We show that the learned vocalizations of male and female large-billed crows (Corvus macrorhynchos) are similar and that their functions and physical features show significant differences from those of other oscine species. We investigate whether the song control nuclei of crows show any sexual differences in size, reflecting differences in their singing behavior, and whether these nuclei are different from those of other songbirds in terms of neural connectivity size and relative to the forebrain. Our Nissl staining results reveal that 1) of the four song nuclei examined (HVC; the robust nucleus of the arcopallium [RA]; Area X; and the dorsolateral medial nucleus [DLM]), HVC, RA, and Area X volumes are significantly larger in males than in females, but DLM volume and body and brain weights show no significant gender differences; and 2) the sizes of song nuclei relative to the forebrain are within the range of other oscines. By injecting a neural tract tracer (DiI) into various song nuclei in brain slices, we found that, as in other songbirds, HVC projects to RA and Area X, while Area X projects to the lateral magnocellular nucleus of the anterior nidopallium (IMAN) and DLM, DLM to IMAN, and IMAN to RA. Our results Indicate that, although the crow has songs very different from those of other oscine species, Its song nuclei and the connections between them are not obviously different.

Song control nuclei have distinct sexual differences in songbirds. However, the mechanism that underlies the sexual differentiation of song nuclei is still not well understood. Using a combination of anatomical, pharmacological, genetic, and behavioral approaches, the present study investigated the role of erbb2 (a homolog of the avian erythroblastic leukemia viral oncogene homolog 2) and the erbb2-interacting gene, erbin, in the sexual differentiation of the song nucleus HVC in the Bengalese finch. We first found that both erbin and erbb2 were expressed in the developing HVC at posthatch day (PHD) 15 in a male-biased fashion using qRT-PCR and in situ hybridization. Following the addition of a pharmaceutical inhibitor of the ErbB2 signaling pathway to the culture medium, cell proliferation in the cultured ventricle zone (VZ) that overlies the developing HVC decreased significantly. After the injection of erbin-or erbb2-interfering lentiviruses into the HVC and its overlying VZ at PHD 15, the cell proliferation in the VZ at PHD 24, the number of the differentiated neurons (Hu 1 /BrdU 1 or NeuN 1 /BrdU 1 ) in the HVC at PHD 31 or PHD 130, and the number of RAprojecting cells at PHD 130 all decreased significantly. Additionally, the adult songs displayed serious abnormalities. Finally, 173 male-biased genes were expressed in the developing HVC at PHD 15 using cDNA microarrays, of which 27.2% were Z-linked genes and approximately 20 genes were involved in the Erbin-or ErbB2-related signaling pathways. Our results provide some specific genetic factors that contribute to neurogenesis and sex differentiation in a song nucleus of songbirds.

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