Germ Plasm Formation in Early Bucky Ball Oocytes and Evolutionary Conservation of the Buc Protein

(A–F) Fluorescent whole-mount in situ hybridization of early Ib oocytes showing mRNAs (red) of cyclinB (A and D), nanos (B and E), and vg1RBP (C and F) in wild-type (A–C) and buc ^p106re mutant oocytes (D–F). Note that the localization of nanos and vg1RBP mRNAs in the Balbiani body is lost in mutants (E and F), but the cyclinB and vg1RBP mRNAs still show polarized localization (D and F). Animal pole to the top. Scale bar represents 25 μm. All mutant images are of the buc^p106re allele.

(G) Unrooted star diagram displaying the phylogenetic conservation of Bucky ball proteins among vertebrates. The scale indicates the number of substitutions per amino acid residue.

(H) Alignment of Buc N terminus in 12 vertebrate species (zebrafish amino acids 23–136). The sequence homology was calculated with Kalign [45], which revealed a conserved peptide (zebrafish amino acids 23–38, red box).

(I) Alignment of conserved peptide (red box in H) with T-Coffee [46]. The color code below the alignment illustrates the level of conservation (cons.) from BAD (blue) to GOOD (red). Note that, with evolutionary distance, the sequence similarity decreases.

Gene-ID from NCBI or Ensembl-genome databases: Zebrafish, Danio rerio: NCBI-locus 560382; Medaka, Oryzias latipes: UTOLAPRE05100115054; Spotted pufferfish, Tetraodon nigroviridis: GSTENT00016539001; Tiger pufferfish, Takifugu rubripes: SINFRUT00000183089; Clawed frog, Xenopus tropicalis: ENSXETT00000045658; Chicken, Gallus gallus: ENSGALT00000019911; Platypus, Ornithorhynchus anatinus: GENSCAN00000155561; Opossum, Monodelphis domestica: GENSCAN00000062731; Cow, Bos taurus: GENSCAN00000042786, GENSCAN00000042790; Pig, Sus scrofa: Ssc.46160, Ssc.28451; Dog, Canis familiaris: GENSCAN00000091809, GENSCAN00000058820; Man, Homo sapiens: EU128483, EU128484

Expression Analysis of buc mRNA

(A–D) Real-time PCR analysis of buc and vasa mRNA relative to expression in the one-cell embryo during oogenesis (A), embryogenesis (B), and in sexually mature adults (C). (D) Real-time PCR comparing the levels of buc, dazl, and vasa mRNA in wild-type (+/-) and buc^p106re mutant ovaries (-/-).

(E–L) Fluorescent whole-mount in situ hybridization of buc (red in all pictures) during stage I (E–H) and stage III (I–L) of oogenesis. Animal to the top in all pictures. (F and J) Double in situ hybridizations showing colocalization of buc and the vegetal dazl mRNA (green) at stage I (F), but not at stage III (J). (G and K) Double in situ hybridizations showing colocalization of buc and foxH1 mRNA (green) at stage I (G) and at stage III (K). (H and L) Animal localization of buc mRNA at stage I (H), but not after 3-fold longer exposure at stage III in buc^p106re oocytes (L). Scale bars represent 25 μm (E–H) and 50 μm (I–L).

buc mRNA Translation Is Required to Assemble Germ Plasm, and Buc-GFP Localizes to the Balbiani Body

(A–E) Buc is required in the oocyte for germ plasm localization. Wild-type (A and C) and buc^p106re mutant oocytes (B and D) after microinjection of wild-type (A and B) or mutant buc^p106re mRNA (C and D) into stage I zebrafish oocytes. (E) Quantification of dazl mRNA localization from three independent experiments in WT oocytes, uninjected (75% ± 6.0%, n = 63), injected with WT buc (84% ± 14%, n = 67), or mutant buc^p106re mRNA (83% ± 12%, n = 47) compared to buc^p106re mutant oocytes, uninjected (6% ± 3.9%, n = 122), injected with WT buc (26% ± 5.1%, n = 103), or mutant buc^p106re mRNA (7% ± 6.4%, n = 26). Note the rescue of dazl mRNA localization in mutant oocytes after injection of wild-type buc mRNA (B and E). Error bars represent SD. ^***p = 0.0008.

(F–I) Buc needs to be translated to assemble germ plasm. buc^p106re mutant stage I oocytes injected with mutant buc^p106re (F and H) or WT buc mRNA (G and I) with control (+ctrl-MO) (F and G) or translation inhibition morpholino (+buc-MO) (H, I), which inhibited translation of Buc-GFP fusion mRNA and, hence, GFP-fluorescence efficiently in embryos (Figure S5).

(A–D and F–I) Whole-mount in situ hybridization of dazl mRNA (blue). Scale bars: 25 μm.

(J) Quantification of dazl mRNA localization in mutant oocytes injected with mutant buc^p106re mRNA plus ctrl-MO (9%, n = 11) or plus buc-MO (10%, n = 21) and WT mRNA plus ctrl-MO (25%, n = 20) or plus buc-MO (11%, n = 27). Note that the rescue of dazl mRNA localization is blocked by a morpholino-inhibiting buc mRNA translation (I and J).

(K–M) Buc localizes to the Balbiani body. Living WT oocytes injected with mRNA encoding a Buc-GFP fusion (green) at mid (K) and late-stage Ib (L), as well as stage II (M) of oogenesis. Scale bars represent 25 μm.

(N) Model of the role of Buc in early-stage I oocytes. Buc inhibits the premature localization of foxH1, vg1RBP, and buc mRNA at the animal pole (A) and concomitantly recruits the germ plasm RNAs dazl, nanos, and vasa in the Balbiani body at the vegetal pole (V).

Buc Colocalizes with Germ Plasm and Induces the Formation of Germ Cells during Embryogenesis

(A and B) Buc-GFP localizes to the germ plasm in eight-cell embryos. One-cell embryos were injected with 200 pg mRNA-encoding Buc-GFP (green) with (A) or without 3′UTR (B). Note the localization of GFP at four cleavage furrows in living eight-cell embryos. Animal view. Scale bars represent 200 μm.

(C and D) buc mRNA is throughout the blastodisc during embryogenesis. Whole-mount in situ hybridization showing buc mRNA in the blastodisc (blue) without specific localization to the germ plasm at the cleavage furrow at four-cell (C) and blastula stages (D) (high stage). Animal view (C), lateral view, animal to the top (D). Scale bars represent 200 μm.

(E–J) Overexpression of Buc in one-cell embryos induces germ cell formation. Dorsal view of living 13 somite stage embryos (15.5 hpf), anterior to the left (E and F). Germ cells are labeled green fluorescent after coinjection with 100 pg GFP-nanos-3′UTR mRNA. In control embryos, germ cells accumulate in the bilateral gonad anlagen (E), whereas in buc-injected embryos, extragonadal germ cells are visible (F). Animal view of oblong stage embryos (3.5 hpf) after whole-mount in situ hybridization with vasa mRNA (blue) injected with 300 pg GFP (G) or buc mRNA at the one-cell stage (H). Note additional vasa-positive cells in buc-injected embryos (85.0% ± 11.5%; n = 343) compared to control injections (5.7% ± 8.7%; n = 280; ^***p = 3.4 x 10^-12). Scale bars represent 200 μm (E–H). (I and J) Real-time PCR analysis of the germ plasm RNAs nanos, vasa, and dazl after control and buc mRNA injection analyzed at oblong stage (3.5 hpf; [I]) and 18 somites stage (18 hpf; [J]). Error bars represent SD (dazl ^*p = 0.028; vasa ^*p = 0.041; Table S3).

(K and L) Experimental scheme for germ cell formation assay. (K) The 16-cell embryos (animal view) were either injected into one corner blastomere (green arrowhead) or into a middle blastomere (blue arrowhead; positive control) containing essential germ plasm (red ovals). (L) Embryos were examined between the 13 and 18 somite stage for the formation of additional germ cells (green dots) in addition to the endogenous germ cells (red dots; not visible in the experiment). Oblique dorsal view, anterior to the left.

(M and N) Live 15 somite stage embryos, similar view as in panel (L), after injection of 100 pg GFP-nanos 3′-UTR mRNA into a corner blastomere (M) or after coinjection of 170 pg buc mRNA into the corner blastomere (N) with 12 ± 5.4 (n = 21) fluorescent cells per embryo. Scale bars represent 200 μm.

(O and P) Buc-induced germ cells express vasa mRNA. Injection of GFP-nanos 3′-UTR into a corner blastomere of a 16-cell embryo does not label germ cells after vasa mRNA in situ hybridization at the 18 somite stage (black; [O]), whereas coinjection of buc mRNA generates additional vasa-positive cells also expressing GFP (green, white arrowheads; [P]). Dorsal view, anterior to the left. Scale bars represent 200 μm.

Egg Polarity and Germ Plasm Formation in Early Bucky Ball Oocytes
(A-H) Fluorescent whole-mount in situ hybridization of stage III (A, B) and early Ib oocytes (C-H). cyclinB (A, B), dazl (C, D), vasa (E, F) (red) and foxH1 (G, H) (green) in wild-type (A, C, E, G) and buc ^p106re mutant stage I oocytes (B, D, F, H).
(I, J) Living staining of mitochondria and endoplasmic reticulum with DiOC₆ (green) in wt (I) and buc ^p106re mutant oocytes (J). Note the absence of signal at the vegetal pole in mutants. Animal pole to the top. Scale bar: 50 μm (A, B), 25μm (C-J).
(K) Quantification of oocytes with localized dazl mRNA. Note dazl mRNA localization in wild type (WT) (94.7±5.5%; n=385; grey bar) but not in buc ^p106re mutants (MUT) (4.8±2.6%; n=402; white bar). Error bars indicate the standard deviation of the average (at least three independent experiments).
(L) Quantitative real-time PCR measuring the relative concentration of vasa (white) and dazl (grey) mRNA in whole ovaries from wt and buc ^p106re mut females.

cyclinB, foxH1, and vg1RBP mRNA Localization in Wild-Type and buc Mutants
(A, B) Whole-mount in situ hybridization with cyclinB (red) and foxH1 mRNA (green) (A) or vg1RBP (green) (B) in stage I and II-III oocytes. Animal to the top. (A) CycB mRNA stays localized at the animal pole in wild type as well as early mutant oocytes. Some of these panels are double stainings from the single channels displayed in Fig. 1 to confirm the localization of the used molecular markers. In wild type foxH1 (A) and vg1RBP mRNA (B) are localized to the Balbiani body at stage I and animal at stage II-III. In buc oocytes foxH1 (A) and vg1RBP (B) are localized animal at stage I. Scale bar: 25 μm for stage I, 50 μm for stage II-III.
(C) CycB (red) and dazl mRNA (green) are localized at opposite poles at the end of stage I. Scale bar: 50 μm.

buc, dazl, and foxH1 mRNA Localization during Oogenesis
Whole-mount in situ hybridization with buc (red) and dazl mRNA (green) or foxH1 mRNA (green). Animal to the top. Some of these panels are double stainings of the single channels displayed in Fig. 2 to confirm the localization of buc mRNA.
(A) In wild type oocytes buc mRNA is localized at the initiation of oogenesis (stage IA) in the Balbiani body, until stage II at the vegetal pole and finally during the transition from stage II to III at the animal pole. In buc mutants buc mRNA is localized at the animal pole until stage II and disappears between stages II and III. Note that the mutant oocyte at stage II-III is three times longer exposed than at stage I.
(B) Until stage II buc and dazl mRNA colocalize (yellow in merged picture, lower row). Then dazl mRNA remains vegetal, whereas buc is detected animal (last column).
(C) During all stages buc and foxH1 mRNA co-localize (yellow in merged pictures, lower row). Scale bar: 25 μm (stage I), 50 μm (all other stages).

Buc-Specific Morpholinos Inhibit Translation of buc-GFP Fusion mRNA
Living embryos at high stage (∼3 hpf). Lateral view, animal to the top. Titration of the buc-specific morpholino (buc-MO) after co-injection of 2 μg/μl buc-GFP mRNA showed only background fluorescence of the vegetal yolk at 0.2 mM like in uninjected embryos, whereas embryos became fluorescent with 0.1 mM buc-MO. Control morpholinos showed no reduction of fluorescence, therefore in all injectionexperiments 0.2 mM morpholino together with 2 μg/μl buc mRNA was used. The results for buc-MO1 (5′-GACGAAGAAAATCAGCACAGGATAC-3′) and buc-MO2 (5′- TTTATTCCTTCCATTTCCTACGAGG-3′) or control-buc-MO1 (5′- GAgGAAcAAAATgAGCAgAGcATAC-3′) and control-buc-MO2 (5′- TTaATTgCTTCgATTTCgTACcAGG-3′) were simlar. Scale bar: 200 μm.

Buc-GFP Overexpression Generates Additional Fluorescent Spots
(A-J) Living embryos, animal view after injection of 200 pg buc-GFP mRNA at the one cell stage shown at the 4-cell (A) 8-cell (B) 8- to 16-cell (C) 16-cell (D) 16- to 32- cell (E) 32-cell (F) 64-cell (G) 128-cell (H) high (I) and oblong stage (J). Note that the oblong stage embryo (J) is overexposed, since the fluorescence is slowly decreasing and becomes invisible around dome stage. Scale bars: 200 μm