scholarly journals Shh and ZRS enhancer co-localisation is specific to the zone of polarizing activity

2016 ◽  
Author(s):  
Iain Williamson ◽  
Laura A. Lettice ◽  
Robert E. Hill ◽  
Wendy A. Bickmore
Keyword(s):  
Developmental Stages ◽  
In Situ Hybridisation ◽  
Super Resolution ◽  
Genomic Region ◽  
Limb Bud ◽  
Fluorescence In Situ Hybridisation ◽  
Chromosome Conformation ◽  
Active Enhancer ◽  
Mouse Limb ◽  
Zone Of Polarizing Activity

AbstractLimb-specific Shh expression is regulated by the (~1 Mb distant) ZRS enhancer. In the mouse, limb bud restricted spatiotemporal expression of Shh occurs from ~E10-E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS co-localisation only in the Shh expressing regions of the limb bud. However, Shh-ZRS spatial distances were consistently shorter than intervening distances to a neural enhancer in all tissues and developmental stages analysed. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS co-localisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh/ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate Shh expression.

scholarly journals Fibroblast growth factors (FGFs) prime the limb specific Shh enhancer for chromatin changes that balance histone acetylation mediated by E26 transformation-specific (ETS) factors

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Silvia Peluso ◽  
Adam Douglas ◽  
Alison Hill ◽  
Carlo De Angelis ◽  
Benjamin L Moore ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Limb Bud ◽  
Fibroblast Growth ◽  
Distal Limb ◽  
Inactive State ◽  
Dynamic Events ◽  
Active Enhancer ◽  
Mouse Limb ◽  
Fgf Signalling ◽  
Zone Of Polarizing Activity

Sonic hedgehog (Shh) expression in the limb bud organizing centre called the zone of polarizing activity is regulated by the ZRS enhancer. Here, we examine in mouse and in a mouse limb-derived cell line the dynamic events that activate and restrict the spatial activity of the ZRS. Fibroblast growth factor (FGF) signalling in the distal limb primes the ZRS at early embryonic stages maintaining a poised, but inactive state broadly across the distal limb mesenchyme. The E26 transformation-specific transcription factor, ETV4, which is induced by FGF signalling and acts as a repressor of ZRS activity, interacts with the histone deacetylase HDAC2 and ensures that the poised ZRS remains transcriptionally inactive. Conversely, GABPα, an activator of the ZRS, recruits p300, which is associated with histone acetylation (H3K27ac) indicative of an active enhancer. Hence, the primed but inactive state of the ZRS is induced by FGF signalling and in combination with balanced histone modification events establishes the restricted, active enhancer responsible for patterning the limb bud during development.

scholarly journals Hierarchical chromatin organization detected by TADpole

2019 ◽  
Author(s):  
Paula Soler-Vila ◽  
Pol Cuscó Pons ◽  
Irene Farabella ◽  
Marco Di Stefano ◽  
Marc A. Marti-Renom
Keyword(s):  
Rapid Development ◽  
Super Resolution ◽  
Region Of Interest ◽  
Principal Component ◽  
Genomic Region ◽  
Mouse Strains ◽  
Bioinformatics Analyses ◽  
Chromosome Conformation ◽  
Resolution Imaging ◽  
Interaction Matrices

ABSTRACTThe rapid development of chromosome conformation capture (3C-based) techniques as well as super-resolution imaging together with bioinformatics analyses has been fundamental for unveiling that chromosomes are organized into the so-called topologically associating domains or TADs. While these TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available computational methods are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide an unsupervised set of significant partitions in a genomic region of interest. TADpole identification of domains is robust to the data resolution, normalization strategy, and sequencing depth. TADpole domain borders are enriched in CTCF and cohesin binding proteins, while the domains are enriched in either H3K36me3 or H3k27me3 histone marks. We show TADpole usefulness by applying it to capture Hi-C experiments in wild-type and mutant mouse strains to pinpoint statistically significant differences in their topological structure.

Maintenance of ZPA signaling in cultured mouse limb bud cells

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1421-1433 ◽  
Author(s):  
R. Anderson ◽  
M. Landry ◽  
K. Muneoka
Keyword(s):  
Culture Technique ◽  
Limb Bud ◽  
In Vivo Studies ◽  
Maintenance Activity ◽  
Vertebrate Limb ◽  
Mouse Limb ◽  
Zone Of Polarizing Activity

The positional signal localized to the posterior (zone of polarizing activity or ZPA) region of the vertebrate limb is transiently expressed during development and a decline in ZPA signaling is accelerated when posterior cells are dissociated and cultured in vitro. The evidence that cultured posterior cells display a precocious decline in ZPA signaling when compared to in vivo studies suggests that a factor present in the limb bud maintains or stabilizes ZPA signaling during limb outgrowth and that this maintenance factor is lost and/or exhausted in in vitro studies. We have developed a new culture technique, ‘microdissociation’, which preserves extracellular components that we have found to be necessary for ZPA signal maintenance. Our data suggest that the limb bud ectoderm produces a maintenance activity that becomes stored in the extracellular matrix where it acts on limb bud cells to stabilize the activity of the ZPA signal. Using our initial characterization of this maintenance activity, we have identified a growth factor, FGF-2 (bFGF), that can replace all of the ZPA signaling maintenance activity observed in microdissociate cultures. The existence of various members of the FGF family in the developing limb strongly argues a role for FGF in stabilizing ZPA signaling in vivo.

scholarly journals Developmentally regulated Shh expression is robust to TAD perturbations

2019 ◽  
Author(s):  
Iain Williamson ◽  
Lauren Kane ◽  
Paul S. Devenney ◽  
Eve Anderson ◽  
Fiona Kilanowski ◽  
...  
Keyword(s):  
Developmental Regulation ◽  
Expression Patterns ◽  
In Situ Hybridisation ◽  
Detectable Effect ◽  
Chromatin Conformation ◽  
Fluorescence In Situ Hybridisation ◽  
Developmentally Regulated ◽  
Enhancer Activity ◽  
Chromosome Conformation

AbstractTopologically Associating Domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD – creating internal deletions, deleting CTCF sites including those at TAD boundaries, as well as larger deletions and inversions of TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used the investigate changes in chromatin conformation that result from these manipulations. Our data suggest that the substantial alteration of TAD structure has no readily detectable effect on Shh expression patterns during development – except where enhancers are deleted - and results in no detectable phenotypes. Only in the case of a larger deletion of one TAD boundary could some ectopic influence of the Shh limb enhancer be detected on a gene - Mnx1 in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.

scholarly journals Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 250
Author(s):  
Rebecca E O’Connor ◽  
Lucas G Kiazim ◽  
Claudia C Rathje ◽  
Rebecca L Jennings ◽  
Darren K Griffin
Keyword(s):  
Semen Analysis ◽  
In Situ Hybridisation ◽  
Economic Loss ◽  
Environmental Damage ◽  
Fluorescence In Situ Hybridisation ◽  
Reciprocal Translocations ◽  
Chromosome Translocations ◽  
Farrowing Rate ◽  
Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

scholarly journals Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Caitlin M. Singleton ◽  
Francesca Petriglieri ◽  
Jannie M. Kristensen ◽  
Rasmus H. Kirkegaard ◽  
Thomas Y. Michaelsen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Wastewater Treatment Plants ◽  
In Situ Hybridisation ◽  
Amplicon Sequencing ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Sequencing Data ◽  
High Quality ◽  
16S Rrna Amplicon Sequencing ◽  
Long Read

AbstractMicroorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

scholarly journals 3D reconstruction of genomic regions from sparse interaction data

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Julen Mendieta-Esteban ◽  
Marco Di Stefano ◽  
David Castillo ◽  
Irene Farabella ◽  
Marc A Marti-Renom
Keyword(s):  
3D Models ◽  
Genomic Region ◽  
Gene Promoters ◽  
Chromosome Conformation ◽  
The Matrix ◽  
Chromatin Structural ◽  
A Cell ◽  
Similar Accuracy ◽  
Genomic Regions ◽  
Interaction Matrices

Abstract Chromosome conformation capture (3C) technologies measure the interaction frequency between pairs of chromatin regions within the nucleus in a cell or a population of cells. Some of these 3C technologies retrieve interactions involving non-contiguous sets of loci, resulting in sparse interaction matrices. One of such 3C technologies is Promoter Capture Hi-C (pcHi-C) that is tailored to probe only interactions involving gene promoters. As such, pcHi-C provides sparse interaction matrices that are suitable to characterize short- and long-range enhancer–promoter interactions. Here, we introduce a new method to reconstruct the chromatin structural (3D) organization from sparse 3C-based datasets such as pcHi-C. Our method allows for data normalization, detection of significant interactions and reconstruction of the full 3D organization of the genomic region despite of the data sparseness. Specifically, it builds, with as low as the 2–3% of the data from the matrix, reliable 3D models of similar accuracy of those based on dense interaction matrices. Furthermore, the method is sensitive enough to detect cell-type-specific 3D organizational features such as the formation of different networks of active gene communities.

Effects of the diploidisation process upon the 5S and 35S rDNA sequences in the allopolyploid species of the Dilatata group of Paspalum (Poaceae, Paniceae)

2019 ◽  
Vol 67 (7) ◽  
pp. 521
Author(s):  
Magdalena Vaio ◽  
Cristina Mazzella ◽  
Marcelo Guerra ◽  
Pablo Speranza
Keyword(s):  
Evolutionary Dynamics ◽  
In Situ Hybridisation ◽  
5S Rdna ◽  
Its Region ◽  
Variable Number ◽  
Rrna Genes ◽  
Fluorescence In Situ Hybridisation ◽  
Rdna Sites ◽  
35S Rdna ◽  
Genome Restructuring

The Dilatata group of Paspalum includes species and biotypes native to temperate South America. Among them, five sexual allotetraploids (x = 10) share the same IIJJ genome formula: P. urvillei Steud, P. dasypleurum Kunze ex Desv., P. dilatatum subsp. flavescens Roseng., B.R. Arrill. & Izag., and two biotypes P. dilatatum Vacaria and P. dilatatum Virasoro. Previous studies suggested P. intermedium Munro ex Morong & Britton and P. juergensii Hack. or related species as their putative progenitors and donors of the I and J genome, respectively, and pointed to a narrow genetic base for their maternal origin. It has not yet been established whether the various members of the Dilatata group are the result of a single or of multiple allopolyploid formations. Here, we aimed to study the evolutionary dynamics of rRNA genes after allopolyploidisation in the Dilatata group of Paspalum and shed some light into the genome restructuring of the tetraploid taxa with the same genome formula. We used double target fluorescence in situ hybridisation of 35S and 5S rDNA probes and sequenced the nrDNA internal transcribed spacer (ITS) region. A variable number of loci at the chromosome ends were observed for the 35S rDNA, from 2 to 6, suggesting gain and loss of sites. For the 5S rDNA, only one centromeric pair of signals was observed, indicating a remarkable loss after polyploidisation. All ITS sequences generated were near identical to the one found for P. intermedium. Although sequences showed a directional homogeneisation towards the putative paternal progenitor in all tetraploid species, the observed differences in the number and loss of rDNA sites suggest independent ongoing diploidisation processes in all taxa and genome restructuring following polyploidy.

Misexpression of Sox9 in mouse limb bud mesenchyme induces polydactyly and rescues hypodactyly mice

2007 ◽  
Vol 26 (4) ◽  
pp. 224-233 ◽  
Author(s):  
Haruhiko Akiyama ◽  
H. Scott Stadler ◽  
James F. Martin ◽  
Takahiro M. Ishii ◽  
Philip A. Beachy ◽  
...  
Keyword(s):  
Limb Bud ◽  
Mouse Limb
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