Characterization of Bombyx embryo segmentation process: expression profiles of engrailed, even-skipped, caudal, and wnt1/wingless homologues.

To gain insight into segmentation processes, the expression at embryonic stages of the silkmoth Bombyx mori homologues of even-skipped (eve), engrailed (en), caudal (cad), and wnt1/wingless (wg) transcripts were examined by whole mount in situ hybridization. Pair-rule eve stripes and segmental en and wnt1/wg stripes were generated sequentially from anterior to posterior, confirming the previous results that showed that Bombyx belongs to short-germ insects. However, unlike in previously described short germ insects, the segmentation of Bombyx occurred without marked germ band elongation: the putative growth zone was expanded compared with previously described short germ insects. This may indicate that Bombyx represents an evolutionarily intermediate state in a transition from short to long germ type. The expressions of cad and wnt1/wg, which are known to be present in the growth zone in short germ insects, initially showed a large median expression domain that, as segmentation proceeded, later retracted to the posterior pole. This is also unique to this insect. Detailed analysis of their relative expressions indicated that wnt1/wg domain retracted faster than the cad domain, and double stain in situ hybridization suggested that the eve stripe appears from cells that have ceased to express wnt1/wg. Another unique aspect of Bombyx embryogenesis is that gastrulation began at later embryonic stage compared with other insects and proceeded slowly from anterior to posterior. On the basis of these results, conserved and divergent aspects of the evolution of insect segmentation mechanisms and germ cell formation are discussed. 2009 Wiley-Liss, Inc.