Cytogenetic Analysis of the Drosophila Testis

In this chapter, I hope to convince the reader that Drosophila spermatogenesis is an ideal system for the cytogeneticist. Spermatogenesis is relatively simple, dispensable for adult viability, and amenable to genetic, cell biological, and biochemical approaches. The stages of spermatogenesis are well defined, the cells are large, easily accessible, and easily identified. Because spermatogenesis initiates with a stem cell division, there is continuous production through the life of the fly....

Fixation of Mitotic Chromosomes

Physiological solution 0.7 NaCl in distilled water. Autoclave and store at 4 C. 2. Hypotonic solution 0.5 Tri-sodium citrate dihydrate in distilled water. Autoclave and store at 4 C. From Methods in Molecular Biology, vol. 247 Drosophila Cytogenetics Protocols Edited by D. S. Henderson Humana Press Inc., Totowa, NJ 3. Fixative solution Methanol acetic acid distilled water in the ratio 11 11 2 (v v v). Methanol is poisonous. It may be harmful by inhalation, ingestion, and skin absorption. Handle...

Fluorescent In Situ Hybridization FISH

Follow steps 1-3 of Subheading 3.2. 2. Transfer a whole ovary to a fresh drop of Ephrussi-Beadle solution. Separate the ovarioles from peritoneal and tracheolated epithelial sheaths, mature eggs, and shelled oocytes, retaining NCs from terminal and subterminal egg chambers. 3. Using tiny needles, transfer five to six selected egg chambers to a drop of 45 acetic acid on a microscope slide. Incubate the egg chambers for a couple of minutes. 4. Cover the drop with a cover slip and make a squash as...

Staining Chromosomes With Antibodies to Proteins

We have tested this protocol using antibodies against Mod (30) and HP1 (Mal'ceva, N. I. and Demakova, O. V., unpublished data Koryakov, D. E., unpublished data). An example of this staining is shown in Fig. 8. This method is based on protocol 30 in ref. 37 and ref. 38, with minor changes. It is important AT ALL STEPS of this protocol, except the last, to KEEP THE SQUASHES WET All solutions for this method should be kept at 4 C during all procedures. 1. Follow Subheading 3.2., steps 1-2. 2....

Immunostaining

Phase-contrast observation of mutant testes can only give a broad indication of the cytological defect in any particular mutant. Immunostaining mutant testes can give information on both cell-type distributions and subcellular structures. At the subcellular level, for example, staining of the meiotic spindle with anti-tubulin antibodies and a DNA dye will reveal much about the cytological mechanism underlying a chromosome nondisjunction phenotype. Antibodies or markers can also reveal which...

Combined FISH and Immunostaining

Proceed as described in Subheadings 3.1-3.5. 2. After completing Subheading 3.5., step 6, rinse the embryos twice with PBST. 3. Block the embryos in approx 1 mL of 10 FCS in PBST for 1 h at room temperature. Mix gently (e.g., on a rotating wheel). 4. Remove the blocking solution and incubate the embryos in 1 10 to 1 100 mouse monoclonal anti-Drosophila lamin antibodies in 10 FCS in PBST for 4-6 h at room temperature or overnight at 4 C (see Note 14). 5. Wash the embryos in five changes of PBST,...

Asymmetric Germ Line Divisions

In the adult ovary, the germ-line stem cells lie at the anterior of the tip of the germarium. Genetic (32) and laser ablation studies (33) showed that each ova-riole contains two to three self-renewing germ-line stem cells that undergo bursts of two to five successive divisions. Germ-line stem cells contain a spectrosome (the fusome precursor), which arises as the embryonic gonad is formed (34). The spectrosome associates with one pole of the metaphase spindle, and so is asymmetrically...

EM In Situ Hybridization to Polytene Chromosomes

In situ hybridization methods have become indispensable tools in molecular cytogenetics because they allow for the localization of specific sequences of DNA and RNA in cells, linking molecular information to structural studies (14,15). In Drosophila, these techniques are especially suitable because of the existence of giant polytene chromosomes, which also show a distinct and reproducible banding pattern. Originally, in situ hybridization studies employed radioactively labeled DNA probes and...

Morphogenesis and Patterning of Follicle Cells

From the point of view of follicle cells, the formation of the egg chamber involves three interdependent processes (1) proliferation of follicle cell-specific stem cells, (2) migration and encapsulation of the 16-cell cyst initially by approx 30 follicle cells, and (3) differentiation of follicular epithelium and follicular stalk to separate the egg chambers (21). Some defects in these processes lead to egg chambers with 2 or more 16-cell cysts. The stem cells of follicle cells are located in...

Materials 21 BrdU Labeling

Store at -80 C. These aliquots are good for several freeze-thaw cycles. Surprisingly, BrdU stock goes bad over time at -80 C and should be remade every couple of months. BrdU solution made fresh works best. BrdU is a mutagen and should be handled with care. 2. Mouse anti-BrdU monoclonal antibody (Becton Dickinson). 3. Cy3-Conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories). 4. Glycerol-based antifade solution for mounting ovaries onto microscope...

Notes

Paraformaldehyde is insoluble polymerized formaldehyde. It is solubilized by depolymerizing it in a reaction catalyzed by base. The resulting solution contains formaldehyde in the form of methylene hydrate, HO-CH2-OH (formaldehyde, HCHO, is a gas). The solution is not stable Formic acid can be produced and the formaldehyde can evaporate or repolymerize (28,30). Electron-microscopy-grade formaldehyde solutions are expensive and are also not stable once the ampoule is opened. It is less expensive...

Mekkara Mandaravally Madhavan and Kornath Madhavan 1 Introduction

The epidermal cells, a derivative of ectoderm during embryogenesis of insects, contribute to the distinct cuticular pattern and form of the different stages that appear during their ontogeny. The type of cuticular products is the result of gene expression of the individual epidermal cells that lie immediately underneath these outgrowths (1). In hemimetabolous insects, the larval epidermal cells (LECs) present at the time of hatching from the egg and their descendants are responsible for the...

Fluorescent Immunostaining of Polytene Chromosomes

Preparation of Third Instar Larvae 1. Add a drop of live baker's yeast and a paper filter moistened with a few drops of water to bottles containing fly food. 2. Let the flies lay eggs just to the point where larvae will hatch under uncrowded conditions (< 100 larvae bottle). This takes 7 d after egg lay at 18 C. We recommend this temperature in order to maximize the degree of polyteny, but higher temperatures can be chosen if needed, with relatively modest loss of size of the...

Appendix A Stages of Oogenesis

The main morphological characteristics of the egg chambers eggs are summarized according to the R.C. King proposed stages of oogenesis (1-3). Duration Size Stage (h) ( im) Characteristics Duration Size Stage (h) ( im) Characteristics Round-shaped egg chamber, situated at the end of the germarium oocyte located at the posterior of the 16-cell cyst follicle cells form a monolayer on the cyst no synaptone-mal complex in the oocyte nucleus follicular stalk differentiates as the egg chamber leaves...

Culture Conditions and Preparation of Squashes

Drosophila Larvae Slide

The best polytene chromosomes spreads are produced from late, third instar larvae that have been well fed with yeast and grown in uncrowded cultures. Culture media can be any one of the three mentioned in Subheading 2. 1. For larger chromosomes, culture second and third instar larvae at 18 C and feed additional yeast (see Note 1.) 2. Using a dissecting needle, select large, third instar larvae that have migrated from the medium but are not about to pupate or have everted their spiracles. Place...

Scatter Refractive Index and the Becke Line

Nonspecific light loss resulting from scatter from differences in refractive index (nD) of a specimen and its mounting medium is often one of the most serious errors affecting cytophotometric determinations of DNA-Feulgen amounts in tissue samples (12,38,41,49,64). Refractive index is a physical property of matter and is a quantitative expression of the degree of change in the velocity of a ray passing through a substance of one refractive index (nD) to another of a different refractive index....

Construction of the

Drosophila Gastrulation

The adult female reproductive system of Drosophila consists of a pair of ovaries, the genital ducts (common and lateral oviducts, uterus, vagina) and their accessory structures (two spermathecae, two accessory glands, and seminal receptacle), and the external genitalia (see Fig. 1). The ovaries are direct descendents of the embryonic gonads, whereas all other genital structures derive from a single genital imaginal disc. The formation of embryonic gonads implies the specification of pole plasm...

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...

Drosophila Cytogenetics Early Milestones

Sutton's 1903 landmark paper, The Chromosomes in Heredity 5 see also ref. 6 , in which he pointed out that the behavior of chromosomes in meiosis parallels the observed patterns of inheritance of Mendelian traits, is considered to mark the beginning of the field of cytogenetics the actual term would be coined years later . Before then, cytology with its focus on animals specimens, and genetics, which consisted of breeding experiments involving mainly plants, had been separate areas of inquiry 6...

Architecture and Morphology of the Polytene Chromosome Where Polytenized and Underrepresented Regions Meet

A plausible and long-held model for the structure of E-H junctions supposes a branched or nested arrangement of static replication forks, sometimes referred to as an onion-skin DNA structure, in which the multiple chromatids of euchromatin are merged with the much fewer chromatids minimally two of heterochromatin see Fig. 2A . Results of recent studies by Glaser and colleagues 188,189 suggest instead that a nonbranched chromosome structure joins regions of high and low polyteny see Fig. 2B ....

Laura Fanti and Sergio Pimpinelli 1 Introduction

Immunostaining of mitotic chromosomes of larval neuroblasts by antibodies directed against specific proteins is a powerful tool for analyzing their distribution in both euchromatin and heterochromatin. This approach is particularly important for the structural analysis of heterochromatin because the high content of repetitive DNA and the absence of meiotic recombination render this material difficult to manipulate by standard genetic and molecular methods. Sensitive chromosome banding...

Analysis of FISH Images and Mounting of Immunostaining and FISH Pictures

We perform this step using Adobe Photoshop software, version 5.0 or higher. Some basic knowledge of this software is needed in order to mount the immuno-FISH pictures. The following instructions are one example of how to do it, but other procedures or imaging software may also be used. 1. Select the best chromosomes for image analysis. In cases where the antibody staining survives FISH, from the digital camera attached to the microscope acquire three pictures for every selected chromosome with...

John Tonzetich 1 Introduction

Acetic orcein staining of polytene chromosomes was introduced in 1941 1 shortly after the initial studies on aceto-carmine-stained chromosomes by Bridges 2 and has remained a standard method of preparation. Orcein dye can be purchased in both its natural form as extracted from two species of lichens, Rocella tinctoria and Lecanora parella, and a synthetic form. The mechanism of staining is not clearly understood because the stain itself is a variety of phenazones, which may interact at an acid...

References

D. 1998 Pairing sites and the role of chromosome pairing in meiosis and spermatogenesis in male Drosophila. Curr. Topics Dev. Biol. 37, 77-115. 2. Maines, J. and Wasserman, S. 1998 Regulation and execution of meiosis in Drosophila males. Curr. Topics Dev. Biol. 37, 301-332. 3. McKim, K. S., Jang, J. K., and Manheim, E. A. 2002 Meiotic recombination and chromosome segregation in Drosophila females. Annu. Rev. Genet. 36, 205-232. 4. Darlington, C. D. 1937 Recent Advances in Cytology....

Microscopic Analysis of Wings

Twin Spots Drosophila

Inspect both the dorsal and the ventral surfaces of the wings under 400x magnification for the presence of single spots mwh or flr phenotype or twin spots a mwh clone adjacent to a flr clone . 2. Note the position of the spots according to the sector of the wing see Fig. 1 . 3. Score clones only in the distal wing compartment. Record the size of each spot the number of affected cells , its type whether it is a mwh or flr spot, or a twin spot , and its frequency see Note 2 . The...

Kami Ahmad and Kent G Golic 1 Introduction

The generation of dicentric chromosomes by site-specific recombination is a technique that has been used to study a number of different chromosomal phenomena, including 1 the segregation of acentric chromosome fragments 1,2 , 2 the behavior and resolution of chromosome bridges during cell division 3,4 , and 3 cellular responses to a broken chromosome end 4 . Its greatest advantage is that it allows quantitative experiments on the behavior and cellular consequences of this class of chromosome...

Polytene Chromosomes in Developing Nurse Cells

Three mutations that block oogenesis, fs 2 B, fs 2 cup, and ovarian tumor otu , also affect the development of NC chromosomes. Some of their alleles result in the formation of giant banded, polytene chromosomes 10-12 . The otu gene has been shown to transcribe at least two mRNAs and the proteins these translate have multiple functions during oogenesis 13-15 . There are 17 mutant aleleles that have been induced by ethyl methane sul-fonate EMS , and these are divided into three major classes...

Mitotic Chromosomes

Chromosomes Drosophila

Diploid nuclei of wild-type D. melanogaster contain eight chromosomes 2n 8 that can be seen most easily in squash preparations of the third instar larval CNS see Fig. 1 . The autosomal complement consists of two pairs of large metacentric chromosomes, designated 2 and 3, and a pair of tiny, spherical fourth chromosomes. Chromosomes 2 and 3 appear morphologically very similar after aceto-orcein or Giemsa staining, but sometimes they can be distinguished Chromosome 3 is slightly larger, and...

Oogenesis and Development of Nurse Cells

Diagram Oogenesis Drosophila

Each of the two paired ovaries of the adult Drosophila female consists of a cluster of parallel ovarioles, where egg chambers are lying in a single-file arrangement see Fig. 1A,B . Each egg chamber contains a branching chain of 16 interconnected cystocytes formed by mitotic division of a germarial cystoblast 2 . Fifteen of the cystocytes differentiate into endopolyploid nurse cells NCs see Fig. 1C . The function of these germ-line-derived cells is to synthesize RNA and protein molecules, which...

King R.c. Rubinson A. C. Smith R. F. 1956 Oogenesis In Adult Drosophila Melanogaster Growth 20 121-157

S. 1933 A new method for the study of chromosome rearrangements and the plotting of chromosome maps. Science 78, 585-586. 2. Koch, E. A., Smith, P. A., and King, R. C. 1967 The division and differentiation of Drosophila cystocytes. J. Morphol. 121, 55-78. 3. Brown, E. H. and King, R. C. 1964 Studies on the events resulting in the formation of an egg chamber in Drosophila melanogaster. Growth 28, 41-81. 4. King, R. C. 1970 Ovarian Development in Drosophila melanogaster, Academic,...

O

Halocarbon Oil 700 Vitelline Membrane

Triple labeling of Drosophila embryonic nuclei using confocal fluorescence microscopy in two stages of the cell cycle. The mAb 1A1, an IgM that recognizes the spindle matrix protein skeletor, was detected by TRITC-conjugated anti-mouse IgM secondary antibody. A rabbit polyclonal antibody raised against lamin generous gift of Dr. P. Fisher was detected by FITC-conjugated anti-rabbit IgG secondary antibody. Hoechst was used to stain the DNA. of xylene. If the embryos do not settle, repeat...

The Feulgen Reaction for DNA

Feulgen Reaction

The Feulgen reaction for DNA was initially described by Feulgen and Rossenbeck in 1924 42 . They found that a mild acid hydrolysis of fixed tissue sections followed by treatment with Schiff's reagent 43 resulted in a dis- Hydrolysis Times in 5 N HCl for Invertebrate Tissues Prepared in Different Fixatives Hydrolysis Times in 5 N HCl for Invertebrate Tissues Prepared in Different Fixatives crete magenta coloring at the sites of DNA in their preparations. Mild acid hydrolysis splits off the...

Brian R Calvi and Mary A Lilly 1 Introduction

The Drosophila ovary has proven to be an excellent model system for addressing many key questions in biology. Among these are questions relating to the cell cycle control of DNA replication and chromosome structure during development. Early studies of ovarian chromosome dynamics employed various histochemical stains and bright-field microscopy 1 . Later, photometric cytometry, 3H-thymidine incorporation, and radioactive in situ DNA hybridization were used to study DNA replication and chromatin...

Characteristics of Polytene Chromosomes from Ovarian Pseudonurse and Nurse Cells of otu Mutants

General Morphology, Chromosome Maps, Puffing Activity, and Protein Localization The PNC and NC chromosomes from otu mutants vary greatly in their length, level of polyteny, and banding pattern see Fig. 3 . All the chromosomes can be classified by length into four major classes pompon chromosomes P see Fig. 3A,D for use of terms, see review by Zhimulev 26 , half-pompons HP see Fig. 3B,C , condensed C see Fig. 3G and normal N see Fig. 3E,F,H . P chromosomes are the shortest, HP are...

Equipment for Culture Chambers

Cover Slips and Cleaning Materials 1. 24 x 24 mm2 cover slips we use Marienfeld, product no. 5370 . 2. Porcelain staining racks for cover slips product no. 8542-E42, Thomas Scientific, www.thomassci.com . 3. Curved forceps A. Dumont inox No. 7 . 4. Long anatomical forceps, 200 mm Bochem 18 8 . 6. Cleaning solution 7X PF 1 in distilled water ICN Biomedicals, Inc., cat. no. 76-671-210 . 8. Ultrapure water double distilled, or Milli-Q . 9. Staining glass box with cup, 90 x 60 x 50 mm3. 10....

Phase Contrast Microscopy of Live Testes

Phase Contrast Microscope Mitochondria

This is the first technique to apply when asking the question Why are my mutant males sterile Because the key stages of spermatogenesis have a very distinctive appearance in wild-type testes see Fig. 1 , it is relatively easy to look at a squash and preliminarily classify the mutant based on the morphologies observed. Table 1 gives a key to the types of defects you may observe these give clues as to what cytological process may be affected and, therefore, will direct your future experiments....

Satellite DNAs and Associated Proteins

Highly repetitive DNAs, often simple reiterations of AT-rich sequences, make up approx 20 of the D. melanogaster genome and therefore the bulk of heterochromatin 72 . These can be separated by centrifugation through cesium gradients into four major satellite bands having buoyant densities distinct from main-band DNA see Table 2 . Satellite classes I, II, and IV each contain a predominant simple repeat sequence together with less abundant repeats distributed in large blocks on one or more...