Abstract
Prophase I is a remarkable stage of meiotic division during which homologous
chromosomes pair together and exchange DNA by meiotic recombination. Fluorescence
microscopy of meiotic chromosome spreads is a central tool in the study of this process, with
chromosome axis proteins being visualised as extended filaments upon which
recombination proteins localise in focal patterns.
Chromosome pairing and recombination are dynamic processes, and hundreds of
recombination foci can be present in some meiotic nuclei. As meiotic nuclei can exhibit
significant variations in staining patterns within and between nuclei, particularly in
mutants, manual analysis of images presents challenges for consistency, documentation, and
reproducibility. Here we share a combination of complementary computational tools which
can be used to partially automate the quantitative analysis of meiotic images. 1) The
segmentation of axial and focal staining patterns, to automatically measure chromosome
axis length and count axis-associated (and non-axis associated) recombination foci; 2)
Quantification of focus position along chromosome axes to investigate spatial regulation; 3)
Simulation of random distributions of foci within the nucleus or along the chromosome axes
to statistically investigate observed foci-axis associations and foci-foci associations; 4)
Quantification of chromosome axis proximity to investigate relationships with chromosome
synapsis/asynapsis; 5) Quantification of and orientation of focus-axis distances. Together
these tools provide a framework to perform routine documentation and analysis of meiotic
images, as well as opening up routes to build on this initial output and perform more
detailed analyses.
chromosomes pair together and exchange DNA by meiotic recombination. Fluorescence
microscopy of meiotic chromosome spreads is a central tool in the study of this process, with
chromosome axis proteins being visualised as extended filaments upon which
recombination proteins localise in focal patterns.
Chromosome pairing and recombination are dynamic processes, and hundreds of
recombination foci can be present in some meiotic nuclei. As meiotic nuclei can exhibit
significant variations in staining patterns within and between nuclei, particularly in
mutants, manual analysis of images presents challenges for consistency, documentation, and
reproducibility. Here we share a combination of complementary computational tools which
can be used to partially automate the quantitative analysis of meiotic images. 1) The
segmentation of axial and focal staining patterns, to automatically measure chromosome
axis length and count axis-associated (and non-axis associated) recombination foci; 2)
Quantification of focus position along chromosome axes to investigate spatial regulation; 3)
Simulation of random distributions of foci within the nucleus or along the chromosome axes
to statistically investigate observed foci-axis associations and foci-foci associations; 4)
Quantification of chromosome axis proximity to investigate relationships with chromosome
synapsis/asynapsis; 5) Quantification of and orientation of focus-axis distances. Together
these tools provide a framework to perform routine documentation and analysis of meiotic
images, as well as opening up routes to build on this initial output and perform more
detailed analyses.
| Original language | English |
|---|---|
| Type | Protocol |
| Publisher | Springer |
| Edition | 1 |
| ISBN (Print) | 9781071639054, 9781071639085 |
| ISBN (Electronic) | 9781071639061 |
| DOIs | |
| Publication status | Published - 11 Aug 2024 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Publisher | Humana Press |
| Volume | 2818 |
| ISSN (Print) | 1064-3745 |