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Developmental Biology - Regeneration

The Genetics of Regeneration

Study uncovers genetic switches controlling process of whole-body regeneration...

When it comes to regeneration, some animals are capable of amazing feats. If you cut the leg off a salamander, it will grow back. When threatened, some geckos drop their tails as a distraction, and regrow them later. Other animals take the process even further. Planarian worms, jellyfish, and sea anemones can actually regenerate their entire bodies after being cut in half.

Led by Assistant Professor Mansi Srivastava PhD, Johns Hopkins University, a team of researchers is shedding new light on how animals pull off regeneration uncovering a number of DNA switches that appear to control genes for whole-body regeneration. The study appears in the journal Science.

Using three-banded panther worms to test regeneration, Mansi Srivastava and Andrew Gehrke, a post-doctoral fellow in Srivastava's lab, found a section of non-coding DNA activates a "master control gene" called early growth response, or EGR. Once active, EGR controls a number of processes by switching genes on or off.

"We found this one master gene comes on and activates genes during regeneration. Basically, non-coding regions are telling coding regions to turn on or off as though they are switches. A lot of those very tightly packed portions of the genome physically become more open, because regulatory switches have to turn genes on or off," explains Gehrke. "One of the big findings in this paper is how the genome is very dynamic during regeneration as different parts are opening and closing."

For the process to work, Gehrke adds, DNA in the worms' cells, normally tightly folded and compacted, has to change to make new areas available for activation.

But, before Gehrke and Srivastava could understand the dynamic nature of the worm's genome, they had to assemble its entire genomic sequence - no simple feat. A big part of the paper is the reveal of the genome of the three-banded panther worm. This is important as it is the first of this phylum to have its full genome sequence available. The three banded panther worm represents a new model system for studying regeneration.

"There are many reasons to work with these new worms, one being they have an important phylogenetic position in their relatedness to other animals, which allows us to make statements about evolution. The other is that they are really great 'lab rats.' Since we've brought them into the lab, they are amenable to a lot more tools than some other systems,"says Srivastava.

Gehrke has identified as many as 18,000 regions that change in regeneration of the banded panther worm. Srivastava adds that EGR acts like a power switch for regeneration - once turned on, other processes can take place, without it - nothing regenerates.

"We were able to decrease the activity of the EGR gene to find that if you don't have EGR, nothing happens. The animals just can't regenerate. All those downstream genes won't turn on, other switches won't work, and the whole house basically goes dark."

Mansi Srivastava PhD, Assistant Professor, Organismic and Evolutionary Biology, Johns Hopkins University, Baltimore, Maryland, USA.

Srivastava: "If you have human cells in a dish and stress them, whether mechanically or by putting toxins on them, they express EGR right away. If humans can turn on EGR, [even] when our cells are injured, why can't we regenerate? The answer may be that our wiring is different than what it is in the three banded panther worm. Andrew has come up with a way to get at this wiring to figure out what those connections are that apply to other animals, including vertebrates who have limited regeneration.

"Now that we know which switches operate regeneration, we are looking at switches involved in [normal] development to see whether they are the same switches. If you compare genomes across all animals, most of the genes that we have are also in the three banded panther worm. So, we think that some answers are probably not going to come from whether or not certain genes are present, but from how they are wired or networked together. That answer can only come from the noncoding portion of the genome."

Mansi Srivastava PhD.
"Only about two percent of the genome makes things like proteins," Andrew Gehrke explains. "We wanted to know: What is the other 98 percent of the genome doing during whole-body regeneration? People have known for some time that many DNA changes cause disease in non-coding DNA regions. But, it is underappreciated as a process, like whole-body regeneration. I think we've only just scratched the surface. We've looked at some switches, but there is another aspect of how the genome interacts on a larger scale, not just how pieces open and close. All of it is important for turning genes on and off; so, I think there are multiple layers to this regulatory nature."

Structured Abstract
INTRODUCTION
Although all animals can heal wounds, some are capable of reconstructing their entire bodies from small fragments of the original organism. Whole-body regeneration requires the interplay of wound signaling, stem cell dynamics, and positional identity, all of which have been investigated at the protein-coding level of the genome. Little is known about how the noncoding portion of the genome responds to wounding to control gene expression and to launch the process of whole-body regeneration. Understanding how these control points (regulatory regions) are activated and then operate during regeneration would uncover how genes connect into networks, ultimately restructuring entire body axes. Networks of transcriptional regulatory genes can reveal important mechanisms for how animals can grow new skin, muscles, or even entire brains.

RATIONALE
To identify regulatory regions involved in whole-body regeneration, we sequenced the genome of the highly regenerative acoel Hofstenia miamia, commonly known as the three-banded panther worm. Equipped with this genome, we reasoned that applying the assay for transposase-accessible chromatin using sequencing (ATAC-seq) would identify regulatory regions that change in response to amputation and during whole-body regeneration. Further, by analyzing the sequence motifs contained within these regulatory regions, we sought to predict which transcription factors (TFs) control regeneration gene networks.

RESULTS
The Hofstenia genome assembly totals 950 megabases of sequence, with sufficient contiguity for functional genomics. ATAC-seq data revealed thousands of chromatin regions that respond dynamically during regeneration. A genome-wide scan for TF binding motifs in these regions identified the EGR (early growth response) motif as the most dynamic. By combining RNA interference (RNAi) and RNA-seq, we predicted a set of EGR target genes in Hofstenia. We found that most of these target genes contained EGR binding motifs in neighboring regions of regeneration-responsive chromatin, which failed to respond under egr-RNAi. This functional validation allowed us to build a gene regulatory network (GRN) with EGR as a direct master regulator of downstream regeneration genes. Lastly, by quantifying the binding probabilities of TFs at individual motifs, we identified targets of TFs further downstream of EGR, extending the regeneration GRN.

CONCLUSION
Using our regulatory data, we inferred a GRN for launching whole-body regeneration in the acoel H. miamia, where the master regulator EGR acts as a putative pioneer factor to directly activate wound-induced genes. This network includes homologs of genes that are involved in regeneration in other species, suggesting that it can serve as a template for direct comparisons of regeneration pathways across distantly related animals. Our approach of combining genome-wide assays for chromatin accessibility with functional studies can be applied to extend the network further in time in Hofstenia regeneration and to construct GRNs for regeneration in other systems.

Authors
Andrew R. Gehrke, Emily Neverett, Yi-Jyun Luo, Alexander Brandt, Lorenzo Ricci, Ryan E. Hulett, Annika Gompers, J. Graham Ruby, Daniel S. Rokhsar, Peter W. Reddien and Mansi Srivastava1.

Acknowledgements
This research was supported with funding from the Milton Fund of Harvard University, the Searle Scholars Program, the Smith Family Foundation, the National Science Foundation, the Helen Hay Whitney Foundation, the Human Frontier Science Program, the National Institutes of Health, the Biomedical Big Training Program, UC Berkeley, the Marthella Foskett Brown Chair in Biological Sciences, and the Howard Hughes Medical Institute.

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Mar 21 2019 Fetal Timeline Maternal Timeline News

The regulatory landscape of whole-body regeneration. Researchers sequenced the genome and used ATAC-seq to identify thousands of regeneration-responsive regions of chromatin. Combining motif analysis, ATAC-seq, and RNAi, they identified EGR as a master regulator of regeneration in Hofstenia [commonly called the three-banded panther worm] and inferred an EGR-controlled GRN for regeneration. Science: 15 Mar 2019:Vol. 363, Issue 6432.