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First published online 4 May 2005
doi: 10.1242/dev.01833

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Identification of Pax2-regulated genes by expression profiling of the mid-hindbrain organizer region

Maxime Bouchard^1,2,*,, David Grote^1,2,*, Sarah E. Craven³, Qiong Sun¹, Peter Steinlein¹ and Meinrad Busslinger¹

¹ Research Institute of Molecular Pathology, Vienna Biocenter, Dr Bohr-Gasse 7, 1030 Vienna, Austria
² McGill Cancer Centre, Biochemistry Department, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, H3G 1Y6, Canada
³ Department of Molecular Biology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA

View larger version (53K): [in a new window]   Fig. 1. Isolation of Pax2-expressing mid-hindbrain cells from mouse embryos. (A) Structure of the Pax2GFP BAC transgene, which was previously described as transgene 30 containing an in-frame GFP insertion in exon 2 of Pax2 (Pfeffer et al., 2002). (B) GFP expression of the Pax2GFP transgene in the mid-hindbrain boundary (mhb) region, otic placode (op) and tail bud (tb) of E8.5 embryos. (C) Representative FACS sorting of Pax2-expressing mid-hindbrain cells. The mid-hindbrain domain (indicated by a bracket in B) was manually dissected from a control wild-type and transgenic Pax2GFP Pax2–/– embryo at E8.5 (seven somites) and dissociated into single cells by trypsin digestion. Live PI– GFP+ cells were isolated from individual embryos by fluorescence-activated cell sorting (FACS). The percentage of GFP+ cells located within the sorting gate is shown for control and transgenic embryos. PI, propidium iodide.

View larger version (75K): [in a new window]   Fig. 2. Expression pattern of the putative Pax2-regulated genes in wild-type embryos. Probes of the indicated genes were used for whole-mount in situ hybridization of embryos at E8.5 (8-10 somites; A,C,E,G,I,K; dorsolateral view) and E9.5 (B,D,F,H,J,L; lateral view). Arrowhead indicates the position of the mid-hindbrain boundary (mhb). anr, anterior neural ridge; lb, limb bud; ms, mesonephros; oc, optic cup; os, optic stalk: ov, otic vesicle; sc, spinal cord.

View larger version (57K): [in a new window]   Fig. 3. Pax2-dependent gene expression in the developing mid-hindbrain region. Pax2+/+ or Pax2+/– control embryos (A,C,E,G,I) and Pax2–/– embryos (B,D,F,H,J) were analyzed by for expression of the indicated genes by in situ hybridization at the five to six somite (A,B,I,J) or seven to eight somite (C-H) stage. Arrowheads indicate the position of the mid-hindbrain boundary. anr, anterior neural ridge; fb, forebrain.

View larger version (18K): [in a new window]   Fig. 4. Sequence conservation and heterogeneous transcription initiation of the Brn1 gene. (A) Conservation of the 5' flanking sequences of mammalian and fish Brn1 genes. The PipMaker program (Schwartz et al., 2000) was used to compare the 10 kb upstream regions of the human and mouse Brn1 genes. Sequence block with 50% to 100% identical sequence (y-axis) are indicated together with their position (x-axis) relative to the translation start codon of Brn1. The conserved elements A,B,C,D and P share 93.2% (D) to 96.5% (B) sequence identity in the two mammalian species (Fig. 6D; see Fig. S1 in the supplementary material). Four of these regions (B, C, D and P) are even conserved between mammals and the pufferfish Fugu rubripes (shown in green). CpG islands with an average GC content of 60% are present in the first 3.8 kb upstream of the Brn1 start codon. (B) Mapping of the transcription initiation region. 5'-RACE amplified PCR fragments of 325 bp from E10.5 head RNA with a primer located downstream of element P (–1098/–1069 relative to start codon). The presence or absence of reverse transcriptase (RT) and the PCR cycles are indicated. (C) Identification of transcription start sites. Sequencing of the 5'-RACE products identified heterogeneous transcriptional start sites within a 17 bp sequence of element P. The number of PCR clones with an identical 5' end is indicated together with the nucleotide positions relative to the start codon.

View larger version (38K): [in a new window]   Fig. 5. Mapping of Pax2-binding sites in the conserved 5' regions of Brn1. (A) Presence of high-affinity Pax2-binding sites in elements D and P. The binding of in vitro translated Pax2 protein to a labeled oligonucleotide containing the Pax2/5/8-binding site 1 of the CD19 promoter (Kozmik et al., 1992) was measured by EMSA in the absence (–) or presence of a 10- or 50-fold molar excess of the indicated competitor DNA. The competition strength of PCR fragments comprising elements C, D and P was compared with that of the high-affinity site 1 of the Blnk promoter (Schebesta et al., 2002). The protein-DNA complexes are shown together with a map, indicating the positions of the conserved elements relative to the Brn1-coding sequence. (B,C) Identification of the sites Dd (B) and Pc (C) as high-affinity Pax2-binding sequences. The same competition assay was used to evaluate the interaction of Pax2 with the indicated restriction fragments (P1-P3) or oligonucleotides containing putative Pax2/5/8-binding sites in element D (Da-Dd) and P (Pa-Pc). Substitution of two base pairs prevents binding of Pax2 to the mutant (m) sites Ddm (B) and Pcm (C). D, DdeI; T, TaqI. (D) Sequence alignment of sites Dd and Pc with the consensus Pax2/5/8 recognition sequence (Czerny and Busslinger, 1995). The positions relative to the Brn1 start codon and the nucleotide substitutions of the mutant sites are indicated.

View larger version (92K): [in a new window]   Fig. 6. Both high-affinity Pax2-binding sites are essential for the initiation of mid-hindbrain-specific Brn1 expression. (A) Expression of the 6.2wt-lacZ transgene at E9.5. A 6.2 kb AflII-NotI fragment from the 5' region of Brn1 (–6267/–59 relative to the start codon) directs expression of a lacZ reporter gene in the posterior forebrain (fb), mid-hindbrain boundary (arrowhead) region and spinal cord (sc), which correspond to a subset of the endogenous Brn1 expression domains at E9.5 (see Fig. 2F). (B,C) Inactivation of the Pax2-binding site Dd or Pc prevents mid-hindbrain-specific expression of the 6.2Ddm-lacZ or 6.2Pcm-lacZ transgene, respectively, while leaving the forebrain and spinal cord expression unaffected. The expression of all transgenes was analyzed by X-gal staining of injected founder (G0) embryos at E9.5. Each transgenic construct gave rise to three lacZ-expressing embryos with a similar ß-galactosidase staining pattern. The embryo shown in B revealed ectopic ß-galactosidase expression in the epidermis (ep) from the forebrain to the hindbrain (hb), which was not seen with other embryos carrying the same 6.2Ddm-lacZ gene. Arrowheads in B,C indicate the midbrain-hindbrain boundary (mhb). (D) Brn1 promoter sequence. The mouse (m) DNA sequence of promoter element P is shown together with the transcription initiation sites, two conserved CCAAT boxes and the functional Pax2/5/8-binding site Pc. Only the divergent nucleotides of the corresponding human (h) Brn1 sequence are indicated.

View larger version (53K): [in a new window]   Fig. 7. Induction of Fgf8 in the hindbrain by ectopic expression of EnR-Brn1. (A) Comparison of chick (c) and mouse (m) Brn1 protein sequences. A partial chick Brn1 cDNA was PCR-cloned from embryo RNA. Numbers refer to the corresponding amino acids of mouse Brn1, for which only the divergent amino acids are shown. Dots indicate five gaps introduced for optimal sequence alignment. (B,C) Brn1 expression in chick embryos at HH stages 14-15 and 19-20. (D-G) Ectopic expression of rat (r) Brn1 proteins together with mouse Otx2 in chick embryos. The expression of endogenous chick (c) Fgf8 (blue) and electroporated rat Brn1 (D,E,G) or chick Pax2 (F) genes (brown) was detected by in situ hybridization. Asterisks indicate ectopic Fgf8 expression in the hindbrain. In G, the strong Fgf8 signal (blue) covered the Brn1 signal (brown) because of colocalization of ectopic EnR-Brn1 and Fgf8 expression. The upper side of the embryo is electroporated, whereas the lower side serves as a control. (H) Hypothetical interactions that may explain Fgf8 induction by the ectopically expressed EnR-Brn1. For explanations, see Discussion.