Bombyxin gene expression in tissues other than brain detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and in situ hybridization

Abstract

Bombyxin is a 5 kDa insulin-related peptide produced in four pairs of medial neurosecretory cells in the brain of the silkmoth Bombyx mori. We demonstrate here the presence of bombyxin mRNA in tissues other than brain: ganglia, epidermis, testis, ovary, fat body, silk gland, Malpighian tubule, midgut, and hindgut of the Bombyx fifth instar larvae. Bombyxin mRNA was detected by Oligotex reverse transcription-polymerase chain reaction (RT-PCR), a rapid and simple procedure of reverse transcription-PCR, and in situ hybridization. The Oligotex RT-PCR method effectively eliminated the contaminating DNA in RNA samples and amplified bombyxin mRNA efficiently. In situ hybridization of the Bombyx ovary clearly demonstrated the localization of the bombyxin mRNA in the ovariole. The present study is the first demonstration of expression of brain neurosecretory peptide in tissues other than the central nervous system in insects at RNA level.

Bombyxin: An Insect Brain Peptide that Belongs to the Insulin Family

“…The brain is the only tissue that expresses the bombyxin gene throughout Bombyx development from the embryonic to adult stages when analyzed by northern hybridization (Adachi et al, 1989;Iwami, 1990). Reverse transcription-PCR analysis, however, demonstrated the presence of bombyxin family A and B mRNAs in all larval tissues examined although the levels of expression were very low (Iwami et al, 1996b). The bombyxin mRNA expression was detected in ganglia, epidermis, testis, ovary, fat body, silk gland, Malpighian tubule, midgut, and hindgut of the fifth instar larvae, which indicates the ubiquitous expression of bombyxin mRNA in the larvae.…”

“…The bombyxin gene is expressed predominantly in the brain and at low levels in a number of other tissues (Iwami et al, 1996b), in contrast to the insulin gene which is expressed in the gastroenteric organs and is almost silent in the brain. The brain is the only tissue that expresses the bombyxin gene throughout Bombyx development from the embryonic to adult stages when analyzed by northern hybridization (Adachi et al, 1989;Iwami, 1990).…”

“…5c (Iwami et al, 1996b). The cells of the inner surface of an ovariole and the cell layers beneath the ovarial capsule also express the bombyxin mRNA.…”

Vitellogenesis and Post-Vitellogenic Maturation of the Insect Ovarian Follicle

“…Bombyxin-binding activity is detected in ovarian follicle membrane extracts, indicating that ovarian tissue is a direct target tissue of bombyxin peptides (Fullbright et al, 1997). Furthermore, the expression of bombyxin mRNAs in ovarian tissue (Iwami et al, 1996) also indicates a possible autocrine or paracrine role for bombyxins during follicle development. A gene encoding a putative receptor for bombyxins, the Bombyx insulin receptor-like (BmIRL) protein, was cloned (Figure 14; I. Lindströ m-Dinnètz and K.I., unpublished data), and the expression pattern of its mRNA during follicle development determined (Swevers f0065 Figure 13 Schematic overview of the expression of the mRNAs of regulatory factors during follicular development in Bombyx mori as detected by Northern blot and quantified by Phosphorimager analysis.…”

Anuran Calcitonins are Diverse in Lower Vertebrates

“…Total RNA was extracted using an RNA isolation kit (Nippon gene, Toyama), and subjected to a RT-PCR kit (Takara, Kyoto) according to the method of Iwami et al (1996). Figure 1A shows locations of primers used in the RT-PCR method.…”