Specific aim #2.
To generate stable transgenic S. tropicalis lines with the goal of streamlining current embryological procedures or creating novel experimental paradigms, specifically
A. to facilitate preparation of genetic mosaics and supplant laborious gene expression assays with rapid in vivo reporter detection, by establishing transgenic reporter lines tissue-specifically expressing Green Fluorescent Protein (GFP) driven by characterized X. laevis regulatory elements (brachyury, cardiac actin, N-tubulin, and goosecoid promoters) with useful domains of expression;
B. to simplify tissue labeling for transplantation, by establishing transgenic lines expressing GFP ubiquitously using the cytoskeletal actin and cytomegalovirus promoters
C. to exert temporal control expression over transgenic gene function, by assaying inducible systems
D. to combine transgenesis with transplantation techniques in order to implement genetic mosaic assays
Rationale:
Stable transgenic lines expressing GFP, a reporter marker which can be assayed in vivo, have the potential to transform embryological research, where much of the labor is currently invested in assaying gene expression. Conventional detection of specific gene expression in whole mount assays (e.g. in situ hybridization, immunocytochemistry) takes several days, considerable labor, and expensive reagents. GFP can be detected instantly in living embryos or explants using fluorescence microscopy.
Transgenic reporter constructs in X. laevis have been shown to faithfully reproduce the endogenous pattern of expression of several promoters1, 2 (see Preliminary Results). Using the short-generation S. tropicalis instead of X. laevis facilitates breeding transgenic founders to generate populations of animals with identical reporter integrations, which should give more consistent reporter patterns for experimental analysis.
A panel of readily-assayable stable transgenic lines will also be invaluable as substrates for chemical mutagenesis (see specific aim #4). Identifying mutations in particular processes, such as eye development and axial patterning, will be much more feasible if workers can visually screen embryos for abnormalities in easily recognizable patterns of relevant gene regulation. The promoters that we propose to use have all been characterized either by plasmid injection or transgenic assays in X. laevis, and are likely to give appropriate and useful non-mosaic tissue-specific expression in S. tropicalis 1, 89. Goosecoid (gsc) is expressed in Spemann's organizer, notochord, and head mesoderm; N-tubulin is a marker for differentiating neurons; the brachyury (Xbra) elements which have been characterized reproduce the ventral mesoderm component of expression; cardiac actin is expressed in developing heart and somites.
Finally, analysis of targeted mouse mutants is often complicated by early embryonic lethality (precluding study of later phenotypes), or by pleiotropy (obscuring primary roles for gene products in particular processes). In other cases, dominance or haploinsufficiency makes propagation of mutants difficult. Even when it is possible to carry mutants as heterozygotes, only 1/4 of embryos from a cross of heterozgyotes will be homozygous; it is frequently neccessary to begin complex manipulations before homozygotes can be phenotypically identified, leading to much wasted effort. Simultaneous analysis of multiple recessive lethal alleles is even more impractical for similar reasons: only one out of sixteen embryos from a cross of two double heterozygotes will be a double homozygote. Such analysis is possible in Drosophila because of the extensive genetic tools available, such as visible markers and balancers. In some cases, the embryological tools available in amphibians may facilitate similar genetic analyses. A set of manipulations, described in section D below, are feasible with the S. tropicalis system should allow us to circumvent these problems in many cases, and underline the potential usefulness of an amphibian genetic model system.
Methods A.
Tissue-specific promoters:
S. tropicalis sperm nuclei will be incubated with DNA constructs fusing previously characterized functional gsc, xbra, cardiac actin, and N-tubulin regulatory regions with a highly-fluorescent mutant of GFP90 followed by an SV40 polyadenylation sequence (SV40pA), and injected into unfertilized eggs according to the transgenic procedure (see Preliminary Data 1). Embryos will be assayed by fluorescence microscopy following the onset of expression of the cognate gene, and embryos displaying tissue-specific reporter patterns comparable to in situ hybridization signals will be cultured to maturity. Populations of marked animals will be generated either by using transgenic females to make gynogenetic diploids, creating a strain bearing identical integrations, or conventionally inbred to fix reporter loci.
Preliminary evidence indicates that occasional transgenic embryos display unexpected tissue-specific expression patterns, possibly through an "enhancer trap" mechanism in which transgenes integrate near cis-acting regulatory regions powerful enough to supersede regulation by transgene promoter sequences (see Preliminary Results, Plate Fig. 6E-G). If stable, these rare events may also provide useful tissue-specific reporter lines.
B. Ubiquitous promoters:
The cytomegalovirus promoter gives high-level expression in plasmid injection and transient transgenic assays, but it is not known whether it will undergo methylation repression in the germ line as do many other viral promoters. Vector construction and transgenesis will be as above. Lines of S. tropicalis expressing GFP under the control of these promoters would be useful in several ways: 1) to unambiguously identify specific tissues for transplantation early in development; 2) to identify changes in cell fate as a result of tissue rearrangements or local expression of genes from injected RNAs; and 3) as easily observed markers to identify mutants of leading edge mesodermal cell patterning or migration (goosecoid), axial and paraxial defects in patterning, convergent extension, and differentiation (brachyury, cardiac actin), and neural patterning and morphogenesis (N-tubulin).
potential pitfalls:
Two complications frequently arise in transgenic analysis of vertebrate cis-acting regulatory sequences. One is failure of the reporter to faithfully reproduce the endogenous gene's expression pattern. The regulatory elements that we are proposing to employ have all been characterized in some assays, but may not contain all the elements neccessary to function in stable transgenics. It may be neccessary to clone additional genomic sequences (see specific aim #3 for genomic library construction and screening) and assay gene fusions bearing more candidate regulatory sequences. A second complication is 'position effect': the genomic site of integration often affects reporter gene expression, with the same cis-acting sequences resulting in different subsets of the 'correct' expression pattern. Promoter analysis in transgenic mice therefore can involve making a series of stable lines for each construct being tested, at considerable expense, in order to assay cis-acting sequences in different integration contexts. This should not be a significant barrier in the current proposal for two reasons: first, it is relatively easy and inexpensive to generate sufficient numbers of transgenic embryos stably bearing a series of constructs; second, the transgenic protocol typically results in integration at multiple sites in a single embryo, so the overall expression pattern is a sum of the different integration sites. Nevertheless, we have begun to investigate one method to consistently incorporate DNA into the same locus using the cre-lox site-specific recombination systems in isolated sperm nuclei91. If successful, this would not only solve position-effect and copy number problems, but might provide more efficient integration and less ancillary chromosomal damage than the current protocol.
Previously characterized X. laevis promoters may not function in S. tropicalis or behave as appropriately in the transgenic assay as they do in plasmid injection assays. In this case, flanking sequences from S. tropicalis genomic libraries (see specific aim #3) could be cloned and used to reconstruct reporter vectors.
C. Inducible systems:
Reliable inducible expression systems circumvent lethality and pleiotropy of early ubiquitous expression of some gene products. These are common problems in mRNA injection assays, but are also likely to complicate transgenic assays for gene function, at least until a suitable 'toolbox' of tissue-specific promoters is assembled. Inducible systems would be particularly useful when combined with transplantation assays: inducibility permits temporal regulation of gene expression; transplantation permits spatial control (see section D below).
We will assay one candidate inducible promoter system by generating a line of transgenic frogs bearing GFP under the control of the binary tetracycline-responsive system (Clontech Tet-on Gene Expression System). Two transcription units must be introduced: a tetracycline response element (seven repeats of the E. coli tet operator) adjacent to a minimal cytomegalovirus promoter fused to the GFP reporter, and a constitutive promoter driving a mutated E. coli tet repressor protein fused to the herpes simplex VP16 transcriptional activator. High-level gene expression from the minimal CMV promoter should be induced only in response to the tetracycline analog doxycycline: the mutant tet repressor responds to doxycycline by binding to the tetracycline response element sites, bringing the VP16 activation domain into proximity with the otherwise silent CMV promoter. This system has been utilized in mammalian cells and in transgenic mice92, 93, and the VP16 transcriptional activation domain functions in Xenopus94,95. This combination has excellent prospects for success in transgenic frogs.
Another inducible system which has already been shown to function in X. laevis can regulate the activity of synthetic transcription factor constructs. In this system, the transcription factor being assayed is fused to the glucocorticoid or estrogen hormone receptor ligand-binding domain, which is thought to inactivate the chimaeric protein in the absence of steroids by complexing with heat shock factors and sequestering it in the cytoplasm96. Addition of dexamethasone or estrogen relaxes this inhibition, permitting transcription factor function. The utility of this system has been demonstrated by mRNA injection.
This hormone-inducible system may also be adapted as a general binary inducible system, by fusing ligand-binding domains of steroid receptors with site-specific transcriptional activators such as GAL4, creating an inducible site-specific transcriptional activator. If an appropriate target promoter, containing a GAL4 binding site (upstream activating sequence (UAS)) adjacent to a minimal promoter, is also provided, introduction of hormone allows GAL4-steroid receptor fusions to bind to the UAS and activate high-level transcription of the test gene from the minimal promoter. This system will also be assayed, using GFP as a reporter.
One concern is that these steroid receptors may respond to endogenous frog steroid hormones and "leak" low-level transcription. A very similar system avoids these problems by using a steroid hormone not found in vertebrates. Instead of glucocorticoid or estrogen receptors, the receptor for the insect hormone ecdysone is fused to transcriptional activators97. The basal level of expression in this system is very low; induction by ecdysone results in an increase in expression level of four orders of magnitude. The creation of stable transgenic line expressing inducible activity in any of these systems would greatly simplify experimental design, by making it unneccessary to add gene products to individual embryos by mRNA injection; it also makes it possible to analyze later roles for genes which cause developmental abnormalities when mis-expressed as RNA at blastomere stages.
D. Transgenesis, transplantation, and genetic mosaic analysis
Transgenesis is a recent development, and relatively few tools, such as tissue-specific or inducible promoters, are currently available. In this section, we will propose experiments that combine well-characterized embryological manipulations, such as transplantation, with multigeneration transgenic analysis using the limited available reagents, in an attempt to demonstrate the practical utility of the system. Development of additional tools for controlling gene expression will make a much wider range of experiments practical.
The tolerance of the amphibian embryo for transplantation makes it possible to create genetic mosaics by replacing a piece of tissue from an embryo of one genotype with that of another. For instance, the cells that will give rise to the germline have been fatemapped to vegetal endoderm, and it is feasible to transplant prospective germline tissue at early stages into a wildtype embryo from one carrying a mutation that will cause a lethal somatic defect. Similar transplantations have been used in the axolotl to rescue the eyeless mutation98. For many loci, wildtype gene function will not be required in the germline, and a mosaic animal, carrying a lethal allele in its germline, will be viable. Gynogenesis can then be used to ensure that all of its progeny express the mutant phenotype. Those mutant embryos can then be assayed directly, or by transplantation of specific tissues into wildtype hosts to circumvent obscuring pleiotropy or lethality.
We propose to test the utility of this approach using available reagents. Understanding the role of transcription factors like Pax-6 in eye determination has been one of our major interests. This gene has been argued to be a master regulator of eye formation99. Homozygous mutant Pax-6 mouse embryos do not develop differentiated eye tissue, but interpretation of the phenotype is complicated by its pleiotropic effect on both retina and lens ectoderm100: failure in lens formation could be directly due to the absence of Pax-6 in the lens itself, or indirectly due to effects on the retina, which is thought to produce lens-inducing signals.
To properly test this hypothesis, transcriptional activation by Pax-6 must be blocked specifically in lens ectoderm, but not retina. Activity of transcriptional activators such as Pax-6 can be blocked by creating a fusion of the DNA-binding domain of the gene being assayed (Pax-6) to the repressor domain of the Engrailed gene, generating a site-specific repressor101. Delivery of this dominant inhibitory construct to lens ectoderm could be accomplished in S. tropicalis by expressing the chimaeric Pax-6-Engrailed gene from a constitutive promoter in donor embryos, then transplanting prospective lens ectoderm to the site of lens formation in wildtype host embryos (see Preliminary Results). Using this transplantation strategy to create genetic mosaics, targeted inactivation of Pax-6 gene function in the prospective lens area should allow us to test whether the inhibition of lens formation is due to an autonomous effect of Pax-6 or not, avoiding the complicating potential retinal pleiotropy.
However, this useful experiment may suffer from another common transgenic complication: the dominant inhibitory form of Pax-6 described here is likely to be lethal in tadpoles, since homozygous Pax-6 mutant mouse pups die shortly after birth102. Lethality might be avoided by conditional expression of dominant inhibitory gene function (see section C above), but at present this technology is not perfected.
A solution to this problem involves making genetic mosaics in a way that is not possible in other vertebrates like mouse or fish. The Pax-6 gene is expressed only in ectodermal tissues72. It should be possible to rescue transgenic embryos bearing dominant inhibitory Pax-6 constructs by exchanging the 'animal cap' ectoderm of the mutant transgenic for wildtype ectoderm, leaving the mutant transgene viably carried in the endodermal germline cells. This animal cap exchange procedure results in normal development, though situs inversus does occur in a fraction of the tadpoles that develop (R.M. Grainger, unpublished;103). Such chimeric animals should develop normally, but since the mutation is present in the germ line, all of their progeny may be used as donors in the lens transplantation experiments described above to assay the tissue autonomy of Pax-6. Note that this general strategy of germline mosaicism by transplantation may simplify analysis of many lethal mutations with neural or mesodermal defects but no germline requirements; the potential to carry homozygous lethal genotypes will also simplify analysis of double mutants.
We will pursue this experimental design using stable lines of transgenic S. tropicalis and not primary transgenic X. laevis for several reasons. First, stable lines give more consistent levels of gene expression, and may be selected for optimal qualities in a given assay. Second, it may be difficult to identify the primary transgenic embryos without assaying for specific gene activity in the transplanted tissue.
We are currently evaluating the hypothesis that Pax-6 activates the early stage of the lens determination process. This proposal is also testable by transplantation experiments assaying the lens-forming ability of Pax-6-expressing ectoderm heterotopically transplanted to wildtype embryos. Lines of S. tropicalis expressing the normal Pax-6 gene in an inducible construct that is available now (see Specific Aim 2.C) could be used in a similar manner to examine the effect of controlled mis-expression of this gene.
In addition to transplantation, transgenesis with tissue-specific promoters may be used to experimentally manipulate spatial and temporal aspects of gene function. The menu of functionally characterized promoters in frogs is currently expanding (see section A and C above); one experiment using available reagents is described briefly. Experiments with dominant negative forms of the extracellular matrix protein fibrillin have suggested an important role in the morphogenetic movements associated with somite and notochord formation, including the boundary between these two tissues (Skoglund et al., 1997 in preparation). To ask whether fibrillin in somites helps form the boundary with notochord, the xbra promoter (see Preliminary Results) will be used to drive somite expression of dominant negative fibrillin constructs in transgenic embryos. Results from RNA injections (ibid) suggest that this will block convergent extension of the somitic mesoderm and perhaps that of the notochordal mesoderm as well, since the notochordal behavior seems dependent on the somitic tissue104. As in the previous example, it is likely to be helpful to use transplantation to produce viable genetic mosaic transgenics carrying the lethal dominant negative fibrillin allele in the germline.
timeframe:
creation of stable tissue-specific and ubiquitous-expressing transgenic lines 4/98; assaying inducible systems 4/98; transplantation mosaics 6/98
implementation:
The Amaya laboratory will develop new inducible systems specifically for the transgenic system; the Grainger and Keller labs will focus on using existing transgenic techniques to create stable lines for embryological experiments concerning eye development and axial patterning, respectively.