Evolutionary Changes in Left-Right Visceral Asymmetry inAstyanaxCavefish

Vertebrates show conserved left-right (L-R) asymmetry of internal organs controlled by Nodal-Pitx2/Lefty signaling [1-3]. Modifications in L-R asymmetry occur in mutants [4] and rarely in humans [5], but little is known about natural L-R changes during evolution. Here we describe changes in L-R asymmetry inAstyanax mexicanus, a teleost with ancestral surface (surface fish) and derived cave (cavefish) morphs [6]. In teleosts, Nodal-Pitx2 signaling is activated in the left lateral plate mesoderm (LPM), the cardiac tube jogs to the left and loops to the right (D-looping), and the liver and pancreas form on opposite sides of the midline. Surface fish show conventional L-R patterning, but cavefish can show Nodal-Pitx2 expression in the right LPM or bilaterally, left (L)-looping hearts, and reversed liver and pancreas asymmetry, and these reversals have no effect on survival. The Lefty1 Nodal antagonist is expressed along the surface fish and cavefish midlines, but expression of the Lefty2 antagonist is absent in the LPM of most cavefish embryos, suggesting a role forlefty2(lft2) in changing organ asymmetry. Although CRISPR-Cas9lft2editing affected D-looping in surface fish, the cavefishlft2gene showed no coding mutations, and was expressed normally during cavefish gastrulation, suggesting downregulation by regulatory changes. Reciprocal hybridization, the fertilization of cavefish eggs with surface fish sperm andvice versa, indicated that the change in cavefish L-R asymmetry is a maternal genetic effect. Our studies reveal natural changes in internal organ asymmetry during evolution and introduceA. mexicanusas a new model to study the underlying mechanisms.