SUPPLEMENTARY MATERIAL AND METHODS
Comparison of AVT expression using transcription profiling
All brains were harvested from euthanized fish after determining standard length
and body mass within minutes after initially disturbing the tank between 13:00 and 15:00
hours. For the whole brain experiment, brains of six T and six NT males were removed
and stored in RNAlater (Ambion, Austin, TX) at -20° C until total RNA was isolated
using a standard Trizol protocol (Invitrogen, Carlsbad, CA). For preoptic area microdissection, the brains of five Ts and five NTs were harvested and embedded in OCT
compound (Sakura Finetek, Torrance, CA), frozen on dry ice, stored at -80° C and
subsequently cryo-sectioned at 16 µm. After fixing the sections in 100% ethanol and
Nissl staining with cresyl violet, the anterior and medial portions of the preoptic area
were harvested from consecutive sections using a laser micro-dissection system (Arcturus
PixCell II). This included the entire region of the brain considered the preoptic area
(Fernald and Shelton 1985), with the exclusion of the gigantocellular portion of the
magnocellular preoptic nucleus (Fernald and Shelton 1985). ). Total RNA was isolated
using a modified Trizol protocol and subjected to amplification in two rounds of in vitro
transcription (Express Art kit, AmpTec GmbH, Hamburg, Germany).
In both experiments, the RNA was then reverse transcribed, fluorescently labelled
and competitively hybridized to a custom-made cichlid cDNA microarray, as previously
described (Renn et al. 2004). The microarray platform contains multiple features
(different library clones whose sequences largely overlap) representing AVT and is
described in detail at NCBI GEO GPL928. Here, we report differences in AVT
expression; other gene expression differences are presented elsewhere (Renn et al. 2008;
Larkins-Ford et al. in preparation).
After removing low intensity features and normalizing the arrays, we used
intensity ratios to determine significant differences in gene expression between T and NT
males using Bayesian analysis of gene expression levels (BAGEL, Townsend and Hartl
2002). BAGEL yields a relative expression level for each gene as well as a Bayesian
posterior probability (PP) of significant differences between individual fish. This
approach is preferred over a traditional Analysis of Variance to determine transcript level
differences, as it is much more robust to missing data and variation in replication
(Meiklejohn and Townsend 2005). We estimated the false-positive rate associated with a
given PP by performing a permutation analysis, which allowed us to determine an
appropriate significance threshold (Renn et al. 2008). In both experiments, all array
features representing AVT (see above) yielded highly concordant measures of gene
expression and a combined probability was determined according to Fisher’s test of
combining probabilities (Sokal and Rohlf 1995).
Comparison of AVT expression using in situ hybridization
A radioactively-labelled antisense AVT probe was generated from a template
within the 3’ untranslated region of the A. burtoni AVT preprohormone cDNA sequence
(Genbank accession no. AF517935). The 380 bp template was generated by PCR
amplification with the following primers: upper primer 5’- GGC GTC CAT CTG CTG
CTC AAG ACC -3’ and lower primer 5’- GTT TTA CCC GGA TGC CTG TAT CAA C
-3’. 35S-labeled riboprobes were generated using a MAXIscript kit (Ambion, Austin, TX).
Three series of 14 μm coronal sections were cut using a cryostat. In situ
hybridization was performed according to an established protocol (Burmeister et al. 2005;
Grens et al. 2005) except that the probe concentration was ~3  106 cpm/ml. After
exposure to nuclear emulsion for 6 h, slides were developed and counterstained with
cresyl violet. A second in situ hybridization run was performed on a second series of
slides containing alternate sections in order to characterize a hypothalamic population of
AVT neurons with low levels of expression (see Results). The procedure was identical to
the above except that probe was diluted to ~5  106 cpm/ml in hybridization solution and
the slides were exposed to emulsion for 7 days.
Slides were visualized on a microscope under brightfield and darkfield
illumination to identify Nissl stained cell bodies and silver grains, respectively.
Neuroanatomy was guided by several published chartings of the diencephalon of
perciform species, including one of A. burtoni (Fernald and Shelton 1985; CerdaReverter et al. 2001). For the hypothalamic population, we chose to follow the
nomenclature used for the sea bass (Cerda-Reverter et al. 2001), rather than that for A.
burtoni (Fernald and Shelton 1985), as it was more consistent with the label ascribed to
this AVT population in previous work (Goodson and Bass 2000).
We used a sampling procedure to measure expression levels in different
populations of AVT neurons. Because magnocellular and parvocellular AVT neurons
form a continuous arc through the preoptic area it was difficult to assign neurons to
distinct populations in the area of overlap. Therefore we restricted our measurements to
portions of these nuclei that were anatomically distinct by sampling the anterior portion
of the parvocellular nucleus and the posterior part of the magnocellular population. For
the parvocellular preoptic nucleus, we scanned the preoptic area of each brain until silver
grains were first detected on the ventral aspect of the brain. Two sections after this first
evidence of expression, we collected images for a total of four subsequent sections. For
the magnocellular portion of the magnocellular preoptic nucleus, we used the anatomy of
the entopeduncular nucleus (Fernald and Shelton 1985) to determine when to start
collecting images. The entopeduncular nucleus first looks like a mass of cells in the
middle of each hemisphere, lateral of the AVT cells in the preoptic area. It then thins into
a narrow band of two approximately parallel lines of cell bodies before the mass of cells
moves dorsally. We collected images on four consecutive sections starting on the section
when the entopeduncular nucleus narrows into a thin slit. For the gigantocellular portion
of the magnocellular preoptic nucleus, we started at the most posterior section containing
gigantocellular AVT cells and took images moving rostrally until this nucleus
disappeared, thereby measuring the entire nucleus. For the hypothalamic population in
the ventral part of the lateral tuberal nucleus (see Results) we captured four images
beginning on the third section after the first evidence of expression along the
ventromedial aspect of the brain posterior to the horizontal commissure.
Digital images of silver grains were taken (SPOT camera, Diagnostic Instruments,
Sterling Heights, MI) using a 100 objective and were centred so the densest staining
would be within the frame of the photograph. We used a blue filter to diminish cresyl
violet stained cell bodies in order to increase the prominence of silver grains. The images
were converted to grey scale. We counted all the silver grains in an image using ImageJ
(National Institutes of Health, Bethesda, MD). Images were subjected to the threshold
function using the same threshold for all images for a given population, and then the
number of particles in the image was counted. The number of particles per section for all
sections on both sides of the brain was summed. For instances of missing data due to
damaged sections, the average value of the intact sections was substituted for the missing
value. For the gigantocellular population, where cells were clearly distinct from one
another, we also recorded the total number of cells expressing AVT.
In several of the AVT nuclei, there were correlations between body size and silver
grain count. Since T and NT males differed in their average standard length (T: range, 7 7.2 cm; mean, 7.09 cm; NT: range, 5.7 - 7 cm ; mean, 6.3 cm; F (1, 10) = 10.5; p < 0.01,
we used the following equation to adjust silver grain values to account for influences of
body size (e.g. White and Fernald 1993; Hofmann and Fernald 2000): SGc = SGu +
([(SGu * SLx / SL) - SGu] * K), where SGc = corrected silver grain count, SGu =
uncorrected silver grain count, SLx = average standard length for all subjects, SL =
standard length for the particular subject, and K = Pearson’s R. The correlation
coefficients between silver grains per section and standard length for each AVT
population were as follows: parvocellular -0.68, magnocellular 0.08, gigantocellular 0.46,
hypothalamic: 0.011. Corrected silver grain values are reported in the text. No qualitative
differences in statistics emerged from this correction.
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