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First published online 13 September 2006
doi: 10.1242/dev.02572

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The role of Tudor domains in germline development and polar granule architecture

¹ Developmental Genetics Program, HHMI, Skirball Institute at New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
² Molecular Structure Division, National Institute for Medical Research, London NW7 1AA, UK.

View larger version (23K): [in a new window]   Fig. 1. Identification of tud missense mutations. (A) Missense and nonsense mutations are placed at their corresponding locations within Tud protein sequence. Tud domains are indicated by blue squares. (B) Western blot of ovary extracts from wild type (wt) and tud mutants probed with TUD-A63 antibody and anti-Dynein antibody to detect Dynein as a loading control. (C) Tud proteins and Dynein were quantified by western blot with TUD-A63 antibody and anti-Dynein antibody, respectively (see Materials and methods). Tud signals were normalized to the Dynein signals and the average percentage relative to the wild-type ±s.e.m. is recorded. For each table entry, 3-4 measurements (n) were used to calculate the average percentage.

View larger version (35K): [in a new window]   Fig. 2. Mutations in Tud domains may affect interactions with polar granule components. (A-C) Structures of (A) Tud domain 10 and (B) Tud domain 1 were predicted based on (C) the known structure of the Tud domain from the Smn protein (Selenko et al., 2001; Sprangers et al., 2003). (A') Predicted structure of Tud domain 10 with an Arg2228Cys change. (B') Predicted structure of Tud domain 1 with an Arg91Trp change. Arg, Cys and Trp residues in Tud domains 1 and 10, and the corresponding Pro residue in the Smn Tud domain, are shown in magenta. Glu134 of the Smn Tud domain plays a crucial role in protein-protein interactions (Selenko et al., 2001) and is indicated in green. The cluster of aromatic amino acids, which form a binding pocket for the Smn Tud domain interacting partners, is shown in yellow.

View larger version (20K): [in a new window]   Fig. 3. Design of tud transgenic constructs and their expression. (A) tud full-length and deletion constructs. Tud domains deleted in the constructs are shown as white squares. (B) Western blot detection of transgenic Tud proteins. Ovary extracts from the wild type, which expresses no transgenes (`Wild-type' and `-HA' lanes), and those from transgenic lines, each of which expresses one copy of the transgene in a tud1/Df(tud) background, were used. Endogenous Tud protein and HA-Tud proteins were detected with TUD-A63 (Tud) and anti-HA (HA) antibodies, respectively. Dynein bands, detected with anti-Dynein antibody, served as loading controls. The full-length transgene is expressed at about 20% of the wild type level. mini-tud 2 and mini-tud 3 are expressed at similar levels to endogenous, wild-type Tud. Expression of the mini-tud 1 transgene was consistently weak (2-3% of the mini-tud 3 amount) in several transgenic lines (lines A-C), probably due to poor protein stability.

View larger version (38K): [in a new window]   Fig. 4. Abdomen development in tudor domain mutants and mini-tud transgenes. (A-H) Left panels: in situ experiments showing localization of nos RNA in preblastoderm embryos. Right panels: dark-field photographs of larval cuticles. anterior left, dorsal up. (A) Wild type. Diagram of full-length Tud with Tud domains indicated with blue squares is shown at the top. (B) tud1, (C) tudA36, (D) tudB42, (E) tudA7, (F) tudB45, (G) mini-tud 3 and (H) mini-tud 2 transgenes. Deleted Tud domains are indicated by the white squares.

View larger version (62K): [in a new window]   Fig. 5. The role of individual Tudor domains for germ cell formation. (A) Cellular blastoderm stage embryos from wild-type (wt), tud mutant (A36, A7, B42 and tud1) and tud1 females that express the mini-tud 3 transgene; anti-Vasa antibody marks germ cells. (B,C) In situ experiments showing gcl (B) and pgc (C) RNA staining in wild type and different tud mutant embryos generated by females transheterozygous for the respective tud allele and Df(2R)PurP133. For all panels, anterior is to the left and dorsal is up.

View larger version (72K): [in a new window]   Fig. 6. Tudor domains are required for protein localization and for proper architecture of polar granules. (A-D'') Preblastoderm embryos from wild-type (wt), Tud domain mutants tudA36 and tudB42, and tudB45 were co-stained with rabbit anti-Tud (A-D, green channel) and rat anti-Vas antibody (A '-D', red channel). Overlay images are shown in A ''-D ''. (A-A'') Wild type; (B-B'') tudA36; (C-C'') tudB42; (D-D'') tudB45. Anterior is to the left and dorsal is up. (E,F,F',H-K) Electron micrographs of germ plasm. Polar granules are indicated with arrows; m, mitochondria; MVB, multivesicular body, frequently observed at the egg cortex (Mahowald et al., 1981). (E) Germ plasm of the wild-type embryos shows distinct round or barrellike electron-dense polar granules. (F,F') Different ultra-thin sections across the same wild-type polar granule demonstrating hollow sphere morphology. (G,G') A simplified diagram of a polar granule. (H) Polar granules of tudA36 mutant frequently show a string- or rod-like architecture. Polar granule remnants of tudB42 (I) and tudB45 (K) are extremely rare. Scale bars: in E, 500 nm for E,H-K; in F, 500 nm for F,F '.

View larger version (40K): [in a new window]   Fig. 7. Tudor localization to the nuage is not required for germ plasm localization and germ cell formation. (A-F) Ovaries (A-C) and preblastoderm embryos (D-F) expressing full-length tud and mini-tud transgenes were stained with anti-HA antibody to determine transgenic Tud protein distribution during oogenesis and early embryogenesis. Nuage localization is indicated with arrows and germ plasm localization in the oocyte with arrowheads. (A,D) Full-length Tud; (B,E) mini-tud 1 protein; (C,F) mini-tud 3 protein.