Role of maternal spiralian-specific homeobox gene SPILE-E in the specification of blastomeres along the animal–vegetal axis during the early cleavage stages of mollusks

Abstract

Spiralians, one of the major clades of bilaterians, share a unique development known as spiralian development, characterized by the formation of tiers of cells called quartets, which exhibit different developmental potentials along the animal–vegetal axis. Recently, spiralian-specific TALE-type homeobox genes (SPILE) have been identified, some of which show zygotic and staggered expression patterns along the animal–vegetal axis and function in quartet specification in mollusks. However, it is unclear which maternal molecular components control the zygotic expression of these transcription factors. In this study, we focused on SPILE-E, a maternal transcription factor, and investigated its expression and function in mollusks. We found that the maternal and ubiquitous expression of SPILE-E in the cleavage stages is conserved in molluskan species, including limpets, mussels, and chitons. We knocked down SPILE-E in limpets and revealed that the expression of transcription factors specifically expressed in the first quartet (1q²; foxj1b) and second quartet (2q; SPILE-B) was abolished, whereas the macromere-quartet marker (SPILE-C) was ectopically expressed in 1q² in SPILE-E morphants. Moreover, we showed that the expression of SPILE-A, which upregulates SPILE-B but represses SPILE-C expression, decreased in SPILE-E morphants. Consistent with changes in the expression pattern of the above transcription factors, SPILE-E-morphant larvae exhibited patchy or complete loss of expression of marker genes of ciliated cells and shell fields, possibly reflecting incomplete specification of 1q² and 2q. Our results provide a molecular framework for quartet specification and highlight the importance of maternal lineage-specific transcription factors in the development and evolution of spiralians.