Developmental Biology Protocols Volume III Edited by Rocky S. Tuan Cecilia W. Lo HUMANA PRESS Methods in Molecular Biology TM HUMANA PRESS Methods in Molecular Biology TM Developmental Biology Protocols Volume III Edited by Rocky S. Tuan Cecilia W. Lo VOLUME 137 Overview 3 3 From: Methods in Molecular Biology, Vol. 137: Developmental Biology Protocols, Vol. III Edited by: R. S. Tuan and C. W. Lo © Humana Press Inc., Totowa, NJ 1 Developmental Biology Protocols Overview III Rocky S. Tuan and Cecilia W. Lo 1. Introduction The marriage of cell and molecular biology with embryology has produced remark- able advances for the field of developmental biology. In this third volume of Develop- mental Biology Protocols, contemporary, practical methods are first presented for the analysis and manipulation of developmental gene expression. To illustrate how such techniques, as well as procedures of experimental embryology including those described in the first two volumes of the series, may be applied in the study of develop- ment, a panoramic collection of experimental models of morphogenesis, development, and cellular differentiation are detailed. Both in vivo and in vitro systems are included. The volume concludes with various examples of developmental models of diseases and their molecular basis. 2. Manipulation of Developmental Gene Expression and Function Drosophila has been and remains one of the most versatile model systems for the manipulation of developmental gene expression. Chapter 2 focuses on a description of the experimental approaches currently used in ectopic gene expression in Drosophila to examine the function of a given gene in the desired tissue. Chapter 3 deals with the utilization of the highly efficient FLP/FRT yeast site-specific recombination system to generate somatic and germline clones in Drosophila for phenotypic analysis and screening. Chapters 3 and 4 address the methods used to alter gene expression as well as gene function in another experimentally highly accessible system, the developing chick embryo. Chapter 3 describes the application of antisense oligonucleotides to “knock down” gene expression in somitic stage chick embryos, whereas Chapter 4 discusses how functional neutralizing monoclonal antibodies may be used to block the activity of a specific gene product, N-cadherin, in the developing chick embryonic limb bud. 3. Analysis of Gene Expression The first step in analyzing the molecular basis of any developmental event is to characterize and compare gene expression profiles, both spatial and temporal, as a function 4 Tuan and Lo of development. A comprehensive list is provided in this section. Classic methods such as Northern blotting is not presented here, because relevant protocols are readily avail- able in many technical manuals of molecular biology. Quantitative methods include ribonuclease protection assay (Chapter 6), and polymerase chain reaction (PCR) based methods (Chapters 7 and 8). In situ hybridization (Chapters 9–15) has gained wide application in visualizing the spatial aspects of gene expression in the developing embryo, particularly in mapping the dynamics of tissue morphogenesis. In particular, the ability to carry out multiple in situ hybridizations (Chapter 14), or sequential in situ hybridization and immunohistochemistry (Chapters 12 and 15), on a given specimen should be invaluable for analyzing the potential roles of genes and gene products in development. The potential of the green fluorescent protein (GFP) of the jellyfish, Aequoria victoria, as a vital recombinant tag for genes of interest has produced a great deal of excitement in developmental biology; Chapter 16 provides a thorough discussion of the principles and techniques in the application of the GFP. Finally, the basic strategy in the application of monoclonal antibodies, one of the most powerful technical advances in modern biomedical research that has enjoyed a distinguished history, in the study of embryonic development is presented (Chapter 17). 4. Models of Morphogenesis and Development This section presents a number of developmental model systems under active inves- tigation to illustrate the multitude of experimental questions currently being addressed in the field of developmental biology. The inductive events of embryogenesis and means for their analyses are described in Chapters 18 and 19. Techniques for whole or partial embryo explant cultures for the somitic stage embryos for the analysis of meso- dermal and neural crest studies are covered in Chapters 20 and 21. Other models of morphogenesis include those for angiogenesis (Chapter 22), vasculogenesis (Chapter 23), and epithelial–mesenchyme interactions (Chapter 24). Specific organogenesis models are also included—limb bud (Chapter 25) and palate (Chapter 26). 5. In Vitro Models and Analysis of Differentiation and Development Regulation of cell differentiation is one of most active research areas of develop- mental biology. With the advent of cell and molecular biology, and the identification of differentiation-associated genes, cell differentiation is often interpreted in terms of regulation of gene expression. Both cis and trans modes of gene expression regulation have been found to operate during cell differentiation, leading to active investigation on structure/function of gene promoters and transcription factors. This section is a collection of many in vitro cell differentiation systems currently under active investigation. Early events in development include fertilization (Chapter 27) and trophoblastic differentiation (Chapter 28). Bone marrow-derived mesechymal pro- genitor cells have received a great deal of recent attention as candidate cells for cell- based tissue engineering. It is generally believed that the differentiation potentials of these cells represent a partial recapitulation of the characteristics of embryonic meso- dermal cells. Techniques for their isolation, culture, and characterization are described in Chapter 29. Another cell type important for studying cell differentiation are germ cells; methods for their isolation and culture are included in Chapter 30. Prostate cell Overview 5 differentiation is discussed in Chapter 30. Cell differentiation in connective tissues is presented in the following chapters: striated muscle differentiation (Chapter 31), somitic myogenesis (Chapter 32), mesenchymal chondrogenesis (Chapters 33–35), and bone cell differentiation (Chapter 36). In addition to specific examples and systems of cellular differentiation, methods for three crucial aspects of cellular activities are also presented. Cell–cell interaction is illustrated in Chapter 39, which deals with cadherin-mediated events. Cell–matrix inter- actions as mediated by hyaluronan binding are discussed in Chapter 40. The dynamic regulation of cytoskeletal architecture, visualized and analyzed by the microinjection of fluorescently-labeled α-actinin into living cells, is presented in Chapter 41. 6. Developmental Models of Diseases The experimental paradigms gained from developmental biology lend readily to the mechanistic analysis of diseases. Several examples are included here. Pax 3, a member of the vertebrate Pax gene family containing a DNA-binding domain known as the paired domain, is important for proper formation of the nervous, cardiovascular, and muscular systems. The molecular analysis of Pax 3 mutations and how the pathways affected lead to the pathogenesis of specific dysmorphogenic consequences is the sub- ject of Chapter 42. Finally, one of the most powerful contributions of molecular devel- opmental biology to the study of diseases is the application of transgenic methodologies to create animal models of human diseases. The three examples included here all deal with various aspects of skeletal defects, including both trunk as well as craniofacial malformations. The methods involve studies utilizing a structural gene (collagen type X, Chapter 43), cell specific promoter (α1(II) procollagen gene, Chapter 44), as well as transcription factors (Msx2, Chapter 44). Ectopic Expression in Drosophila 9 9 From: Methods in Molecular Biology, Vol. 137: Developmental Biology Protocols, Vol. III Edited by: R. S. Tuan and C. W. Lo © Humana Press Inc., Totowa, NJ 2 Ectopic Expression in Drosophila Elizabeth L. Wilder 1. Introduction Ectopic expression in Drosophila has been used extensively to examine the capa- bilities of a given gene in virtually any tissue. Three general approaches are described here, and the choice of which to use is determined by the needs of the particular experi- ment. Certain aspects of each approach can also be combined, providing powerful tools for the examination of gene function. Because ectopic expression does not involve a protocol, but rather generation of certain types of transgenic strains, this chapter focuses on a description of the approaches and in what circumstances each is likely to be useful. 2. Materials For each of the methods of ectopic expression described here, the production of transgenic strains is required. The vectors that are widely used in these experiments are available (1–3). 3. Methods 3.1. Expression Through Defined Promoters The simplest means of ectopic expression is through the construction of a promoter- cDNA fusion in which a gene of interest is driven by a defined promoter or enhancer. Transgenic strains carrying this construct then ectopically express the gene of interest in the defined pattern. One of the most commonly used promoters for this purpose is the heat shock protein 70 (hsp70) promoter (1). This promoter allows ubiquitous expression to be induced in any tissue of the fly through a simple heat shock at 37°C. The inducible nature of this approach is a great advantage. However, basal levels of expression can be prob- lematic, and heat shock itself can induce developmental defects. In addition, short bursts of ectopic expression ubiquitously is often not ideal. Therefore, sustained expression in defined domains may be preferred. To achieve ectopic expression within a defined domain, transcriptional regulatory regions from characterized genes have been linked to genes of interest (4,5). The advan- tage of this approach is its simplicity. Its primary limitation is that lethality can result from the ectopic expression. This makes it impossible to establish stable transgenic 10 Wilder lines. Enhancers that drive expression during late stages of development or in tissues that are nonessential have been particularly useful, because lethality owing to ectopic expression is avoided. The lethality associated with sustained expression of transgenes during develop- ment, the effort required to generate transgenic strains in which the transgene is expressed in multiple patterns, and the lack of defined enhancers driving expression in certain tissues prompted the development of alternative strategies for ectopic expression. 3.2. The GAL4 System The identification of the yeast transcriptional activator, GAL4, as a highly active, specific transcription factor that can activate transcription in Drosophila (6) led to the development of a system of ectopic expression referred to as the GAL4 system (2). This two-part system is shown in Fig. 1 and involves a cross between a fly expressing GAL4 in particular cells and a fly carrying a gene of interest under the transcriptional control of the GAL4 upstream activating sequence, or UAS. In the progeny of such a cross, the gene of interest will be expressed in cells where GAL4 is synthesized. Tar- geted ectopic expression of the gene of interest can therefore be achieved by choosing among many strains that express GAL4 in defined patterns. Three vectors are generally useful for investigators using this system (2). pGaTB/N provides either a BamHI site or a NotI site upstream of GAL4, allowing a defined promoter to drive GAL4 expression. The second, pGawB, is an enhancer-trapping vec- tor that directs GAL4 expression from genomic enhancers. Finally, pUAST includes multiple cloning sites behind five copies of an ideal GAL4 binding sequence. Genes of interest are easily cloned into this vector for GAL4-mediated expression. Fig. 1. The GAL4 system of ectopic expression (modified from ref. 2). This system allows the ectopic expression of any gene of interest (Gene X) in a pattern determined by the expres- sion of the transcriptional activator, GAL4. Hundreds of lines in which GAL4 is expressed in a variety of patterns have been generated through enhancer trapping or by linking the GAL4 coding sequence to defined regulatory elements. These are crossed to flies carrying the gene of interest under the transcriptional control of the GAL4 Upstream Activating Sequence (UAS). The progeny of this cross express the gene of interest in the pattern of choice. Ectopic Expression in Drosophila 11 Hundreds of GAL4 strains have been generated through the process of enhancer trapping. These strains have been characterized by crossing newly generated lines to a UAS-LacZ strain and characterizing the expression pattern. Many of these strains are now available through the Drosophila Stock Center at Bloomington, IN. The expres- sion patterns that have been detected through these enhancers vary from very broad expression to highly specific patterns. They, thus, offer the possibility of driving ectopic expression in virtually any tissue. In addition to the strains generated through enhancer trapping, many lines have been generated by fusing the GAL4 coding sequence to defined promoters, such as the hsp70 promoter. The latter offers the advantage mentioned above of inducible expression. The construction of strains expressing GAL4 in defined domains allows any UAS transgene to be examined within the particular region of interest. The GAL4 system has contributed to the utility of the FLP-FRT system of inducing mutant clones (see Chapter 3) (7). In this system, mitotic recombination is induced via flip recombinase (FLP), which is under the control of a heat shock promoter. The result- ing mutant clones are then generated in all mitotically active cell populations. How- ever, if FLP is placed under the control of GAL4-UAS, mutant clones are only generated within the GAL4 expression domain. This allows the investigator to deter- mine whether a particular gene has an endogenous function within cells defined by GAL4 expression. The GAL4 system addresses many of the problems associated with simple transgenes. First, since the UAS transgenic lines are produced in the absence of GAL4 activity, ectopic expression of the transgene does not occur. Therefore, lethality associated with ectopic expression is avoided until the transgenic flies are crossed to a GAL4 express- ing strain. Second, defined enhancers are not required for expression in a particular set of cells. Sites of expression are only limited by the number of enhancer trapped strains available, the number of which is continually growing. Finally, the GAL4 system allows ectopic expression in any number of patterns and conditions with the construction of only a single UAS transgene. This system of ectopic expression is extremely powerful for these reasons, but it does have limitations. First, for undefined reasons, GAL4 does not seem to function in the germline (A. Brand, personal communication). For experiments where germline expression is needed, other methods must be used. A more universal limitation of the GAL4 system is the fact that it is not inducible. Many enhancers drive expression dur- ing early phases of development, so that GAL4-mediated ectopic expression of certain UAS transgenes results in embryonic lethality. For investigators interested in later aspects of development, this has been a serious limitation of the GAL4 system. This problem can be partially addressed through modulation of temperature. The optimal temperature for GAL4 activity appears to be the ambient temperature for yeast, which is 30°C. By rearing flies at lower temperature, GAL4 activity is reduced (8,9), and in some instances, early lethality associated with higher levels of ectopic expres- sion from the UAS transgene is avoided. The flies can be shifted at later stages of development to increase GAL4-mediated expression. In at least one instance, an inductive ability has been added to the GAL4 system through the construction of a UAS transgene carrying a cDNA encoding a temperature sensitive protein (9). Thus, progeny of the GAL4-UAS cross are maintained at the 12 Wilder restrictive temperature during embryogenesis and shifted to the permissive tempera- ture at the relevant stages. This permits ectopic activity to begin at the desired stage. However, since temperature sensitive lesions have not been defined for most genes, the inability to control expression temporally remains a problem with the GAL4 system in analysis of postembryonic development. 3.3. Ectopic Expression in Clones The temporal control of ectopic expression has been critical for the analysis of gene activity during imaginal development. An ingenious method of ectopically expressing genes in any region of the imaginal discs was developed by Struhl and Basler (3) (Fig. 2) and has come to be called the flip-out system. This method involves the generation of random clones in which the coding region of a gene of interest comes to lie adjacent to a ubiquitous promoter. In these clones, the gene is ectopically expressed, whereas in the surrounding tissue, a gene encoding a visible marker is adjacent to the ubiquitous promoter, separating it from the gene of interest. This is accomplished through the use of flip recombinase target (FRT) sites flanking the marker gene. In the presence of the recombinase, the marker is removed, bringing the promoter and the gene of interest together. The resulting clone of cells is marked by the absence of the marker, which is ubiquitously present elsewhere in the fly. This technique requires the generation of a construct in which the gene of interest is placed within the context of the promoter-FRT-marker-FRT construct (3,10,11). Two vectors are available that utilize either the Actin-5C promoter or the β-Tubulin pro- Fig. 2. The Flip-out system of ectopic expression (see ref. 3). Flip recombinase (FLP) target sites (FRTs) are arranged as direct repeats flanking a visible marker. The expression of this marker is under the control of the promoter element. However, in the presence of FLP, recom- bination between the FRTs is induced, resulting in deletion of the marker gene. The gene of interest is now juxtaposed to the promoter element, resulting in ectopic expression of the gene of interest. This is an efficient but stochastic process, resulting in clones of cells that express the gene. The area over which the clones are induced is defined by the region in which the promoter is active. Ectopic Expression in Drosophila 13 moter. Both of these produce ubiquitous expression, so clones can be generated in any tissue. Levels of expression produced by the Actin-5C promoter are generally higher than those produced by the β-Tubulin promoter. A third vector uses the Ultrabithorax (Ubx) promoter, which produces clones in a more restricted pattern. Transgenic lines carrying the flip-out construct as well as a FLP transgene under the control of the hsp70 promoter (hs-FLP) must be generated. This is done through standard genetic manipula- tions using any of a number of hsFLP insertions on various chromosomes. A variation on this method of ectopic expression involves a combination of the GAL4 system and the flip-out system (12). The promoter-driving expression of the FRT cassette, in this instance, is the GAL4 UAS. Clones induced via hs-FLP, there- fore, fall only within the domain of GAL4 expression. The advantage of this combina- tion lies in the strength of GAL4 as a transcriptional activator. Clones induced in this way express very high levels of the gene of interest. The strengths of the flip-out technique are as follows. 1. The clones are efficiently generated randomly throughout the animal. By analyzing a num- ber of animals, it is very likely that clones will be found in a region of interest. 2. Ectopic expression is completely inducible. Lethality because of early expression is avoided. 3. The clones are marked molecularly by the ectopic expression of the gene of interest, and they are marked in the adult cuticle by the absence of the visible marker. As with any form of clonal analysis, this technique is limited to mitotically active cells, because cell division is required to generate a clone. A second limitation is that randomly generated clones are not reproducible; therefore, clones analyzed in the imaginal discs cannot be analyzed later in the adult cuticle. This contrasts with GAL4- driven expression that generates reproducible phenotypes. In this instance, one can precisely correlate imaginal disc phenotypes with the later phenotypes produced in the adult. Although these limitations need to be considered, the strengths of the flip-out system make it a very useful way to analyze gene activity during imaginal development. 4. Notes The foregoing approaches provide enormous temporal and spatial control over ectopic expression in Drosophila, allowing investigators to analyze gene activity in virtually any cell at any stage of development. However, in addition to the caveats mentioned for each of these methods, a few general concerns should be noted. 1. Positional effects can alter the levels of ectopic expression produced from any transgene. Thus, a transgene under the control of a given regulatory element may not express at the same level as a different transgene under the control of the same element. Therefore, multiple transgenic strains should be generated for any experiment to control for posi- tional effects. 2. Variability in phenotypes produced by ectopic expression is common. The reason for this is apparent with the flip-out system, because clones are randomly generated. Variation can be controlled, however, by inducing the clones within a narrow window of develop- ment. By collecting embryos over a short period before aging them and inducing the clones, clone size is kept more constant, as is the timing of ectopic expression relative to other developmental events. Variation in phenotypes using the GAL4 system is less pro- nounced, but can still be a problem. This can be minimized by rearing flies at a consistent temperature and by maintaining cultures in uncrowded conditions. 14 Wilder References 1. Struhl, G. (1985) Near-reciprocal phenotypes caused by inactivation or indiscriminate expression of the Drosophila segmentation gene ftz. Nature 318, 677–680. 2. Brand, A. and Perrimon, N. (1992) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415. 3. Struhl, G. and Basler, K. (1993) Organizing activity of wingless protein in Drosophila. Cell 72, 527–540. 4. Zuker, C. S., Mismer, D., Hardy, R., and Rubin, G. M. (1988) Ectopic expression of a minor Drosophila opsin in the major photoreceptor cell class: distinguishing the role of primary receptor and cellular context. Cell 53, 475–482. 5. Parkhurst, S. M. and Ish-Horowicz, D. (1991) Mis-regulating segmentation gene expres- sion in Drosophila. Development 111, 1121–1135. 6. Fischer, J. A., Giniger, E., Maniatis, T., and Ptashne, M. (1988) GAL4 activates tran- scription in Drosophila. Nature 332, 853–856. 7. Duffy, J. B., Harrison, D. A., and Perrimon, N. (1998) Identifying loci required for folli- cular patterning using directed mosaics. Development 125, 2263–2271. 8. Staehling-Hampton, K., Jackson, P. D., Clark, M. J., Brand, A. H., and Hoffmann, F. M. (1994) Specificity of bone morphogenesis protein (BMP) related factors: cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 60A. Cell Growth Diff. 5, 585–593. 9. Wilder, E. L. and Perrimon, N. (1995) Dual functions of wingless in the Drosophila leg imaginal disc. Development 121, 477–488. 10. Diaz-Benjumea, F. J. and Cohen, S. M. (1995) Serrate signals through Notch to establish a Wingless-dependent organizer at the dorsal/ventral compartment boundary of the Droso- phila wing. Development 121, 4215–4225. 11. Zecca, M., Basler, K., and Struhl, G. (1995) Sequential organizing activities of engrailed, hedgehog, and decapentaplegic in the Drosophila wing. Development 121, 2265–2278. 12. Nellen, D., Burke, R., Struhl, G., and Basler, K. (1996) Direct and Long Range action of a Dpp morphogen Gradient. Cell 85, 357–368. [...]... developing From: Methods in Molecular Biology, Vol 137: Developmental Biology Protocols, Vol III Edited by: R S Tuan and C W Lo © Humana Press Inc., Totowa, NJ 15 16 Rooke, Theodosiou, and Xu Fig 1 (A) Use of the FLP–FRT system or X-rays to induce mitotic recombination and clone formation Mutant clones are identifiable by concomitant loss of a marker gene (B) Because X-rays induce recombination at random... zebrafish and chicken In this chapter, we describe protocols we have developed for the application of antisense ODNs to developing chick embryos by two routes of administration—topical treatment and microinjection—for the study of somite development From: Methods in Molecular Biology, Vol 137: Developmental Biology Protocols, Vol III Edited by: R S Tuan and C W Lo © Humana Press Inc., Totowa, NJ 23 24 Alexander,... D., and Tuan, R S (1996) Valproic acid-induced somite teratogenesis in the chick embryo: relationship with Pax-1 gene expression Teratology 54, 93–102 24 Barnes, G L., Alexander, P G., Hsu, C W., Mariani, B D., and Tuan, R S (1997) Cloning and characterization of chicken Paraxis: a regulator of paraxial mesoderm development and somite formation Dev Biol 189, 95–111 36 Alexander, Barnes, and Tuan 25 Love,... High High Low P[mini w+; FRT]42B P[ry+, hs-neo; FRT]42B P[ry+, hs-neo; FRT]42C P[ry+, hs-neo; FRT]42D P[ry+, hs-neo; FRT]43D P[ry+, hs-neo; FRT]50B 2R FRT101 FRT11A FRT 9-2 FRT18A FRT19A FRT19F P[ry+, hs-neo; FRT]29D P[ry+, hs-neo; FRT]34B P[ry+, hs-neo; FRT]40A 2L Code P[mini w+; FRT]14A-B P[ry+, hs-neo; FRT]11A P[mini w+; FRT]18E-F P[ry+, hs-neo; FRT]18A P[ry+, hs-neo; FRT]19A P[ry+, hs-neo; FRT]19F... solution contains 120 mM NaCl, 56 mM KCl, and 2 mM CaCl2 in ddH2O We prepare 20X stock solutions 10 Large weigh boats (Fisher cat no 0 2-2 02D) 11 Parafilm (Fisher cat no 1 3-3 4 7-1 0) 12 35 mm Petri dishes (Fisher cat no 0 8-7 5 7-1 1YZ) 13 100 mm Petri dishes (Fisher cat no 0 8-7 5 7-1 2) 14 150 mm Petri dishes (Fisher cat no 0 8-7 5 7-1 4) 15 Low-melt agarose (Fisher cat no BP136 0-1 00) 16 Sterilized Erlenmeyer flasks... Chemical Co., St Louis, MO) added to nine parts PBS will yield 10% formalin buffered with PBS Formaldehyde should be used within 3 mo of purchase 4 Vital dye Nile Blue sulfate; add to antibody solution just prior to use From: Methods in Molecular Biology, Vol 137: Developmental Biology Protocols, Vol III Edited by: R S Tuan and C W Lo © Humana Press Inc., Totowa, NJ 37 38 Oberlender and Tuan 5 Mineral... commercially from Sigma and diluted to a concentration of 10 mg/mL in PBS at physiological pH NCD-2, a rat-derived monoclonal antibody directed against Functional Blocking Antibodies 3 4 5 6 7 8 9 39 the extracellular binding region of N-cadherin and capable of blocking N-cadherin-mediated homophilic interaction, was purified from an NCD-2 rat hybridoma cell line (2) using a standard procedure involving... Foe, V E., Odell, G M., and Edgar, B A (1993) Mitosis and morphogenesis in the Drosophila embryo: Point and counterpoint, in The Development of Drosophila melanogaster (Bate, M and Martinez-Arias, A., eds.), Cold Spring Harbor, New York, pp 149–300 16 Cohen, S M (1993) Imaginal disc development, in The Development of Drosophila melanogaster (Bate, M and Martinez-Arias, A., eds.), Cold Spring Harbor,... Theodosiou, and Xu Table 2 FRT Elements Chromosomes Frequencies of recombination FRT29D FRT34B FRT40A ND ND High b FRT2R-G13 FRT42B FRT42C FRT42D FRT43D FRT50B High Low Low Medium High ND a,c FRT69A FRT72D FRT3L-2A FRT80B ND High High Medium b P[ry+, hs-neo; FRT]82B P[ry+, hs-neo; FRT]89B P[ry+, hs-neo; FRT]93D 3R a,c P[ry+, hs-neo; FRT]69A P[ry+, hs-neo; FRT]72D P[mini w+; FRT]79D-F P[ry+, hs-neo; FRT]80B... background of a wild-type red eye Only very large clones or clones located at the edge of the eye will appear white 3.2 Induction of Germline Clones by FLP/FRT or X-rays 1 Set up crosses at 25°C such that progeny will be trans-heterozygous for a dominant female-sterile mutation (such as OvoD1) and the mutant gene or marker of interest (see Notes 2, 3, and 8) 2 Collect eggs for 24 h at 25°C 3a Heat-shock vials . Theodosiou, and Xu Table 5 Strains for Clones in Developing and Internal Tissues Chromosomes Strains Footnotes X w P[mini -w+ hsπM]5A, 10D FRT18A; hsFLP3, MKRS/TM6B a w P[mini -w+ hsNM]8A FRT18A a w. Developmental Biology Protocols Volume III Edited by Rocky S. Tuan Cecilia W. Lo HUMANA PRESS Methods in Molecular Biology TM HUMANA PRESS Methods in Molecular Biology TM Developmental Biology Protocols Volume. Theodosiou, and Xu developmental profile of the tissue (s) you wish to study (see Fig. 3). For Ey-FLP or GAL4/ UAS-FLP, FLP is expressed and will get large clones. 6. When heat-shocking or X-raying