Analyzing negative feedback using a synthetic gene network expressed in the Drosophila melanogaster embryo

Jermusyk, Ashley; Murphy, Nicholas P.; Reeves, Gregory T.

doi:10.1186/s12918-016-0330-z

Cited by 5 publications

(2 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The intensity of mRNA expression and total Dl were calculated as average intensity within an annulus ∼18 μm wide around the perimeter of the embryo. A description of the image analysis of whole-mount embryos can be found in the Supplementary Materials and Methods and in Jermusyk et al (2016).…”

Section: Discussionmentioning

confidence: 99%

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

et al. 2017

Self Cite

View full text Add to dashboard Cite

The transcription factor NF-κB plays an important role in the immune system, apoptosis and inflammation. Dorsal, a homolog of NF-κB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IκB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. Here, we use modeling and experimental studies to show that this accumulation is caused by facilitated diffusion, or shuttling, of the Dorsal/Cactus complex. We also show that active Toll receptors are limiting in wild-type embryos, which is a key factor in explaining global Dorsal gradient formation. Our results suggest that shuttling is necessary for viability of embryos from mothers with compromised levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also has a secondary role in shuttling. Given that this mechanism has been found in other, independent, systems, we suggest that it might be more prevalent than previously thought.

show abstract

Section: Discussionmentioning

confidence: 99%

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…As a proof-of-concept, we next wanted to test these tools in a multicellular system. We chose to work with Drosophila embryos as we have previously used this system to study synthetic networks (40). Thus, we generated transgenic fly lines carrying these ribozyme constructs.…”

Section: Resultsmentioning

confidence: 99%

Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems

Jacobsen

Asafen

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

ABSTRACTAdvancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5’ untranslated region (UTR) vs. 3’UTR) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.

show abstract

Determination of novel members in theDrosophila melanogasteranteriorposterior patterning system using natural variation

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

The anterior-posterior axis of the developing Drosophila melanogaster embryo is patterned by a well-studied gene regulatory network called the Gap Gene Network. This network acts to buffer the developing pattern against noise, thereby minimizing errors in gene expression and preventing patterning defects.In this paper, we sought to discover novel regulatory regions and transcription factors acting in a subset of the Gap network using a selection of wild-caught fly lines derived from the Drosophila Genetic Reference Panel (DGRP). The fly lines in the DGRP contain subtle genomic differences due to natural variation; we quantified the differences in positioning of gene expression borders of two anterior-poster patterning genes, Krüppel (Kr) and Even-skipped in 13 of the DGRP lines. The differences in the positions of Krüppel andEven-skipped were then correlated to specific single nucleotide polymorphisms and insertions/deletions within the select fly lines. Putative enhancers containing these genomic differences were validated for their ability to produce expression using reporter constructs and analyzed for possible transcription factor binding sites. The identified transcription factors were then perturbed and the resulting Eve and Kr positioning was determined. In this way, we found medea, ultraspiracle, glial cells missing, and orthopedia effect Kr and Eve positioning in subtle ways, while knock-down of pangolin produces significant shifts in Kr and subsequent Eve expression patterns. Most importantly this study points to the existence of many additional novel members that have subtle effects on this system and the degree of complexity that is present in patterning the developing embryo.

show abstract

Analyzing negative feedback using a synthetic gene network expressed in the Drosophila melanogaster embryo

Cited by 5 publications

References 49 publications

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems

Determination of novel members in theDrosophila melanogasteranteriorposterior patterning system using natural variation

Contact Info

Product

Resources

About