Homologous Chromosome Pairing in Drosophila melanogaster Proceeds through Multiple Independent Initiations Jennifer C. Fung,* Wallace F. Marshall,‡ Abby Dernburg,‡ David A. Agard,‡i and John W. Sedat‡ *Graduate Group in Biophysics and ‡Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0554; and iThe Howard Hughes Medical Institute, San Franciso, California 94143 Abstract. The dynamics by which homologous chro- pairing kinetics of histone loci during nuclear cycle 14. mosomes pair is currently unknown. Here, we use fluo- By measuring changes of nuclear length and correlating rescence in situ hybridization in combination with these changes with progression of time during cycle 14, three-dimensional optical microscopy to show that ho- we were able to express the pairing frequency and dis- mologous pairing of the somatic chromosome arm 2L tance between homologous loci as a function of time. in Drosophila occurs by independent initiation of pair- Comparing the experimentally determined dynamics of ing at discrete loci rather than by a processive zippering pairing to simulations based on previously proposed Downloaded from of sites along the length of chromosome. By evaluating models of pairing motion, we show that the observed the pairing frequencies of 11 loci on chromosome arm pairing kinetics are most consistent with a constrained 2L over several timepoints during Drosophila embry- random walk model and not consistent with a directed onic development, we show that all 11 loci are paired motion model. Thus, we conclude that simple random very early in Drosophila development, within 13 h after contacts through diffusion could suffice to allow pairing egg deposition. To elucidate whether such pairing oc- of homologous sites. jcb.rupress.org curs by directed or undirected motion, we analyzed the n eukaryotes, a chromosome is sometimes aligned and isms as Antirrhinum majus (Bollman et al., 1991) and on August 2, 2017 associated with its homologue over part or all of its Neurospora crassa (Aramayo and Metzenberg, 1996). In I length. Although this chromosome alignment is prom- these cases, either suppression or enhancement of a phe- inent in meiosis (Roeder, 1995), for some organisms such notype is observed when pairing is disrupted by chromo- as Dipteran insects, this association, termed homologous somal rearrangements. In cases where it has been explic- chromosome pairing, is observed to be a normal part of itly tested (e.g., Goldsborough and Kornberg, 1996), the nuclear organization (Metz, 1916). Although in other or- effects can be accounted for by changes in levels of tran- ganisms, less extensive homologous pairing is seen (e.g., scription that accompany the disruption of pairing. An- Vourc’h et al., 1993; Lewis et al., 1993). Nevertheless, a other area where homologous pairing might play an im- number of intriguing biological phenomena center around portant role is in paramutation, an interaction between homologous chromosome pairing. alleles that leads to a directed heritable change at a locus Pairing of homologous chromosomes can also influence at high frequency (Patterson and Chandler, 1995). Simi- gene regulation. In Drosophila melanogaster, gene expres- larly, methylation transfer, believed to be important for sion can be modulated by physical pairing of homologous many epigenetic phenomena, seems to require pairing of loci in what are termed transvection and trans-sensing ef- homologues as an initial and crucial step in the process fects (reviewed in Tartof and Henikoff, 1991). Similar ef- (Colot et al., 1996). fects have been reported in such widely divergent organ- Although progress is being made in determining the bio- logical relevance of homologous pairing, relatively little is known about the mechanism by which homologues be- Address correspondence to John W. Sedat, Department of Biochemistry come paired. For both somatic and meiotic homologous and Biophysics, University of California, San Francisco, San Francisco, pairing, this is primarily a consequence of the inability to CA 94143-0554. Tel.: (415) 476-2489. Fax: (415) 476-1902. E-mail: sedat@ directly monitor two homologous sites as pairing proceeds. msg.ucsf.edu Thus, many fundamental questions about the dynamics of Jennifer C. Fung and Wallace F. Marshall’s present address is MCDB Dept., Yale University, New Haven, CT 06520. how homologous chromosomes come together remain un- Abby Dernburg’s present address is Department of Developmental Bi- answered. Is the homology search carried out by discrete ology, Stanford University School of Medicine, Palo Alto, CA 94305. sites or simultaneously along the entire length of the chro- The Rockefeller University Press, 0021-9525/98/04/5/16 $2.00 The Journal of Cell Biology, Volume 141, Number 1, April 6, 1998 5–20 http://www.jcb.org 5 mosome? How do those sites that undergo homology the differentiated groups of cells can give us information search locate each other in the nucleus? Once homologous on what factors influence a chromosome’s ability to pair. sites have located each other, does the pairing of adjacent Also aiding in the pairing analysis is that the embryonic sites proceed processively? Furthermore, numerous mod- developmental stages described here are all extremely els have been put forth for how this pairing might take amenable to the three-dimensional (3-D)1 imaging tech- place (as reviewed in Loidl, 1990). In general, these mod- niques needed to properly observe the pairing. Particu- els can be characterized as those emphasizing active move- larly useful is that many of these nuclear cycles can be im- ment of chromatin to bring homologous regions into con- aged as they proceed in vivo, which is important for tact or those relying mainly on fortuitous encounters of developing time courses necessary for determining the homologous sites brought about by random movements of mechanisms governing the homology search. the chromatin by diffusion (also referred to here as ran- To elucidate how pairing proceeds during Drosophila dom walk motion). Further complicating this issue is what development, we used fluorescence in situ hybridization role, if any, does nuclear organization play in facilitating (FISH) and immunofluorescence under conditions that the homology search. This question has been raised by nu- preserve nuclear and chromosomes substructure com- merous meiotic studies suggesting that prealignment through bined with high resolution 3-D optical microscopy. The bouquet formation (an alignment of telomeres and chro- pairing frequencies of 11 loci distributed over chromo- mosomes within meiotic nuclei; reviewed in Dernburg et some arm 2L were evaluated at several timepoints during al., 1995; Scherthan, 1996), Rabl orientation (a centromere Drosophila development. This analysis revealed that all 11 to telomere polarity found in interphase nuclei; Rabl, loci are paired within 13 h after egg deposition (AED), 1865; Fussell, 1987) or juxtaposition of chromosomes dur- demonstrating that pairing along the entire length of the ing metaphase congression (Maguire, 1983a) can reduce chromosome is attained very early in the Drosophila life the volume over which homology search takes place and is cycle. More crucially, the frequencies of pairing at differ- thus a necessary part in the meiotic pairing process. In this ent sites indicates that side-by-side alignment is achieved Downloaded from report, we attempt to address these questions and related through the independent initiation of pairing at discrete ones by following the kinetics of somatic homologue pair- loci rather than by a processive zippering of sites along the ing in Drosophila embryos. length of chromosome. Drosophila melanogaster offers a unique system in which We measured the time course of pairing of the histone to study homologous pairing. Drosophila chromosomes locus through a cycle of interphase and compared our ob- are thought to be homologously associated in the early servations to computer simulations of pairing based upon stages of development in addition to their association different models of chromosome motion. Through quanti- jcb.rupress.org observed during meiosis. For instance, the giant polytene tative kinetic analyses, we provide evidence that simple chromosomes found in larval tissue exhibit a close synap- random contacts through diffusion are sufficient to allow sis of homologues along their entire lengths. Observations pairing of homologous sites at the observed rates. of a side-by-side juxtaposition of metaphase chromosomes in squashed neuroblast preparations lend further support Materials and Methods on August 2, 2017 that homologues pair early in development in this organ- ism (Metz, 1916). Furthermore, examination of homolo- Drosophila Stocks gous pairing during embryogenesis of a single site has indi- cated that the attainment of homologous pairing for Wild-type flies were obtained from an Oregon-R stock maintained at 313 Drosophila as determined by the pairing of the histone lo- UCSF. The lt stock (Wakimoto and Hearn, 1990) was provided by B. Wakimoto (University of Washington, Seattle, WA). All stocks were cus occurs very early (Hiraoka et al., 1993). maintained at 248C. There are several advantages for studying the dynamics of homologue pairing during the well-characterized stages Preparation of DNA Probes of embryonic development (reviewed in Foe, 1993). Be- Probes were prepared as described in Hiraoka et al. (1993) for the histone tween the 10–13th nuclear cycles, nuclei