The role of rice HEI10 in the formation of meiotic crossovers

Abstract

HEI10 was first described in human as a RING domain-containing protein that regulates cell cycle and cell invasion. Mice HEI10(mei4) mutant displays no obvious defect other than meiotic failure from an absence of chiasmata. In this study, we characterize rice HEI10 by map-based cloning and explore its function during meiotic recombination. In the rice hei10 mutant, chiasma frequency is markedly reduced, and those remaining chiasmata exhibit a random distribution among cells, suggesting possible involvement of HEI10 in the formation of interference-sensitive crossovers (COs). However, mutation of HEI10 does not affect early recombination events and synaptonemal complex (SC) formation. HEI10 protein displays a highly dynamic localization on the meiotic chromosomes. It initially appears as distinct foci and co-localizes with MER3. Thereafter, HEI10 signals elongate along the chromosomes and finally restrict to prominent foci that specially localize to chiasma sites. The linear HEI10 signals always localize on ZEP1 signals, indicating that HEI10 extends along the chromosome in the wake of synapsis. Together our results suggest that HEI10 is the homolog of budding yeast Zip3 and Caenorhabditis elegans ZHP-3, and may specifically promote class I CO formation through modification of various meiotic components.

Figure 1. Organization of the HEI10 gene and protein alignment.

(A) Schematic representation of HEI10 gene and the location of hei10 mutation. Translated regions are represented by black boxes and untranslated regions are indicated by white boxes. HEI10A and HEI10B indicate long and short transcripts, respectively.

Figure 2. Meiosis in the hei10-1 mutant.

(A) Pachytene. (B) Diakinesis with both bivalents and univalents. (C) Metaphase I with five bivalents and fourteen univalents. (D) Metaphase I with four bivalents and sixteen univalents. Thirteen univalents and all bivalents align on the equatorial plate. (E) Anaphase I. Many univalents undergo a precocious segregation of sister chromatids. (F) Telophase II. Tetrads with several randomly distributed chromosomes. Chromosomes were stained with DAPI. Scale bars, 5 µm.

Figure 3. Chiasma distribution in WT, hei10, and mer3hei10.

(A) Chiasma distribution in the WT. The observed distribution of chiasmata deviates from a Poisson distribution. (B) In hei10, the observed distribution of chiasmata is consistent with a Poisson distribution. (C) In mer3hei10, the observed distribution of chiasmata is consistent with a Poisson distribution. Triangles indicate observed distribution, whereas squares show predicted Poisson distribution.

Figure 4. Immunolocalization of REC8, MER3, and ZEP1 in hei10-1.

(A) The localization of REC8 and MER3 at late leptotene. (B) The localization of REC8 and ZEP1 at zygotene. (C) The localization of REC8 and ZEP1 at pachytene. Scale bars, 5 µm.

Figure 5. Immunolocalization of REC8 and HEI10 in WT rice.

(A) Early leptotene, HEI10 appears as distinct foci. (B) Late leptotene. (C) Zygotene. (D) Late zygotene/Early pachytene, arrows indicate the gaps of HEI10 linear signals. (E) Middle pachytene. (F) Late pachytene, prominent foci localize on the chromosomes. (G) Diplotene. (H) Diakinesis, HEI10 foci locate at the chiasmata position. Scale bars, 5 µm.

Figure 6. Analysis of the distribution of HEI10 bright foci in WT meiocytes.

(A) Dual immunolocalization of WT scattered chromosomes with REC8 (red) and HEI10 (green) antibodies. Scale bar: 5 µm. (B) Dual immunolocalization of CENH3 and HEI10 at late pachytene. (C) No HEI10 signals were detected at metaphase I, chromosomes are stained with DAPI (blue). Scale bars: 5 µm. (D) Histogram of the observed distances between two adjacent HEI10 bright foci on the shortest chromosomes (percentages of the length of the synaptonemal complex). The gray bars show the observed relative frequencies of inter-HEI10 foci distances. The red curve presents the best fit of the distance to the gamma distribution. ν is the interference parameter in the gamma model. is the ν value (estimated SE) for which the best fit of the observed distances to the gamma model was got. Here, the estimated is 8.39, which indicates a strong interference among HEI10 bright foci on the shortest chromosome in rice (if there is no interference, ν is 1).

Figure 7. Dual immunolocalization of HEI10 and MER3 in WT meiocytes.

(A) Leptotene, HEI10 foci and MER3 foci show a high co-localization. (B) Zygotene, MER3 foci localize on HEI10 foci or short stretches. (C) Pachytene. Arrows indicate the co-localization of HEI10 foci and remaining MER3 foci. Scale bars, 5 µm.

Figure 8. Dual immunolocalization of HEI10 and ZEP1 in WT meiocytes.

(A) Early zygotene. (B) Late zygotene, HEI10 signals localize on ZEP1 linear signals. (C) Early pachytene, HEI10 signals overlap well with ZEP1 signals. (D) Late pachytene, prominent HEI10 foci localize on ZEP1 linear signals. Scale bars, 5 µm.

Figure 9. The localization of HEI10 in pair3 and zep1 mutants.

(A) Leptotene, faint HEI10 foci and bright spots are detected in pair3. Arrow indicates HEI10 spot. (B) Zygotene, faint HEI10 foci in pair3 become diffused. (C) Pachytene, bright spot in pair3 become diffused. (D) Zygotene, HEI10 consistently appears as foci in most zep1 cells. (E) Zygotene, short HEI10 lines are found in zep1 cells that contained compact chromosomes. (F) Late pachytene, zep1 shows an increased number of HEI10 prominent foci. Scale bars, 5 µm.

Figure 10. The localization of HEI10 in the mer3 mutant.

(A) Late leptotene, HEI10 foci localize normally. (B) Late pachytene, prominent HEI10 foci form normally. (C) Diakinesis, some prominent HEI10 foci locate on chromosomes while some disassociate from chromosomes. Chromosomes are stained with CENH3 signals (red) and DAPI (blue). Scale bars, 5 µm.