BMP inhibition by DAN in Hensen's node is a critical step for the establishment of left–right asymmetry in the chick embryo

Abstract

During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.

Expression and Functional Study of Extracellular BMP Antagonists during the Morphogenesis of the Digits and Their Associated Connective Tissues

“…Previous analysis in different systems has provided controversial information concerning the targets of DAN [38], [51], [52]. It has been proposed that the production of bioactive DAN protein is cell-specific [52].…”

The DAN family: Modulators of TGF‐β signaling and beyond

“…For the known, strong BMP antagonists in the DAN family, including Gremlin, PRDC, and Coco (Grem1, Grem2, and Grem3, respectively), their target affinity and ability to inhibit BMP signaling and downstream SMAD 1/5/8 phosphorylation is robust, showing preference for BMP2, BMP4, and BMP7 . In addition, Nbl1 also shows preference for BMP2 and BMP4, as well as GDF5 (growth and differentiation factor 5 or BMP14), although with reduced affinity . For the less robust BMP antagonists within the DAN family, results and conclusions are often conflicting, bringing distortion to our perception of their true activities.…”

Opposing Nodal and BMP Signals Regulate Left–Right Asymmetry in the Sea Urchin Larva

“…For example, bmp4 is expressed on the right side of Hensen's node in the chick embryo and initiates a right-sided signaling cascade [9]. Although bmp genes are expressed symmetrically in the LPM of the mouse and chick embryos, BMP activity is greater on the right side due to the presence of BMP antagonists on the left side [10]–[13]. A BMP/ALK2/Smad-mediated signaling pathway is also proposed to be active on the right side of the Xenopus embryo [14].…”