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First published online August 2, 2004
doi: 10.1242/10.1242/dev.01282

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Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins

Omar Abdel Samad^1,*, Marc J. Geisen^1,*, Giuliana Caronia², Isabelle Varlet^3,, Vincenzo Zappavigna^2,4, Johan Ericson⁵, Christo Goridis⁶ and Filippo M. Rijli^1,

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR7104, BP 10142-67404 Illkirch Cedex, CU de Strasbourg, France
² DIBIT, H San Raffaele, Via Olgettina 58, 20132 Milano, Italy
³ Laboratoire de Génétique et Physiologie du Développement, IBDM, CNRS/INSERM/Université de la Méditerranée, Campus de Luminy, 13288 Marseille Cedex 9, France
⁴ Department of Animal Biology, University of Modena and Reggio Emilia, via Campi 213/D, 41100 Modena, Italy
⁵ Department of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden
⁶ CNRS UMR8542, Ecole Normale Supérieure, Département de Biologie, 75005 Paris, France

View larger version (58K): [in a new window]   Fig. 1. Analysis of Phox2b regulatory regions in transgenic mice. (A) The Phox2b locus and lacZ reporter constructs used in transgenic mouse assay. Black boxes represent Phox2b exons I, II, and III; the red box represents the ß-globin (ß-glo) minimum promoter. (B,D) Dorsal (B) and lateral (D) views of E10.5 embryos carrying the P2b_10/lacZ construct. lacZ expression is detected by whole-mount in situ hybridization. (C,E) Dorsal (C) and lateral (E) views of E10.5 embryos carrying the P2b_2.8/lacZ construct, stained for ß-gal activity. Expression of P2b_10/lacZ recapitulates most of Phox2b expression pattern in hindbrain and sensory ganglia (Pattyn et al., 1997) (B,D), whereas that of the P2b_2.8/lacZ is selectively restricted to ventral r4 (C) and not expressed in the ganglia (E). r, rhombomere; nIII, oculomotor nucleus; nIV, trochlear nucleus; lc, locus cruleus; gVII, gIX and gX, geniculate, petrose and nodose ganglia of cranial nerves, respectively.

View larger version (68K): [in a new window]   Fig. 2. A Phox2b enhancer that contains conserved Pbx-Hox and Prep/Meis sites. (A) The mouse Phox2b locus highlighting (red box) a 376 nucleotide (nt) proximal enhancer (P2b_0.38). (B) Sequence alignment between mouse, rat, human, zebrafish and fugu, showing high conservation. PH and P/M binding elements are boxed in red and green, respectively. (C) Comparison between PH and P/M sequences in Phox2b, Hoxb1 (ARE repeat 2) (Popperl et al., 1995) and Hoxb2 (Maconochie et al., 1997) r4 enhancers. The Phox2b and Hoxb1 PH and P/M core sequences are identical.

View larger version (73K): [in a new window]   Fig. 3. The PH and P/M sites are both essential for Phox2b enhancer regulation in cell culture and ventral r4. (A,B) Fold activation of luciferase activity assayed from P19 cells transiently co-transfected with combinations of Hoxb1, HOXB2, Pbx1a or Prep1 vectors along with P2b_0.38/Luc (A) or mutant P2b_0.38mPH/Luc (B) reporter constructs. The box in B shows the nucleotide changes in the PH site of P2b_0.38mPH/Luc. Note that Hox, Pbx and Prep synergistic activity depends on an intact PH site. (C-J) Dorsolateral views (anterior towards the left) of stage 17-18 chick embryo hindbrains electroporated with P2b_0.38/lacZ (C), P2b_0.38mPH/lacZ (D), P2b_0.38PM/lacZ (H) or P2b_0.38mPM/lacZ (J) constructs. P2b_0.38mPH/lacZ carries the same mutation as P2b_0.38mPH/Luc. P2b_0.38PM/lacZ and P2b_0.38mPM/lacZ carry P/M site mutations shown in H and J, respectively. (C) High reporter expression is restricted to r4 and, to a lesser extent, to r2. (D,H,J) Overall ß-gal levels decrease and ventral r4 expression is lost (arrows). Thus, both PH and P/M sites are required for ventral r4 expression. Co-electroporation of Hoxb1 (E), HOXB2 (G) or Hoxa2 (I) vectors significantly enhances expression from wild type P2b_0.38/lacZ but not mutated P2b_0.38mPH/lacZ (F). ov, otic vesicle.

View larger version (65K): [in a new window]   Fig. 4. Binding of a Hox-Pbx-Prep ternary complex requires intact PH and P/M sites. (A,B) In vitro synthesized proteins were subjected to EMSA with different radiolabeled probes (drawn below the gels) containing the PH and/or P/M sites. (A) Pbx-Prep (arrow in lanes 4,5), but not Pbx-Hox, heterodimers are formed on the 30 bp probe. (B) A retarded band (arrow in lane 5) forms only in the presence of Hoxb1, Pbx1a and Prep1 proteins on a 233 bp probe containing PH and P/M sites in their native context (left panel), but not on a probe carrying a PH site deletion (middle panel) or a P/M site mutation (same as that in Fig. 3J) (right panel). The ternary complex is selectively inhibited by adding specific antibodies (lanes 6-8). Asterisks indicate unspecific bindings. RRL, unprogrammed rabbit reticulocyte lysate; FP, free probe.

View larger version (48K): [in a new window]   Fig. 6. Nkx2-mediated derepression of the Phox2b enhancer enhances Hox-dependent regulation at the PH site. (A-D) Fold activation of luciferase activity assayed from P19 cells co-transfected with different vector combinations (as indicated below the graphs) along with P2b_0.38/Luc (A) or p3xPH/Luc (C) carrying three copies of the PH site, or their mutated versions P2b_0.38mPH/Luc (B) and p3xmPH/Luc (D). The nucleotide changes in the PH site are shown in the boxes in B and D. Cooperative activation by Hoxb1, Pbx1a and Prep1 is further enhanced by Nkx2.2 or Nkx2.2HD-EnR repressor proteins (A,C), and it requires an intact PH site (B,D). Co-transfection of P2b_0.38/Luc with Nkx2.2HD-VP16 does not activate reporter expression (A), suggesting that Nkx2.2 HD does not bind to the enhancer. Co-transfection of Nkx2.2TN, carrying a deletion of the Groucho-interacting domain (Muhr et al., 2001), almost abolishes Nkx2-dependent transcriptional cooperation (C). (E-G) Dorsolateral views (rostral to the left) of stage 17-18 chick embryo hindbrains electroporated with p3xPH/lacZ carrying three copies of the PH site (E), and with Nkx2.2 (F), or Nkx2.2HD-EnR (G) vectors. p3xPH/lacZ is weakly active and does not display spatially restricted reporter expression (E). (F,G) p3xPH/lacZ expression is enhanced throughout the hindbrain by Nkx2.2 (F) or Nkx2.2HD-EnR (G) repressors. (H,I) Model for integration of Hox-dependent activation and Nkx2-mediated derepression on the Phox2b enhancer. (H) In the ventral neural tube, dorsal to the pMNv domain (i.e. in the absence of Nkx2 factors) Phox2b is repressed, despite the presence of Hox activators and their Pbx and Prep co-factors, through the binding of a putative repressor (R) at, or in the vicinity of, the PH site. (I) In the pMNv domain, the repressor activity of Nkx2.2/Groucho (Gro) inhibits R, either directly or transcriptionally. After derepression, a Hox-Pbx-Prep ternary complex can bind to the PH and P/M sites and stimulate high levels of Phox2b transcription. Shh, sonic hedgehog; ov, otic vesicle.

View larger version (115K): [in a new window]   Fig. 5. The PH site contributes to mouse Phox2b regulation in ventral r4 progenitors. (A,B) Dorsal views of whole-mount E10.5 mouse embryos carrying (A) P2b_10/lacZ or (B) P2b_10PH/lacZ, containing an 11 bp deletion of the PH site (same as in Fig. 4B). No overt differences are apparent between mutated and wild-type constructs. (C,D) Cross-sections through ventral r4 of transgenic embryos carrying P2b_10/lacZ (C) or P2b_10PH/lacZ (D). (D) Reporter expression is severely reduced in the ventricular zone (VZ) (delimited by the broken line) of the pMNv domain but not in the mantle layer (ML). lacZ expression is detected by in situ hybridization. The summary represents the expression pattern differences at E10.5 in ventral r4 of wild type P2b_10/lacZ and mutant P2b_10PH/lacZ transgenic embryos. Red circles, strong expression; pink circle, weak residual expression. FP, floorplate.

View larger version (88K): [in a new window]   Fig. 7. Generation of ectopic Phox2b+ motoneurons requires the combined activities of Hox and Nkx2 factors. Dorsal views of whole-mounts (A-E) or cross-sections through r1 (G-I) of stage 21-22 chick embryos electroporated, on the right-hand side, with the vectors indicated above each panel, and assayed for Phox2b in situ hybridization (A-E,G) or Isl1/2 immunohistochemistry (H). Hoxa2 (A) or Hoxb1 (B) misexpression induces ectopic Phox2b expression only in ventral r1. Conversely, Nkx2.2 misexpression (C) induces ectopic Phox2b expression at dorsal levels, though not in r1. Co-electroporation of Nkx2.2 with Hoxa2 (D) or Hoxb1 (E) additionally induces ectopic Phox2b+ (G), Isl1/2+ (H) motoneurons in dorsal r1. (I) GFP fluorescence, showing the dorsoventral distribution of electroporated cells. (F) Summary showing, on AP and DV coordinate axes, the requirement for the combined activities of Hox and Nkx2.2 proteins to induce ectopic Phox2b expression in the hindbrain. GOF, gain of function; r, rhombomere.