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The activation and maintenance of Pax2 expression at the mid-hindbrain boundary is controlled by separate enhancers

Peter L. Pfeffer^*, Bernhard Payer, Gerlinde Reim, Marina Pasca di Magliano and Meinrad Busslinger

Research Institute of Molecular Pathology, Vienna Biocenter, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
^* Present address: AgResearch, Crown Research Centre, Private Bag 3123, Hamilton, New Zealand
Present address: Wellcome CRC Institute of Cancer and Developmental Biology, Tennis Court Road, Cambridge CB2 1QR, UK
Present address: Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany

View larger version (22K): [in a new window]   Fig. 1. Conservation of upstream sequences between vertebrate Pax2 genes. (A) The promoter and proximal (P), intermediate (I) and distal (D) homology regions of the human, mouse and Fugu rubripes Pax2 genes are shown with their degree of sequence identity and nucleotide positions relative to the transcription start sites. The Fugu gene was identified as Pax2.1 by comparison of its exon 1 with those of the two zebrafish Pax2 genes (Pfeffer et al., 1998). (B,C) Conservation of the box D (B) and promoter of Pax2 (C). Only those nucleotides of the Fugu (f) Pax2.1 gene that differ from the mouse (m) sequence are shown. A high-affinity Pax-binding site in box D is aligned with the Pax2/5/8 consensus sequence (Czerny and Busslinger, 1995) and with element E1 of the MHB-specific enhancer of Pax5 (Pfeffer et al., 2000). An arrowhead points to the insertion of 26 nucleotides in the Fugu promoter, and a red arrow indicates the region of heterogeneous transcription initiation of mouse Pax2 (Ryan et al., 1995).

View larger version (84K): [in a new window]   Fig. 2. Upstream sequences of the mouse and Fugu Pax2 genes direct expression at the MHB. (A) Schematic diagram of the transgenes and statistical overview of the ß-galactosidase staining patterns observed in injected founder embryos or permanent transgenic lines. The indicated DNA fragments of the mouse or Fugu Pax2 gene were linked to the minimal promoter and lacZ gene of pTrap. The number of independent transgenic (tg) and ß-galactosidase (ß-gal)-positive embryos (a), analyzed between E8.5 and E10.5, is shown together with the number of embryos exhibiting ß-galactosidase staining at the MHB (b), in the developing kidney (c) or only in ectopic locations (d). B, BsrD1; Ba, BamHI; H, HindIII; K, KpnI; Sa, SacI. (B,C) ß-Galactosidase staining of Pax2lacZ/+ embryos. The MHB is indicated by arrowheads in B-G,I. (D,E) Broad expression of transgene #1 throughout the midbrain and hindbrain. X-gal staining was performed for 20 minutes or 4 hours to detect expression in the brain region (D,E) or developing kidney (insert in E). (F,G) Expression of the Fugu transgene #2 at the MHB of transgenic mouse embryos. (H,I) Later onset and more restricted expression of transgene #3 at the MHB. All embryos are shown in lateral view except for D,H (dorsal view). A, anterior; ba, branchial arch; ms, mesonephros; mt, metanephros; nd, nephric duct; os, optic stalk; ov, otic vesicle; P, posterior; pn, pronephros; sc, spinal chord; som, somites.

View larger version (58K): [in a new window]   Fig. 3. Characterization of the proximal homology region of Pax2. (A) Constructs and statistical overview of transgenic embryos. Transgene #6 was expressed in the MHB region and kidney of midgestation embryos both in the presence (#6a) or absence (#6b) of the Pax2 promoter (broken lines). A, ApaI; Af, AflII; Av, AvaI; B, BsrD1; K, KpnI; N, NarI; S, SphI; Sc, ScaI; Ps, PshAI; X, XmnI. (B-F) X-Gal staining of representative transgenic embryos. Strong staining was observed at the MHB for all transgenes up to construct #11 (B,C), whereas the minimal 410 bp NarI-ApaI fragment of transgene #14 (E) gave rise to reduced and more variable staining at the MHB. The activity of the MHB-specific enhancer was lost upon removal of the Pax2/5/8-binding site (S1) by either 3' deletion (D) or point mutation (F). Ectopic staining was frequently observed in the hindlimb (hl) and mesenchymal tissue of the trunk. fl, forelimb.

View larger version (62K): [in a new window]   Fig. 4. Binding of Pax2/5/8 proteins to the proximal enhancer. (A) EMSA analysis of extracts prepared from dissected trunk or MHB tissue of 2-day-old and 3-day-old chick embryos (Pfeffer et al., 2000) with a 0.3 kb AvaI-ApaI probe of transgene #11 (Fig. 3A). In vitro synthesized Pax2b was used as a control. (B) Preferential binding of Pax2 and Pax5 to the proximal enhancer. Equimolar amounts of in vitro translated Pax proteins (quantitated by [35S]Met incorporation) were analyzed by EMSA for binding to the 0.3 kb AvaI-ApaI probe. (C) Identification of a high-affinity Pax-binding site. Binding of Pax2b to the end-labeled S1 and S1m oligonucleotides was investigated by EMSA in the absence or presence of a 100-fold molar excess of the indicated oligonucleotides. (D) Alignment of the proximal enhancer sequences S1 and S2 with the consensus Pax2/5/8-binding site (Czerny and Busslinger, 1995). The S1m mutations are indicated.

View larger version (58K): [in a new window]   Fig. 5. Presence of a second late MHB enhancer in the Pax2 upstream region. (A) Transgenic constructs and statistics. Ap, ApoI; B, BsrD1; Bh, BssHII; Bs, BsmI; K, KpnI; Sc, ScaI; Sp, SapI; Sm, SmaI; Ss, SspI. (B,C) Transgene #17 lacking the proximal enhancer was still expressed in the MHB region (arrowhead). (D,E) Four (* in A) of five embryos expressed transgene #19 only in the ventral MHB region at E10 to E10.5, indicating that the conserved 102 bp sequence in the distal homology region of mammalian and Fugu Pax2 genes (Fig. 1B) is required for maintaining lacZ expression in the dorsal region of the MHB. Embryos are shown in frontal (B), dorsolateral (D) or lateral (C,E) view.

View larger version (58K): [in a new window]   Fig. 6. Identification of the early Pax2 enhancer. (A) Transgenic constructs and statistics. ‘Early’ refers to expression in the anterior neural plate of presomitic embryos and ‘late’ to expression at the MHB beyond the four-somite stage. B, BsrD1; Bu, Bsu36I; Bs, BsmI; Ps, PshAI; Sc, ScaI. (B,C) Transgenes containing the intermediate homology region of Pax2 are expressed already in the neural plate of presomitic embryos. (D,E) Loss of the early (D) but not late (E) MHB-specific expression in a permanent transgenic line lacking the intermediate homology region. (F,G) The 120 bp intermediate homology region together with the Pax2 promoter suffices for anterior neural plate expression (F), but fails to maintain expression in the MHB region (G, arrowhead) at later stages of the same permanent transgenic line. All embryos are shown in lateral view. op, otic placode.

View larger version (59K): [in a new window]   Fig. 7. Mutational analysis of the early Pax2 enhancer. (A) Binding of homeodomain proteins to the early enhancer. In vitro translated proteins and a MHB extract were analyzed for binding to a 157 bp probe containing the wild-type enhancer (SX[wt], from –3833 to –3677) or the corresponding sequence mutated at all four TAAT sites (SX[TAAT]). Oct1 was analyzed at a 10-fold lower concentration. (B) Interaction of in vitro translated Oct1 and Oct3/4 proteins with a wild-type (wt) or mutated (M1) probe (see C) containing the distal two TAAT motifs. X, nonspecific DNA-binding activity. (C) Sequence of the early enhancer. Recognition sequences for homeo (HD) and POU-specific (POUS) domains and binding sites for Otx and Sp1-like zinc finger (Zn) proteins are shown together with an alignment of the E2 element present in the MHB-specific enhancer of Pax5 (Pfeffer et al., 2000). (D-F) Inactivation of the early enhancer by mutation of the TAAT motifs in the context of transgene #1. The introduced mutations are indicated together with the statistical analysis (D) and representative examples of transgenic embryos (E,F). Posterior refers to ectopic expression in the posterior region of late gastrula embryos (D). The mid-hindbrain (mh) territory is indicated by a bracket (E,F).

View larger version (66K): [in a new window]   Fig. 8. Functional analyses of the early and late enhancers in the context of the Pax2 locus. (A) Structure of the Pax2 BAC #30, which contains an in-frame GFP insertion in exon 2 and extends at least 30 kb upstream of Pax2. A structurally different Pax2-GFP BAC (#76) gave rise to the same expression pattern as BAC #30. All mutant transgenes (#31-34) were derived from BAC #30. Nucleotide positions relative to the transcription start site indicate the extent of deletion. n.d., not determined. (B-F) Temporal expression pattern of the parental BAC #30. Strong GFP expression was observed in the metanephros (F) at E16.5. (G-K) The proximal but not distal enhancer of Pax2 is essential for late expression at the MHB (arrowhead). An oblique section (G) through the mesonephros of an E10 embryo revealed normal expression of transgene #32 in both the nephric duct (nd) and tubules (tub). (L-N) Before the four-somite stage, the early enhancer in the intermediate homology region is required for Pax2 expression in the MHB region. ad, adrenal gland; nt, neural tube; tb, tail bud.