HTP-1 coordinates synaptonemal complex assembly with homolog alignment during meiosis in C. elegans

Abstract

During meiosis, the mechanisms responsible for homolog alignment, synapsis, and recombination are precisely coordinated to culminate in the formation of crossovers capable of directing accurate chromosome segregation. An outstanding question is how the cell ensures that the structural hallmark of meiosis, the synaptonemal complex (SC), forms only between aligned pairs of homologous chromosomes. In the present study, we find that two closely related members of the him-3 gene family in Caenorhabditis elegans function as regulators of synapsis. HTP-1 functionally couples homolog alignment to its stabilization by synapsis by preventing the association of SC components with unaligned and immature chromosome axes; in the absence of the protein, nonhomologous contacts between chromosomes are inappropriately stabilized, resulting in extensive nonhomologous synapsis and a drastic decline in chiasma formation. In the absence of both HTP-1 and HTP-2, synapsis is abrogated per se and the early association of SC components with chromosomes observed in htp-1 mutants does not occur, suggesting a function for the proteins in licensing SC assembly. Furthermore, our results suggest that early steps of recombination occur in a narrow window of opportunity in early prophase that ends with SC assembly, resulting in a mechanistic coupling of the two processes to promote crossing over.

Figure 1.

Time-course analysis of pairing levels in htp-1 and htp-1; htp-2(RNAi) mutant germlines. Whole-mounted gonads were divided into five equivalently sized intervals along their distal–proximal axes (45–50 μm each) as in Couteau et al. (2004), each predominantly containing a population of nuclei corresponding to the following stages: (I) mitotic/premeiotic, (II) leptotene–zygotene, (III–V) early, mid-, and late pachytene. Single-copy probes were used to monitor the pairing of the left ends of chromosomes I and X (LG-IL and LG-XL), and a 5S rDNA repetitive sequence probe was used to follow the pairing of the right arm of chromosome V (LG V). Histograms illustrate the level of pairing attained for each probe used in the genotypes tested. In wild type, homolog alignment initiated in the transition zone (zone II), and pairing levels rapidly increased and persisted throughout pachytene. (zones III–V). In htp-1 and htp-1; htp-2(RNAi) mutants, autosomal pairing levels were not different from the backgrounds levels observed in the mitotic/premeiotic region (zone I), except in late pachytene for LG IL in htp-1 mutants (p = 0.002). A robust level of X-chromosome pairing was observed in htp-1 mutants, albeit at slightly reduced levels in comparison to wild type in zone II (p = 0.0218), zone III, and zone IV (p < 0.0001). The alignment is, however, stabilized over time and finally reaches a level that is not statistically different from wild type in zone V (p = 0.45). In htp-1; htp-2(RNAi) germlines, X-chromosome pairing is at wild-type levels, but fails to persist and decreases over time to return to background levels in late pachytene (zone V). The numbers of scored nuclei and percentage of paired FISH signals are presented in Supplementary Table 1.

Figure 2.

Defective synapsis in htp-1 and htp-1; htp-2(RNAi) mutants. (A) In wild-type germlines, parallel DAPI-stained tracts corresponding to two synapsed chromosome axes show precise colocalization of the meiotic chromosome axis component HIM-3 and the SC component SYP-1 in mid-pachytene nuclei. In htp-1 mutants, DAPI-stained chromosomes are aligned to a distance compatible with synapsis (inset) and HIM-3 and SYP-1 colocalize, suggesting the presence of SC. In htp-1; htp-2(RNAi) depleted nuclei, HIM-3 and SYP-1 extensively localize to chromosomes; however, nuclei are disorganized in appearance; no alignment of DAPI-stained chromosomes is evident, and chromatin masses are dispersed throughout the nucleus. (B) Squashed preparations of pachytene nuclei of htp-1 mutants reveal the presence of unsynapsed axes; arrows indicate a single unaligned DAPI-stained stretch, corresponding to an unsynapsed chromosome segment. HIM-3 is localized to the unsynapsed core, and SYP-1 is detectable as a discontinuous and thin thread that follows the HIM-3 pattern. Bars, 4 μm.

Figure 3.

SYP-1 loading is uncoupled from X-chromosome pairing in htp-1 mutants. Time-course analysis of X-chromosome pairing and SYP-1 recruitment on whole-mounted, three-dimensionally preserved gonads of wild-type controls and htp-1 mutants. For scoring, germlines were divided into five equivalently sized zones as described in Figure 1. For each nucleus, the status of X-chromosome alignment was first ascertained (paired or unpaired FISH signals), and then the DAPI-stained chromosome region to which the FISH signal mapped was examined for SYP-1 colocalization. In the wild-type germlines, two predominant classes of nuclei are observed: those with unpaired FISH signals, neither of which was associated with detectable SYP-1 tracts, and those with a paired FISH signals associated with an SYP-1 tract. In htp-1 mutant germlines, a third category of nuclei appears at a high frequency: those in which FISH signals are unpaired, but at least one of them lies on a tract of SYP-1. The numbers of scored nuclei in each category are presented in Supplementary Table 2.

Figure 4.

Coordination of axis formation with SYP-1 recruitment. HIM-3 and SYP-1 localization in leptotene–zygotene nuclei of whole-mounted gonads. Nuclei corresponding to the leptotene–zygotene stage (transition zone) were identified in wild-type, htp-1, and htp-1; htp-2(RNAi) mutant germlines by position in the gonad (zone II) and by the clustering of the DAPI-stained chromatin to one side of the nucleus. Similar to him-3(gk149)-null mutants (Couteau et al. 2004), no chromosome clustering is observed in htp-1(gk174) him-3(gk149) double mutants, and zone II nuclei expressing SYP-1 are shown. In wild-type germlines, HIM-3 localizes to developing chromosome cores, and SYP-1 localizes to a subset of HIM-3 stretches. A similar level and pattern of HIM-3 localization is present in htp-1 mutants; however, SYP-1 staining is generally more abundant relative to HIM-3 staining, and contiguous SYP-1 stretches can already be detected at chromosome axes where HIM-3 localization is still punctate (arrows). In both htp-1; htp-2(RNAi) and htp-1 him-3, mutants, SYP-1 fails to localize to chromosomes and instead appears as the single nuclear aggregate observed in him-3 mutants in which no synapsis occurs (Couteau et al. 2004). Bars, 4 μm.

Figure 5.

Formation of RAD-51-marked recombination intermediates increases in asynaptic htp-1; him-3 mutants and in htp-1; htp-2(RNAi).(A) Histograms showing the quantitation of RAD-51 foci from germlines of animals of the indicated genotypes. Nuclei were classified into five categories depending on the number of RAD-51 foci that were scored; category >3 includes nuclei showing 4–11 RAD-51 foci, but the vast majority of the nuclei in this category show 4–6 RAD-51 foci/nucleus in every genetic background tested. (B) Early pachytene nuclei of control and γ-irradiated htp-1 mutant germlines stained with DAPI (red) and α-RAD-51 (green). Bars, 4 μm.