Figure 1.
Levels of 12 Fmr1 transcript isoforms in adult C57/BL6 wild-type whole brain.
Total RNA was isolated from each brain and a portion was reverse transcribed using a gene specific primer for Fmr1 that is present in all splice variants and a MuP0 gene-specific primer. All values are normalized to MuP0 transcript from each brain (Materials and Methods). [A] Schematic representation of Fmr1 alternative splicing patterns in mouse. [B] Quantification of transcript levels for higher abundance isoforms. Error bars represent standard deviations for biological triplicates. [C] Quantification of transcript levels for lower abundance isoforms. Error bars represent standard deviations of values of triplicate measurements of a single brain. Please see Table 1 for numerical data.
Table 1.
Numerical values for transcript isoform levels in adult mouse braina.
Figure 2.
Polyribosome profiles of the longest and shortest Fmr1 transcript isoforms.
An adult mouse was euthanized by CO2 asphyxiation and decapitated. The brain was quickly removed and placed into liquid nitrogen. The brain was then later homogenized (Materials and Methods) and a portion was loaded onto a linear 15–60% sucrose gradient. Fractions were collected by upward displacement. [A] A254 profiles were determined using an ISCO fractionator. [B] Carrier E.coli RNA and UT2 internal standard mRNA was added to each fraction prior to RNA extraction. The amount of RNA lost during extraction was determined by qRT-PCR of the UT2 mRNA (Materials and Methods). Fmr1 isoforms 1 and 12, the longest and shortest forms, were quantified across the sucrose gradient by qRT-PCR. Error bars represent variation in replicate measurements from one brain.
Figure 3.
Fmr1 transcript isoforms enriched on neuron-specific ribosomes from adult mouse brain.
The neuron-specific, ribosome-bound transcripts were enriched using the RiboTag methodology as described in the text. (A) Immunohistochemistry on paraformaldehyde fixed coronal brain sections from Eno2-Cre:RiboTag mice demonstrating neural specific expression of Rpl22-HA (HA staining); sections are stained with the neuronal marker NeuN and counterstained with the glial specific marker GFAP. Overlap of NeuN and HA staining and absence of overlapping HA and GFAP staining supports neuron-specific labeling of ribosomes. This particular section corresponds to the dentate gyrus area of the hippocampus. Immunohistochemistry in cortex, striatum and other brain regions was similar to the image shown, suggesting that HA staining is specific to neurons. Scale Bar = 40 microns. (B) The Fmr1 transcript isoforms were isolated from immuno-purified polyribosomes and quantified by qRT-PCR as in Fig. 1. The results are averaged values from five independent, neuron-tagged mice. The average degree of neuronal enrichment of the tagged ribosomes over these five experiments was 3.2±0.7 fold, as estimated using the neuron-specific transcript from the NeuN gene (Fig. S4).
Figure 4.
Expression of the major Fmr1 isoforms during mouse brain development.
Brains were harvested in triplicate from male littermates and Fmr1 transcript isoforms were quantified as described in Materials and Methods. The data for all time points (except P7) are averages of biological triplicates and error bars represent the standard deviation in the biological triplicates. The values for P7 are an average of two brains, and the error bars represent the range in data.
Figure 5.
Exon 12 in Fmr1 transcripts from mouse brain and cultured progenitor cells.
The levels of the abundant Fmr1 transcripts were measured in [A] embryonic day 9 (E9), postnatal day 40 (P40) and adult mouse brains, in [B] cultured neural stem/progenitor cells, and in [C] dissected regions of adult mouse brain [cerebellum (Cb), cortex (Cx), hippocampus (Hip), and olfactory bulb (Ob)]. The data are presented as the fractions of Fmr1 transcripts containing (gray bars) and lacking (black bars) Exon 12 (eg. “+ Exon 12″ is the sum of Isoforms 1–3 divided by the sum of Isoforms 1–3 plus 7–9). Error bars for the individual isoforms are found in Fig. 1 for adult whole brain, in Fig. 4 for E9 and P40, in Fig. 6 for cultured neural stem/progenitor cells, and in Figs. 7 and 8 for individual brain sections.
Figure 6.
The abundant Fmr1 transcript isoforms in cultured neural stem/progenitor cells
. Neural stem/progenitor cells were maintained as described in Materials and Methods. An unstained phase contrast micrograph of the cultured cells is shown in [A]. Levels of the most abundant transcript isoforms were determined in triplicate biological samples and the error bars represent standard deviations of the means [B].
Figure 7.
Highly expressed Fmr1 transcript isoforms in eleven brain regions.
The amygdala, brainstem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, midbrain, nucleus accumbens, olfactory bulb and thalamus were dissected from three adult mouse brains. The levels of the abundant Fmr1 transcripts are shown with standard deviations in biological triplicates.
Figure 8.
Low expressed Fmr1 transcript isoforms in eleven brain regions.
Data for isoforms 4–6 and 10–12 are as indicated. The brain regions are defined in the legend to Fig. 7.