A pathway for synapsis initiation during zygotene in Drosophila oocytes


Pairs of meiotic chromosomes, or homologs, are brought together in an elaborate pairing process which culminates with synapsis, where bivalents are held together along their entire length by the synaptonemal complex (SC).   Formation of the SC between homologs is essential for crossing over and chromosome segregation but how the homologs are paired and SC assembly initiates is poorly understood.   We investigated the requirements for SC assembly in Drosophila oocytes and found that there are three temporally and genetically distinct stages of synapsis initiation (Figure 1).  First, early zygotene cells have one or two patches of SC that colocalizes with a cluster of centromeres, suggesting synapsis initiates first at the centromeres.  Second, oocytes at mid-zygotene contain the centromere SC plus several euchromatic sites.  The centromeric and first euchromatic SC initiation sites depend on the cohesin protein ORD.  Third, late zygotene contains many more sites of SC initiation and these depend on the Kleisin-like protein C(2)M.  Surprisingly, the synapsis initiation events in late zygotene are independent of the earlier mid-zygotene events but all synapsis depend on the cohesin subunit SMC3.  Our results show that cohesin proteins have an important role in SC initiation.  Based on the observation that ORD and SMC3 are enriched at centromeres and promote their clustering and synapsis, we suggest that the enrichment of cohesin proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly.  ORD is also required for most crossing over, suggesting that SC initiation at mid-zygotene may also trigger the formation of DSBs that will be repaired as crossovers.



Figure 1: Cohesin proteins acSynapsis Modelcumulate at certain sites, like the centromeres and a few other locations, and are required for sister chromatid cohesion.  Synapsis initiates first at sites enriched for cohesin proteins like ORD.  Double strand breaks occur at these sites (not shown) and ORD is also required for crossing over and thus, these synapsis initiation sites may also be sites of future crossovers.  This is followed by additional synapsis initiation sites which depend on C(2)M.  These two types of synapsis initiation can occur independently.  Although not shown in the model, there are more of the C(2)M dependent initiation sites than ORD-dependent.  Both types of synapsis site depend on SMC3.  Whether SMC3 is enriched at specific sites during zygotene is not known.  While this model shows the initial loading of SC components, the process is dynamic involving the continual addition, and likely removal, of subunits. 



Chromosome axis defects induce a checkpoint-mediated delay and interchromosomal effect on crossing over during Drosophila meiosis


Crossovers mediate the accurate segregation of homologous chromosomes during meiosis but how their frequency and location is regulated is poorly understood. Checkpoints regulate cellular processes including those affecting chromosome dynamics and DNA repair.  We have found that the widely conserved pch2 gene of Drosophila melanogaster is required for a pachytene checkpoint that delays prophase progression when genes necessary for DSB repair and crossover formation are defective. However, the underlying process that the pachytene checkpoint is monitoring remains unclear. We investigated the relationship between chromosome structure and the pachytene checkpoint and found that disruptions in chromosome axis formation, caused by mutations in axis components or chromosome rearrangements, trigger a pch2-dependent delay. Accordingly, the global increase in crossovers caused by chromosome rearrangements, known as the ‘interchromosomal effect of crossing over,’ is also dependent on pch2.  Checkpoint-mediated effects require the histone deacetylase Sir2, revealing a conserved functional connection between PCH2 and Sir2 in monitoring meiotic events from Saccharomyces cerevisiae to a metazoan. These findings suggest a model in which the pachytene checkpoint monitors the structure of chromosome axes and may function to promote an optimal number of crossovers. Interestingly, above we described how axis proteins regulate the initiation of synapsis and crossover formation. (Figure 1)  The Pch2-dependent pachytene checkpoint may monitor this process of specifying synapsis initiation sites that also has a role in crossover determination.


The chromosomal passenger complex directs meiotic spindle assembly and chromosome bi-orientation


Chromosome segregation at meiosis I requires two distinct processes, the generation of crossover and the assembly of a bipolar spindle.  During meiosis in the females of many species, spindle assembly naturally occurs in the absence of the microtubule-organizing centers called centrosomes.  In the absence of centrosomes, spindle assembly initiates around the chromosomes, but how spindle bipolarity is established and how this bipolarity is transmitted to the chromosomes to promote proper segregation is not known.  We have found that early in meiotic spindle assembly of Drosophila oocytes, the chromosomal passenger complex (CPC) interacts with chromosomes in a ring that is aligned with both the axis of the spindle and the orientation of homologous centromeres.  The CPC includes the Aurora B kinase which regulates the activity of several target proteins involved in spindle assembly.  Furthermore, in the absence of the CPC components Aurora B or Incenp, microtubules do not assemble around the chromosomes, suggesting that the CPC is the chromosome-based signal which initiates spindle assembly.  The CPC is also required for recruitment of Subito, which is required for organizing a bipolar spindle and the bi-orientation of homologous chromosomes at metaphase of meiosis I.  Thus, the CPC is a key regulator of both meiotic spindle assembly and chromosome segregation.  We propose that the CPC promotes meiotic spindle assembly by interacting with chromosomes in a ring, and that the orientation of the ring establishes bipolarity in the acentrosomal meiotic spindle, leading to proper chromosome segregation (Figure 2).


Spindle Model

Figure 2: A model for the relationship between the central spindle, spindle bipolarity and homologous centromere orientation during prometaphase I.  A) Prior to NEB, the homologous centromeres (white circles) are paired in one more clusters or chromocenters.  The blue ovals represent each chromosomes in the karyosome.  B) Following NEB, the CPC (red circles) interacts with the chromatin. This stage is hypothetical and may be ephemeral as we have not observed oocytes with only CPC proteins around the karyosome.  The CPC is recruited by interacting with either the chromosomes or cooperatively with the chromosomes and the microtubules (Tseng et al., 2010).  C) A complex of the Kinesin 6 Subito and the CPC (red circles) interacts with antiparallel microtubules (green).  These antiparallel bundles may predict the eventual bipolarity of the spindle and either directly or through bundling with kinetochore fibers, could direct the orientation of homologous centromeres.  A ring of Subito (not shown) could facilitate the interaction of all kinetochore fibers with interpolar fibers.  D) Early prometaphase spindles with tapered poles and both interpolar (bundled by Subito) and kinetochore microtubule fibers.  E) Late prometaphase or metaphase spindle in which most of the microtubules appear to be in interpolar bundles.  One mechanism for this is that the kinetochore fibers are bundled with the interpolar fibers by a crosslinking motor like NCD (black).