摘要: Cilia are microtubule-based, hair-like organelles involved in sensory function or motility, playing critical roles in many physiological processes such as reproduction, organ development, and sensory perception. In insects, cilia are restricted to certain sensory neurons and sperms, being important for chemical and mechanical sensing, and fertility. Although great progress has been made regarding the mechanism of cilia assembly, the formation of insect cilia remains poorly understand, even in the insect model organism Drosophila. Intraflagellar transport (IFT) is a cilia-specific complex that traffics protein cargos bidirectionally along the ciliary axoneme and is essential for most cilia. Here we investigated the role of IFT52, a core component of IFT-B, in cilia/flagellar formation in Drosophila. We show that Drosophila IFT52 is distributed along the sensory neuronal cilia, and is essential for sensory cilia formation. Deletion of Ift52 results in severe defects in cilia-related sensory behaviors. It should be noted that IFT52 is not detected in spermatocyte cilia or sperm flagella of Drosophila. Accordingly, ift52 mutants can produce sperms with normal motility, supporting a dispensable role of IFT in Drosophila sperm flagella formation. Altogether, IFT52 is a conserved protein essential for sensory cilia formation and sensory neuronal function in insects.

摘要: The Notch signaling pathway plays a central role in the development of various organisms. However, dysregulation of microRNAs (miRNAs), which are crucial regulators of gene expression, can disrupt signaling pathways at all stages of development. Although Notch signaling is involved in wing development in Drosophila, the mechanism underlying miRNA-based regulation of the Notch signaling pathway is unclear. Here, we report that loss of Drosophila miR-252 increases the size of adult wings, whereas the overexpression of miR-252 in specific compartments of larval wing discs leads to patterning defects in the adult wings. The miR-252 overexpression-induced wing phenotypes were caused by aberrant Notch signaling with intracellular accumulation of the full-length Notch receptor during development, which could be due to defects in intracellular Notch trafficking associated with its recycling to the plasma membrane and autophagy-mediated degradation. Moreover, we identified Rab6 as a direct target of miR-252-5p; Rab6 encodes a small Ras-like GTPase that regulates endosomal trafficking pathways. Consistent with this finding, RNAi-mediated downregulation of Rab6 led to similar defects in both wing patterning and Notch signaling. Notably, co-overexpression of Rab6 completely rescued the wing phenotype associated with miR-252 overexpression, further supporting that Rab6 is a biologically relevant target of miR-252-5p in the context of wing development. Thus, our data indicate that the miR-252-5p-Rab6 regulatory axis is involved in Drosophila wing development by controlling the Notch signaling pathway.

摘要: N-6-methyladenosine (m(6)A) RNA is the most abundant modification of mRNA, and has been demonstrated in regulating various post-transcriptional processes. Many studies have shown that m(6)A methylation plays key roles in sex determination, neuronal functions, and embryonic development in Drosophila and mammals. Here, we analyzed transcriptome-wide profile of m(6)A modification in the embryonic development of the destructive agricultural pest Spodoptera frugiperda. We found that the 2 key mRNA m(6)A methyltransferases SfrMETTL3 and SfrMETTL14 have high homologies with other insects and mammals, suggesting that SfrMETTL3 and SfrMETTL14 may have conserved function among different species. From methylated RNA immunoprecipitation sequencing analysis, we obtained 46 869 m(6)A peaks representing 8 587 transcripts in the 2-h embryos after oviposition, and 41 389 m(6)A peaks representing 9 230 transcripts in the 24-h embryos. In addition, 5 995 m(6)A peaks were differentially expressed including 3 752 upregulated and 2243 downregulated peaks. Functional analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes suggested that differentially expressed m(6)A peak-modified genes were enriched in cell and organ development between the 2- and 24-h embryos. By conjoint analysis of methylated RNA immunoprecipitation-seq and RNA-seq data, we found that RNA m(6)A methylation may regulate the transcriptional levels of genes related to tissue and organ development from 2- to 24-h embryos. Our study reveals the role of RNA m(6)A epigenetic regulation in the embryonic development of S. frugiperda, and provides new insights for the embryonic development of insects.

摘要: DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the -223 and -190 nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the -223 and -190 nt region of its promoter. Simultaneous RNAi-mediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.

摘要: 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.