A diagram of the mutagenesis and crossing strategy used by Nüsslein-Volhard and Wieschaus to generate and maintain segmentation mutants is shown. Male flies were exposed to EMS (a mutagen) and mated to a non-mutagenized female. How did the researchers ensure that the offspring of Cross B (i.e., the offspring seen in generation C in the diagram) were heterozygotes that carried a mutagenized chromosome? Please select all that apply.

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CyO homozygotes die, so offspring in generation C with curly wings must be heterozygotes. This means that the offspring could have either the mutagenized chromosome (with markers cn, bw, sp here) or the other parental chromosome (with markers b, pr, can, sca, DTS-91, here). To ensure that the only surviving flies were those carrying a mutagenized chromosome, the researchers used a temperature sensitive allele on the non-mutagenized chromosome (DTS-91 here). The researchers raised the temperature, and flies with the DTS-91 allele died. This means that the only surviving flies were heterozygous flies with CyO and the mutagenized chromosome. Although it is true that homozygous flies with a severe segmentation mutation would likely die, at this stage the researchers did not know which of their mutations were lethal or which were segmentation mutants - in fact, the majority of mutants would not have an aberrant segmentation phenotype. In any case, no offspring of cross B would be homozygous for the mutagenized chromosome. Here, male flies were mutagenized, but this did not have an impact on selection of heterozygotes in generation

A Diagram of the Mutagenesis and Crossing Strategy Used by Nüsslein-Volhard