Androgen Modulation of Foxp1 and Foxp2 in the Developing Rat Brain: Impact on Sex Specific Vocalization

NEUROENDOCRINOLOGY Androgen Modulation of Foxp1 and Foxp2 in the Developing Rat Brain: Impact on Sex Specific Vocalization J. Michael Bowers, Miguel Perez-Pouchoulen, Clinton R. Roby, Timothy E. Ryan, and Margaret M. McCarthy Department of Pharmacology (J.M.B., M.P.-P., C.R.R., M.M.M.), University of Maryland School of Medicine and Programs in Neuroscience (M.M.M.) and Medicine (T.E.R.), University of Maryland School of Medicine, University of Maryland, Baltimore, Baltimore, Maryland 21201 Sex differences in vocal communication are prevalent in both the animals and humans. The mechanism(s) mediating gender differences in human language are unknown, although, sex hormones, principally androgens, play a central role in the development of vocalizations in a wide variety of animal species. The discovery of FOXP2 has added an additional avenue for exploring the origins of language and animal communication. The FOXP2 gene is a member of the forkhead box P (FOXP) family of transcription factors. Prior to the prenatal androgen surge in male fetuses, we observed no sex difference for Foxp2 protein levels in cultured cells. In contrast, 24 hours after the onset of the androgen surge, we found a sex difference for Foxp2 protein levels in cultured cortical cells with males having higher levels than females. Furthermore, we observed the potent nonaromatizable androgen dihydrotestosterone altered not only Foxp2 mRNA and protein levels but also Foxp1. Androgen effects on both Foxp2 and Foxp1 were found to occur in the striatum, cerebellar vermis, and cortex. Immunofluorescence microscopy and coimmunoprecipitation demonstrate Foxp2 and the androgen receptor protein interact. Databases for transcription factor binding sites predict a consensus binding motif for androgen receptor on the Foxp2 promoter regions. We also observed a sex difference in rat pup vocalization with males vocalizing more than females and treatment of females with dihydrotestosterone eliminated the sex difference. We propose that androgens might be an upstream regulator of both Foxp2 and Foxp1 expression and signaling. This has important implications for language and communication as well as neuropsychiatric developmental disorders involving impairments in communication. (Endocrinology 155: 4881– 4894, 2014) L anguage in humans and vocal communication in animals is a phenomenon that has intrigued scientists for more than a century (1). The discovery of the FOXP2 gene has added an additional avenue for exploring the origins of language and animal communication. The FOXP2 gene is a member of the forkhead box P (FOXP) family of transcription factors, which also includes FOXP1, FOXP3, and FOXP4 (for review see reference 2). Of these four FOXP genes, most is known about FOXP2 due to the identification of mutations more than a decade ago in the large intergenerational KE family (3). The affected mem- bers have a mutation in the DNA-binding domain of FOXP2 and exhibit profound deficits in cognition and language (4, 5). FOXP2 is expressed in several areas of the brain involved in motor control, sensory integration, and vocal communication (6 – 8). Furthermore, FOXP2 transcriptionally regulates genes involved in neuronal development, neurite outgrowth, dendritic branching, and axonal morphology (9, 10). A note about nomenclature is that nucleotide sequences are italicized, proteins are not, human forms are in uppercase letters (eg, FOXP2), murine forms are in lowercase letters (Foxp2) and those of other ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received June 17, 2014. Accepted September 16, 2014. First Published Online September 23, 2014 Abbreviations: AR, androgen receptor; ASD, autism spectrum disorder; Co-IP, coimmunoprecipitation; DHT, dihydrotestosterone; E, embryonic day; FOXP, forkhead box P; PBS-T, glycine in Triton X-100; PN, postnatal day; qRT-PCR, quantitative RT-PCR; USV, ultrasonic vocalization. doi: 10.1210/en.2014-1486 Endocrinology, December 2014, 155(12):4881– 4894 endo.endojournals.org 4881 4882 Bowers et al Androgen Modulation of Foxp1and Foxp2 species, such as birds, contain both uppercase and lowercase letters (eg, FoxP2) (11). Rodents communicate through the use of ultrasonic vocalizations (12, 13). Ultrasonic vocalizations (USVs) are produced during social encounters and function as a shortdistance communication method (14, 15). It is hypothesized that USVs became the preferred mode of communication for rodents because many predators have a limited hearing capacity for ultrasonic tones. Thus, providing rodents an ideal method for communicating with intended receivers while minimizing the threat of predation (12, 15). USVs emerge early in infancy in many rodent species, (ie, within the first few days of life for mice and rats) (13, 16) and are triggered by aversive events such as maternal separation and hypothermia (17, 18). This specific type of USV (ie, maternal separation calls or pup isolation calls) is the most widely used method for eliciting and analyzing vocalizations in young rodents (19 –21). A number of Foxp2 mutant mice have been developed to investigate how this gene influences brain development and animal vocalizations (for a complete review, see references 6 and 22). Foxp2 mutant mice show a range of deficient neuronal and behavioral developments ranging from reductions in neurite outgrowth (23), decreased or atypical vocalizations (24, 25), and impairments in learning (26). FoxP2 has also been studied in the highly vocal bird with FoxP2 expressed in brain regions comparable with the mammalian expression pattern (27). FoxP2 levels change during song acquisition (28), further highlighting the important role for FoxP2 in learned vocal behavior. Together the research from both rodent and songbird emphasizes the vital role this transcription factor has on the development of neural circuitry underlying vocalization and behavior. Sex differences in vocal communication are prevalent in both the animals (29) and human language (30 –32). Several studies have found language processing in women involves more bilateral hemispheric activation, whereas men are more strongly lateralized to the left hemisphere in their language processing (33–36). There is also a consistent trend for girls to acquire language earlier than boys (31, 37), and girls begin to use gestural communication earlier than boys (38) as well as produce their first words (39) and first sentences (40) at a younger age than boys. Nonetheless, the existence of gender differences in human speech and language processing are highly controversial (for review see reference 41). Potential mechanism(s) mediating gender differences in human language have not been identified, although sex hormones, principally androgens, play a central role in the development of vocalizations in a wide variety of animal species including primates (42– 46). Interestingly, men Endocri (...truncated)