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On the organisation of the regulatory region of the zebrafish deltaD gene

Institut für Entwicklungsbiologie, Universität zu Köln, 50923 Köln, Germany

View larger version (59K): [in a new window]   Fig. 1. Whole-mount in situ hybridisation to show the distribution of deltaD transcripts. (A) 30% epiboly. deltaD is transcribed in the marginal zone (black arrowhead). (B) 70% epiboly. The shield region is devoid of transcripts (white arrow). (C) 95% epiboly. deltaD expression in a V-shaped expression domain (1) anteriorly, in two longitudinal stripes (IS) in the trunk region of the neural plate, and in one transverse, band-like domain (white arrowhead) in the hypoblast. (D) Tailbud stage embryo. Expression domains in the neural plate comprise two stripes in an intermediate position (IS) and two further lateral and caudal stripes (LS). deltaD is also expressed in the primordium of the trigeminal ganglion (asterisk) and in a horseshoe-like domain (2). (E) Two-somite stage embryo. Two medial stripes (MS) in the neural plate at the level of the trunk. In addition, two further expression domains (3 + 4) appear in the prospective brain region. (F) Five-somite stage embryo. Intermediate and lateral stripes are displaced due to neurulation movements. The white arrowhead indicates the presomitic expression band. (G) 12-somite stage embryo. In addition to presomitic bands (white arrowhead), the anterior halves of the somites contain deltaD transcripts (black arrows). (H) 20-somite stage embryo. Expression domain 2 has shifted to a new position, whereas expression domain 3 is still in place. In addition deltaD transcripts can be detected in the anlage of the epiphysis (E). The white arrowhead indicates the presomitic expression band.

View larger version (28K): [in a new window]   Fig. 2. (A) The structure of the deltaD locus was deduced from the sequence of 12.5 kb of genomic DNA isolated from two overlapping phage clones. The structures of the various constructs used for transformation are also shown. The transcriptional start point is indicated by an arrow. The 11 exons and the 5'- and the 3'-untranslated regions are indicated by large and small white boxes, respectively. The two conserved regions Homology I (HI) and Homology II (HII) are represented by dark-grey boxes. The light-grey box at the end of the construct deltaD:myc represents exons 10 and 11 derived from the deltaD cDNA and including five myc epitopes fused in frame with the genomic deltaD DNA (see Materials and Methods). (B) Number of independent insertions and their classification according to the gfp expression level. Strong expression indicates the detection of fluorescent GFP; weakly expressed insertions do not produce fluorescent GFP, but gfp transcripts are detectable by in situ hybridisation. (C) Names of the transgenic lines used in this study.

View larger version (99K): [in a new window]   Fig. 3. Comparison of after eightAR33 (aeiAR33) and aeiAR33; Tg(dlD:myc)kca5 embryos. (A,A') 14-somite stage embryos, arrows indicate the last formed somites. (A) aeiAR33 mutant. No normal somites form beyond the eighth somite. (A') aeiAR33; Tg(dlD:myc)kca5 embryos have normal somites. (B) Western blot loaded with protein extracts from 24 hpf embryos of different genotypes, probed with an anti-myc antibody. Lane 1, wild-type; lane 2, progeny of aeiAR33; Tg(dlD:myc)kca5 x wild type; lane 3, progeny of aeiAR33; Tg(dlD:myc)kca5 x aeiAR33; Tg(dlD:myc)kca5; lane 4, progeny of deltaD:Gal4 x UAS:myc:notch1a-intra (positive control). The size of the deltaD:myc protein is approximately 91 kDa, that of notch-intra:myc 92 kDa. The left and the right lanes show the See Blue Pre-Stained Standards marker (Novex). (C,C',C'') 14-somite stage. The pattern of expression of her1 is aberrant in aeiAR33 (C) and indistinguishable from that in aeiAR33; Tg(dlD:myc)kca5 (C') or in wild type (C''). (D,D',D'') Four-somite stage. The number of primary sensory neurones (islet1 in situ hybridisation) is increased in aeiAR33 (D), but normal in the transformants (D') and the wild-type embryos (D'').

View larger version (92K): [in a new window]   Fig. 4. GFP expression driven by the deltaD promoter in transgenic embryos. Living embryos carrying the 3.5gfp+3' construct at tailbud stage (A,A'), 14-somite (B,B'), four-somite (C,C') and 22-somite (D,D') stages show GFP expression in neural and mesodermal domains. This pattern is nearly identical to that of the endogenous gene with the exception of the somitic expression, which is much weaker in the transgenic embryos. (E,E') Strong somitic GFP expression at the 20-somite stage in an embryo carrying the 61.8-1.3gfp construct. (F) Subcellular distribution of GFP within the trunk neural tube of an embryo carrying the construct 3.5gfp+3'. GFP fluorescence was detected by stereomicroscopy in A-E'; (F) GFP fluorescence analysed by laser confocal microscopy. Tailbud, black arrowhead; presomitic expression, white arrowhead; somitic expression, black arrows; intermediary and medial expression stripes, IS and MS; epiphysis, E; telencephalic and diencephalic expression domains, 2 and 3.

View larger version (134K): [in a new window]   Fig. 5. Distribution of gfp transcripts in mesodermal derivatives of embryos carrying different gfp constructs (B-D) in comparison with the expression of the endogenous gene (A). 14-somite stage embryos. (A) deltaD transcripts are visible in the tailbud (black arrowhead), in one to two transverse bands in the presomitic mesoderm (white arrowhead) and in the anterior halves of the somites (black arrows) decreasing in intensity from caudal to rostral. (B) Embryos carrying the 0.5gfp+3' construct reveal strong expression in the tailbud and in the posterior half of the somites from the eighth somite onwards. (C) Embryos carrying the 63.5-0.5gfp construct show gfp transcripts only in newly formed somites. (D) Embryos carrying the 1.3gfp construct also reveal gfp transcription in newly formed somites and in the presomitic mesoderm in a stripe pattern, whereas the tailbud is transcript free.

View larger version (138K): [in a new window]   Fig. 6. Neuroectodermal distribution of gfp transcripts in embryos carrying different constructs (B-E,G-J,L-O) in comparison with transcripts of the endogenous gene (A,F,K). Tailbud stage (A-E), two-somite stage (F-J) and 12-somite stage embryos (K-O). Embryos carrying the 6gfp construct (B,G,L) show all the neural expression domains of deltaD with the exception of the early expression of domain 2 and anterior region of domain 3 (A,F,K). Embryos carrying the 63.5-1.3gfp construct show no neural gfp expression at all (C,H,M). gfp expression in neural domains in embryos carrying the 0.5gfp+3' construct (D,I,N) shows a striking delay in comparison with the endogenous gene. Expression domain 2 requires sequence elements located both in the upstream and the downstream regions, as only progeny of transformants carrying the 3.5gfp+3' (E,J,O) and 6gfp+3' construct show gfp transcripts here. Prosencephalic, mesencephalic and rhombencephalic expression domains, 1-4; developing trigeminal ganglion, asterisk; epiphysis, E; medial, intermediary and lateral expression stripes, MS, IS and LS.

View larger version (91K): [in a new window]   Fig. 7. Comparison of the distribution of gfp transcripts driven by 61.8-1.3gfp and 63.5-1.8gfp with that of neurogenin 1 (ngn1), zash1a and zash1b. (A-E) Two-somite stage and (F-J) 12-somite stage embryos. Neural gfp expression driven by the 61.8-1.3gfp construct (A,F) comprises gfp transcripts in the primordium of the trigeminal ganglion (asterisk), in domains 1 and 4 and in the lateral, intermediate and medial stripes (LS, IS, MS) of the neural plate, but lack expression in domains 2 and 3 and the epiphysis anlage (E). This expression is similar to that of neurogenin 1 (B,G). Embryos carrying the 63.5-1.8gfp construct (C,H) reveal a complementary expression pattern in the distribution of gfp transcripts, which is highly reminiscent of the expression of zash1a (D,I) and zash1b (E,J).

View larger version (22K): [in a new window]   Fig. 8. (A) CAT reporter gene constructs. Structures are as in Fig. 2. (B-E) Assays of transient CAT expression in zebrafish embryos. (B) In the presence of neurogenin 1, constructs with HI (3.5CAT and 6CAT) express a high level of CAT, whereas constructs without HI (0.5CAT, 1.3CAT, 1.8CAT and 63.5-1.8 CAT) show a much weaker response. (C) In the presence of zash1a, constructs without HII (0.5CAT, 1.3CAT and 61.8-1.3 CAT) express little CAT protein and constructs with HII (1.8CAT, 3.5CAT and 6CAT) generate larger amounts. (D) HI is sufficient for a strong CAT response in the presence of neurogenin 1 (HI 0.5 CAT). Mutation of either the distal (HI mut1 0.5 CAT) or the proximal E-box (HI mut2 0.5 CAT) leads to a decrease in the amount of CAT produced. When both E-boxes are mutated (HI mut1+2 0.5 CAT) CAT production drops to the basal level. (E) In the presence of zash1a, mutation of the distal E-box in HII (asterisk in 6 HII mut1 CAT) has no apparent effect on the levels of CAT, whereas mutation of the proximal E-box (asterisk in 6 HII mut2 CAT) results in a decrease in the amount of CAT. Mutation of both E-boxes simultaneously (asterisks in 6 HII mut1+2 CAT) leads to no further reduction.

View larger version (35K): [in a new window]   Fig. 9. Organisation of cis-regulatory regions of the deltaD locus. Six regulatory regions and two quantitative enhancers (en) were identified within 12.5 kb of the deltaD locus. Representative in situ hybridisation experiments have been allocated to the corresponding genomic regions with respect to expression in neuroectodermal (above) or mesodermal derivatives (below). Regulatory regions sufficient to direct expression in specific expression domains are indicated by the surrounding bars, whereas regulatory regions necessary for transgene expression (HI and HII) are indicated by boxes.