Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds

Zinzow-Kramer, Wendy M.; Horton, Brent M.; Maney, Donna L.

doi:10.1016/j.yhbeh.2014.04.011

Cited by 59 publications

(35 citation statements)

References 63 publications

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“…We used MxPro software (v.4.10, Agilent, Santa Clara, CA, USA) to analyze gene expression with the comparative Ct method, where abundance of transcripts from individual samples is expressed against a calibrator sample, and normalized against expression of a housekeeping gene (RPL4). RPL4 is one of the most stable housekeeping genes in testes of passerines (Zinzow-Kramer et al, 2014), and its expression did not differ between the sedentary urban and migrant juncos in our study (Mann-Whitney U = 32.50, n urban = 19, n migrant = 6, p = 0.234 two-tailed). Primer sequences are reported in the Supplementary Material.…”

Section: Mrna Extraction and Qpcrmentioning

confidence: 45%

Mechanisms Associated with an Advance in the Timing of Seasonal Reproduction in an Urban Songbird

et al. 2017

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The colonization of urban environments by animals is often accompanied by earlier breeding and associated changes in seasonal schedules. Accelerated timing of seasonal reproduction in derived urban populations is a potential cause of evolutionary divergence from ancestral populations if differences in physiological processes that regulate reproductive timing become fixed over time. We compared reproductive development in free-living and captive male dark-eyed juncos deriving from a population that recently colonized a city (∼35 years) and ceased migrating to that of conspecifics that live in sympatry with the urban population during winter and spring but migrate elsewhere to breed. We predicted that the earlier breeding sedentary urban birds would exhibit accelerated reproductive development in the spring along the hypothalamic-pituitary-gonadal (HPG) axis as compared to migrants. We found that free-living sedentary urban and migrant juncos differed at the level of the pituitary when measured as baseline luteinizing hormone (LH) levels, but not in increased LH when challenged with Gonadotropin-Releasing Hormone (GnRH). Among captives held in a common garden, and at the level of the gonad, we found that sedentary urban birds produced more testosterone in response to GnRH than migrants living in the same common environment, suggesting greater gonadal sensitivity in the derived urban population. Greater gonadal sensitivity could arise from greater upstream activation by LH or FSH or from reduced suppression of gonadal development by the adrenal axis. We compared abundance of gonadal transcripts for LH receptor (LHR), follicle stimulating hormone receptor (FSHR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) in the common-garden, predicting either more abundant transcripts for LHR and FSHR or fewer transcripts for GR and MR in the earlier breeding sedentary urban breeders, as compared to the migrants. We found no difference in the expression of these genes. Together these data suggest that advanced timing of reproduction in a recently derived urban population is facilitated by earlier increase in upstream baseline activity of the HPG and earlier release from gonadal suppression Fudickar et al. Timing of Reproduction following Urbanizationby yet-to-be-discovered mechanisms. Evolutionarily, our results suggest that potential for gene flow between seasonally sympatric populations may be limited due to urban-induced advances in the timing of reproduction and resulting allochrony with ancestral forms.

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Section: Mrna Extraction and Qpcrmentioning

confidence: 45%

Mechanisms Associated with an Advance in the Timing of Seasonal Reproduction in an Urban Songbird

et al. 2017

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“…We designed exon-spanning primers (Table S2) for use with probes from the Roche Universal Probe Library for OTR, V1aR, and V2R, then verified the amplified sequences via cloning and sequencing. qPCR was performed using a Roche LightCycler 480 Real-Time PCR System, in triplicate for each sample on a 384-well plate as previously described (Zinzow-Kramer et al, 2014). The target genes (OTR, V1aR, and V2R) and reference genes were always run on the same plate for each sample, and sex and age were balanced across plates.…”

Section: Autoradiographymentioning

confidence: 99%

“…We normalized the expression of each gene of interest to the geometric mean of two reference genes (Pfaffl, 2001;Vandesompele et al, 2002). The reference genes were glyceraldehyde 3-phosphate dehydrogenase (GADPH) and peptidylprolyl isomerase A (PPIA), which have been previously validated for use in zebra finch brain tissue and are stably expressed across sex (Zinzow-Kramer et al, 2014). For samples with a coefficient of variance above 21.8% within triplicates, indicating technical error, we identified and removed one outlying point within the triplicate (Bookout et al, 2006) using a modified Dixon's q test (Dean & Dixon, 1951).…”

Section: Autoradiographymentioning

confidence: 99%

Expression of oxytocin receptors in the zebra finch brain during vocal development

Davis

Grogan

Maney

2019

Preprint

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Juvenile male zebra finches memorize and learn to sing the song of a male caregiver, or "tutor", during a complex vocal learning process. Juveniles are highly motivated to interact socially with their tutor, and these interactions are required for effective vocal learning. It is currently unknown what neurological mechanisms underlie attraction to tutors, but social motivation and affiliation in this and other species may be mediated by oxytocin and related nonapeptides. Here, we used qPCR to quantify expression of oxytocin receptor (OTR) mRNA in the lateral septum, auditory forebrain, and regions of the song control system in zebra finches throughout post-hatch development and vocal learning. We found that zebra finches express OTR mRNA in these regions from post-hatch day 5 to adulthood, encompassing the entire period of auditory and sensorimotor learning. We also mapped the binding of 125 I-ornithine vasotocin, an oxytocin receptor antagonist that binds to oxytocin receptors in songbird brain, to understand the neuroanatomical distribution of oxytocin-like action during vocal development. This study provides the groundwork for the use of zebra finches as a model for understanding the mechanisms underlying social motivation and its role in vocal development.

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“…The amplified sequence for ESR2 was verified via agarose gel and Sanger sequencing. qPCR was performed using a Roche LightCycler 480 Real-Time PCR System in triplicate for each sample on 384-well plates as previously described 47 . Using the LightCycler 480 Software Version 1.5.0, we calculated crossing point (Cp) values using the Abs Quant/2nd Derivative Max method.…”

Section: Effects Of Esr1 Knockdown On Aggressionmentioning

confidence: 99%

“…Using the LightCycler 480 Software Version 1.5.0, we calculated crossing point (Cp) values using the Abs Quant/2nd Derivative Max method. We normalized the expression of each gene of interest to two reference genes: peptidylprolyl isomerase A (PPIA) and glyceraldehyde-3phosphate dehydrogenase (GAPDH), which have been previously validated for use in white-throated sparrow brain tissue 47 . We performed GLMs, as chosen on the basis of AIC values, 43 to test for effects of morph and oligo type (antisense or scrambled) on the expression of ESR1, and then followed up the significant interaction with GLMs testing for an effect of oligo type on expression of ESR1 within each morph.…”

Section: Effects Of Esr1 Knockdown On Aggressionmentioning

confidence: 99%

A behavioral polymorphism caused by a single gene inside a supergene

Merritt

Grogan

Zinzow-Kramer

et al. 2020

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Behavioral evolution relies on genetic changes, yet few social behaviors can be traced to specific genetic sequences in vertebrates. Here, we show experimental evidence that differentiation of a single gene has contributed to divergent behavioral phenotypes in the white-throated sparrow, a common North American songbird. In this species, one of two alleles of ESR1, encoding estrogen receptor a (ERa), has been captured inside a differentiating supergene that segregates with an aggressive phenotype, such that ESR1 expression predicts aggression. Here, we show that the aggressive phenotype associated with the supergene is prevented by ESR1 knockdown in a single brain region. Next, we show that in a free-living population, aggression is predicted by allelic imbalance favoring the supergene allele. Cis-regulatory variation between the two alleles affects transcription factor binding sites, DNA methylation, and rates of transcription. This work provides a rare illustration of how genotypic divergence has led to behavioral phenotypic divergence in a vertebrate.

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Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds

Cited by 59 publications

References 63 publications

Mechanisms Associated with an Advance in the Timing of Seasonal Reproduction in an Urban Songbird

Mechanisms Associated with an Advance in the Timing of Seasonal Reproduction in an Urban Songbird

Expression of oxytocin receptors in the zebra finch brain during vocal development

A behavioral polymorphism caused by a single gene inside a supergene

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