FundingKelly A. Smith was supported by a Concordia fellowship from the Friends of the Hubrecht Institute. Research in the Jeroen Bakkers laboratory was supported by the Netherlands Organisation for Scientific Research (NWO/ALW). The bmpr1absa0028 zebrafish mutant was generated as part of the ZF-MODELS Integrated Project in the 6th Framework Programme, funded by the European Commission.

CitationBmp and Nodal Independently Regulate lefty1 Expression to Maintain Unilateral Nodal Activity during Left-Right Axis Specification in Zebrafish. Kelly A. Smith, Emily Noël, Ingrid Thurlings, Holger Rehmann, Sonja Chocron, Jeroen Bakkers. PLoS Genetics 7(9): e1002289. doi:10.1371/journal.pgen.1002289
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Abstract

In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning.

Author Summary

Although vertebrates are bilaterally symmetric when observed from the outside, inside the body cavity the organs are positioned asymmetrically with respect to the left and right sides. Cases where all the organs are mirror imaged, known as situs inversus, are not associated with any medical defects. Severe medical problems occur however in infants with a partial organ reversal (situs ambigious or heterotaxia), which arises during embryonic development. Left-right asymmetry in the embryo is established by unilateral expression of Nodal, a member of the Tgf-ß superfamily of secreted growth factors, a role that has been conserved from human to snails. By performing a genetic screen in zebrafish for laterality mutants, we have identified the linkspoot mutant, which displayed partial defects in asymmetric left-right positioning of the internal organs. The gene disrupted in the linkspoot mutant encodes a receptor for bone morphogenetic proteins (Bmp), another member of the Tgf-ß superfamily of secreted growth factors. Further analysis of Bmp over-expression or knock-down models demonstrate that Bmp signalling is required for unilateral Nodal expression, through the initiation and maintenance of an embryonic midline barrier. Our results demonstrate a novel and important mechanism by which left-right asymmetry in the vertebrate embryo is established and regulated.