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.

Effect of TGF-ß2 on the Mechanical Properties of Posterior Scleral Fibroblasts in Experimental Myopia.

Objective: The effects of TGF-ß2 on mechanical properties of sclerotic desmocytes isolated from healthy and myopic guinea pigs were investigated in order to further understand the pathogenesis of myopia. To study the effect of TGF-ß2 on the mechanical properties of posterior scleral fibroblasts in experimental myopia. Methods: A lens-induced myopia (LIM) animal model was developed in 12 guinea pigs, with the opposite eye serving as a self-control (SC). Five untreated guinea pigs served as normal controls. Lenses were removed 30 days after model onset. Primary scleral fibroblasts were isolated and passaged twice and then treated with vehicle control or 1, 10, or 100 ng/mL TGF-ß2. After 24 h, micropipette aspiration was used to investigate the viscoelastic properties of the cells. Results: Scleral fibroblasts from LIM exhibited significantly higher equilibrium moduli and apparent viscosities relative to SC without TGF-ß2 treatment. Treatment of LIM or SC scleral fibroblasts with 1 or 10 ng/mL TGF-ß2 led to significantly different (p < 0.05) equilibrium moduli and apparent viscosities compared with vehicle control, whereas no significant differences were observed upon treatment with 100 ng/mL TGF-ß2. LIM cells treated with 1 and 10 ng/mL TGF-ß2 exhibited lower equilibrium moduli and apparent viscosities compared with similarly treated SC cells, but LIM cells and SC cells treated with 100 ng/mL TGF-ß2 had similar mechanical properties. Conclusions: The addition of 1 and 10 ng/mL TGF-ß2 can lower the equilibrium modulus and apparent viscosity of scleral fibroblasts in the normal eye.

DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori.

Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression. However, the mechanisms by which DNA methylation regulates these processes in insects remain unclear. Here, we studied the impacts of DNA methylation on early embryonic development in Bombyx mori. Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5' region of protein metabolism-related genes. The transcription factor gene zinc finger protein 615 ( ZnF615) was methylated by DNA methyltransferase 1 (Dnmt1) to be up-regulated and bind to protein metabolism-related genes. Dnmt1 RNA interference (RNAi) revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615. The same sites in the ZnF615 gene were methylated in ovaries and embryos. Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout. Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo. Thus, Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.

Study of the effects of rabbit scleral fibroblasts on cellular biomechanical properties and MMP-2 expression using two modes of riboflavin/ultraviolet A wave collagen cross-linking.

OBJECTIVE: The aim of this study is to evaluate the cellular biomechanical properties and MMP-2 expression changes in rabbit scleral fibroblasts using two modes of riboflavin and ultraviolet A (UVA) collagen cross-linking (CXL). METHODS: Twenty-four New Zealand white rabbits were randomly divided into two groups, A and B. The left eye was chosen for the experimental group and the right eye for the control group. In group A, the eyes were irradiated for 30 min, with a power density of 3.0 mW/cm2. In group B, the eyes were irradiated for 9 min, with a power density of 10.0 mW/cm2. One week after CXL, full-field electroretinography was performed. Sixty days after CXL, the rabbits were sacrificed, and scleral fibroblasts were extracted from the CXL-treated sclera area and corresponding parts of control sclera and cultured. Cellular biomechanical properties were evaluated using the micropipette aspiration technique, and the MMP-2 protein expression was determined by Western blot analysis. RESULTS: There was no statistical difference in the amplitude and latency of the dark adaptation 3.0 and light adaptation 3.0 between the CXL and control eyes of groups A and B (P > 0.05). Compared with the control groups, the Young's modulus of the fibroblasts and apparent viscosity of the experimental eyes in groups A and B were increased after CXL (P < 0.05), but there was no significant difference between the two groups under different irradiation modes (P > 0.05). The MMP-2 expression in scleral fibroblasts from experimental eyes was significantly higher than that in scleral fibroblasts from control eyes in groups A and B. Under the two different irradiation modes, the MMP-2 expression in the scleral fibroblasts from experimental eyes in group A was significantly higher than that in the scleral fibroblasts from experimental eyes in group B. CONCLUSION: The riboflavin-UVA scleral CXL conducted in two different modes produced no significant side effects on the retina and could strengthen the cell biomechanical properties as well as increase the MMP-2 expression of scleral fibroblasts significantly.

20E-mediated regulation of BmKr-h1 by BmKRP promotes oocyte maturation.

BACKGROUND: Krüppel homolog 1 (Kr-h1) is a critical transcription factor for juvenile hormone (JH) signaling, known to play a key role in regulating metamorphosis and adult reproduction in insects. Kr-h1 can also be induced by molting hormone 20-hydroxyecdysone (20E), however, the underlying mechanism of 20E-induced Kr-h1 expression remains unclear. In the present study, we investigated the molecular mechanism of Kr-h1 induction by 20E in the reproductive system of a model lepidopteran insect, Bombyx mori. RESULTS: Developmental and tissue-specific expression analysis revealed that BmKr-h1 was highly expressed in ovaries during the late pupal and adult stages and the expression was induced by 20E. RNA interference (RNAi)-mediated depletion of BmKr-h1 in female pupae severely repressed the transcription of vitellogenin receptor (VgR), resulting in the reduction in vitellogenin (Vg) deposition in oocytes. BmKr-h1 specifically bound the Kr-h1 binding site (KBS) between - 2818 and - 2805 nt upstream of BmVgR and enhanced the transcription of BmVgR. A 20E cis-regulatory element (CRE) was identified in the promoter of BmKr-h1 and functionally verified using luciferase reporter assay, EMSA and DNA-ChIP. Using pull-down assays, we identified a novel transcription factor B. mori Kr-h1 regulatory protein (BmKRP) that specifically bound the BmKr-h1 CRE and activated its transcription. CRISPR/Cas9-mediated knockout of BmKRP in female pupae suppressed the transcription of BmKr-h1 and BmVgR, resulting in arrested oogenesis. CONCLUSION: We identified BmKRP as a new transcription factor mediating 20E regulation of B. mori oogenesis. Our data suggests that induction of BmKRP by 20E regulates BmKr-h1 expression, which in turn induces BmVgR expression to facilitate Vg uptake and oogenesis.

Transcriptomic analysis reveals the regulation network of BmKrüppel homolog 1 in the oocyte development of Bombyx mori.

Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor, is involved in the metamorphosis and adult reproduction of insects. However, the role of Kr-h1 in reproduction of holometabolic insects remains to be elucidated. The regulation network of Kr-h1-associated genes in the reproduction in Bombyx mori was investigated in this study. The higher expression level of BmKr-h1 in the ovaries was detected during the late pupal stage and adults. RNA interference (RNAi)-mediated depletion of BmKr-h1 in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment, which showed less yolk protein deposition and partially transparent chorion. RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-h1 caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae. Totally, 27 genes coding for transcription factors were down-regulated, while six genes coding for other transcription factors were up-regulated. BmKr-h1 bound to the Kr-h1 binding site of the transcription factors AP-1 (activating protein-1) and FOXG1 to increase their messenger RNA transcripts in the BmN cells, respectively. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes. These data suggested that BmKr-h1 might directly regulate the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and /or FOXG1 to regulate oocyte development.