Regulation of the Pax6 : Pax6(5a) mRNA ratio in the developing mammalian brain
© Pinson et al; licensee BioMed Central Ltd. 2005
Received: 06 May 2005
Accepted: 19 July 2005
Published: 19 July 2005
Early in mammalian brain development cell proliferation generates a population of progenitor cells whose subsequent divisions produce increasing numbers of postmitotic neurons. Pax6 affects both processes and it has been suggested that this changing role is due at least in part to changes in the relative concentrations of its two main isoforms, (i) Pax6 and (ii) Pax6(5a), created by insertion of a 42 bp exon (exon 5a) into one of the two DNA-binding domains. Crucially, however, no previous study has determined whether the ratio between Pax6 and Pax6(5a) transcripts alters during mammalian neurogenesis in vivo.
Using RNase protection assays, we show that Pax6 transcripts are 6–10 times more prevalent than Pax6(5a) transcripts early in neurogenesis in the murine telencephalon, diencephalon and hindbrain and that the ratio later falls significantly to about 3:1 in these regions.
These changes in vivo are similar in magnitude to those shown previously to alter target gene activity in vitro and might, therefore, allow the single mammalian Pax6 gene to carry out different functions at different times in mammalian brain development.
Pax6 affects early progenitor cell proliferation and later neuronal differentiation in the developing brain [4, 5]. This changing role might be due at least in part to a shift in the relative concentrations of Pax6 and Pax6(5a) during development but there has been no previous report of such alterations during neurogenesis in the brain in vivo. We anticipated that even relatively small changes in the Pax6 : Pax6(5a) ratio might be important since stronger effects on gene activity via P6CON and 5aCON are observed if Pax6 and Pax6(5a) are introduced into cultured cell lines at ratios of 1:1 or 8:1 than at ratios of 2:1, 4:1 or 16:1 . We selected a direct method for quantification of the ratio (RNase protection assays) and carried out multiple assays so as to obtain statistically analysable data on key Pax6-expressing brain tissues at a range of ages throughout neurogenesis. We found changes in the ratio similar in magnitude to those shown previously to alter target gene activity in vitro.
Results and Discussion
Mean Pax6: Pax6(5a) ratios ± SEMs. "Combined" data are from telencephalon, diencephalon and hindbrain considered together at each age. For each tissue and for combined brain tissues, significant differences between values at E12.5 and values at subsequent ages are shown (unpaired Student's t-tests; * = p < 0.05, ** = p < 0.01). Numbers of individual assays are indicated in brackets.
6.25 +/- 0.68 (5)
2.12 +/- 0.77** (2)
2.63 +/- 1.52* (3)
2.53 +/- 0.37** (4)
9.72 +/- 3.37 (3)
3.63 +/- 0.63 (3)
3.00 +/- 0.63 (2)
7.66 +/- 3.68 (4)
3.87 +/- 1.62 (2)
1.66 +/- 0.35 (2)
2.23 +/- 0.12 (3)
7.59 +/- 1.41 (12)
3.27 +/- 0.55* (7)
2.10 +/- 0.72** (6)
2.53 +/- 0.21** (9)
6.81 +/- 2.08 (4)
9.52 +/- 3.64 (3)
5.95 +/- 1.90 (3)
3.55 +/- 1.30 (3)
There is evidence that Pax6 and Pax6(5a) have different functions in vivo in both vertebrates [9–11] and invertebrates [12, 13]. Studies of Pax6- and Pax6(5a)-related genes in Drosophila melanogaster, ey/ toy and eyg/ toe, have shown that they promote, respectively, differentiation and proliferation of eye precursor cells [12, 13]. Overexpression of Pax6 and Pax6(5a) can alter the expression of different sets of genes in mammals [10, 11]. Mammalian brain cells reduce their proliferation in response to overexpression of Pax6 or Pax6(5a) and increase their neurogenesis in response to overexpression of Pax6 . It is possible that a reduction in the Pax6 : Pax6(5a) mRNA ratio from E14.5 is involved in programming progenitor cells to initiate processes that occur later in embryogenesis. Such processes are potentially numerous; they might, for example, include the transition from predominantly neurogenesis to the major phase of gliogenesis or the development of specific sets of later-generated neurones such as the superficial layers of the cerebral cortex.
We conclude that the Pax6 : Pax6(5a) ratio falls in the telencephalon, diencephalon and hindbrain during neurogenesis and, moreover, the magnitude of the change is in the range that alters target gene expression in vitro . This finding allows the possibility that changes in the relative expression levels of isoforms of a single Pax6 gene might result in changes in the functions of this gene in mammalian brain development.
For each RNase protection assay, wild-type mice (CD-1) were time-mated [the day of conception was designated embryonic day 0.5 (E0.5)] and killed on E12.5, E14.5, E16.5 or E18.5. The telencephalon, diencephalon, hindbrain, eyes and feet (used as a Pax6 non-expressing control tissue) were dissected from each embryo. Tissues from multiple embryos were combined so as to obtain sufficient material for each assay. Total RNA was isolated from tissue snap-frozen in liquid nitrogen, its integrity was checked by agarose gel electrophoresis and its concentration was measured using a fluorimeter. The riboprobe was designed to span the Pax6 mRNA from exon 3 to exon 5a so as to protect Pax6 and Pax6(5a) mRNAs (Fig. 1A). Oligonucleotides 5'-AAG TGG ACG TAT ATC CCA GTT CTC-3' and 5'-AGC ACC TGG ACT TTT GCA TC-3' were used to amplify sequence from mouse Pax6 cDNA. Pax6 cDNA was cloned into pCR-BluntII-TOPO and sequenced across the Pax6 insert in both directions to identify clones with Pax6 sequence in the appropriate orientation to generate antisense probe. The plasmid pTRI-GAPDH (Ambion) was used for synthesis of a murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe. Riboprobe synthesis was carried out using the Maxiscript SP6 in vitro transcription kit (Ambion) in the presence of 40 μCi α-32P-UTP 800 Ci/mmol (Amersham). Riboprobes were purified using a MicroSpin G25 column (Amersham Biosciences).
RNase protection assays were performed on 1 μg total RNA from E12.5 and E14.5 embryonic tissues, or 4 μg total RNA from E16.5 and E18.5 embryonic tissues, using the Hyb Speed kit (Ambion). The quality the undigested full-length riboprobe was examined and that of all total RNA samples was assessed using a GAPDH riboprobe (e.g. Fig. 1B). Products were resolved on 6% polyacrylamide, 8 M urea gels that were then fixed (15% methanol, 5% acetic acid for 30 minutes), dried and exposed to film with an intensifying screen. Where GAPDH signal was weak, this was a sign that protein or DNA contamination had led to inaccurate RNA quantification, and these samples were excluded from subsequent analyses. Gel bands were quantitated using a GS-710 densitometer and the Quantity One software package (BioRad: background subtraction was performed using the rolling disk method ; Fig. 1C). Each assay was repeated several times (n values for the number of assays for each tissue at each age are in Table 1).
Many thanks to Karen Chapman for advice on RNase protection, and to Tom Pratt and Jane Quinn for help with dissections. This work was funded by the Wellcome Trust.
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