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First published online 20 October 2004
doi: 10.1242/dev.01455

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Conserved and acquired features of neurogenin1 regulation

Patrick Blader^*,, Chen Sok Lam^*, Sepand Rastegar^*, Raffaella Scardigli, Jean-Christophe Nicod, Nicolas Simplicio, Charles Plessy, Nadine Fischer, Carol Schuurmans^¶, François Guillemot and Uwe Strähle^**,

Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, CU de Strasbourg, France

View larger version (52K): [in a new window]   Fig. 1. The –8.4ngn1:gfp zebrafish transgene recapitulates the pattern of endogenous ngn1 transcripts and drives telencephalic expression in the zebrafish embryo. (A) The 8.4 kb upstream zebrafish ngn1 regulatory sequence drives GFP expression in the telencephalon (arrow) and in the diencephalon. (B,C) Comparison of the endogenous ngn1 gene (B) and the gfp transgene (C) by in situ hybridisation indicates that the gfp transgene is capable of recapitulating the endogenous telencephalic (t, indicated by arrows) and diencephalic expression of ngn1. Expression is detected in at least six regions of the diencephalon, comprising the epiphysis (e), pretectum (pre), dorsal thalamus (dt), ventral thalamus (vt), preoptic area (po) and posterior tuberculum (pt). Transcripts are also localised in the midbrain tegmentum (tg). Zebrafish embryos are at stage 28 hpf. Panels show lateral views of whole-mounted embryos oriented anterior to the left and dorsal up.

View larger version (43K): [in a new window]   Fig. 2. Identification of the regulatory elements required for telencephalic expression of the –8.4ngn1:gfp transgene in the zebrafish. (A,B) Analysis of zebrafish embryos transgenic for truncated versions of the –8.4ngn1:gfp reporter fragment. Transgene expression in the telencephalon (arrows) is no longer detected in embryos carrying transgenes with 5.3 kb of ngn1 sequence. (C) Overlapping deletions spanning the region between 6.9 and 5.3 kb reveal a 200-bp element, the LSE, that is required for telencephalic expression of the –8.4 ngn1:gfp transgene. The heads of embryos in A are seen from the dorsal aspect, with the telencephalon up, and are staged between 28 and 30 hpf. Boxes in B, corresponding to the LSE (blue) and LATE (green), indicate regions of homology between the zebrafish, mouse and human ngn1 loci. Each construct was analysed in one to three transgenic lines. Note that expression patterns from the overlapping deletions provided an independent further confirmation of the results. Transgenic lines varied in the intensity of reporter expression, but no significant variations in the expression pattern due to integration site effects were observed.

View larger version (97K): [in a new window]   Fig. 3. Recapitulating the Ngn1 expression pattern in the mouse requires two zebrafish ngn1 gene regulatory regions. (A) The 8.4 kb sequence upstream of zebrafish ngn1 drives reporter expression in the mouse telencephalon (arrow). (B,C) Comparison of the expression of the transgene with the expression of the endogenous Ngn1 gene in coronal sections (dorsal up) shows that this fragment recapitulates fully the endogenous telencephalic expression of Ngn1. The sharp ventral boundary of Ngn1 and transgene expression is indicated by a line. (D) Whereas the complete –8.4ngn1:gfp transgene recapitulates the endogenous pattern of Ngn1 (C), reporter activity is lost dorsally (arrow), but remains laterally (arrowhead), in transgenic lines with the LSE deleted [–8.4(del3)ngn1:gfp]. (E) Expression in the lateral telencephalon (arrowhead) is driven by an element within 3.1 kb of the ngn1 regulatory region (–3.1ngn1:nlacZ). (F) Deletion of LATE [–3.1(del LATE)ngn1:nlacZ] abolishes the lateral activity of the –3.1ngn1:nlacZ transgene. Thus, two regulatory regions of the zebrafish transgene control the spatial expression of ngn1 in the mouse pallium. (G) Summary of the activities of LATE and LSE in the dorsal telencephalon of mouse. m, mantle zone; v, ventricular zone. Mouse embryos are at 12.5 dpc. Two transgenic lines were analysed per construct. With the expception of A, which is a lateral view of a whole-mounted mouse embryo, panels show coronal sections through the telencephalon, with dorsal up.

View larger version (116K): [in a new window]   Fig. 7. The LATE region is conserved between tetrapods and zebrafish. (A) Sequence comparison of the LATE regulatory region of zebrafish ngn1 with that of human, mouse, chicken and Xenopus tropicalis. The aligned sequences comprise nucleotides –1775 and –1368 in the zebrafish ngn1 genomic regulatory region. In each column, the nucleotides are printed on a black background when they are all identical, or on grey background when one base is present in more than half of the sequences. Boxed regions indicate regions where the zebrafish sequence is similar to the Pax6 consensus-binding site (Epstein et al., 1994). Three sites (A, B and C) were scored. (B) Comparison of the three putative Pax6-binding sites with the consensus-binding sequence.

View larger version (125K): [in a new window]   Fig. 4. The LATE region is required for expression in the diencephalon and hindbrain of zebrafish embryos. (A) The –3.1ngn1:gfp transgene, harbouring LATE, drives gfp expression extensively in the diencephalon and in the hindbrain. This pattern is partially reminiscent of the expression of the two zebrafish pax6 genes (see Fig. 5D,E). (B) Upon deletion of LATE in –3.1(delLATE)ngn1:gfp transgenic embryos, expression of gfp mRNA in the diencephalon and in the hindbrain is strongly reduced. (C,D) Deletion of LATE in the –8.4ngn1:gfp transgenics reduced reporter expression in the diencephalon (arrowhead) but not in the telencephalon (t). Thus, the activity of LATE is required for transgene expression in the diencephalon. Embryos were oriented with anterior directed towards the left and dorsal up.

View larger version (95K): [in a new window]   Fig. 5. LATE requires the activity of Pax6 in the mouse. (A) Schematic of the expression domain of Pax6 in the telencephalon of the mouse [reproduced, with permission, from Stoykova et al. (Stoykova et al., 2000)]. (B) Control embryo carrying the –3.1ngn1:nlacZ transgene. (C) Pax6sey/sey embryo carrying the –3.1ngn1:nlacZ transgene, showing the loss of reporter expression, indicating that LATE activity depends on Pax6. Coronal sections through the telencephalon with dorsal up. (D,E) Lateral view of the two zebrafish pax genes, pax6.1 (D) and pax6.2 (E) shows overlapping expression in the fore- and hindbrain (insets). Staining is detected mainly in the dorsal diencephalon, reminiscent of the –3.1ngn1:gfp transgene (see Fig. 4A). m, mantle zone; v, ventricular zone.

View larger version (113K): [in a new window]   Fig. 6. Pax6 is required for ngn1 and transgene expression in the diencephalon of the zebrafish embryo. (A-F) Control (A,C,E) and morpholino-injected (B,D,F) embryos hybridised to ngn1 (A-D) and gfp (E,F) antisense probes. Expression of the endogenous ngn1 mRNA and the transgene (–8.4ngn1:gfp) is reduced in the diencephalon (arrowheads) but not in the telencephalon (t). Embryos were injected with a cocktail of morpholino oligonucleotides directed against pax6.1 and pax6.2. Embryos are 28 hpf. (A,B) Dorsal views of embryos with anterior oriented upwards; (C-F) lateral views. e, eye.

View larger version (67K): [in a new window]   Fig. 8. Pax6 binds to site-C of the LATE region. Electromobility-shift assays with recombinant mouse Pax6 protein. Radioactively labelled oligonucleotide containing site-C of the LATE region was incubated with Pax6 protein without competitor oligonucleotide, or with a 50-fold molar excess of cold site-C oligonucleotide (sC), or with an oligonucleotide that contained a cluster of point mutations (msC) in the Pax6-binding site homology domain. The site-C competitor abolished the shifted band, whereas the complex formation was not affected by the presence of the mutated oligonucleotide, demonstrating that the interaction of Pax6 protein is dependent on an intact Pax6-binding site. As a positive control, an oligonucletide (Control) harbouring the Pax6-binding site described by Czerny et al. (Czerny et al., 1993) was used.

View larger version (45K): [in a new window]   Fig. 9. The cis-regulatory elements of mouse LATE and LSE direct GFP expression in the zebrafish diencephalon and the telencephalon, respectively. The left panels show a cumulative map of the expression (blue dots; n, number of embryos analysed) of the reporter and the right panels are representative images of embryos injected with the construct indicated in the right bottom corner. Reporter gene expression was revealed by hybridising embryos to the antisense gfp probe. Blue and red boxes in the schematic drawings indicate zebrafish and mouse enhancers, respectively. (A-C) Embryos injected with –3.1ngn1:gfp, with mutant derivatives without the zebrafish LATE [–3.1ngn1(delLATE):gfp], or with a replacement with the mouse LATE [–3.1ngn1(msLATE):gfp]. Mouse LATE drives expression in the zebrafish diencephalon in a pattern very similar to zebrafish LATE. Like its zebrafish homologue, mouse LATE does not mediate expression in the zebrafish telencephalon (indicated by arrows). (D-F) Embryos were injected with the –8.4ngn1:gfp transgenes, with mutants without the LSE [–8.4ngn1(delLSE):gfp], or with the mouse LSE in place of the homologous zebrafish sequence [–8.4ngn1(msLSE):gfp]. As shown in stable expression experiments, the absence of the LSE significantly reduced the expression of the reporter in the telencephalon. The replacement of the zebrafish LSE with the homologous mouse LSE restored reporter expression in the telencephalon.