A large-scale insertional mutagenesis screen in zebrafish

Abstract

It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

Figure 1

Schematic diagram of the protocol for the large-scale screen. We have finished generating founder fish. As of 8/99, 3800 F₁ families have been born, 2500 have been genotyped by fin clips, and 950 F₂ families have been born.

Figure 2

Southern analysis of the top eight fish from two different F₁ families. Note that in family A, the majority of the insertions are the same from fish to fish, implying that most of them came from the same germ cell from one of the founders. In this case, we would keep fish 3 (seven inserts), fish 6 (three new inserts), and no others, as none would give more than two new inserts. Family B has much greater diversity of inserts. In this case, we would keep fish 6 (nine inserts), fish 7 (seven new inserts), and fish 2 (five new inserts).

Figure 3

Isolation of genomic sequence flanking mutagenic insertions from multiple insert families. The schematic at top indicates the structure of the provirus along with the position of Southern blot probes and PCR primers.

Figure 4

Photographs of wild-type vs. mutant embryos or adult fish for 9 of the 11 insertional mutants described. (A) Ten-week-old wild-type (top) vs. dominant mutant hiD862 with long fins. (B) Wild-type (left) vs. bubble brain at day 2. (Arrowhead) Region of enlarged ventricle in mutants. (C) Wild-type (top) vs. two no knack mutant embryos at day 4. (D) Nine-week-old wild-type (top) vs. a nearly normal sibling, one of ∼10% of the homozygotes that survived. (E) Wild-type (top) vs. hi37 mutant at day 4. (F) Wild-type (top) vs. hi43 at day 5. (Arrowhead) Liver that is abnormal in the mutant. (G) Closer view of wild-type (top) vs. hi43 liver region. (H) Wild-type (top) vs. hi63 mutant at day 3. (I) Wild-type (left) vs. hi96 mutant embryo at day 4. (Arrowhead) Unusual edema with pooled blood around eye. Edema around body of mutant is also visible. (J) Wild-type (left) vs. bleached blond mutant at day 4. (K) Closer view of eyes of bleached blond at day 4 showing mottled appearance.