Male germline development

duo1.jpg
Wild type (WT) and mutant (duo1) pollen stained with the DNA stain DAPI.

In genetic screens for pollen cell division mutants in the model Arabidopsis thaliana we isolated a novel class which produces pollen with a single germ cell instead of two sperm. These duo pollen (duo) mutants provided genetic evidence for germline-specific control of the cell cycle. For  two of these mutants (duo1 and duo3) we showed that mutant germ cells complete S-phase of the cell cycle, but skip G2-to-M phase transition and fail to differentiate [Durbarry et al., 2005].

DUO1 and DUO3 are essential regulators of male germline development

duodapi.jpg
Sperm cell-specific expression of mRFP1 under control of the DUO1 promoter (left) and DAPI-stained pollen (right). Sperm nuclei are indicated with arrows, the vegetative nucleus with an arrowhead.

DUO1 and DUO3 both encode regulatory proteins with essential roles in cell cycle progression and sperm differentiation. DUO1 is an R2R3 MYB transcription factor and its promoter is sufficient to direct RFP expression specifically to germ cells in pollen [Rotman et al., 2005].  The cell cycle defects in duo1 are accompanied by incomplete differentiation such that mutant germ cells are unable to complete fertilisation.The duo1 mutant phenotype arises because DUO1 is required to coordinate  the accumulation of mitotic cyclins, which promote entry into mitosis, with the expression of germline differentiation genes [Brownfield et al., 2009a].

proMGH3:H2B-GFP
Wild‐type (right) and mutant duo1 (left) pollen showing absence of HTR10 promoter activity (proHTR10:H2B‐GFP) in single duo1 germ cells.

 

 

DUO1 activates the expression of a large network of genes associated with sperm cell differentiation including those involved in transcription, protein fate, signalling and transport [Borg et al.2011]. We showed that DUO1 binds to a well-defined DNA sequence motif present in the promoters of its target genes. Direct target genes of DUO1 encode proteins (GEX2 and HAP2/GCS1) required for gamete adhesion and fusion during fertilisation and chromatin associated proteins such as the male gamete-specific histone H3 variant H3.10.

DUO1 Model Simple
Simple model illustrating the essential role of DUO1 in coordinating germ cell division and sperm cell specification.

 

 

 

 

 

 

 

 

DUO3 is another essential nuclear protein required to coordinate germ cell division with gamete specification. DUO3 regulates the expression of a number of germline differentiation genes in common with DUO1, but the mechanism by which DUO3-deficiency inhibits G2-to-M phase transition is distinct from that in duo1 [Brownfield et al., 2009b].

We are currently investigating germline regulatory networks using integrated approaches involving transcriptome analysis of isolated cells, conditional expression, advanced light microscopy and cell imaging.

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