Expression of fragile X mental retardation protein within the vocal control system of developing and adult male zebra finches

Abstract

Individuals with fragile X syndrome (FXS) are cognitively impaired and have marked speech delays and deficits. Our goal was to characterize expression of fragile X mental retardation protein (FMRP), encoded by Fmr1 fragile X mental retardation 1 gene or transcript (FMR1), in an animal model that learns to vocalize, namely the zebra finch Taeniopygia guttata (Tgu). We cloned and sequenced the zebra finch ortholog of FMR1 (TguFmr1) and developed an antibody that recognizes TguFmrp specifically. TguFmrp has structural features similar to its human ortholog FMRP. Because FXS patients exhibit sensorimotor deficits, we examined TguFmrp expression prior to, during, and after sensorimotor song learning in zebra finches. We found that TguFmrp is expressed throughout the brain and in four major song nuclei of the male zebra finch brain, primarily in neurons. Additionally, prior to sensorimotor learning, we observed elevated TguFmrp expression in the robust nucleus of the arcopallium (RA) of post-hatch day 30 males, compared with the surrounding telencephalon, suggesting a preparation for this stage of song learning. Finally, we observed variable TguFmrp expression in the RA of adolescent and adult males: in some males it was elevated and in others it was comparable to the surrounding telencephalon. In summary, we have characterized the zebra finch ortholog of FMRP and found elevated levels in the premotor nucleus RA at a key developmental stage for vocal learning.

Fig. 1. Map of the zebra finch ‘song circuit’

HVC: letter-based name; DLM: DorsoLateral Medial nucleus of the thalamus; LMAN: Lateral Magnocellular nucleus of the Anterior Nidopallium; nXIIts: nucleus trachiosyringealis of cranial nerve XII; RA: Robust nucleus of the Arcopallium. Anterior Forebrain Pathway (AFP) shown in blue; Posterior Pathway (PP) in red. The AFP traditionally has been recognized as important for song learning, while the PP for song production. LMAN is the output nucleus for the AFP. Recent data show that LMAN input into RA induces variability in song in both juveniles and adults, indicating that both pathways contribute to the motor production of song (Kao et al., 2005, Ölveczky et al., 2005, Thompson and Johnson, 2007, Aronov et al., 2008). (Rostral [R] left; Ventral [V] down. Not drawn to scale.)

Fig. 2. Alignment of Fmr orthologs of mouse, human, zebra finch, and chicken Fmr proteins

The alignment was performed using ClustalW. Stars indicate identical residues; dots denote similarity (residues with two dots are more similar than those with one). The human sequence is accession number NP_002015; mouse is NP_03257. Zebra finch cDNA sequence (see methods) was translated using DNA Strider. The chicken cDNA sequence (accession number XM_420363) was translated using DNA Strider. Colors: KH1 and KH2 are shown in brown and the RGG box in purple according to (Siomi et al., 1993); anti-TguFmrp epitope is shown in green; the isoleucine converted to asparagines in one severe case of FXS (De Boulle et al., 1993) is underlined and in bold font within the KH2 domain; the primary phosphorylation site of FMRP and Fmrp (Ceman et al., 2003) is shown in blue.

Fig. 3. TguFmr1 mRNA runs at 3.4Kb

Total RNA was prepared from songbird brain and 20ug was resolved on a 1.2% agarose/formaldehyde gel and probed with a digoxigenin-labeled riboprobe specific to TguFmr1. Markers indicating sizes in kilobases (Kb) are shown on the left. To measure the transcript length, a standard curve was generated using ribosomal RNA (rRNA) of subunits as size markers. Avian rRNA subunits were measured against murine rRNA and Fmr1 mRNA and calculated to measure 4.25 and 2.0Kb (data not shown). Using these values, we were able to calculate the length of TguFmr1 mRNA as 3.4Kb, with a minor band at 5.0Kb.

Fig. 4. Anti-TguFmrp is specific to TguFmrp

A. Lysate from Cos-7 cells mock-transfected (M) and transfected (TxF) with TguFmrp-EGFP, immunoblotted with anti-TguFmrp 24 and anti-EGFP. Addition of the 28 kDa EGFP increased the size of the fusion construct to ∼98kDa. eIF5 shown here as a loading control. B. M and TxF lysate was immunoprecipitated with anti-TguFmrp 23 and 24 and the immunoprecipitates were analyzed by western blot using anti-EGFP. C. Protein lysate from zebra finch muscle (Mu) and brain (Br) tissue, as well as lysate from (A) was analyzed by western blot using anti-TguFmrp 24. The blot was followed with the 1a antibody to visualize endogenous Cos-7 Fmrp as well as both zebra finch and Cos-7 Fxr1. Endogenous TguFmrp runs at 75kDa. For A and C, similar results were obtained using anti-TguFmrp 23 (data not shown). D. Cos-7 cells transfected with TguFmr1-EGFP were fixed 24 hours post-transfection and stained with anti-TguFmrp 23. Shown are EGFP expression (green), 23 immunoreactivity (red), and DAPI-labeled nuclei (blue). The overlay of all three exposures is shown in the lower right. Similar results were obtained with anti-TguFmrp 24 (data not shown). Bar = 20μm

Fig. 5. TguFmrp is expressed in neurons in the male zebra finch brain

Sagital brain sections containing A. HVC (letter-based name) and RA and B. LMAN (Lateral MAgnocellular nucleus of the Nidopallium) and Area X were stained for anatomy with cresyl violet (bar = 1000μm) (upper left). Upper right is an accompanying sketch with the significant anatomical features indicated. Adjacent sections were co-stained with the anti-TguFmrp antibody 24 (red), the neuronal marker NeuN (green), and DAPI (blue). In the overlay, a yellow signal indicates co-fluorescence for red and green. Bar = 20μm. Bst: Brainstem; Cb: Cerebellum; LFM: Lamina frontalis suprema; LFS: Lamina frontalis superior; LH: Lamina hyperstriatica; LMD: Lamina medullaris dorsalis; TeO: Optic Tectum. Shown are images from a P30 brain; P60 and Adult males showed similar results (data not shown).

Fig. 6. TguFmrp is consistently elevated in the RA nucleus of a P30 male zebra finch and variably expressed in P60 and Adult males

A-C. Representative fluorescent-IHC using anti-TguFmrp 24 on a male P30 (A) P60 (B) and Adult (C) zebra finch RA. Shown are TguFmrp immunoreactivity (red), NeuN stain (green), and DAPI-labeled nuclei (blue), along with the overlay. Arrows denote ventral border of RA. Bar = 100μm. D-F. Representative DAB-IHC using anti-TguFmrp 24 on a male P30 (D) P60 (E) and Adult (F) zebra finch RA. Bar = 200μm.