Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

Julien Rougot, Naomi D Chrispijn, Marco Aben, Dei M Elurbe, Karolina M Andralojc, Patrick J Murphy, Pascal WTC Jansen, Michiel Vermeulen, Bradley R Cairns, Leonie M Kamminga

Posted on: 7 January 2019 , updated on: 18 January 2019

Preprint posted on 12 November 2018

Article now published in Development at http://dx.doi.org/10.1242/dev.178590

Though ezh2-mutant zebrafish form grossly normal body plans, key developmental factors are dysregulated early on, which dooms the embryo to later failure of organogenesis and lethality.

Selected by Yen-Chung Chen

Categories: cell biology, developmental biology, genomics

Background and context

Polycomb repressive complex 2 (PRC2) mediates methylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is a chromatin mark associated with gene repression, and the disruption of PRC2 results in grave disruption of development. In flies, body segments are organized wrongly when H3K27me3 is disrupted, and mouse embryos die before gastrulation, presumably as a result of failed differentiation. These severe consequences have made H3K27me3 one of the best studied histone modifications in development, and several critical genes governing segmentation or fate specification have been shown to be under the control of PRC2 via H3K27me3 [Reviewed in 1 and 2].

Because mammalian embryos fail to gastrulate without PRC2, the function of PRC2 in development is mostly studied by differentiating stem cells in vitro. Although the in vivo functions of PRC2 are well characterized in flies, the mode of action of Drosophila PRC2 is starkly different from vertebrate PRC2. In a previous study from the Kamminga lab, they used a germ cell transplantation technique in zebrafish to generate a PRC2 loss-of-function model by mutating ezh2, a core component of PRC2. They found out that even without ezh2, the fish remains grossly normal through segmentation until organogenesis [3]. The power of this model enables Rougot et al. to investigate the consequences of the absence of H3K27me3 in gene regulation in vivo in the current preprint.

Key findings

Ezh2 mutant embryos — *Zebra fish provides a novel and versatile model to study the consequences of the loss of H3K27me3 (Figures are adapted from the manuscript under a CC-BY-NC-ND 4.0 International license).*
*(A) ezh2-mutant embryo develops largely normal up to 48 hours post-fertilization (from Figure 1A)*
*(B) ezh2-mutants are steadily deprived of ezh2 during early embryogenesis (from Figure 1B).*
*(C) The landscape of histone marks and the binding of writers are perturbed (from Figure 1G).*
*(D) Spatial expression of histone mark-regulated genes is altered in the absence of ezh2 (from Figure 6A).*

Rougot et al. showed that in maternal-zygotic ezh2mutant zebrafish, the level of H3K27me3 decreases to a level barely detectable with ChIP-seq. Interestingly, besides the absence of H3K27me3, rnf2, the core component of PRC1, failed to find its normal targets in ezh2 mutants, though the subunits of PRC1 are present in ezh2mutants. It has been unclear whether the action of PRC1 is dependent on the disposition of H3K27me3, and the current study provides a new line of evidence that in the early stages of zebrafish development, PRC1 requires functional PRC2 to work properly.

In the absence of H3K27me3, H3K4me3, a histone modification associated with transcriptionally active state, is enriched in otherwise H3K27me3-occupied gene loci. Consistent with the important role H3K27me3 plays in developmental gene regulation, genes regulating developmental processes gain H3K4me3 and are upregulated in the absence of H3K27me3. The authors further performed mass spectrometry to check if these epigenetic and transcriptomic changes resulted in altered proteome. Indeed, the proteomic study shows an up-regulation of both a subset of genes occupied by H3K27me3 in the wildtype and the genes gaining H3K4me3 in the ezh2 mutant.

To further examine segment-specific effects of H3K27me3 disruption, the authors first performed RT-qPCR for different body segments to evaluate the expression pattern of hox genes, which are expressed with a clear segmental pattern and govern the head-to-tail body plane. In ezh2 mutants, thoracic hox genes (e.g. hoxa9a and hoxa9b) are up-regulated in the rostral part of body. More caudal hox genes (e.g. hoxa11b and hoxa13b) are also up-regulated but to a lesser extent. These caudal hoxgenes might be repressed by other mechanisms in addition to H3K27me3. Then, the authors performed in situhybridization of several transcription factors with known spatial expression patterns, including the tbx family, isl1, and gsc. Ectopic expression of every transcription factor examined in mutants. The most extreme change is observed in gsc. The normal expression pattern for gsc is confined in telencephalon and diencephalon, branchial arches, and otic vesicle, while in ezh2 mutants, the expression becomes ubiquitous despite being weak.

In short, Rougot et al. used ezh2 mutant zebrafish to show that the early developmental process is robust against the absence of H3K27me3 and proceeds in a grossly normal pattern without H3K27me3. Nonetheless, dysregulation of expression of developmental regulators is detected at the RNA and protein level at 24 hpf, and the spatial patterns of these developmental regulators are perturbed to various extent without H3K27me3.

Why I like this preprint

Histone modifications have long been proposed as an important factor in development to ensure precise coordination of differentiation processes. Indeed, mechanistic studies corroborate the roles of histone modifications in gene regulation and identify many processes they are involved in. Nevertheless, many studies on histone modification-mediated regulation in mammals are performed in vitro, and phenotypic and mechanistic discrepancies between in vivo and in vitromodels are not uncommon. Besides, although the well-defined and scalable nature of cell models enables detailed characterization of the mechanism and a great variety of manipulations, the lack of higher order organization and interactions between different cell types in cell models poses a great challenge for researchers hoping to assess the crosstalk between different regulatory mechanisms. For example, it is common that a striking mutant phenotype in cell models is less severe and presumably compensated in vivobecause environmental cues might provide information that compensates the dysfunction. Thus, it is an invaluable resource to have a vertebrate model of PRC2 loss of function, and this study shows the degree of erosion of spatial expression pattern differs a lot among the genes examined, which might suggest H3K27me3 contributes differently to the repression of H3K27me3-marked genes.

Open questions

H3K27me3 is reported to safeguard cell identities. Different cell identities are often controlled by antagonizing developmental cues and undergo transcriptomic changes in opposite direction. Would it be possible to examine each germ layer individually to see if the cells gain more plasticity or dual identity in the absence of H3K27me3? This could also rule out the possibility that the transcriptomic changes in different germ layers of ezh2mutant cancel out each other when an embryo is profiled as a whole.
It is still unclear how PRC2 finds its target loci to tri-methylate. Would it be feasible to re-introduce ezh2 later in development in an ezh2mutant zebrafish to see if H3K27me3 at certain loci could be established properly to see whether the guiding signal that leads PRC2 to its target loci is transient or not?

References

Kassis, J. A., Kennison, J. A. & Tamkun, J. W. Polycomb and Trithorax Group Genes in Drosophila. Genetics. 206,1699–1725 (2017).
Aloia, L., Di Stefano, B. & Di Croce, L. Polycomb complexes in stem cells and embryonic development. Development 140, 2525 LP-2534 (2013).
San, B. et al.Normal formation of a vertebrate body plan and loss of tissue maintenance in the absence of ezh2. Rep. 6,24658 (2016).

Tags: epigenetics, h3k27me3, prc2, zebra fish

doi: https://doi.org/10.1242/prelights.6996

Read preprint

(No Ratings Yet)

Author's response to the question raised in this highlight

Leonie M Kamminga shared

This is an interesting thought. We performed our analysis on whole embryo lysates at 24hpf, a stage when many different cell types are already present. By using germ layer specific transgenes or markers it would be possible to study the individual germ layers. However, in case the identity of cells changed because of loss of Ezh2, these cells might have lost the germ layer specific identity and will therefore not be picked up. In addition, and the same goes for single cell techniques, in case cell identity has changed, it will be difficult to reconstruct the embryo based on expression profiles. A good alternative, in this case, would be an approach using tomo-seq. This technique ensures the spatial information of gene expression will be maintained and changes between wildtype and mutant embryos will be better and easier to compare.
The way PRC2 finds its target loci is still under debate. A recent study shows Mtf2 is involved in recruitment of PRC2. The authors of the paper describing this work, propose that recruitment of PRC2 is dependent on the helical shape of the DNA. There are different methods to reintroduce Ezh2 later during development in MZezh2mutants to get insight into recruitment of PRC2. One of the approaches would be to cross a germ line mutant female with a heterozygous male, which results in having only zygotic expression of ezh2. However, with this method it is difficult to control when ezh2starts to express. Another method, which allows control of timing of ezh2expression involves a transgene with an inducible promoter, for instance a heat shock promoter.

Have your say Cancel reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Also in the cell biology category:

Motor Clustering Enhances Kinesin-driven Vesicle Transport

Rui Jiang, Qingzhou Feng, Daguan Nong, et al.

Selected by 16 November 2024

Sharvari Pitke

Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis

Joshua Hawley, Robert Lea, Veronica Biga, et al.

Selected by 15 November 2024

Ankita Walvekar

Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water

Adebayo J. Bello, Omorilewa B. Ebunoluwa, Rukayat O. Ayorinde, et al.

Selected by 14 November 2024

Safieh Shah, Benjamin Dominik Maier

Discussion

Also in the developmental biology category:

Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis

Joshua Hawley, Robert Lea, Veronica Biga, et al.

Selected by 15 November 2024

Ankita Walvekar

Actin-based deformations of the nucleus control multiciliated ependymal cell differentiation

Marianne Basso, Alexia Mahuzier, Syed Kaabir Ali, et al.

Selected by 30 October 2024

Ryan Harrison

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos

Estefania Sanchez-Vasquez, Marianne E. Bronner, Magdalena Zernicka-Goetz

Selected by 11 October 2024

Anchel De Jaime Soguero

Also in the genomics category:

A fine kinetic balance of interactions directs transcription factor hubs to genes

Apratim Mukherjee, Samantha Fallacaro, Puttachai Ratchasanmuang, et al.

Selected by 23 July 2024

Deevitha Balasubramanian

Enhancer-driven cell type comparison reveals similarities between the mammalian and bird pallium

Nikolai Hecker , Niklas Kempynck , David Mauduit, et al.

Selected by 02 July 2024

Rodrigo Senovilla-Ganzo

Modular control of time and space during vertebrate axis segmentation

Ali Seleit, Ian Brettell, Tomas Fitzgerald, et al.

AND

Natural genetic variation quantitatively regulates heart rate and dimension

Jakob Gierten, Bettina Welz, Tomas Fitzgerald, et al.

Selected by 24 June 2024

Girish Kale, Jennifer Ann Black

preLists in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

Share this:

Have your say Cancel reply

Sign up to customise the site to your preferences and to receive alerts

Also in the cell biology category:

Motor Clustering Enhances Kinesin-driven Vesicle Transport

Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis

Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water

Also in the developmental biology category:

Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis

Actin-based deformations of the nucleus control multiciliated ependymal cell differentiation

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos

Also in the genomics category:

A fine kinetic balance of interactions directs transcription factor hubs to genes

Enhancer-driven cell type comparison reveals similarities between the mammalian and bird pallium

Modular control of time and space during vertebrate axis segmentation

Natural genetic variation quantitatively regulates heart rate and dimension

preLists in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

‘In preprints’ from Development 2022-2023

preLights peer support – preprints of interest

The Society for Developmental Biology 82nd Annual Meeting

CSHL 87th Symposium: Stem Cells

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

9th International Symposium on the Biology of Vertebrate Sex Determination

Alumni picks – preLights 5th Birthday

CellBio 2022 – An ASCB/EMBO Meeting

Fibroblasts

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

FENS 2020

Planar Cell Polarity – PCP

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

Cell Polarity

TAGC 2020

3D Gastruloids

ECFG15 – Fungal biology

ASCB EMBO Annual Meeting 2019

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Autophagy

Lung Disease and Regeneration

Cellular metabolism

BSCB/BSDB Annual Meeting 2019

MitoList

Biophysical Society Annual Meeting 2019

ASCB/EMBO Annual Meeting 2018

Also in the developmental biology category:

BSDB/GenSoc Spring Meeting 2024

GfE/ DSDB meeting 2024

‘In preprints’ from Development 2022-2023

preLights peer support – preprints of interest

The Society for Developmental Biology 82nd Annual Meeting

CSHL 87th Symposium: Stem Cells

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

9th International Symposium on the Biology of Vertebrate Sex Determination

Alumni picks – preLights 5th Birthday

CellBio 2022 – An ASCB/EMBO Meeting

2nd Conference of the Visegrád Group Society for Developmental Biology

Fibroblasts

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

EMBL Conference: From functional genomics to systems biology

Single Cell Biology 2020

Society for Developmental Biology 79th Annual Meeting

FENS 2020

Planar Cell Polarity – PCP

Cell Polarity

TAGC 2020

3D Gastruloids

ASCB EMBO Annual Meeting 2019

EDBC Alicante 2019

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

SDB 78th Annual Meeting 2019

Lung Disease and Regeneration

Young Embryologist Network Conference 2019

Pattern formation during development

BSCB/BSDB Annual Meeting 2019

Zebrafish immunology

Also in the genomics category:

BSCB-Biochemical Society 2024 Cell Migration meeting

9th International Symposium on the Biology of Vertebrate Sex Determination

Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University