Introduction 1997 Drosophila engrailed en wingless wg 1987 1988 1988 hedgehog hh 1990 1993 wg en 1993 en wg en hh en wg 1989 1994 1994 1994 1996 1998 2002 2003 2004 Drosophila wg Tribolium en wg–en 1999 wg en Oncopeltus faciatus 2005 Apis mellifera 2002 Lucilia sericata 2006 Tribolium 2006 wg hh Gryllus 2004 armadillo wg wg Drosophila 1991 1993 hedgehog hh patched ptc smoothened smo cubitus interruptus ci en wg 2006 Tribolium Drosophila, 1992 1992 1993 1989 1989 1996 1996 1996 1998 1997 1998 Cubitus Interruptus Ci 1995 ci 1997 ci wg ptc gooseberry decapentaplegic 1996 1996 1997 2001 hh ci smo ptc Tribolium. Tribolium wg ptc Tribolium en–wg–hh Materials and methods Beetle husbandry Tribolium castaneum 1989 Tribolium tc-hh tc-ci tc-smo tc-ptc Tribolium Tribolium hh smo ci ptc In situ hybridization and immunostaining in situ 1989 Tribolium 1994 2001 RNA interference (RNAi) 2007 1 4 2002 Microscopy and imaging Stained embryos and larval cuticles were documented with a Nikon Digital DXM 1200F camera on an Olympus BX50 microscope using Nikon ACT-1 version 2.62 software. Brightness and contrast of all images were adjusted and some were placed on a white background using Adobe Photoshop 7.0.1 software. Table 1 Summary of RNAi effects Gene DsRNA μg/μl Class I (%) Class II (%) Class III (%) Undeveloped (%) Wild type (%) n Tchh 3.0 0 6 83 11 0 464  1.5 18 73 0 9 0 477 Tcsmo 3.0 0 0 89 11 0 579  1.5 0 43 43 14 0 477  0.75 38 44 0 20 0 230 Tcci 3.0 0 33 61 5 0 694  1.5 0 30 63 7 0 405  0.75 73 19 0 8 0 293 Tcptc 4.0 0 76 – 24 0 271  2.0 13 68 – 19 0 189 Embryos were collected every 48 h for the first 2 weeks after injection. Phenotypic classes are unique for each gene. See text for details. Percentages have been rounded up. Results Tc-hh, Tc-smo Tc-ci Tc-hh 1 Tc-hh 1 1 Tc-hh 1 1 1 Tc-hh 1 1 1 Tc-hh Tc-hh Tc-hh 1 Fig. 1 Tc hh Tc-ci Tc-smo Tribolium a c Tc-hh d f Tc-hh purple Tc-En gold g h Tc-smo i k Tc-ci l m Tc-ci purple Tc-En gold a Tc-hh arrowhead b arrowheads c d gold Tc-hh purple Tc-hh e f Tc-hh arrowheads Tc-hh blue arrowhead g Tc-smo h Tc-smo arrowheads i Tc-ci arrowhead j k Tc-ci l gold Tc-ci purple m l a Tc-ci arrowhead  Tc-smo 1 Tribolium 1 smo Drosophila 1996 Tc-ci 1 Tc-ci 1 1 1 Tc-ci 1 Tc-ci Tc-ci 1 ci Bombyx mori 2002 Cupiennius salei 2002 Glomeris 2004 ci Tc-ci Tc-ci l ci Drosophila wg 1990 1997 ci 2002 Bombyx Glomeris Tribolium Tc-hh Tc-smo, Tc-ci Tc-hh Tc-smo Tribolium Tc-ci Tc-hh Tc-hh Tc-smo 1 1 2 Tc-hh Tc-smo 2 2 Tc-hh Tc-smo 2 Tc-hh Tc-smo 2 Tc-hh Tc-smo Tc-hh Tc-smo 2 Fig. 2 Tc-hh Tc-smo Tc-ci a d Tc-hh e h Tc-smo i l Tc-ci a Tc-hh b c arrowhead d e Tc-smo f g arrowhead h Tc-smo Tc-hh i Tc-ci j k Tc-hh Tc-smo Tc-ci  Tc-ci Tc-hh Tc-smo Tc-ci 2 Tc-ci 2 Tc-hh Tc-smo Tc-ci 2 2 Tc-hh Tc-smo 2 ci Drosophila hh 1999 3 3 3 3 Tc-hh Tc-smo Tc-ci 3 2 3 3 3 Drosophila 2006 Tc-wg Tc-wg Fig. 3 Tc-hh Tc-smo Tc-ci a c Tc-hh d f k Tc-smo g h Tc-ci i j a Tc-hh blue arrowhead black arrowhead b c d Tc-smo blue arrowhead black arrowhead e f g Tc-ci Tc-hh Tc-smo blue arrowhead black arrowhead h arrowhead i j k Tc-smo  hedgehog Drosophila 1980 wg hh smo Tc-hh Tc-smo Tribolium 2 Drosophila Tribolium Tc-ptc Tc-ptc 4 Tc-ptc 4 Tc-ptc 4 Tc-ptc Tc-ptc 4 ptc Drosophila 1989 1990 Fig. 4 Tc-ptc Tribolium. a c Tc-ptc d f Tc-ptc h m Tc-ptc a Tc-ptc b c gold Tc-ptc black lines Inset Tc-ptc arrowhead d Tc-ptc e f Tc-ptc g gold Tc-wg purple h Tc-wg Tc-ptc Tc-wg i Tc-wg j blue arrowhead k arrowhead h l arrowheads Tc-wg i m blue arrowhead black arrowhead j  Drosophila Tribolium Tc-ptc 1 4 4 4 Tc-ptc Tc-wg Tc-ptc Tc-wg 4 Tc-wg 4 Tc-wg 4 Tc-wg Tc-wg Tc-wg 4 Tc-wg 4 Tribolium Tc-wg Drosophila wg Drosophila ptc wg 1989 Drosophila Tribolium Discussion Tribolium Tribolium Drosophila Drosophila Tribolium, Tc-hh Tc-hh Tc-hh Tc-hh Tc-hh Tc-hh Tc-hh hh Drosophila 1992 1992 1993 Tc-hh Drosophila Tribolium 2007 Drosophila ptc ptc ptc ptc Tc-ptc Tc-ptc Tc-ptc Tribolium Drosophila Tribolium Tc-ci 1 Drosophila ci ci Tribolium Tc-ci Tc-ci Tc-ci Tribolium Tc-ci Tc-ci Tc-ci Drosophila ci ci 1995 1995 ptc Tribolium Drosophila Tc-ci Tribolium Functional analysis of Hh pathway component genes supports conserved roles in segment boundary formation. Drosophila hh smo ci 1980 Tribolium Tc-hh Tc-smo Tc-ci Tc-hh Tc-smo ci94 Drosophila 1995 1999 hh hh ci94 hh ci ci hh 2001 Drosophila pangolin wg 1998 1998 hh smo ci ci Tribolium Tc-ci Tc-hh Drosophila 1985 1989 Tribolium hh smo ci 2 Tc-wg Tc-wg Tribolium Tc-wg 2006 Tribolium 1994 Tc-hh Tc-smo Tc-ci Drosophila ptc 1980 Tribolium Tc-ptc Drosophila Tribolium ptc Drosophila 1988 Tribolium, Drosophila ptc 1988 ptc wg wg wg Tc-ptc Tribolium Tc-wg Tc-En Tc-ptc Tc-ptc Drosophila Tribolium Tc-ptc Drosophila ptc hh Gryllus bimaculatus 2004 hh hh ptc Tribolium Tc-wg hh–wg–en hh wg en Drosophila Tribolium Drosophila smo hh ci wg 2006 Tribolium Tc-wg Tc-hh Tc-smo and Tc-ci Tc-hh Tc-smo Tc-ci Tc-ptc Tribolium 2004 2005 2007 Tribolium Drosophila 2006 Drosophila Tribolium 2006 en wg Tribolium