An evolutionary-conserved Wnt3/β-catenin/Sp5 feedback loop restricts head organizer activity in Hydra

AbstractThe Hydra polyp regenerates its head by transforming the gastric tissue below the wound into a head organizer made of two antagonistic cross-reacting components. The activator, previously characterized as Wnt3, drives apical differentiation by acting locally and auto-catalytically. The uncharacterized inhibitor, produced under the control of the activator, prevents ectopic head formation. By crossing RNA-seq data obtained in a β-catenin(RNAi) screen performed in planarians and a quantitative analysis of positional and temporal gene expression in Hydra, we identified Sp5 as a transcription factor that fulfills the head inhibitor properties: a Wnt/β-catenin inducible expression, a graded apical-to-basal expression, a sustained up-regulation during head regeneration, a multi-headed phenotype when knocked-down, a repressing activity on Wnt3 expression. In mammalian cells, Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also auto-activates its expression, possibly via β-catenin and/or Tcf/Lef1 interaction. This work identifies Sp5 as a novel potent feedback loop inhibitor of Wnt/β-catenin signaling across eumetazoans.

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Single-Cell RNA-Seq Reveals Dynamic, Random Monoallelic Gene Expression in Mammalian Cells

Science ◽

10.1126/science.1245316 ◽

2014 ◽

Vol 343 (6167) ◽

pp. 193-196 ◽

Cited By ~ 696

Author(s):

Q. Deng ◽

D. Ramskold ◽

B. Reinius ◽

R. Sandberg

Keyword(s):

Gene Expression ◽

Single Cell ◽

Mammalian Cells ◽

Rna Seq ◽

Expression In Mammalian Cells

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Faculty Opinions recommendation of Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718233329.793490974 ◽

2014 ◽

Author(s):

Michael Rudnicki ◽

Alessandra Pasut

Keyword(s):

Gene Expression ◽

Single Cell ◽

Mammalian Cells ◽

Rna Seq ◽

Expression In Mammalian Cells

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Faculty Opinions recommendation of Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718233329.793490333 ◽

2014 ◽

Author(s):

Marc S Williams

Keyword(s):

Gene Expression ◽

Single Cell ◽

Mammalian Cells ◽

Rna Seq ◽

Expression In Mammalian Cells

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Massively parallel RNA device engineering in mammalian cells with RNA-Seq

Nature Communications ◽

10.1038/s41467-019-12334-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 10

Author(s):

Joy S. Xiang ◽

Matias Kaplan ◽

Peter Dykstra ◽

Michaela Hinks ◽

Maureen McKeague ◽

...

Keyword(s):

Mammalian Cells ◽

Cell Types ◽

Massively Parallel ◽

Rna Seq ◽

High Activation ◽

Conserved Sequence ◽

Regulatory Processes ◽

Basal Expression ◽

Wide Range ◽

Gene Regulatory

Abstract Synthetic RNA-based genetic devices dynamically control a wide range of gene-regulatory processes across diverse cell types. However, the limited throughput of quantitative assays in mammalian cells has hindered fast iteration and interrogation of sequence space needed to identify new RNA devices. Here we report developing a quantitative, rapid and high-throughput mammalian cell-based RNA-Seq assay to efficiently engineer RNA devices. We identify new ribozyme-based RNA devices that respond to theophylline, hypoxanthine, cyclic-di-GMP, and folinic acid from libraries of ~22,700 sequences in total. The small molecule responsive devices exhibit low basal expression and high activation ratios, significantly expanding our toolset of highly functional ribozyme switches. The large datasets obtained further provide conserved sequence and structure motifs that may be used for rationally guided design. The RNA-Seq approach offers a generally applicable strategy for developing broad classes of RNA devices, thereby advancing the engineering of genetic devices for mammalian systems.

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Orthogonal control of mean and variability of endogenous genes in a human cell line

Nature Communications ◽

10.1038/s41467-020-20467-8 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Alain R. Bonny ◽

João Pedro Fonseca ◽

Jesslyn E. Park ◽

Hana El-Samad

Keyword(s):

Mammalian Cells ◽

Human Cell Line ◽

Transcriptional Level ◽

Clear Understanding ◽

Gene Expression Noise ◽

Endogenous Gene ◽

Basal Expression ◽

Stochastic Fluctuations ◽

Human Genes ◽

Synthetic Circuit

AbstractStochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.

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