We have isolated suppressors of the deletion allele of δ-tubulin, uni3-1, in the biflagellate green alga Chlamydomonas reinhardtii. The deletion of δ-tubulin produces cells that assemble zero, one or two flagella and have basal bodies composed primarily of doublet rather than triplet microtubules. Flagellar number is completely restored in the suppressed strains. Most of the uni3-1 suppressors map to the TUA2 locus, which encodes α2-tubulin. Twelve independent tua2 mutations were sequenced. Amino acids D205 or A208, which are nearly invariant residues in α-tubulin, were altered. The tua2 mutations on their own have a second phenotype - they make the cells colchicine supersensitive. Colchicine supersensitivity itself is not needed for suppression and colchicine cannot phenocopy the suppression. The suppressors partially restore the assembly of triplet microtubules. These results suggest that the δ-tubulin plays two roles: it is needed for extension or stability of the triplet microtubule and also for early maturation of basal bodies. We suggest that the mutant α-tubulin promotes the early maturation of the basal body in the absence of δ-tubulin, perhaps through interactions with other partners, and this allows assembly of the flagella.

The specific Ca2+ binding site that triggers contraction of molluscan muscle requires the presence of an essential light chain (ELC) from a Ca2+ binding myosin. Muscle myosins are highly conserved in overall morphology and subunit composition, consisting of two heavy chains, two essential light chains (ELCs), and two regulatory light chains (RLCs). The C-terminal portions of the heavy chains are wrapped around each other in an a-helical coiled-coil "tail," and the N termini form two pear-shaped globular "heads." Each head contains the actin binding sites, ATPase activity, and binding sites for one of each type of light chain. Despite these similarities, myosins from different muscle types are functionally distinct. A distinguishing feature of molluscan myosins is that each head possesses one specific, high-affinity

Numerous disorders that affect proper development, including the structure and function of the nervous system, are associated with altered embryonic movement. Ongoing challenges are to understand in detail how embryonic movement is generated and to understand better the connection between proper movement and normal nervous system function. Controlled manipulation of embryonic limb movement and neuronal activity to assess short- and long-term outcomes can be difficult. Optogenetics is a powerful new approach to modulate neuronal activity in vivo. In this study, we have used an optogenetics approach to activate peripheral motor axons and thus alter leg motility in the embryonic chick. We used electroporation of a transposon-based expression system to produce ChIEF, a channelrhodopsin-2 variant, in the lumbosacral spinal cord of chick embryos. The transposon-based system allows for stable incorporation of transgenes into the genomic DNA of recipient cells. ChIEF protein is detectable within 24 h of electroporation, largely membrane-localized, and found throughout embryonic development in both central and peripheral processes. The optical clarity of thin embryonic tissue allows detailed innervation patterns of ChIEF-containing motor axons to be visualized in the living embryo in ovo, and pulses of blue light delivered to the thigh can elicit stereotyped flexures of the leg when the embryo is at rest. Continuous illumination can disrupt full extension of the leg during spontaneous movements. Therefore, our results establish an optogenetics approach to alter normal peripheral axon function and to probe the role of movement and neuronal activity in sensorimotor development throughout embryogenesis.

Our goal is to understand the effects of altered embryonic movement and sensation on proper neural development. Previously, we have shown in chick embryos that persistent expression of channelrhodopsin in peripheral axons of the leg can be achieved via electroporation of a modified transposon‐based exrpression system and that illumination of the leg with a blue LED can activate leg movements and modulate spontaneous leg motility. We now extend this work to include the ability to achieve persistent expression in targeted neuronal populations including peripheral sensory or motor axons by controlling the age of the embryo electroporated and the directionality of the applied current field. Also, we have developed an inexpensive system for focusing light from brighter LEDs to confine illumination to selected nerves and to activate them at later stages of development as superficial tissues thicken. This allows us to assay the effects of selective activation of sensory or motor axons in specific nerves in the leg. Lastly, we are testing transposon‐based expression of Arch and Halorhodopsin in chick embryos for the ability to suppress excitation in peripheral nerves.