Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.
As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.
Hydra has been used to study regeneration, pattern formation, and stem cells for approximately 250 years2. Hydra has a simple body plan consisting of three cell lineages: ectodermal epithelial, endodermal epithelial, and interstitial. The tubular body is formed by the ectodermal and endodermal epithelial lineages, each of which is a single cell layer. All of the epithelial cells in the body column are mitotic. When epithelial cells are displaced into the extremities3, the head (mouth and tentacles) at the oral end or the foot (basal disc) at the aboral end, they arrest in the G2 phase of the cell cycle and change cell fate4. The cells of the interstitial lineage reside within the interstices between the epithelial cells. This lineage is supported by multipotent stem cells that are located in the ectodermal epithelial layer of the body column5. The interstitial stem cells give rise to three somatic cell types (nerves, gland cells, and nematocytes) and the germ cells6,7.
As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Until recently, these efforts were impeded by the lack of reliable methods for the perturbation of gene function. However, with the development of transgenic methodology1, we are now able to take full advantage of Hydra to gain a better understanding of the basic mechanisms common to multicellular animals, such as stem cell function, regeneration, and patterning. Transgenic Hydra lines are established by injection of plasmid DNA into embryos, which results in random integration and chimeric expression in a substantial frequency of hatchlings. A line with uniform expression in a particular lineage can be established by asexual propagation. The ability to clonally propagate transgenic Hydra lines is an advantage over the majority of animal models, which can be propagated only by sexual reproduction. In addition, transgenic cells can be tracked easily in vivo due to the transparency of the animal and the absence of endogenous fluorescent proteins 8.
In the seven years since the first transgenic Hydra lines were made1, such lines have been used for a variety of applications. Expression of fluorescent proteins in different cell types has made it possible to track cell movement, observe changes in cell shape, and track cell fates both in wild type conditions and after chemical perturbation1,5,9-12. In addition, expression of different fluorescent proteins in the various lineages allows for FACS isolation of specific cell populations. This technique has been used for the sequencing of stem-cell specific mRNAs and lineage-specific small RNAs13,14. While the promoter of one of the two Hydra actin genes has been most widely used, a few cell-type specific promoters have been identified and used to drive expression of GFP in transgenic Hydra9,11,15,16. In the future, cell-type specific promoters will allow for the observation and collection of any specific cell type. In addition, a transgenic approach was successfully used to define the cis-acting regulatory elements of the Wnt3 promoter17.
The development of transgenic methods in Hydra provides a robust approach for testing the function of genes by ectopic expression, overexpression, and knockdown. Transgenic animals have been made that express fluorescently-tagged proteins in order to examine both function and cellular localization18-20. In addition, the expression of RNA hairpins in the 3’UTR of a GFP transgene leads to knockdown of target genes21,22. In these approaches GFP is required to identify and track the transgenic tissue during the creation of the transgenic line. However, it is likely that in some cases the GFP molecule would interfere with the function of the tagged protein. A recent study demonstrates that Hydra genes can be arranged in an operon configuration, i.e., polycistronic transcripts are made, which are then separated by trans-spliced leader addition and translated separately23. By placing a gene encoding a protein or an RNA hairpin in the upstream position of an operon and a fluorescent protein gene in the downstream position, one can track transgenic tissue without having to tag the gene encoding the protein or RNA hairpin. This method has been used to express an RNA hairpin in an operon configuration with DsRed2 in order to achieve gene knockdown14.
1. Preparation of Plasmid DNA, Needles, and Injection Dishes
2. Preparation of Embryos for Injection
3. Microinjection of Plasmid DNA
4. Culturing and Hatching of Embryos
Establishing transgenic Hydra lines
Feed transgenic hatchlings every 2-3 days with Artemia nauplii. Hatchlings sometimes do not eat for a day or two after hatching. Some new hatchlings will never eat, and thus will not survive. If the hatchling is transgenic in either the ectodermal (Figure 1A) or endodermal epithelial tissue, allow the animal to bud and collect the buds that have the most transgenic tissue (Figure 1B, C). Continue to do this with the new buds until a transgenic line is established with uniform expression of the transgene in either the ectodermal (Figure 1D) or endodermal epithelial lineage. Simultaneously, a second line can be established that does not contain the transgene but is otherwise genetically identical (Figure 1B). This line serves as a negative control for future experiments. If the transgenic tissue does not move into a bud, it is sometimes possible to cut the animal and allow it to regenerate such that the transgenic tissue will be in the new budding zone. However, if the transgenic tissue is too close to the extremities it will likely be lost as it is displaced during the normal growth of the animal. Often there is nothing that can be done in this case. In our hands, if the transgene is neutral (i.e., has no impact on biological function) we are able to establish a uniform epithelial line from approximately 30% of Hydra that hatch with epithelial transgenic tissue. The remaining 70% either die or the transgenic tissue is lost. An endodermal line is approximately 2-3 times as common as an ectodermal line. If a transgene is being used that disrupts biological function, this may have an impact on the feasibility of establishing a uniform line. In such cases, inducible promoters will be essential additions to the toolkit.
If the hatchling is transgenic in the interstitial lineage, allow the animal to bud and collect the hatchlings with an increasing number of transgenic cells in the interstitial lineage. Be aware that sometimes it is not immediately clear that an animal is transgenic in the interstitial lineage, especially if it is also transgenic in an epithelial lineage. It is highly unlikely that a line completely transgenic in the interstitial lineage will be established simply by budding. If it is required that the interstitial lineage be fully transgenic, this can be accomplished by cloning in aggregates from interstitial stem cell-depleted animals such that the entire lineage is established from a single transgenic stem cell7.
In cases where a tissue- or cell type-specific promoter is used to drive expression of a fluorescent protein, transgenic animals may not be evident initially. For example if a promoter that is active only in the tentacles is used and the initial patch of transgenic tissue is in the body column, no fluorescent cells will be seen in the hatchling. So all hatchlings need to be propagated to allow transgenic tissue to be displaced into the tentacles and become evident.
Figure 1. Establishing a transgenic line with uniform ectodermal epithelial expression of DsRed2. (A) A hatchling with chimeric expression of a DsRed2 transgene under control of an actin promoter in a patch of ectodermal epithelial cells. (B) A first-generation bud from the hatchling in panel A is now producing two new buds. The bud labeled with an asterisk has no transgenic tissue and was used as the founding animal for a control line that is genetically identical to the transgenic line, except for the presence of the transgene. (C) A second-generation bud produced from the Hydra in panel B. (D) An example of a polyp from the transgenic line that was established with DsRed2 expression throughout the ectoderm. Please click here to view a larger version of this figure.
Hydra routinely reproduces asexually, but requires environmental stimuli to begin producing gametes. These stimuli are not well-defined for most Hydra species and may differ from strain to strain. A significant hurdle to the production of transgenic Hydra is obtaining embryos on a regular basis because it can be difficult in a laboratory setting to induce Hydra to become sexual. The AEP strain25, however, produces gametes readily in the laboratory and this is the only strain that has been used so far for making transgenic lines. The most common method for inducing gamete production is by diet manipulation. As previously described, the animals should be fed daily for three weeks, starved for 5 days, and then fed twice a week, during which time gametes will be produced1. It has also been our experience that culturing AEP Hydra on vertical plates in an aquarium26, perhaps simulating a more natural environment, leads to egg production even when animals are fed at regular intervals. In addition, lines obtained from AEP self-crosses sometimes produce gametes more regularly than the parent strain. Thus establishing an F1 line of AEP is another possible method for obtaining a more reliable source of embryos.
The percentage of embryos that survive injection and develop to the cuticle stage depends on the health of the embryos, the amount of solution injected, and the amount of damage done by the injection. With the constant flow injection set-up described in this protocol, it is difficult to control the amount of solution that is injected into the embryo. In our hands, approximately half of the embryos injected as described here successfully complete embryogenesis and form a cuticle. Of these, 50-75% hatch and of those that hatch, approximately 50% will have at least some transgenic tissue. Therefore, 10-20% of the initially injected embryos yield an F1 Hydra with transgenic tissue. The amount of solution injected can be precisely controlled with more expensive equipment such as the IM-300 microinjector. The original transgenic lines were made with equipment from Eppendorf, which allows a great deal of precision in manipulating and injecting the embryo. While this level of accuracy may give a higher survival rate of injected embryos, this expense is not necessary for routine establishment of transgenic lines.
The integration of the transgene is random and could potentially occur in multiple locations in the genome or in a tandem array in a single location. Based on Southern blot analysis, the number of integrations was estimated at five in one epithelial transgenic line created previously1. In an interstitial lineage transgenic line in which the transgene undergoes germline transmission, it has been demonstrated by genome sequencing that only a single copy of the transgene was integrated (C. E. Dana and R. E. Steele, unpublished observation). However, aside from these two examples, there is no information available regarding transgene integration sites and copy number. Since it is possible that a transgene could interrupt gene function by insertional mutagenesis, more than one transgenic line should be made when analyzing phenotypes from, for example, RNAi or overexpression constructs. It is also important to note that transgene expression is constitutive when driven by the commonly used actin gene promoter, and thus transgenes that lead to serious or lethal phenotypes when expressed constitutively will not be maintained. For the future, it will be important to develop an inducible system of transgene expression to circumvent this problem.
The authors have nothing to disclose.
This work was supported by a G. Harold & Leila Y. Mathers Award to H.L. and an NIH grant (R24 GM080527) to R.E.S. C.E.J. was an NRSA Postdoctoral Fellow (NIH F32GM9037222) and is currently supported by a Mentored Research Scientist Development Award from the National Institute on Aging (K01AG04435). We would like to thank the reviewers for helpful comments.
AEP Hydra Strain | NA | NA | Email: Celina Juliano at celina.juliano@yale.edu or Hiroshi Shimizu bubuchin2006@yahoo.co.jp |
High Speed Maxi Kit | Qiagen | 12662 | |
100 x 15 mm Petri Dishes | BD Falcon | 351029 | |
75 x 50 mm Glass Microscope Slide | Sigma | CLS294775X50 | |
Microinjection Fish Mold | IBI Scientific | FM-600 | |
Borosilicate Glass with Filament | Sutter Instrument | BF100-50-10 | O.D.: 1.0 mm, I.D.: 0.50 mm, 10 cm length |
Flaming/Brown Micropipette Puller | Sutter Instrument | P-97 | |
Phenol Red | Sigma | P3532 | |
Jewelers Forceps, Durmont #5 | Sigma | F6521 | |
Scalpel Blade #15 | Fisher | 50-822-421 | |
Mineral oil | Sigma | M8410-500ML | |
Microinjector | Narishige | IM-9B | |
Magnetic Stand | Narishige | GJ-1 | |
Iron Plate | Narishige | IP | |
Joystick Micromanipulator | Narishige | MN-151 |