Alternative splicing of POUM2 regulates embryonic cuticular formation and tanning in Bombyx mori

Insect cuticle is an apical extracellular matrix produced by the epidermis,tracheal,hind-and foregut epithelia during embryogenesis and renewed during molting and metamorphosis.However,the underlying regulatory mechanism for embryonic cuticle formation remains largely unclear.Here,we investigate the function of the transcription factor POUM2 in the embryonic cuticular formation in Bombyx mori,a model lepidopteran insect.Clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein-9-mediated knockout of POUM2 resulted in the defect of cuticular deposition,pigmentation,and sclerotization in the embryos.Differentially expressed transcripts analysis of 7-d-old embryos identified 174 up-or downregulated cuticular protein transcripts,8 upregulated chitin degradation transcripts,2 downregulated chitin synthesis transcripts and 48 up-or downregulated transcription factor transcripts in the POUM2−/−embryos.The expression levels of the key factors of the tyrosine metabolic pathway,such as tyrosine hydroxylase(Th),Dopa decarboxylase(DDC),and arylalkylamine N-acetyltransferase(aaNAT),were significantly decreased in the POUM2−/−embryos.POUM2 isoform POUM2-L specifically bound the POU cis-regulatory element(CRE)in the Th promoter and increased the transcription of Th,whereas POUM2-S could not bind the POU CRE,although it also increased the transcription of Th.Heterogeneous nuclear ribonucleoprotein Squid-1 directly bound the POUM2 pre-mRNA(messenger RNA)and inhibited the alternative splicing of POUM2-L to POUM2-S mRNA.These results suggest that POUM2 participates in the cuticular formation by regulating the chitin and cuticular protein synthesis and metabolism,and the cuticular pigmentation and sclerotization by regulating tyrosine metabolism during embryogenesis.This study provides new insights into novel function of POUM2 in embryogenesis.