Three-Dimensional Structure of Eukaryotic Chromosomes 
the other known components of the nuclear pe- 
riphery (pore complexes, chromatin) relative to 
this network? Second, what, if anything, occupies 
these large, lamin-empty regions? Third, how are 
these structures assembled as the NE re-forms 
during telophase? 
Chromatin in the nuclear periphery displays an 
^mteresting structural paradox in that a large frac- 
tion appears to be aligned beneath the lamin net- 
work, but with very little contacting lamins di- 
rectly. The majority of it seems to be at a distance 
of about 0.2 /xm. This result is consistent with 
much indirect evidence for a strong interaction 
between chromatin and the nuclear lamina, but 
strongly suggests that a direct physical contact is 
not involved. 
We injected lamins and lamin-specific mono- 
clonal antibody Fab fragments, both fluores- 
cently labeled, into early Drosophila embryos to 
study four-dimensional lamin-NE dynamics. In 
these experiments the embryos develop normally 
and hatch on time. We observe a highly discon- 
tinuous lamin network in vivo, with interlamin 
fiber spacings at least as large as those observed in 
fixed samples. From these experiments, new 
four-dimensional data, spanning prophase to 
metaphase in the cell cycle, show a surprisingly 
complex series of lamin structural rearrange- 
ments. Lamins do not completely disassemble 
and disperse at the onset of mitosis, but remain 
well localized with complex structural dynamics 
until well into the mitotic process. Further lamin 
structural changes take place at anaphase and 
telophase. 
These studies emphasize that lamins appear es- 
sential for the nuclear structural reorganizations 
that take place at all points of the cell cycle. If, 
however, we inject fluorescently labeled inter- 
phase lamins, a very different picture results. 
Arrested nuclear structures leading to chromo- 
somal/nuclear aggregates are seen. These studies 
suggest that structural/functional assays will be 
required for proper interpretation of the 
biochemistry. 
Three-Dimensional in situ Hybridization 
We are continuing our study of nuclear organi- 
zation, using three-dimensional fluorescence in 
situ hybridization. The studies, described in our 
previous report, were performed in Drosophila 
embryos, primarily at the histone gene locus, a 
500-kb, tandemly repeated gene cluster. We have 
extended our techniques to other whole-mount 
tissues from Drosophila, including developing 
imaginal tissues (which will later form the adult 
fly). We have also improved our hybridization 
protocols and can now detect chromosomal 
probes as small as 1 2 kb with high signal-to-noise 
resolution. Using these techniques, we are in- 
vestigating the arrangement of chromosomes 
throughout the cell cycle and as a function of 
development. 
A focus of our studies is the question of homolo- 
gous chromosome pairing (one chromosome 
from the male parent, the other from the female) . 
This is of particular interest in Drosophila biol- 
ogy because genetic evidence has indicated that 
different alleles at certain homologous loci can 
influence one another, implying that the loci 
communicate in some fashion. Such phenomena 
have been termed transvections, or more gener- 
ally, "trans-sensing effects." By determining the 
nuclear positions of the histone gene cluster in 
developing embryos, we have shown that the ho- 
mologous chromosomes bearing this locus are for 
the most part spatially distinct, or unpaired, 
throughout most of early embryonic develop- 
ment, but that a transition occurs just prior to 
cellularization, a distinct time point in develop- 
ment, after which the locus is seen to be paired at 
high frequency. 
We are currently extending this analysis to 
other genetic loci, to determine whether this un- 
paired/paired transition occurs simultaneously at 
all chromosomal positions. We have preliminary 
evidence that loci more distant from the centro- 
mere than the histone locus may show different 
pairing behavior in early embryos. We are particu- 
larly interested in carrying out this type of analy- 
sis for loci that are known to have transvection or 
trans-sensing effects. 
This general methodology has a number of po- 
tential applications to problems of cell lineage, 
neural architecture, and pattern formation in de- 
velopment. We are pursuing some of these inter- 
ests in collaboration with other laboratories. 
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