first, trap your enhancer

MATTHEW FRE~EMAN ENHANCER TRAPPING

First; trap your enhancer s O&

Enhancer trapping is a powerful new way of isolating developmental ‘@A@’ genes tlhat may not have been detectable by conventional approaches. It has been pioneered in Drosophila but can be adapted to other organisms.

The ability to isolate and characterize genes with important roles M the development of higher eukaryotes has al- lowed us to begin to unravel the molecular mechanisms underlying the phenomena described by classical embry- ologists. The question put simply is: how does a corn- plex organism with many cell types organized in a precise architecture, develop from a single-cell egg? At the forefront of this explosion of interest in molecular genetics of development has been the fruit fly, Drosopbihz melunoguster [l]. The standard approach to studying dtielopment in ~~osophih has been to identify mutations that disrupt the process of interest, and then to characterize the gene thus identified - both genetically, and by cloning it and studying it at the molecular level [2-d]. Even though this approach has been extraordi- narily successful in identifying genes with a role in many developmental processes [2-51, relying on the phenotypes of mutations in genes of interest does have its shortcomings.

The main problem is that genes can have functions at more than one time in development, and mutations in such genes may only produce phenotypes associated with one of those functions. This is well illustrated by the development of the adult Drosophila eye, which is a relatively late-developing part of the nervous system. Several genes (for example, seven-up 161 and rhomboi4 MF, BE Kimmel and GM Rubin, unpublished data) have now been found that play a part in eye development, but which also have an important function in embryonic nervous system formation. Mutations in these genes are often lethal at the embryonic stage; such mutants there- fore never reach the stage where eye development be- gins. This means that genetic screens for adult flies with abnormal eye phenotypes will not detect such mutations and a whole class of genes involved in eye development will be missed. A second problem is that the phenotypes associated with some mutations may simply be too sub- tle for us to detect. This is particularly likely to be the case when studying such complex structures as the central nervous system (CNS). Mutations that cause the mis- specification or n&-routing of a small number of cells in the CNS are unlikely to be detected in a screen for visible embryonic abnormalities. A third general problem with the classic mutational approach is that of redundancy of function. It may be that in the absence of a functional gene product another one can at least partially replace the lost function; in this case, a mutation in such a gene may not give a detectable phenotype. It is d8icult to imagine how fully redundant functions could be selected for Idtiring evolution, but there is evidence for at least partially redundant functions: for example

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mutations in the gene encoding the neural adhesion molecule, fasciclin 1, only show detectable phenotypes in combination with mutations in other genes [7].

Recently, a powerful new tool has been added to the Drosophila researcher’s bag of tricks. This ‘enhancer trap’ technique surmounts the problems discussed above, and is destined to have a major impact on the analysis of Drosopbih development. Furthermore, the enhancer trap principle can potentially be used in other organisms, even ones with less well characterized genetics than Drosophila. Initially described by O’Kane and Gehring [8], and since modilied in various ways [9-121, the principle of the enhancer trap is as follows. Instead of identifying genes by means of the phenotype caused by a mutation, they are identified by their pattern of expression. The underlying assumption is that a developmentally important gene will show a specific temporal and spatial expression pattern related to its function. This assumption is well supported by many examples.

Enhancer trapping is done by -constructing a transposon from a DrosoplG P-element - a well characterized transposable element that can be induced to hop around the genome at a high frequency. Within this synthetic transposable element, the gene encoding Escberikhh coli P-galactosidase (p-gal) is pIaced under the control of a weak constitutive promoter. The element is transformed into the Drosophila genome, but the p-galgene is effec- tively inactive in the fly unless it randomly integrates near a transcriptional enhancer, in which case, the enhancer stimulates transcription from the weak promoter (Fig. l), and $-gal is expressed in the same pattern as the ad- jacent gene that is normally regulated by that enhancer. The presence of $-gal in any tissue is easily detected using a histological stain, which produces a blue product (Fig. 1). The element also carries a dominant eye colour gene (W+ ) to identify flies that contain it, and plasmid sequences that facilitate the cloning of the Llanking genomic DNA; it is bounded by P-element sequences, which allow it to transpose in the presence of P transposase (Fig. 1). Note that as enhancers work independently of orienta- tion and position [13], the element can insert anywhere in the vicinity of the gene. The insertion may or may not be mutagenic itself, depending upon its precise location with respect to the gene. A screen for genes expressed in interesting patterns can be undertaken by producing many lines of flies, each representing a single random insertion of the enhancer trap element, and screening the ilies for expression of p-gal This basic approach has been made easier by the use

@ 1991 Current Biology

Fig. 1. How to entrap an enhancer. The enhancer (E) drives expression of a gene (coloured green). After the nearby insertion of an enhancer trap element, the enhancer drives expression of the p-gal gene (coloured blue). Below, p-gal expression in an ‘enhancer trap’ line: ventral view of a Drosophila embryo. In this line the enhancer trap detects a gene that is expressed in a subset of the cells within the central nervous system.

of a stable genomic source of P-element transposase en- zyme [14,15]. This allows new enhancer trap lines to be produced by genetic crosses instead of by embryo mi- croinjection. Flies already carrying the enhancer trap element, which does not encode transposase, are crossed to flies that carry a disabled P-element, which encodes transposase but cannot itself move. The enhancer trap transposons are thus mobilized in the progeny, and genetic markers are then used to select individuals that contain an element at a new genomic site, and which do not contain the transposase gene. In this way stable lines of flies, each representing a single, random insertion event, are established. Using this method, the limiting factor for the number of lines that can be screened is the histological staining: in practice several hundred a week can be analysed.

Having identified a gene of interest by means of its expression pattern, it is then relatively straightforward to

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clone it, as the enhancer trap element can be used as a molecular tag for the genomic region. There are now several examples of genes that have been identilied and cloned using enhancer traps (for example, [6,16-181).

It is worth outlining some of the assumptions that were made when this technique was being pioneered, as these will be relevant when considering the possibility of adapt- ing enhancer traps to other animals. For the technique to be widely useful it was necessary that the promoter used could be activated by enhancers in all cell types. Various promoters have been tried [12], and they differ in their response, so the choice of a good general promoter is important. Another uncertainty was whether there would be many spurious enhancers in the genome - regions that drove tissue-specific expression of p-gal even though there was no nearby gene with a similar pattern. In fact, in most cases so far identified, a gene with the expected expression pattern has been found near the insertion. A third consideration was the numbers involved: how many enhancer trap insertions would show specific expression patterns? This, too, turned out better than most people would have dared to hope. For example, Bellen et al. [9] found that about 65% of lines were expressed in tissue- specific patterns in the embryo; Bier et al. [IO] found that about 35% of their lines were expressed in the embryonic nervous system, and we have found that 5-10% of lines are specifically expressed behind the morphogenetic furrow in the developing eye imaginal disc (Fig. 2) (MF, U Gaul, JS Heilig, IS Higgins, M Mlodzik and GM Ru- bin, unpublished observations; see [19] for explanation of eye development). This surprisingly high frequency of tissue-specific expression patterns may reflect a number of different features [ 111: the Drosophila genome is rather compact, with the average spacing of genes being between 10 and 30 kilobases (kb); enhancers can act at long distances, so the insertion can be quite far from the enhancer and still be influenced by it; P-elements seem to show some preference for inserting near the 5’ ends of genes, and they don’t usually insert within heterochro- matin. It.is also surprising that there are not more ubiq- uitously expressed insertions. The reason for this is not clear, but it may represent some specificity of insertion site inherent in P-elements. It is also possible that constitutive genes do not use the same kind of enhancer as genes with more complex regulation.

Only about 10% of Drosophih enhancer trap lines contain insertions that are mutagenic themselves, but a feature of P-element transposition can be exploited to produce small deletions around the insertion site, with the aim of deleting the nearby gene of interest. In this man ner, the phenotype caused by the removal of the newly identified gene can be assessed, and all the power of genetics - with its ability to elucidate gene functions and identify interacting genes - can be used to characterize the gene. This approach is possible because the enhancer trap element carries a dominant eye-colour marker, whose loss can be easily scored, and because P- elements often take a small amount of flanking genomic DNA with them when they jump from a locus. Thus, an excision screen can be carried out on a Drosophila line that already contains an enhancer trap transposon. The

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element is mobilized by crossing this line with flies that express the transposase, and resulting lines are scored for the absence of the marker gene. Some of those lines that have lost the 8enhancer trap transposon will have small deletions around the former insertion site, and can then be tested for lethality, or any other phenotype that may be associated with the excision of the nearby gene. Potential deletions of the gene are confirmed by Southern blot analysis.

Second generation enhancer traps TO exploit the basic enhancer trap principle even further, ‘second generation’ enhancer trap systems have been de- veloped (A Brand and N Perrimon, personal communication). In these, the lp-gal reporter gene is replaced by the gene encoding the yeast transcription factor, GAL4 This transcription factor can function in Drosophila, where it only activates the expression of genes that have the GAL4 binding site, known as the upstream activating site (MS), linked to their promoters [20]. These second generation enhancer traps allow one to express any cloned marker gene in the cells in which a particular GAL4 enhancer trap is active. This is achieved by transforming flies with the marker gene of interest, positioned downstream from a UAS, and mating these transformants with the GAL4 enhancer trap line. Tlhe progeny of these crosses will con- tam both the GAL4 enhancer trap and the UAS-marker gene, and will there:fore express the marker in all the cells in which the GAL4 enhancer trap is active. The advantage ,of this system is that different markers can be tested in a single GAL4 enhancer trap line: for example, it might be useful to cross a cell-surface marker into an enhancer trap line that expresses GAL4 in neurons, thereby allow- ing the axonal processes to be traced. An increasing number of suitable markers are becoming available, including efficient markers for the nucleus, the cytoplasm, the cell surface, and axonatl processes. This range allows precise characterization of the position and morphology of cells of many kinds. Furthermore, the gene attached to the UAS need not simply encode a marker. By linking a UAS to a gene involved in development, the consequences of ectopically expressing that gene in the cells in which the enhancer trap is active can be assayed.

One of the most powerful uses of this feature will be the ability to ablate specific cells by expressing cell- autonomous toxin genes under the control of a UAS. As more and more enhancer traps expressing GAL4 become available, the range of specific ablations possible wiU also increase. The technology for this kind of ablation already exists, using the toxic subunits of diphtheria toxin and ricin, without the membrane transport subunits ([21] ; A Brand, J Haseloff, N Perrimon, H Goodman, personal communication; C O’Kane, personal communication).

Enhancer traps in the nervous system One of the most challenging problems in biology is the elucidation of the: mechanisms that underlie the development and function of the nervous system. Even in Drosophila, very :kttle is yet known about the nervous system, especially the CNS. The standard mutational approach simply has not produced the required number

of relevant phenotypes - presumably for the reasons discussed above: pleiotropy, complexity and redundancy. Enhancer traps might be a practicable way to approach this difficult problem, and many groups are now isolating enhancer trap lines expressed in the nervous system (for example [22,23] >. Even before genes are cloned, enhancer traps can provide some of the most useful tools of a developmental biologist: cell markers. By isolating lines in which the reporter gene is expressed in subsets of cells in, for example, the developing brain, it will be possible to distinguish morphologically similar cells, and to follow their fates during development. This will help enormously in the characterization of the nervous system. Furthermore, enhancer traps can also be used to study nervous system function. When it is possible to identify small groups of cells in the nervous system, and then ablate them; and when genes Specific for those cells can be mutated, one has some powerful ways of analysing how the nervous system works.

Fig. 2. The P-gal expression pattern in an ‘enhancer trap’ line. An eye imaginal disc from a Drosophila larva. In this line the enhancer trap detects a gene expressed transiently in the developing photoreceptor neurons in the eye imaginal disc. Its expression is ‘limited to posterior of the morphogenetic furrow.

Enhancer trapping in other organisms It is clear that enhancer traps are an important new technique available to those working on Drosophila, but one of the exciting features about them is that they should be adaptable, at least in part, to other organisms. No other organism has the same combination of sophisticated genetics, a well characterized transposable element system that can be regulated, and easy germline transformation. But the only absolute requirement for an enhancer trap system is the ability to transform foreign DNA into the germline and have it stably expressed. There are many organisms in which that is now feasible. Indeed, several enhancer trap (and related ‘gene trap’) lines have been reported in mice (for example [2&26]). AS it may soon be possible to mutate mouse genes at will, by homolo- gous recombination, the ability to identify developmen-

380 @ 1991 Current Biology

tally regulated genes by means of enhancer traps may be very useful. In the nematode, Caenorbabditk elegam, an approach has been taken in which random genomic sequences have been cloned upstream of a reporter gene, and transformed back into the worms: several tissue-specific patterns have thus been identified [27]. One of the newly favoured organisms for developmental biologists is the zebra&h. It has been shown that it is possible to inte- grate DNA into the zebrafish germline [28], so enhancer traps may soon be available. An enhancer trap system has also been recently reported in plants [29].

In conclusion, enhancer trapping is an important new tool available to biologists studying a broad range of questions about development. For example, it may at last provide a way to begin to understand the development and function of the brain at the molecular and genetic level. Although Drosqfh&z offers a unique set of features that make enhancer trapping easy and eflicient, one of the bonuses of the technique is that it may provide a valuable way of identifying and analysing important developmental genes in a variety of other organisms - even those that are genetically less amenable than the fly.

Acknowt’edgemenb I thank Cahir O’Kane and David BowteE for use fuI advice about enhancer traps, Uhike Gaul and Rose Taylor for ctiti- caIly reading this manuscript, and Gerry R&m, in whose laboratory I have worked on enhancer trapping.

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Matthew Freeman, Howard Hughes Medical Institute, De- partment of Molecular and Cell Biology, University of Cal- ifornia, Berkeley, California 94720, USA (From March 1st 1992: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, UK.)

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first, trap your enhancer

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