A Simultaneous Genetic Screen for Zygotic and Sterile Mutants in a Hermaphroditic Vertebrate (Kryptolebias marmoratus)

Abstract

The mangrove killifish, Kryptolebias marmoratus, is unique among vertebrates due to its self-fertilizing mode of reproduction involving an ovotestis. As a result, it constitutes a simplistic and desirable vertebrate model for developmental genetics as it is easily maintained, reaches sexual maturity in about 100 days, and provides a manageable number of relatively clear embryos. After the establishment and characterization of an initial mutagenesis pilot screen using N-ethyl-N-nitrosourea, a three-generation genetic screen was performed to confirm zygotic mutant allele heritability and simultaneously score for homozygous recessive mutant sterile F2 fish. From a total of 307 F2 fish screened, 10 were found to be 1° males, 16 were sterile, 92 wild-type, and the remaining 189, carriers of zygotic recessive alleles. These carriers produced 25% progeny exhibiting several zygotic phenotypes similar to those previously described in zebrafish and in the aforementioned pilot screen, as expected. Interestingly, new phenotypes such as golden yolk, no trunk, and short tail were observed. The siblings of sterile F2 mutants were used to produce an F3 generation in order to confirm familial sterility. Out of the 284 F3 fish belonging to 10 previously identified sterile families, 12 were found to be 1° males, 69 were wild-type, 83 sterile, and 120 were classified as */+ (either wild-type or carriers) with undefined genotypes. This screen provides proof of principle that K. marmoratus is a powerful vertebrate model for developmental genetics and can be used to identify mutations affecting fertility.

Keywords: ENU mutagenesis; Kryptolebias marmoratus; forward genetic screen; sterile mutant; zygotic mutant.

Figure 1

Summary of zygotic confirmation scheme. F₁ fish previously identified as heterozygous for a zygotic mutation are allowed to self-cross. In their F₂, they are expected to produce 25% zygotic mutant embryos, which were originally identified as embryonic lethal. Therefore, surviving F₂ fish are composed of two carriers (m/+): one wild-type (+/+) ratio with the remaining fish displaying a zygotic defect leading to lethality. F₂ fish are allowed to self-cross to confirm the zygotic phenotype into the F₃ and assess proper transmission of wild-type phenotypes.

Figure 2

Sterile mutant identification and confirmation scheme. A fish from the F₂ generation is identified as sterile (s/s) when it is raised to adulthood and has no viable progeny belonging to any of two predicted types (see text for details). Among the types are three categories of predicted embryos: (1) black circle with white halo (fertilized but nonviable); (2) tan circle with no white halo (nonfertilized); and (3) tan circle with white halo and red X (no progeny laid). The sterile F₂ siblings are composed of two carriers (+/s): one wild-type (+/+). These siblings are self-crossed to collect F₃ progeny that are in turn raised to sexual maturity. Wild-type F₃ siblings (+/+) will lay 100% wild-type F₄ viable embryos (tan circle with white halo). F₂ fish carrying the sterile allele (+/s) will give rise to 25% F₃ sterile progeny that, when raised to sexual maturity, will lay 100% defective embryos or be devoid of F₄ progeny. Example given is a nonviable maternal effect mutant that arrests during early cleavage (all black circles with halo, see Figure 7).

Figure 3

New zygotic phenotypes identified across different developmental stages in the confirmation screen. (A) Wild-type fertilized embryo. (B) Wild-type embryo (14 dpf). (C) Wild-type hatched juvenile fish. (D) Golden yolk phenotype (R058 family). (E) No trunk phenotype (R109 family). (F) Short tail phenotype (R228 family). (G) One eye phenotype (R182). (H) Dwarf/eyes forward phenotype (R196 family). (I) Downward curled tail phenotype (R217 family).

Figure 4

Short tail phenotype across two F₂ clonal lines of the R228 family (14 dpf). (A) Wild-type embryo. (B) F₃ embryos descended from R228-6 clonal line (left anterior view, right posterior view). (C) F₃ embryo descended from R228-8 clonal line.

Figure 5

No trunk defect phenotype across multiple F₂ clonal lines of R109 family (14 dpf). (A) Wild-type embryo. (B) F₃ embryo descended from R109-3 F₂ parent. (C) F₃ embryo descended from R109-4 F₂ parent. (D) F₃ embryo descended from R109-8 F₂ parent.

Figure 6

Golden yolk phenotype across development from the R058-2 F₃ family. (A) Fertilized wild-type embryo with clear oil droplets. B–L. Golden yolk phenotype from embryo to adult among F₄ offspring. (B) One-cell stage (Stage 1). (C) Eight-cell stage (Stage 4). (D) Early gastrula (Stage 11). (E) Midgastrula (Stage 12). (F) Optic vesicle and somite formation (Stage 17). (G) Dorsal view at liver formation (Stage 26). (H) Side view as pigmentation and body movement increases (Stage 27A). (I) Dorsal view as caudal fin forms (Stage 28). (J) Jaw formation (Stage 30). (K) Juvenile fish. (L) Adult fish with yellow pectoral fin phenotype. Developmental stages are indicated according to Mourabit et al. (2011). Arrows indicate location of golden yolk or pigment remnants at later stages of development.

Figure 7

Early embryonic developmental arrest of F₄ embryos from the confirmation screen (R152-1 family). (A) Wild-type fertilized embryo. (B) Fertilized F₄ mutant embryo. (C) Within 4 hr development halts at the 2-cell stage and yolk begins to degenerate. (D) Within 24 hr the mutant is completely arrested in development. Arrow indicates 2-cell blastula.

Figure 8

Sterile embryo holder phenotype in F₃ fish from the R171 family (9 months old). (A) Dorsal view of enlarged abdomen. (B) Side view cloacal damage from inability to lay embryos. (C) Side ventral view of abdomen. (D) Enlarged view of (C) displays embryos in advanced stages of development.

Figure 9

Pedigree of the R010 sterile mutant family displays the heritability pattern of the s allele. Diamonds represent various genotypes of adult hermaphroditic fish within the family (aged >6 months).