How to make an egg?
Meiosis
Meiosis is a special kind of cell division were by one cycle of chromosome duplication is followed by two rounds of segregations to reduce the chromosome number by half, and thereby create the haploid sperm and eggs. In the first meiotic division (meiosis I) homologous chromosome segregate, while in the second division (meiosis II) sister chromatid segregate.
During the prophase stage of meiosis I, the chromosome undergo unique interactions which include homologous chromosome paring, forming of the synaptonemal complex, DNA double strand break induction and repair into crossover recombination.
Correct execution of these processes is required for the faithful segregation of chromosomes in young and old females.
To find master regulators of meiotic progression we have previously made use of the C. elegans nematode in which the nuclei are ordered in the gonad from the stem-cells to the mature oocyte. We conducted RNASeq analysis on microscope dissected gonad segments, as small as single oocytes. This has allowed us to create the gonad spatial transcriptome database.
Spatial transcriptomics: Illustration of the C. elegans gonad and the 10 consecutive sections we captured using Laser Capture Microdissection. Following RNASeq the stdandardized expression (y axis) was color coded for each gene in the autosomes and the X chromosome along the 10 sections (x axis).
​
We are using this database to find the genes which are activated or repressed at critical points along oogenesis. One such genes is ogr-2 which we found to be activated at the entry into meiosis, and at the onset of the final differentiation stage:
ogr-2 expression pattern: Normalized levels of ogr-2 transcripts, as extracted from our transcriptomic map, along 10 stages of oogensis from the germ stem cells to the mature oocyte stages.
​
Using genome engineering, we completely deleted ogr-2 sequence from the genome:
​
Experimental strategy to completely delete ogr-2 : Cas-9 was expressed togayher with gRNAs directed to both sides of the gene.
In worms without OGR-2 meiotic progression was altered, and the activation of MAPK signaling along the gonad changed:
ogr-2 deletion leads to aberrant MPK-1 activation: A: IF images of WT and ogr-2 gonads stained with DAPI (blue) and activated MPK-1 (red). B. Quantification of activated MPK-1 staining along the gonad.
Genetic analysis indicated that this role is mediated by controlling the transcription of the phosphatase LIP-1. Not surprisingly OGR-2 localization is enriched on chromatin:
OGR-2 is localized to germline chromatin: Pachytene nuclei in a strain we anginered a FLAG tag at the ogr-2 endogenous site before the STOP codon stained with DAPI (blue) and anti-FLAG (red).
Open questions and current work:
-
What is the role of OGR-2 on the chromatin?
-
Who are OGR-2’s binding partners?
-
Why is OGR-2 enriched on one chromosome during pachytene?
