Gamma ray mutagenesis. We have completed our pilot F3 g-ray mutagenesis screen using over 120 F1 founders representing four different doses of g-ray mutagen (http://tropicalis.berkeley.edu/home/mission/RMH-Mutagenesis.html). We find that 600 rads of g-ray irradiation is optimal for generating a maximal mutation rate (1% hit rate) that still leads to fertile F1 frogs. We have confirmed 8 genetic mutations and identified another 4 potential genetic mutations (http://tropicalis.berkeley.edu/home/genetic_resources/mutants/mutants.html).

The first pilot screen turns out to have used sub-optimal rates of mutatgenesis for most of the stocks. We did discover the fairly narrow range of radiation dose that yields high rates of mutagenesis without excessive lethality. Among the mutations isolated, six of the eight confirmed mutations have been shown to be present in the genetic background of the Nigerian line, while the other two confirmed mutations may be induced. We found that the two lowest doses of mutagen used (150 and 300 rads) were ineffective in generating sufficient numbers of mutations. The highest dose of mutagen used (900 rads) resulted in an unacceptably high rate of infertility without a significant gain in mutation hit rate compared to 600 rads. As mentioned in our last report, we used three independently segregating loci to determine a specific locus mutagenesis rate. We found that a dose of 600 rads produced a mutagenesis rate of 1% at a specific locus, as measured by loss of a GFP transgene, or induction of a curly or grinch phenotype in an F1 screen. Furthermore, this dose of 600 rads does not result in high rates of infertility and has therefore been determined to be the optimal dose.

We have generated frog lines that are free from background mutations and have used these for a large g-ray mutagenesis screen. We are currently raising 1,037 F1 frogs exposed to 600 rads and will be generating F2 families in April. Based on our optimized husbandry rates (3 months to sexual maturity for males), we should begin screening F2 families this summer starting around July.

ENU mutagenesis. We are initiating ENU mutagenesis screens using frog lines that have so far been shown to be free from background genetic mutations. We are mutagenizing sperm as well as spermatogonia.

As mentioned for our g-irradiation screen, we are using three independently segregating loci to determine a specific locus mutagenesis rate for both ENU sperm and spermatogonia mutagenesis. We do not yet know whether the loss of function of the GFP transgene will be informative. Although we showed it is at a single locus, it may be a tandem array, which is unlikely to be effectively eliminated by a point mutagen. We intend to optimize both sperm and spermatogonial mutagenesis. In discussion with the group, it appears that an ENU mutagen dose between 0.2-10 mM for 10 minutes will be optimal.

We will then also test spermatogonial mutagenesis using the same genetic loci. Based on results from the Grainger group, it appears that a dose of mutagen of 100mg/kg given as an injection to adult males between one to three times weekly may be optimal between survival, fertility, and mutation load. We will test each of these three doses and identify which is optimal.

Once the optimal dose is determined, we will initiate a large ENU mutagenesis screen both targeting sperm and spermatogonia that will be performed in a similar fashion as to our strategy being used for the g-ray mutagenesis. Animals generated from the sperm mutagenesis will also be prepared for re-sequencing defined loci to identify desired mutated loci and also quantify hit rates.

2. screening strategies that you are using and the phenotypes that you've

identified

We are doing a standard F3 screen analogous to the zebrafish screens done in Tubingen and Boston, but with a backcross variation. For screening F1 females, testes from six F2 males are used for in vitro fertilization of eggs from the F1 female. For screening F1 males, six F2 females will be screened by natural mating to the F1 male. Embryos from these backcross matings are raised through to swimming tadpoles (4 days postfertilization) and any phenotypes scored by morphology. Those embryos without obvious morphological phenotypes will undergo multi-tissue in situ analysis to determine if more subtle defects are present. Because sperm mutagenesis can produce mosaic founders, we will be screening six F2 females and six F2 males by backcross matings only. However, with spermatogonial mutagenesis we will be able to screen far fewer animals and still be able to effectively identify mutants while increasing screening efficiency (http://tropicalis.berkeley.edu/home/mission/phenotyping.html).

We have already identified mutations in cardiovascular development, craniofacial formation, melanocyte/cell migration, eye development, germ layer differentiation and early viability.

3. a list and description of the mutants that you've identified

Mutations on the Nigerian line:

grinch: confirmed background mutation in cardiovascular development

curly: confirmed background mutation in dorsal/ventral axis formation

bubblehead: confirmed background mutation in craniofacial development

bub9 (bigeye): confirmed background mutation in craniofacial development

agnatha: confirmed background mutation in craniofacial development

beaver: confirmed background mutation in embryo viability

rubenesque: confirmed mutation in pigmentation, cardiovascular function and cell migration

hourglass: confirmed mutation in eye development

dexter: potential background mutation in left:right patterning

owl: potential mutation in craniofacial development

clearview: potential mutation in melanocyte development

Mutations on the Ivory Coast line:

curly: confirmed background mutation in dorsal/ventral axis formation. This has been identified in an individual from the Ivory Coast line developed from Virginia stock 239 and was shown by complementation testing to be allelic to the Nigerian curly mutation.

bubbletrouble: potential background mutation in craniofacial development. This was found through inbreeding a TGA Ivory Coast pair derived from the colony in France.

4. an estimate of the mutagenesis rate

Results from the initial pilot screen were greatly confounded by epistatic effects from background mutations and the practical difficulty of carrying out the screen with this background. However, results from testing 600 rads g-ray mutagenesis suggest a single locus is mutated in approximately 1 in 100 embryos that survive past gastrulation and produces fertile F1 animals. In the next few months, we will obtain mutagenesis rates for ENU mutagenesis.

5. a description of how people can get your mutant strains

We are ready to distribute curly and grinch mutants. We will soon be able to distribute bubblehead, agnatha, beaver, and hourglass, as well. In addition, we are ready to distribute non-mutant inbred animals from five different frog lines: Nigerian, Ivory Coast (Virginia), Ivory Coast (TGA from France), Population A, and Golden (http://tropicalis.berkeley.edu/home/genetic_resources/Inbred-strains/inbred-strains.html). Requests should be made to harland@socrates.berkeley.edu.

6) time required to get animals to sexual maturity, and procedure used to

achieve the shortest time.

We have been able to cut the rates at which animals reach sexual maturity in half using optimized husbandry techniques and selective breeding for fast rates of sexual maturation. We still find that females take longer than males to mature: females in 5-7 months; males in 3 months. For our husbandry methods, see http://tropicalis.berkeley.edu/home/. We have also published our husbandry protocols (TC Grammer, MK Khokha, MA Lane, K Lam and RM Harland. Identification of Mutants in Inbred Xenopus tropicalis. Mech Dev. 2005 Mar 122(3):263-72.)

7) Recent publications supported by this grant

A Mve-Obiang, RE Lee, ES Umstot, KATrott, TC Grammer, JM Parker, B Ranger, R Grainger, and PLC Small. A newly discovered mycobacterial pathogen isolated from lethal infections in laboratory colonies of Xenopus species produces a novel form of the M. ulcerans macrolide toxin, mycolactone. Infection and Immunity (in press)

TC Grammer, MK Khokha, MA Lane, K Lam and RM Harland. Identification of Mutants in Inbred Xenopus tropicalis. Mech Dev. 2005 Mar;122(3):263-72.

MK Khokha, J Yeh, TC Grammer and RM Harland. Depletion of Three BMP Antagonists from Spemann's Organizer Leads to a Catastrophic Loss of Dorsal Structures. Dev Cell (2005) Mar;8(3):401-411.

KA Trott, BA Stacy, BD Lifland, HE Diggs, RM Harland, MK Khokha, TC Grammer, and JM Parker Characterization of a Mycobacterium ulcerans-like infection in a colony of African Tropical Clawed Frogs (Xenopus tropicalis) Comp. Med. (2004) 54(3), 309-317.

Gamma-Ray Mutagenesis of the X. tropicalis genome

Progress report for May 2005

Timothy C. Grammer, Mustafa K. Khokha, and Richard M. Harland

Department of Molecular and Cell Biology

University of California-Berkeley

401 Barker Hall

Berkeley CA 94720

Gamma-Ray Mutagenesis

Pilot study has been completed

To mutagenize the X. tropicalis genome, we exposed sperm to g-rays using a Cesium-137 source, and used the mutagenized sperm to fertilize wildtype eggs, which generated F1 animals. Please refer to previous progress reports on toxicity of g-ray mutagen and numbers of mutagenized F1 animals that were generated in our pilot study.

600 rads has been determined to be the optimal dose

We have now completed our pilot study and have effectively determined our mutagenesis efficiency using three independently segregating genetic loci. We used females that are curly/grinch double heterozygotes. Both of these mutations are homozygous embryonic lethal mutations that we identified in the background of the inbred Nigerian strain (Grammer, et.al, Mech Dev. 2005 Mar 122(3):263-72). Also we have inbred the transgenic g-crystallin GFP line and have homozygous transgenic animals.

To evaluate mutagenesis efficiency, we isolated sperm from g-crystallin GFP homozygotes and irradiated with different doses of g-rays (0 rads, 150 rads, 300 rads, 600 rads, 900 rads). These were the same doses that we used in our pilot screen to generate F1 animals. Irradiated sperm was applied to eggs from grinch/curly double heterozygotes (non-transgenic) for in vitro fertilization. We determined the next day that the g-ray exposure was effective based on the stereotypical lethal gastrulation defect that occurs in a dose-dependent fashion. We grew the surviving embryos to approximately stage 41 (two days after fertilization), and we evaluated a number of criteria. First, GFP should be expressed in the lens of all of the embryos unless this locus is mutated. Second, if either the curly locus or grinch locus is mutated in the sperm, a phenotype will develop in the half of these embryos that also receive a mutated allele from the mother (curly and grinch are recessive lethal).

The picture to the right demonstrates the phenotypes scored (from top to bottom): normal GFP+, GFP-, grinch, and curly.

Four independent experiments showed that the 600 rad dose is the optimal mutagen dose. We saw a consistent loss of GFP in approximately 1.5 in 100 embryos and curly or grinch in 1 in 200 embryos. Since any mutation at the curly or grinch locus will be masked 50% of the time by a wildtype maternal allele, the mutation rate is twice the rate at which we see the phenotype. Therefore, we predict a dose of 600 rads of g-ray irradiation to produce a single locus mutagenesis rate of approximately 1-2 in 100.

For the lower g-ray doses, no GFP-, curly, or grinch phenotypes were induced by the doses of 150 and 300 rads. Consistent with this, in our pilot screen, all potential induced mutations were from the 600 rad group and no induced mutations were observed in the 150 and 300 rad F1 groups. For the highest dose of 900 rads, the generation of GFP-, curly, and grinch embryos was similar to that of 600 rads, but with much higher levels of unscorable early embryonic lethals. Furthermore, our pilot screen showed that most of the F1 animals of the 900 rad group were sterile. Therefore, we conclude that 600 rads is the optimal dose for a g-ray sperm mutagenesis screen.

Initiation of optimized g-irradiation mutagenesis screen

Inbreeding mutant-free frogs

Now that we have identified the optimal dose of mutagen, we have initiated a much larger screen using this dose of 600 rads. Our first priority was to identify frogs that did not harbor background genetic mutations.

First, we identified embryonic lethal-free lines. Our pilot screen was severely hampered by background mutations that are present in the Nigerian inbred strain. Two recessive embryonic lethal mutations appear highly prevalent (>70%) in our population of inbred Nigerians: curly and grinch. In addition, we have confirmed the bubblehead, agnatha, and beaver mutations in the background as well, albeit at a lower frequency in our general population (each present in about 1-5%). Unfortunately, these background mutations masked other phenotypes that might have been present. Therefore, we have identified Nigerian inbred F8 founders that do not have either curly or grinch and have produced F9 offspring from these founders. We are screening them for other background mutations. As soon as we confirm that they are free of embryonic-lethal mutations, we will make them available for use.

As an alternative to the Virginia-derived inbred Nigerian line, we are breeding another line of Nigerian frogs starting from a pair of frogs brought to UC Berkeley by Enrique Amaya and maintained here by Dr. Tyrone Hayes. We obtained these frogs and used them to generate what we have called our “Golden” line, based on the light golden color of the original P0 founders. We confirmed that these frogs are free of all of the mutations identified to date in the Virginia-derived Nigerian line. We have now bred this line to an F3 generation using selective breeding for fast rates of sexual maturation, high fertility rates, and the lack of recessive lethal alleles (http://tropicalis.berkeley.edu/home/genetic_resources/Inbred-strains/Goldens/goldens.html). We are sending mating pairs of this line to Dr. Nicolas Pollet in France and Dr. Masanori Taira in Japan for independent maintenance of the line.

Large g-ray mutagenesis screen has been started

We used the Golden line as the starting point for our large g-ray mutagenesis screen. Using four independent founding mothers, we fertilized their eggs with sperm that had been exposed to 600 rads g-irradiation (using our previously reported protocols).

Having now conducted eight separate mutagenesis trials (two for the pilot study and six for the large study), we consistently find two periods of mass lethality due to the mutagenesis. The first period of large-scale embryo lethality is seen at gastrula stages. Approximately 50% of embryos suffer severe exogastrula defects and die at the dose of 600 rads of g-ray mutagenesis. The surviving embryos survive well until metamorphosis (4 weeks of age) when again we see about a 40% death rate. The affected metamorphosing tadpoles develop abdominal bloating causing them to swim upside down. They die within one to two days of this symptom. We have been unable to fix this defect, which is remarkably uniform in its phenotype amongst the dying tadpoles. It is not due to husbandry or “gas bubble” disease since it is only seen in our mutagenized clutches and never in nonmutagenized clutches being raised simultaneously in the same facility. Likewise, it is not treatable by transferring the tadpoles to static water or hyperosmotic buffers (which we have used successfully to treat gas bubble diseases before).

We predict that the two stages of mass lethality are due to dominant lethal mutations. The first wave of death being seen in early embryonic development around the time of zygotic transcription and the second being seen at metamorphosis when large numbers of previously silent genes are being turned on. Fortunately, since the fecundity of the parent Golden females is high, we started with many thousands of fertilized eggs and have been able to raise over 1,037 F1 frogs past these critical stages of development. The first set of F1s are now five months old (>100 frogs) and the second set (937 frogs) are now two months old.

Screening strategy

We have modified our original screening protocol in order to maximize space utilization and address concerns of potential mosaicism in our mutant F1s (refer to the diagram below for general screening outline).

In order to maximize space, we will be sibling mating F1 pairs to generate F2 clutches. Each F2 clutch will therefore represent two F1 animals, saving half of the required housing space. Our first group of F1s are now 5 months old and we are beginning to set up sibling matings now to generate F2 clutches. We expect our females will be sexually mature by 6-8 months of age.

We predict that since we did sperm mutagenesis, our mutations may not be fixed in the first cell division of the F1 embryo and may result in a mosaic animal. This mosaicism can decrease the production of F2 mutant animals, decreasing the likelihood of random F2 sibling matings to reveal a recessive mutation. Therefore, we will not use sibling matings of F2 frogs to generate F3 embryos for screening. Instead, we will only use backcross matings for screening (see diagram).

For screening F1 females, we will continue to use the three male backcross. Three fertile F2 males (expect 3-4 months of age) are sacrificed and used to fertilize the eggs of an F1 female. We will do this twice (one month apart to allow the female to rest), thereby screening six F1 males. This approach will identify carriers of mutations with 98.4% efficiency if no mosaicism is present. If there is mosaicism, we feel six backcross matings will be sufficient in detecting most of the latent mutations. F1 males will be screened by natural matings to six F2 females (two weeks rest for the male between matings, starting when the females are about 6-8 months of age). This also will identify carriers with an estimated 98.4% efficiency. Therefore, each F2 family screened this way will represent 2 X (0.984) or roughly 1.97 mutant genomes screened. If we screen all 1037 F1s, we will have screened approximately over 1000 mutant genomes. With our estimate of a 1% single locus mutagenesis rate for 600 rads of irradiation, we hope to identify a large number of mutants.

We will screen for phenotypes in the embryos produced by the backcrosses as we did for our pilot screen, based mostly on morphology. We will raise 200-600 embryos from each backcross mating through to stage 44 (4 days old). Due to the potential mosaicism in the F1 parent’s germline, we will consider any phenotype that is seen in greater than 10% of the embryos as being of interest. If positive, the phenotype will be documented and the entire clutch of embryos will be raised to adulthood (becoming F2.5 frogs). These F2.5 frogs will be tested for the inheritance of the mutation by sibling matings and by backcrossing to the F2 parent. In both cases, the mutation will be fixed (nonmosaic) and should behave in a typical mendelian fashion.

During the initial screening, F2.5 embryos that have no obvious morphological phenotype will be fixed and taken through in situ analysis as we have described previously. If no phenotype is discovered, the F1 and F2 parents will be culled.

Haploid screen

We had originally planned to screen haploid progeny in our pilot mutagenesis screen (see previous progress reports). Unfortunately, our inbred Nigerian line has not produced good haploids in our experience. Even control animals that received no gamma irradiation produce haploids that cannot be screened even after multiple attempts. Therefore we converted our pilot screen to a standard diploid F3 screen. Now that we are using our Golden line for our large mutagenesis screen, we hope that this line of frogs will produce good haploid embryos. We are in the process of selectively breeding Golden females that generate good haploid embryos and will readdress in our large mutagenesis screen whether the haploid strategy is feasible.

There will be a 3 month interval between the time of generating the F2 embryos and when the F2 males will be sexually mature enough for backcross matings. During this interval, we will try screening the F1 females by making haploid embryos.

Haploids will be produced by UV irradiation of sperm. The irradiated sperm can still activate the egg to begin development but does not contribute any genetic material. "Good" haploids are indistinguishable from diploid embryos through neurula stages. In the mid-20's, haploids develop a stereotypical "haploid syndrome." The axis is shortened and they appear stubby. By the 30's, haploids are very obvious with edema and bent tails. Although the morphology at later stages may be altered, transcripts characteristic of differentiated tissues are present so an in situ based screen is feasible.

We will UV irradiate sperm isolated from our g-crystallin GFP+ homozygous transgenic males as a control for haploidization. If any GFP+ tadpoles are seen, we know that the UV irradiation was inadequate in destroying the sperm’s genetic contribution and that the embryos are not haploid.

If the haploid screen is deemed unfeasible, we will have lost no time in our F3 screening strategy. In fact, we expect the multiple ovulations of the F1 female during this haploid screening strategy will help remove immature oocytes in these young females that we know can lead to epigenetic phenotypes (see Mutants section below). This will help them produce higher quality eggs by the time the F2 backcross matings are initiated.

Mutants

Go to

http://tropicalis.berkeley.edu/home/genetic_resources/mutants/mutants.html for pictures of mutants

Non-Genetic Phenotypes

Initial ovulations can produce epigenetic, stereotyped developmental defects

We have found that the first few ovulations of a female often produce poor quality eggs, regardless of her age. Females over 1 year of age at the time of an initial ovulation often produce defective eggs similar to much younger females at their initial ovulation. These epigenetic effects should not be confused with genetic defects.

We have discovered that young females in their first few ovulations produce a stereotypical defect that we have called Narrowminded. In these embryos a range of phenotypes are seen including microcephaly, cyclopia, anterior truncations, and in the most severe cases, complete ventralization of the embryo. This phenotype is not genetic and the severity decreases in subsequent matings. We typically see this phenotype persist in the first two matings of a female and then get progressively better with less severe phenotypes in subsequent matings. Compounding this, the effect was sometimes obtained in Mendelian ratios, erroneously suggesting a genetic basis. Therefore the phenotype of cyclopia, anterior truncations, or ventralization in embryos from initial ovulations should be viewed skeptically before assigning it as a genetic mutation.

Many initial ovulations will result in eggs with obvious gross abnormalities such as excessively large jelly coats often laid in strings, mottled pigmentation in animal hemispheres, or white eggs that lack clear animal/vegetal hemisphere distinctions. These almost always result in unsuccessful fertilizations. Because initial ovulations are consistently poor, we ovulate females at 5 to 6 months of age and discard the eggs. We then start natural matings with the females (typically at 6 to 7 months of age) one month after their first successful laying of normal looking eggs.

Two types of defects are typically seen in embryos generated in the first several matings of immature females: ventralization and anterior truncations with cyclopia. In the most severe cases of hyperventralization, there is a complete loss of dorsal structures. The more common nongenetic phenotype seen in initial ovulations is a defect in early dorsal midline structures that produces anterior truncations and cyclopia in later stages. We showed that the defect can be initially seen molecularly at the neurula stages (Grammer, et.al, Mech Dev. 2005 Mar 122(3):263-72). Morphologically, the embryos look relatively normal through gastrula stages, but develop obvious anterior truncations by the end of neurulation and early 20 stages often accompanied by ventral edema around the blood forming region.

The percentage of embryos developing these dorsoventral and mediolateral defects can vary widely. They are often present in less than 10% of the clutch, but a few matings produced nearly 25% defective embryos as would be expected for a recessive mutation. However, in over five hundred matings that have shown these defects, subsequent matings show a continual decline in the numbers of embryos developing these phenotypes. Therefore, these phenotypes can be epigenetic.

While these effects can alter the viability of early embryos and complicate an evaluation of phenotypes in a forward genetic screen, a significant number of embryos can be unaffected and survive. Therefore, this epigenetic effect may not extend the generation time of X. tropicalis.

Identified Genetic mutations

Mutations on the Nigerian line:

grinch: confirmed background mutation in cardiovascular development

curly: confirmed background mutation in dorsal/ventral axis formation

bubblehead: confirmed background mutation in craniofacial development

bub9 (bigeye): confirmed background mutation in craniofacial development

agnatha: confirmed background mutation in craniofacial development

beaver: confirmed background mutation in embryo viability

rubenesque: confirmed mutation in pigmentation, cardiovascular function and cell migration

hourglass: confirmed mutation in eye development

dexter: potential background mutation in left:right patterning

owl: potential mutation in craniofacial development

clearview: potential mutation in melanocyte development

Mutations on the Ivory Coast line:

bubbletrouble: potential background mutation in craniofacial development. This was found through inbreeding a TGA Ivory Coast pair derived from the colony in France.

Nigerian Background mutants

Six mutations are in the background of the inbred Nigerian line: grinch, curly, bubblehead, bub9(bigeye), agnatha, and beaver.

In the case of grinch, curly, and bubblehead, these have been found in many nonmutagenized animals confirming their presence as background mutations. We have identified single, double, and triple mutants in all combinations of the three mutations. We used this as both a tool for quantitating our mutagenesis rates (as explained above in the g-ray mutagenesis section) as well as demonstrating the utility of Xenopus tropicalis genetics. We demonstrated that the mutations segregate just as expected for a diploid genome and that the large numbers of embryos obtained in each mating allow us to easily study genetic mutations in hybrid, dihybrid, and trihybrid matings. We recently published this work (TC Grammer, MK Khokha, MA Lane, K Lam and RM Harland. Identification of Mutants in Inbred Xenopus tropicalis. Mech Dev. 2005 Mar 122(3):263-72.) and our results are summarized below.

Identification of grinch, curly, and bubblehead mutants during inbreeding

During our inbreeding, we discovered that some of our inbred animals are carriers of mutations. We have designated three of the Nigerian strain mutants as grinch, curly, and bubblehead. All three are recessive embryonic lethals, can be identified morphologically by the late 30 stages and cause death by the late 40s.

grinch

At stage 38, grinch mutants show signs of pericardial edema correlating with the onset of heartbeat. The edema around the heart worsens and compresses the heart, while spreading ventrally until the entire ventral side is filled with fluid by the early stage 40s. The embryos eventually die by rupturing around stage 47.

We have identified over 200 carriers of grinch, confirmed that they belong to the same complementation group, and have passed the mutation through four generations. Hybrid matings of grinch heterozygous parents produce results as expected for a single recessive allele (25% mutant). We found that an F7 male, the son of the holotype female whose dna is being sequenced by the JGI genome project, is a carrier of grinch. We have been inbreeding the holotype line and are now identifying frogs free from this mutation.

We are now characterizing the grinch mutation and have plenty of mature heterozygous carriers to distribute to the community. Meanwhile, we have outbred the grinch mutation to both the Ivory Coast and the Population A lines of frogs in anticipation of mapping the mutation once polymorphic markers are identified by Amy Sater’s group.

curly

Until the mid 30s, curly mutants appear morphologically normal but then develop dorsal curvatures in the body axis that get progressively worse. The tail curvature is accompanied by pericardial edema. By the early 40s, curly embryos show severe dorsal curvature, ventral edema, endodermal defects, and small body size. The curly embryos die by the late 40 stages.

We have identified over 120 carriers of curly and have passed the mutation through four generations. Complementation testing confirms that the mutations in our various clutches are at the same locus. Matings of curly heterozygotes produce mutant numbers expected for a single recessive allele.

We are now characterizing the curly mutation and have plenty of mature heterozygous carriers to distribute to the community. In addition, we have outbred the curly mutation to both the Ivory Coast and the Population A lines of frogs in anticipation of mapping the mutation. For the Ivory Coast outbreeding, we chose to outcross to the Ivory Coast TGA line from France because of fears that our Ivory Coast line derived from Virginia may have Nigerian DNA as well (see next section for more details).

We also want to test if the curly mutation is allelic to the rocinante mutant identified in the Grainger laboratory. We tried a complementation test with a rocinante pair we received from Virginia but this failed to be conclusive. We received two rocinante frogs (a male and female). The female died before we could do a complementation test, but the male is confirmed by complementation testing to be curly positive. However, we cannot conclusively say that the mutations are allelic since we cannot rule out that he is a curly/rocinante double heterozygote. We were originally able to mate the rocinante pair when they first arrived to confirm the rocinante mutation. They showed very nice 25% rocinante phenotype with a dramatic downward curvature of the tail at stage 40. Our curly mutants always develop tail curvatures either upward or in a zig-zag pattern along the anteroposterior axis and we do not see the curly embryos show the strong downward axis defect seen with the original rocinante pair. However, we want to rule this possibility out.

curly in the Ivory Coast (Virginia) line

We obtained two Ivory Coast mating pairs from the Virginia colony that were ICF5s (stock 239). We have been inbreeding these frogs to further this line (http://tropicalis.berkeley.edu/home/genetic_resources/Inbred-strains/IC/IC.html). One of the original pairs was sacrificed by us and sent to Amy Sater to use in her mapping experiments. The other pair is still in our facility. The male of this pair has been shown to have the curly allele that we've identified in our Nigerian frogs. We are certain that the frog has not been misidentified because it has a unique body size and coloration that our Nigerian frogs do not. Thus, unless mutations in this gene are widespread in the Xenopus tropicalis population, it is possible that Nigerian DNA found its way into the Ivory Coast stock at some point. We have not found the mutation in any of the Ivory Coast frogs we obtained from France (via Nicholas Pollet) or from the Population A or Golden lines.

We have been in contact with both Rob Grainger and Amy Sater about the possibility of there being a contamination of the Ivory Coast line with the Nigerian line. This seemed important because the Ivory Coast frogs we identified the potential problem in are from the same stock being used for the genetic mapping effort. Rob and Amy have concluded that the mapping data has not shown that there is a major problem and that Amy’s group does see strain-specific alleles or sets of alleles. We hope to work with Amy to identify some diagnostic markers to distinguish these animals as well as identifying which are the most polymorphic lines.

bubblehead

The bubblehead phenotype appears by the late 30s, when small eyes develop. By the mid-stage 40s, bubblehead embryos show craniofacial abnormalities and small body size. The bubblehead embryos also have gut looping defects in which the gut fails to turn and coil. The embryos die by the late 40s.

We have identified over 40 carriers of bubblehead and have passed the mutation through three generations. Matings of bubblehead heterozygous parents produce phenotypic ratios expected for a single recessive allele. We are now beginning to analyze this mutant.

bub9(bigeye)

We have identified a mutation similar to the bubblehead mutant which is consistently seen in conjunction with low levels of a phenotype we refer to as bigeye. The bigeye mutants are most easily distinguished from normal embryos at the mid-30 stages when dorsal edema and tailfin defects develop along with severe swelling of the eyes. We have identified five individuals from the same clutch family that all have this bubblehead-like (bub) phenotype with the bigeye phenomenon. Because of the coincident findings of bigeye in conjuction with a bubblehead-like phenotype, the family that harbors this mutation may represent a separate mutant locus which we have designated as bub9. We are now in the process of performing complementation testing to identify if the bubblehead and bub9 mutations represent a single locus.

agnatha

We have identified a recessive embryonic lethal mutation that causes gut coiling defects and most notably a failure to form proper ventral craniofacial structures (lower jaw, basihyoidal, and ceratohyoidal cartilages). We identified this during our mutagenesis screen in three separate F2 clutches that were originally designated as agnatha, diamondback, and agnatha-like mutations. We have confirmed by complementation testing that they are all allelic and we are now calling the mutation agnatha. Because we found the same mutation in three separate families, we believe that it is in the genetic background of the Nigerian line. This stresses the importance of identifying and propagating mutation-free lines of frogs for future genetic screens.

We have identified 16 agnatha carriers and are currently analyzing the mutation. The agnatha phenotype is strikingly similar to the Sucker-class of zebrafish mutants which tend to be mutations in genes in the Endothelin signaling pathway whose mutations result in a loss of ventral craniofacial structures, most notably the lower jaw.

We have outcrossed this mutant for future mapping and are currently analyzing it.

In addition, we noticed that the agnatha mutants are similar to the jaws and haggis mutants identified by Lyle’s group and posted on their website. We have obtained frozen sperm of these mutants from Lyle and will be doing complementation tests using them to fertilize eggs from our agnatha females. We have Mike Sargents sperm freezing/thawing protocol working nicely in our lab (http://tropicalis.berkeley.edu/home/obtaining_embryos/sperm-freezing/sperm-freeze.html) and will be doing the complementation tests over the next few weeks.

beaver

We found a mutation in two separate clutches of our mutagenized F2s. Because we found the same mutation in two separate families (confirmed to be allelic by complementation testing), we believe that it may be in the genetic background of the Nigerian line. We have designated this mutation as beaver because of the dramatic accumulation of what appear to be undifferentiated cells at the tip of the tail.

The beaver embryos develop normally until the early 20s when the embryos appear to undergo a developmental arrest. All early molecular markers appear normal, but all development past stage 25 is defective and the embryos undergo a dramatic degeneration by the early 30s. All cells lose their adherence from each other and the embryos disintegrate by the mid-30s. This mutation may be similar to the Class II zebrafish phenotypes described in Kane et al. 1996:Development, in which tissue types form initially, followed by developmental arrest, then degeneration, particularly in the nervous system.

We have outcrossed this mutant for future mapping and are currently analyzing it.

In addition, we noticed that the beaver mutants are similar to the snowwhite and gene of mass destruction (gmd) mutants identified by Lyle’s group and posted on their website. We have obtained frozen sperm of these mutants from Lyle and will be doing complementation tests using them to fertilize eggs from our agnatha females.

Nigerian Induced mutants

The following mutations appear to be induced, meaning that we have not seen these mutations in matings of unirradiated frogs and/or between unrelated parents.

Hourglass

We identified a mutation we call hourglass that has a defect in the formation of the pigmented retinal epithelia. The ventral surface of the eye doesn’t form and the pigmented retinal epithelia stretches back to maintain contact with the diencephalon of the forebrain.

There is no overt mutation seen other than the eye defect, but all attempts to raise these tadpoles has failed. They always die within the first week of life. Therefore, we predict that there is more than just a defect in eye formation.

We have propagated the mutation through two generations. We have also outcrossed this mutant for future mapping and are currently studying it in more detail.

Owl

We identified a mutation we call owl that has a defect in craniofacial development. The phenotype first appears by the late 30s, when the ventral surface of the eye is rotated out and can be seen from the dorsal viewpoint. By the mid-stage 40s, owl embryos show craniofacial abnormalities and smaller body and head size. The owl embryos die by the late 40s.

We are raising F1 outcrosses of owl and will soon be screening these to identify carriers and to begin analyzing it.

Clearview

We identified a mutation we call clearview that has a defect in the formation of melanocytes. The mutation is most easily seen in early 40s as a lack of normal pigmention. The embryos are not albino, but have a dramatic reduction in the number of melanocytes as well as a lack of pigmentation granules in the outer regions of the stellate melanocyte cell bodies.

We are raising F1 outcrosses of owl and will soon be screening these to identify carriers and to begin analyzing it.

Dexter

We identified a mutation we call dexter that has a defect in the formation of the left side of the body. The right side appears to develop normally, but the left side develops poorly resulting in anything from a mild phenotype, most noticeably seen as a left eye usually smaller in size or absent, to a severe defect with the left side of the body not developing normally at all.

We are raising F1 outcrosses of dexter and will soon be screening these to identify carriers and to begin analyzing it.

Ivory Coast Background mutants

Bubbletrouble

We identified a potential background mutation in craniofacial development while inbreeding a TGA Ivory Coast pair derived from the colony in France. Besides the craniofacial deformation, the mutant embryos also develop gutlooping defects and internal swellings around the posterior side of the proctodeum. The defect was seen in numbers very consistent with a recessive mutation.

We are retesting the parents and will determine if it repeats. We will then propagate it through successive generations.

Husbandry and Website Updates

We have made a lot of progress in optimizing husbandry protocols and have made the information available on our website (http://tropicalis.berkeley.edu/home/). Our protocol for raising tadpoles and frogs was recently published (Grammer, et.al, Mech Dev. 2005 Mar 122(3):263-72). Our website has several new sections and updates including protocols and information on the following:

Inbred lines: we have updated genealogies and information on the various inbred lines we are propagating.

Shipping frogs: procedures for shipping and receiving animals

Frog tags: identification techniques, microchips, polymers, alphanumeric tags

Disease: new updates on mycobacteria including our new manuscript

A Mve-Obiang, RE Lee, ES Umstot, KATrott, TC Grammer, JM Parker, B Ranger, R Grainger, and PLC Small. A newly discovered mycobacterial pathogen isolated from lethal infections in laboratory colonies of Xenopus species produces a novel form of the M. ulcerans macrolide toxin, mycolactone. Infection and Immunity (in press)

Sperm freezing/storage: we have links to the published protocol of Sargent and Mohun, as well as some modifications that we tested and use.

Morpholino oligos: we have updates on the use of morpholinos and the strength of using Xenopus tropicalis for morpholino experiments as outlined in our recent paper:

MK Khokha, J Yeh, TC Grammer and RM Harland. Depletion of Three BMP Antagonists from Spemann's Organizer Leads to a Catastrophic Loss of Dorsal Structures. Dev Cell (2005) Mar;8(3):401-411.

Cartilage staining: we are optimizing cartilage staining and analysis protocols for Xenopus tropicalis and will continue updating this section as we now are focusing on developing good staining and flatmounting techniques.

Tropicalis plasmid library: we have now identified and collected 287 clones of important early developmental genes, many of which were not annotated or identified by name in the various available clone collections.

Isolating Genomic DNA: we have protocols for isolating genomic DNA from tadpoles, adult tissues, and red blood cells.

Mutagenesis strategies: updates on the progress of g-ray and ENU mutagenesis.

Mutants: updates on the mutants we’ve identified

Comparative Genomics: updated links to valuable genomic resource sites