ABSTRACT
As daughter centrioles assemble during G2, they recruit conserved Ana3/RTTN followed by its partner Rcd4/PPP1R35. Together, this contributes to the subsequent recruitment of Ana1/CEP295, required for the centriole's conversion to a centrosome. Here, we show that Rcd4/PPP1R35 is also required to maintain 9-fold centriole symmetry in the Drosophila male germline; its absence causes microtubule triplets to disperse into a reduced number of doublet or singlet microtubules. rcd4-null mutant spermatocytes display skinny centrioles that elongate normally and localize centriolar components correctly. Mutant spermatocytes also have centrioles of normal girth that splay at their proximal ends when induced to elongate by Ana1 overexpression. Skinny and splayed spermatid centrioles can still recruit a proximal centriole-like (PCL) structure marking a capability to initiate features of centriole duplication in developing sperm. Thus, stable 9-fold symmetry of microtubule triplets is not essential for centriole growth, correct longitudinal association of centriole components, and aspects of centriole duplication.
Subject(s)
Centrioles , Drosophila Proteins , Microtubules , Spermatocytes , Centrioles/metabolism , Centrioles/ultrastructure , Centrioles/genetics , Animals , Male , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Spermatocytes/metabolism , Microtubules/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Spermatids/metabolism , Spermatids/cytology , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/genetics , Mutation , DrosophilaABSTRACT
In this chapter, we detail the experimental protocol leading to the generation of stem cell-based mouse embryo-like structures termed "ETiX-embryoids." ETiX-embryoids are formed from combined embryonic stem cells, trophoblast stem cells, and embryonic stem cells transiently induced to express Gata4. Cells are seeded into AggreWell dishes where they form aggregates that develop to resemble post-implantation mouse embryos following 4 days of culture. ETiX-embryoids establish an anterior signaling center and undergo gastrulation over the following 2 days. By day 7, ETiX-embryoids undergo neurulation and form an anterior-posterior axis with head folds at one end and a tail bud on the other. On day 8, they develop a brain and form a heart-like structure and a gut tube.
Subject(s)
Embryo, Mammalian , Embryonic Development , Mice , Animals , Gastrulation , Embryonic Stem Cells , TrophoblastsABSTRACT
Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. Here, we show that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. We find that neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.
