Investigating the role of Rab23 in vertebrate left-right patterning

Abstract

Proper asymmetric development of internal organs, such as the heart and lungs, depends on the formation of a left-right axis. In vertebrate embryos, patterning of the left-right axis requires expression of the TGF-beta Nodal in the left lateral plate mesoderm (LPM). Nodal expression in the LPM is dependent on Nodal expression in the node. While the exact function of Nodal produced in the node is unknown, recent evidence suggests that Nodal protein travels directly from the node to the LPM to induce its own expression. In this work, I demonstrate that the small GTPase Rab23 is involved in these early left-right patterning events that transfer asymmetric information generated in the node to the left LPM.

Rab23 mutant mouse embryos express Nodal in the node but fail to express Nodal or any Nodal target genes in the LPM. The requirement for Rab23 in establishing asymmetric gene expression is unexpected, given that the only known function of Rab23 is as an antagonist of the Hedgehog (Hh) signaling pathway. Rab23 mutant mice improperly activate the Hh pathway, yet increased levels of Hh signaling have not been shown to affect left-right patterning. To address whether Rab23 functions in the Hh pathway during left-right axis specification, I examined expression of Hh target genes in Rab23 mutants, and analyzed Nodal expression in embryos mutant for both Rab23 and the Hh pathway components Gli2 or Kif3a. My findings suggest that Rab23's role in left-right patterning is unrelated to its role in Hh signaling later in development. Further studies indicate that Rab23 is not required to generate asymmetric flow in the node, but functions downstream of nodal flow to regulate Nodal signaling. Injection of Nodal protein or transfection of a Nodal expression vector in Rab23 mutants did not activate Nodal appropriately in the LPM, indicating a possible role for Rab23 in Nodal signal production. Furthermore, perturbing Rab23 levels in zebrafish embryos resulted in abnormal initiation and possibly propagation of the Nodal homolog southpaw in the LPM, suggesting that Rab23 also regulates Nodal signaling in left-right patterning of the zebrafish.