A serine protease homologue Bombyx mori scarface induces a short and fat body shape in silkworm.

Body shape is one of the most prominent and basic characteristics of any organism. In insects, abundant variations in body shape can be observed both within and amongst species. However, the molecular mechanism underlying body shape fine-tuning is very complex and has been largely unknown until now. In the silkworm Bombyx mori, the tubby (tub) mutant has an abnormal short fat body shape and the abdomen of tub larvae expands to form a fusiform body shape. Morphological investigation revealed that the body length was shorter and the body width was wider than that of the Dazao strain. Thus, this mutant is a good model for studying the molecular mechanisms of body shape fine-tuning. Using positional cloning, we identified a gene encoding the serine protease homologue, B. mori scarface (Bmscarface), which is associated with the tub phenotype. Sequence analysis revealed a specific 312-bp deletion from an exon of Bmscarface in the tub strain. In addition, recombination was not observed between the tub and Bmscarface loci. Moreover, RNA interference of Bmscarface resulted in the tub-like phenotype. These results indicate that Bmscarface is responsible for the tub mutant phenotype. This is the first study to report that mutation of a serine protease homologue can induce an abnormal body shape in insects.

Ultrabithorax and abdominal-A specify the abdominal appendage in a dosage-dependent manner in silkworm, Bombyx mori.

In insects, there is a considerable diversity in leg distribution on the body, including number, segmental arrangement, morphological identity and consequent function, but the genetic basis for these differences is not well understood. Here by positional cloning, we showed that a ~355 kb region, including Bombyx mori Ultrabithorax (BmUbx) and abdominal-A (Bmabd-A), was responsible for the silkworm mutant Kh-extra-crescents-like (EKh-l) that displayed additional thoracic limb-like legs on the first abdominal segment (A1) and occasionally on the second abdominal segment (A2). We found that BmUbx gene was downregulated at both messenger RNA level and protein level in EKh-l embryo, while its expression domain in the EKh-l embryo was almost the same as that in the wild type. Whereas Bmabd-A was upregulated at both levels and was ectopically overexpressed on the supernumerary leg-bearing segments in EKh-l. Compared with the previously reported Ecs-l mutant in which increased expression of both BmUbx and Bmabd-A gave rise to ectopic proleg-like appendages on the same segments, we propose that overexpressed Bmabd-A gene is capable to promote the outgrowth of extra leg appendages on A1 and A2 segments, whereas BmUbx gene is required to specify accurate morphologies of the ectopic legs in a dosage-dependent manner in silkworm. These results provide insights into how these hox genes regulate the leg morphologic diversity on the same segments.

Morphological characterization and molecular mapping of an irradiation-induced Speckled mutant in the silkworm, Bombyx mori.

Speckled (Spc), an X-ray-induced lethal mutant of Bombyx mori, exhibits a mosaic dark-brown-spotted larval epidermis in both sexes and egg-laying problems only in females. Here, we report the morphological characterization and molecular mapping of the Spc mutant. Morphological investigations revealed that the epidermal ultrastructure of the small, dark-brown spots was more dense than that of the white regions in both Spc/+ mutants and wild type, and that the lethality of the Spc/Spc mutants occurred during early embryogenesis. Furthermore, the ovarioles and ovipositor were disconnected in approximately 85.5% of Spc/+ females, a further 2.5% had a connection between the ovarioles and ovipositor that was too narrow to lay eggs. The remaining females showed a normal connection similar to that of the wild type. We successfully narrowed down the location of the Spc mutation to a region on chromosome 4 that was ∼1041 kb long. Gene-prediction analysis identified 25 candidate genes in this region. Chromosome structure analysis indicated that a ∼305 kb deletion was included in the mapping region. Temporal and spatial reverse transcription PCR (RT-PCR) analysis showed that several genes in the mapped region are associated with the Spc mutant. Although the genes responsible for the Spc mutation were not definitively identified, our results further the current understanding of the complex mechanism underlying the multiple morphological defects in Spc mutants.

Analysis of interaction between Bmhrp28 and BmPSI in sex-specific splicing of Bombyx mori Bmdsx gene.

Bombyx mori BmHRP28 and BmPSI, which belong to the family of RNA-binding proteins, have been identified binding to the female-specific exon 4 of the sex-determining gene Bmdsx pre-mRNA. However, the relationships between BmHRP28 and BmPSI still remain unclear. In this study, we carried out yeast two-hybrid (Y2H) and co-immunoprecipitation (Co-IP) analyses to address them. Y2H analysis showed that there was little or no direct binding between the BmHRP28 and BmPSI proteins. Also, the Co-IP experiments revealed that BmHRP28 and BmPSI coexisted in a multiprotein complex. Our results suggested that BmHRP28 and BmPSI form a muliprotein complex to regulate the splicing of Bmdsx pre-mRNA, but are not directly bound to each other. In an effort to find other regulatory factors in the multiprotein complex, we constructed a silkworm Y2H cDNA library of male early embryo. By Y2H screening, we identified an RNA-binding protein BmSPX, a putative component of the spliceosome, binding to BmPSI. These results indicated that BmHRP28 and BmPSI make up a spliceosome complex to regulate Bmdsx splicing and that BmSPX is another potential protein involved in this process. Our study provides some clues to better understand the mechanism of sex determination in the silkworm.

Fine mapping of a supernumerary proleg mutant (E(Cs) -l) and comparative expression analysis of the abdominal-A gene in silkworm, Bombyx mori.

Patterning and phenotypic variations of appendages in insects provide important clues on developmental genetics. In the silkworm Bombyx mori, morphological variations associated with the E complex, an analogue of the Drosophila melanogaster bithorax complex, mainly determine the shape and number of prolegs on abdominal segments. Here, we report the identification and characterization of the allele responsible for the supernumerary crescents and legs-like (E(Cs) -l) mutant, a model derived from spontaneous mutation of the E complex, with supernumerary legs and extra crescents. Fine mapping with 1605 individuals revealed a ∼68 kb sequence in the upstream intergenic region of B. mori abdominal-A (Bmabd-A) clustered with the E(Cs) -l locus. Quantitative real-time PCR (qRT-PCR) and Western blotting analyses disclosed a marked increase in Bmabd-A expression in the E(Cs) -l mutant at both the transcriptional and translational levels, compared to wild-type Dazao. Furthermore, we observed ectopic expression of the Bmabd-A protein in the second abdominal segment (A2) of the E(Cs) -l mutant. Our results collectively suggest that the 68 kb region contains important regulatory elements of the Bmabd-A gene, and provide evidence that the gene is required for limb development in abdominal segments in the silkworm.

Antennapedia is involved in the development of thoracic legs and segmentation in the silkworm, Bombyx mori.

Homeotic genes, which are associated closely with body patterning of various species, specify segment identity. The Wedge eye-spot (Wes) is a new homeotic mutant located on the sixth linkage group. Homozygous Wes/Wes embryos are lethal and display a pair of antenna-like appendages under the mouthparts as well as fused thoracic segments. These mutants also exhibit a narrower eye-spot at the larval stage compared with the wild type. By positional cloning, we identified the candidate gene of the Wes locus, Bombyx mori Antennapedia (BmAntp). Two BmAntp transcripts were identified in the homozygote of the Wes mutant, including a normal form and an abnormal form with a 1570-bp insertion. Our data showed that the insertion element was a long interspersed nuclear element (LINE)-like transposon that destroyed the original open reading frame of BmAntp. Quantitative RT-PCR analysis showed that the expression levels of normal BmAntp transcripts were increased markedly in the Wes heterozygous larvae compared with the wild type. Furthermore, we performed RNAi of BmAntp and observed fused thoracic segments and defective thoracic legs in the developing embryos. Our results indicated that BmAntp is responsible for the Wes mutant and has an important role in determining the proper development of the thoracic segments. Our identification of a homeotic mutation in the silkworm is an important contribution to our understanding of the regulation of Hox genes at different levels of expression.

Identification and function of Abdominal-A in the silkworm, Bombyx mori.

Abdominal-A (adb-A) is a key gene in the development of insects. To understand its function in the silkworm, we cloned 1193 bp of the abd-A gene of Bombyx mori (Bmabd-A), including the complete coding sequence and part of the 3' untranslated region sequence. Bmabd-A has at least three mRNA splice variants with coding sequences of lengths 1032, 1044 and 1059 bp, encoding 343, 347 and 352 amino acids, respectively. Each splice variant of Bmabd-A has three exons and differs only in second exon size. Bmabd-A was expressed at low levels in unfertilized eggs, but increased gradually in fertilized eggs after laying 22 h. Bmabd-A expression decreased in ant silkworms (newly hatched silkworms). After RNA interference for Bmabd-A, the embryos had two mutant phenotypes, either completely or partially absent abdominal feet from the third to sixth abdominal segments, suggesting that Bmabd-A is responsible for normal development of the third to sixth abdominal segments during embryonic development.

Identification and expression of the achaete-scute complex in the silkworm, Bombyx mori.

Recently, the study of achaete-scute (AS-C) homologues has contributed enormously to understanding of gene duplication and function evolution, particularly in Diptera. We identified four AS-C homologue genes in the silkworm, Bombyx mori, referred to as BmASH, BmASH2, BmASH3, and Bmase. The complex displayed tandem array structure in the genome. Analysis of spatial expression profiles showed that they all were expressed in obviously higher levels in wing disc than in other tissues, suggesting that they might play important roles in the development of the wing. Furthermore, we found that their expression profiles in the wing discs were mostly correlated with the development of the scales, especially the BmASH gene. RNA interference results further indicated that BmASH was necessary for scale formation in silkworm wing.

[Construction of silkworm RAPD molecular linkage map].

In this research, a RAPD linkage map of Bombyx mori was constructed with Dazao/C108 and their F2 generation. The map consists of 182 RAPD loci, of which 103 loci come from Dazao and from the first 23 linkage groups and the other 79 loci come from C108 and form the second 16 linkage groups. This map covered a total genetic distance of over 1,148.3 cM (centimorgan). It could be integrated with the SADF map of the same materials constructed in our laboratory and the corresponding RFLP linkage map.

[Effect of scald on gene transcription and content of endothelin-1 in supraoptic nucleus of rat hypothalamus].

Endothelin-1 (ET-1) gene transcription and endothelin-1-immunoreactivity (ET-1-ir) in the supraoptic nucleus (SON) of rat hypothalamus were respectively observed by in situ hybridization and immunohistochemistry after scald. Intensity of ET-1 mRNA and endothelin-1-immunoreactivity (ET-1-ir) was quantified by image analysis. Compared with the control (sham scald) group, no significant change in the intensity of ET-1 mRNA positive hybridization signals in SON was found 15 min post-scald, while there was a 35.1% increase in the positive hybridization signal intensity 60 min post-scald (P<0.05) and a 62.4% increase 180 min post-scald (P<0.01). The content of ET-1-ir in SON decreased significantly to 8.5% of the control 15 min post-scald (P<0.01), and gradually recovered to 31.5% and 52.4% of the control 60 min and 180 min post-scald respectively, though still significantly lower than the control (P<0.01). Pre- and post-scald ET-1 gene transcription in rat hypothalamus was also measured by Northern blot hybridization. No significant difference in the quantity of ET-1 mRNA was found between 15 min post-scald data and those of the control. The quantity increased to a significantly higher level 60 min post-scald (P<0.05) and further increased to 2.5 creased to 2.5 fold of the control 180 min post-scald (P<0.05). In addition, the Northern blot hybridization showed that the post-scald size of ET-1 mRNA remained unchanged despite of the increase in quantity. In view of the neuroendocrine role of SON, the changes in ET-1 mRNA and ET-1-ir in SON resulting from scald suggest that ET-1 may play an important role in neuroendocrine reactions following scald.

Detection and analysis of alternative splicing in the silkworm by aligning expressed sequence tags with the genomic sequence.

We identified 277 alternative splice forms in silkworm genes based on aligning expressed sequence tags with genomic sequences, using a transcript assembly program. A large fraction (74%) of these alternative splices are located in protein-coding regions and alter protein products, whereas only 26% are in untranslated regions. From the alternative splices located in protein-coding regions, some (43%) affect protein domains that bind various biological molecules. The vast majority of the detected alternative forms in this study appear to be novel, and potentially affect biologically meaningful control of function in silkworm genes. Our results indicate that alternative splicing in silkworm largely produces protein diversity and functional diversity, and is a widely used mechanism for regulating gene expression.

[Effects of burn on synthesis and secretion of endothelin-1 in rat paraventricular nucleus of hypothalamus].

AIM AND METHODS: In order to investigate the pathophysiological role of ET-1 in paraventricular nucleus of hypothalamus (PVH) under burn, changes in synthesis and secretion of endothelin-1 (ET-1) in PVH after burn were observed by in situ hybridization and immunohistochemistry. The intensity of ET-1 mRNA and ET-1-immunoreactivity (ET-1-ir) were quantized by image analysis. RESULTS: Compared with the control group (sham burn, 100% +/- 25%), no significant change in the intensity of ET-1 mRNA positive hybridization signal in PVH was found at 15 min postburn, while the intensity increased significantly both at 60 min (138% +/- 26%, P < 0.05) and at 180 min postburn (167% +/- 18%, P < 0.01). Intensity of ET-1-ir in the neurons in PVH decreased significantly at 15 min postburn to 6.3% +/- 1.5% of the control (P < 0.01) and gradually recovered to 23.1% +/- 2.9% and to 44.1% +/- 3.8% at 60 min and 180 min postburn respectively, while still significantly lower than that in control (P < 0.01). CONCLUSION: The results demonstrated that the synthesis and secretion of ET-1 in rat PVH increased significantly after burn in rats, suggesting that ET-1 in PVH played an important pathophysiological role under burn.