Genomic resources for songbird research and their use in characterizing gene expression during brain development.

Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3' ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30-50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis.

Contribution of neurons born during embryonic, juvenile, and adult life to the brain of adult canaries: regional specificity and delayed birth of neurons in the song-control nuclei.

Neurogenesis occurs in adult song birds, which suggests that neurons born after hatching may contribute to histogenesis and plasticity of the avian brain. However, little is known about the overall contribution to the mature brain of neurons born in juveniles and adults, and how this process affects different regions of the avian brain. In fact, studies of the histogenesis of the avian forebrain have made the classical assumption that neuronal birth ends before hatching. Here we determined the contribution of neurons born before and after hatching to different regions throughout the adult canary brain. Male canaries were injected with [3H]-thymidine at different times during embryonic, juvenile, and adult life. The position of labeled neurons was mapped in parasagittal brain sections. Because all birds were killed as adults, results indicate the time of birth of neurons that survived to adulthood in different structures of the avian brain. Injection at embryonic day (E) 5 or E9 resulted in labeled neurons in all regions of the neuroaxis. The vast majority of neurons outside of the telencephalon were born before E9. One exception was a discrete region in the dorsal thalamus, a part of the song-control circuit, where neurons continued to be born after E9. Most regions of the telencephalon had a high proportion of its neurons labeled by the embryonic injections. In particular, archistriatum, anterior neostriatum, and the hippocampus had most of their neurons labeled before hatching. This indicates that many of the telencephalic neurons born in the embryo are long lived and are not replaced by other neurons that continue to be added to the telencephalon after hatching. Neurons labeled by [3H]-thymidine injections after hatching were restricted to the telencephalon and contributed importantly to many regions. In particular, the avian striatum (lobus parolfactorius, LPO) received a large number of its neurons during the first 20 days of life, but continued to incorporate new neurons throughout juvenile and adult life. Neurons continued to be added to the telencephalon of adults (even in 4-year-old birds). The distribution of labeled neurons after [3H]-thymidine injections in adults was similar to that observed in latter stages of juvenile development. The contribution of neurons born at different ages from embryonic development to adulthood varied among different anatomical subdivisions of the canary brain. this could, in part, explain differences in the cytoarchitecture and plasticity between brain regions. Neurogenesis after hatching may allow the modification of selected brain circuits as the bird matures and ages.