RESEARCH ARTICLE 3373 Development 139, 3373-3382 (2012) doi:10.1242/dev.083873 © 2012. Published by The Company of Biologists Ltd INTRODUCTION Bone morphogenetic proteins (BMPs) are important players in the dorsoventral differentiation of animal embryos (Niehrs, 2010), but how BMP signaling evolves with changes in tissue specification is not well understood. Flies (Diptera) provide an excellent opportunity to address this question because the BMP pathway of Drosophila melanogaster embryos has been studied in much detail (O’Connor et al., 2006; Umulis et al., 2009) and because extra-embryonic tissue specification, presumably under the control of the BMP pathway, has changed in dipteran evolution (Fig. 1) (Schmidt-Ott et al., 2010). Schizophoran flies, which include D. melanogaster, develop a single extra-embryonic tissue called the amnioserosa. This tissue is specified along the dorsal midline of the blastoderm and closes the dorsal side of the germband. In non-schizophoran dipterans, dorsal blastoderm folds over the gastrulating embryo and differentiates into two tissues: a cuticle-secreting serosa underneath the eggshell and an amnion that either lines the ventral side or closes the dorsal side of the germband (Goltsev et al., 2007; Rafiqi et al., 2008; Goltsev et al., 2009). Dorsal patterning of the dipteran embryo must have changed with the evolutionary transition from two extra-embryonic tissue types to one, but whether the transition involved altered BMP activity or genetic changes downstream or independent of this signaling pathway is unknown. In D. melanogaster, high levels of BMP activity are required to induce amnioserosa formation (Ray et al., 1991; Arora and Nusslein-Volhard, 1992; Ferguson and Anderson, 1992a; Wharton et al., 1993). This activity is induced by the extracellular ligands Decapentaplegic (Dpp) and Screw (Scw) (Padgett et al., 1987; Arora et al., 1994; Shimmi et al., 2005), which are secreted into the perivitelline space and transported in the presence of antagonists towards the dorsal midline (Francois et al., 1994; Ashe and Levine, 1999; Decotto and Ferguson, 2001; Eldar et al., 2002; Shimmi et al., 2005; Wang and Ferguson, 2005). Dorsally, BMP dimers are released from their antagonists to the transmembrane receptor proteins Thickveins (Tkv) and Saxophone (Sax), triggering phosphorylation of the transcription factor Mad and thereby BMP-dependent transcriptional gene regulation (Shimell et al., 1991; Ferguson and Anderson, 1992b; Marques et al., 1997; Shimmi et al., 2005; Wang and Ferguson, 2005). The BMP transport and release mechanism results in a shallow gradient of BMP activity, which broadly influences patterning in the dorsal ectoderm (Mizutani et al., 2006). Through a positive-feedback loop, the initially shallow gradient of BMP activity is transformed into a narrow and sharply delineated domain of high BMP activity (Wang and Ferguson, 2005; Umulis et al., 2006; Umulis et al., 2010), which, at this stage, becomes crucial for the expression of zerknüllt (zen) and hence amnioserosa specification (Rushlow et al., 2001; Liang et al., 2008). Dpp is essential for BMP activity and therefore controls the specification of all tissues that develop under the control of the BMP pathway in the early embryo, including the amnioserosa and dorsal ectoderm (Ferguson and Anderson, 1992b; Wharton et al., 1993). scw, a diverged paralog of glass bottom boat (gbb) (Van der Zee et al., 2008; Fritsch et al., 2011), boosts BMP activity along the dorsal midline and is required to generate high BMP activity for amnioserosa specification (Arora et al., 1994). University of Chicago, Department of Organismal Biology and Anatomy, CLSC 1061C, 920 E. 58th Street, Chicago, IL 60637, USA. *Present address: McGill University, Department of Biology, 1205 Dr Penfield Avenue, Montréal, Québec, H3A 1B1, Canada ‡ Present address: University of Heidelberg, Centre for Organismal Studies, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany § These authors contributed equally to this work ¶ Author for correspondence (uschmid@uchicago.edu) Accepted 21 June 2012 SUMMARY Bone morphogenetic protein (BMP) signaling is an essential factor in dorsoventral patterning of animal embryos but how BMP signaling evolved with fundamental changes in dorsoventral tissue differentiation is unclear. Flies experienced an evolutionary reduction of extra-embryonic tissue types from two (amniotic and serosal tissue) to one (amnionserosal tissue). BMP-dependent amnioserosa specification has been studied in Drosophila melanogaster. However, the mechanisms of serosal and amniotic tissue specification in less diverged flies remain unknown. To better understand potential evolutionary links between BMP signaling and extra-embryonic tissue specification, we examined the activity profile and function of BMP signaling in serosa and amnion patterning of the scuttle fly Megaselia abdita (Phoridae) and compared the BMP activity profiles between M. abdita and D. melanogaster. In blastoderm embryos of both species, BMP activity peaked at the dorsal midline. However, at the beginning of gastrulation, peak BMP activity in M. abdita shifted towards prospective amnion tissue. This transition correlated with the first signs of amnion differentiation laterally adjacent to the serosa anlage. Marker-assisted analysis of six BMP signaling components (dpp, gbb, scw, tkv, sax, sog) by RNA interference revealed that both serosa and amnion specification of M. abdita are dependent on BMP activity. Conversely, BMP gain-of-function experiments caused sharpened expression boundaries of extra-embryonic target genes indicative of positive feedback. We propose that changes in the BMP activity profile at the beginning of gastrulation might have contributed to the reduction of extra-embryonic tissue types during the radiation of cyclorrhaphan flies. KEY WORDS: Evolutionary development, Bone morphogenetic protein, Diptera BMP-dependent serosa and amnion specification in the scuttle fly Megaselia abdita Ab. Matteen Rafiqi*, Chee-Hyurng Park § , Chun Wai Kwan § , Steffen Lemke ‡ and Urs Schmidt-Ott ¶ DEVELOPMENT