Daryl S Henderson 1 Introduction

Whole-mount fluorescence in situ hybridization (FISH) to chromosomes of Drosophila embryos is used to pinpoint the position of a chromosomal segment of interest, specified by the DNA probe(s), within a "preserved" three-dimensional nuclear structure. This technique has been used to (1) visualize the relative positions of homologous loci during the onset of somatic chromosome pairing (1,2), (2) establish whether separation of sister chromatids had occurred in mutants arrested at the metaphase/anaphase transition (3-5), (3) karyotype zygotes (6), and (4) ascertain the ploidy of nuclei of a checkpoint mutant (7). The basic embryo-FISH technique can be combined with antibody staining to permit simultaneous detection of both a hybridized DNA probe and a specific subcellular structure, such as the nuclear envelope, permitting spatial relationships to be defined (8,9). Protocols for both techniques are included in this chapter.

Probably the most important consideration when planning an embryo-FISH experiment is the choice of DNA probe. Although the exact selection will depend heavily on the biological question that is to be addressed, some general comments about probes can be made (see also Chapter 18).

The longer the target sequence available to the probe, the better the signal that will be produced. Thus, satellite DNA repeats, which in genomic DNA can extend for megabases (Mb), can make excellent probes. Simple satellite DNA probes have another advantage: They can be produced by automated synthesis (e.g., as a 50-60-mer oligonucleotide consisting of 10-12 repeats of a 5-nt sequence). Be aware, however, that many satellite sequences and

From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ

other repetitive DNAs reside at multiple chromosomal loci (10), which may limit their experimental utility. Some single-locus repetitive DNAs are listed in Table 1.

Several embryo-FISH studies have used unique DNAs as probes, typically derived from approx 80- to 100-kb genomic fragments cloned in P1 vectors (8,9,14). Such large DNAs must be broken into small fragments, either by digestion with multiple "4-base cutter" restriction enzymes (e.g., AluI, HaelII, Msel, MspI, RsaI, and Sau3AI; 14) or by sonication (8), to enable their penetration into the embryo. Probe fragments having a mean size of 200-300 bp are reported to work best (1). Unique genomic sequences totaling <10 kb have also been used successfully for embryo-FISH, although in those cases, the fluorescence signals were detected using a microscope equipped with a highly sensitive cooled charge-coupled device (CCD) camera (15,16).

Chromosomes in syncitial nuclei adopt a polarized or Rabl orientation in interphase, in which centromeric regions are localized to one side the nucleus (apically, toward the embryo surface) and telomeric regions are positioned toward the opposite side (basally, toward the embryo interior; see refs. 17 and 18). In effect, the anaphase configuration of the previous mitosis is maintained until prophase of the next mitosis. Consequently, in interphase, each gene locus tends to occupy a particular axial position in the nucleus. Highly repetitive DNAs most often reside in pericentric regions of chromosomes, and probes to such repeats will produce FISH signals apically (9), although YL-linked satellites would be expected to show an axial distribution in accordance with their positions along the chromosome arm. In the case of the AAGAG satellite cluster associated with the bwD mutation at the tip of the right arm of chromosome 2, in syncitial embryos it assumes a basal position in the nucleus, characteristic of the chromosome end, and shows preferential association with the nuclear envelope (NE) (14). However, the characteristic associations of het-erochromatic DNAs with the nuclear periphery and nucleolus seen in interphase nuclei of postsyncitial developmental stages are not observed in precelluarized embryos, most probably because heterochromatin has not yet formed. Moreover, certain DNA-NE associations that have been observed in the interphase of cycle 13 syncitial embryos do not persist into telophase, and in telophase new DNA-NE associations can form (9).

Probe DNA can be labeled using a variety of enzymatic methods that incorporate either hapten- or fluorophore-coupled deoxyribonucleotide triphosphates (dNTPs) into the probe fragments. The most commonly used labeling methods are random priming (19,20) (see Subheading 3.1.), nick-translation (21), and 3'-end labeling ("tailing") catalyzed by terminal deoxyribonucleotidyl transferase (TdT) (22). FISH methods using hapten-coupled dNTPs offer greater versatility and are generally more sensitive than direct methods using

Table 1

Examples of Single-Locus Repetitive DNAs

Table 1

Examples of Single-Locus Repetitive DNAs

Chromosome

DNA probe

Locus

Comments

Ref.

X

359-bp satellite

X heterochromatin

Some labeling of chromosomes 2 and 3 on metaphase squashes showing heavy labeling of X (10).

10

Yl

AATAC satellite

h5-h6

~3.5 Mb

10,11

YS

AATAAAC satellite

h22

~1.6 Mb

10,11

2L

Histone gene cluster

39D-E

100-150 copies per chromosome

1

2R

AACAC satellite

Pericentric heterochromatin

11,12

2R

5S rDNA

56F1,2

dodeca satellite

Pericentric heterochromatin

13

4

See Note 1

fluorophore-coupled dNTPs. The most widely used haptens are biotin and digoxigenin (DIG). After hybridization to chromosomes, biotin moieties of the probe are detected using fluorescently labeled avidin or streptavidin, both of which bind biotin with high affinity. DIG moieties are detected using anti-DIG antibodies (from Roche). Commonly used fluorochromes include fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC), and various cyanine dyes (e.g., Cy2, Cy3, Cy5, and Alexa® Fluors). A wide range of fluo-rochrome-conjugated avidin and streptavidin reagents are available from numerous vendors (e.g., Vector Laboratories, Sigma-Aldrich, Jackson ImmunoResearch, Molecular Probes), whereas the range of fluorophore-conjugated anti-DIG antibodies is fairly limited (e.g., FITC, TRITC, and amino-methylcoumarinacetic acid [AMCA]). Although the exact choice of fluorophores will depend on both your microscope's light sources and filter sets, typically even basic laser scanning confocal microscopes are configured to allow imaging of both green-fluorescent (e.g., FITC, Cy2) and red-fluorescent (e.g., TRITC, Cy3) dyes.

An emerging technology, quantum dots (QDs), may some day supplant organic dyes as fluorophores for biological imaging. Core-shell QDs consist of a luminescent semiconductor dot core (e.g., CdSe) capped by a thin inorganic shell (e.g., ZnS) that prevents surface quenching and increases photosta-bility of the core (e.g., refs. 23 and 24). QDs emit at different wavelengths depending on dot size, but different color dots can be excited at a single wavelength, which has important implications for multicolor FISH. Furthermore, QDs are intensely fluorescent at concentrations comparable to organic dyes and can be visualized by both conventional fluorescence and confocal microscopy. They are highly photostable and do not require antifade reagents. At least two QD-streptavidin conjugates are now available commercially, from Quantum Dot Corporation (Hayward, CA [www.qdot.com]). Qdot™ 585 would substitute for lissamine rhodamine B or TRITC conjugates, and Qdot™ 605 would substitute for Texas Red.

Protocols for preparation of embryos are given in Subheadings 3.2. and 3.3. The FISH method (Subheadings 3.4-3.6.) is essentially that of Hiraoka et al. (1) incorporating minor modifications of Sigrist et al. (3). The protocol for combined immunostaining and FISH (Subheading 3.7.) is modified from Gemkow et al. (8).

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