We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.
Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.
Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.
All experimental procedures were approved by the University of Virginia Institutional Animal Care and Use Committee.
Note: Figure 1 shows a schematic overview of the experimental procedures.
1. Preparation of Oocytes
2. Injection of Library Transcripts
3. Animal Cap Assay
4. Analysis of Response in Ectoderm by In situ Hybridization (ISH)
5. Sib Selection and Cloning
In response to expression of mRNA injected into oocytes, responding animal cap tissue was assayed for expression of otx2 by in situ hybridization (Figure 2 and Table 1); otx2 is expressed in the presumptive lens ectoderm (PLE) from neural tube closure through lens placode thickening19. However, since otx2 is also expressed in the anterior neural ectoderm as well as non-neural head ectoderm outside the PLE, it is associated with both neural and placodal responses. The use of foxe3 to screen the library for a gene product capable of producing a lens-inductive response in lens-competent animal cap ectoderm allowed a more specific approach to the goal of the expression cloning, since foxe3 is expressed in the PLE from neural plate stages and throughout lens vesicle formation20, present in adjacent placodal regions but absent from neuroectoderm. Using the expression cloning and sib selection protocol above and injecting pools of library transcripts, a gene capable of producing foxe3 expression in the animal caps was isolated (Table 2). Following isolation of the clone, 179 additional animal cap assays using oocytes injected with library transcripts were screened for expression of foxe3; 50 were positive (28%). Of 140 animal cap pieces placed on uninjected oocytes, 0 were positive (Figure 3).
Figure 1. Oocyte-animal Cap Assay and Expression Cloning. Schematic overview of the protocol: transcripts are prepared from the clone library and injected into oocytes, animal cap ectoderm is cultured with oocytes and then assayed for induced gene expression by in situ hybridization. Please click here to view a larger version of this figure.
Figure 2. Typical Results of Animal Cap Assay following mRNA Injection and in situ Hybridization for Otx2. (A-B) Representative result of inductive response to dorsalized stage 14 poly(A)+ RNA in animal caps, assayed for otx2 expression by whole mount in situ hybridization. (A) Animal caps placed on uninjected oocytes at stage 10.5 and cultured to stage 25. (B) Stage 10.5 animal caps placed on oocytes injected with 10 ng RNA and cultured to stage 25. otx2 expression observed in 6/7 cases and indicated by arrowheads. Bar = 500 μm. Please click here to view a larger version of this figure.
Figure 3. Typical Results of Animal Cap Assay following in situ Hybridization for Foxe3. (A-C) Representative animal caps from oocyte-animal cap assays, tested for expression of foxe3 by in situ hybridization. (A) Stage 11-11.5 animal caps placed on ldb1-injected oocytes and cultured to stage 23. foxe3 expression is indicated by arrowheads. (B) Stage 11-11.5 animal caps placed on uninjected oocytes and cultured to stage 23; no foxe3 expression detected. (C) Section through foxe3-positive induced animal cap from A showing expression in inner and outer layers of ectoderm. Bars = 500 μm. Please click here to view a larger version of this figure.
Injected RNA | Positive cases | Gene | % |
Stage 14 dorsalized mRNA, 10 ng | 12/24 | otx2 | 50 |
Library pools of 105 clones, 20 ng | 3/9 | otx2 | 33 |
Library pools of 105 to 100 clones, 20 ng | 50/179 | foxe3 | 28 |
None | 0/140 | foxe3 | 0 |
Table 1. Oocyte-Animal Cap Assay Results. Results of animal cap assay assessed by in situ hybridization with otx2 and foxe3, using oocytes injected with mRNA or with transcripts synthesized from cDNA library pools; or uninjected oocytes.
Injected RNA | Pool designation/selection | Positive foxe3 expression |
Library pools of 105 clones, 20 ng | A | 2/4 |
B* | 4/28 | |
C | 4/44 | |
Library pools of 104 clones, 20 ng | 1 | 0/11 |
2 | 0/10 | |
3 | 3/14 | |
4 | 0/12 | |
5 | 0/15 | |
6 | 1/20 | |
7 | 3/23 | |
8 | 0/16 | |
9 | 0/16 | |
10* | 5/20 | |
Library pools of 5,000 clones, 20 ng | 1 | 0/7 |
2* | 5/16 | |
3 | 1/7 | |
4 | 0/7 | |
5 | 0/7 | |
Library pools of 400 clones, 20 ng | 1 | 0/10 |
2 | 0/10 | |
3 | 0/10 | |
4 | 0/10 | |
5* | 8/26 | |
6 | 0/11 | |
7 | 0/10 | |
8 | 0/10 | |
9 | 0/10 | |
10 | 0/8 | |
Library pools of 70-200 colonies, 20 ng | 1 | 0/8 |
2 | 0/10 | |
3* | 1/10 | |
4* | 1/10 | |
5 | 0/10 | |
6 | 0/9 | |
7 | 0/10 | |
8 | 0/10 | |
Library pools of 20 colonies, 20 ng | 1 | 0/10 |
2 | 0/10 | |
3 | 0/10 | |
4* | 7/21 | |
5 | 0/10 | |
6 | 0/10 | |
7 | 0/10 | |
8 | 0/10 | |
9 | 0/10 | |
10 | 0/10 | |
Library pools of 6 – 7 colonies, 20 ng | K2-6, L1 | 0/10 |
L2-6, M7 | 0/10 | |
M8-12, N7-8 | 0/10 | |
K5-6, L1-4 | 1/10 | |
L5-6, M7-10 | 0/10 | |
M11-12, N7-8, K2-4 | 0/10 | |
K2, K5, L2, L5, M8, M11 | 0/10 | |
K3, K6, L3, L6, M9, M12 | 0/9 | |
K4, L1, L4, M7, M10, N7, N8 | 1/9 | |
Library RNAs, 20 ng | K6 | 0/10 |
L1* | 3/10 | |
L3 | 0/10 | |
L4 | 0/10 | |
Library RNA, 20 ng | L1 (ldb1) confirmation | 50/179 |
* indicates selected pool |
Table 2. Sib Selection and Expression Cloning Results. The pool with the highest level of response in each animal cap assay (ranging between 10% and 36% positive for foxe3 expression) was selected for use in the next experiment to narrow down activity to one clone. Asterisk indicates selected pool.
The method described here for the functional cloning of genes capable of inducing a response in competent ectoderm can be used to identify a wide range of gene products. This method expands upon past work by combining tissue-inducing assays with expression cloning techniques. We utilize the metabolic pathways of the Xenopus oocyte as a source of production of inducing factors, directly or indirectly, following RNA injection. This, in combination with the use of established methods for cloning a gene of interest6,7 using expression of transcripts generated from a cDNA library or other collection of clones, provides a valuable approach for those seeking to identify genes of new interest involved in embryonic induction. The broad applicability of this method to functional identification of new genes is a useful complement to exciting new reverse genetic approaches, and could also be used to functionally test transcripts identified using high-throughput methods (such as RNA-seq)21.
Controls are critical in monitoring the metabolic function of oocytes used in the cap assay. Both to ensure the health of each batch of oocytes and to establish the usefulness of this system to detect low-abundance transcripts, varying concentrations of mRNA of the mesoderm inducer INHBB were tested; this gene is unrelated to the lens-induction pathway and is a well-documented independent control14. Muscle-inducing activity, assayed by the expression of muscle-specific antigens in animal cap ectoderm with the 12/101 antibody, was observed with 2 – 200 pg INHBB mRNA, even in the presence of 500-fold excess stage 14 poly(A)+ RNA. The system is thus useful over a very wide range of injected RNA quantity, and oocytes are able to stably produce protein and remain viable following cytoplasmic injection of volumes of 50 nl and higher.
One consideration for the use of a cDNA library in this protocol is the necessity for full-length or nearly full-length clones. Prior to use in the protocol, the library should be examined by Northern analysis to determine the size of known transcripts in the library and therefore reduce the possibility that dominant-negative effects are obtained from potentially truncated injected transcripts. Since a full-length cDNA pool is important, the use of EST clones22 or optimally, the Xenopus ORFeome23 (which provides a complete set of validated full-length clones) are preferred to a traditional cDNA library unless a stage- and tissue-specific source of cDNA are sought and used for the library construction. Another consideration is the presence of β-globin and poly(A) sequences in the library vector intended to augment mRNA stability in injected transcripts. While this is desirable to increase the quantity of protein produced from injected synthetic mRNA, it also raises the possibility of producing a dominant-negative effect from mRNA that is higher in stability and activity than in vivo. A low-copy number mRNA may not exert the same effects as its endogenous counterpart in the background of a large pool of transcripts; one that is stabilized in the cloning and transcription process may persist to allow detection in the cap assay. A third issue is pool size; considerable reduction of pool size (10 clones or fewer) has been demonstrated to be advantageous in recent gain-of-function screening projects in embryos24 and is likely to provide clearer results and easier identification of candidate genes. A smaller pool size than the 103 – 104 recommended in this protocol is achievable if the total complexity of the collection of clones is less than 104, as is the case with the ORFeome23; one could screen the ~9,000 clones beginning with 10 pools of 900, though it may be prohibitively labor-intensive to process more than 10 pools in an experiment.
Determination of the type of gene product (by sequence analysis) made by the gene identified in the screen determines the nature of tests of its function. Since a nuclear transcriptional cofactor was identified in our screen8, it was necessary to confirm the role of the nucleus in the inductive process triggered by introduction of ldb1. Enucleated oocytes have fully functioning protein synthetic machinery and are viable and capable of producing secreted factors from injected transcripts. However, the absence of the nucleus will abolish functioning of transcription factors, cofactors, or other indirectly-acting gene products. Subsequent analyses of genes identified in the screen include determination of developmental expression pattern by in situ hybridization and gain-of-function and loss-of-function tests. Overexpression by injection of RNA into zygotes or by limiting injection to specific blastomeres to restrict its effects to particular regions of the embryo can provide insights, as well as knockdown of gene function through injection of morpholino oligonucleotides directed against the in vivo transcript.
Direct visualization of an inductive effect in the responding animal cap ectoderm using a transgenic line with a reporter gene (such as GFP) may greatly expedite the screening process and eliminate the need for the analysis of RNA expression by in situ hybridization. Similarly, the use of antibodies as quicker means of screening is desirable if an appropriate antibody (such as the 12/101 for muscle response discussed above) is available. Albino embryos may be used for the animal caps, which although more difficult to stage accurately at gastrula stages, streamline the in situ hybridization process by eliminating the need for any bleaching of wild-type pigmentation to better visualize the color reaction product.
Finally, it is important to consider that for several reasons, a large number of trials may need to be conducted to identify a given gene. For one, the number of cases one may reasonably process in a given trial is limited by the development (and eventual loss of competence) that occurs over time even given a large batch of embryos and a range of temperatures at which to culture them, as well as the loss that may occur of small pieces of ectoderm in processing. For another, success rates of expression of a chosen marker in the ectoderm may be very low and require many cases to observe a statistically significant effect.
The authors have nothing to disclose.
This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.
12/101 Antibody | Developmental Studies Hybridoma Bank | 12/101 | Monoclonal antibody for detection of muscle tissue |
20X SSC Buffer | Sigma | S6639 | for ISH |
Acetic anhydride | Sigma | A6404 | for ISH |
Anti-Dig-AP | Roche | 11093274910 | for ISH |
Aurum Plasmid Mini Kit | Bio-Rad | 732-6400 | Plasmid DNA purification |
Blocking Reagent | Roche | 11096176001 | for ISH |
BM Purple | Roche | 11442074001 | for ISH |
Boekel Hybridization Oven | Fisher Scientific | 13-245-121 | for ISH |
Bouin's Solution | Sigma | HT10132 | for ISH |
BSA | Sigma | A9647 | for OCM |
CHAPS | Sigma | C3023 | for ISH |
Collagenase A | Roche | 10103578001 | Defolliculation of oocytes |
Cysteine | Sigma | C121800 | Dejelly embryos |
DEPC-H2O | Fisher Scientific | BP5611 | for ISH |
Dig-RNA Labeling Mix | Roche | 11277073910 | for ISH probes |
Dumont #5 forceps | World Precision Instruments | 500233 | for Vitelline envelope removal |
Ethyl 3-aminobenzoate | Sigma | A5040 | MS222 anesthetic |
Ficoll PM 400 | Sigma | F4375 | for Injection media |
Formamide | Sigma | F9037 | for ISH |
Gentamicin sulfate | Sigma | G1914 | for OCM |
Glass capillaries | World Precision Instruments | 4878 | 3.5" long, I,D, 0.530mm |
Glass sample vials | Fisher Scientific | 06-408B | for ISH |
Hair loop | Hair affixed in pasteur pipette for tissue manipulation | ||
Heparin sodium salt | Sigma | H4784 | for ISH |
Injector Nanoliter 2010 | World Precision Instruments | Nanoliter 2010 | Microprocessor-controlled microinjector |
Instant Ocean | Carolina | 972433 | Aquarium Salt for frog recovery |
IRBG XGC Xenopus verified full-length cam cDNA | Source Bioscience | 989_IRBG | cDNA library |
LB Agar plates with 100 µg/mL Ampicillin | Teknova | L5004 | 150mm pre-poured LB-Amp plates for sib selection |
LB Luria Broth | Teknova | L8650 | LB for collecting colonies in sib selection from plates and dilution of cultures |
Magnetic mRNA Isolation Kit | New England BioLabs | S1550S | for isolation of poly(A)-enriched RNA |
Maleic Acid | Sigma | M0375 | for ISH |
Manual Microfil Micromanipulator | World Precision Instruments | M3310R | Manual micromanipulator |
Nutating Mixer | Fisher Scientific | 22-363-152 | Rocker for ISH |
Permoplast | Nasco | SB33495M | Clay for injection and dissection dishes |
Phosphate Buffered Saline | Sigma | P5368 | for ISH |
PMSG | Sigma | G4877 | to stimulate oocyte development |
Polyvinylpyrrolidone | Sigma | PVP40 | for ISH |
Programmable Puller | World Precision Instruments | PUL-1000 | Micropipette needle puller |
Proteinase K | Sigma | P6556 | for ISH |
pTnT Vector | Promega | L5610 | cDNA library construction |
Riboprobe Combination System | Promega | P1450 | in vitro transcription |
Superscript Full Length cDNA Library Construction Kit | Life Technologies | 18248013 | kit for cDNA library construction |
Sutures, 3-0 silk | Fisher Scientific | 19-037-516 | Suture thread and needle for post-oocyte removal |
Torula RNA | Sigma | R3629 | for ISH |
Triethanolamine | Sigma | T1502 | for ISH |
Tween 20 | Sigma | P9416 | for ISH |
Universal RiboClone cDNA Synthesis System | Promega | C4360 | alternative kit for cDNA library construction |
Xenopus Full ORF Entry Clones – ORFeome Collaboration | Source Bioscience | 5055_XenORFeome | ORFeome Clones |
XL2-Blue Ultracompetent Cells | Agilent Technologies | 200150 | cells for transformation of cDNA library |