Cross-Regulation and Integration of Nodal and Bmp Signals Restricts Asymmetric Nodal and Establishes Cardiac Asymmetries During Left-Right Patterning in Zebrafish

Abstract

The establishment of differences along the left-right (L/R) axis is critically important for proper placement and morphogenesis of vertebrate organs and it is known that left-restricted Nodal pathway activation plays a conserved role in this process. However, the regulations that restrict Nodal asymmetrically throughout development and the mechanisms by which this molecular difference along the L/R axis is interpreted by tissues to result in morphological asymmetries are not well understood. To gain insight into these questions, I have studied Nodal pathway regulation and cardiac morphogenesis during L/R patterning in zebrafish. I have identified two molecular barriers to Nodal propagation across the L/R axis of the embryo that act in parallel with the known midline barrier function of the Nodal antagonist, Lefty1. In the posterior, Bmp signaling is required to limit Nodal responsiveness in tissue that connects the left and right LPMs, thereby preventing spread of Nodal pathway activation across the midline of the embryo. In the anterior, the Nodal antagonist Lefty2 is necessary in the left cardiac field to prevent ectopic Nodal propagation through the cardiac tissue at the midline and down the right LPM. In addition, I find that this left-restriction of Nodal signaling is necessary for correct establishment of cardiac asymmetries. Through time lapse imaging of cardiac cell movements, I identified an asymmetric migration event required to convert the symmetric "cardiac cone" into the asymmetric linear heart tube and show that the L/R directionality of this migration is dependent upon the laterality of Nodal signaling in the LPM. This migration event leads to a conversion of the L/R axis of the cone into the dorsal-ventral axis of the linear heart tube. Fate mapping experiments have revealed that the original L/R axis is later restored in the heart prior to the conserved asymmetric event of dextral looping through a second rotation of the cardiac tissue. Finally, I have found that Nodal provides the dominant signal for cardiac laterality by increasing migration velocities asymmetrically on the left while Bmp signaling plays a secondary role in this process by limiting migration rates on the right. Interestingly, both pathways require the activity of the "Nodal" transcription factor FoxH1, as midway mutants lacking FoxH1 activity exhibit loss of Nodal target gene expression, as well as Bmp pathway activation, in the cardiac tissue. This work has provided new insights into the regulations necessary to restrict Nodal activity to the left of the embryo and have shed light on the mechanism by which the Nodal pathway instructs asymmetric morphogenesis.