Chromosome Painting Reveals Asynaptic Full Alignment of Homologs and HIM-8–Dependent Remodeling of X Chromosome Territories during Caenorhabditis elegans Meiosis
[['The success of sexual reproduction relies on the ability of diploid germ cells to generate haploid gametes through the specialized cell division program of meiosis .', 'Haploidization relies on the faithful segregation of chromosomes from their homologous partners , which in turn relies on an ability to sort chromosomes into homologous pairs and establish temporary associations between them .', 'It is now well established that both recombinational interactions at the DNA level and assembly of a meiosis-specific proteinaceous structure known as the synaptonemal complex ( SC ) play roles in stabilizing associations between homologous chromosomes .', 'However , how homologs become colocalized and how initial recognition is accomplished to establish these associations remains poorly understood .', 'Substantial progress has been made recently in illuminating a conserved mechanism that mediates chromosome movements that likely contribute to the chromosome sorting process in diverse organisms ( for reviews , see [1]–[3] ) .', 'The common feature of these large-scale spatial reorganization mechanisms involves tethering of one or two specified site ( s ) on a chromosome to conserved nuclear envelope ( NE ) - spanning protein complexes , thereby coupling the chromosomes to the cytoskeletal motility apparatus that can transmit forces to drive chromosome movement .', 'Such forces and movements have been proposed to enhance the efficiency of chromosome sorting both by providing opportunities for homology assessment and by destabilizing interactions between incorrect partners .', 'However , our understanding remains limited regarding how localized chromosome tethering sites might mediate recognition and bring about colocalization along the entire lengths of chromosomes .', 'One reason for this limitation is that many studies of early prophase chromosome reorganization have focused on assessing the behavior of a few specified individual chromosomal loci .', 'While such approaches have been highly fruitful , they do not provide information regarding the spatial and morphological organization of whole chromosomes within intact nuclei prior to and during the pairing process .', 'Intermediate events in the pairing process are particularly difficult to investigate using single-locus visualization methods , as only two pairing states ( paired or unpaired ) can be captured at each locus .', 'Thus in order to comprehensively study the pairing process , including intermediate states , it is necessary to study pairing in the context of whole chromosomes .', 'One cytological approach that has been applied to address this problem is the use of “chromosome paints” , i . e . , fluorescence in situ hybridization ( FISH ) probes that allow visualization of whole chromosomes or large chromosome segments .', 'Several previous studies have applied whole chromosome paints to investigate meiotic pairing , e . g . to visualize endogenous chromosomes in human spermatocytes [4]–[6] or “alien” chromosomes in hybrid plants [7]–[9] .', 'Such studies have indeed succeeded in revealing aspects that would be difficult to demonstrate without whole chromosome visualization , such as intermediate pairing states along the chromosome before synapsis [10] and a correlation between pairing activity and morphological changes in heterochromatin in plants [9] , [11]–[13] .', 'However , technical challenges such as limited availability of material ( e . g . , human ) or difficulty visualizing native chromosome complements rather than exogenously-derived chromosomes ( e . g . , plants ) have hindered widespread adoption of the paint approach .', 'In the current work , we have applied a multi-color whole-chromosome painting approach to visualize spatial and morphological reorganization of chromosome territories during meiotic prophase in the nematode Caenorhabditis elegans , which has several features that make it especially amenable to reaping the benefits of the painting approach .', 'Germ cells comprise more than half of the cells in the adult hermaphrodite and are organized in a spatial-temporal gradient along the longitudinal axis of the gonad , facilitating analysis of large numbers of nuclei undergoing the meiotic pairing process [14] , [15] .', 'Further , the germ line is organized as an optically clear single-layer epithelial tube , enabling visualization of chromosome territories in the context of well-preserved 3D nuclear architecture in whole mount gonads .', 'Finally , these cytological advantages can be exploited in combination with a rich history of genetic analysis of meiosis that has identified both cis- and trans-acting factors required to establish and maintain homolog pairing ( Reviewed in [16] ) .', 'A central player driving the chromosome sorting process in C . elegans is the meiotic pairing center ( PC ) , a cis-acting domain located near one end of each chromosome [17] , [18] .', 'PCs have been demonstrated to play roles both in promoting local stabilization of pairing and in promoting SC assembly; these dual roles of PCs together ensure that synapsis occurs specifically between homologous chromosomes [19] , [20] .', 'PC function depends on members of a family of Zn finger DNA binding proteins ( HIM-8 and ZIM-1 , -2 , -3 ) , each of which becomes concentrated at the PCs of a specific subset of chromosomes [21]–[23] .', 'Recent work has shown that PCs are the sites at which C . elegans chromosomes associate with conserved NE-spanning protein complexes ( comprised of the SUN-1 and ZYG-12 proteins ) that mediate tethering of chromosomes to the cytoskeletal motility apparatus to enable chromosome movement during meiotic prophase [24]–[26] .', 'Further , PCs have been proposed to participate in checkpoint-like mechanisms that function , in a manner analogous to the spindle-assembly checkpoint , to prevent licensing of SC assembly until successful homologous associations have been achieved [25] , [27] , [28] .', 'While PCs represent a key focal point for activities that drive chromosome movements , mediate associations between prospective pairing partners , and couple SC assembly to homology verification , however , it is not clear how the known functions and properties of PCs and PC binding proteins might contribute to homolog recognition per se .', 'Further , our current understanding of PCs does not explain how chromosomal regions outside of the PCs might contribute to homology recognition .', 'In the current work , we fill a major gap in our knowledge regarding the spatial and morphological organization of chromosomes within C . elegans germ cell nuclei prior to meiotic entry and during the process of homolog pairing .', 'Visualization of whole chromosome territories has allowed us to exclude several possible mechanisms as primary drivers of chromosome sorting .', 'Further , our analysis has revealed both a robust capacity for synapsis-independent full-lengthwise alignment between homologous chromosomes as well as a dramatic longitudinal extension of chromosome territories that could enable this alignment .', 'Moreover , we demonstrate unanticipated roles for the X chromosome PC and PC binding protein HIM-8 in promoting territory elongation .', 'These and other observations together paint a picture in which longitudinal restructuring of chromosome territories collaborates with other functions of meiotic pairing centers to bring about efficient , stable and productive interactions between homologous chromosomes , ultimately leading to the production of haploid gametes .'], ['During meiotic prophase , chromosomes are spatially reorganized within the nucleus to establish productive associations between homologous chromosomes .', 'In order to study this reorganization process , we used a chromosome painting approach to visualize complete chromosomal territories in the context of preserved 3D nuclear architecture in whole mount gonads of C . elegans .', 'This method provides a means to trace the full length of a chromosome , making it particularly useful for analyzing the spatial organization of chromosomes in premeiotic nuclei and during intermediate steps in chromosome alignment .', 'Figure 1 summarizes representative observations from an experiment in which chromosome II was visualized with a paint probe generated from YAC clones tiled along the length of the chromosome ( see Materials and Methods ) .', 'For this probe , three different fluorescent dyes were assigned to three distinct segments of the chromosome; in addition , the left- and right-most YACs were double-labeled with a second fluorophore in order to clearly locate chromosome ends .', 'We exploited the stereotyped spatio-temporal organization of nuclei within the C . elegans gonad and the appearance of DAPI-stained chromatin to identify premeiotic nuclei and nuclei at different stages of meiotic prophase .', 'Several basic observations are introduced briefly here and will be expanded upon in subsequent sections: 1 ) In premeiotic nuclei ( located in the distal region of the germ line ) , the chromosome II paint probe typically labels two relatively compact ovoid territories , often widely separated within the nucleus ( Figure 1a ) .', '2 ) As nuclei move proximally in the gonad , they enter meiotic prophase and initiate homolog pairing in a region known as the transition zone .', 'The transition zone contains nuclei in the leptotene and zygotene stages of meiotic prophase , and is characterized by an asymmetric clustering of chromosomes within the nucleus that reflects active chromosome movement driven by connections of chromosomes to the cytoskeletal motility apparatus [25] , [26] .', 'Nuclei within the transition zone frequently exhibit highly elongated chromosome territories; such elongated chromosome territories may be unassociated ( Figure 1b ) , partially aligned ( Figure 1c–1f ) or completely aligned along their entire lengths ( Figure 1g ) .', 'At this and later stages , chromosome paint signals sometimes exhibit a discontinuous “beads-on-a-string” appearance , even for chromosome regions whose sequence is fully represented by YAC clones in the paint probe .', '3 ) Nuclei exit the transition zone upon entry into the pachytene stage , during which homologous chromosomes are fully aligned and stably associated with their homologous partners via the synaptonemal complex ( SC ) , and chromosomes are redispersed around the nuclear periphery .', 'During the pachytene stage , chromosome paints reveal a single elongated chromosome territory reflecting complete alignment and association of homologous chromosomes along their entire lengths ( Figure 1h , 1i ) .', 'In principle , a prior non-random arrangement of chromosomes within the nucleus could potentially contribute mechanistically to the process of homolog pairing during meiosis .', 'It was previously shown that homologous chromosomes are not aligned prior to meiotic entry in C . elegans , as pairing between homologous loci in premeiotic nuclei is rarely observed by conventional locus-specific FISH ( e . g . [19] , [29] , [30] ) .', 'However , it had not been addressed whether territories of homologous chromosomes might exhibit preferential proximity compared to non-homologous territories .', 'Therefore , we compared the spatial relationships between homologous and non-homologous territories by simultaneously visualizing two pairs of chromosomes in a subset of the pre-meiotic zone ( 1–15 cell diameters from the distal tip ) that does not include pre-meiotic S phase .', 'These experiments used four fluorescent dyes ( two assigned to each chromosome ) to identify each chromosome and to distinguish their left and right halves .', 'Spatial relationships between chromosome territories were examined in 3D reconstructed images ( Figure 2A ) .', 'We defined four categories of spatial proximity for this analysis .', 'If we observed any overlap or association between two territories , territories were scored as “touching” .', 'Other spatial relationships between two territories were classified into three categories based on visual inspection of 3D-rendered images .', 'We approximated the distances between the closest edges of the territories ( d ) using the width of chromosome territory as a scale unit ( D ) , since these territories are highly uniform in their width ( 0 . 67 µm±SD 0 . 05 µm , n\u200a=\u200a16 ) ; distances were classified as: close ( 0<d≤D ) , intermediate ( D<d≤2D ) or far ( 2D≤d ) .', 'Several features of premeiotic nuclear organization were revealed by simultaneous analysis of chromosome I and II territories ( Figure 2B ) .', 'First , homologous chromosome territories were frequently widely spaced within the nucleus , with more than half of the distances in the intermediate and far categories .', 'Second , both homologous pairs ( I-I and II-II ) exhibited similar distributions among the different spatial relationship categories .', 'Third , heterologous pairs of chromosome territories ( I-II ) did not exhibit a significantly different distribution among the spatial categories compared with homologous ( I-I or II-II ) pairs ( p\u200a=\u200a0 . 34 and p\u200a=\u200a0 . 26 ) .', 'This indicates that these chromosomes are not “presorted” in most of the pre-meiotic stage and suggests that premeiotic nuclear organization is not the primary factor driving meiotic homolog pairing .', 'Simultaneous analysis of chromosome I and X chromosome territories revealed both similarities and differences ( Figure 2C ) .', 'On one hand , the X chromosomes were no more or less likely than chromosomes I or II to be in close proximity to or associated with their homologous partners , as the distributions among the spatial categories did not exhibit any significant differences ( p\u200a=\u200a0 . 79 , p\u200a=\u200a0 . 36 ) .', 'This reinforces the view that no chromosome pair is in a preferentially pre-associated state in the pre-meiotic stage .', 'On the other hand , however , heterologous pairs of chromosome territories ( I-X ) did exhibit a significantly different distribution among the spatial categories when compared with homologous ( I-I or X-X ) pairs ( p<0 . 0001 ) , reflecting a tendency of I and X to be separated by larger distances .', 'This observation raises the possibility that the X chromosomes , while no more likely than autosomes to be closely associated with each other , may nevertheless be spatially segregated from autosomes in pre-meiotic nuclei .', 'Such a feature could help explain the higher proficiency of X chromosome pairing under some conditions where autosomal pairing is severely abrogated [27] , [28] , [31]–[34] .', 'One possible reason why the X chromosomes might be spatially segregated from the autosomes premeiotically is that X chromosome PCs exhibit strong association with the HIM-8 protein and the nuclear envelope in premeiotic nuclei , whereas the ZIM proteins do not show strong association with the autosomal PCs until meiotic entry [21] , [22] .', 'We tested whether HIM-8 might be responsible for X-A spatial segregation by examining the spatial organization of pre-meiotic territories in the him-8 ( e1489 ) mutant , which lacks detectable HIM-8 protein .', 'We found that the tendency for the X chromosome to be spatially segregated from chromosome I was diminished in the him-8 ( e1489 ) mutant ( Figure 2D ) , as the distribution among spatial categories for heterologous pairs ( I-X ) did not differ significantly from the distribution observed for X-X pairs ( p\u200a=\u200a0 . 21 ) and exhibited only a modest difference from that observed for I-I pairs ( p\u200a=\u200a0 . 03 ) .', 'Thus , we conclude that association of the X chromosomes with the HIM-8 protein and/or the nuclear envelope does indeed contribute to the spatial segregation of X chromosomes from autosomes in premeiotic nuclei .', 'The same sets of paints ( 4-color , 2-chromosome ) were used to examine chromosome organization in transition zone nuclei , where homolog pairing is established .', 'This analysis enabled us to investigate the possibility that temporal heterogeneity in chromosome organization or behavior might play a role in chromosome sorting during the pairing phase of meiotic prophase .', 'Specifically , for each transition zone nucleus we assessed whether each homologous chromosome pair was unaligned , partially aligned or fully aligned ( Figure 3 ) .', 'In the majority of transition zone nuclei , we found that either both pairs of chromosomes were unaligned or both pairs were completely aligned , presumably reflecting nuclei that had either not yet begun or had already completed the homolog alignment process .', 'Thus , to address the issue of relative timing , we specifically considered the subset of nuclei that were in the process of achieving homolog alignment , i . e . , those nuclei in which either 1 ) both chromosome pairs exhibited partial alignment or 2 ) the two chromosome pairs exhibited disparate alignment states ( Figure 3A ) .', 'Importantly , there was no pattern among these nuclei that would indicate a specific temporal hierarchy in the order of chromosome pairing .', 'We observed nuclei in which chromosome I was partially or fully aligned and chromosome II was unaligned , and we likewise found nuclei in which chromosome II was partially or fully aligned and chromosome I was unaligned .', 'Similar results were obtained for I and X . Thus , these data demonstrate that all of the chromosomes initiate and complete homologous alignment in a very similar time window .', 'Moreover , these experiments rule out the possibility that different chromosomes within the nucleus might pair in a reproducible temporal order .', 'During wild-type C . elegans meiosis , successful pairing of homologous chromosomes is quickly stabilized by assembly of the SC , making it difficult to determine the extent to which full alignment between homologs can occur in the absence of synapsis .', 'Mutants lacking the SC central region proteins ( syp mutants ) provide an opportunity to address this issue , as such mutants not only lack synapsis but also prolong the period of chromosome clustering and chromosome mobility associated with the onset of homolog pairing .', 'Previous work using locus-specific FISH probes had shown that syp mutants achieve substantial levels of pairing at the pairing center ( PC ) ends of their chromosomes , revealing a role for PCs in promoting local synapsis-independent stabilization of pairing [19] .', 'A subset of chromosomes in this prior analysis displayed paired FISH signals at both PC and non-PC ends , consistent with the possibility of full alignment , but the status of interstitial chromosomal loci was ambiguous .', 'Images of wild-type leptotene/zygotene chromosome spreads showing parallel tracks of foci of chromosome axis protein HIM-3 are also highly suggestive of presynaptic alignment [35] , but such images lack information regarding chromosome identity , orientation and extent of the proposed alignment .', 'Thus , to directly evaluate the capacity of C . elegans chromosomes to achieve full lengthwise alignment in the absence of synapsis , we applied chromosome painting to track chromosome territories in the syp-1 ( me17 ) mutant , using the three-color chromosome II paint introduced in Figure 1 .', 'Figure 4A shows images of the most prominent classes of chromosome association configurations observed during meiotic prophase in the syp-1 mutant; Figure 4B shows a quantification of the frequencies of these and other association types among nuclei from different zones along the distal/proximal axis of the gonad ( representing a time course encompassing meiotic entry and meiotic prophase progression through the end of the pachytene stage ) .', 'This analysis revealed that when homologous chromosome pairs are associated in the syp-1 mutant , the two most common configurations are: 1 ) a “V-shaped” configuration in which homologs are associated only at the end of the chromosomes harboring PC domain ( Figure 4A , a ) , or 2 ) a configuration in which the homologous territories are closely juxtaposed along their entire lengths ( Figure 4A , c ) .', 'When the two homologous territories are intimately aligned from end to end , they are visualized as a single chromosome territory that is indistinguishable in appearance from the synapsed chromosome pairs observed in wild type nuclei at the pachytene stage ( Figure 1h , 1i ) , with distinct boundaries between chromosomal segments labeled with different fluorophores clearly indicating alignment in register along the entire length of the chromosomes .', 'The high incidence of full alignment demonstrates that C . elegans chromosomes have a considerable propensity to align and associate closely along their entire lengths without the aid of SC formation .', 'Interestingly , there was a relatively low abundance of homolog pairs exhibiting a partial longitudinal association ( “Y-shaped” configuration , Figure 4A ,', 'b ) compared to the V-shaped and fully-aligned configurations .', 'This dearth of intermediate alignment states suggests that the PC-end-only association state and the full alignment state may represent more stable configurations and/or that there may be a relatively rapid transition between them .', 'Our chromosome painting strategy revealed and allowed us to document an extensive elongation of chromosome territories that occurs in nuclei in the transition zone ( Figure 1b–1d; Figure 5 ) .', 'This longitudinal extension is mostly seen after meiotic entry .', 'As shown in Figure 5A , most chromosomes are in compact territories before nuclei enter the transition zone ( Figure 5A left , inset 1 ) , but once nuclei are in the transition zone ( Figure 5A , a , b middle ) , longitudinal extension of chromosome territories is clearly observed in a subset of nuclei ( Figure 5A , insets 2–4 ) .', 'For purposes of quantitation , we defined “highly extended” chromosome territories as those exhibiting an extremely elongated thread-like morphology that was clearly distinguishable from the compact ovoid shape of territories in the pre-meiotic region .', 'Such territories have a slenderness ratio ( the ratio of the approximate length to the approximate width of the painted chromosome ) of >6 .', 'Half of chromosome I territories and 25–30% of X chromosome territories were scored as “highly extended” in wild-type transition zone nuclei ( see below ) .', 'Our analysis indicates that longitudinal extension of chromosome territories initiates before homologous association .', 'Among nuclei exhibiting highly extended chromosome territories prior to full alignment , there was no obvious relationship between the elongation state and the association status of a homolog pair; extended chromosome territories were seen for non-associated , end-associated and partially aligned chromosomes ( Figure 5B ) .', 'Quantitation of association/alignment states in these nuclei revealed that between 42% ( chromosome I ) and 68% ( X chromosomes ) of such nuclei showed no evidence of homologous association between the assayed chromosome pair .', 'Therefore , neither alignment nor association between homologous chromosomes is a prerequisite for the longitudinal extension of chromosome territories .', 'This observation indicates that chromosome territories extend from a compact shape to an elongated shape in early meiotic prophase , before initiating homologous alignment .', 'Among the chromosome I and chromosome II territories analyzed in Figure 5B , association of chromosome territories only at the PC end or only at the non-PC end ( V-PC or V-NPC configuration ) was observed with similar frequencies .', 'In contrast , partial alignment of chromosome territories along the PC half of the chromosome ( the Y-PC configuration ) occurred much more frequently than the Y- NPC configuration ( which was rarely observed ) .', 'This observation suggests that even though the non-PC terminus of a chromosome has some potential to engage in homologous association very early in meiotic prophase , this activity is much weaker than the robust pairing-stabilization activity at the PC terminus , which is capable of propagating pairing into adjacent chromosome regions .', 'Elongation of chromosome territories revealed a beads-on-string appearance of the chromosome paint signals: chromosomal segments of high signal intensity are interspersed with regions of low intensity or gaps .', 'Further , while the number of “beads” depends on the composition of the probe , an increase in the degree of extension generally correlates with an increase of the number of discernable painted segments per chromosome ( Figure 6A , 6B ) .', 'Thus , counting of painted segments provides a means to quantify and compare the elongation states of the chromosomes at different stages .', 'Specifically , we counted the numbers of these “painted segments” in three-dimensionally rendered images , as illustrated in Figure 6A , 6B ( see Videos S1 and S2 and Materials and Methods ) .', 'This analysis was conducted for chromosome I and for the X chromosome , for nuclei from three distinct zones within the gonad: the pre-meiotic zone , the transition zone , and the early pachytene zone .', 'Further , for plotting of the data ( Figure 6C ) we sorted nuclei from the transition zone into two classes: those that had not yet achieved full alignment ( TZ-NF ) , and those that had achieved full alignment ( TZ-Full , which likely includes nuclei that had reached the pachytene stage ) .', 'This quantitative treatment provided strong statistical support for the observations described above .', 'For both chromosome I and X , there was an extremely significant increase in the number of painted chromosomal segments between pre-meiotic nuclei and TZ-NF nuclei ( p<0 . 0001 ) , reflecting a substantial elongation of chromosome territories following meiotic entry .', 'Moreover , we also detected an extremely significant increase in the number of painted segments per chromosome between TZ-NF nuclei and TZ-Full nuclei ( p<0 . 0001 ) .', 'This increase indicates an ongoing elongation of chromosome territories within the TZ as homolog alignment proceeds to completion .', 'In contrast , we did not detect a significant difference in the number of painted chromosomal segments between TZ-Full and pachytene nuclei for either chromosome .', 'In part , this observation likely reflects inclusion of some pachytene nuclei in the TZ-Full category .', 'However , it also suggests that while the segment-counting assay clearly reports on one aspect of chromosome elongation , it may not capture the full extent of pachytene chromosome extension .', 'The timing of the longitudinal extension coincides temporally with a period of active chromosome movement that is mediated by association of chromosome pairing centers ( PCs ) with mobile patches of the NE-spanning SUN-1/ZYG-12 protein complexes .', 'In order to test whether PC–SUN-1/ZYG-12-mediated movement of chromosomes is required for chromosome elongation , we evaluated territory elongation in mutants defective for chk-2 ( which encodes a protein kinase required for homolog pairing and nuclear reorganization during meiotic prophase [30] ) , him-3 ( which encodes a major chromosome axis component required for synapsis [36] ) , and syp-1 ( which encodes a major component of the SC central region [19] . Previous work has shown that: 1 ) NE-associated PC–SUN-1/ZYG-12 aggregates do not form and SUN-1 is not phosphorylated in chk-2 mutants [22] , [26]; 2 ) him-3 mutants are defective for formation of autosomal PC–SUN-1/ZYG-12 aggregates [37]; and 3 ) in a syp mutant , the movement of SUN-1 aggregates is slower and more spatially constrained than during wild-type meiosis [37] .', 'We used three-color paints to assess chromosome II territory morphology in pre-meiotic nuclei and in nuclei within an early meiotic prophase zone ( between 40 and 50 rows from the distal tip ) that corresponds to early pachytene in the wild type controls .', 'The images shown in Figure 7A and the quantitation of painted chromosome segments in Figure 7B both indicate that substantial elongation of chromosome II territories occurs in all of these mutants .', 'chk-2 mutants exhibited an extremely significant increase in the number of painted chromosomal segments ( p<0 . 0001 ) between the premeiotic and pachytene stages .', 'Moreover , the number of painted segments in pachytene nuclei in the chk-2 mutant was indistinguishable from wild-type controls ( p\u200a=\u200a0 . 122 ) , suggesting a normal degree of territory elongation .', 'him-3 and syp-1 mutants similarly exhibited extremely significant increases in the number of painted segments between the premeiotic and pachytene stages ( p<0 . 0001 ) ; however , these mutants had modest but significant differences from wild type at the pachytene stage ( p\u200a=\u200a0 . 0006 ) , suggesting a slight reduction in the extent of elongation .', 'The apparently normal chromosome elongation observed in the chk-2 mutant indicates that phosphorylation of SUN-1 and association of PCs with mobile SUN-1/ZYG-12 patches at the NE are not required to accomplish elongation of chromosome territories during early meiotic prophase .', 'This in turn implies that chromosome movements mediated by PC–SUN-1 linkages are not the primary driver of territory elongation .', 'Further , the substantial territory elongation observed in the him-3 and syp-1 mutants also indicates that a high degree of elongation can be achieved without SC assembly .', 'Although chromosome movement mediated by PC–SUN-1/ZYG-12 linkages does not seem to be required for the territory elongation , PCs have been shown previously to play several distinct roles in promoting homolog synapsis .', 'Therefore , we tested the possibility that PCs might contribute to elongation in other ways .', 'Specifically , we investigated the importance of X chromosome PC function in the process of chromosome territory elongation .', 'We first evaluated the potential contribution of HIM-8 , a zinc-finger protein that concentrates at the PC domain of the X chromosome and is required for PC function [21] , by assessing X chromosome territory elongation in him-8 mutants .', 'Because impaired X pairing in him-8 mutants results in an expanded zone of nuclei with a clustered chromosome distribution , we used the alignment status of chromosome I to define the boundary between transition zone and pachytene for this analysis .', 'Specifically , we defined the transition zone as the region of the gonad in which nuclei exhibited a clustered distribution of chromosomes and a mixture of unaligned , partially aligned and fully aligned chromosome I territories ( Figure 8B ) .', 'The length of the transition zone defined by these criteria is very similar among the strains tested ( Materials and Methods and Figure S1 ) .', 'Nuclei in the pachytene zone , adjacent to the transition zone , mostly contained fully aligned chromosome I territories .', 'In wild type controls , X chromosome territories extended longitudinally in the transition zone ( Figure 8A ) , which was quantitatively reflected as an increase of the number of painted chromosomal segments ( Figure 8C; p<0 . 0001 ) .', 'In the pachytene zone , we saw full alignment of X chromosomes ( Figure 8A , 8B ) and a further elongation of the X chromosome territory ( Figure 8A ) , reflected by a corresponding increase in the number of painted segments ( Figure 8C; p<0 . 0001 ) .', 'In contrast , we did not observe any obvious longitudinal extension of X chromosome territories in transition zone nuclei in the him-8 ( e1489 ) mutant ( Figure 8A ) , which is a putative null allele that encodes a mutant HIM-8 protein with a disrupted DNA binding domain that does not localize to the X chromosomes [21] .', 'A severe impairment of X chromosome elongation was clearly reflected both in quantitation of painted chromosomal segments , which did not detect any significant difference between premeiotic and transition zone nuclei in the him-8 ( e1489 ) mutant ( Figure 8C; p\u200a=\u200a0 . 27 ) , and in the frequency of transition zone nuclei exhibiting highly extended X chromosome territories ( Figure 8D ) , which was greatly reduced relative to the wild-type control ( p<0 . 0001 ) .', 'Chromosome territories also remained relatively compact in the pachytene region of him-8 ( e1489 ) mutant germ lines , even at the late pachytene stage where extension of chromosome territories is most obvious in wild-type germ cells ( Figure 8A and data not shown ) .', 'Further , although we did detect a modest increase in the
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['During early meiotic prophase , a nucleus-wide reorganization leads to sorting of chromosomes into homologous pairs and to establishing associations between homologous chromosomes along their entire lengths .', 'Here , we investigate global features of chromosome organization during this process , using a chromosome painting method in whole-mount Caenorhabditis elegans gonads that enables visualization of whole chromosomes along their entire lengths in the context of preserved 3D nuclear architecture .', 'First , we show that neither spatial proximity of premeiotic chromosome territories nor chromosome-specific timing is a major factor driving homolog pairing .', 'Second , we show that synaptonemal complex-independent associations can support full lengthwise juxtaposition of homologous chromosomes .', 'Third , we reveal a prominent elongation of chromosome territories during meiotic prophase that initiates prior to homolog association and alignment .', 'Mutant analysis indicates that chromosome movement mediated by association of chromosome pairing centers ( PCs ) with mobile patches of the nuclear envelope ( NE ) –spanning SUN-1/ZYG-12 protein complexes is not the primary driver of territory elongation .', 'Moreover , we identify new roles for the X chromosome PC ( X-PC ) and X-PC binding protein HIM-8 in promoting elongation of X chromosome territories , separable from their role ( s ) in mediating local stabilization of pairing and association of X chromosomes with mobile SUN-1/ZYG-12 patches .', 'Further , we present evidence that HIM-8 functions both at and outside of PCs to mediate chromosome territory elongation .', 'These and other data support a model in which synapsis-independent elongation of chromosome territories , driven by PC binding proteins , enables lengthwise juxtaposition of chromosomes , thereby facilitating assessment of their suitability as potential pairing partners .']
['Successful sexual reproduction relies on the ability of germ cells to faithfully segregate homologous chromosomes in meiosis , which requires accurate sorting of chromosomes into homologous pairs and alignment of homologs along their entire lengths .', 'The mechanisms underlying homolog sorting and alignment are not well understood , partly because of a scarcity of studies investigating homolog alignment at the level of whole chromosomes .', 'This study provides a global view of the organization of chromosome territories during early meiotic prophase in the nematode Caenorhabditis elegans .', 'We applied chromosome painting to visualize the entire lengths of chromosomes .', 'Our study provides several conceptual advances .', 'First , our study excluded several possible mechanisms as primary drivers of chromosome sorting .', 'Second , our analysis has revealed both a robust capacity for full-lengthwise alignment between homologous chromosomes prior to the stabilization of pairing by the synaptonemal complex as well as a dramatic elongation of chromosome territories that could enable this alignment .', 'We also identified a factor required for the elongation of chromosome territories .', 'Elongation of chromosome territories could enable lengthwise juxtaposition of chromosomes , thereby facilitating assessment of their suitability as potential pairing partners by promoting utilization of information about chromosome identity that is distributed along the length of a chromosome .']
['meiosis', 'cell biology', 'chromosome biology', 'biology', 'molecular cell biology']