Functional Analysis Of Bombyx Wnt1 During Embryogenesis

Wnt signalling is required for a wide range of developmental processes, including the development of embryogenesis, planar cell polarity, pigmentation patterning. The developmental process of segmengtation, appendages, pigmentation production and wings are controlled by Wnt signalling in Drosophila melanogaster(D. melanogaster). The domesticated silkworm, Bombyx mori(B. mori), is an economically valuable lepidopteran model insect. In B.mori, the research of Wnt signalling in B.mori is conducive to the understanding of the roles of Wnt signalling duing the process of embryogenesis and pigmentation patterning. Recently established, custom-designed nuclease technologies such as the Clustered Regularly Interspaced Short Palindromic Repeat(CRISPR) and associated Cas system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on D. melanogaster and the B. mori, little success has been reported, which is largely due to lack of effective genetic manipulation tools that can be used in other insect orders.To performe a functional analysis of Wnt signalling in B. mori, we establish a simple and effective method of gene knockout system, especially for dissecting gene functioning during insect embryogenesis using Cas9-sgRNA mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into B.mori embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. In addition, a large deletion of approximately 18 Kb was generated by combining two separate sgRNAs targeting BmWnt1. Quantitative real-time PCR(qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 deletion, which indicated that Wnt might control the segmentation and pigmentation through the Hox genes.Furthermore, we investigated the biological role of Ubx, Abd-A, Abd-B. The pigmentation patterning was disrupted after Ubx, Abd-A and Abd-B knockout. The current study not only expands our understanding of the biological function of Wnt signaling during the embryogenesis of B.mori, but also demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis.