Shaking hands is a homeodomain transcription factor that controls axon outgrowth of central complex neurons in the insect model Tribolium.

Gene regulatory mechanisms that specify subtype identity of central complex (CX) neurons are the subject of intense investigation. The CX is a compartment within the brain common to all insect species and functions as a 'command center' that directs motor actions. It is made up of several thousand neurons, with more than 60 morphologically distinct identities. Accordingly, transcriptional programs must effect the specification of at least as many neuronal subtypes. We demonstrate a role for the transcription factor Shaking hands (Skh) in the specification of embryonic CX neurons in Tribolium. The developmental dynamics of skh expression are characteristic of terminal selectors of subtype identity. In the embryonic brain, skh expression is restricted to a subset of neurons, many of which survive to adulthood and contribute to the mature CX. skh expression is maintained throughout the lifetime in at least some CX neurons. skh knockdown results in axon outgrowth defects, thus preventing the formation of an embryonic CX primordium. The previously unstudied Drosophila skh shows a similar embryonic expression pattern, suggesting that subtype specification of CX neurons may be conserved.

A Protocol for Double Fluorescent In Situ Hybridization and Immunohistochemistry for the Study of Embryonic Brain Development in Tribolium castaneum.

The red flour beetle, Tribolium castaneum, is an emerging model system well suited to the study of embryonic brain development and evolution (see Chapters 11 and 13 ). Brain genesis is driven by specific gene products whose expression underlies a tight spatiotemporal control. Therefore, the analysis of gene expression in time and space provides valuable insights into the molecular mechanisms that govern brain development. Since Tribolium-specific antibodies are scarce, fluorescent RNA in situ hybridization is the method of choice to determine the dynamics of individual gene expression. We have modified common RNA in situ protocols to facilitate the concomitant detection of two gene-specific expression patterns (double fluorescent RNA in situ). In addition, we describe a procedure which combines fluorescent single RNA in situ and immunostaining with gene-specific antibodies. Conventional in situ using RNA probes that are complementary to mature mRNAs often produce diffuse signals. We demonstrate that RNA in situ probes complementary to intronic gene sequences facilitate single cell resolution because the fluorescent signal is restricted to the nucleus. We believe our protocols can be adapted easily to suit the analysis of brain development in other insect species.