Chiral blastomere arrangement dictates zygotic left–right asymmetry pathway in snails

Most animals display internal and/or external left–right asymmetry. Several mechanisms for left–right asymmetry determination have been proposed for vertebrates^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} and invertebrates^{1, 2, 4, 9, 11, 12, 13, 14} but they are still not well characterized, particularly at the early developmental stage. The gastropods Lymnaea stagnalis and the closely related Lymnaea peregra have both the sinistral (recessive) and the dextral (dominant) snails within a species and the chirality is hereditary, determined by a single locus that functions maternally^{15, 16, 17, 18}. Intriguingly, the handedness-determining gene(s) and the mechanisms are not yet identified. Here we show that in L. stagnalis, the chiral blastomere arrangement at the eight-cell stage (but not the two- or four-cell stage) determines the left–right asymmetry throughout the developmental programme, and acts upstream of the Nodal signalling pathway. Thus, we could demonstrate that mechanical micromanipulation of the third cleavage chirality (from the four- to the eight-cell stage) leads to reversal of embryonic handedness. These manipulated embryos grew to 'dextralized' sinistral and 'sinistralized' dextral snails—that is, normal healthy fertile organisms with all the usual left–right asymmetries reversed to that encoded by the mothers' genetic information. Moreover, manipulation reversed the embryonic nodal expression patterns. Using backcrossed F₇ congenic animals, we could demonstrate a strong genetic linkage between the handedness-determining gene(s) and the chiral cytoskeletal dynamics at the third cleavage that promotes the dominant-type blastomere arrangement. These results establish the crucial importance of the maternally determined blastomere arrangement at the eight-cell stage in dictating zygotic signalling pathways in the organismal chiromorphogenesis. Similar chiral blastomere configuration mechanisms may also operate upstream of the Nodal pathway in left–right patterning of deuterostomes/vertebrates.

ational design of a structural and functional nitric oxide reductase

Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally^{1, 2, 3}, it is more difficult to design functional proteins^{4, 5, 6, 7, 8}. In comparison to recent successes in designing non-metalloproteins^{4, 6, 7, 9, 10}, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes^{5, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20}. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem Fe_B centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.