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The Forkhead transcription factor FOXP2 is implicated in speech perception and production. The avian homolog, FoxP21 contributes to song learning and production in birds. In human cell lines, transcriptional activity of FOXP2 requires homo-dimerization or dimerization with paralogs FOXP1 or FOXP4. Whether FoxP dimerization occurs in the brain is unknown. We recently showed that FoxP1, FoxP2 and FoxP4 (FoxP1/2/4) proteins are co-expressed in neurons of Area X, a song control region in zebra finches. We now report on dimer- and oligomerization of zebra finch FoxPs and how this affects transcription. In cell lines and in the brain we identify homo- and hetero-dimers, and an oligomer composed of FoxP1/2/4. We further show that FoxP1/2 but not FoxP4 bind to the regulatory region of the target gene Contactin-associated protein-like 2 (CNTNAP2). In addition, we demonstrate that FoxP1/4 bind to the regulatory region of very low density lipoprotein receptor (VLDLR), as has been shown for FoxP2 previously. Interestingly, FoxP1/2/4 individually or in combinations regulate the promoters for SV40, zebra finch VLDLR and CNTNAP2 differentially. These data exemplify the potential for complex transcriptional regulation of FoxP1/2/4, highlighting the need for future functional studies dissecting their differential regulation in the brain.

Osteoblast induction and differentiation in developing long bones is dynamically controlled by the opposing action of transcriptional activators and repressors. In contrast to the long list of activators that have been discovered over past decades, the network of repressors is not well-defined. Here we identify the expression of Foxp1/2/4 proteins, comprised of Forkhead-box (Fox) transcription factors of the Foxp subfamily, in both perichondrial skeletal progenitors and proliferating chondrocytes during endochondral ossification. Mice carrying loss-of-function and gain-of-function Foxp mutations had gross defects in appendicular skeleton formation. At the cellular level, over-expression of Foxp1/2/4 in chondroctyes abrogated osteoblast formation and chondrocyte hypertrophy. Conversely, single or compound deficiency of Foxp1/2/4 in skeletal progenitors or chondrocytes resulted in premature osteoblast differentiation in the perichondrium, coupled with impaired proliferation, survival, and hypertrophy of chondrocytes in the growth plate. Foxp1/2/4 and Runx2 proteins interacted in vitro and in vivo, and Foxp1/2/4 repressed Runx2 transactivation function in heterologous cells. This study establishes Foxp1/2/4 proteins as coordinators of osteogenesis and chondrocyte hypertrophy in developing long bones and suggests that a novel transcriptional repressor network involving Foxp1/2/4 may regulate Runx2 during endochondral ossification.

The Forkhead transcription factor FoxP2 is important both for human speech and for bird song learning. In vitro, transcriptional activity of FoxP2 requires dimerization, either with itself of with other members of the Forkhead P family, FoxP1 and FoxP4. In vivo, the brain expression patterns of FoxP1, FoxP2 and FoxP4 have not been systematically compared for regional or cellular co-localization. To provide the means for future functional studies I cloned FoxP4 from zebra finches and compared both the mRNA and protein expression patterns of FoxP1, FoxP2 and FoxP4 at different ages. I found overlapping expression of FoxP1, FoxP2 and FoxP4 in striatum and there, in Area X, a nuclei important for song learning. HVC and RA, two important nuclei of the motor pathway of the song system, express FoxP1 and FoxP4. All FoxP subfamily members studied had a specific pattern of expression, shown in regions of overlapping expression and regions were there are either expressed alone or with another FoxP member. I further characterize HVC neurons expressing FoxP1 and FoxP4 as Area X and RA projecting neurons. To address whether specific combinations of FoxP expression existed I analyzed co-expression in the most important song nuclei using double in situ hybridization and triple immunohistochemistry. I provide the first evidence that FoxP subfamily members can be co-express in the same neurons. In Area X and striatum, I found neurons expressing all combinations of FoxP expression consistently during the different ages assessed. Surprisingly I found few cells that expressed only FoxP2, the majority of cells were FoxP1+/FoxP2+/FoxP4+, FoxP1+/FoxP4+ or FoxP1 alone. In addition all Purkinje cells express FoxP2 and FoxP4, HVC projecting neurons express FoxP1 and FoxP4 as well as RA neurons. A second part of my work focus on the interaction of zebra finch FoxP1, FoxP2 and FoxP4 proteins and what the functional implication of this interaction would be. I first show in vitro that zebra finch FoxP1/2/4 proteins are able to homo- and hetero- dimerize as shown for their mouse counterparts. I further shown that in vivo hetero-dimerization occurs. Last I show that cells expressing only FoxP2 are not as repressed as cells expressing FoxP2 and FoxP1 or FoxP4. These results imply that a variety of regulatory possibilities exist via dimerization of the FoxP members in cells expressing them. In the last part of my work I assessed the functional consequence of a reduction of FoxP1 and FoxP4 in Area X using lentiviral mediated RNAi. I observed a similar phenotype with FoxP1 and FoxP4 to the one observed with FoxP2. Birds did not copy completely the tutor song and had a lower similarity and sequential match if compared to control birds. This data corroborates recent data on FoxP1 involvement in speech deficits in humans and opens the question if FoxP4 might mutations in humans might lead to a similar phenotype as the one seen with FoxP1 and FoxP2. This is the first evidence that the auditory-guided vocal learning in the basal ganglia requires all FoxP subfamily members, together as a family., Der Transkriptionsfaktor FOXP2, dessen Mutationen mit einer erblichen Sprachstörung, Childhood Apraxia of Speech, CAS, (auch Developmental Verbal Dyspraxia, DVD genannt) assoziiert sind, spielt auch beim Erwerb des Vogelgesangs eine wichtige Rolle. Es ist bereits durch in vitro Studien bekannt, dass FoxP2 um an DNA binden zu können, als Dimer vorliegen muss, entweder als Homodimer (FoxP2/FoxP2) oder in Kombination mit FoxP1 oder FoxP4. In vivo, im Gehirn des Zebrafinken, wurde bisher nicht systematisch untersucht ob FoxP1, FoxP2 und FoxP4 regional und zellulär koexpremiert werden. Um die Basis für zukünftige funktionelle Studien zu schaffen, klonierten wir das FoxP4 Gen des Zebrafinken und untersuchten die Expressionsmuster von FoxP1, FoxP2 und FoxP4 mRNA und Protein in Zebrafinken unterschiedlichen Alters. Regionale Koexpression von FoxP1, Foxp2 und FoxP4 konnte im Striatum und dort in Area X, einer für das Gesangslernen essentiellen Struktur nachgewiesen werden. HVC und RA, zwei Kerne der motorischen Bahn des Gesangssystems zeigten eine Koexpression von FoxP1 und FoxP4. Alle FoxP Mitglieder die wir analysiert haben zeigten regional spezifische Expressionsmuster mit teilweiser Überlappung. Weiterhin konnten wir zeigen, dass FoxP1 und FoxP4 koexpremierende Neurone des HVC entweder zu Area X oder RA projizieren. Um der Frage nachzugehen, in welche Kombinationen FoxP1, FoxP2 und FoxP4 in Neuronen von Area X koexpremiert werden, führten wir dreifach-fluoreszente Immunhistochemie und duale in situ Hybridizierungen durch, wobei simultan zwei verschiedene Sonden unterschiedlich fluoreszensmarkiert nachgewiesen werden. Wir konnten so zeigen, dass alle Kombinationen von FoxP1, FoxP2 und FoxP4 in Neuronen koexpremiert werden können. In Area X sind die meisten Neurone FoxP1+/FoxP2+/FoxP4+, FoxP1+/FoxP4+ oder FoxP1+ positiv. Es gab sehr wenige Neurone die nur FoxP2 expremieren. Purkinjezellen zeigten sehr starke Expression von FoxP2 und FoxP4, während Projektionsneurone in HVC und Neurone des RA FoxP1 und FoxP4 expremierten. Im zweiten Teil meiner Dissertation habe ich die Interaktionen zwischen FoxP1, FoxP2 und FoxP4 funktionell untersucht. Anhand von in vitro Studien konnten wir belegen, dass FoxP1, FoxP2 und FoxP4 des Zebrafinken, wie auch schon für die Mausorthologen gezeigt wurde, als Homodimere und Heterodimere vorliegen können. In vivo, im Gehirn von Zebrafinken, konnten wir Heterodimere von FoxP1/FoxP2 und FoxP2/FoxP4 nachweisen. Zuletzt konnten wir zeigen, dass FoxP2 Zielgene in vitro stärker reprimiert werden, wenn FoxP1 und FoxP4 koexpremiert wurden. Diese Ergebnisse weisen darauf hin, dass differentielle Genregulation durch FoxP´s in verschiedenen Zelltypen durch die Zusammensetzung von FoxP Dimeren beeinflusst werden kann. Abschliessend zeigte die experimentelle Reduktion von FoxP1 und FoxP4 eine Beeinträchtigung des Gesangslernens, ähnlich derer, die schon für FoxP2 beschrieben wurde. Im Gegensatz zu Kontrollvögeln konnten Vögel mit RNAi-vermittelter Verminderung der FoxP1 oder FoxP4 in Area X verschiedene Gesangsmerkmale des Tutorgesanges nicht akkurat kopieren. Diese Ergebnisse deuten darauf hin, dass FoxP1, FoxP2 und FoxP4 notwendig für auditorisch geleitetes, vokales Lernen sind.