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doi: 10.1242/10.1242/dev.00582

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Brn3a regulation of TrkA/NGF receptor expression in developing sensory neurons

¹ Center for Developmental Biology and Kent Waldrep Foundation Center for Basic Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX 75390-9133, USA
² San Diego VA Medical Center, University of California, San Diego, La Jolla, CA 92093, USA
³ Department of Psychiatry, University of California, San Diego, La Jolla, CA 92093, USA

View larger version (98K): [in a new window]   Fig. 1. Reduced TrkA mRNA in P0 Brn3a-null or double-null trigeminal ganglia. Low (A1-A4) and high (B1-B4) magnification pictures of TrkA mRNA in situ hybridization signal, showing that Brn3a-null (A3,B3) and double-null (A4,B4) ganglia have reduced TrkA mRNA compared with control (A1,B1) and Bax-null ganglia (A2,B2), respectively. As a control, (C1-C4) NCAM mRNA signal is similar in all four genotypes.

View larger version (148K): [in a new window]   Fig. 2. Reduced TrkA protein in P0 Brn3a-null or double-null trigeminal ganglia. Low (A1-A4) and high (B1-B4) magnification pictures of peripherin immunostaining, showing that all four genotypes have similar levels of peripherin in trigeminal ganglia. Low (C1-C4) and high (D1-D4) magnification pictures of TrkA immunostaining, showing that Brn3a-null (C3,D3) and double-null (C4,D4) ganglia have reduced TrkA protein compared with control (C1,D1) and Bax-null (C2,D2) ganglia, respectively. (E1-E4) Brn3a immunostaining showing that Brn3a-null (E3) and double-null (E4) ganglia lack nuclear Brn3a protein compared with control (E1) and Bax-null (E2) ganglia. Double-null ganglia are always larger and have more neurons than Brn3a-null ganglia.

View larger version (136K): [in a new window]   Fig. 3. Reduced TrkA enhancer activity in P0 Brn3a-null or double-null trigeminal ganglia. Low (A1-A4) and high (B1-B4) magnification pictures of ß-galactosidase activity driven by the TrkA minimal enhancer, showing that Brn3-null (A3,B3) and double-null (A4,B4) ganglia have reduced ß-galactosidase activity compared with control (A1,B1) and Bax-null (A2,B2) ganglia, respectively. (C1-C4, arrows) Individual ß-galactosidase positive cells from each genotype show apparently reduced ß-galactosidase activity in cell bodies of Brn3a-null (C3) and double-null (C4) ganglia, but normal activity in control (C1) and Bax-null (C2) ganglia. (D1-D4, arrows) A second set of ß-galactosidase-positive trigeminal ganglia neurons from an independent group of mice, showing that reduced TrkA enhancer activity in Brn3a-null (D3) and double-null (D4) ganglia is consistent, compared with control (D1) and Bax-null (D2) ganglia.

View larger version (14K): [in a new window]   Fig. 5. Summary of transgenic analysis of the wild-type TrkA minimal enhancer and TrkA minimal enhancers with one or both Brn3a-binding-site mutations. Note that the minimal enhancer that contains both Brn3a-binding-site mutations has apparently reduced activity in trigeminal ganglia. *The reported numbers include results from three stable TrkA-minimal-enhancer transgenic lines.

View larger version (87K): [in a new window]   Fig. 6. The TrkA minimal enhancer containing both Brn3a-binding-site mutations has reduced activity in trigeminal ganglia. Representative trigeminal ganglia ß-galactosidase activity driven by the wild-type TrkA minimal enhancer (A,E), by a TrkA minimal enhancer bearing a 5' Brn3a-binding-site mutation (B,F), by a TrkA minimal enhancer bearing a 3' Brn3a-binding-site mutation (C,G), and by a TrkA minimal enhancer bearing both Brn3a-site mutations (D,H,I) at E13.5 and E17.5. Only the TrkA minimal enhancer with both mutations caused severe reduction in enhancer activity at E13.5 and E17.5.

View larger version (40K): [in a new window]   Fig. 4. Gel-shift and footprint assays define two Brn3a-binding sites in the TrkA minimal enhancer. (A) Illustration of six, overlapping, 100 bp fragments covering TrkA minimal enhancer and (B) gel-shift using a GST-Brn3a POU-domain fusion protein indicating that only the first and sixth fragments can bind Brn3a. (C,D) DNAse I footprint assays to define the Brn3a-binding sequences in the TrkA minimal enhancer. The 5' protected sequence is 5'-TCTAAGAGATCTATTAATTTCTC-3' (C) and the 3' protected sequence is 5'-TCACCTAACTTATTCCAAGTGACATGCACACCCTCTTAA-3' (D). (E,F, left panels) Gel-shift assays showing that the protected sequences in (C,D) compete with the first and sixth 100 bp fragments in (A,B) for GST-Brn3a POU-domain fusion-protein binding. (E,F, top panels) Protected sequences from (C,D) share (red boxes) an A/T-rich core sequence. The mutant sequences used in the competitive gel-shift assays (left panels) and the following transgenic analysis (Figs 5, 6) are shown in red. (E,F, right panels) Competitive gel-shift assays using either the wild-type or mutated Brn3a-binding sites, showing that mutated Brn3a-binding sites do not bind the GST-Brn3a POU-domain fusion protein (lane 8) and, unlike wild-type sites (lanes 1-4), do not compete with wild-type sites for binding (lanes 5-7). Arrows in E,F indicate the DNA-protein complexes.