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First published online 21 January 2004
doi: 10.1242/dev.00975

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Identification of minimal enhancer elements sufficient for Pax3 expression in neural crest and implication of Tead2 as a regulator of Pax3

Cardiovascular Division, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

View larger version (22K): [in a new window]   Fig. 1. Identification of minimal cis-acting neural crest enhancer regions in the proximal Pax3 genomic region. Constructs 1-10 used for the creation of transgenic mice (not to scale, see Materials and methods for construct details). Neural crest enhancer elements, NCE1 and NCE2, are in red. The Tead binding site within NCE2 is indicated and the mutant version of this site used in subsequent experiments is shown. The number of genotype positive E10.5-11.5 embryos obtained from injection of each construct is shown and those expressing ß-galactosidase are indicated.

View larger version (65K): [in a new window]   Fig. 2. ß-galactosidase expression in Pax3 transgenic mice. (A) The 6.1 kb upstream region of the proximal Pax3 genomic sequence (construct 1) partially recapitulates Pax3 expression in the hindbrain (hb), the dorsal root ganglia (drg), and the neural tube (nt). (B) Transgenic expression from the 1.6 kb upstream region and intron 1 (construct 2) yields a similar expression pattern to the 6.1 kb construct. (C) Deletion of 5' upstream region (construct 4) maintains the neural crest expression pattern. (D) Deletion of sequences 5' and 3' to NCE1 and NCE2 as well as the 156 bp internal region (resulting in construct 9) maintains the expression pattern seen in A, although hindbrain and neural tube expression is somewhat weaker.

View larger version (45K): [in a new window]   Fig. 3. Tead2 binds to a Tead binding site within the Pax3 neural crest enhancer. An electrophoretic mobility shift assay is shown with Tead2 protein and an oligonucleotide derived from the NCE2 sequence. Tead2 binds to this site as seen in lane 1. Mutation of the Tead binding site abolishes binding (lane 2). When the known Tead co-factor YAP65 is added to the reaction, a supershift of the Tead2/DNA complex is observed (lane 3). Mutation of the Tead site abolishes the observed supershift (lane 4). All samples were run on the same EMSA gel.

View larger version (136K): [in a new window]   Fig. 4. Tead2 and Pax3 are co-expressed in the dorsal neural tube. In situ hybridization was performed at E11.5. (A) Pax3 expression is dorsally restricted in the neural tube (arrow). (B) Tead2 expression overlaps that of Pax3 and extends ventrally. (C) YAP65 expression mimics that of Tead2. (D,E) Tead1 (D) and Tead3 (E) are expressed weakly in the neural tube. (F) Tead4 expression is not detected in the neural tube. (G-I) Sagittal sections through the brain showing expression of Pax3 in the ventricular zone (G), overlapping with that of Tead2 (H) and YAP65 (I). c, cerebellar pimordium; IV, fourth ventricle.

View larger version (10K): [in a new window]   Fig. 5. Tead2 activates transcription of a reporter construct containing NCE2. Co-transfection of a reporter construct containing NCE2 with Tead2 results in minimal activation (bar 2) over baseline transfection with pcDNA3 (bar 1). Addition of the Tead co-activator YAP65 (bar 3) results in a 3.5-fold activation over baseline. Mutation of the Tead binding site in NCE2 (bar 4) abolishes activation. YAP65 alone (bar 5) is unable to activate luciferase expression. Results are normalized for transfection efficiency and expressed as the mean ± standard deviation of three experiments, each performed in triplicate.

View larger version (79K): [in a new window]   Fig. 6. Mutation of the Tead2 binding site in the NCE2 enhancer abolishes transgenic expression in the neural tube and neural crest. (A) Transgenic embryo carrying a construct that includes a somite-specific Pax3 enhancer and the neural crest enhancers with a mutation in the Tead site of NCE2 is shown. No expression is evident in the dorsal neural tube or neural crest, while somite expression is maintained. (B) Transgenic embryo with the proximal 15 kb proximal Pax3 region including intact somites and neural crest enhancers demonstrates expression in both the somites and neural crest. (C) Whole-mount in situ hybridization demonstrating the wild-type Pax3 expression pattern. (D) Magnification of transgenic embryo shown in B.

View larger version (68K): [in a new window]   Fig. 7. Expression of dominant negative Tead2 affects Pax3 expression. A Tead2 fusion protein including the Engrailed repressor domain was expressed in the neural tube using the Wnt1 promoter. (A) Wild-type embryo with normal morphology and Pax3 protein expression (green) in the dorsal neural tube (nt), dorsal root ganglia (drg, dotted line) and somites. (B) Adjacent Haematoxylin and Eosin-stained wild-type section. (C) Transgenic embryo with an abnormal dorsal neural tube (nt) and small dorsal root ganglia (drg, dotted line) with diminished Pax3 expression in neural tube and drg. Pax3 protein expression in the somites is unaffected. (D) Adjacent Haematoxylin and Eosin stained transgenic section. (E) Wild-type E10.5 embryo stained with anti-neurofilament antibody (arrows) showing strong expression adjacent to neural tube (nt) and dorsal root ganglia and weaker expression by enteric ganglia (eg) in the hindgut (hg). (F) Neurofilament expression in transgenic embryo is weak in regions adjacent to dorsal root ganglia (arrows) and absent from hindgut. (G) Tead2-Engrailed inhibits Tead2 and YAP65-mediated activation of a luciferase reporter construct containing NCE2 (NCE2-luciferase) in a dose-dependent manner. Fold activation of luciferase activity is corrected for transfection efficiency and performed as in Fig. 5. (H) Retinoic acid induces endogenous Pax3 protein expression (red; detected by immunohistochemistry) in P19 cells transfected with a GFP expression vector (green). (I) Co-transfection of a Tead2-Engrailed expression vector with GFP inhibits Pax3 expression such that those cells expressing GFP do not express Pax3. Nuclei are stained blue.