Sexually Dimorphic Neurocalcin Expression in the Developing Zebra Finch Telencephalon

Sexually Dimorphic Neurocalcin Expression in the Developing Zebra Finch Telencephalon Sean L. Veney, 1,2 Camilla Peabody, 1,2 George W. Smith, 3 Juli Wade 1,2,4 1 Neuroscience Program, Michigan State University, East Lansing, Michigan 48824 2 Department of Psychology, Michigan State University, East Lansing, Michigan 48824 3 Departments of Animal Science & Physiology, Michigan State University, East Lansing, Michigan 48824 4 Department of Zoology, Michigan State University, East Lansing, Michigan 48824 Received 6 January 2003; accepted 3 April 2003 ABSTRACT: Differential display RT-PCR was used on RNA isolated from the zebra finch telencephalon to identify gene products potentially involved in its development, including the sexually dimorphic nuclei responsible for song learning and production. A cdna identified only in juvenile females was cloned and sequenced. It shares homology with neurocalcin, a calcium binding protein. Northern blots indicated three neurocalcin species. A 10.6 kb transcript was present in males and most females throughout development and in adulthood. Smaller 6.2 and 3.3 kb species were detected almost exclusively in females and primarily between posthatching days 18 25. In situ hybridization, using a probe that identified all three mrna species, indicated a broad distribution in the telencephalon of both sexes, with particularly high levels in the song nucleus RA. Across regions examined, neurocalcin expression was enhanced in females compared to males, probably reflecting the presence of the two smaller transcripts. However, within RA, neurocalcin expression was statistically equivalent between the sexes. These data indicate that calcium signaling via neurocalcin may be involved in telencephalic development, but suggest that sexually dimorphic expression of this gene exists on a level too general to specifically regulate masculine or feminine development of song control regions. Neurocalcin might: broadly influence functional differentiation, including areas that are not morphologically distinct between the sexes; be a benign consequence of general dimorphisms, such as those due to sex chromosomes; or involve a compensatory mechanism, which allows function of the juvenile female telencephalon to equal that of males, despite fundamental physiological differences. 2003 Wiley Periodicals, Inc. J Neurobiol 56: 372 386, 2003 Keywords: calcium binding; sex difference; differential display; neural development; songbird INTRODUCTION In many species of vertebrates, early gonadal secretions organize sex differences in neural morphology Correspondence to: S. Veney (veney@msu.edu). Contract grant sponsor: NIH; contract grant number: MH55488 (J.W.). Contract grant sponsor: NSF; contract grant number: DBI- 0001973 (S.V.). 2003 Wiley Periodicals, Inc. DOI 10.1002/neu.10246 and/or function (Feder, 1981; Yahr, 1988; Meisel and Sachs, 1994). These differences, particularly in brain areas important for reproduction, often relate to the frequency with which masculine or feminine behaviors are displayed (Cooke et al., 1998; Madeira and Lieberman, 1995). However, not all sex differences can easily be explained by the actions of gonadal steroids (Arnold, 1997; Wade, 2001; Carruth et al., 2002). In addition, sex differences in morphology need not always bias function; differences in the brains of males and females on a cellular (or molecular) level may in some cases even facilitate similar 372

Zebra Finch Neurocalcin 373 function of brain regions despite inherent differences between the sexes (De Vries and Boyle, 1998). The development of sex differences in the brain and behavior of zebra finches is particularly intriguing (Balthazart and Adkins-Regan, 2002). Song learning and production are controlled by a series of interconnected regions within the telencephalon. Area X and the lateral portion of the magnocellular nucleus of the anterior neostriatum (lman) are involved in song learning (Bottjer et al., 1984; Scharff and Nottebohm, 1991). The high vocal center (HVC) and robust nucleus of the archistriatum (RA) form the motor pathway for song production (Nottebohm et al., 1976; Simpson and Vicario, 1990). HVC projects to RA, which in turn projects to the tracheosyringeal portion of the hypoglossal nucleus (nxiits), which contains motoneurons that innervate the vocal organ, or syrinx (Nottebohm et al., 1976). Sexual dimorphisms exist at a number of levels within this system (Arnold, 1992; Wade, 1999). For example, only males sing, and the volumes of HVC and RA as well as the soma size and number of neurons within these nuclei are greater in males than in females. The projection from HVC to RA is more robust in males than in females. And Area X, which is easily identified in males, is not visible in females using standard Nissl stains. These neural dimorphisms, which are assumed to permit song production in males and inhibit it in females, develop between approximately 1 week and 2 months of age, with the most striking rate of differentiation occurring between days 15 and 35 posthatching (Bottjer et al., 1985; Kirn and DeVoogd, 1989; Nixdorf-Bergweiler, 1996). Similar to many other sexually dimorphic systems, both the structure and function of the zebra finch song system can be permanently modified by steroid hormones. For example, treatment of females with estradiol in the first few weeks after hatching can partially masculinize several morphological features of the neural song system as well as the capacity for females to sing in adulthood (Gurney, 1981, 1982; Nordeen et al., 1986; Simpson and Vicario, 1991a, b; Grisham and Arnold, 1995). However, some data are inconsistent with the hypothesis that gonadal secretions normally induce development of the more robust song system in males compared to females. Studies using aromatase inhibitors or estrogen receptor antagonists in males, or the induction of functional testicular tissue in genetic females, have produced either negative data or results opposite to those predicted if gonadal sex steroids normally induce masculinization (Mathews et al., 1988; Mathews and Arnold, 1990; Wade and Arnold, 1994, 1996; Wade et al., 1996, 1999). In contrast, estrogen produced in the brain masculinizes the projection from HVC to RA (Holloway and Clayton, 2001). Thus, while it seems that axonal growth is regulated by neural estradiol, other aspects of sexual differentiation may not be. Whether or not hormones trigger the process, it is likely that differential gene/protein expression is important in the development of male and female brains. The present study was therefore conducted to identify gene products associated with that process. The approach was to use differential display reverse transcription-polymerase chain reaction (ddrt-pcr) to identify patterns of RNA expression that are present in the telencephalon of either juvenile females or males, but not both, and are absent in adults of both sexes. A cdna encoding the calcium binding protein, neurocalcin, was identified from juvenile female zebra finch tissue using this technique. Northern blot analysis was then used to determine in more detail the age- and tissue-specificity of neurocalcin expression, and in situ hybridization was employed to characterize the distribution of neurocalcin mrna in the brain. MATERIALS AND METHODS Tissue Collection and RNA Isolation for Differential Display and Northern Blot Analysis The telencephalon of each zebra finch was removed following rapid decapitation, immediately frozen on dry ice, and stored at 80 C. Tissue was collected to obtain RNA for use in differential display from 19 25-day-old males (n 4) and females (n 3), as well as from adults of both sexes (greater than 120 days of age; n 4 of each sex). For the Northern blot analyses, telencephalic tissue was collected from males and females from posthatching day 5 through adulthood (specific ages and sample sizes are indicated in Table 1). The cerebellum, liver, and syrinx were also collected from posthatching day 22 males and females. Total RNA was isolated from individually homogenized samples with Trizol (Gibco Life Technologies, Grand Island, NY). Each sample was checked for concentration, and run on a gel to confirm purity and RNA integrity. Zebra finch RNA samples used for differential display were then pooled within sex and age group (see above) and brought to a final concentration of 100 ng/ L. Samples for use on Northern blots were processed individually, with the exception of RNA from the syrinx that was pooled within sex (n 2 females and n 3 males) due to the small amount of product available. All samples were maintained in DEPC H 2 0at 80 C until use. ddrt-pcr The four samples of pooled RNA (juvenile males and females; adult males and females) were treated with Deoxyri-

374 Veney et al. Table 1 Transcript Ratio of Individuals Expressing the Three Neurocalcin Transcripts in Telencephalic Tissue d5 d15 d18 d19 d20 d21 d22 d23 d24 M F M F M F M F M F M F M F M F M F 10.6 kb 4/4 6/6 2/2 3/3 2/2 2/2 1/1 1/1 2/2 3/4 1/1 1/1 9/9 13/13 1/1 1/1 1/1 1/1 6.2 kb 0/4 0/6 0/2 0/3 1/2 1/2 0/1 0/1 0/2 2/4 0/1 0/1 0/9 4/13 0/1 1/1 0/1 1/1 3.3 kb 0/4 0/4 0/2 0/3 0/2 0/2 0/1 0/1 0/2 2/4 0/1 0/1 0/9 4/13 0/1 0/1 0/1 0/1 Transcript d25 d30 d38 d45 d52 d60 d70 Adults Total M F M F M F M F M F M F M F M F M F 10.6 kb 1/1 1/1 2/2 4/4 2/2 3/3 2/2 3/3 2/2 3/3 3/3 4/4 2/2 3/3 2/2 3/3 39/39 55/56 6.2 kb 0/1 1/1 0/2 1/4 0/2 0/3 0/2 0/3 0/2 1/3 0/3 1/4 0/2 0/3 0/2 0/3 1/39 13/56 3.3 kb 0/1 0/1 0/2 1/4 0/2 0/3 0/2 0/3 0/2 1/3 0/3 1/4 0/2 0/3 0/2 0/3 0/39 9/56 d posthatching day, M male, F female. bonuclease I (Gibco) per manufacturer s instructions. cdna was synthesized using Superscript II RNase H Reverse Transcriptase (Gibco) as follows. RNA (620 ng) and 3.75 M T 12 GC anchored primer in DEPC H 2 O were heated for 10 min at 70 C and chilled on ice. The first strand buffer, 0.01 M dithioreitol (DTT), 20 U RNasin (Promega, Madison WI), and 0.5 mm dntp mix were added according to manufacturer s recommendation and incubated at 42 C for 2 min. Superscript II (200U) was added and the incubation continued at 42 C for an additional 50 min. The reaction was heat inactivated at 70 C for 15 min. Samples were chilled on ice, and 20 L of DEPC H 2 O was added. Reactions were stored at 4 C. The cdna was diluted (1:5) and added to PCR buffer, 1.5 mm MgCl 2, 0.625 M arbitrary primer (TACAAC- GAGG; Bauer et al., 1993), 2.8 M anchored T 12 GC primer, 2 M dntps, 1.1 Ci 33 P-dATP (New England Nuclear, Boston, MA), 1 U Taq polymerase and DEPC H 2 O to a total volume of 20 L. Samples were amplified under the following conditions: five cycles of 94 C for 30 s, 40 C for 1 min, 72 C for 1 min; 35 cycles of 94 C for 30 s, 45 C for 1 min, 72 C for 1 min; and an extension at 72 C for 5 min. Reactions were maintained at 4 C until further use. Each sample was concentrated (9 L to2 L) using a SpeedVac (Savant, Holbrook, NY) and heated for 2 min at 95 C following the addition of 1 L denaturing buffer (95% formamide, 0.025% xylene cyanol, 0.025% bromphenol blue, 0.05% EDTA, and 0.027% SDS). Samples were then chilled on ice and loaded onto a 0.2 mm 6% polyacrylamide denaturing sequencing gel and separated by electrophoresis for 15 h at 850 V. The gel was dried for1hat80 C,allowed to cool, and exposed to film (Kodak Biomax MR1; Eastman Kodak, Rochester, NY). Cloning and Sequencing A number of sex-specific bands were detected. However, the present study is limited to one of interest that was evaluated in detail. The band was excised from the gel, eluted in TE, and reamplified under the PCR conditions described above using 5 L excised DNA, PCR buffer, 3 mm MgCl 2, 50 M dntp mix, 0.2 M anchored and arbitrary primers (TACAACGAGG and T 12 GC; Bauer et al., 1993), 2.5 U Taq polymerase, and sterile H 2 O to a total volume of 50 L. Amplified cdna was cloned into pgem- T Easy Vector (Promega), and the individual strands were sequenced (PerkinElmer 373A DNA sequencer) with vector primers flanking the cdna insert using the dye primer method. The data were analyzed with the Genbank BLAST search program. The cdna shared a high degree of similarity with the 3 end of a sequence for chicken neurocalcin (see below). To verify that it coded for zebra finch neurocalcin, a cdna containing the complete open reading frame was amplified from a 20-day-old female. The RT conditions outlined above were used, followed by amplification with primers that were designed based on the sequence of chicken neurocalcin and the partial zebra finch neurocalcin cdna (corresponding to bases 41 60 and 759 780 of the chicken neurocalcin cdna, Genbank accession #U91630). The cdna (1 L) was amplified in a 20 L reaction with PCR buffer, 1.5 mm MgCl 2, 0.2 mm dntps, 0.25 M forward and reverse primers, and 1 U Taq polymerase under the following conditions: 94 C for 5 min; 30 cycles of 94 C for 30 s, 65 C for 30 s, 72 C for 1 min; and an extension at 72 C for 1 min. The amplified cdna was cloned into pgem-t Easy Vector (Promega), and sequences were initially obtained using dye terminator procedures with vector specific primers. The remaining portions of the sequence were obtained using custom-designed internal primers corresponding to positions 216 to 236 and 354 to 376 of the zebra finch neurocalcin cdna (see below). All of the sequence information reported was confirmed in both directions. Northern Blot Analysis Total RNA (15 g/lane) was electrophoretically separated on a 1% agarose/formaldehyde gel, transferred onto a nylon membrane (Zeta Probe; BioRad, Hercules, CA), and crosslinked (UV Stratalinker; Stratagene, La Jolla, CA). RNasefree DNase I digestion and PCR experiments confirmed the absence of genomic DNA contamination in RNA samples

Zebra Finch Neurocalcin 375 utilized for Northern analyses. A probe that coded for the 3 untranslated region was synthesized from the plasmid containing the original neurocalcin ddrt-pcr product. Once the complete open reading frame had been sequenced, a second probe was made to the 5 end of zebra finch neurocalcin. This probe corresponded to bases 73 to 141 of cdna and was cloned into pbluescript II SK (Stratagene). The 5 end zebra finch probe was highly specific for neurocalcin; the 214 bases were 93% identical to chicken neurocalcin (Genbank accession #U91630), but shared 14% or less nucleotide sequence identity with other neuron specific calcium sensor family members. Similarly, while the portion of the probe corresponding to the 5 untranslated region contained sequence apparently unique to birds, the 141 bases corresponding to the coding region were 85% identical to human neurocalcin delta (#AF251061) and 84% identical to bovine neurocalcin (#D10884). The cdna fragments were labeled with [ - 32 P]dCTP as follows. Appropriate sense and antisense primers (0.25 M) were added to PCR buffer, 2.5 mm MgCl 2, 1.55 M dntps (-dctp) with 50 Ci [ - 32 P]dCTP, 100 pg cdna template, and 1.5 U Taq polymerase. The 32 P cdna was amplified using the following conditions: 95 C for 5 min; 40 cycles of 94 C for 30 s, 54 C for 1 min, 72 C for 1.5 min; and an extension at 72 C for 10 min. Membranes were prehybridized at 42 C for a minimum of 6hin50%formamide, 5X SSC, 5X Denhardts, 0.1% SDS, 50 mm NaPO 4, and 250 g/ml of heat-denatured herring sperm DNA (Boehringer Mannheim, Indianapolis, IN). Probes were heat denatured for 10 min at 100 C and then individually hybridized to the membranes for a minimum of 16 h at 42 C in a buffer containing 50% formamide, 5X SSC, 1X or 5X Denhardt s, 0.1% SDS, 20 or 50 mm NaPO 4, 10% dextran sulfate, and 100 g/ml heat-denatured herring sperm DNA. Membranes were washed in decreasing concentrations of SSC and 0.1% SDS two or three times at 42 C. If necessary, they were then washed in 0.1X SSC and 0.1% SDS at increasing temperatures (up to 70 C) until the background radioactivity averaged 250 counts per minute or less. Blots were exposed to film (Kodak X-OMAT AR; Eastman Kodak) and were stripped in 0.1X SSC and 0.5% SDS at 95 C for 20 40 min before reprobing. A ribosomal protein L19 probe (360 bp) was also synthesized, and used as a loading control on each blot under the same hybridization conditions as neurocalcin. To verify the presence and relative abundance of the three neurocalcin transcripts detected on Northern blots by visual observation, optical density measurements for each of the tissues (telencephalon, cerebellum, liver, and syrinx) were obtained using Scion Image (NIH software). Under consistent illumination, images were captured from the films using a video camera mounted over a light box. Separately for each transcript, a box covering the area of the smallest band (least hybridization) was drawn, and the average optical density was calculated. A box of the same size was then used to measure the average optical density of bands across the row. In lanes without detectable bands, a box was drawn in the blank area where the band would be expected, and the optical density determined. For each lane, a ratio was calculated of the average optical density of each of the neurocalcin bands, divided by the average optical density of the loading control. For bands detectable by visual observation, the ratios were between 0.92 and 1.22. Optical density ratios were between 0.24 and 0.85 in areas where no bands were detectable by eye. For most blots that were analyzed, no overlap existed between bands that were identified as present or not present by eye. However, a total of three bands (on two different blots) appeared ambiguous by visual inspection. The optical density ratios all fell within the range of bands that were not detectable, and were therefore considered as such. In Situ Hybridization Following rapid decapitation, brains were collected from 22-day-old males and females (n 7 each sex), quick frozen in chilled methyl-butane, and stored at 80 C. Frozen brains were coronally sectioned at 20 m, thaw mounted onto SuperFrost Plus slides (Fisher Scientific), and stored at 80 C until processing. Adjacent sections were placed on separate slides so that sense and antisense probes could be used on the same brain. At the time of processing, the brain sections were thawed to room temperature and fixed in 10% phosphate buffered formalin for 10 min. After fixation, sections were briefly rinsed in 2X SSC, followed by a 10 min incubation in 0.1 M triethanolamine and 0.25% acetic anhydride. Sections were then washed in 2X SSC, dehydrated through a series of graded alcohols, delipidated in chloroform, rinsed in 95% ethanol, and air dried. The 35 S-labeled crna riboprobes (antisense and sense) for zebra finch neurocalcin were generated from the 5 neurocalcin cdna used for Northern analyses. The antisense probe was transcribed in a reaction mixture containing 2 g of DNA template (linearized with Xho 1), 1X transcription buffer (Stratagene), 1.2 mm rntps (-rutp), 30 mm DTT, 62.5 Ci 35 S-UTP, 1 U RNase inhibitor, 3UT3 RNA polymerase, and DEPC H 2 O. The reaction mixture was heated at 37 C for 1 h, after which 2 L of RNasefree-DNase was added, and the reaction mixture heated for an additional 15 min. Unincorporated nucleotides were removed by spin-column filtration through Sephadex G50 beads. The sense control probe was labeled in an identical manner with the exception of using BAMH 1 to linearize and3ut7rnapolymerase. Both probes were diluted in hybridization buffer (50% formamide, 0.3 M NaCl, 10 mm Tris, 1 mm EDTA, 1X Denhardts, 50 mm DTT, 0.05 mg/ml yeast trna, and 10% dextran sulfate) to yield a final concentration of 1.0 10 6 c.p.m./50 L of hybridization buffer. Diluted probe was applied to each slide and the sections were covered with a strip of parafilm. Slides were then placed in Plexiglas boxes lined with DEPC-H 2 O-moistened filter paper, sealed with plastic wrap, and placed at 55 C for 20 24 h. Following hybridization, the coverslips were floated off, and the slides washed in 2X SSC. To digest all nonspecifically hybridized

376 Veney et al. RNA, sections were then incubated in RNase A (50 g/ml) for1hat37 C. Slides were rinsed in decreasing concentrations of SSC containing 0.1% -mercaptoethanol, followed by dehydration in a graded series of alcohols, and allowed to air dry. Finally, the slides were dipped into Kodak NTB-2 autoradiographic emulsion, air dried, and exposed for 2 weeks at 4 C. Slides were developed in Dektol, fixed (Eastman Kodak), counterstained with the Nissl stain thionin, dehydrated, cleared in xylene, and coverslipped. The distribution of cells labeled with silver grains was characterized under bright and dark field microscopy using an Olympus BX-60 microscope. A canary atlas was used to match comparable sections from each animal and to identify the brain regions (Stokes et al., 1974). To get an impression of the overall distribution and relative levels of neurocalcin expression, an observer blind to the sex of the animal ranked the intensity of labeling on a 4-point scale (0, 1, 2, 3). These ratings were based on visual inspection and were designed to be approximately evenly divided over the intensity range, with 0 representing labeling similar to background (silver grain density in sections treated with the sense probe) and 3 representing intense labeling comparable to that in RA of females (see below). The following regions, spanning the entire rostral-caudal continuum of the brain, were analyzed: cerebellum, RA, archistriatum dorsale, archistriatum, preoptic/hypothalamic area, parolfactory lobe/area X, hippocampus, HVC, and paleostriatum augmentatum. The neostriatum, neostriatum caudale, and hyperstriatum ventrale were also investigated, but the approximate rostral and caudal halves of each were rated separately because the intensity of labeling clearly increased from anterior to posterior. As no nonparametric test is available for this type of design, data were analyzed by a mixed model two-way ANOVA to compare the effects of sex (between subjects) and brain areas (within subjects). While not used extensively in biological research, employing ANOVAs for this type of ordinal data is routine in some other disciplines, including various fields of psychology (e.g., some intelligence testing and the evaluation of data collected by questionnaires that use scales of even fewer values than ours). This statistical analysis is appropriate as long as the difference between each level (e.g., 0 1 and 1 2) is roughly equivalent (Diekhoff, 1992; Maxwell and Delaney, 1990). Using this method, we noted that the pattern of expression was highly consistent across individuals, but the overall intensity of neurocalcin mrna expression was increased in females compared to males. To determine whether the sexual dimorphism that was so obvious across the telencephalon as a whole was likely to be biologically relevant in a brain region of particular interest, we analyzed silver grain density in two structures, RA and the cerebellum. We chose RA because the labeling appeared more intense than in other regions, and this area is part of the highly sexually dimorphic song circuit. The cerebellum was examined as a control, because it was not expected to be sexually dimorphic in either structure or function, and the minimal amount of labeling that it contained did not appear more intense in females. For each of the seven male and female brains on which in situ hybridization was conducted, bright field images were captured in Scion Image without knowledge of animal s sex. A box of the same size was centered in each of the two brain regions, and the area ( m 2 ) covered by silver grains was determined using the density slice function (Rosen et al., 2002). An average of four measurements (left and right in two sections) were determined for RA; two adjacent sections were used from the center of the cerebellum. This process was used for sections exposed to both the antisense and sense probes. The background silver grain area (from the sense-treated sections) was subtracted from that covering the antisense-treated sections and then divided by the size of the box to determine the percentage of area specifically labeled for neurocalcin. Data were analyzed separately for RA and the cerebellum by t tests. RESULTS Initial Identification of a Sexually Dimorphic cdna Expressed in the Developing Zebra Finch Telencephalon A band that appeared in only the pooled juvenile female sample was detected using ddrt-pcr (Fig. 1). Initial sequence analyses (3 end) tentatively identified the cdna as coding for neurocalcin. Sequence analysis of a cdna containing the complete open reading frame confirmed its identity. Similar to other calcium binding proteins in the family, zebra finch neurocalcin has four potential calcium binding EFhands (Fig. 2), and contains the consensus myristoylation site at the N-terminus. The deduced amino acid sequence derived from the zebra finch neurocalcin cdna (GenBank accession #AF272896) is identical to that of chicken neurocalcin, and of the 193 amino acids in bovine neurocalcin and human neurocalcin delta, the zebra finch sequence differs in only one position (Fig. 3; Okazaki et al., 1992; Terasawa et al., 1992; Wang et al., 2001). The zebra finch amino acid sequence shares 89% identity with Drosophila neurocalcin and is similar to other members of the neuronal calcium sensor family, including human hippocalcin (Fig. 3; Teng et al., 1994; Nef, 1996). Characterization of Neurocalcin RNA Species: Sex-, Age-, and Tissue- Specificity Northern blot analyses were used to characterize the pattern of neurocalcin RNA expression in the telencephalon, using total RNA samples collected from posthatching males and females. RNA from males and females of the same ages were always run to-

Zebra Finch Neurocalcin 377 Figure 1 Autoradiograph of electrophoretically separated ddrt-pcr products amplified from pooled samples of juvenile and adult males and females. The four lanes on the right utilized the primers identified in the Materials and Methods section. The arrow indicates a band that was present only in the juvenile female tissue, and was excised for cloning. The four lanes on the left contain cdna from the same samples, but used a different upstream primer. gether, and samples were processed such that several different ages were represented on each blot. A total of seven blots were run with the sample sizes and ages represented in Table 1. In the telencephalon, at least three distinct transcripts were detected with two different probes that coded for the 5 and 3 ends of the open reading frame of zebra finch neurocalcin (Fig. 4). A 10.6 kb transcript was detected in both sexes at all ages examined, with the exception of one 20-day-old female. Fisher Exact tests confirmed that there was no sex difference in the proportion of individuals expressing this transcript (p.999). Two smaller transcripts (6.2 and 3.3kb) were sexually dimorphic in their expression (Fisher Exact; p.006 and p.010, respectively). They also appeared to be developmentally regulated. That is, neither was detected in the first 2 weeks after hatching or in animals 70 days of age or older. Within the 18 25 day age range, 42% of females expressed the 6.2 kb transcript and 25% expressed the 3.3 kb transcript. These two transcripts were also weakly detected in one female at each of days 30, 52, and 60. The 6.2 kb transcript was also expressed in one 18- day-old male, but the 3.3 kb transcript was never detected in males at any age (Table 1). Expression of the 10.6 kb transcript was comparable across all samples. However, in four females that expressed the two smaller transcripts (at days 22, 52, and 60), it appeared diminished. The most dramatic example of this and the absence in the day 20 female are depicted in Figure 4. The tissue-specificity of neurocalcin expression was examined in RNA from six females and six males. Each animal was 22 days of age, and all samples were run together on a single blot. Only the 10.6 kb transcript was detected in all telencephalic and cerebellar samples. However, consistent with results of the in situ hybridization (see below), the optical density ratio of this band in the cerebellar samples was approximately half that detected in telencephalic samples. None of the neurocalcin transcripts were detected in samples from the liver or syrinx (data not shown). Distribution and Relative Intensity of Neurocalcin Expression in Specific Regions of Male and Female Zebra Finch Brain In situ hybridization indicated broad, specific labeling of neurocalcin mrna throughout the telencephalon in day 22 posthatching males and females. The expression of one or more of the transcripts (the antisense probe recognized all three) overlapped with several song control nuclei, but was particularly high in RA (Fig. 5). Outside of the telencephalon, expression was much lower (Fig. 6), as indicated by a significant effect of brain region [F(14, 168) 16.57; p.001]. Overall, neurocalcin expression was significantly greater in females than in males [F(1, 12) 5.57; p.04], but there was no interaction between sex and brain region [F(14, 168) 1.19; p

378 Veney et al. Figure 2 Nucleotide and deduced amino acid sequence of zebra finch neurocalcin. The locations of the four potential calcium binding sites (EF hands) are underlined. The DNA sequence of zebra finch neurocalcin has been deposited with GenBank under accession number AF272896.

Zebra Finch Neurocalcin 379 Figure 3 Amino acid sequence of zebra finch neurocalcin (NCa) compared to that of chicken, human, bovine, and Drosophila neurocalcins. Human hippocalcin (HippoCa), a related protein in the neuron calcium sensor family, is also shown (Takamatsu et al., 1994). Amino acid residues identical to those in zebra finch neurocalcin are depicted in capital letters..33]. While the female-biased expression was consistent across regions of the telencephalon (Fig. 7), within RA silver grain density was not significantly different between the sexes (t 0.51, p.62). Labeling also did not differ statistically between the sexes in the cerebellum (t 1.73, p.11). In both cases, the direction of the difference was consistent with the ordinal data (greater ex- Figure 4 Northern blot analysis of neurocalcin RNA expression in the telencephalon. Total RNA from females and males at days 20 60 posthatching was used. Hybridization to the 32 P-labeled 3 zebra finch neurocalcin probe is depicted, although an identical pattern was detected with the 5 neurocalcin probe.

380 Veney et al. Figure 5 Photomicrographs of neurocalcin mrna labeling in the song control nucleus RA of a posthatching day 22 female hybridized with (A) antisense and (B) sense probes. Also pictured are age-matched sections from a male treated with the same (C) antisense and (D) sense probes. Scale bar 50 m. pression in the female RA, greater expression in the male cerebellum). DISCUSSION General Summary and Conclusions In mammals and in birds, sex differences in gene expression may contribute to overall differences in morphology and behavior (Arnold, 2002; Balthazart and Adkins-Regan, 2002). This article documents the cloning and sequencing of sexually dimorphic zebra finch neurocalcin, originally generated using ddrt- PCR procedures. Northern blot analyses indicated at least three distinct transcripts. One large (10.6 kb) neurocalcin RNA species was expressed in the telencephalon of males and most females from posthatching day 5 to adulthood. Two smaller (6.2 and 3.3 kb) RNA species were detected overwhelmingly in females primarily between the ages of 18 25 days posthatching. The expression of neurocalcin mrna in both sexes was broadly distributed throughout the telencephalon and overlapped with several nuclei of the song control circuit. The enhanced expression of neurocalcin mrna detected in females was most likely due to the presence of one or both of the two smaller transcripts, because the large transcript was generally comparable in both sexes at all ages. However, it is possible that the 6.2 and 3.3 kb species may be spliced from the 10.6 kb RNA, because expression of the large transcript was diminished when the two smaller ones were present in some individuals. It will be important in the future to isolate and characterize the individual transcripts to assess their origin and potential function. However, several pieces of evidence confirm that multiple transcripts do exist. For example, identical expression patterns were detected with two different probes generated from opposite ends of the neurocalcin cdna. Enhanced expression of neurocalcin in juvenile females was

Zebra Finch Neurocalcin 381 Figure 6 Camera lucida sketches depicting the distribution and relative intensity of neurocalcin mrna expression. The pattern was very similar in both sexes. Coronal sections, 250 m apart, are arranged rostral to caudal (top left to bottom right). Increasing intensity of gray represents more neurocalcin mrna labeling. Abbreviations: A, archistriatum; Ad, archistriatum dorsale; APH, area parahippocampalis; Cb, cerebellum; CO, chiasma opticum; CoA, commissura anterior; HA, hyperstriatum accessorium; HD, hyperstriatum dorsale; HV, hyperstriatum ventrale; HP, hippocampus; HVC, high vocal center; Ico, nucleus intercollicularis; lman, lateral magnocellular nucleus of the anterior neostriatum; LH, lamin hyperstriatica; LPO (X), lobus parolfactorius/area X; N, neostriatum; NC, neostriatum caudale; PA, paleostriatum augmentatum; RA, robust nucleus of the archistriatum; Rt, nucleus rotundus; TeO, tectum opticum; V, ventricle.

382 Veney et al. Figure 7 Average intensity of neurocalcin mrna labeling in each sex (0 least intense, 3 most intense). Overall, mrna expression was greater in females than in males. Abbreviations: A, archistriatum; Ad, archistriatum dorsale; c.hv, caudal hyperstriatum ventrale; c.n, caudal neostriatum; c.nc, caudal neostriatum caudale; Cb, cerebellum; HP, hippocampus; HVC, high vocal center; LPO (X), lobus parolfactorius/area X; PA, paleostriatum augmentatum; POA/Hyp, preoptic/ hypothalamic; r.hv, rostral hyperstriatum ventrale; r.n, rostral neostriatum; r.nc, rostral neostriatum caudale; RA, robust nucleus of the archistriatum. detected by both Northern blot and in situ hybridization. DNase treatment, reducing the gel concentration to increase separation of the RNA, and modifying the hybridization conditions to decrease the amount of nonspecific binding on the Northerns did not affect detection of the transcripts. Additionally, RNA was extracted from samples in random order, so it is highly unlikely that some complication in processing induced a female-biased artifact. The timing and expression patterns are consistent with the idea that neurocalcin is involved in the development of the telencephalon, including circuits within the song system and areas of the brain responsible for song perception (e.g., caudomedial neostriatum) (Mello and Clayton, 1994; Mello and Ribeiro, 1998; Kruse et al., 2000; Bailey et al., 2002) and memory (hippocampus) (Biegler et al., 2001). However, it is clear that sexual differentiation of the song system is not specifically affected by neurocalcin for the following reasons: the mrna expression is too widespread; the silver grain analysis did not detect a significant sex difference in RA; and our more general analysis indicated that the average difference in labeling between males and females was even smaller in HVC, lman, and the lobus parolfactorius (which contains Area X in males). It is possible that a more complete analysis of silver grain density in specific regions might have revealed a statistically significant difference between the sexes (such as in the preoptic area or hippocampus), but the most striking aspect of the data is the consistency of the female-biased expression across the telencephalon and at least one portion of the diencephalon (hypothalamus). While clearly sexually dimorphic, the expression of the 6.2 and 3.3 kb transcripts was variable among juvenile females (see Table 1). One possible explanation is that the expression of the 6.2 and 3.3 kb transcripts is generally restricted to a finite developmental period. All females may express the transcripts, but typically do so during a narrow developmental window, which we could have missed for many of the birds. Animals that are developmentally less mature, for example ones part of a large nest of siblings, may express the transcripts at an age later than when we collected the tissue. In contrast, the weak expression of the two smaller transcripts in two females at days 52 and 60 might indicate a regulatory

Zebra Finch Neurocalcin 383 mechanism for neurocalcin that can occur at a wide range of juvenile stages. Calcium Binding Proteins and the Influence of Calcium in the Nervous System Neurocalcin is a calcium binding protein, a member of the EF-hand superfamily, which contains more than 250 individual proteins. Multiple isoforms of neurocalcin (,, 1, 2, ) have been identified in several species, and one or more is expressed in various tissues, including bovine cerebrum, cerebellum and brainstem, rat hippocampus, cerebellum, inner ear and olfactory bulb, the human temporal cortex, ovary and testis, the abdominal ganglion of Aplysia and the central nervous system of Drosophila (Terasawa et al., 1992; Hidaka and Okazaki, 1993; Iino et al., 1995; Kato et al., 1998; Wang et al., 2001; Braunewell and Gundelfinger, 1999). As in other species, zebra finch neurocalcin has four potential calcium binding EF-hands, although in at least bovine neurocalcin, only three are functional (Terasawa et al., 1992; Vijay-Kumar and Kumar, 1999). Specific functions of neurocalcin are not known in any species (Burgoyne and Weiss, 2001). As a whole, however, the family of calcium binding proteins appears to be involved in buffering intracellular calcium and/or facilitating communication between calcium and other second messenger systems (Heizmann, 1992; Clapham, 1995; Braunewell and Gundelfinger, 1999; Burgoyne and Weiss, 2001). Recently, neurocalcin delta was determined to interact specifically with clathrin heavy chain, - and -tubulin, and -actin, suggesting potential functions in the control of vesicle release and cytoskeletal changes (Ivings et al., 2002). Consistent with that idea, calcium regulation is known to be critical for mediating changes in neural structure and function, including neurite extension, gene transcription, and neurotransmitter release (Mattson, 1992; Burgoyne and Morgan, 1995; Braunewell and Gundelfinger, 1999). Calcium regulation by calbindin-d 28K and calretinin may influence sexual differentiation of the mammalian preoptic area by affecting cell survival (Brager et al., 1999; Stuart and Lephardt, 1999). Neurocalcin may mediate forebrain development via one or more of these mechanisms, but more work is needed to address this possibility. However, a clue to its function may come from its anatomical distribution in zebra finches. The mrna expression in juveniles overlaps with that of aromatase enzyme and estrogen receptor alpha in several broad regions of the forebrain during development, including the preoptic area, caudomedial neostriatum, HVC, and RA (Jacobs et al., 1999). Therefore, it is conceivable that neurocalcin and estradiol interact to influence neural development. Again, any interaction among these proteins is unlikely to directly mediate sexual differentiation of the song circuit. In addition to the lack of a specific sex difference in neurocalcin expression in song control nuclei, brain aromatase (Schlinger and Arnold, 1992; Wade et al., 1995; Saldanha et al., 2000), plasma estradiol (Adkins-Regan et al., 1990; Schlinger and Arnold, 1992), and estrogen receptors (Gahr, 1996; Jacobs et al., 1999) are generally equivalent in developing male and female zebra finch brain. However, the extent of the overlap of neurocalcin and estrogen-related proteins during development suggests that their interaction should be investigated. A Sex Difference that Promotes Similarity? It will clearly be important to learn the sequence and distribution of the female specific transcripts, because we do not currently know if they are translated into functional protein or where they are specifically expressed. If the smaller transcripts are not functional, then neurocalcin may be generally equivalent between the sexes, and the smaller RNAs may simply be a benign consequence of some other fundamental difference between the males and females, such as those due to sex chromosomes. However, if these transcripts that are expressed almost exclusively in females code for protein(s) that do bind calcium, then functional differences between the sexes are far more widespread than typically considered and certainly span areas that are not morphologically distinct between the sexes. If it exists, the enhanced calcium binding in females would indicate a fundamental sex difference in the developing telencephalon that includes, but is not limited to, the song control system. This calcium buffering or signaling might influence neurogenesis, cell migration, survival, or even apoptosis in the developing telencephalon. Alternatively, because of the widespread nature of the neurocalcin expression, we should also consider the possibility that rather than the smaller transcripts enhancing a function in females during development, they may actually be compensating for inherent physiological differences between the sexes (De Vries and Boyle, 1998). That is, the increased neurocalcin expression in females might reflect a level of calcium binding/ signaling that simply allows basic telencephalic development to occur in a manner similar to males. We thank Lori Buhlman and Stephanie Mott for taking excellent care of the birds, Leanne Bakke, Christina Be-

384 Veney et al. sonen, Carolyn Cassar, Mark Dow, and Isam Qahwash for technical assistance, and Dr. Debby Kashy for assistance with statistics. REFERENCES Adkins-Regan E, Abdelnabi M, Mobarak M, Ottinger MA. 1990. Sex steroid levels in developing and adult male and female zebra finches (Poephila guttata). Gen Comp Endocrinol 78:93 109. Arnold AP. 1992. Developmental plasticity in neural circuits controlling birdsong: sexual differentiation and the neural basis of learning. J Neurobiol 23:1506 1528. Arnold AP. 1997. Sexual differentiation of the zebra finch song system: Positive evidence, negative evidence, null hypotheses, and a paradigm shift. J Neurobiol 33:572 584. Arnold AP. 2002. Concepts of genetic and hormonal induction of vertebrate sexual differentiation in the twentieth century, with special reference to the brain. In: Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, editors. Hormones, brain and behavior. San Diego: Academic Press, p 105 135. Bailey DJ, Rosebush JC, Wade J. 2002. The hippocampus and caudomedial neostriatum show selective responsiveness to conspecific song in the female zebra finch. J Neurobiol 52:43 51. Balthazart J, Adkins-Regan E. 2002. Sexual differentiation of brain and behavior in birds. In: Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, editors. Hormones, brain and behavior. San Diego: Academic Press, p 223 301. Bauer D, Müller H, Reich J, Riedel H, Ahrenkiel V, Warthoe P, Strauss M. 1993. Identification of differentially expressed mrna species by an improved display technique (DDRT-PCR). Nucleic Acids Res 21:4272 4280. Biegler R, McGregor A, Krebs JR, Healy SD. 2001. A larger hippocampus is associated with longer lasting spatial memory. Proc Natl Acad Sci USA 98:6941 6944. Bottjer SW, Glaessner SL, Arnold AP. 1985. Ontogeny of brain nuclei controlling song learning and behavior in zebra finches. J Neurosci 5:1556 1561. Bottjer SW, Miesner EA, Arnold AP. 1984. Forebrain lesions disrupt development but not maintenance of song in passerine birds. Science 224:901 903. Brager DH, Sickel MJ, McCarthy MM. 1999. Developmental sex differences in calbindin-d 28K and calretinin immunoreactivity in the neonatal rat hypothalamus. J Neurobiol 42:315 322. Braunewell K-H, Gundelfinger ED. 1999. Intracellular neuronal calcium sensor proteins: a family of EF-hand calcium-binding proteins in search of a function. Cell Tissue Res 295:1 12. Burgoyne RD, Morgan A. 1995. Ca 2 and secretory vesicle dynamics. Trends Neurosci 18:191 196. Burgoyne RD, Weiss JL. 2001. The neuronal calcium sensor family of Ca 2 -binding proteins. Biochem J 353:1 12. Carruth LL, Reisert I, Arnold AP. 2002. Sex chromosome genes directly affect brain sexual differentiation. Nat Neurosci 5:933 934. Clapham DE. 1995. Calcium signaling. Cell 80:259 268. Cooke B, Hegstrom CD, Villeneuve LS, Breedlove SM. 1998. Sexual differentiation of the vertebrate brain: principles and mechanisms. Front Neuroendocrinol 19:323 362. De Vries GJ, Boyle PA. 1998. Double duty for sex differences in the brain. Behav Brain Res 92:205 213. Diekhoff G. 1992. Statistics, data, and research design. In: Lange M, editor. Statistics for the social and behavioral sciences: univariate, bivariate, multivariate. Dubuque: W.C. Brown, p 3 13. Feder H. 1981. Perinatal hormones and their role in the development of sexually dimorphic behaviors. In: Adler NT, editor. Neuroendocrinology of Reproduction. New York: Plenum Press, p 127 157. Gahr M. 1996. Developmental changes in the distribution of oestrogen receptor mrna expressing cells in the forebrain of female, male and masculinized female zebra finches. NeuroReport 7:2469 2473. Grisham W, Arnold AP. 1995. A direct comparison of the masculinizing effects of testosterone, androstenedione, estrogen, and progesterone on the development of the zebra finch song system. J Neurobiol 26:163 170. Gurney ME. 1981. Hormonal control of cell form and number in the zebra finch song system. J Neurosci 1:658 673. Gurney ME. 1982. Behavioral correlates of sexual differentiation in the zebra finch song system. Brain Res 231: 153 172. Heizmann CD. 1992. Calcium binding proteins: basic concepts and clinical implications Gen Physiol Biophys 11: 411 425. Hidaka H, Okazaki K. 1993. Neurocalcin family: a novel calcium-binding protein abundant in bovine central nervous system. Neurosci Res 16:73 77. Holloway CC, Clayton DF. 2001. Estrogen synthesis in the male brain triggers development of the avian song control pathway in vitro. Nat Neurosci 4:170 175. Iino S, Kobayashi S, Okazaki K, Hidaka H. 1995. Immunohistochemical localization of neurocalcin in the rat inner ear. Brain Res 680:128 134. Ivings L, Pennington SR, Jenkins R, Weiss JL, Burgoyne RD. 2002. Identification of Ca 2 -dependent binding partners for the neuronal calcium sensor protein neurocalcin : Interaction with actin, clathrin and tubulin. Biochem J 363:599 608. Jacobs EC, Arnold AP, Campagnoni AT. 1999. Developmental regulation of the distribution of aromatase- and estrogen-receptor-mrna-expressing cells in the zebra finch brain. Dev Neurosci 21:453 472. Kato M, Watanabe Y, Iino S, Takaoka Y, Kobayashi S, Haga T, Hidaka H. 1998. Cloning and expression of a

Zebra Finch Neurocalcin 385 cdna encoding a new neurocalcin isoform (neurocalcin ) from bovine brain. Biochem J 331:871 876. Kirn JR, DeVoogd TJ. 1989. Genesis and death of vocal control neurons during sexual differentiation in the zebra finch. J Neurosci 9:3176 3187. Kruse AA, Stripling R, Clayton DF. 2000. Minimal experience required for immediate-early gene induction in zebra finch neostriatum. Neurobiol Learn Mem 74:179 184. Madeira MD, Lieberman AR. 1995. Sexual dimorphism in the mammalian limbic system. Prog Neurobiol 45:275 333. Mathews GA, Arnold AP. 1990. Antiestrogens fail to prevent the masculine ontogeny of the zebra finch song system. Gen Comp Endocrinol 80:48 58. Mathews GA, Brenowitz EA, Arnold AP. 1988. Paradoxical hypermasculinization of the zebra finch song system by an antiestrogen. Horm Behav 22:540 551. Mattson MP. 1992. Calcium as sculptor and destroyer of neural circuitry. Exp Gerontol 27:29 49. Maxwell SE, Delaney HD. 1990. Introduction to model comparisons: One-way between-subjects design. In: King K, editor. Designing experiments and analyzing data: A model comparison perspective. Belmont: Wadsworth, p 63 128. Meisel RL, Sachs BD. 1994. The physiology of male sexual behavior. In: Knobil E, Neill JD, editors. The physiology of reproduction. New York: Raven Press, p 3 105. Mello CV, Clayton DF. 1994. Song-induced ZENK gene expression in auditory pathways of songbird brain and its relation to the song control system. J Neurosci 14:6652 6666. Mello CV, Ribeiro S. 1998. ZENK protein regulation by song in the brain of songbirds. J Comp Neurol 393:426 438. Nef P. 1996. Neuron specific calcium sensors (the NCS subfamily). In: Celio MR, Pauls TL, Schwaller B, editors. Guidebook to the calcium-binding proteins. Oxford: Oxford University Press, p 94 98. Nixdorf-Bergweiler BE. 1996. Divergent and parallel development in volume sizes of telencephalic song nuclei in male and female zebra finches. J Comp Neurol 375:445 456. Nordeen KW, Nordeen EJ, Arnold AP. 1986. Estrogen establishes sex differences in androgen accumulation in zebra finch brain. J Neurosci 6:734 738. Nottebohm F, Stokes TM, Leonard CM. 1976. Central control of song in the canary, Serinus canarius. J Comp Neurol 165:457 486. Okazaki K, Watanabe M, Ando Y, Hagiwara M, Terasawa M, Hidaka H. 1992. Full sequence of neurocalcin, a novel calcium-binding protein abundant in central nervous system. Biochem Biophys Res Commun 185:147 153. Rosen G, O Bryant E, Matthews J, Zacharewski T, Wade J. 2002. Distribution of androgen receptor mrna expression and immunoreactivity in the brain of the green anole lizard. J Neuroendocrinol 14:19 28. Saldanha CJ, Turek MJ, Kim Y-H, Fernandes AO, Arnold AP, Schlinger BA. 2000. Distribution and regulation of telencephalic aromatase expression in the zebra finch revealed with a specific antibody. J Comp Neurol 423: 619 630. Scharff C, Nottebohm F. 1991. A comparative study of the behavioral deficits following lesions of various parts of the zebra finch song system: Implications for vocal learning. J Neurosci 11:2896 2913. Schlinger BA, Arnold AP. 1992. Plasma sex steroids and tissue aromatization in hatchling zebra finches: Implications for the sexual differentiation of singing behavior. Endocrinol 130:289 299. Simpson HB, Vicario DS. 1990. Brain pathways for learned and unlearned vocalizations differ in zebra finches. J Neurosci 10:1541 1556. Simpson HB, Vicario DS. 1991a. Early estrogen treatment alone causes female zebra finches to produce learned, male-like vocalizations. J Neurobiol 22:755 776. Simpson HB, Vicario DS. 1991b. Early estrogen treatment of female zebra finches masculinizes the brain pathway for learned vocalizations. J Neurobiol 22: 777 793. Stokes TM, Leonard CM, Nottebohm F. 1974. The telencephalon, diencephalon, and mesencephalon of the canary, Serinus canaria, in stereotaxic coordinates. J Comp Neurol 156:337 374. Stuart E, Lephardt ED. 1999. Dimorphic expression of medial basal hypothalamic-preoptic area calbindin-d 28K mrna during perinatal development and adult distribution of calbindin-d 28K mrna in Sprague-Dawley rats. Mol Brain Res 73:60 67. Takamatsu K, Kobayashi M, Saitoh S, Fujishiro M, Noguchi T. 1994. Molecular cloning of human hippocalcin cdna and chromosomal mapping of its gene. Biochem Biophys Res Commun 200:606 611. Teng DH-F, Chen C-K, Hurley JB. 1994. A highly conserved homologue of bovine neurocalcin in Drosophila melanogaster is a Ca 2 -binding protein expressed in neuronal tissues. J Biol Chem 269:31900 31907. Terasawa M, Nakano A, Kobayashi R, Hidaka H. 1992. Neurocalcin: a novel calcium-binding protein from bovine brain. J Biol Chem 267:19596 19599. Vijay-Kumar S, Kumar VD. 1999. Crystal structure of recombinant bovine neurocalcin. Nat Struct Biol 6:80 88. Wade J. 1999. Sexual dimorphisms in avian and reptilian courtship: Two systems that do not play by mammalian rules. Brain Behav Evol 54:15 27. Wade J. 2001. Zebra finch sexual differentiation: The aromatization hypothesis revisited. Microsc Res Tech 54: 354 363. Wade J, Arnold AP. 1994. Post-hatching inhibition of aromatase activity does not alter sexual differentiation of the zebra finch song system. Brain Res 639:347 350.