We have developed a method for dechorionation and devitellinization of the silkworm eggs without damage, to facilitate the analysis of gene expression during embryonic development of Bombyx mori. Making use of antibodies available from heterologous systems, the spatio-temporal expression patterns of peroxidase and proliferating cell nuclear antigen have been directly visualized in whole mount embryos at various stages of development without the need for generating transformed ·lines carrying specific reporter constructs. The B. mori system, previously unamenable for such studies, could thus serve as an attractive model for molecular analysis of insect development.
The attention lavished on Drosophila melanogaster as the insect species par excellence for the genetic and molecular analysis of development, has eclipsed the earlier investigations on most other insects for a multitude of challenging biological phenomena. The mulberry silkworm, Bombyx mori has a genetic legacy comparable to that of Drosophila, but most of the efforts on the silkworm were channelled towards the improvement of the race through breeding, due to its economic importance.
The extended period of embryonic development of B. mori (10 days as compared to 24 h in Drosophila and l 00 h in Manduca sexta, another widely studied Lepidopteran insect) provides an advantage to discretely analyse without overlap, the events leading to pattern formation. The early investigations on B. mori were confined to the problems of diapause and storage of embryos or towards the elucidation of morphological landmarks by electron microscopy and fluorescent vital dyes. Recently the fate mapping of B. mori embryo has been carried out following laser irradiation. The investigations on gene expression during early embryonic development of silkworm have lagged behind because the dechorionation and devitellinization of embryos proved to be major hurdles. Besides, the embryonic development could not be examined due to the nonavailability of cell type-specific molecular markers as well as the lack of transgenic methodologies. In such situations the immunocytochemical approaches provide an alternative. Antibodies against gene products can be exploited as tissue- and cell-specific markers to analyze phenotypes in mutant embryos. Recent studies have implicated evolutionary conservation, based on sequence similarity of developmentally important genes, in a range of distantly related species. As a consequence, immunologic probes from heterologous sources could prove to be useful in detailed tracking of individual gene products during development. There is a distinct need. however, to adopt the existing methodologies to the B. mori system, where a major hurdle exists due to the presence of a thick chorion layer encasing the egg which affects the permeability of the embryo to external probes.
Here, we have improvised a protocol for the dechorionation and devitellinization of B. mori embryos to facilitate the analysis of spatio-temporal expression of genes during development. We have also exploited the heterologous antibodies against Drosophila gene products to examine the pattern of gene expression during embryonic development of silkworm and demonstrate that the system is now amenable for such analysis.
Available at: http://works.bepress.com/amit_singh/5/