The Bombyx mori ortholog of protein-cysteine N-palmitoyltransferase (Rasp) is essential for the development of adult organs.

The gap (apterous and rudimentary gonads) mutation is a spontaneous mutation that causes defects in adult organs in the silkworm Bombyx mori. Although gap larvae exhibit normal external morphology, gap mutants exhibit severe defects in adult organs, suggesting that the gap gene is required for the development of imaginal discs and the primordia of adult organs. Genetic analysis of the gap mutation provides an opportunity to understand the molecular mechanisms underlying the development of adult organs. In this study, we performed RNA-seq analysis of ovaries from gap mutants. De novo assembly of RNA-seq reads identified the insertion of a retrotransposon in the gene encoding the Bombyx ortholog of protein-cysteine N-palmitoyltransferase (Rasp), which is required for Hedgehog signaling in Drosophila. PCR and subsequent sequencing analyses revealed that an LTR transposon is inserted into exon 1 of the BmRasp gene. To verify the involvement of BmRasp in the gap phenotype, we generated a knockout allele of BmRasp (BmRaspKO) using the CRISPR/Cas9 system. Both homozygous knockout mutants and F1 pupae with the gap/BmRaspKO genotype exhibited gap-like phenotypes characterized by apterous pupa, indicating that BmRasp corresponds to the gap locus. To investigate why zygotic BmRasp expression is dispensable during embryogenesis but essential for the development of adult organs, we analyzed BmRasp expression levels in eggs and larval tissues. Furthermore, testis transplantation experiments demonstrated that BmRasp is dispensable for spermatogenesis, despite the fact that the testis exhibits the highest expression level of BmRasp among the tissues analyzed. Finally, we discuss the physiological roles of Hedgehog signaling in embryogenesis and in the development of imaginal discs in B. mori.