock RNAs and calculate their variability among neighboring cells. This approach is easily adaptable to count RNA numbers of any gene and calculate transcriptional variability among neighboring cells in diverse biological settings. For complete details on the use and execution of this protocol, please refer to Keskin et al. (2018),1 Zinani et al. (2021),2 and Zinani et al. (2022).3.

Nature ; 613(7942): 153-159, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36517597


Sequential segmentation creates modular body plans of diverse metazoan embryos1-4. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation4. However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1-Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish5. Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species3,4,6-8. Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients.

Padronização Corporal , MAP Quinases Reguladas por Sinal Extracelular , Periodicidade , Somitos , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Somitos/efeitos dos fármacos , Somitos/embriologia , Somitos/enzimologia , Somitos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo , Relógios Biológicos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo
iScience ; 25(7): 104579, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35789861


Timely progression of a genetic program is critical for embryonic development. However, gene expression involves inevitable fluctuations in biochemical reactions leading to substantial cell-to-cell variability (gene expression noise). One of the important questions in developmental biology is how pattern formation is reproducibly executed despite these unavoidable fluctuations in gene expression. Here, we studied the transcriptional variability of two paired zebrafish segmentation clock genes (her1 and her7) in multiple genetic backgrounds. Segmentation clock genes establish an oscillating self-regulatory system, presenting a challenging yet beautiful system in studying control of transcription variability. In this study, we found that a negative feedback loop established by the Her1 and Her7 proteins minimizes uncorrelated variability whereas gene copy number affects variability of both RNAs in a similar manner (correlated variability). We anticipate that these findings will help analyze the precision of other natural clocks and inspire the ideas for engineering precise synthetic clocks in tissue engineering.

Trends Genet ; 38(1): 73-81, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34376301


Coordinated spatiotemporal expression of large sets of genes is required for the development and homeostasis of organisms. To achieve this goal, organisms use myriad strategies where they form operons, utilize bidirectional promoters, cluster genes, share enhancers among genes by DNA looping, and form topologically associated domains and transcriptional condensates. Coexpression achieved by these different strategies is hypothesized to have functional importance in minimizing gene expression variability, establishing dosage balance to ensure stoichiometry of protein complexes, and minimizing accumulation of toxic intermediate metabolites. By combining gene-editing tools with computational modeling, recent studies tested the advantages of adjacent genes located in pairs and clusters. We propose that with the advancement of gene editing, single-cell sequencing, and imaging tools, one could readily test the functional importance of different coexpression strategies in a variety of biological processes.

Edição de Genes , Regiões Promotoras Genéticas/genética
Nature ; 589(7842): 431-436, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33361814


Gene expression is an inherently stochastic process1,2; however, organismal development and homeostasis require cells to coordinate the spatiotemporal expression of large sets of genes. In metazoans, pairs of co-expressed genes often reside in the same chromosomal neighbourhood, with gene pairs representing 10 to 50% of all genes, depending on the species3-6. Because shared upstream regulators can ensure correlated gene expression, the selective advantage of maintaining adjacent gene pairs remains unknown6. Here, using two linked zebrafish segmentation clock genes, her1 and her7, and combining single-cell transcript counting, genetic engineering, real-time imaging and computational modelling, we show that gene pairing boosts correlated transcription and provides phenotypic robustness for the formation of developmental patterns. Our results demonstrate that the prevention of gene pairing disrupts oscillations and segmentation, and the linkage of her1 and her7 is essential for the development of the body axis in zebrafish embryos. We predict that gene pairing may be similarly advantageous in other organisms, and our findings could lead to the engineering of precise synthetic clocks in embryos and organoids.

Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Padronização Corporal/genética , Proteínas CLOCK/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Relógios Biológicos/genética , Mutação , Análise de Célula Única