Co-evolution between an endosymbiont and its nematode host: Wolbachia asymmetric posterior localization and AP polarity establishment

Abstract

While bacterial symbionts influence a variety of host cellular responses throughout development, there are no documented instances in which symbionts influence early embryogenesis. Here we demonstrate that Wolbachia, an obligate endosymbiont of the parasitic filarial nematodes, is required for proper anterior-posterior polarity establishment in the filarial nematode B. malayi. Characterization of pre- and post-fertilization events in B. malayi reveals that, unlike C. elegans, the centrosomes are maternally derived and produce a cortical-based microtubule organizing center prior to fertilization. We establish that Wolbachia rely on these cortical microtubules and dynein to concentrate at the posterior cortex. Wolbachia also rely on PAR-1 and PAR-3 polarity cues for normal concentration at the posterior cortex. Finally, we demonstrate that Wolbachia depletion results in distinct anterior-posterior polarity defects. These results provide a striking example of endosymbiont-host co-evolution operating on the core initial developmental event of axis determination.

Figure 1. Unfertilized, mature B. malayi oocytes contain a polar MTOC.

Mature oocytes (A,C) in meiosis I with bivalents associated with the anterior cortex, and embryos in first zygotic metaphase (B,D) are stained for DNA (blue), α-tubulin (red), and either the PCM marker Zyg-9 (A, B) or γ-tubulin (C,D) (green). In the oocyte, Wolbachia are associated with both poles and distributed in the cytoplasm. By the first zygotic division, Wolbachia are associated with the posterior pole. Scale bar = 5 µm.

Figure 2. γ –tubulin dynamics during fertilization suggest a de novo origin of centrosomes in B.malayi.

Fertilized eggs stained for actin (red), DNA (blue), and γ-tubulin (green). (A) Fertilization: the arrow points to the sperm in an egg in meiosis I, γ-tubulin foci (small green dots) are present throughout the cytoplasm. Arrowhead highlights Wolbachia (larger blue structures). (B) Pronuclei apposition. γ-tubulin foci concentrate around the pronuclei. Note the increased number of γ-tubulin foci compared to (A). (C) Metaphase: γ-tubulin foci form poles of metaphase spindle. Scale bar = 5 µm. (D) Comparison of zygote formation in B. malayi versus C. elegans. Yellow arrows point to the paternal pronuclei. In B. malayi, no MTOC is associated with the paternal pronucleus, while a cortical, maternal MTOC is present at the pole. γ-tubulin distributes around the pronuclei and precedes a de novo centrosome formation.

Figure 3. Absence of sperm-associated MTOC during fertilization.

Two fertilized B. malayi eggs in meiosis I, stained for total DNA (red), and for either α –tubulin (A, green) or γ-tubulin (B, green). Arrows point to the sperm derived chromatin. Note in (A) the five paternal chromosomes still condensed. Arrowheads point to the maternal MTOC. There is no MTOC associated with the sperm-derived chromatin. All eggs are oriented with the anterior to the left, scale bar = 5 µm.

Figure 4. Wolbachia dynamics from fertilization to the two-cell stage in B. malayi.

Whole mount eggs and embryos stained with propidium iodide to reveal the DNA (first column and red), and with an anti-α tubulin highlighting microtubules (second column and green). Wolbachia appear as DNA positive cytoplasmic foci (red). The dotted purple line highlights the equator from (A) to (I), and the asymmetry between blastomeres in (L). The red dotted line shows establishment of asymmetric spindle movement in late anaphase in (I) to (J). Anterior to the left, based on localization of polar bodies. (A) Prior to fertilization and (B) Fertilization (arrow points to the sperm/male pronucleus). (C) Pronuclei migration and condensation. (D) and (E) Prophase. (F) and (G) Metaphase. (H) Early anaphase. (I) and (J) Late anaphase. (K) Two-cell stage. (L) Two-cell stage in division. Scale bar = 5 µm.

Figure 5. Wolbachia concentrate in the vicinity of host microtubules.

B. malayi eggs soon after fertilization (A) or during the first anaphase (B), stained for DNA (red) and microtubules (green). In (A), the five paternal chromosomes are clustered in the center of the egg (yellow arrow), while meiosis I is being resumed. (A′) and (B′) are enlargements showing a close association between Wolbachia (red foci) and MTOC-derived microtubules (green). All eggs are oriented with the anterior pole to the left, scale bar = 5 µm.

Figure 6. Host dynein is required for Wolbachia posterior concentration.

Zygotes extracted after 48 hr-in vitro culture of (A) control adult females, or (B) B.m. dhc-1 hsiRNA treated adult females stained for DNA (green) and α-tubulin (red). In hsiRNA-Dynein knockdown embryos, Wolbachia fail to concentrate at the posterior pole, but rather occupy randomly the egg cytoplasm. (C) Zygote in metaphase stained for DNA (red), and with an anti- B.m. dhc-1 antibody (green). (C′) Enlargement of the posterior pole in (C) as indicated by the white box. Arrowhead points to the chromosome-associated dynein; arrow to the dynein co-localized with Wolbachia. Scale bar = 5 µm.

Figure 7. B.m. Par-1 and Par-3 are required for asymmetric segregation of Wolbachia in the two-cell embryo.

(A) Two-cell embryos from either non-treated, or B.m. par-1 or par-3 hsiRNA-treated B. malayi females, stained for α-tubulin (“MT” red), DNA (green), and for actin (blue). Classic C. elegans par1 and par3 spindle rotation mutant phenotypes are produced. (B) Proportion of Wolbachia endosymbionts present in the posterior blastomere at the two-cell-stage (the posterior being defined whenever the polar bodies allow identification of AB and P1). For wild type embryos, n>100. For par-1 and par-3 hsiRNA-treated embryos showing division synchrony, n = 15. Scale bar = 5 µm.

Figure 8. Loss of Wolbachia leads to A-P polarity defects.

(A) Proportion of A-P polarity defects in dividing two-cell B. malayi embryos in presence (WT) or absence of Wolbachia (Wb(-)). Class I, normal, asynchronous division patterns. Class II, abnormal synchronous divisions and P1 spindle rotation failure. Class III, abnormal synchronous divisions and AB spindle rotation. (B) Wild-type and (C) Wb(-) embryos, stained for DNA (red) and α-tubulin (green). Scale bar = 5 µm.

Figure 9. A model for Wolbachia asymmetric inheritance in the filarial egg.

Schematic view of the key cytoplasmic and nuclear events and Wolbachia distribution after the fertilization (red arrowhead). I, fertilized egg in meiosis I; II, completion of meiosis; III, pronuclei apposition; and IV, mitosis.