~ I E C H N I C A L

T h e precise localization of gene transcripts is a crucial element in the description of any sequence that is involved in differentiation, morphogenesis or pattern formation. This spatial information can locate tran- scripts within a cell, provide evidence for cellular heterogeneity within a tissue, or even identify domains of expression that subdivide otherwise uniform regions of an embryo. For example, the localization of bicoid transcripts in the Drosophila egg gave a vital clue as to how embryonic polarity is establishedL Likewise, the different expression boundaries of members of the Hox gene family observed along the rostrocaudal axis of the mouse embryo, or the differ- ent domains of expression seen within the limb bud, prompt a new focus for dissecting mechanisms of pattern formation 2,3.

Until recently, all information about embryonic gene expression has come from the analysis of two- dimensional sections. However, embryos are complex three-dimensional entities, and non-isotopic in situ hybridization to whole, unsectioned embryos has become a useful and often more informative alterna- tive for localizing transcripts. First applied to analysis of Drosophila embryo#, this methodology has now been extended to the embryos of a number of other organisms, including sea urchins, amphibians, teleost fish and rodents n-17. This review focuses on the recent application of whole-mount in situ hybridization to the mouse embryo.

Radioactive versus nonradioactive whole.mount techniques

All in situ hybridization techniques are founded on the original autoradiographic methods in which 3sS-labelled riboprobes are hybridized to sectioned embryonic material~,5. However, radioisotopic in situ hybridization, besides being a lengthy process that may take several weeks, suffers from several other limitations. Autoradiography is only appropriate for monolayers of cells or sectioned material, and this makes it difficult to reconstruct accurate three- dimensional information about gene expression pat- terns. In addition, despite the sensitivity of radioactive detection, it is almost impossible to achieve unequi- vocal single-cell resolution. From a practical point of view, isotopic probes are not very convenient: they have a limited shelf-life and require special handling.

The introduction of nonradioisotopic labelling sys- tems, such as those using digoxigenin and biotin, has allowed the development of rapid techniques for studying the distribution of mRNA in intact, unsec- tioned material6.7. Whole-mount in situ hybridization of mouse embryos overcomes many of the problems associated with their three-dimensional structure. For example, during much of early postimplantation embryogenesis, when the embryo is acquiring its definitive pattern, the anteroposterior axis is either U- or S-shaped. As a result, it is virtually impossible to section through the entire length of an embryo while maintaining the same axial plane. Using whole-mount techniques, gene expression can be directly visualized in the intact embryo without resorting to cumbersome three-dimensional reconstruction from histological

©i993 Elsevier Science Publishers lad (UK) 0168 - 9525/93/$06.00

~ ' ~ O C U S

Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions BARRY ROSEN AND ROSA S.P. BEDDINGTON

Non-isotopic whole-mount in situ hybridization of mRNA is a novel technique that has greatlgfacllitated the precise three.dimensional localization of transcripts from genes whose expression is important during development. This methodology has recently been applied to the study of the mouse embryo and offers particular advantages over conventional procedures.

sections that are often distorted or incomplete. What is more, the opportunity for inspecting histological detail is not lost, since embryos can be sectioned after staining.

Whole-mount procedures also save time and labour compared with autoradiography of sections. Large numbers of intact embryos, of the same or different developmental stage or genotype, can be analysed simultaneously. Therefore, gene expression can be charted in a population of embryos rather than in multiple sections from two or three embryos, making it easier to ascertain the reproducibility of results. It is also easier to identify very restricted sites of gene expression by examining intact embryos than by look- ing at large numbers of serial sections. These consider- ations are particularly relevant when mapping the expression pattern of novel genes through the various stages of development.

Furthermore, because the ultimate detection method is histochemical (or fluorescent) rather than radioactive, it is possible to localize transcripts at the level of the single cell. Gene expression in dispersed individual cells, such as migrating germ cells or neural crest cells, can be readily identified and more easily related to known landmarks in the embryo. The sub- cellular distribution of transcripts can also be deter- mined, as illustrated by studies in Drosophila on the mechanisms of cytoplasmic RNA localization and the relationship between nascent RNA stability and pro- gression through the cell cycle9a 0.

The improved resolution of whole-mount RNA in situ hybridization offers a unique opportunity to study the localization of RNA transcripts TM. Non-random localization of cytoplasmic RNA species (such as actin mRNA) and nuclear RNA species (such as snRNAs) has been reported, and accurate moni- toring of other RNAs may reveal unforeseen polariz- ation in the localization of mRNA within cells19. 20. Apart from its importance for studies of basic cell biology, whole-mount in situ allows the investigation of possible asymmetries within embryonic cells which could, like the polarized distribution of transcripts in the syncytium of the eady Drosophila embryo, play an important role in tissue diversification.

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R E C H N I C A L [ R O C U S

The relative sensitivity of nonradioactive whole- mount in situ hybridization versus radioactive in situ hybridization to sections is unknown, however the former has successfully been used to localize a number of nonabundant RNAs within the mouse embryol6,212 2. Visualizing the signal in three dimensions can often help to detect less intense signals, which are more apparent in contiguous blocks of tissue than in iso- lated thin sections.

A tool for molecular genetic analysis Mapping the altered patterns of gene expression in

mutant embryos can yield valuable information about the mutant phenotype and elucidate the genetic inter- actions that are involved. For example, detailed exam- ination of the expression of the Engrailed-1 (En-1) gene in homozygous Wnt-1 mutant mouse embryos 23 has shown that they appear to lack populations of cells in the midbrain that normally express En-1. This result suggests that the Wnt-1 mutant phenotype may be partly caused by the loss of En-1 expression, and supports the idea that cells expressing these genes may interact in the wild-type embryo. The ability to analyse large numbers of embryos may also help to resolve the relationship between specific gene ex- pression patterns and observed variations in phenotype, or the unexpected absence of abnormalities in some mutants.

The ease and speed with which patterns of gene expression can be visualized by whole-mount in situ hybridization makes it an attractive technique for screening large numbers of candidate clones for region- or tissue-specific expression. As methods for making cDNA libraries from early mouse embryos improve, this application will become increasingly important24, 25 and should generate an important armoury of new genetic markers with which embry- onic pattern formation can be dissected.

A further application, and one that may prove particularly important in resolving genetic pathways involved in differentiation, is the detection in situ of transcription in populations of cells grown in vitro (Fig. 1). In the mouse, embryonic stem (ES) cells rep- resent a unique population of pluripotent cells, whose differentiation can mimic certain aspects of normal development 26,27. Whole-mount in situ hybridiz- ation enables the detection of significant changes in gene expression that may occur only in small sub- populations of cells: such subtle heterogeneity cannot be identified by methods that analyse RNA from total cell populations. Consistent coexpression of two or more genes in the same cell, or expression of a gene in one cell invariably followed by expression of a sec- ond gene in an adjacent cell, might provide a valuable pointer to molecular pathways. In the case of ES cells, this kind of ~malysis can be extended to studying RNA localization in three-dimensional embryoid bodies 2a.

Outline of the technique The whole-mount in situ hybridization protocols

that have been developed for use in different species have been optimized to suit the individual character- istics of each organism. For example, the fatty yolk found in amphibian early embryos presents unique

F1Gil

Hybridization of digoxigenin-labelled T(Brachyury) probe to ES cells grown in gelatin-coated plastic multiwells. Fixation and treatments were essentially identical to those used for mouse embryos, the cells remaining ,"ttached to the dish through the procedure. A digoxigenin-labeUed antisense riboprobe was synthesized with T7 RNA polymerase from BamHI-linearized plasmid pSK75 (courtesy of B. Herrmann, TObingen). The group of cells expressing T may represent an area of mesoderm-like differentiation of the ES cell monolayer. The staining reaction was developed for 2 h.

methodological problems. Developing methods suit- able for mouse embryos has been a fairly straight- forward process. These procedures are derived from whole-mount protocols (for both in situ hybridization and immunocytochemistry) used in lower organisms such as amphibians and insects. The limited numbers of mouse embryos (particularly mutant ones) that can be collected compared to amphibian or invertebrate embryos necessitates more careful handling of the material. The technique has been used successfully mainly on intact mouse embryos at the earlier stages of development - blastocysts and 5.5--10.5 days post coitum (d.p.c.) - but isolated organs and regions from older embryos can also be suitable 29. A number of protocols have been developed 15,16,21,2z and while there are major similarities between procedures, each has its own advantages and pitfalls. A short survey follows.

Preparation, fixation and storage Antibody and/or probe may become trapped

in cavities and cause high background staining. Therefore, it is critical to dissect away (as in the case of Reichert's membrane) or puncture (as in the case of the amnion) extraembryonic membranes. In embryos older than 9 d.p.c., cavities such as the brain ventricles and the heart chambers must be punctured for optimal

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~ '~ECHNICAL r~OCUS

proteinase K, we have used it to reliably detect the expression of a variety of genes, including int-2, Krox-20 and T(Brachyury) (B. Rosen and R. Bedding- ton, unpublished; Fig. 2). After either detergent treat- ment or proteinase K digestion, it is critical to include a postfixation step with the more powerful fixative glutaraldehyde. Other pretreatments have included the use of sodium borohydride to inactivate residual fixatives, and acetic anhydride to block positively charged groups that may bind probe, but their use is not essential.

FIGItl

Brachyury(T) expression is seen in the primitive streak (extensive region staining in posterior) and the notochord

(continuous narrow group of cells along the midline) of 8.5 d.p.c. mouse embryos. Probe was prepared as for Fig. 1. The staining

reaction was developed for 25 min.

results. Paraformaldehyde, a mild fixative, is used as the fixing agent in all protocols. Dehydrated embryos can be stored for weeks at -20°C in methanol or ethanol. A number of protocols include a bleaching step with H20 2 to reduce background but the effec- tiveness of this has not been critically evaluated.

Permeabilization and unmasking treatments Optimal sensitivity, giving a strong signal with low

background, in whole-mount in situ hybridization is achieved by treatments that unmask the mRNA target and/or make the embryo permeable to probe and detection reagents. In very thick specimens, such as whole embryos, tilere is a trade-off between the effec- tiveness of a given treatment in enhancing signal and the degradation of the embryonic structure. One com- mon procedure is to digest embryos with proteinase K. While proteinase treatment increases the sensitivity of the procedure, especially in embryos older than 10 d.p.c., this step can also be a source of variability and can damage embryos. Different embryonic stages and regions of individual embryos have various digestion requirements, so digestion conditions must be care- fully optimized. For example, if gastrulating embryos are subjected to prolonged proteinase treatment, the normal pattern of hybridization of ubiquitous mRNAs (such as that for ~-actin) in the outer cell layer may be lost, and the expression appears to be germ-layer specific (B. Rosen and R. Beddington, unpublished).

An alternative method, introduced in our laboratory, has been to incubate embryos in a cocktail of ionic and non-ionic detergents (RIPA). This milder pre- treatment gives consistent results on embryos from blastocyst to 10 d.p.c. Although the RIPA technique may be intrinsically less sensitive than protocols that use

Hybridization and washes Antisense riboprobes containing a nonradioisotopic

hapten group, typically biotin or digoxigenin, are syn- thesized by bacteriophage polymerases as run-off tran- scripts. Some protocols have used hydrolysed probes, while others use probes of up to 1 kb. Nonisotopic probes are used at higher concentrations than radio- active probes (approximately 0.1-1.0 Jig ml-O and this may contribute to the sensitivity of the nonradioactive techniques. Most protocols specify very stringent hybridization parameters (70°C, 50% formamide) and/or acid pH, conditions which were designed for Drosophila embryos. Individual protocols differ most in the hybridization conditions used, but no direct comparisons have been made. They also vary as to whether ionic or non-ionic detergents, carrier nucleic acids, or blocking agents such as heparin are included in the hybridization mix. Hybridization is followed by washes of varying stringencies and durations. A num- ber of protocols include a posthybridization digestion with ribonuclease A to remove unhybridized probe, a step which may reduce background.

Detection The hapten-labelled riboprobe is detected in situ

with conjugates of highly specific recognition reagents and reporter enzymes. There are no reports that directly compare the digoxigenin- and biotin-labelling techniques, although the former reagent has been used more extensively. Digoxigenin-labelled probes are visualized with a conjugate of anti-digoxigenin Fab and calf intestinal alkaline phosphatase 8 (any endogen- ous alkaline phosphatase is inactivated by heat). Digoxigenin, which is a natural plant product as opposed to an animal one, may ensure particularly low background. Streptavidin-~-galactosidase com- plexes have been used to detect biotinylated probes~. Reporter molecules that are directly conjugated to detection reagents produce lower background than attempts to amplify the signal by using layers of reagents (such as antibodies). The enzyme activity of the reporter is subsequently localized by the precipi- tation of a colorimetric substrate, such as X-Gal or BCIP-NBT (see Box 1). The colorimetric and fluor- escent substrate Vector Red30 has also been used with alkaline phosphatase substrate reporters. Stained embryos can be cleared in glycerol and stored refriger- ated for weeks.

Future d i ~ o n s A major challenge for the future will be to develop

automated methods for recording, analysing, storing

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~ I E C H N I C A L [ ] ~ O C U S

Box 1. Protocol for non-isotopic whole-mount in sttu hybridization of mouse embryos

Controls should be performed using: (1) a heterologous probe (2) antibody conjugate without probe (3) neither probe nor antibody added (control for endogenous alkaline phosphatase activity) and, ideally, (4) a probe from a separate region of the mRNA. The specificity of a signal can also be demonstrated by competition of the hybridization signal with an identical but unlabelled transcript.

Washes are carried out by allowing embryos to sink to the bottom of the container and carefully removing the liquid with a pipet. Always leave a small volume of liquid on the embryos, because even brief drying increases background. Reactions are performed in 10 ml glass Reacti-Vials (Pierce) with steep conical bottoms or in conical plastic tubes. Vials should be acid- washed, siliconized and DEPC-treated before use. Blastocysts are best handled by serial transfer to shallow glass dishes that contain the appropriate solutions. It is important to perform experiments on batches of ten or more embryos, to both allow for accidental losses and simplify the interpretation of results. It is also critical that all solutions used before and during the hybridization are free of contaminating RNase activity. If possible, autoclave and/or DEPC treat (0.1% overnight, then auto- dave) solutions. Heat inactivation is carried out by heating to 70°C for 30 min.

Solutions PBS Phosphate buffered saline. PBT PBS, 0.1% Tween-20. RIPA Detergent mix: 150 m i NaCl, 1% Nonidet-P--40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mi EDTA, 50 mm Tris

pH 8.0. PG-PBT Fresh 4% paraformaldehyde, 0.2% EM grade glutaraldehyde in PBT. HB Hybridization buffer: 50°/0 ultrapure formamide, 5 x SSC pH 4.5 (use a 20 x SSC stock solution, acidified with

citric acid), 50 tag m1-1 heparin, 0.1% Tween-20. SSC-FT 2 x SSC pH 4.5, 50OA formamide, 0.1% Tween-20. TBST Dilute from a 10 x stock: to make 100 ml of stock solution use 8 g NaCI, 0.2 g KCI, 25 ml 1 i Tris pH 7.5, 10 ml

10% Tween-20. APB Alkaline phosphate buffer: 100 trim NaCI, 50 rnM MgCI 2, 0.1% Tween-20, 100 m i Tris pH 9.5; make daily from stocks. NBT 4-nitroblue tetrazolium chloride: stock is 75 mg m1-1 in 70O~ DMF (N,N-dimethyl formamide); store at -20°C and

dilute as required. BCIP 5-bromo-4-chloro-3-indoyl-phosphate: stock is 50 mg ml -t in 100O,~ DMF; store at -20°C and dilute as required.

1. Dissection and fixation (i) Dissect embyros at room temperature in medium containing 10% fetal calf serum. Dissect away all extraembryonic

membranes and the anmion. (ii) Place embryos on ice and wash twice with cold PBS. (iii) Fix embryos overnight at 4°C in freshly prepared 4% paraformaldehyde in PBS. (iv) Wash embryos twice with cold PBT. Dehydrate through one change each of 25%, 50% and 75% Methanol-PBS, and

finally in two changes of 100% methanol, 5 rain each wash, on ice. Embryos can then be stored for several months at -20°C.

2. Pretreatments (i) Rehydrate embryos on ice through 75%, 50% and 25% Methanol-PBS. (ii) Wash embryos three times with PBT at room temperature. (iii) Wash embryos with three changes of RIPA at room temperature (30 rain per wash). (iv) Postfix embryos for 20 win in PG-PBT at room temperature with occasional mixing. (v) Wash embryos with five changes of PBT at room temperature (5 rain per wash).

3. Prehybrldimtion and hybridization (i) Wash embryos for 15 rain with a 1:1 mix of HB and PBT at room temperature. (ii) Wash embryos once with HB at room temperature. (iii) Prehybridize for 1-5 h at 70°C in HB containing 100 tag ml-I tRNA and 100 tag ml -t sheared denatured herring sperm

DNA (both phenol extracted). (iv) Denature the digoxigenin-labelled riboprobe (see below for preparation) by heating to 95°C for several minutes and

placing on ice. (v) Add HB (typically 0.5-1.0 mD containing 100 tag ml-I tRNA and denatured DNA. Mix well. Hybridize overnight at

70"(; in a chamber humidified with 50% formamide in water. (vi) Wash embryos once for 10 min with HB at 70°C; then twice for 5 min with SSC--FT at room temperature: then three

times for 30 rain with SSC--FT at 65°C. ," (vii) Cool embryos to room temperature. Wash three times at room temperature with TBST.

4. Probe synthesis (i) We synthesize antisense riboprobes as run-off transcripts from linearized plasmid templates using bacteriophage RNA

polymerases (T3, T7, SP6 ) under standard condition#. We include 11-digoxigenin UTP (Boehringer Mannheim) in trte probe synthesis reaction at a 1:4 molar ratio with UTP.

(ii) Plasmids are prepared by the Qiagen method and phenol extracted after restriction enzyme digestion. Probes 250-1500 bp long have been used successfully. Probe hydrolysis is not necessary.

(iii) Digest the reaction mixtures with RNase-free DNase and ethanol precipitate in the presence of LiCI. Resuspend in DEPC-treated water. Probes are stable for many months at -20°C.

(iv) Optimal probe concentration is in the 0.1-1 tag ml -l range, and should be determined empirically.

TIG MAY 1993 VOL. 9 NO. 5

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El ECHNICAL III ecus

5. Antibody bhd@g (i) Block embryos by incubating for 1 h at room temperature in 10% heat-inactivated sheep serum in TBST. (ii) Replace with a 1:2000 dilution of freshly preadsorbed anti-digoxigenin Fab-alkaline phosphatase conjugate

(Boehringer Mannheim) in TSBT with 1% heat-inactivated sheep serum. Mix well. (iii) Pre-adsorb the antibody conjugate by incubating with agitation for 1 h at 4°C in 1% heat-inactivated sheep

serum-TBST with several mgs of an acetone powder prepared from 11.5-12.5 d.p.c. embryos%. The powder is heat inactivated in TBST before use.

(iv) Continue antibody conjugate incubation overnight at 4OC.

6. w~~andco~urdevelopment (i) Remove antibody conjugate. Wash with TBST at room temperature: three times for 5 min, then four times for 30 min. (ii) Wash three times for 10 min with APB. (iii) Transfer embryos to a glass dish for staining in a solution of 4.5 fl ml-1 NBT and 3.5 fl ml-1 BCIP in APB. Mix

embryos well, then place in the dark at room temperature. (iv) Observe the progress of the reaction with a dissecting microscope for brief intervals. Staining is dark purple in colour.

Signals corresponding to abundant RNA species (e.g. actin) should be visible within 30 min. Incubations can be continued for up to 24 h.

(v) Stop staining reactions by rinsing embryos three times in PBS containing 1 mu EDTA. Store stained embryos in this solution at 4OC. Staining is stable for at least a month.

The major complication encountered when using this protocol is background caused by trapping of antibody and/or probe in embryonic cavities such as the amniotic cavity, heart chambers or brain ventricles. Background problems become particularly acute in embryos older than 9 d.p.c. Such diiculties can be alleviated by dissecting cavities open after fition or performing the procedure on the isolated organ or region of interest.

and retrieving the large amount of complex three- dimensional information generated by whole-mount in situ hybridization. One approach is the digitization and computer processing of images collected through high resolution black and white or colour TV cameras. Recent technological advances have resulted in excel- lent, inexpensive systems that can be used in conjunc- tion with standard personal microcomputers. Using such systems, it should also be possible to build com- prehensive and readily accessible databases of patterns of mRNA localization in embryos. However, this ap- proach is limited by the production of a two-dimen- sional, albeit sharpened, image of a three-dimensional embryo. A superior instrument for the analysis of RNA hybridization patterns in intact embryos is the confocal microscope3rJ2, which can record a series of detailed optical sections through intact specimens that are no thicker than a few millimeters33~34. Information is collected in digitized form, thereby simplifying sub- sequent image processing, which can make a detailed three-dimensional reconstruction of the signal. At pres- ent confocal microscopy can detect only fluorescently labelled probes, and therefore its potential for the analysis of whole-mount in situ data will only be fully realized when new fluorescent substrates for reporter enzymes become available.

Another important way in which the technique could be refined is by the development of protocols

several different fluorescent probes to be localized simultaneously 35. Although the choice of detection reagents for whole mount protocols is more restricted, since standard amplification methods that entail large complexes are unsuitable for thick specimens, the advent of new fluorescent substrates is likely to over- come this limitation, Whole-mount mRNA in situ hybridization procedures cc~rld also be used in con- junction with molecules that are currently used as lineage markers or reporters of gene expression, such as P-galactosidase.

conclusions In the past year the feasibility of using whole-

mount in s&r hybridization to mRNA has been dem- onstrated in mouse embryos. Given the complex anatomy of embryos during early organogenesis, this technique greatly simplifies the detection and interpret- ation of gene expression patterns. In addition, it offers a powerful means of comparing the expression patterns of several genes within an individual embryo, be this mutant or wild type.

Acknowledgements We thank B. Skames, V. Wilson and P. Rashbsrss for valu-

able discussion.

References 1 Berleth, T. et al. (1988) EMBOJ. 7, 1749-1756

McGinnis, W. and Krumlauf, R. (1992) Cell68, 283302 Dolle, P. et al. (1989) Nutum 342, 767-772 Wilkinson, D.G. and Green, J. (1990) in Partimpluntution Mammalian Embga: A Pnacticul Appmucb (Copp, A.J. and Cockcroft, D.L., eds), pp. 155-171, Oxford University Press Cox, K.H., Deleon, D.V., Angerer, L.M. and Angerer, R.C. (1984) Dar. Biol. 101, 485-502 Langer, P.R., Waldrop, A.A. and Ward, D.C. (1981) Proc. Nut1 Acud. Sci. USA 78, 6633-6637 Nonrudicactiue In Situ Hybridizution Applicution Manual (1992) Boehringer Mannheim Biachemicals

that allow more than one mRNA sequen;e to be 2 detected within the same specimen. This approach would simplify the simultaneous localization of tran-

i

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I

probes to alternate serial sections. Furthermore, co- localization of novel tissue-specific markers might help

6

to identify novel compartments of embryonic cells. 7 Techniques used in DNA in situ hybridization allow

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~EVIEWS

8 Tautz, D. and Pfeifle, C. (1989) Cbromosoma 98, 81--85 9 Davis, I. and Ish-Horowicz, D. (1991) Cell 67, 927-940

10 Shermoen, A.W. and O'Farreli, P.H. (1991) Cell67, 303-310

11 Lepage, T., Sardet, C. and Gache, C. (1992) Dev. Biol. 150, 23-32

12 Hemmati-Brivanlou, A. et al. (1990) Development 110, 325-330

13 Hadand, R.M. (1991) Methods CellBiol. 36, 675--685 14 Krauss, S., Johansen, T., Korzh, V. and Fjose, A. (1991)

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1275-1279 20 Carmo-Fonesca, M. etal. (1991) EMBOJ. 10, 195-206 21 Wilkinson, D.G. (1992) in In Situ Hybridization: A

Practical Approach (Wilkinson, D.G., ed.), pp. 75--83, Oxford University Press

22 Conlon, R.A. and Rossant, J. (1992) Development 116, 357-368

23 McMahon, A.P., Joyner,A.L., Bradley, A. and McMahon, J.A. (1992) Cell 69, 1-20

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26 Doetschman, T.C. et al. (1985)J. Embryol. Exp. MorphoL 87, 27-45

27 Robertson, E.J. and Bradley A. (1986) in Experimental Approaches to Mammalian Embryonic Development (Rossant, J. and Pedersen, R.A., eds), pp. 475-508, Cambridge University Press

28 Becker, S., Cassanova, J. and Grabel, L. (1992) Mech. Dev. 37, 3-12

29 Moens, C.B. etaL (1992) GenesDev. 6, 691-704 30 Murdoch, A., Jenkinson, E.J., Johnson, G.D. and Owen

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Laboratory Manual, p. 633, Cold Spring Harbor Laboratory Press

l i ROSF~' PdvO R.•P. BEDDINGTON ARE IN THE AFRC C~mTCE FOR GENOM£ RF.SF.ARClg UNBrERSffg OF EDINBURGH, KING'S BUl~ll~a~ WEST MAINS ROAI~ EDINBURGH, gig EH9 3JQ

N u m e r o u s environmental factors influence plant development. Temperature, light, touch, water and gravity are among the stimuli that serve as signals for the activation of endogenous developmental programs. Of these, light has an especially important role, not only as a substrate for photosynthesis, but also as a stimulus for many developmental processes, including chloroplast biogenesis, differentiation of the leaf meri- stem, floral induction and coordinate expression of several light-regulated nuclear and chloroplast-encoded genes. This light-dependent development, a complex process called photomorphogenesis, is controlled by the combined-action of several photoreceptor systemsL

Responses to light are mediated by several classes of photoreceptors including protochlorophyllide, blue and UV receptors, and the red/far-red light absorbing receptors, the phytochromesL Phytochrome, the only receptor that has been identified and characterized, is a chromoprotein that exists in two spectrally distinct, photointerconvertible forms: Pr, the red-absorbing form, and Pfr, the far-red absorbing form 1,2. The spec- tral properties of purified Pr and Pfr result from the combined properties of the 120 kDa apoprotein and its thioether-linked linear tetrapyrrole chromophore. Photoconversion of Pr to Pfr induces a diverse array of morphogenetic processes, whereas reconversion of Pfr to Pr cancels this induction. Thus, Pfr is considered the active and Pr the inactive form. The mechanism of action of Pfr in the developing seedling is not known. A further complication is that phytochrome is actually a family of photoreceptors; for instance, in the small mustard plant, Arabidopsis tbaliana, there are at least

Out of darkness: mutants reveal pathways controlling light-regulated development in plants JOANNE CHORY

Plant growth and deveiopme~ am gov_,,rned by. complex interactions between environmental signals and ~.qulogenous developmental programs. Mutations that disrupt light signal perceptton or transdt~tion have provided an important resource to begin dissection of the pathways that control light.regulated development in young seedlings.

five expressed phytochrome apoprotein genes 2 (PHYA, B, C, D and E). Their individual roles during develop- ment are largely unknown.

The downstream light-regulated responses include changes in gross and subcellular morphology, gene expression and physiologyL The way in which light regulates plant growth and development can best be illustrated by contrasting the morphologies of light- and dark-grown plants (Fig. 1A). Dark-grown (etiolated) dicotyledonous seedlings have elongated hypocotyls, small folded cotyledons and undeveloped chloroplasts. There is little or no expression of the light-regulated genes, such as the nuclear genes that

TIG MAY 1993 VOt. 9 NO. 5

©1993 Elsevier Science Publishers Ltd (UK) 0168 - 9525/93/$06.00 /