Robert J. Agate

Neural, not gonadal, origin of brain sex differences in a gynandromorphic finch

In mammals and birds, sex differences in brain function and disease are thought to derive exclusively from sex differences in gonadal hormone secretions. For example, testosterone in male mammals acts during fetal and neonatal life to cause masculine neural development. However, male and female brain cells also differ in genetic sex; thus, sex chromosome genes acting within cells could contribute to sex differences in cell function. We analyzed the sexual phenotype of the brain of a rare gynandromorphic finch in which the right half of the brain was genetically male and the left half genetically female. The neural song circuit on the right had a more masculine phenotype than that on the left. Because both halves of the brain were exposed to a common gonadal hormone environment, the lateral differences indicate that the genetic sex of brain cells contributes to the process of sexual differentiation. Because both sides of the song circuit were more masculine than that of females, diffusible factors such as hormones of gonadal or neural origin also likely played a role in sexual differentiation.

Transgenic songbirds offer an opportunity to develop a genetic model for vocal learning

Zebra finches are widely used for studying the basic biology of vocal learning. The inability to introduce genetic modifications in these animals has substantially limited studies on the molecular biology of this behavior, however. We used an HIV-based lentivirus to produce germline transgenic zebra finches. The lentivirus encoded the GFP regulated by the human ubiquitin-C promoter [Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) Science 295:868–872], which is active in a wide variety of cells. The virus was injected into the very early embryo (blastodisc stage) to target the primordial germline cells that later give rise to sperm and eggs. A total of 265 fertile eggs were injected with virus, and 35 hatched (13%); 23 of these potential founders (F0) were bred, and three (13%) produced germline transgenic hatchlings that expressed the GFP protein (F1). Two of these three founders (F0) have produced transgenic young at a rate of 12% and the third at a rate of 6%. Furthermore, two of the F1 generation transgenics have since reproduced, one having five offspring (all GFP positive) and the other four offsping (one GFP positive).

A cDNA microarray from the telencephalon of juvenile male and female zebra finches.

Studies over roughly the last decade have emphasized the importance of gene expression in the development of structure and function of the songbird forebrain. However, few tools have been available to efficiently identify the critical factors. To that end, we have produced a normalized cDNA library from juvenile zebra finch telencephalon, and have spotted inserts from 2400 randomly selected cDNA clones on microarrays (1664 unique sequences). We have also added several previously cloned cDNAs of interest, including three representing genes encoded on sex chromosomes. Hybridizations comparing Cy3- and Cy5-labeled cDNA from the telencephalon of day 25 male and female zebra finches confirmed sexually dimorphic expression of the Z- and W-linked genes, demonstrating the utility of these microarrays for detecting differential expression and providing information about the relative expression of these genes in the brains of juveniles of this age.

Cloning and Expression of Zebra Finch (Taeniopygia guttata) Steroidogenic Factor 1: Overlap with Hypothalamic but Not with Telencephalic Aromatase

Abstract The zebra finch (Taeniopygia guttata) brain is highly sexually dimorphic. The organization and production of sex-specific song is considerably influenced by estrogens and androgens. Because the brain itself expresses several steroidogenic enzymes, the local production of sex steroids may contribute to sex differences in neural development. Sex steroid production in gonads is directed by a master regulatory factor, steroidogenic factor 1 (SF1). We have identified a cDNA encoding the homologue of SF1 in the zebra finch and utilized reverse transcription-polymerase chain reaction and in situ hybridization to examine early and late developmental expression of SF1 in brain and in early gonadal development. We found that SF1 is expressed early in embryonic development in the Rathke pouch, beginning at stage 15 and extending to at least stage 27 in both males and females. The earliest expression of SF1 in gonads was found at stage 17 for both males and females and extended to at least stage 27. In brain, we assessed SF1 mRNA expression in posthatch and adult telencephalon, and we compared SF1 and aromatase mRNA expression in adult hypothalamus. In the telencephalon and hippocampus, aromatase was expressed independently of SF1, whereas in the hypothalamus, aromatase and SF1 expression were more closely associated. Expression of SF1 and of aromatase overlapped in restricted regions of the hypothalamus, suggesting that SF1 may regulate aromatase expression in these regions. These findings suggest that steroidogenesis in the zebra finch brain may be regulated by both SF1-dependent and SF1-independent mechanisms. No sex differences were detected in SF1 expression in brain.

FnTm2, a novel brain-specific transcript, is dynamically expressed in the song learning circuit of the zebra finch

Zebra finch males learn their song by imitation, a process influenced by social variables. The neural pathways for acquisition and production of learned song are known, but the cellular and molecular underpinnings are not. Here we describe a novel gene named “FnTm2” (“Phantom 2”) that is predicted to encode a small protein (220 aa) with a single fibronectin type III domain and a single transmembrane domain. This gene shows great variability in its expression in song system neurons of the anterior forebrain pathway (AFP), a circuit that influences song discrimination and is necessary for normal song acquisition. AFP nuclei that express FnTm2 include the nucleus HVC (its Area X‐projecting neurons), Area X, and LMAN (core and shell). FnTm2 expression does not correlate with singing behavior like the immediate early gene ZENK. It is expressed variably during sleeping hours and is not dependent on an intact song circuit. FnTm2′s expression is sensitive to hearing, because in deafened birds its expression is substantially reduced in the core of LMAN. Furthermore, a comparison of FnTm2 expression between mice and zebra finches revealed a conserved pattern of expression in the “limbic system.” We suggest that FnTm2 may be sensitive to affective and/or attentional states and thus may provide insights on how social variables influence the production and discrimination of learned vocalizations. J. Comp. Neurol. 504:127–148, 2007.

Erratum to “A cDNA microarray from the telencephalon of juvenile male and female zebra finches”: [J. Neurosci. Methods 138 (2004) 199–206]

a Departments of Psychology and Zoology and Neuroscience Program, Michigan State University, 108 Giltner Hall, East Lansing, MI 48824, USA b Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA c Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA d W.M. Keck Center for Comparative and Functional Genomics, University of Illinois, Urbana, IL 61801, USA e Department of Physiological Science, University of California, Los Angeles, CA 90095, USA