Credit: Photodisc

Imai et al. have identified a role for Pinhead gene transcription in specifying ventral fate in the sea squirt Ciona intestinalis. Furthermore, the genomic position of Pinhead — that is, adjacent to the bone morphogenic protein (Bmp) ligand antidorsalizing morphogenetic protein (Admp) — was found to be important for this specification, and this feature that looks likely to be conserved in animals.

Bmp signalling in dorsal–ventral specification is mediated by the spatially opposed expression of dorsal Admp and ventral Bmp2/4. Bmp2/4 locally induces ventral fate, whereas Admp migrates ventrally to induce ventral fate. To understand Bmp signalling further, the authors scanned the C. intestinalis genome for genes encoding cysteine-knot domains that are important in Bmp signalling. This scan identified the Pinhead gene, which is directly upstream of the Admp gene. During early dorsal–ventral axis specification, Pinhead was expressed ventrally and overlapped with Bmp2/4 expression but not with Admp. By carrying out morpholino antisense knockdowns of these three genes, the authors then showed them to be essential for specifying ventral fate and for regulating each other. The authors also showed that Pinhead protein directly interacts with Admp in vivo, thus it might act as an antagonist by sequestering this protein.

To investigate the regulatory relationship between Pinhead and Admp further, Imai et al. cloned a region that included both adjacent genes and their putative regulatory regions, replacing Pinhead with an RFP reporter gene and Admp with a GFP reporter gene. Deletion of the Admp upstream region recapitulated endogenous expression of Admp and Pinhead. However, deletion of the Pinhead upstream region resulted in ectopic ventral GFP expression and decreased RFP expression, indicating that Pinhead transcription interferes with Admp transcription. Finer deletions identified an enhancer upstream of Admp that they termed 'G', which enhanced Pinhead expression, and an enhancer they termed 'A', which was adjacent to this and essential for Admp expression. A 'P' enhancer was also identified upstream of Pinhead that is controlled by Admp and Bmp2/4. A similar construct in Medaka recapitulated these results, indicating conservation of a cis-acting mechanism.

To examine the three-dimensional conformation of the Pinhead and Admp region, the authors carried out chromosome conformation capture (3C) on embryos that were modified to have inverse upregulation and downregulation of Pinhead and Admp. When Pinhead was upregulated, the authors observed a strong interaction between the Pinhead upstream promoter region and the A and G enhancers. Thus, when Pinhead is transcribed, the Pinhead upstream region contacts the G enhancer, which in turn sequesters the A enhancer, which is essential for Admp expression.

The authors' data thus indicate dual negative feedback between Admp and Pinhead. Admp migrates ventrally to activate Pinhead, and in turn Pinhead transcription sequesters the Admp enhancer to prevent its transcription in ventral regions. Pinhead protein interacts with Admp protein to reinforce its repression. The conservation of the genomic architecture of Pinhead and Admp from arthropods to vertebrates suggests that this dual negative feedback is conserved.